U.S. patent application number 11/891027 was filed with the patent office on 2008-08-07 for immunodeficiency recombinant poxvirus.
Invention is credited to William I. Cox, Genoveffa Franchini, Robert Gallo, Enzo Paoletti, James Tartaglia.
Application Number | 20080188640 11/891027 |
Document ID | / |
Family ID | 26918187 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080188640 |
Kind Code |
A1 |
Paoletti; Enzo ; et
al. |
August 7, 2008 |
Immunodeficiency recombinant poxvirus
Abstract
Attenuated recombinant viruses containing DNA encoding an
immunodeficiency virus and/or CTL antigen, as well as methods and
compositions employing the viruses, expression products therefrom,
and antibodies generated from the viruses or expression products,
are disclosed and claimed. The recombinant viruses can be NYVAC or
ALVAC recombinant viruses. The DNA can code for at least one of:
HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef (BRU)CTL,
pol(IIIB)CTL, ELDKWA or LDKW epitopes, preferably HIV1gag(+pro)
(IIIB), gp120(MN) (+transmembrane), two (2) nef(BRU)CTL and three
(3) pol(IIIB)CTL epitopes; or two ELDKWA in gp120 V3 or another
region or in gp160. The two (2) nef(BRU)CTL and three (3)
pol(IIIB)CTL epitopes are preferably CTL1, CTL2, pol1, pol2 and
pol3. The recombinant viruses and gene products therefrom and
antibodies generated by the viruses and gene products have several
preventive, therapeutic and diagnostic uses. DNA from the
recombinant viruses are useful as probes or, for generating PCR
primers or for immunization. Also disclosed and claimed are HIV
immunogens and modified gp160 and gp120.
Inventors: |
Paoletti; Enzo; (Delmar,
NY) ; Tartaglia; James; (Schenectady, NY) ;
Cox; William I.; (East Greenbush, NY) ; Gallo;
Robert; (Baltimore, MD) ; Franchini; Genoveffa;
(Washington, DC) |
Correspondence
Address: |
Robert Yoshida;Sanofi Pasteur Inc.
Intellectual Property-Knerr Building, One Discovery Drive
Swiftwater
PA
18370
US
|
Family ID: |
26918187 |
Appl. No.: |
11/891027 |
Filed: |
August 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441788 |
May 20, 2003 |
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11891027 |
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09136159 |
Aug 14, 1998 |
6596279 |
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10441788 |
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08417210 |
Apr 5, 1995 |
5863542 |
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09136159 |
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08223842 |
Apr 6, 1994 |
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08417210 |
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07897382 |
Jun 11, 1992 |
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08223842 |
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07715921 |
Jun 14, 1991 |
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07897382 |
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08105483 |
Aug 12, 1993 |
5494807 |
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07715921 |
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07847951 |
Mar 6, 1992 |
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08105483 |
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07713967 |
Jun 11, 1991 |
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07847951 |
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07666056 |
Mar 7, 1991 |
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07713967 |
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Current U.S.
Class: |
530/329 ;
530/330; 530/350; 536/23.72 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2710/24122 20130101; C12N 2710/24143 20130101; C07K 2319/00
20130101; C12N 2740/16122 20130101; C12N 2760/12022 20130101; A61P
37/02 20180101; C12N 2750/14322 20130101; C12N 2770/24022 20130101;
C07K 14/005 20130101; A61P 37/04 20180101; C12N 2740/13022
20130101; A61P 31/12 20180101; C12N 2740/16222 20130101; C12N
2760/18722 20130101; C12N 2770/24143 20130101; C12N 2710/16622
20130101; C12N 2770/24122 20130101; A61P 31/18 20180101; C12N
2740/15022 20130101; C12N 2710/24043 20130101; C12N 2740/16322
20130101; C12N 2710/16222 20130101; C12N 2760/18122 20130101; C12N
2760/20122 20130101; C12N 2760/20022 20130101; C12N 2760/16122
20130101; C12N 2710/16722 20130101; C07K 14/33 20130101; C12N
2730/10122 20130101; A61K 2039/5254 20130101; C12N 2710/24022
20130101; A61K 39/00 20130101 |
Class at
Publication: |
530/329 ;
530/330; 530/350; 536/23.72 |
International
Class: |
C07K 7/00 20060101
C07K007/00; C07K 14/00 20060101 C07K014/00; C07H 21/04 20060101
C07H021/04 |
Claims
1-35. (canceled)
36. A protein having an amino acid sequence comprising the amino
acid sequence of HIV1 gp120, wherein the amino acid sequence of the
gp120 is modified so as to contain an epitope not naturally
occurring in HIV1 gp120.
37. The protein of claim 36, wherein the epitope is a B-cell
epitope.
38. The protein of claim 36, wherein the amino acid sequence of the
gp120 is modified in the V3 loop so as to contain the epitope.
39. The protein of claim 38, wherein the epitope is a B-cell
epitope.
40. The protein of claim 38, wherein the epitope is ELDKWA or
LDKW.
41. The protein of claim 36, wherein the amino acid sequence of the
gp120 is modified to contain at least one of HIV1gag(+ pro) (IIIB),
gp120(MN) (+ transmembrane), nef(BRU)CTL, pol(IIIB)CTL, and ELDKWA
or LDKW epitopes.
42. The protein of claim 41, wherein the amino acid sequence of the
gp120 is modified in the V3 loop to contain the epitope.
43. The protein of claim 38, comprising the amino acid sequence
identified as SEQ ID NO: 137, SEQ ID NO: 140 or SEQ ID NO: 143.
44. The protein having the amino acid sequence identified as SEQ ID
NO: 137, SEQ ID NO: 140 or SEQ ID NO: 143.
45. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 36.
46. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 37.
47. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 38.
48. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 39.
49. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 40.
50. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 41.
51. A nucleic acid molecule comprising a nucleic acid sequence
which encodes the protein of claim 42.
52. A nucleic acid molecule comprising the nucleic acid sequence
which encodes the amino acid sequence identified as SEQ ID NO: 137,
SEQ ID NO: 140 or SEQ ID NO: 143.
53. The nucleic acid molecule of claim 52 selected from the group
consisting of the nucleic acid molecules identified as SEQ ID NO:
135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 141
and SEQ ID NO: 142.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/441,788, filed May 20, 2003, which in turn is a divisional of
application Ser. No. 09/136,159, filed Aug. 14, 1998, now U.S. Pat.
No. 6,596,279, which in turn is a divisional of application Ser.
No. 08/417,210, filed Apr. 5, 1995, now U.S. Pat. No. 5,863,542,
which in turn is a continuation-in-part of application Ser. No.
08/223,842, filed Apr. 6, 1994, abandoned, which in turn is a
continuation-in-part of application Ser. No. 07/897,382, filed Jun.
11, 1992, abandoned, which in turn is a continuation-in-part of
application Ser. No. 07/715,921, filed Jun. 14, 1991, abandoned.
This application is also a continuation-in-part of application Ser.
No. 08/105,483, filed Aug. 12, 1993, now U.S. Pat. No. 5,494,807
which in turn is a continuation of application Ser. No. 07/847,951,
filed Mar. 6, 1992, abandoned, which in turn is a
continuation-in-part of application Ser. No. 07/713,967, filed Jun.
11, 1991, abandoned, which in turn is a continuation in part of
application Ser. No. 07/666,056, filed Mar. 7, 1991, abandoned.
Mention is also made of co-pending application Ser. No. 08/184,009,
filed Jan. 19, 1994, now U.S. Pat. No. 5,833,975, as a
continuation-in-part of application Ser. No. 08/007,115, filed Jan.
21, 1993, abandoned. Each of the aforementioned and
above-referenced applications is hereby incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a modified poxvirus and to
methods of making and using the same. More in particular, the
invention relates to improved vectors for the insertion and
expression of foreign genes for use as safe immunization vehicles
to elicit an immune response against immunodeficiency virus. Thus,
the invention relates to a recombinant poxvirus, which virus
expresses gene products of immunodeficiency virus and to
immunogenic compositions which induce an immunological response
against immunodeficiency virus infections when administered to a
host, or in vitro (e.g. ex vivo modalities) as well as to the
products of expression of the poxvirus which by themselves are
useful for eliciting an immune response e.g., raising antibodies,
which antibodies are useful against immunodeficiency virus
infection, in either seropositive or seronegative individuals, or
are useful if isolated from an animal or human for preparing a
diagnostic kit, test or assay for the detection of the virus or
infected cells.
[0003] Several publications are referenced in this application.
Full citation to these references is found at the end of the
specification immediately preceding the claims or where the
publication is mentioned; and each of these publications is hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] Vaccinia virus and more recently other poxviruses have been
used for the insertion and expression of foreign genes. The basic
technique of inserting foreign genes into live infectious poxvirus
involves recombination between pox DNA sequences flanking a foreign
genetic element in a donor plasmid and homologous sequences present
in the rescuing poxvirus (Piccini et al., 1987).
[0005] Specifically, the recombinant poxviruses are constructed in
two steps known in the art and analogous to the methods for
creating synthetic recombinants of poxviruses such as the vaccinia
virus and avipox virus described in U.S. Pat. Nos. 4,769,330,
4,772,848, 4,603,112, 5,100,587, and 5,179,993, the disclosures of
which are incorporated herein by reference.
[0006] First, the DNA gene sequence to be inserted into the virus,
particularly an open reading frame from a non-pox source, is placed
into an E. coli plasmid construct into which DNA homologous to a
section of DNA of the poxvirus has been inserted. Separately, the
DNA gene sequence to be inserted is ligated to a promoter. The
promoter-gene linkage is positioned in the plasmid construct so
that the promoter-gene linkage is flanked on both ends by DNA
homologous to a DNA sequence flanking a region of pox DNA
containing a nonessential locus. The resulting plasmid construct is
then amplified by growth within E. coli bacteria (Clewell, 1972)
and isolated (Clewell et al., 1969; Maniatis et al., 1982).
[0007] Second, the isolated plasmid containing the DNA gene
sequence to be inserted is transfected into a cell culture, e.g.
chick embryo fibroblasts, along with the poxvirus. Recombination
between homologous pox DNA in the plasmid and the viral genome
respectively gives a poxvirus modified by the presence, in a
nonessential region of its genome, of foreign DNA sequences. The
term "foreign" DNA designates exogenous DNA, particularly DNA from
a non-pox source, that codes for gene products not ordinarily
produced by the genome into which the exogenous DNA is placed.
[0008] Genetic recombination is in general the exchange of
homologous sections of DNA between two strands of DNA. In certain
viruses RNA may replace DNA. Homologous sections of nucleic acid
are sections of nucleic acid (DNA or RNA) which have the same
sequence of nucleotide bases.
[0009] Genetic recombination may take place naturally during the
replication or manufacture of new viral genomes within the infected
host cell. Thus, genetic recombination between viral genes may
occur during the viral replication cycle that takes place in a host
cell which is co-infected with two or more different viruses or
other genetic constructs. A section of DNA from a first genome is
used interchangeably in constructing the section of the genome of a
second co-infecting virus in which the DNA is homologous with that
of the first viral genome.
[0010] However, recombination can also take place between sections
of DNA in different genomes that are not perfectly homologous. If
one such section is from a first genome homologous with a section
of another genome except for the presence within the first section
of, for example, a genetic marker or a gene coding for an antigenic
determinant inserted into a portion of the homologous DNA,
recombination can still take place and the products of that
recombination are then detectable by the presence of that genetic
marker or gene in the recombinant viral genome. Additional
strategies have recently been reported for generating recombinant
vaccinia virus.
[0011] Successful expression of the inserted DNA genetic sequence
by the modified infectious virus requires two conditions. First,
the insertion must be into a nonessential region of the virus in
order that the modified virus remain viable. The second condition
for expression of inserted DNA is the presence of a promoter in the
proper relationship to the inserted DNA. The promoter must be
placed so that it is located upstream from the DNA sequence to be
expressed.
[0012] Vaccinia virus has been used successfully to immunize
against smallpox, culminating in the worldwide eradication of
smallpox in 1980. In the course of its history, many strains of
vaccinia have arisen. These different strains demonstrate varying
immunogenicity and are implicated to varying degrees with potential
complications, the most serious of which are post-vaccinial
encephalitis and generalized vaccinia (Behbehani, 1983).
[0013] With the eradication of smallpox, a new role for vaccinia
became important, that of a genetically engineered vector for the
expression of foreign genes. Genes encoding a vast number of
heterologous antigens have been expressed in vaccinia, often
resulting in protective immunity against challenge by the
corresponding pathogen (reviewed in Tartaglia et al., 1990a).
[0014] The genetic background of the vaccinia vector has been shown
to affect the protective efficacy of the expressed foreign
immunogen. For example, expression of Epstein Barr Virus (EBV)
gp340 in the Wyeth vaccine strain of vaccinia virus did not protect
cottontop tamarins against EBV virus induced lymphoma, while
expression of the same gene in the WR laboratory strain of vaccinia
virus was protective (Morgan et al., 1988).
[0015] A fine balance between the efficacy and the safety of a
vaccinia virus-based recombinant vaccine candidate is extremely
important. The recombinant virus must present the immunogen(s) in a
manner that elicits a protective immune response in the vaccinated
animal but lacks any significant pathogenic properties. Therefore
attenuation of the vector strain would be a highly desirable
advance over the current state of technology.
[0016] A number of vaccinia genes have been identified which are
non-essential for growth of the virus in tissue culture and whose
deletion or inactivation reduces virulence in a variety of animal
systems.
[0017] The gene encoding the vaccinia virus thymidine kinase (TK)
has been mapped (Hruby et al., 1982) and sequenced (Hruby et al.,
1983; Weir et al., 1983). Inactivation or complete deletion of the
thymidine kinase gene does not prevent growth of vaccinia virus in
a wide variety of cells in tissue culture. TK.sup.- vaccinia virus
is also capable of replication in vivo at the site of inoculation
in a variety of hosts by a variety of routes.
[0018] It has been shown for herpes simplex virus type 2 that
intravaginal inoculation of guinea pigs with TK.sup.- virus
resulted in significantly lower virus titers in the spinal cord
than did inoculation with TK.sup.+ virus (Stanberry et al., 1985).
It has been demonstrated that herpesvirus encoded TK activity in
vitro was not important for virus growth in actively metabolizing
cells, but was required for virus growth in quiescent cells
(Jamieson et al., 1974).
[0019] Attenuation of TK.sup.- vaccinia has been shown in mice
inoculated by the intracerebral and intraperitoneal routes (Buller
et al., 1985). Attenuation was observed both for the WR
neurovirulent laboratory strain and for the Wyeth vaccine strain.
In mice inoculated by the intradermal route, TK.sup.- recombinant
vaccinia generated equivalent anti-vaccinia neutralizing antibodies
as compared with the parental TK.sup.+ vaccinia virus, indicating
that in this test system the loss of TK function does not
significantly decrease immunogenicity of the vaccinia virus vector.
Following intranasal inoculation of mice with TK.sup.- and TK.sup.+
recombinant vaccinia virus (WR strain), significantly less
dissemination of virus to other locations, including the brain, has
been found (Taylor et al., 1991a).
[0020] Another enzyme involved with nucleotide metabolism is
ribonucleotide reductase. Loss of virally encoded ribonucleotide
reductase activity in herpes simplex virus (HSV) by deletion of the
gene encoding the large subunit was shown to have no effect on
viral growth and DNA synthesis in dividing cells in vitro, but
severely compromised the ability of the virus to grow on serum
starved cells (Goldstein et al., 1988). Using a mouse model for
acute HSV infection of the eye and reactivatable latent infection
in the trigeminal ganglia, reduced virulence was demonstrated for
HSV deleted of the large subunit of ribonucleotide reductase,
compared to the virulence exhibited by wild type HSV (Jacobson et
al., 1989).
[0021] Both the small (Slabaugh et al., 1988) and large (Schmidtt
et al., 1988) subunits of ribonucleotide reductase have been
identified in vaccinia virus. Insertional inactivation of the large
subunit of ribonucleotide reductase in the WR strain of vaccinia
virus leads to attenuation of the virus as measured by intracranial
inoculation of mice (Child et al., 1990).
[0022] The vaccinia virus hemagglutinin gene (HA) has been mapped
and sequenced (Shida, 1986). The HA gene of vaccinia virus is
nonessential for growth in tissue culture (Ichihashi et al., 1971).
Inactivation of the HA gene of vaccinia virus results in reduced
neurovirulence in rabbits inoculated by the intracranial route and
smaller lesions in rabbits at the site of intradermal inoculation
(Shida et al., 1988). The HA locus was used for the insertion of
foreign genes in the WR strain (Shida et al., 1987), derivatives of
the Lister strain (Shida et al., 1988) and the Copenhagen strain
(Guo et al., 1989) of vaccinia virus. Recombinant HA vaccinia virus
expressing foreign genes have been shown to be immunogenic (Guo et
al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al.,
1987) and protective against challenge by the relevant pathogen
(Guo et al., 1989; Shida et al., 1987).
[0023] Cowpox virus (Brighton red strain) produces red
(hemorrhagic) pocks on the chorioallantoic membrane of chicken
eggs. Spontaneous deletions within the cowpox genome generate
mutants which produce white pocks (Pickup et al., 1984). The
hemorrhagic function (u) maps to a 38 kDa protein encoded by an
early gene (Pickup et al., 1986). This gene, which has homology to
serine protease inhibitors, has been shown to inhibit the host
inflammatory response to cowpox virus (Palumbo et al., 1989) and is
an inhibitor of blood coagulation.
[0024] The u gene is present in WR strain of vaccinia virus (Kotwal
et al., 1989b). Mice inoculated with a WR vaccinia virus
recombinant in which the u region has been inactivated by insertion
of a foreign gene produce higher antibody levels to the foreign
gene product compared to mice inoculated with a similar recombinant
vaccinia virus in which the u gene is intact (Zhou et al., 1990).
The u region is present in a defective nonfunctional form in
Copenhagen strain of vaccinia virus (open reading frames B13 and
B14 by the terminology reported in Goebel et al., 1990a,b).
[0025] Cowpox virus is localized in infected cells in cytoplasmic A
type inclusion bodies (ATI) (Kato et al., 1959). The function of
ATI is thought to be the protection of cowpox virus virions during
dissemination from animal to animal (Bergoin et al., 1971). The ATI
region of the cowpox genome encodes a 160 kDa protein which forms
the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al.,
1987). Vaccinia virus, though containing a homologous region in its
genome, generally does not produce ATI. In WR strain of vaccinia,
the ATI region of the genome is translated as a 94 kDa protein
(Patel et al., 1988). In Copenhagen strain of vaccinia virus, most
of the DNA sequences corresponding to the ATI region are deleted,
with the remaining 3' end of the region fused with sequences
upstream from the ATI region to form open reading frame (ORF) A26L
(Goebel et al., 1990a,b).
[0026] A variety of spontaneous (Altenburger et al., 1989; Drillien
et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al.,
1985; Panicali et al., 1981) and engineered (Perkus et al., 1991;
Perkus et al., 1989; Perkus et al., 1986) deletions have been
reported near the left end of the vaccinia virus genome. A WR
strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et
al., 1981; Panicali et al., 1981) was shown to be attenuated by
intracranial inoculation in mice (Buller et al., 1985). This
deletion was later shown to include 17 potential ORFs (Kotwal et
al., 1988b). Specific genes within the deleted region include the
virokine N1L and a 35 kDa protein (C3L, by the terminology reported
in Goebel et al., 1990a,b). Insertional inactivation of N1L reduces
virulence by intracranial inoculation for both normal and nude mice
(Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L
into the medium of vaccinia virus infected cells. The protein
contains homology to the family of complement control proteins,
particularly the complement 4B binding protein (C4 bp) (Kotwal et
al., 1988a). Like the cellular C4 bp, the vaccinia 35 kDa protein
binds the fourth component of complement and inhibits the classical
complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa
protein appears to be involved in aiding the virus in evading host
defense mechanisms.
[0027] The left end of the vaccinia genome includes two genes which
have been identified as host range genes, K1L (Gillard et al.,
1986) and C7L (Perkus et al., 1990). Deletion of both of these
genes reduces the ability of vaccinia virus to grow on a variety of
human cell lines (Perkus et al., 1990).
[0028] Two additional vaccine vector systems involve the use of
naturally host-restricted poxviruses, avipoxviruses. Both
fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to
express foreign gene products. Fowlpox virus (FPV) is the
prototypic virus of the Avipox genus of the Poxvirus family. The
virus causes an economically important disease of poultry which has
been well controlled since the 1920's by the use of live attenuated
vaccines. Replication of the avipox viruses is limited to avian
species (Matthews, 1982) and there are no reports in the literature
of avipoxvirus causing a productive infection in any non-avian
species including man. This host restriction provides an inherent
safety barrier to transmission of the virus to other species and
makes use of avipoxvirus based vaccine vectors in veterinary and
human applications an attractive proposition.
[0029] FPV has been used advantageously as a vector expressing
antigens from poultry pathogens. The hemagglutinin protein of a
virulent avian influenza virus was expressed in an FPV recombinant
(Taylor et al., 1988a). After inoculation of the recombinant into
chickens and turkeys, an immune response was induced which was
protective against either a homologous or a heterologous virulent
influenza virus challenge (Taylor et al., 1988a). FPV recombinants
expressing the surface glycoproteins of Newcastle Disease Virus
have also been developed (Taylor et al., 1990; Edbauer et al.,
1990).
[0030] Despite the host-restriction for replication of FPV and CPV
to avian systems, recombinants derived from these viruses were
found to express extrinsic proteins in cells of nonavian origin.
Further, such recombinant viruses were shown to elicit
immunological responses directed towards the foreign gene product
and where appropriate were shown to afford protection from
challenge against the corresponding pathogen (Tartaglia et al.,
1993a,b; Taylor et al., 1992; 1991b; 1988b).
[0031] In 1983, human immunodeficiency virus type 1 (HIV1) was
identified as the causative agent of AIDS. Twelve years later,
despite a massive, worldwide effort, an effective HIV1 vaccine is
still not available. Recently, however, several reports have
suggested that an efficacious HIV1 vaccine may be attainable. For
example, macaques have been protected against a simian
immunodeficiency virus (SIV) challenge by a vaccination protocol
involving a primary immunization with a vaccinia virus recombinant
expressing the SIV gp160 glycoprotein and a booster immunization
with purified SIV gp160 glycoprotein (Hu et al., 1992). In
addition, chimpanzees have been protected against an HIV1 challenge
with an HIV1 gp120 subunit vaccine (Berman et al, 1990). Chimps
have also been protected against an HIV1 challenge by a vaccination
protocol involving multiple injections of either inactivated HIV1,
gp160 and/or V3 peptide or gp160, p17 (a Gag protein) and/or V3
peptide (Girard et al., 1991). A similar protocol involving
multiple injections of gp160, p17, p24 (a Gag protein), Vif, Nef
and/or V3 peptide has also protected chimps against a challenge of
HIV1-infected cells (Fultz et al., 1992). Furthermore, chimps have
been passively protected by the infusion of HIV1 V3-specific
antibodies (Emini et al., 1992).
[0032] Most of these vaccination protocols have focused on
eliciting an immune response against the HIV1 or SIV envelope
glycoprotein, or more specifically, against the V3 epitope of the
envelope glycoprotein. Unfortunately, different strains of HIV1
exhibit extensive genetic and antigenic variability, especially in
the envelope glycoprotein. Therefore, an effective HIV1 vaccine may
need to elicit an immune response against more than one HIV1
antigen, or one epitope of one HIV1 antigen.
[0033] Contrary to the extensive sequence variability observed in
B-cell epitopes, T-cell epitopes are relatively conserved. For
example, cytotoxic T-lymphocytes (CTL) clones, isolated from an
HIV1-seronegative individual vaccinated with a vaccinia virus
recombinant expressing HIV1 gp160 (LAI strain) and boosted with
purified HIV1 gp160 (LAI), lyse target cells expressing the HIV1 MN
or RF envelope glycoprotein as efficiently as cells expressing the
HIV1 LAI envelope glycoprotein (Hammond et al., 1992). Therefore, a
vaccine that elicits an immune response against relatively
conserved T-cell epitopes may not only be more efficacious against
a homologous challenge, but also more efficacious against a
heterologous challenge.
[0034] HIV1-seronegative individuals have been vaccinated with an
ALVAC recombinant (vCP125) expressing HIV1 gp160, in a prime-boost
protocol similar to the regimen used to vaccinate macaques against
SIV. These ALVAC-based protocols demonstrated the ability of vCP125
to elicit HIV1 envelope-specific CD8.sup.+ CTLs and to enhance
envelope-specific humoral responses observed following a subunit
booster (Pialoux et al., 1995). These results justify the rationale
for a recombinant ALVAC-based HIV1 vaccine.
[0035] Individuals infected with human immunodeficiency virus type
1 (HIV1) initially generate a relatively dynamic and extensive
antiviral immune response, including HIV1-specific neutralizing
antibodies and HIV1-specific CTLs. Despite these responses,
however, the vast majority of HIV1-infected people eventually
succumb to HIV1-associated diseases. Since the immune response
generated by most HIV1-infected people is not protective,
generation of an effective immune response may necessitate that the
immune response be modulated or redirected against HIV1 epitopes
that are not normally or efficiently seen by HIV1-infected
individuals.
[0036] Approximately 40% of the HIV1-specific antibody in
HIV1-seropositive individuals capable of binding HIV1-infected
cells is specific to the third variable region (V3) of the HIV1
envelope glycoprotein (Spear et al, 1994). These results indicate
that the V3 loop is 1) highly immunogenic and 2) exposed on the
surface of infected cells. The amino acid sequence of the V3 loop
varies considerably between different HIV1 isolates. Therefore, a
moderate level of sequence variation does not appear to alter the
structure or immunogenicity of this region of the envelope
glycoprotein. Since the V3 loop is highly immunogenic and its
structure and immunogenicity is not severely affected by sequence
variation, this region of the envelope glycoprotein may be useful
as an immunogenic platform for presenting normally non-immunogenic
linear HIV1 epitopes or heterologous epitopes to the immune
system.
[0037] Sera from HIV1-seropositive individuals can neutralize
lab-adapted strains of HIV1. These sera can also neutralize primary
HIV1 isolates (although 100.times. higher titers are required).
Conversely, sera from individuals vaccinated with HIV1 gp120 can
neutralize lab-adapted strains of HIV1 (although 10.times. higher
titers relative to sera from seropositive individuals are
required), but can not neutralize (at assayable levels) primary
isolates (Hanson, 1994). A significant portion of the neutralizing
activity found in sera from seropositive and gp120-vaccinated
individuals appears to be specific to the V3 loop (Spear et al.,
1994; Berman et al., 1994). Since the V3 loop is hypervariable and
since antibodies against this region may not neutralize primary
isolates or heterologous strains of HIV1, it may be necessary to
develop vaccines that elicit an immune response against epitopes
other than the V3 loop, epitopes that can neutralize a broad
spectrum of HIV1 strains, including primary isolates.
[0038] A monoclonal antibody capable of neutralizing primary HIV1
isolates, as well as a broad spectrum of lab-adapted HIV1 strains,
has been isolated (Conley et al., 1994; Katinger et al., 1992). The
epitope recognized by this monoclonal antibody has been mapped
between amino acids 662 and 667 of HIV1 gp41 and has the amino acid
sequence, ELDKWA (Buchacher et al, 1994). Approximately 80% of the
HIV1 strains from which sequence information has been derived,
including strains from the various HIV1 clades, express the core
binding sequence of this epitope, LDKW (Conley et al., 1994).
Therefore, unlike the V3 loop, this epitope appears to be
relatively well conserved. Unfortunately, this epitope does not
appear to be very immunogenic in its normal configuration. Only
approximately 50% of HIV1-seropositive individuals have a
detectable antibody response to the region of gp41 containing this
epitope (Broliden et al., 1992).
[0039] It can thus be appreciated that provision of an
immunodeficiency virus recombinant poxvirus, and of an immunogenic
composition which induces an immunological response against
immunodeficiency virus infections when administered to host,
particularly a composition having enhanced safety such as NYVAC or
ALVAC based recombinants containing coding for any or all of
HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL
epitopes, pol(IIIB)CTL epitopes; for instance, HIV1gag(+pro)
(IIIB), gp120(MN) (+transmembrane), nefCTL1, nefCTL2,
pol1(PolCTL1), pol2(PolCTL2), pol3(PolCTL3), ELDKWA or LDKW
epitopes (SEQ ID NOS: 147 and 148), especially in an immunogenic
configuration, or any combination thereof, for example all of them
in combination, would be a highly desirable advance over the
current state of technology.
OBJECTS AND SUMMARY OF THE INVENTION
[0040] It is therefore an object of this invention to provide
modified recombinant viruses, which viruses have enhanced safety,
and to provide a method of making such recombinant viruses.
[0041] It is an additional object of this invention to provide a
recombinant poxvirus antigenic, vaccine or immunological
composition having an increased level of safety compared to known
recombinant poxvirus vaccines.
[0042] It is a further object of this invention to provide a
modified vector for expressing a gene product in a host, wherein
the vector is modified so that it has attenuated virulence in the
host.
[0043] It is another object of this invention to provide a method
for expressing a gene product in a cell cultured in vitro using a
modified recombinant virus or modified vector having an increased
level of safety.
[0044] These and other objects and advantages of the present
invention will become more readily apparent after consideration of
the following.
[0045] In one aspect, the present invention relates to a modified
recombinant virus having inactivated virus-encoded genetic
functions so that the recombinant virus has attenuated virulence
and enhanced safety. The functions can be non-essential, or
associated with virulence. The virus is advantageously a poxvirus,
particularly a vaccinia virus or an avipox virus, such as fowlpox
virus and canarypox virus. The modified recombinant virus can
include, within a non-essential region of the virus genome, a
heterologous DNA sequence which encodes an antigen or epitope
derived from immunodeficiency virus and/or CTL epitope such as,
e.g., HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane),
nef(BRU)CTL, pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination
thereof, preferably all of them in combination.
[0046] In another aspect, the present invention relates to an
antigenic, immunological or vaccine composition or a therapeutic
composition for inducing an antigenic or immunological response in
a host animal inoculated with the composition, said vaccine
including a carrier and a modified recombinant virus having
inactivated nonessential virus-encoded genetic functions so that
the recombinant virus has attenuated virulence and enhanced safety.
The virus used in the composition according to the present
invention is advantageously a poxvirus, particularly a vaccinia
virus or an avipox virus, such as fowlpox virus and canarypox
virus. The modified recombinant virus can include, within a
non-essential region of the virus genome, a heterologous DNA
sequence which encodes an antigenic protein, e.g., derived from
immunodeficiency virus and/or CTL such as, HIV1gag(+pro) (IIIB),
gp120 (MN) (+transmembrane), nef(BRU)CTL, pol(IIIB)CTL, ELDKWA,
LDKW epitopes or any combination thereof, preferably all of them in
combination.
[0047] In yet another aspect, the present invention relates to an
immunogenic composition containing a modified recombinant virus
having inactivated nonessential virus-encoded genetic functions so
that the recombinant virus has attenuated virulence and enhanced
safety. The modified recombinant virus includes, within a
non-essential region of the virus genome, a heterologous DNA
sequence which encodes an antigenic protein (e.g., derived from an
immunodeficiency virus and/or CTL such as, HIV1gag(+pro) (IIIB),
gp120(MN) (+transmembrane), nef(BRU)CTL, pol(IIIB)CTL, ELDKWA, LDKW
epitopes or any combination thereof, preferably all of them in
combination) wherein the composition, when administered to a host,
is capable of inducing an immunological response specific to the
antigen.
[0048] In a further aspect, the present invention relates to a
method for expressing a gene product in a cell (e.g. peripheral
blood mononuclear cells (PBMCs) or lymph node mononuclear cells
(LNMC) in vitro by introducing into the cell a modified recombinant
virus having attenuated virulence and coenhanced safety. The
modified recombinant virus can include, within a nonessential
region of the virus genome, a heterologous DNA sequence which
encodes an antigenic protein, e.g. derived from an immunodeficiency
virus such as HIV/gag (+pro) (IIIB), gp120(MN) (+transmembrane),
nef (BRU)CTL, pol (IIIB)CTL, ELDKWA, LDKW epitopes or any
combination thereof, preferably all of them in combination. The
cells can then be reinfused directly into the individual or used to
amplify specific CD8.sup.+ CTL reactivities for reinfusion (Ex vivo
therapy).
[0049] In a further aspect, the present invention relates to a
method for expressing a gene product in a cell cultured in vitro by
introducing into the cell a modified recombinant virus having
attenuated virulence and enhanced safety. The modified recombinant
virus can include, within a non-essential region of the virus
genome, a heterologous DNA sequence which encodes an antigenic
protein, e.g., derived from a immunodeficiency virus such as
HIV1gag (+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL,
pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination thereof,
preferably all of them in combination. The product can then be
administered to individuals or animals to stimulate an immune
response. The antibodies raised can be useful in individuals for
the prevention or treatment of immunodeficiency virus and, the
antibodies from animals can be used in diagnostic kits, assays or
tests to determine the presence or absence in a sample such as sera
of immunodeficiency virus or CTL antigens (and therefore the
absence or presence of the virus of an immune response to the virus
or antibodies).
[0050] In a still further aspect, the present invention relates to
a modified recombinant virus having nonessential virus-encoded
genetic functions inactivated therein so that the virus has
attenuated virulence, and wherein the modified recombinant virus
further contains DNA from a heterologous source in a nonessential
region of the virus genome. The DNA can code for an
immunodeficiency virus and/or CTL antigen such as HIV1gag(+pro)
(IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL, pol(IIIB)CTL,
ELDKWA, LDKW epitopes or any combination thereof, preferably all of
them in combination. In particular, the genetic functions are
inactivated by deleting an open reading frame encoding a virulence
factor or by utilizing naturally host restricted viruses. The virus
used according to the present invention is advantageously a
poxvirus, particularly a vaccinia virus or an avipox virus, such as
fowlpox virus and canarypox virus. Advantageously, the open reading
frame is selected from the group consisting of J2R, B13R+B14R,
A26L, A56R, C7L-K1L, and I4L (by the terminology reported in Goebel
et al., 1990a,b); and, the combination thereof. In this respect,
the open reading frame comprises a thymidine kinase gene, a
hemorrhagic region, an A type inclusion body region, a
hemagglutinin gene, a host range gene region or a large subunit,
ribonucleotide reductase; or, the combination thereof. The modified
Copenhagen strain of vaccinia virus is identified as NYVAC
(Tartaglia et al., 1992). However, the COPAK strain can also be
used in the practice of the invention.
[0051] Most preferably, in recombinant viruses of the invention,
the exogenous DNA codes for HIV1gag(+pro) (IIIB), gp120(MN)
(+transmembrane), two (2) nef(BRU)CTL and three (3) pol(IIIB)CTL
epitopes; or, the exogenous DNA codes for the ELDKWA or LDKW
epitopes, and, is inserted so as to be expressed in a region of
gp120 or gp160 (i.e., the exogenous DNA codes for a ELDKWA or LDKW
modified gp120 or gp160, for instance ELDKWA or LDKW or repeats of
either or both in the V3 loop) such that the epitope is expressed
in an immunogenic configuration. In this most preferred embodiment
it is even more preferred that the two (2) nef(BRU)CTL and three
(3) pol(IIIB)CTL epitopes are CTL1, CTL2, poll, pol2, and pol3. In
another most preferred embodiment the exogenous DNA codes for HIV1
gp120+TM in which the V3 loop has been modified to contain at least
one, and preferably two ELDKWA epitopes.
[0052] In further embodiments, the invention comprehends HIV
immunogens and modified gp160 or gp120. Thus, the invention
includes an HIV immunogen preferably selected from the group
consisting of: HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane),
nef (BRU)CTL, pol(IIIB)CTL, and ELDKWA or LDKW epitopes. The HIV
immunogen of the invention can be part of gp160 or gp120. Thus the
HIV immunogens ELKDKWA or LDKWA, for example, can be a part of a
region of go120 or a region of gp160; for instance, part of
gp120V3. Accordingly, the invention comprehends a gp120 or gp160
modified so as to contain an epitope not naturally occurring in
gp160. The epitope can be a B-cell epitope. The epitope, more
specifically, can be at least one of HIV1gag(+pro) (IIIB),
gp120(MN) (+transmembrane), nef(BRU)CTL, pol(IIIB)CTL, and ELDKWA
or LDKW epitopes. The gp120 can be modified in the V3 loop. The
immunogen and modified gp120 or gp160 can be synthesized by any
suitable vector, including a poxvirus, such as a recombinant of the
invention; or, by any suitable chemical synthesis method such as
the Merrifield Synthesis Method.
[0053] The invention in yet a further aspect relates to the product
of expression of the inventive recombinant poxvirus and uses
therefor, as well as to uses for the inventive immunogens and
modified gp120 and sp160, such as to form antigenic, immunological
or vaccine compositions for treatment, prevention, diagnosis or
testing. The invention in still a further embodiment relates to the
uses of DNA from the recombinants as probes for detecting the
presence or absence of HIV DNA in a sample or for DNA immunization
using an appropriate expression plasmid.
[0054] These and other embodiments are disclosed or are obvious
from and encompassed by the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0056] FIG. 1 schematically shows a method for the construction of
plasmid pSD460 for deletion of thymidine kinase gene and generation
of recombinant vaccinia virus vP410;
[0057] FIG. 2 schematically shows a method for the construction of
plasmid pSD486 for deletion of hemorrhagic region and generation of
recombinant vaccinia virus vP553;
[0058] FIG. 3 schematically shows a method for the construction of
plasmid pMP494.DELTA. for deletion of ATI region and generation of
recombinant vaccinia virus vP618;
[0059] FIG. 4 schematically shows a method for the construction of
plasmid pSD467 for deletion of hemagglutinin gene and generation of
recombinant vaccinia virus vP723;
[0060] FIG. 5 schematically shows a method for the construction of
plasmid pMPCK1.DELTA. for deletion of gene cluster [C7L-K1L] and
generation of recombinant vaccinia virus vP804;
[0061] FIG. 6 schematically shows a method for the construction of
plasmid pSD548 for deletion of large subunit, ribonucleotide
reductase and generation of recombinant vaccinia virus vP866
(NYVAC);
[0062] FIG. 7 schematically shows a method for the construction of
plasmid pRW842 for insertion of rabies glycoprotein G gene into the
TK deletion locus and generation of recombinant vaccinia virus
vP879;
[0063] FIG. 8 shows the DNA sequence (SEQ ID NO:66) of a canarypox
PvuII fragment containing the C5 ORF.
[0064] FIGS. 9A and 9B schematically show a method for the
construction of recombinant canarypox virus vCP65 (ALVAC-RG);
[0065] FIG. 10 shows schematically the ORFs deleted to generate
NYVAC;
[0066] FIG. 11 shows the nucleotide sequence (SEQ ID NO:67) of a
fragment of TROVAC DNA containing an F8 ORF;
[0067] FIG. 12 shows the DNA sequence (SEQ ID NO:68) of a 2356 base
pair fragment of TROVAC DNA containing the F7 ORF;
[0068] FIGS. 13A to 13D show graphs of rabies neutralizing antibody
titers (RFFIT, IU/ml), booster effect of HDC and vCP65 (10.sup.5.5
TCID.sub.50) in volunteers previously immunized with either the
same or the alternate vaccine (vaccines given at days 0, 28 and
180, antibody titers measured at days 0, 7, 28, 35, 56, 173, 187
and 208);
[0069] FIG. 14A to 14C shows the nucleotide sequence of the
H6-promoted HIV1 gp120 (+transmembrane) gene and the I3L-promoted
HIV1gag(+pro) gene contained in pHIV32 (SEQ ID NOS: 78 and 79);
[0070] FIG. 15A to 15F shows the nucleotide sequence of the C3
locus in pVQH6CP3L (SEQ ID NOS: 80 and 81);
[0071] FIG. 16 shows the nucleotide sequence of the I3L-promoted
nef CTL2 epitope and H6-promoted nef CTL1 epitope contained in
p2-60-HIV.3 (SEQ ID NOS: 93-96);
[0072] FIG. 17A to 17C shows the nucleotide sequence of the C6
locus in pC6L (SEQ ID NOS: 97 and 98);
[0073] FIG. 18A to 18B shows the nucleotide sequence of the
I3L-promoted pol2 epitope, H6-promoted poll epitope and
42K-promoted pol3 epitope contained in pC5POLT5A (SEQ ID NOS:
111-115);
[0074] FIG. 19A to 19C shows the nucleotide sequence of the C5
locus in pNC5L-SP5 (SEQ ID NOS: 116 and 117);
[0075] FIG. 20 shows the rabbit antibody responses to the HIV
envelope glycoprotein following immunization with ALVAC, vCP205, or
with peptide CLTB-36;
[0076] FIG. 21 shows the rabbit antibody responses to the HIV MN V3
loop following immunization with ALVAC, vCP205, or with peptide
CLTB-36;
[0077] FIG. 22 shows the guinea pig antibody responses to the HIV
envelope glycoprotein following immunization with ALVAC, vCP205, or
with peptide CLTB-36;
[0078] FIG. 23 shows the guinea pig antibody responses to the HIV
MN V3 loop following immunization with ALVAC, vCP205, or with
peptide CLTB-36;
[0079] FIG. 24 shows in vitro stimulation of HIV-1-specific CTLs
from PBMCs of an HIV-seropositive individual--Patient 1;
[0080] FIG. 25 is as in FIG. 24 but with Patient 2;
[0081] FIG. 26a-c, shows the nucleotide sequence of the H6-promoted
HIV1 gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 135 and 136)
contained in pHIV59 and vCP1307 and the protein expressed (SEQ ID
NO: 137);
[0082] FIG. 27 shows FACS analysis of vCP1307-infected cells (FACS
analysis was performed on HeLa cells infected with ALVAC, vP1286 or
vCP1307 with sera from HIV1-seropositve humans (upper panel) or a
human monoclonal antibody specific for the ELDKWA epitope,
IAM41-2F5 (lower panel));
[0083] FIG. 28a-c shows the nucleotide sequence of the H6-promoted
HIV1 gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 138 and 139)
contained in pHIV60 and vP1313 and the protein expressed (SEQ ID
NO: 140);
[0084] FIG. 29 shows FACS analysis of vP1313-infected cells (FACS
analysis was performed on HeLa cells infected with NYVAC, vP1286 or
vP1313 with sera from HIV1-seropositve humans (upper panel) or a
human monoclonal antibody specific for the ELDKWA epitope,
IAM41-2F5 (lower panel)).
[0085] FIG. 30a-c shows the nucleotide sequence of the H6-promoted
HIV1 gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 141 to 142)
contained in pHIV61 and vP1319 and the protein expressed (SEQ ID
NO: 143);
[0086] FIG. 31 shows the FACS analysis of vP1319-infected cells
(FACS analysis was performed on HeLa cells infected with WR, vP1286
or vP1319 with sera from HIV1-seropositve humans (upper panel), a
human monoclonal antibody specific for the ELDKWA epitope,
IAM41-2F5 (middle panel) or a mouse monoclonal antibody specific
for the V3 loop, 50.1 (lower panel));
[0087] FIGS. 32, 33a, 33b, 33c, 34, 35, 36, 37a, 37b, 37c, 38a,
38b, 38c show comparative body weights (FIG. 32), blood counts
(FIG. 33a-c), creatinine (FIG. 34), SGOT (35), SGPT (FIG. 36),
ELISA (Anti-gp160 MN/BR, -v3MN, -p24, FIGS. 37a-c, 38a-c) of
monkeys inoculated with vCP205 and placebo (FIG. 32). upper
panel=monkeys 1-4, placebo; lower panel=monkeys 5-8 vCP205;
monkeys: 1=open square, 2=open diamond, 3=open triangle, 4=open
circle, 5=darkened square, 6=darkened diamond, 7=darkened triangle,
8--darkened circle; plots of Kg (wt) vs. weeks (inoculations
indicated with arrow). FIG. 33a: leucoytes: left top and bottom
panels=monkeys 1-4, placebo; right top and bottom panels=monkeys
5-8, vCP205; top panels individual WBC counts, key same as FIG. 32
except small darkened circle is mean (m); lower panels differential
cell counts, darkened square=granulo, open square=lympho, darkened
diamond=mono. FIG. 33b: same layout and keying as FIG. 33a, with
upper panels indicating erythrocytes and lower panels indicating
mean corpuscle volume and mean indicated by smaller darkened
circle. FIG. 33c: same layout as FIG. 33b with upper panels
indicating hematocrite and lower panels indicating hemoglobin. FIG.
34: upper bar graphs=monkeys 1-4, placebo; lower bar graph=monkeys
5-8, vCP205; mg/l vs. days, arrow indicates inoculation; monkeys 1
and 5=dark bars, monkeys 2 and 6=double stippling bars (slanted
lines in opposite directions), monkeys 3 and 7=dotted bars, monkeys
4 and 8=single stippling bar (slant lines in one direction), mean
is darkened circles. FIG. 35: same keying as FIG. 34, except IU/l
vs. days. FIG. 36: same keying as FIG. 35. FIGS. 37a-c and 38a-c:
ELISA in placebo administered monkeys (FIGS. 37a-c) and in vCP205
administered monkeys (FIGS. 38a-c), titer (log) vs. weeks, arrow
indicates injection; FIGS. 37a and 38a=anti-gp160 MN/BRU, FIGS. 37b
and 38b=anti-V3MN, FIGS. 37c and 38C=anti-p24; monkeys 1 and 5=open
circle; monkeys 2 and 6=darkened circle; monkeys 3 and 7=open
inverted triangle; monkeys 4 and 8=darkened inverted triangle);
[0088] FIG. 39 shows anti-HIV1 (MN) neutralizing antibodies in
monkeys inoculated with vCP205 (keying same as FIG. 38a-c);
and,
[0089] FIGS. 40, 41a, 41b, 41c, 42, 43a, 43b, 43c, 43d, 44a, 44b,
45a, 45b, 46a, 46b, 47a, and 47b show leucocyte counts (FIG. 40),
blood counts (erythrocytes FIG. 41a, hematocrite FIG. 41b,
reticulocytes FIG. 41c), prothrombin (FIG. 42), biochemical results
(total cholesterol, total proteins, glucose FIG. 43a; sodium,
potassium FIG. 43b; creatinine, bilirubin FIG. 43c;
SGOTransaminase, SGPTransaminase, alkaline phosphatase FIG. 43d),
gp160 MN/BRU ELISA (control FIG. 44a, test animals FIG. 44b), V3 MN
ELISA (control FIG. 46a, test animals FIG. 46b), and nef ELISA
(control FIG. 47a, test animals FIG. 47b) in monkeys inoculated
with vCP300 and a placebo (FIG. 40: layout same as FIG. 33a; keying
same as FIG. 33a, except in upper panels, mean is dotted circle
(left) and open circle (right) and in lower panels decimal instead
of percentage and darkened square=neutro, open diamond=eosino, and
darkened triangle=baso. FIG. 41a: layout same as FIG. 33b, keying
same as FIG. 33a, except mean is dotted circle (left) and open
circle (right). FIG. 41b: layout same as FIG. 33c, keying same as
FIG. 41a. FIG. 41c: layout and keying same as FIG. 41b, upper
panels=reticulocytes, lower panels=thrombocytes. FIG. 42: upper
panel=placebo, lower panel=vCP300, keying same as FIG. 41c. FIG.
43a: top=cholesterol, middle=proteins, lower=glucose; open
circle=placebo, darkened circle=vCP300. FIG. 34b: top=sodium,
lower=potassium; keying same as FIG. 43a. FIG. 43c: top=creatinine,
lower=bilirulain; keying same as FIG. 43a. FIG. 43d: top=SGOT,
middle=SGPT, lower=alkaline phosphatases; keying same as FIG. 43a.
FIGS. 44a, 44b: gp160 MN/BRU ELISA, control and vCP300,
respectively; keying same as FIGS. 37a and 38a, respectively. FIGS.
45a, 45b: V3 MN ELISA, control and vCP300, respectively; keying
same as FIGS. 37b and 38b, respectively. FIGS. 46a, 46b: p34 ELISA,
control and vCP300, respectively; keying same as FIGS. 37c and 38c,
respectively. FIG. 47a, 47b, nef ELISA, control and vCP300,
respectively; keying as in FIGS. 44a-46b).
DETAILED DESCRIPTION OF THE INVENTION
[0090] To develop a new vaccinia vaccine strain, NYVAC (vP866), the
Copenhagen vaccine strain of vaccinia virus was modified by the
deletion of six nonessential regions of the genome encoding known
or potential virulence factors. The sequential deletions are
detailed below. All designations of vaccinia restriction fragments,
open reading frames and nucleotide positions are based on the
terminology reported in Goebel et al., 1990a,b.
[0091] The deletion loci were also engineered as recipient loci for
the insertion of foreign genes.
[0092] The regions deleted in NYVAC are listed below. Also listed
are the abbreviations and open reading frame designations for the
deleted regions (Goebel et al., 1990a,b) and the designation of the
vaccinia recombinant (vP) containing all deletions through the
deletion specified: [0093] (1) thymidine kinase gene (TK; J2R)
vP410; [0094] (2) hemorrhagic region (u; B13R+B14R) vP553; [0095]
(3) A type inclusion body region (ATI; A26L) vP618; [0096] (4)
hemagglutinin gene (HA; A56R) vP723; [0097] (5) host range gene
region (C7L-K1L) vP804; and [0098] (6) large subunit,
ribonucleotide reductase (I4L) vP866 (NYVAC).
[0099] NYVAC is a genetically engineered vaccinia virus strain that
was generated by the specific deletion of eighteen open reading
frames encoding gene products associated with virulence and host
range. NYVAC is highly attenuated by a number of criteria including
i) decreased virulence after intracerebral inoculation in newborn
mice, ii) inocuity in genetically (nu.sup.+/nu.sup.+) or chemically
(cyclophosphamide) immunocompromised mice, iii) failure to cause
disseminated infection in immunocompromised mice, iv) lack of
significant induration and ulceration on rabbit skin, v) rapid
clearance from the site of inoculation, and vi) greatly reduced
replication competency on a number of tissue culture cell lines
including those of human origin. Nevertheless, NYVAC based vectors
induce excellent responses to extrinsic immunogens and provided
protective immunity.
[0100] TROVAC refers to an attenuated fowlpox that was a
plaque-cloned isolate derived from the FP-1 vaccine strain of
fowlpoxvirus which is licensed for vaccination of 1 day old chicks.
ALVAC is an attenuated canarypox virus-based vector that was a
plaque-cloned derivative of the licensed canarypox vaccine, Kanapox
(Tartaglia et al., 1992). ALVAC has some general properties which
are the same as some general properties of Kanapox. ALVAC-based
recombinant viruses expressing extrinsic immunogens have also been
demonstrated efficacious as vaccine vectors (Tartaglia et al., 1993
a,b). This avipox vector is restricted to avian species for
productive replication. On human cell cultures, canarypox virus
replication is aborted early in the viral replication cycle prior
to viral DNA synthesis. Nevertheless, when engineered to express
extrinsic immunogens, authentic expression and processing is
observed in vitro in mammalian cells and inoculation into numerous
mammalian species induces antibody and cellular immune responses to
the extrinsic immunogen and provides protection against challenge
with the cognate pathogen (Taylor et al., 1992; Taylor et al.,
1991). Recent Phase I clinical trials in both Europe and the United
States of a canarypox/rabies glycoprotein recombinant (ALVAC-RG)
demonstrated that the experimental vaccine was well tolerated and
induced protective levels of rabiesvirus neutralizing antibody
titers (Cadoz et al., 1992; Fries et al., 1992). Additionally,
peripheral blood mononuclear cells (PBMCs) derived from the
ALVAC-RG vaccinates demonstrated significant levels of lymphocyte
proliferation when stimulated with purified rabies virus (Fries et
al., 1992).
[0101] NYVAC, ALVAC and TROVAC have also been recognized as unique
among all poxviruses in that the National Institutes of Health
("NIH") (U.S. Public Health Service), Recombinant DNA Advisory
Committee, which issues guidelines for the physical containment of
genetic material such as viruses and vectors, i.e., guidelines for
safety procedures for the use of such viruses and vectors which are
based upon the pathogenicity of the particular virus or vector,
granted a reduction in physical containment level: from BSL2 to
BSL1. No other poxvirus has a BSL1 physical containment level. Even
the Copenhagen strain of vaccinia virus--the common smallpox
vaccine--has a higher physical containment level; namely, BSL2.
Accordingly, the art has recognized that NYVAC, ALVAC and TROVAC
have a lower pathogenicity than any other poxvirus.
[0102] Both NYVAC- and ALVAC-based recombinant viruses have been
shown to stimulate in vitro specific CD8.sup.+ CTLs from human
PBMCs (Tartaglia et al., 1993a). Mice immunized with NYVAC or ALVAC
recombinants expressing various forms of the HIV-1 envelope
glycoprotein generated both primary and memory HIV specific CTL
responses which could be recalled by a second inoculation
(Tartaglia et al., 1993a; Cox et al., 1993). ALVAC-env and
NYVAC-env recombinants (expressing the HIV-1 envelope glycoprotein)
stimulated strong HIV-specific CTL responses from peripheral blood
mononuclear cells (PBMC) of HIV-1 infected individuals (Tartaglia
et al., 1993a; Cox et al., 1993). Acutely infected autologous PBMC
were used as stimulator cells for the remaining PBMC. After 10 days
incubation in the absence of exogenous IL-2, the cells were
evaluated for CTL activities. NYVAC-env and ALVAC-env stimulated
high levels of anti-HIV activities in mice.
[0103] Applicants have generated an ALVAC recombinant, vCP300
(ALVAC-MN120TMGNP), that expresses numerous HIV1 antigens and HIV1
T-cell epitopes. vCP300 expresses the HIV1 (IIIB) gag (and
protease) proteins. (Expression of the protease protein allows the
gag polyprotein to be correctly processed.) vCP300 also expresses a
form of the HIV1 (MN) envelope glycoprotein in which gp120 is fused
to the transmembrane anchor sequence derived from gp41. vP300 also
expresses two (2) HIV1 (BRU) nef CTL epitopes and three (3) HIV1
(IIIB) pol CTL epitopes. vCP300 does not, however, express a
functional reverse transcriptase activity. vCP300 also does not
express a functional nef gene product; a protein associated with
pathogenicity in the SIV-macaque model system and HIV1 virulence
(Miller et al, 1994; Spina et al, 1994). Therefore, vCP300
expresses immunologically important antigens and/or epitopes from
gag, env, pol and nef, but does not express the potentially
detrimental enzymatic and/or pathogenic activities associated with
pol and nef.
[0104] As previously mentioned, vCP300 expresses a form of HIV1
envelope glycoprotein in which the vast majority of the gp41
sequence is deleted. Since most of the immunologically important
epitopes associated with the HIV1 envelope glycoprotein are found
on gp120, rather than gp41, it is assumed that the immunogenicity
of the envelope glycoprotein expressed by this recombinant is not
adversely affected. In fact, in a side-by-side analysis, an HIV1
gp120 subunit vaccine was able to protect chimpanzees against an
HIV1 challenge, whereas an HIV1 gp160 subunit vaccine was not
(Berman et al., 1990). It is not known why the efficacy of these
two vaccines is different. However, it is known that antibodies
against an epitope gp41 can enhance HIV1 infection in vitro
(Robinson et al., 1990). Furthermore, it is known that antibodies
to a putative immunosuppressive region of gp41 are associated with
the absence of AIDS in HIV1-seropositive individuals, suggesting a
potential role in pathogenicity for this region (Klasse et al.,
1988). In addition, it is known that antibodies to the C-terminal
region of gp41 can cross-react with HLA class II antigens (Golding
et al., 1988) and inhibit antigen-specific lymphoproliferative
responses (Golding et al., 1989). Since the envelope glycoprotein
expressed by vCP300 does not contain any gp41 sequence, except for
the 28 amino acids associated with the transmembrane region, the
potentially detrimental effects associated with gp41 are avoided.
Furthermore, the envelope glycoprotein expressed by vCP300 does not
contain the immunodominant epitope on gp41 that is recognized by
antisera from every HIV1-seropositive individual from every stage
of an HIV1 infection (Shafferman et al., 1989). Therefore,
diagnostic tests based upon reactivity against this epitope can be
used to distinguish between vaccinated and infected individuals.
The ability to differentiate vCP300-vaccinated individuals from
HIV1-infected individuals with a gp41 antibody assay is important
because the most commonly used diagnostic kit (which assays for the
presence of HIV1 p24 antibodies) would be useless, since
vCP300-vaccinated individuals would be expected to have a high
level of p24 antibodies. Alternatively, HIV-1 infected individuals
would be expected to make anti-gp41 antibodies but those vaccinated
with vCP205 or vCP300 would not since gp41 is absent from vCP205 or
vCP300.
[0105] Rabbits and guinea pigs have been inoculated with an ALVAC
recombinant (vCP205; ALVAC-MN120TMG) expressing the same cell
surface-associated form of HIV1 gp120 (120.TM.) and Gag/pro as
expressed by vCP300. Rabbits and guinea pigs have also been
inoculated with vCP205 and boosted with an HIV1 T-B peptide. Both
ALVAC-based protocols were able to elicit HIV1 gp160- and V3
loop-specific antibodies, thereby indicating that an ALVAC
recombinant expressing the cell surface form of HIV1 gp120 induces
an HIV1-specific immune response.
[0106] vCP300 expresses the HIV1 Gag proteins, a cell
surface-associated form of the HIV1 gp120 envelope glycoprotein,
two (2) regions from HIV1 nef containing CTL epitopes and three (3)
regions from HIV1 pol containing CTL epitopes. The expression of an
HIV1 envelope glycoprotein that does not contain gp41 allows
vaccinated individuals to be differentiated from HIV1-infected
individuals via an assay for gp41 antibodies and eliminates
potentially detrimental responses associated with various gp41
epitopes. Since a previous ALVAC recombinant expressing HIV1 gp160
has been shown to elicit HIV1-specific humoral and cellular immune
responses in humans (Pialoux et al., 1995), the addition of Gag and
the Pol and Nef epitopes (and the deletion of the potentially
detrimental gp41 epitopes) heightens and broadens the immune
response elicited by vP300, relative to vCP125, and, may provide an
efficacious HIV1 vaccine, or immunological or antigenic
composition.
[0107] In Macaca fascicularis (monkeys; macaques) immunized with
vCP205 or vCP300, an antibody response (anti-HIV) was observed,
thereby further demonstrating the utility and efficacy of these
recombinants.
[0108] Since the ELDKWA or LDKW epitope does not appear to be very
immunogenic in its normal configuration, to increase its
immunogenicity, recombinants of the invention present it to the
immune system in a more immunogenic setting, such as within the V3
loop of gp120 or within other regions of gp120 and/or as part of an
intact gp160 envelope.
[0109] ALVAC recombinant (vCP1307), NYVAC recombinant (vP1313) and
COPAK recombinant (vP1319) express a form of the HIV1 gp120+TM gene
product in which the V3 loop has been modified to contain two
copies of the ELDKWA epitope. The ELDKWA epitopes of this gp120+TM
(with ELDKWA epitopes) gene product are expressed on the surface of
vCP1307-, vP1313- and vP1319-infected cells.
[0110] The V3 loop of HIV1 gp120+TM (or gp160) can be used as an
immunological platform for any linear epitope, not just linear HIV1
epitopes. The gp120+TM (with epitopes of interest) protein
generated by these recombinants can also be isolated from
poxvirus-infected cells and used to inoculate individuals in a
subunit vaccine configuration (composition, or an antigenic or
immunological composition). The proteins generated by the
recombinants and antibodies elicited therefrom can also be used in
assays to detect the presence or absence of HIV. Accordingly, the
invention comprehends HIV immunogens and modified gp120 and gp160.
Further, such envelope-based immunogens (HIV immunogens or
unmodified gp120 or gp160 (can be derived from any eukaryotic or
prokaryotic expression vector and used as subunit preparations or
can be administered through DNA immunization using an appropriate
expression plasmid. Techniques for DNA immunization are known in
the art. With respect to techniques for DNA immunization, mention
is particularly made of Nabel and Felgner, "Direct gene transfer
for immunotherapy and immunization", Tibtech, May 1993, 11;
211-215, and Webster et al, "protection of ferrets against
influenza challenge with a DNA vaccine to the haemagglutinin",
vaccine, 1994, 12(16): 1495-1498, incorporated herein by reference.
Also, the DNA from the recombinants vP1313, vP1319 and vCP1307 can
be used to probe for the presence of HIV DNA in a sample of
interest using known hybridization techniques, or, to generate PCR
primers using known techniques.
[0111] Clearly based on the attenuation profiles of the NYVAC,
ALVAC, and TROVAC vectors and their demonstrated ability to elicit
both humoral and cellular immunological responses to extrinsic
immunogens (Tartaglia et al., 1993a,b; Taylor et al., 1992; Konishi
et al., 1992) such recombinant viruses offer a distinct advantage
over previously described vaccinia-based recombinant viruses.
[0112] The administration procedure for recombinant virus,
immunogen, modified gp120 or gp160, DNA or expression product
compositions of the invention such as immunological, antigenic or
vaccine compositions or therapeutic compositions can be via a
parenteral route (intradermal, intramuscular or subcutaneous). Such
an administration enables a systemic immune response.
[0113] More generally, the inventive antigenic, immunological or
vaccine compositions or therapeutic compositions (compositions
containing the poxvirus recombinants, expression products,
immunogens, DNA, modified gp120 or gp160 of the invention) can be
prepared in accordance with standard techniques well known to those
skilled in the pharmaceutical art. Such compositions can be
administered in dosages and by techniques well known to those
skilled in the medical arts taking into consideration such factors
as the age, sex, weight, and condition of the particular patient,
and the route of administration. The compositions can be
administered alone, or can be co-administered or sequentially
administered with compositions of the invention or with other
immunological, antigenic or vaccine or therapeutic compositions in
seropositive individuals. The compositions can be administered
alone, or can be co-administered or sequentially administered with
compositions of the invention or with other antigenic,
immunological, vaccine or therapeutic compositions in seronegative
individuals. Such other compositions can include purified antigens
from immunodeficiency virus or from the expression of such antigens
by a recombinant poxvirus or other vector system or, such other
compositions can include a recombinant poxvirus which expresses
other immunodeficiency antigens or biological response modifiers
(e.g. cytokines; co-stimulating molecules). Again,
co-administration is performed by taking into consideration such
known factors as the age, sex, weight, and condition of the
particular patient, and, the route of administration.
[0114] Examples of compositions of the invention include liquid
preparations for orifice, e.g., oral, nasal, anal, vaginal, etc.,
administration such as suspensions, syrups or elixirs; and,
preparations for parenteral, subcutaneous, intradermal,
intramuscular or intravenous administration (e.g., injectable
administration) such as sterile suspensions or emulsions. In such
compositions the recombinant poxvirus, expression product,
immunogen, DNA, or modified gp120 or gp160 may be in admixture with
a suitable carrier, diluent, or excipient such as sterile water,
physiological saline, glucose or the like.
[0115] Further, the products of expression of the inventive
recombinant poxviruses can be used directly to stimulate an immune
response in either seronegative or seropositive individuals or in
animals. Thus, the expression products can be used in compositions
of the invention instead or in addition to the inventive
recombinant poxvirus in the aforementioned compositions. The
immunogens of the invention can be similarly used.
[0116] Additionally, the inventive recombinant poxvirus and the
expression products therefrom and immunogens and modified gp120 or
gp160 of the invention stimulate an immune or antibody response in
humans and animals. From those antibodies or by techniques
well-known in the art, monoclonal antibodies can be prepared and,
those monoclonal antibodies, can be employed in well known antibody
binding assays, diagnostic kits or tests to determine the presence
or absence of particular immunodeficiency virus antigen(s) and
therefore the presence or absence of the virus, or to determine
whether an immune response to the virus or antigen(s) has simply
been stimulated. Those monoclonal antibodies can also be employed
in immunoadsorption chromatography to recover immunodeficiency
virus or expression products of the inventive recombinant
poxvirus.
[0117] Monoclonal antibodies are immunoglobulins produced by
hybridoma cells. A monoclonal antibody reacts with a single
antigenic determinant and provides greater specificity than a
conventional, serum-derived antibody. Furthermore, screening a
large number of monoclonal antibodies makes it possible to select
an individual antibody with desired specificity, avidity and
isotype. Hybridoma cell lines provide a constant, inexpensive
source of chemically identical antibodies and preparations of such
antibodies can be easily standardized. Methods for producing
monoclonal antibodies are well known to those of ordinary skill in
the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265,
issued Apr. 1, 1989, incorporated herein by reference.
[0118] Uses of monoclonal antibodies are known. One such use is in
diagnostic methods, e.g., David, G. and Greene, H. U.S. Pat. No.
4,376,110, issued Mar. 8, 1983; incorporated herein by reference.
Monoclonal antibodies have also been used to recover materials by
immunoadsorption chromatography, e.g., Milstein, C. 1980,
Scientific American 243:66, 70, incorporated herein by
reference.
[0119] Furthermore, the inventive recombinant poxvirus or
expression products therefrom or the inventive immunogens or
modified gp120 or gp160 can be used to stimulate a response in
cells such as lymphocytes or CTLs in vitro or ex vivo for
subsequent reinfusion into a patient. If the patient is
seronegative, the reinfusion is to stimulate an immune response,
e.g., an immunological or antigenic response such as active
immunization. In a seropositive individual, the reinfusion is to
stimulate or boost the immune system against immunodeficiency
virus.
[0120] Additionally, the DNA from inventive recombinants can be
used as probes to detect the presence of HIV DNA in a sample or, to
generate PCR primers, or for DNA immunization using an appropriate
expression plasmid, by methods known in the art. (See Nabel and
Felger and Webster et al, Supra)
[0121] Accordingly, the inventive recombinant poxvirus has several
utilities: In antigenic, immunological or vaccine compositions such
as for administration to seronegative individuals. In therapeutic
compositions in seropositive individuals in need of therapy to
stimulate or boost the immune system against immunodeficiency
virus. In vitro to produce antigens or the inventive immunogens or
the inventive modified gp120 or gp160 which can be further used in
antigenic, immunological or vaccine compositions or in therapeutic
compositions. To generate antibodies (either by direct
administration or by administration of an expression product of the
inventive recombinant poxvirus) which can be further used: in
diagnosis, tests or kits to ascertain the presence or absence of
antigens in a sample such as sera, for instance, to ascertain the
presence or absence of immunodeficiency virus or CTLs in a sample
such as sera or, to determine whether an immune response has
elicited to the virus or, to particular antigen(s); or, in
immunoadsorption chromatography (the inventive immunogens and
modified gp120 or gp160 can also be used to generate antibodies
which can be also so further used). To generate DNA for use as
hybridization probes or to prepare PCR primers or for DNA
immunization. And, the inventive recombinant poxvirus, expression
products therefrom, immunogens and modified gp120 or gp160 can be
used to generate stimulated cells which can be further used
(reinfused) to stimulate an immune response (antigenic, or
immunological response; or active immunization) or, to boost or
stimulate the immune system (for instance, of an immunocompromised
or seropositive individual). Other utilities also exist for
embodiments of the invention.
[0122] A better understanding of the present invention and of its
many advantages will be had from the following examples, given by
way of illustration.
EXAMPLES
[0123] DNA Cloning and Synthesis. Plasmids were constructed,
screened and grown by standard procedures (Maniatis et al., 1982;
Perkus et al., 1985; Piccini et al., 1987). Restriction
endonucleases were obtained from Bethesda Research Laboratories,
Gaithersburg, Md., New England Biolabs, Beverly, Mass.; and
Boehringer Mannheim Biochemicals, Indianapolis, Ind. Klenow
fragment of E. coli polymerase was obtained from Boehringer
Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase
were obtained from New England Biolabs. The reagents were used as
specified by the various suppliers.
[0124] Synthetic oligodeoxyribonucleotides were prepared on a
Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as
previously described (Perkus et al., 1989). DNA sequencing was
performed by the dideoxy-chain termination method (Sanger et al.,
1977) using Sequenase (Tabor et al., 1987) as previously described
(Guo et al., 1989). DNA amplification by polymerase chain reaction
(PCR) for sequence verification (Engelke et al., 1988) was
performed using custom synthesized oligonucleotide primers and
GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk,
Conn.) in an automated Perkin Elmer Cetus DNA Thermal Cycler.
Excess DNA sequences were deleted from plasmids by restriction
endonuclease digestion followed by limited digestion by BAL-31
exonuclease and mutagenesis (Mandecki, 1986) using synthetic
oligonucleotides.
[0125] Cells, Virus, and Transfection. The origins and conditions
of cultivation of the Copenhagen strain of vaccinia virus has been
previously described (Guo et al., 1989). Generation of recombinant
virus by recombination, in situ hybridization of nitrocellulose
filters and screening for B-galactosidase activity are as
previously described (Piccini et al., 1987).
[0126] The origins and conditions of cultivation of the Copenhagen
strain of vaccinia virus and NYVAC has been previously described
(Guo et al., 1989; Tartaglia et al., 1992). Generation of
recombinant virus by recombination, in situ hybridization of
nitrocellulose filters and screening for B-galactosidase activity
are as previously described (Panicali et al., 1982; Perkus et al.,
1989).
[0127] The parental canarypox virus (Rentschler strain) is a
vaccinal strain for canaries. The vaccine strain was obtained from
a wild type isolate and attenuated through more than 200 serial
passages on chick embryo fibroblasts. A master viral seed was
subjected to four successive plaque purifications under agar and
one plaque clone was amplified through five additional passages
after which the stock virus was used as the parental virus in in
vitro recombination tests. The plaque purified canarypox isolate is
designated ALVAC.
[0128] The strain of fowlpox virus (FPV) designated FP-1 has been
described previously (Taylor et al., 1988a). It is an attenuated
vaccine strain useful in vaccination of day old chickens. The
parental virus strain Duvette was obtained in France as a fowlpox
scab from a chicken. The virus was attenuated by approximately 50
serial passages in chicken embryonated eggs followed by 25 passages
on chicken embryo fibroblast cells. The virus was subjected to four
successive plaque purifications. One plaque isolate was further
amplified in primary CEF cells and a stock virus, designated as
TROVAC, established.
[0129] NYVAC, ALVAC and TROVAC viral vectors and their derivatives
were propagated as described previously (Piccini et al., 1987;
Taylor et al., 1988a,b). Vero cells and chick embryo fibroblasts
(CEF) were propagated as described previously (Taylor et al.,
1988a,b).
Example 1
Construction of Plasmid pSD460 for Deletion of Thymidine Kinase
Gene (J2R)
[0130] Referring now to FIG. 1, plasmid pSD406 contains vaccinia
HindIII J (pos. 83359-88377) cloned into pUC8. pSD406 was cut with
HindIII and PvuII, and the 1.7 kb fragment from the left side of
HindIII J cloned into pUC8 cut with HindIII/SmaI, forming pSD447.
pSD447 contains the entire gene for J2R (pos. 83855-84385). The
initiation codon is contained within an NlaIII site and the
termination codon is contained within an SspI site. Direction of
transcription is indicated by an arrow in FIG. 1.
[0131] To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI
fragment was isolated from pSD447, then digested with NlaIII and a
0.5 kb HindIII/NlaIII fragment isolated. Annealed synthetic
oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:1/SEQ ID NO:2)
TABLE-US-00001 SmaI MPSYN43 5' TAATTAACTAGCTACCCGGG 3' MPSYN44 3'
GTACATTAATTGATCGATGGGCCCTTAA 5' NlaIII EcoRI
were ligated with the 0.5 kb HindIII/NlaIII fragment into pUC18
vector plasmid cut with HindIII/EcoRI, generating plasmid
pSD449.
[0132] To obtain a restriction fragment containing a vaccinia right
flanking arm and pUC vector sequences, pSD447 was cut with SspI
(partial) within vaccinia sequences and HindIII at the pUC/vaccinia
junction, and a 2.9 kb vector fragment isolated. This vector
fragment was ligated with annealed synthetic oligonucleotides
MPSYN45/MPSYN46 (SEQ ID NO:3/SEQ ID NO:4)
TABLE-US-00002 HindIII SmaI MPSYN45 5'
AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA MPSYN46 3'
AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT NotI SspI
ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45
TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5' MPSYN46
generating pSD459.
[0133] To combine the left and right flanking arms into one
plasmid, a 0.5 kb HindIII/SmaI fragment was isolated from pSD449
and ligated with pSD459 vector plasmid cut with HindIII/SmaI,
generating plasmid pSD460. pSD460 was used as donor plasmid for
recombination with wild type parental vaccinia virus Copenhagen
strain VC-2. .sup.32P labelled probe was synthesized by primer
extension using MPSYN45 (SEQ ID NO:3) as template and the
complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:5) (5'
TTAGTTAATTAGGCGGCCGC 3') as primer. Recombinant virus vP410 was
identified by plaque hybridization.
Example 2
Construction of Plasmid pSD486 for Deletion of Hemorrhagic Region
(B13R+B14R)
[0134] Referring now to FIG. 2, plasmid pSD419 contains vaccinia
SalI G (pos. 160, 744-173,351) cloned into pUC8. pSD422 contains
the contiguous vaccinia SalI fragment to the right, SalI J (pos.
173, 351-182,746) cloned into pUC8. To construct a plasmid deleted
for the hemorrhagic region, u, B13R-B14R (pos. 172, 549-173,552),
pSD419 was used as the source for the left flanking arm and pSD422
was used as the source of the right flanking arm. The direction of
transcription for the u region is indicated by an arrow in FIG.
2.
[0135] To remove unwanted sequences from pSD419, sequences to the
left of the NcoI site (pos. 172,253) were removed by digestion of
pSD419 with NcoI/SmaI followed by blunt ending with Klenow fragment
of E. coli polymerase and ligation generating plasmid pSD476. A
vaccinia right flanking arm was obtained by digestion of pSD422
with HpaI at the termination codon of B14R and by digestion with
NruI 0.3 kb to the right. This 0.3 kb fragment was isolated and
ligated with a 3.4 kb HincII vector fragment isolated from pSD476,
generating plasmid pSD477. The location of the partial deletion of
the vaccinia u region in pSD477 is indicated by a triangle. The
remaining B13R coding sequences in pSD477 were removed by digestion
with ClaI/HpaI, and the resulting vector fragment was ligated with
annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID
NO:6/SEQ ID NO:7)
TABLE-US-00003 ClaI BamHI HpaI SD22mer 5' CGATTACTATGAAGGATCCGTT 3'
SD20mer 3' TAATGATACTTCCTAGGCAA 5'
generating pSD479. pSD479 contains an initiation codon (underlined)
followed by a BamHI site. To place E. coli Beta-galactosidase in
the B13-B14 (u) deletion locus under the control of the u promoter,
a 3.2 kb BamHI fragment containing the Beta-galactosidase gene
(Shapira et al., 1983) was inserted into the BamHI site of pSD479,
generating pSD479BG. pSD479BG was used as donor plasmid for
recombination with vaccinia virus vP410. Recombinant vaccinia virus
vP533 was isolated as a blue plaque in the presence of chromogenic
substrate X-gal. In vP533 the B13R-B14R region is deleted and is
replaced by Beta-galactosidase.
[0136] To remove Beta-galactosidase sequences from vP533, plasmid
pSD486, a derivative of pSD477 containing a polylinker region but
no initiation codon at the u deletion junction, was utilized. First
the ClaI/HpaI vector fragment from pSD477 referred to above was
ligated with annealed synthetic oligonucleotides SD42mer/SD40mer
(SEQ ID NO:8/SEQ ID NO:9)
TABLE-US-00004 ClaI SacI XhoI HpaI SD42mer 5'
CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3' SD40mer 3'
TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5' BglII SmaI BamHI
generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia
junction was destroyed by digestion of pSD478 with EcoRI followed
by blunt ending with Klenow fragment of E. coli polymerase and
ligation, generating plasmid pSD478E.sup.-. pSD478E.sup.- was
digested with BamHI and HpaI and ligated with annealed synthetic
oligonucleotides HEM5/HEM6 (SEQ ID NO:10/SEQ ID NO:11)
TABLE-US-00005 BamHI EcoRI HpaI HEM5 5' GATCCGAATTCTAGCT 3' HEM6 3'
GCTTAAGATCGA 5'
generating plasmid pSD486. pSD486 was used as donor plasmid for
recombination with recombinant vaccinia virus vP533, generating
vP553, which was isolated as a clear plaque in the presence of
X-gal.
Example 3
Construction of Plasmid pMP494.DELTA. for Deletion of ATI Region
(A26L)
[0137] Referring now to FIG. 3, pSD414 contains SalI B cloned into
pUC8. To remove unwanted DNA sequences to the left of the A26L
region, pSD414 was cut with XbaI within vaccinia sequences (pos.
137,079) and with HindIII at the pUC/vaccinia junction, then blunt
ended with Klenow fragment of E. coli polymerase and ligated,
resulting in plasmid pSD483. To remove unwanted vaccinia DNA
sequences to the right of the A26L region, pSD483 was cut with
EcoRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated,
forming plasmid pSD484. To remove the A26L coding region, pSD484
was cut with NdeI (partial) slightly upstream from the A26L ORF
(pos. 139,004) and with HpaI (pos. 137,889) slightly downstream
from the A26L ORF. The 5.2 kb vector fragment was isolated and
ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID
NO:12/SEQ ID NO:13)
TABLE-US-00006 NdeI ATI3 5'
TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAA ATI4 3'
ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTT BglII EcoRI HpaI
AAAATAAGTTATATAAATAGATCTGAATTCGTT 3' ATI3
TTTTATTCAATATATTTATCTAGACTTAAGCAA 5' ATI4
reconstructing the region upstream from A26L and replacing the A26L
ORF with a short polylinker region containing the restriction sites
BglII, EcoRI and HpaI, as indicated above. The resulting plasmid
was designated pSD485. Since the BglII and EcoRI sites in the
polylinker region of pSD485 are not unique, unwanted BglII and
EcoRI sites were removed from plasmid pSD483 (described above) by
digestion with BglII (pos. 140,136) and with EcoRI at the
pUC/vaccinia junction, followed by blunt ending with Klenow
fragment of E. coli polymerase and ligation. The resulting plasmid
was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcORV (pos.
139,048) fragment from pSD489 containing the A26L ORF was replaced
with the corresponding 0.7 kb polylinker-containing ClaI/EcoRV
fragment from pSD485, generating pSD492. The BglII and EcoRI sites
in the polylinker region of pSD492 are unique.
[0138] A 3.3 kb BglII cassette containing the E. coli
Beta-galactosidase gene (Shapira et al., 1983) under the control of
the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et
al., 1990) was inserted into the BglII site of pSD492, forming
pSD493KBG. Plasmid pSD493KBG was used in recombination with
rescuing virus vP553. Recombinant vaccinia virus, vP581, containing
Beta-galactosidase in the A26L deletion region, was isolated as a
blue plaque in the presence of X-gal.
[0139] To generate a plasmid for the removal of Beta-galactosidase
sequences from vaccinia recombinant virus vP581, the polylinker
region of plasmid pSD492 was deleted by mutagenesis (Mandecki,
1986) using synthetic oligonucleotide MPSYN177 (SEQ ID NO:14) (5'
AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3'). In the
resulting plasmid, pMP494.DELTA., vaccinia DNA encompassing
positions [137,889-138,937], including the entire A26L ORF is
deleted. Recombination between the pMP494L and the
Beta-galactosidase containing vaccinia recombinant, vP581, resulted
in vaccinia deletion mutant vP618, which was isolated as a clear
plaque in the presence of X-gal.
Example 4
Construction of Plasmid pSD467 for Deletion of Hemagglutinin Gene
(A56R)
[0140] Referring now to FIG. 4, vaccinia SalI G restriction
fragment (pos. 160, 744-173,351) crosses the HindIII A/B junction
(pos. 162,539). pSD419 contains vaccinia SalI G cloned into pUC8.
The direction of transcription for the hemagglutinin (HA) gene is
indicated by an arrow in FIG. 4. Vaccinia sequences derived from
HindIII B were removed by digestion of pSD419 with HindIII within
vaccinia sequences and at the pUC/vaccinia junction followed by
ligation. The resulting plasmid, pSD456, contains the HA gene,
A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4
kb of vaccinia sequences to the right. A56R coding sequences were
removed by cutting pSD456 with RsaI (partial; pos. 161,090)
upstream from A56R coding sequences, and with EagI (pos. 162,054)
near the end of the gene. The 3.6 kb RsaI/EagI vector fragment from
pSD456 was isolated and ligated with annealed synthetic
oligonucleotides MPSYN59 (SEQ ID NO:15), MPSYN62 (SEQ ID NO:16),
MPSYN60 (SEQ ID NO:17), and MPSYN61 (SEQ ID NO:18)
TABLE-US-00007 RsaI MPSYN59 5'
ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT- MPSYN62 3'
TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA- MPSYN59
AGTTGATAGAACAAAATACATAATTT 3' MPSYN62 TCAACTATCT 5' MPSYN60 5'
TGTAAAAATAAATCACTTTTTATA- MPSYN61 3'
TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT- BglII SmaI PstI EagI
MPSYN60 CTAAGATCTCCCGGGCTGCAGC 3' MPSYN61
GATTCTAGAGGGCCCGACGTCGCCGG 5'
reconstructing the DNA sequences upstream from the A56R ORF and
replacing the A56R ORF with a polylinker region as indicated above.
The resulting plasmid is pSD466. The vaccinia deletion in pSD466
encompasses positions [161,185-162,053]. The site of the deletion
in pSD466 is indicated by a triangle in FIG. 4.
[0141] A 3.2 kb BglII/BamHI (partial) cassette containing the E.
coli Beta-galactosidase gene (Shapira et al., 1983) under the
control of the vaccinia 11 kDa promoter (Bertholet et al., 1985;
Guo et al., 1989) was inserted into the BglII site of pSD466,
forming pSD466 KBG. Plasmid pSD466 KBG was used in recombination
with rescuing virus vP618. Recombinant vaccinia virus, vP708,
containing Beta-galactosidase in the A56R deletion, was isolated as
a blue plaque in the presence of X-gal.
[0142] Beta-galactosidase sequences were deleted from vP708 using
donor plasmid pSD467. pSD467 is identical to pSD466, except that
EcoRI, SmaI and BamHI sites were removed from the pUC/vaccinia
junction by digestion of pSD466 with EcoRI/BamHI followed by blunt
ending with Klenow fragment of E. coli polymerase and ligation.
Recombination between vP708 and pSD467 resulted in recombinant
vaccinia deletion mutant, vP723, which was isolated as a clear
plaque in the presence of X-gal.
Example 5
Construction of Plasmid pMPCSK1.DELTA. for Deletion of Open Reading
Frames [C7L-K1L]
[0143] Referring now to FIG. 5, the following vaccinia clones were
utilized in the construction of pMPCSK1.DELTA.. pSD420 is SalI H
cloned into pUC8. pSD435 is KpnI F cloned into pUC18. pSD435 was
cut with SphI and religated, forming pSD451. In pSD451, DNA
sequences to the left of the SphI site (pos. 27,416) in HindIII M
are removed (Perkus et al., 1990). pSD409 is HindIII M cloned into
pUC8.
[0144] To provide a substrate for the deletion of the [C7L-K1L]
gene cluster from vaccinia, E. coli Beta-galactosidase was first
inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as
follows. To eliminate the BglII site in pSD409, the plasmid was cut
with BglII in vaccinia sequences (pos. 28,212) and with BamHI at
the pUC/vaccinia junction, then ligated to form plasmid pMP409B.
pMP409B was cut at the unique SphI site (pos. 27,416). M2L coding
sequences were removed by mutagenesis (Guo et al., 1990; Mandecki,
1986) using synthetic oligonucleotide
TABLE-US-00008 MPSYN82 (SEQ ID NO: 19) BglII 5'
TTTCTGTATATTTGCACCAATTTAGATCTT-ACTCAAAATATGTAAC AATA 3'
The resulting plasmid, pMP409D, contains a unique BglII site
inserted into the M2L deletion locus as indicated above. A 3.2 kb
BamHI (partial)/BglII cassette containing the E. coli
Beta-galactosidase gene (Shapira et al., 1983) under the control of
the 11 kDa promoter (Bertholet et al., 1985) was inserted into
pMP409D cut with BglII. The resulting plasmid, pMP409DBG (Guo et
al., 1990), was used as donor plasmid for recombination with
rescuing vaccinia virus vP723. Recombinant vaccinia virus, vP784,
containing Beta-galactosidase inserted into the M2L deletion locus,
was isolated as a blue plaque in the presence of X-gal.
[0145] A plasmid deleted for vaccinia genes [C7L-K1L] was assembled
in pUC8 cut with SmaI, HindIII and blunt ended with Klenow fragment
of E. coli polymerase. The left flanking arm consisting of vaccinia
HindIII C sequences was obtained by digestion of pSD420 with XbaI
(pos. 18,628) followed by blunt ending with Klenow fragment of E.
coli polymerase and digestion with BglII (pos. 19,706). The right
flanking arm consisting of vaccinia HindIII K sequences was
obtained by digestion of pSD451 with BglII (pos. 29,062) and EcoRV
(pos. 29,778). The resulting plasmid, pMP581CK is deleted for
vaccinia sequences between the BglII site (pos. 19,706) in HindIII
C and the BglII site (pos. 29,062) in HindIII K. The site of the
deletion of vaccinia sequences in plasmid pMP581CK is indicated by
a triangle in FIG. 5.
[0146] To remove excess DNA at the vaccinia deletion junction,
plasmid pMP581CK, was cut at the NcoI sites within vaccinia
sequences (pos. 18,811; 19,655), treated with Bal-31 exonuclease
and subjected to mutagenesis (Mandecki, 1986) using synthetic
oligonucleotide MPSYN233 (SEQ ID NO:20)
5'-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3'. The resulting
plasmid, pMPCSK18, is deleted for vaccinia sequences positions
18,805-29,108, encompassing 12 vaccinia open reading frames
[C7L-K1L]. Recombination between pMPCSK1.DELTA. and the
Beta-galactosidase containing vaccinia recombinant, vP784, resulted
in vaccinia deletion mutant, vP804, which was isolated as a clear
plaque in the presence of X-gal.
Example 6
Construction of Plasmid pSD548 for Deletion of Large Subunit,
Ribonucleotide Reductase (I4L)
[0147] Referring now to FIG. 6, plasmid pSD405 contains vaccinia
HindIII I (pos. 63, 875-70,367) cloned in pUC8. pSD405 was digested
with EcoRV within vaccinia sequences (pos. 67,933) and with SmaI at
the pUC/vaccinia junction, and ligated, forming plasmid pSD518.
pSD518 was used as the source of all the vaccinia restriction
fragments used in the construction of pSD548.
[0148] The vaccinia I4L gene extends from position 67,371-65,059.
Direction of transcription for I4L is indicated by an arrow in FIG.
6. To obtain a vector plasmid fragment deleted for a portion of the
I4L coding sequences, pSD518 was digested with BamHI (pos. 65,381)
and HpaI (pos. 67,001) and blunt ended using Klenow fragment of E.
coli polymerase. This 4.8 kb vector fragment was ligated with a 3.2
kb SmaI cassette containing the E. coli Beta-galactosidase gene
(Shapira et al., 1983) under the control of the vaccinia 11 kDa
promoter (Bertholet et al., 1985; Perkus et al., 1990), resulting
in plasmid pSD524 KBG. pSD524 KBG was used as donor plasmid for
recombination with vaccinia virus vP804. Recombinant vaccinia
virus, vP855, containing Beta-galactosidase in a partial deletion
of the I4L gene, was isolated as a blue plaque in the presence of
X-gal.
[0149] To delete Beta-galactosidase and the remainder of the I4L
ORF from vP855, deletion plasmid pSD548 was constructed. The left
and right vaccinia flanking arms were assembled separately in pUC8
as detailed below and presented schematically in FIG. 6.
[0150] To construct a vector plasmid to accept the left vaccinia
flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with
annealed synthetic oligonucleotides 518A1/518A2
TABLE-US-00009 (SEQ ID NO: 21/SEQ ID NO: 22) BamHI RsaI 518A1 5'
GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACT 518A2 3'
GACTCATGAAACATTATATTACTATATATAAAAGTGA BglII EcoRI
TTATCTCATTTGAGAATAAAAAGATCTTAGG 3'
AATAGAGTAAACTCTTATTTTTCTAGAATCCTTAA 5' 518A1 518A2
forming plasmid pSD531. pSD531 was cut with RsaI (partial) and
BamHI and a 2.7 kb vector fragment isolated. pSD518 was cut with
BglII (pos. 64,459)/RsaI (pos. 64,994) and a 0.5 kb fragment
isolated. The two fragments were ligated together, forming pSD537,
which contains the complete vaccinia flanking arm left of the I4L
coding sequences.
[0151] To construct a vector plasmid to accept the right vaccinia
flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with
annealed synthetic oligonucleotides 518B1/518B2
TABLE-US-00010 (SEQ ID NO: 23/SEQ ID NO: 24) BamHI BglII SmaI 518B1
5' GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCAT 518B2 3'
GTCTAGAGGCCCCTTTTTTTAATAAATTGAAAAGTA RsaI EcoRI
TAATAG-GGATTTGACGTATGTAGCGTACTAGG 3'
ATTATC-CCTAAACTGCATACTACGCATGATCCTTAA 5' 518B1 518B2
forming plasmid pSD532. pSD532 was cut with RsaI (partial)/EcoRI
and a 2.7 kb vector fragment isolated. pSD518 was cut with RsaI
within vaccinia sequences (pos. 67,436) and EcoRI at the
vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two
fragments were ligated together, forming pSD538, which contains the
complete vaccinia flanking arm to the right of I4L coding
sequences.
[0152] The right vaccinia flanking arm was isolated as a 0.6 kb
EcoRI/BglII fragment from pSD538 and ligated into pSD537 vector
plasmid cut with EcoRI/BglII. In the resulting plasmid, pSD539, the
I4L ORF (pos. 65, 047-67,386) is replaced by a polylinker region,
which is flanked by 0.6 kb vaccinia DNA to the left and 0.6 kb
vaccinia DNA to the right, all in a pUC background. The site of
deletion within vaccinia sequences is indicated by a triangle in
FIG. 6. To avoid possible recombination of Beta-galactosidase
sequences in the pUC-derived portion of pSD539 with
Beta-galactosidase sequences in recombinant vaccinia virus vP855,
the vaccinia I4L deletion cassette was moved from pSD539 into
pRC11, a pUC derivative from which all Beta-galactosidase sequences
have been removed and replaced with a polylinker region (Colinas et
al., 1990). pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment
isolated. This fragment was ligated into pRC11 cut with EcoRI/PstI
(2.35 kb), forming pSD548. Recombination between pSD548 and the
Beta-galactosidase containing vaccinia recombinant, vP855, resulted
in vaccinia deletion mutant vP866, which was isolated as a clear
plaque in the presence of X-gal.
[0153] DNA from recombinant vaccinia virus vP866 was analyzed by
restriction digests followed by electrophoresis on an agarose gel.
The restriction patterns were as expected. Polymerase chain
reactions (PCR) (Engelke et al., 1988) using vP866 as template and
primers flanking the six deletion loci detailed above produced DNA
fragments of the expected sizes. Sequence analysis of the PCR
generated fragments around the areas of the deletion junctions
confirmed that the junctions were as expected. Recombinant vaccinia
virus vP866, containing the six engineered deletions as described
above, was designated vaccinia vaccine strain "NYVAC."
[0154] NYVAC was deposited under the terms of the Budapest Treaty
on Mar. 6, 1997 with the American Type Culture Collection (ATCC),
P.O. Box 1549, Manassas, Va. 20108 USA under ATCC accession number
VR-2559.
Example 7
Insertion of a Rabies Glycoprotein G Gene into NYVAC
[0155] The gene encoding rabies glycoprotein G under the control of
the vaccinia H6 promoter (Taylor et al., 1988a,b) was inserted into
TK deletion plasmid pSD513. pSD513 is identical to plasmid pSD460
(FIG. 1) except for the presence of a polylinker region.
[0156] Referring now to FIG. 7, the polylinker region was inserted
by cutting pSD460 with SmaI and ligating the plasmid vector with
annealed synthetic oligonucleotides VQ1A/VQ1B (SEQ ID NO:25/SEQ ID
NO:26)
TABLE-US-00011 SmaI BglII XhoI PstI NarI BamHI VQ1A 5'
GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3' VQ1B 3'
CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5'
to form vector plasmid pSD513. pSD513 was cut with SmaI and ligated
with a SmaI ended 1.8 kb cassette containing the gene encoding the
rabies glycoprotein G gene under the control of the vaccinia H6
promoter (Taylor et al., 1988a,b). The resulting plasmid was
designated pRW842. pRW842 was used as donor plasmid for
recombination with NYVAC rescuing virus (vP866). Recombinant
vaccinia virus vP879 was identified by plaque hybridization using
.sup.32P-labelled DNA probe to rabies glycoprotein G coding
sequences.
[0157] The modified recombinant viruses of the present invention
provide advantages as recombinant vaccine vectors. The attenuated
virulence of the vector advantageously reduces the opportunity for
the possibility of a runaway infection due to vaccination in the
vaccinated individual and also diminishes transmission from
vaccinated to unvaccinated individuals or contamination of the
environment.
[0158] The modified recombinant viruses are also advantageously
used in a method for expressing a gene product in a cell cultured
in vitro by introducing into the cell the modified recombinant
virus having foreign DNA which codes for and expresses gene
products in the cell.
Example 8
Construction of TROVAC-NDV Expressing the Fusion and
Hemagglutinin-Neuraminidase Glycoproteins of Newcastle Disease
Virus
[0159] This example describes the development of TROVAC, a fowlpox
virus vector and, of a fowlpox Newcastle Disease Virus recombinant
designated TROVAC-NDV and its safety and efficacy. A fowlpox virus
(FPV) vector expressing both F and HN genes of the virulent NDV
strain Texas was constructed. The recombinant produced was
designated TROVAC-NDV. TROVAC-NDV expresses authentically processed
NDV glycoproteins in avian cells infected with the recombinant
virus and inoculation of day old chicks protects against subsequent
virulent NDV challenge.
[0160] Cells and Viruses. The Texas strain of NDV is a velogenic
strain. Preparation of cDNA clones of the F and HN genes has been
previously described (Taylor et al., 1990; Edbauer et al., 1990).
The strain of FPV designated FP-1 has been described previously
(Taylor et al., 1988a). It is a vaccine strain useful in
vaccination of day old chickens. The parental virus strain Duvette
was obtained in France as a fowlpox scab from a chicken. The virus
was attenuated by approximately 50 serial passages in chicken
embryonated eggs followed by 25 passages on chicken embryo
fibroblast cells. The virus was subjected to four successive plaque
purifications. One plaque isolate was further amplified in primary
CEF cells and a stock virus, designated as TROVAC, established. The
stock virus used in the in vitro recombination test to produce
TROVAC-NDV had been subjected to twelve passages in primary CEF
cells from the plaque isolate.
[0161] TROVAC was deposited under the terms of the Budapest Treaty
on Feb. 6, 1997 with the American Type Culture Collection (ATCC),
P.O. Box 1549, Manassas, Va. 20108 USA under ATCC accession number
VR-2553.
[0162] Construction of a Cassette for NDV-F. A 1.8 kbp BamHI
fragment containing all but 22 nucleotides from the 5' end of the F
protein coding sequence was excised from pNDV81 (Taylor et al.,
1990) and inserted at the BamHI site of pUC18 to form pCE13. The
vaccinia virus H6 promoter previously described (Taylor et al.,
1988a,b; Guo et al., 1989; Perkus et al., 1989) was inserted into
pCE13 by digesting pCE13 with SalI, filling in the sticky ends with
Klenow fragment of E. coli DNA polymerase and digesting with
HindIII. A HindIII-EcoRV fragment containing the H6 promoter
sequence was then inserted into pCE13 to form pCE38. A perfect 5'
end was generated by digesting pCE38 with KpnI and NruI and
inserting the annealed and kinased oligonucleotides CE75 (SEQ ID
NO:27) and CE76 (SEQ ID NO:28) to generate pCE47.
TABLE-US-00012 CE75:
CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGA TCCCGGTAC CE76:
CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGA TATCG.
In order to remove non-coding sequence from the 3' end of the NDV-F
a SmaI to PstI fragment from pCE13 was inserted into the SmaI and
PstI sites of pUC18 to form pCE23. The non-coding sequences were
removed by sequential digestion of pCE23 with SacI, BamHI,
Exonuclease III, SI nuclease and EcoRI. The annealed and kinased
oligonucleotides CE42 (SEQ ID NO:29) and CE43 (SEQ ID NO:30) were
then inserted to form pCE29.
TABLE-US-00013 CE42: AATTCGAGCTCCCCGGG CE43: CCCGGGGAGCTCG
The 3' end of the NDV-F sequence was then inserted into plasmid
pCE20 already containing the 5' end of NDV-F by cloning a PstI-SacI
fragment from pCE29 into the PstI and SacI sites of pCE20 to form
pCE32. Generation of pCE20 has previously been described in Taylor
et al., 1990.
[0163] In order to align the H6 promoter and NDV-F 5' sequences
contained in pCE47 with the 3' NDV-F sequences contained in pCE32,
a HindIII-PstI fragment of pCE47 was inserted into the HindIII and
PstI sites of pCE32 to form pCE49. The H6 promoted NDV-F sequences
were then transferred to the de-ORFed F8 locus (described below) by
cloning a HindIII-NruI fragment from pCE49 into the HindIII and
SmaI sites of pJCA002 (described below) to form pCE54.
Transcription stop signals were inserted into pCE54 by digesting
pCE54 with SacI, partially digesting with BamHI and inserting the
annealed and kinased oligonucleotides CE166 (SEQ ID NO:31) and
CE167 (SEQ ID NO:32) to generate pCE58.
TABLE-US-00014 CE166: CTTTTTATAAAAAGTTAACTACGTAG CE167:
GATCCTACGTAGTTAACTTTTTATAAAAAGAGCT
A perfect 3' end for NDV-F was obtained by using the polymerase
chain reaction (PCR) with pCE54 as template and oligonucleotides
CE182 (SEQ ID NO:33) and CE183 (SEQ ID NO:34) as primers.
TABLE-US-00015 CE182: CTTAACTCAGCTGACTATCC CE183:
TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC
[0164] The PCR fragment was digested with PvuII and HpaI and cloned
into pCE58 that had been digested with HpaI and partially digested
with PvuII. The resulting plasmid was designated pCE64. Translation
stop signals were inserted by cloning a HindIII-HpaI fragment which
contains the complete H6 promoter and F coding sequence from pCE64
into the HindIII and HpaI sites of pRW846 to generate pCE71, the
final cassette for NDV-F. Plasmid pRW846 is essentially equivalent
to plasmid pJCA002 (described below) but containing the H6 promoter
and transcription and translation stop signals. Digestion of pRW846
with HindIII and HpaI eliminates the H6 promoter but leaves the
stop signals intact.
[0165] Construction of Cassette for NDV-HN. Construction of plasmid
pRW802 was previously described in Edbauer et al., 1990. This
plasmid contains the NDV-HN sequences linked to the 3' end of the
vaccinia virus H6 promoter in a pUC9 vector. A HindIII-EcoRV
fragment encompassing the 5' end of the vaccinia virus H6 promoter
was inserted into the HindIII and EcoRV sites of pRW802 to form
pRW830. A perfect 3' end for NDV-HN was obtained by inserting the
annealed and kinased oligonucleotides CE162 (SEQ ID NO:35) and
CE163 (SEQ ID NO:36) into the EcoRI site of pRW830 to form pCE59,
the final cassette for NDV-HN.
TABLE-US-00016 CE162:
AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATT
TAATTTTTATAAGCTTG CE163:
AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTA
GTAAAGGAACGATCCTG
[0166] Construction of FPV Insertion Vector. Plasmid pRW731-15
contains a 10 kb PvuII-PvuII fragment cloned from genomic DNA. The
nucleotide sequence was determined on both strands for a 3660 bp
PvuII-EcoRV fragment and is shown in FIG. 11 (SEQ ID NO:67). The
limits of an open reading frame designated here as F8 were
determined. Plasmid pRW761 is a sub-clone of pRW731-15 containing a
2430 bp EcoRV-EcoRV fragment. The F8 ORF was entirely contained
between an XbaI site and an SspI site in pRW761. In order to create
an insertion plasmid which on recombination with TROVAC genomic DNA
would eliminate the F8 ORF, the following steps were followed.
Plasmid pRW761 was completely digested with XbaI and partially
digested with SspI. A 3700 bp XbaI-SspI band was isolated from the
gel and ligated with the annealed double-stranded oligonucleotides
JCA017 (SEQ ID NO:37) and JCA018 (SEQ ID NO:38).
TABLE-US-00017 JCA017: 5'
CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGG
GATCCTTATACGGGGAATAAT JCA018: 5'
ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATA
TTAAAAAACATAAAGTGT
The plasmid resulting from this ligation was designated
pJCA002.
[0167] Construction of Double Insertion Vector for NDV F and HN.
The H6 promoted NDV-HN sequence was inserted into the H6 promoted
NDV-F cassette by cloning a HindIII fragment from pCE59 that had
been filled in with Klenow fragment of E. coli DNA polymerase into
the HpaI site of pCE71 to form pCE80. Plasmid pCE80 was completely
digested with NdeI and partially digested with BglII to generate an
NdeI-BglII 4760 bp fragment containing the NDV F and HN genes both
driven by the H6 promoter and linked to F8 flanking arms. Plasmid
pJCA021 was obtained by inserting a 4900 bp PvuII-HindIII fragment
from pRW731-15 into the SmaI and HindIII sites of pBSSK+. Plasmid
pJCA021 was then digested with NdeI and BglII and ligated to the
4760 bp NdeI-BglII fragment of pCE80 to form pJCA024. Plasmid
pJCA024 therefore contains the NDV-F and HN genes inserted in
opposite orientation with 3' ends adjacent between FPV flanking
arms. Both genes are linked to the vaccinia virus H6 promoter. The
right flanking arm adjacent to the NDV-F sequence consists of 2350
bp of FPV sequence. The left flanking arm adjacent to the NDV-HN
sequence consists of 1700 bp of FPV sequence.
[0168] Development of TROVAC-NDV. Plasmid pJCA024 was transfected
into TROVAC infected primary CEF cells by using the calcium
phosphate precipitation method previously described (Panicali et
al., 1982; Piccini et al., 1987). Positive plaques were selected on
the basis of hybridization to specific NDV-F and HN radiolabelled
probes and subjected to five sequential rounds of plaque
purification until a pure population was achieved. One
representative plaque was then amplified and the resulting TROVAC
recombinant was designated TROVAC-NDV (vFP96).
[0169] Immunofluorescence. Indirect immunofluorescence was
performed as described (Taylor et al., 1990) using a polyclonal
anti-NDV serum and, as mono-specific reagents, sera produced in
rabbits against vaccinia virus recombinants expressing NDV-F or
NDV-HN.
[0170] Immunoprecipitation. Immunoprecipitation reactions were
performed as described (Taylor et al., 1990) using a polyclonal
anti-NDV serum obtained from SPAFAS Inc., Storrs, Conn.
[0171] The stock virus was screened by in situ plaque hybridization
to confirm that the F8 ORF was deleted. The correct insertion of
the NDV genes into the TROVAC genome and the deletion of the F8 ORF
was also confirmed by Southern blot hybridization.
[0172] In NDV-infected cells, the F glycoprotein is anchored in the
membrane via a hydrophobic transmembrane region near the carboxyl
terminus and requires post-translational cleavage of a precursor,
F.sub.0, into two disulfide linked polypeptides F.sub.1 and
F.sub.2. Cleavage of F.sub.0 is important in determining the
pathogenicity of a given NDV strain (Homma and Ohuchi, 1973; Nagai
et al., 1976; Nagai et al., 1980), and the sequence of amino acids
at the cleavage site is therefore critical in determining viral
virulence. It has been determined that amino acids at the cleavage
site in the NDV-F sequence inserted into FPV to form recombinant
vFP29 had the sequence Arg-Arg-Gln-Arg-Arg (SEQ ID NO: 149) (Taylor
et al., 1990) which conforms to the sequence found to be a
requirement for virulent NDV strains (Chambers et al., 1986; Espion
et al., 1987; Le et al., 1988; McGinnes and Morrison, 1986; Toyoda
et al., 1987). The HN glycoprotein synthesized in cells infected
with virulent strains of NDV is an uncleaved glycoprotein of 74
kDa. Extremely avirulent strains such as Ulster and Queensland
encode an HN precursor (HNo) which requires cleavage for activation
(Garten et al., 1980).
[0173] The expression of F and HN genes in TROVAC-NDV was analyzed
to confirm that the gene products were authentically processed and
presented. Indirect-immunofluorescence using a polyclonal anti-NDV
chicken serum confirmed that immunoreactive proteins were presented
on the infected cell surface. To determine that both proteins were
presented on the plasma membrane, mono-specific rabbit sera were
produced against vaccinia recombinants expressing either the F or
HN glycoproteins. Indirect immunofluorescence using these sera
confirmed the surface presentation of both proteins.
[0174] Immunoprecipitation experiments were performed by using
(.sup.35S) methionine labeled lysates of CEF cells infected with
parental and recombinant viruses. The expected values of apparent
molecular weights of the glycosylated forms of F.sub.1 and F.sub.2
are 54.7 and 10.3 kDa respectively (Chambers et al., 1986). In the
immunoprecipitation experiments using a polyclonal anti-NDV serum,
fusion specific products of the appropriate size were detected from
the NDV-F single recombinant vFP29 (Taylor et al., 1990) and the
TROVAC-NDV double recombinant vFP96. The HN glycoprotein of
appropriate size was also detected from the NDV-HN single
recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV. No NDV
specific products were detected from uninfected and parental TROVAC
infected CEF cells.
[0175] In CEF cells, the F and HN glycoproteins are appropriately
presented on the infected cell surface where they are recognized by
NDV immune serum. Immunoprecipitation analysis indicated that the
F.sub.0 protein is authentically cleaved to the F.sub.1 and F2
components required in virulent strains. Similarly, the HN
glycoprotein was authentically processed in CEF cells infected with
recombinant TROVAC-NDV.
[0176] Previous reports (Taylor et al., 1990; Edbauer et al., 1990;
Boursnell et al., 1990a,b,c; Ogawa et al., 1990) would indicate
that expression of either HN or F alone is sufficient to elicit
protective immunity against NDV challenge. Work on other
paramyxoviruses has indicated, however, that antibody to both
proteins may be required for full protective immunity. It has been
demonstrated that SV5 virus could spread in tissue culture in the
presence of antibody to the HN glycoprotein but not to the F
glycoprotein (Merz et al., 1980). In addition, it has been
suggested that vaccine failures with killed measles virus vaccines
were due to inactivation of the fusion component (Norrby et al.,
1975). Since both NDV glycoproteins have been shown to be
responsible for eliciting virus neutralizing antibody (Avery et
al., 1979) and both glycoproteins, when expressed individually in a
fowlpox vector are able to induce a protective immune response, it
can be appreciated that the most efficacious NDV vaccine should
express both glycoproteins.
Example 9
Construction of ALVAC Recombinants Expressing Rabies Virus
Glycoprotein G
[0177] This example describes the development of ALVAC, a canarypox
virus vector and, of a canarypox-rabies recombinant designated as
ALVAC-RG (vCP65) and its safety and efficacy.
[0178] Cells and Viruses. The parental canarypox virus (Rentschler
strain) is a vaccinal strain for canaries. The vaccine strain was
obtained from a wild type isolate and attenuated through more than
200 serial passages on chick embryo fibroblasts. A master viral
seed was subjected to four successive plaque purifications under
agar and one plaque clone was amplified through five additional
passages after which the stock virus was used as the parental virus
in in vitro recombination tests. The plaque purified canarypox
isolate is designated ALVAC.
[0179] ALVAC was deposited under the terms of the Budapest Treaty
with the American Type Culture Collection (ATCC), 12301 Parklawn
Drive, Rockville, Md., 20852, USA: ALVAC under ATCC accession
number VR-2547 on Nov. 14, 1996.
[0180] Construction of a Canarypox Insertion Vector. An 880 bp
canarypox PvuII fragment was cloned between the PvuII sites of pUC9
to form pRW764.5. The sequence of this fragment is shown in FIG. 8
(SEQ ID NO:66) between positions 1372 and 2251. The limits of an
open reading frame designated as C5 were defined. It was determined
that the open reading frame was initiated at position 166 within
the fragment and terminated at position 487. The C5 deletion was
made without interruption of open reading frames. Bases from
position 167 through position 455 were replaced with the sequence
(SEQ ID NO:39) GCTTCCCGGGAATTCTAGCTAGCTAGTTT. This replacement
sequence contains HindIII, SmaI and EcoRI insertion sites followed
by translation stops and a transcription termination signal
recognized by vaccinia virus RNA polymerase (Yuen et al., 1987).
Deletion of the C5 ORF was performed as described below. Plasmid
pRW764.5 was partially cut with RsaI and the linear product was
isolated. The RsaI linear fragment was recut with BglII and the
pRW764.5 fragment now with a RsaI to BglII deletion from position
156 to position 462 was isolated and used as a vector for the
following synthetic oligonucleotides:
TABLE-US-00018 RW145 (SEQ ID NO: 40):
ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA RW146 (SEQ ID NO:
41): GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT
Oligonucleotides RW145 and RW146 were annealed and inserted into
the pRW 764.5 RsaI and BglII vector described above. The resulting
plasmid is designated pRW831.
[0181] Construction of Insertion Vector Containing the Rabies G
Gene. Construction of pRW838 is illustrated below. Oligonucleotides
A through E, which overlap the translation initiation codon of the
H6 promoter with the ATG of rabies G, were cloned into pUC9 as
pRW737. Oligonucleotides A through E contain the H6 promoter,
starting at NruI, through the HindIII site of rabies G followed by
BglII. Sequences of oligonucleotides A through E ((SEQ ID
NO:42)-(SEQ ID NO:46)) are:
TABLE-US-00019 A (SEQ ID NO: 42):
CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTAATGGT
TCCTCAGGCTCTCCTGTTTGT B (SEQ ID NO: 43):
CATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTCAG C (SEQ ID NO: 44):
ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATTCCCTATTTACACGA
TCCCAGACAAGCTTAGATCTCAG D (SEQ ID NO: 45):
CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATAGGGAATTTCCCAAAAC A E (SEQ ID NO:
46): CAACGGAAAAACCAGAAGGGGTACAAACAGGAGAGCCTGAGGAAC
The diagram of annealed oligonucleotides A through E is as
follows:
##STR00001##
[0182] Oligonucleotides A through E were kinased, annealed
(95.degree. C. for 5 minutes, then cooled to room temperature), and
inserted between the PvuII sites of pUC9. The resulting plasmid,
pRW737, was cut with HindIII and BglII and used as a vector for the
1.6 kbp HindIII-BglII fragment of ptg155PRO (Kieny et al., 1984)
generating pRW739. The ptg155PRO HindIII site is 86 bp downstream
of the rabies G translation initiation codon. BglII is downstream
of the rabies G translation stop codon in ptg155PRO. pRW739 was
partially cut with NruI, completely cut with BglII, and a 1.7 kbp
NruI-BglII fragment, containing the 3' end of the H6 promoter
previously described (Taylor et al., 1988a,b; Guo et al., 1989;
Perkus et al., 1989) through the entire rabies G gene, was inserted
between the NruI and BamHI sites of pRW824. The resulting plasmid
is designated pRW832. Insertion into pRW824 added the H6 promoter
5' of NruI. The pRW824 sequence of BamHI followed by SmaI is (SEQ
ID NO:47): GGATCCCCGGG. pRW824 is a plasmid that contains a
nonpertinent gene linked precisely to the vaccinia virus H6
promoter. Digestion with NruI and BamHI completely excised this
nonpertinent gene. The 1.8 kbp pRW832 SmaI fragment, containing H6
promoted rabies G, was inserted into the SmaI of pRW831, to form
plasmid pRW838.
[0183] Development of ALVAC-RG. Plasmid pRW838 was transfected into
ALVAC infected primary CEF cells by using the calcium phosphate
precipitation method previously described (Panicali et al., 1982;
Piccini et al., 1987). Positive plaques were selected on the basis
of hybridization to a specific rabies G probe and subjected to 6
sequential rounds of plaque purification until a pure population
was achieved. One representative plaque was then amplified and the
resulting ALVAC recombinant was designated ALVAC-RG (vCP65) (see
also FIGS. 9A and 9B). The correct insertion of the rabies G gene
into the ALVAC genome without subsequent mutation was confirmed by
sequence analysis.
[0184] Immunofluorescence. During the final stages of assembly of
mature rabies virus particles, the glycoprotein component is
transported from the golgi apparatus to the plasma membrane where
it accumulates with the carboxy terminus extending into the
cytoplasm and the bulk of the protein on the external surface of
the cell membrane. In order to confirm that the rabies glycoprotein
expressed in ALVAC-RG was correctly presented, immunofluorescence
was performed on primary CEF cells infected with ALVAC or ALVAC-RG.
Immunofluorescence was performed as previously described (Taylor et
al., 1990) using a rabies G monoclonal antibody. Strong surface
fluorescence was detected on CEF cells infected with ALVAC-RG but
not with the parental ALVAC.
[0185] Immunoprecipitation. Preformed monolayers of primary CEF,
Vero (a line of African Green monkey kidney cells ATCC # CCL81) and
MRC-5 cells (a fibroblast-like cell line derived from normal human
fetal lung tissue ATCC # CCL171) were inoculated at 10 pfu per cell
with parental virus ALVAC and recombinant virus ALVAC-RG in the
presence of radiolabelled .sup.35S-methionine and treated as
previously described (Taylor et al., 1990). Immunoprecipitation
reactions were performed using a rabies G specific monoclonal
antibody. Efficient expression of a rabies specific glycoprotein
with a molecular weight of approximately 67 kDa was detected with
the recombinant ALVAC-RG. No rabies specific products were detected
in uninfected cells or cells infected with the parental ALVAC
virus.
[0186] Sequential Passaging Experiment. In studies with ALVAC virus
in a range of non-avian species no proliferative infection or overt
disease was observed (Taylor et al., 1991b). However, in order to
establish that neither the parental nor recombinant virus could be
adapted to grow in non-avian cells, a sequential passaging
experiment was performed.
[0187] The two viruses, ALVAC and ALVAC-RG, were inoculated in 10
sequential blind passages in three cell substrates: [0188] (1)
Primary chick embryo fibroblast (CEF) cells produced from 11 day
old white leghorn embryos; [0189] (2) Vero cells--a continuous line
of African Green monkey kidney cells (ATCC # CCL81); and [0190] (3)
MRC-5 cells--a diploid cell line derived from human fetal lung
tissue (ATCC # CCL171). The initial inoculation was performed at an
m.o.i. of 0.1 pfu per cell using three 60 mm dishes of each cell
substrate containing 2.times.10.sup.6 cells per dish. One dish was
inoculated in the presence of 40 .mu.g/ml of Cytosine arabinoside
(Ara C), an inhibitor of DNA replication. After an absorption
period of 1 hour at 37.degree. C., the inoculum was removed and the
monolayer washed to remove unabsorbed virus. At this time the
medium was replaced with 5 ml of EMEM+2% NBCS on two dishes
(samples t0 and t7) and 5 ml of EMEM+2% NBCS containing 40 .mu.g/ml
Ara C on the third (sample t7A). Sample t0 was frozen at
-70.degree. C. to provide an indication of the residual input
virus. Samples t7 and t7A were incubated at 37.degree. C. for 7
days, after which time the contents were harvested and the cells
disrupted by indirect sonication.
[0191] One ml of sample t7 of each cell substrate was inoculated
undiluted onto three dishes of the same cell substrate (to provide
samples t0, t7 and t7A) and onto one dish of primary CEF cells.
Samples to, t7 and t7A were treated as for passage one. The
additional inoculation on CEF cells was included to provide an
amplification step for more sensitive detection of virus which
might be present in the non-avian cells.
[0192] This procedure was repeated for 10 (CEF and MRC-5) or 8
(Vero) sequential blind passages. Samples were then frozen and
thawed three times and assayed by titration on primary CEF
monolayers.
[0193] Virus yield in each sample was then determined by plaque
titration on CEF monolayers under agarose. Summarized results of
the experiment are shown in Tables 1 and 2.
[0194] The results indicate that both the parental ALVAC and the
recombinant ALVAC-RG are capable of sustained replication on CEF
monolayers with no loss of titer. In Vero cells, levels of virus
fell below the level of detection after 2 passages for ALVAC and 1
passage for ALVAC-RG. In MRC-5 cells, a similar result was evident,
and no virus was detected after 1 passage. Although the results for
only four passages are shown in Tables 1 and 2 the series was
continued for 8 (Vero) and 10 (MRC-5) passages with no detectable
adaptation of either virus to growth in the non-avian cells.
[0195] In passage 1 relatively high levels of virus were present in
the t7 sample in MRC-5 and Vero cells. However this level of virus
was equivalent to that seen in the t0 sample and the t7A sample
incubated in the presence of Cytosine arabinoside in which no viral
replication can occur. This demonstrated that the levels of virus
seen at 7 days in non-avian cells represented residual virus and
not newly replicated virus.
[0196] In order to make the assay more sensitive, a portion of the
7 day harvest from each cell substrate was inoculated onto a
permissive CEF monolayer and harvested at cytopathic effect (CPE)
or at 7 days if no CPE was evident. The results of this experiment
are shown in Table 3. Even after amplification through a permissive
cell substrate, virus was only detected in MRC-5 and Vero cells for
two additional passages. These results indicated that under the
conditions used, there was no adaptation of either virus to growth
in Vero or MRC-5 cells.
[0197] Inoculation of Macaques. Four HIV seropositive macaques were
initially inoculated with ALVAC-RG as described in Table 4. After
100 days these animals were re-inoculated to determine a booster
effect, and an additional seven animals were inoculated with a
range of doses. Blood was drawn at appropriate intervals and sera
analyzed, after heat inactivation at 56.degree. C. for 30 minutes,
for the presence of anti-rabies antibody using the Rapid
Fluorescent Focus Inhibition Assay (Smith et al., 1973).
[0198] Inoculation of Chimpanzees. Two adult male chimpanzees (50
to 65 kg weight range) were inoculated intramuscularly or
subcutaneously with 1.times.10.sup.7 pfu of vCP65. Animals were
monitored for reactions and bled at regular intervals for analysis
for the presence of anti-rabies antibody with the RFFI test (Smith
et al., 1973). Animals were re-inoculated with an equivalent dose
13 weeks after the initial inoculation.
[0199] Inoculation of Mice. Groups of mice were inoculated with 50
to 100 .mu.l of a range of dilutions of different batches of vCP65.
Mice were inoculated in the footpad. On day 14, mice were
challenged by intracranial inoculation of from 15 to 43 mouse
LD.sub.50 of the virulent CVS strain of rabies virus. Survival of
mice was monitored and a protective dose 50% (PD.sub.50) calculated
at 28 days post-inoculation.
[0200] Inoculation of Dogs and Cats. Ten beagle dogs, 5 months old,
and 10 cats, 4 months old, were inoculated subcutaneously with
either 6.7 or 7.7 log.sub.10 TCID.sub.50 of ALVAC-RG. Four dogs and
four cats were not inoculated. Animals were bled at 14 and 28 days
post-inoculation and anti-rabies antibody assessed in an RFFI test.
The animals receiving 6.7 log.sub.10 TCID.sub.50 of ALVAC-RG were
challenged at 29 days post-vaccination with 3.7 log.sub.10 mouse
LD.sub.50 (dogs) or 4.3 log.sub.10 mouse LD.sub.50 (cats) of the
NYGS rabies virus challenge strain.
[0201] Inoculation of Squirrel Monkeys. Three groups of four
squirrel monkeys (Saimiri sciureus) were inoculated with one of
three viruses (a) ALVAC, the parental canarypox virus, (b)
ALVAC-RG, the recombinant expressing the rabies G glycoprotein or
(c) vCP37, a canarypox recombinant expressing the envelope
glycoprotein of feline leukemia virus. Inoculations were performed
under ketamine anaesthesia. Each animal received at the same time:
(1) 20 .mu.l instilled on the surface of the right eye without
scarification; (2) 100 .mu.l as several droplets in the mouth; (3)
100 .mu.l in each of two intradermal injection sites in the shaven
skin of the external face of the right arm; and (4) 100 .mu.l in
the anterior muscle of the right thigh.
[0202] Four monkeys were inoculated with each virus, two with a
total of 5.0 log.sub.10 pfu and two with a total of 7.0 log.sub.10
pfu. Animals were bled at regular intervals and sera analyzed for
the presence of antirabies antibody using an RFFI test (Smith et
al., 1973). Animals were monitored daily for reactions to
vaccination. Six months after the initial inoculation the four
monkeys receiving ALVAC-RG, two monkeys initially receiving vCP37,
and two monkeys initially receiving ALVAC, as well as one naive
monkey were inoculated with 6.5 log.sub.10 pfu of ALVAC-RG
subcutaneously. Sera were monitored for the presence of rabies
neutralizing antibody in an RFFI test (Smith et al., 1973).
[0203] Inoculation of Human Cell Lines with ALVAC-RG. In order to
determine whether efficient expression of a foreign gene could be
obtained in non-avian cells in which the virus does not
productively replicate, five cell types, one avian and four
non-avian, were analyzed for virus yield, expression of the foreign
rabies G gene and viral specific DNA accumulation. The cells
inoculated were: [0204] (a) Vero, African Green monkey kidney
cells, ATCC # CCL81; [0205] (b) MRC-5, human embryonic lung, ATCC #
CCL 171; [0206] (c) WISH human amnion, ATCC # CCL 25; [0207] (d)
Detroit-532, human foreskin, Downs's syndrome, ATCC # CCL 54; and
[0208] (e) Primary CEF cells.
[0209] Chicken embryo fibroblast cells produced from 11 day old
white leghorn embryos were included as a positive control. All
inoculations were performed on preformed monolayers of
2.times.10.sup.6 cells as discussed below.
A. Methods for DNA Analysis.
[0210] Three dishes of each cell line were inoculated at 5 pfu/cell
of the virus under test, allowing one extra dish of each cell line
un-inoculated. One dish was incubated in the presence of 40
.mu.g/ml of cytosine arabinoside (Ara C). After an adsorption
period of 60 minutes at 37.degree. C., the inoculum was removed and
the monolayer washed twice to remove unadsorbed virus. Medium (with
or without Ara C) was then replaced. Cells from one dish (without
Ara C) were harvested as a time zero sample. The remaining dishes
were incubated at 37.degree. C. for 72 hours, at which time the
cells were harvested and used to analyze DNA accumulation. Each
sample of 2.times.10.sup.6 cells was resuspended in 0.5 ml
phosphate buffered saline (PBS) containing 40 mM EDTA and incubated
for 5 minutes at 37.degree. C. An equal volume of 1.5% agarose
prewarmed at 42.degree. C. and containing 120 mM EDTA was added to
the cell suspension and gently mixed. The suspension was
transferred to an agarose plug mold and allowed to harden for at
least 15 min. The agarose plugs were then removed and incubated for
12-16 hours at 50.degree. C. in a volume of lysis buffer (1%
sarkosyl, 100 .mu.g/ml proteinase K, 10 mM Tris HCl pH 7.5, 200 mM
EDTA) that completely covers the plug. The lysis buffer was then
replaced with 5.0 ml sterile 0.5.times.TBE (44.5 mM Tris-borate,
44.5 mM boric acid, 0.5 mM EDTA) and equilibrated at 4.degree. C.
for 6 hours with 3 changes of TBE buffer. The viral DNA within the
plug was fractionated from cellular RNA and DNA using a pulse field
electrophoresis system. Electrophoresis was performed for 20 hours
at 180 V with a ramp of 50-90 sec at 15.degree. C. in
0.5.times.TBE. The DNA was run with lambda DNA molecular weight
standards. After electrophoresis the viral DNA band was visualized
by staining with ethidium bromide. The DNA was then transferred to
a nitrocellulose membrane and probed with a radiolabelled probe
prepared from purified ALVAC genomic DNA.
B. Estimation of Virus Yield.
[0210] [0211] Dishes were inoculated exactly as described above,
with the exception that input multiplicity was 0.1 pfu/cell. At 72
hours post infection, cells were lysed by three successive cycles
of freezing and thawing. Virus yield was assessed by plaque
titration on CEF monolayers.
C. Analysis of Expression of Rabies G Gene.
[0211] [0212] Dishes were inoculated with recombinant or parental
virus at a multiplicity of 10 pfu/cell, allowing an additional dish
as an uninfected virus control. After a one hour absorption period,
the medium was removed and replaced with methionine free medium.
After a 30 minute period, this medium was replaced with
methionine-free medium containing 25 uCi/ml of .sup.35S-Methionine.
Infected cells were labelled overnight (approximately 16 hours),
then lysed by the addition of buffer A lysis buffer.
Immunoprecipitation was performed as previously described (Taylor
et al., 1990) using a rabies G specific monoclonal antibody.
[0213] Results: Estimation of Viral Yield. The results of titration
for yield at 72 hours after inoculation at. 0.1 pfu per cell are
shown in Table 5. The results indicate that while a productive
infection can be attained in the avian cells, no increase in virus
yield can be detected by this method in the four non-avian cell
systems.
[0214] Analysis of Viral DNA Accumulation. In order to determine
whether the block to productive viral replication in the non-avian
cells occurred before or after DNA replication, DNA from the cell
lysates was fractionated by electrophoresis, transferred to
nitrocellulose and probed for the presence of viral specific DNA.
DNA from uninfected CEF cells, ALVAC-RG infected CEF cells at time
zero, ALVAC-RG infected CEF cells at 72 hours post-infection and
ALVAC-RG infected CEF cells at 72 hours post-infection in the
presence of 40 pg/ml of cytosine arabinoside all showed some
background activity, probably due to contaminating CEF cellular DNA
in the radiolabelled ALVAC DNA probe preparation. However, ALVAC-RG
infected CEF cells at 72 hours post-infection exhibited a strong
band in the region of approximately 350 kbp representing
ALVAC-specific viral DNA accumulation. No such band is detectable
when the culture is incubated in the presence of the DNA synthesis
inhibitor, cytosine arabinoside. Equivalent samples produced in
Vero cells showed a very faint band at approximately 350 kbp in the
ALVAC-RG infected Vero cells at time zero. This level represented
residual virus. The intensity of the band was amplified at 72 hours
post-infection indicating that some level of viral specific DNA
replication had occurred in Vero cells which had not resulted in an
increase in viral progeny. Equivalent samples produced in MRC-5
cells indicated that no viral specific DNA accumulation was
detected under these conditions in this cell line. This experiment
was then extended to include additional human cell lines,
specifically WISH and Detroit-532 cells. ALVAC infected CEF cells
served as a positive control. No viral specific DNA accumulation
was detected in either WISH or Detroit cells inoculated with
ALVAC-RG. It should be noted that the limits of detection of this
method have not been fully ascertained and viral DNA accumulation
may be occurring, but at a level below the sensitivity of the
method. Other experiments in which viral DNA replication was
measured by .sup.3H-thymidine incorporation support the results
obtained with Vero and MRC-5 cells.
[0215] Analysis of Rabies Gene Expression. To determine if any
viral gene expression, particularly that of the inserted foreign
gene, was occurring in the human cell lines even in the absence of
viral DNA replication, immunoprecipitation experiments were
performed on .sup.35S-methionine labelled lysates of avian and
non-avian cells infected with ALVAC and ALVAC-RG. The results of
immunoprecipitation using a rabies G specific monoclonal antibody
illustrated specific immunoprecipitation of a 67 kDa glycoprotein
in CEF, Vero and MRC-5, WISH and Detroit cells infected with
ALVAC-RG. No such specific rabies gene products were detected in
any of the uninfected and parentally infected cell lysates.
[0216] The results of this experiment indicated that in the human
cell lines analyzed, although the ALVAC-RG recombinant was able to
initiate an infection and express a foreign gene product under the
transcriptional control of the H6 early/late vaccinia virus
promoter, the replication did not proceed through DNA replication,
nor was there any detectable viral progeny produced. In the Vero
cells, although some level of ALVAC-RG specific DNA accumulation
was observed, no viral progeny was detected by these methods. These
results would indicate that in the human cell lines analyzed the
block to viral replication occurs prior to the onset of DNA
replication, while in Vero cells, the block occurs following the
onset of viral DNA replication.
[0217] In order to determine whether the rabies glycoprotein
expressed in ALVAC-RG was immunogenic, a number of animal species
were tested by inoculation of the recombinant. The efficacy of
current rabies vaccines is evaluated in a mouse model system. A
similar test was therefore performed using ALVAC-RG. Nine different
preparations of virus (including one vaccine batch (J) produced
after 10 serial tissue culture passages of the seed virus) with
infectious titers ranging from 6.7 to 8.4 log.sub.10 TCID.sub.50
per ml were serially diluted and 50 to 100 .mu.l of dilutions
inoculated into the footpad of four to six week old mice. Mice were
challenged 14 days later by the intracranial route with 300 .mu.l
of the CVS strain of rabies virus containing from 15 to 43 mouse
LD.sub.50 as determined by lethality titration in a control group
of mice. Potency, expressed as the PD.sub.50 (Protective dose 50%),
was calculated at 14 days post-challenge. The results of the
experiment are shown in Table 6. The results indicated that
ALVAC-RG was consistently able to protect mice against rabies virus
challenge with a PD.sub.50 value ranging from 3.33 to 4.56 with a
mean value of 3.73 (STD 0.48). As an extension of this study, male
mice were inoculated intracranially with 50 .mu.l of virus
containing 6.0 log.sub.10 TCID.sub.50 of ALVAC-RG or with an
equivalent volume of an uninfected cell suspension. Mice were
sacrificed on days 1, 3 and 6 post-inoculation and their brains
removed, fixed and sectioned. Histopathological examination showed
no evidence for neurovirulence of ALVAC-RG in mice.
[0218] In order to evaluate the safety and efficacy of ALVAC-RG for
dogs and cats, a group of 14, 5 month old beagles and 14, 4 month
old cats were analyzed. Four animals in each species were not
vaccinated. Five animals received 6.7 log.sub.10 TCID.sub.50
subcutaneously and five animals received 7.7 log.sub.10 TCID.sub.50
by the same route. Animals were bled for analysis for anti-rabies
antibody. Animals receiving no inoculation or 6.7 log.sub.10
TCID.sub.50 of ALVAC-RG were challenged at 29 days post-vaccination
with 3.7 log.sub.10 mouse LD.sub.50 (dogs, in the temporal muscle)
or 4.3 log.sub.10 mouse LD.sub.50 (cats, in the neck) of the NYGS
rabies virus challenge strain. The results of the experiment are
shown in Table 7.
[0219] No adverse reactions to inoculation were seen in either cats
or dogs with either dose of inoculum virus. Four of 5 dogs
immunized with 6.7 log.sub.10 TCID.sub.50 had antibody titers on
day 14 post-vaccination and all dogs had titers at 29 days. All
dogs were protected from a challenge which killed three out of four
controls. In cats, three of five cats receiving 6.7 log.sub.10
TCID.sub.50 had specific antibody titers on day 14 and all cats
were positive on day 29 although the mean antibody titer was low at
2.9 IU. Three of five cats survived a challenge which killed all
controls. All cats immunized with 7.7 log.sub.10 TCID.sub.50 had
antibody titers on day 14 and at day 29 the Geometric Mean Titer
was calculated as 8.1 International Units.
[0220] The immune response of squirrel monkeys (Saimiri sciureus)
to inoculation with ALVAC, ALVAC-RG and an unrelated canarypox
virus recombinant was examined. Groups of monkeys were inoculated
as described above and sera analyzed for the presence of rabies
specific antibody. Apart from minor typical skin reactions to
inoculation by the intradermal route, no adverse reactivity was
seen in any of the monkeys. Small amounts of residual virus were
isolated from skin lesions after intradermal inoculation on days
two and four post-inoculation only. All specimens were negative on
day seven and later. There was no local reaction to intra-muscular
injection. All four monkeys inoculated with ALVAC-RG developed
anti-rabies serum neutralizing antibodies as measured in an RFFI
test. Approximately six months after the initial inoculation all
monkeys and one additional naive monkey were re-inoculated by the
subcutaneous route on the external face of the left thigh with 6.5
log.sub.10 TCID.sub.50 of ALVAC-RG. Sera were analyzed for the
presence of anti-rabies antibody. The results are shown in Table
8.
[0221] Four of the five monkeys naive to rabies developed a
serological response by seven days post-inoculation with ALVAC-RG.
All five monkeys had detectable antibody by 11 days
post-inoculation. Of the four monkeys with previous exposure to the
rabies glycoprotein, all showed a significant increase in serum
neutralization titer between days 3 and 7 post-vaccination. The
results indicate that vaccination of squirrel monkeys with ALVAC-RG
does not produce adverse side-effects and a primary neutralizing
antibody response can be induced. An anamnestic response is also
induced on re-vaccination. Prior exposure to ALVAC or to a
canarypox recombinant expressing an unrelated foreign gene does not
interfere with induction of an anti-rabies immune response upon
re-vaccination.
[0222] The immunological response of HIV-2 seropositive macaques to
inoculation with ALVAC-RG was assessed. Animals were inoculated as
described above and the presence of anti-rabies serum neutralizing
antibody assessed in an RFFI test. The results, shown in Table 9,
indicated that HIV-2 positive animals inoculated by the
subcutaneous route developed anti-rabies antibody by 11 days after
one inoculation. An anamnestic response was detected after a
booster inoculation given approximately three months after the
first inoculation. No response was detected in animals receiving
the recombinant by the oral route. In addition, a series of six
animals were inoculated with decreasing doses of ALVAC-RG given by
either the intra-muscular or subcutaneous routes. Five of the six
animals inoculated responded by 14 days post-vaccination with no
significant difference in antibody titer.
[0223] Two chimpanzees with prior exposure to HIV were inoculated
with 7.0 log.sub.10 pfu of ALVAC-RG by the subcutaneous or
intra-muscular route. At 3 months post-inoculations both animals
were re-vaccinated in an identical fashion. The results are shown
in Table 10.
[0224] No adverse reactivity to inoculation was noted by either
intramuscular or subcutaneous routes. Both chimpanzees responded to
primary inoculation by 14 days and a strongly rising response was
detected following re-vaccination.
TABLE-US-00020 TABLE 1 Sequential Passage of ALVAC in Avian and
non-Avian Cells. CEF Vero MRC5 Pass 1 Sample to.sup.a 2.4 3.0 2.6
t7.sup.b 7.0 1.4 0.4 t7A.sup.c 1.2 1.2 0.4 Pass 2 Sample to 5.0 0.4
.sup. N.D..sup.d t7 7.3 0.4 N.D. t7A 3.9 N.D. N.D. Pass 3 Sample to
5.4 0.4 N.D. t7 7.4 N.D. N.D. t7A 3.8 N.D. N.D. Pass 4 Sample to
5.2 N.D. N.D. t7 7.1 N.D. N.D. t7A 3.9 N.D. N.D. .sup.aThis sample
was harvested at zero time and represents the residual input virus.
The titer is expressed as log.sub.10 pfu per ml. .sup.bThis sample
was harvested at 7 days post-infection. .sup.cThis sample was
inoculated in the presence of 40 .mu.g/ml of Cytosine arabinoside
and harvested at 7 days post infection. .sup.dNot detectable
TABLE-US-00021 TABLE 2 Sequential Passage of ALVAC-RG in Avian and
non-Avian Cells CEF Vero MRC-5 Pass 1 Sample t0.sup.a 3.0 2.9 2.9
t7.sup.b 7.1 1.0 1.4 t7A.sup.c 1.8 1.4 1.2 Pass 2 Sample t0 5.1 0.4
0.4 t7 7.1 .sup. N.D..sup.d N.D. t7A 3.8 N.D. N.D. Pass 3 Sample t0
5.1 0.4 N.D. t7 7.2 N.D. N.D. t7A 3.6 N.D. N.D. Pass 4 Sample t0
5.1 N.D. N.D. t7 7.0 N.D. N.D. t7A 4.0 N.D. N.D .sup.aThis sample
was harvested at zero time and represents the residual input virus.
The titer is expressed as log.sub.10 pfu per ml. .sup.bThis sample
was harvested at 7 days post-infection. .sup.cThis sample was
inoculated in the presence of 40 .mu.g/ml of Cytosine arabinoside
and harvested at 7 days post-infection. .sup.dNot detectable.
TABLE-US-00022 TABLE 3 Amplification of residual virus by passage
in CEF cells CEF Vero MRC-5 a) ALVAC Pass 2.sup.a .sup. 7.0.sup.b
6.0 5.2 3 7.5 4.1 4.9 4 7.5 .sup. N.D..sup.c N.D. 5 7.1 N.D. N.D.
b) ALVAC-RG Pass 2.sup.a 7.2 5.5 5.5 3 7.2 5.0 5.1 4 7.2 N.D. N.D.
5 7.2 N.D. N.D. .sup.aPass 2 represents the amplification in CEF
cells of the 7 day sample from Pass 1. .sup.bTiter expressed as
log.sub.10 pfu per ml .sup.cNot Detectable
TABLE-US-00023 TABLE 4 Schedule of inoculation of rhesus macaques
with ALVAC-RG (vCP65) Animal Inoculation 176L Primary: 1 .times.
10.sup.8 pfu of vCP65 orally in TANG Secondary: 1 .times. 10.sup.7
pfu of vCP65 plus 1 .times. 10.sup.7 pfu of vCP82.sup.a by SC route
185 L Primary: 1 .times. 10.sup.8 pfu of vCP65 orally in Tang
Secondary: 1 .times. 10.sup.7 pfu of vCP65 plus 1 .times. 10.sup.7
pfu of vCP82 by SC route 177 L Primary: 5 .times. 10.sup.7 pfu SC
of vCP65 by SC route Secondary: 1 .times. 10.sup.7 pfu of vCP65
plus 1 .times. 10.sup.7 pfu of vCP82 by SC route 186L Primary: 5
.times. 10.sup.7 pfu of vCP65 by SC route Secondary: 1 .times.
10.sup.7 pfu of vCP65 plus 1 .times. 10.sup.7 pfu of vCP82 by SC
route 178L Primary: 1 .times. 10.sup.7 pfu of vCP65 by SC route
182L Primary: 1 .times. 10.sup.7 pfu of VCP65 by IM route 179L
Primary: 1 .times. 10.sup.6 pfu of vCP65 by SC route 183L Primary:
1 .times. 10.sup.6 pfu of vCP65 by IM route 180L Primary: 1 .times.
10.sup.6 pfu of vCP65 by SC route 184L Primary: 1 .times. 10.sup.5
pfu of vCP65 by IM route 187L Primary 1 .times. 10.sup.7 pfu of
vCP65 orally .sup.avCP82 is a canarypox virus recombinant
expressing the measles virus fusion and hemagglutinin genes.
TABLE-US-00024 TABLE 5 Analysis of yield in avian and non-avian
cells inoculated with ALVAC-RG Sample Time Cell Type t0 t72
t72A.sup.b Expt 1 CEF .sup. 3.3.sup.a 7.4 1.7 Vero 3.0 1.4 1.7
MRC-5 3.4 2.0 1.7 Expt 2 CEF 2.9 7.5 <1.7 WISH 3.3 2.2 2.0
Detroit-532 2.8 1.7 <1.7 .sup.aTiter expressed as log.sub.10 pfu
per ml .sup.bCulture incubated in the presence of 40 .mu.g/ml of
Cytosine arabinoside
TABLE-US-00025 TABLE 6 Potency of ALVAC-RG as tested in mice Test
Challenge Dose.sup.a PD.sub.50.sup.b Initial seed 43 4.56 Primary
seed 23 3.34 Vaccine Batch H 23 4.52 Vaccine Batch I 23 3.33
Vaccine Batch K 15 3.64 Vaccine Batch L 15 4.03 Vaccine Batch M 15
3.32 Vaccine Batch N 15 3.39 Vaccine Batch J 23 3.42
.sup.aExpressed as mouse LD.sub.50 .sup.bExpressed as log.sub.10
TCID.sub.50
TABLE-US-00026 TABLE 7 Efficacy of ALVAC-RG in dogs and cats Dogs
Cats Dose Antibody.sup.a Survival.sup.b Antibody Survival 6.7 11.9
5/5 2.9 3/5 7.7 10.1 N.T. 8.1 N.T. .sup.aAntibody at day 29 post
inoculation expressed as the geometric mean titer in International
Units. .sup.bExpressed as a ratio of survivors over animals
challenged
TABLE-US-00027 TABLE 8 Anti-rabies serological response of Squirrel
monkeys inoculated with canarypox recombinants Monkey Previous
Rabies serum-neutralizing antibody.sup.a # Exposure -196.sup.b 0 3
7 11 21 28 22 ALVAC.sup.c .sup. NT.sup.g <1.2 <1.2 <1.2
2.1 2.3 2.2 51 ALVAC.sup.c NT <1.2 <1.2 1.7 2.2 2.2 2.2 39
vCP37.sup.d NT <1.2 <1.2 1.7 2.1 2.2 N.T..sup.g 55
vCP37.sup.d NT <1.2 <1.2 1.7 2.2 2.1 N.T..sup. 37
ALVAC-RG.sup.e 2.2 <1.2 <1.2 3.2 3.5 3.5 3.2 53
ALVAC-RG.sup.e 2.2 <1.2 <1.2 3.6 3.6 3.6 3.4 38
ALVAC-RG.sup.f 2.7 <1.7 <1.7 3.2 3.8 3.6 N.T..sup. 54
ALVAC-RG.sup.f 3.2 <1.7 <1.5 3.6 4.2 4.0 3.6 57 None NT
<1.2 <1.2 1.7 2.7 2.7 2.3 .sup.aAs determined by RFFI test on
days indicated and expressed in International Units .sup.bDay -196
represents serum from day 28 after primary vaccination
.sup.cAnimals received 5.0 log.sub.10 TCID.sub.50 of ALVAC
.sup.dAnimals received 5.0 log.sub.10 TCID.sub.50 of vCP37
.sup.eAnimals received 5.0 log.sub.10 TCID.sub.50 of ALVAC-RG
.sup.fAnimals received 7.0 log.sub.10 TCID.sub.50 of ALVAC-RG
.sup.gNot tested.
TABLE-US-00028 TABLE 9 Inoculation of rhesus macaques with
ALVAC-RG.sup.a Route of Primary Inoculation Days post- or/Tang SC
SC SC IM SC IM SC IM OR Inoculation 176L.sup.b 185L 177L 186L 178L
182L 179L 183L 180L 184L 187L.sup.b -84 -- -- -- -9 -- -- -- -- --
-- 3 -- -- -- -- 6 -- -- .+-. .+-. 11 -- -- .sup. 16.sup.d 128 19
-- -- 32 128 -- -- 35 -- -- 32 512 59 -- -- 64 256 75 -- -- 64 128
-- -- .sup. 99.sup.c -- -- 64 256 -- -- -- -- -- -- 2 -- -- 32 256
-- -- -- -- -- -- -- 6 -- -- 512 512 -- -- -- -- -- -- -- 15 16 16
512 512 64 32 64 128 32 -- -- 29 16 32 256 256 64 64 32 128 32 --
-- 55 32 32 32 16 -- 57 16 128 128 16 16 -- .sup.aSee Table 9 for
schedule of inoculations. .sup.bAnimals 176L and 185L received 8.0
log.sub.10 pfu by the oral route in 5 ml Tang. Animal 187L received
7.0 log.sub.10 pfu by oral route not in Tang. .sup.cDay of
re-vaccination for animals 176L, 185L, 177L and 186L by S.C. route,
and primary vaccination for animals 178L, 182L, 179L, 183L, 180L,
184L and 187L. .sup.dTiters expressed as reciprocal of last
dilution showing inhibition of fluorescence in an RFFI test.
TABLE-US-00029 TABLE 10 Inoculation of chimpanzees with ALVAC-RG
Weeks post- Animal 431 Animal 457 Inoculation I.M. S.C. 0 .sup.
<8.sup.a <8 1 <8 <8 2 8 32 4 16 32 8 16 32
12.sup.b/0.sup. 16 8 13/1 128 128 15/3 256 512 20/8 64 128 26/12 32
128 .sup.aTiter expressed as reciprocal of last dilution showing
inhibition of fluorescence in an RFFI test .sup.bDay of
re-inoculation
Example 10
Immunization of Humans Using Canarypox Expressing Rabies
Glycoprotein (ALVAC-RG; vCP65)
[0225] ALVAC-RG (vCP65) was Generated as Described in Example 9 and
FIGS. 9A and 9B. For scaling-up and vaccine manufacturing ALVAC-RG
(vCP65) was grown in primary CEF derived from specified pathogen
free eggs. Cells were infected at a multiplicity of 0.1 and
incubated at 37.degree. C. for three days.
[0226] The vaccine virus suspension was obtained by ultrasonic
disruption in serum free medium of the infected cells; cell debris
were then removed by centrifugation and filtration. The resulting
clarified suspension was supplemented with lyophilization
stabilizer (mixture of amino-acids), dispensed in single dose vials
and freeze dried. Three batches of decreasing titer were prepared
by ten-fold serial dilutions of the virus suspension in a mixture
of serum free medium and lyophilization stabilizer, prior to
lyophilization.
[0227] Quality control tests were applied to the cell substrates,
media and virus seeds and final product with emphasis on the search
for adventitious agents and inocuity in laboratory rodents. No
undesirable trait was found.
[0228] Preclinical data. Studies in vitro indicated that VERO or
MRC-5 cells do not support the growth of ALVAC-RG (vCP65); a series
of eight (VERO) and 10 (MRC) blind serial passages caused no
detectable adaptation of the virus to grow in these non avian
lines. Analyses of human cell lines (MRC-5, WISH, Detroit 532, HEL,
HNK or EBV-transformed lymphoblastoid cells) infected or inoculated
with ALVAC-RG (vCP65) showed no accumulation of virus specific DNA
suggesting that in these cells the block in replication occurs
prior to DNA synthesis. Significantly, however, the expression of
the rabies virus glycoprotein gene in all cell lines tested
indicating that the abortive step in the canarypox replication
cycle occurs prior to viral DNA replication.
[0229] The safety and efficacy of ALVAC-RG (vCP65) were documented
in a series of experiments in animals. A number of species
including canaries, chickens, ducks, geese, laboratory rodents
(suckling and adult mice), hamsters, guinea-pigs, rabbits, cats and
dogs, squirrel monkeys, rhesus macaques and chimpanzees, were
inoculated with doses ranging from 10.sup.5 to 10.sup.8 pfu. A
variety of routes were used, most commonly subcutaneous,
intramuscular and intradermal but also oral (monkeys and mice) and
intracerebral (mice).
[0230] In canaries, ALVAC-RG (vCP65) caused a "take" lesion at the
site of scarification with no indication of disease or death.
Intradermal inoculation of rabbits resulted in a typical poxvirus
inoculation reaction which did not spread and healed in seven to
ten days. There was no adverse side effects due to canarypox in any
of the animal tests. Immunogenicity was documented by the
development of anti-rabies antibodies following inoculation of
ALVAC-RG (vCP65) in rodents, dogs, cats, and primates, as measured
by Rapid Fluorescent Focus Inhibition Test (RFFIT). Protection was
also demonstrated by rabies virus challenge experiments in mice,
dogs, and cats immunized with ALVAC-RG (vCP65).
[0231] Volunteers. Twenty-five healthy adults aged 20-45 with no
previous history of rabies immunization were enrolled. Their health
status was assessed by complete medical histories, physical
examinations, hematological and blood chemistry analyses. Exclusion
criteria included pregnancy, allergies, immune depression of any
kind, chronic debilitating disease, cancer, injection of
immunoglobins in the past three months, and seropositivity to human
immunodeficiency virus (HIV) or to hepatitis B virus surface
antigen.
[0232] Study design. Participants were randomly allocated to
receive either standard Human Diploid Cell Rabies Vaccine (HDC)
batch no E0751 (Pasteur Merieux Serums & Vaccine, Lyon, France)
or the study vaccine ALVAC-RG (vCP65).
[0233] The trial was designated as a dose escalation study. Three
batches of experimental ALVAC-RG (vCP65) vaccine were used
sequentially in three groups of volunteers (Groups A, B and C) with
two week intervals between each step. The concentration of the
three batches was 10.sup.3.5, 10.sup.4.5, 10.sup.5.5 Tissue Culture
Infectious Dose (TCID.sub.50) per dose, respectively.
[0234] Each volunteer received two doses of the same vaccine
subcutaneously in the deltoid region at an interval of four weeks.
The nature of the injected vaccine was not known by the
participants at the time of the first injection but was known by
the investigator.
[0235] In order to minimize the risk of immediate hypersensitivity
at the time of the second injection, the volunteers of Group B
allocated to the medium dose of experimental vaccine were injected
1 h previously with the lower dose and those allocated to the
higher dose (Group C) received successively the lower and the
medium dose at hourly intervals.
[0236] Six months later, the recipients of the highest dosage of
ALVAC-RG (vCP65) (Group C) and HDC vaccine were offered a third
dose of vaccine; they were then randomized to receive either the
same vaccine as previously or the alternate vaccine. As a result,
four groups were formed corresponding to the following immunization
scheme: 1. HDC, HDC-HDC; 2. HDC, HDC-ALVAC-RG (vCP65); 3. ALVAC-RG
(vCP65), ALVAC-RG (vCP65)-HDC; 4. ALVAC-RG (vCP65), ALVAC-RG
(vCP65), ALVAC-RG (vCP65).
[0237] Monitoring of Side Effects. All subjects were monitored for
1 h after injection and re-examined every day for the next five
days. They were asked to record local and systemic reactions for
the next three weeks and were questioned by telephone two times a
week.
[0238] Laboratory Investigators. Blood specimens were obtained
before enrollment and two, four and six days after each injection.
Analysis included complete blood cell count, liver enzymes and
creatine kinase assays.
[0239] Antibody assays. Antibody assays were performed seven days
prior to the first injection and at days 7, 28, 35, 56, 173, 187
and 208 of the study.
[0240] The levels of neutralizing antibodies to rabies were
determined using the Rapid Fluorescent Focus Inhibition test
(RFFIT) (Smith et al., 1973). Canarypox antibodies were measured by
direct ELISA. The antigen, a suspension of purified canarypox virus
disrupted with 0.1% Triton X100, was coated in microplates. Fixed
dilutions of the sera were reacted for two hours at room
temperature and reacting antibodies were revealed with a peroxidase
labelled anti-human IgG goat serum. The results are expressed as
the optical density read at 490 nm.
[0241] Analysis. Twenty-five subjects were enrolled and completed
the study. There were 10 males and 15 females and the mean age was
31.9 (21 to 48). All but three subjects had evidence of previous
smallpox vaccination; the three remaining subjects had no typical
scar and vaccination history. Three subjects received each of the
lower doses of experimental vaccine (10.sup.3.5 and 10.sup.4.5
TCID.sub.50), nine subjects received 10.sup.5.5 TCID.sub.50 and ten
received the HDC vaccine.
[0242] Safety (Table 11). During the primary series of
immunization, fever greater than 37.7.degree. C. was noted within
24 hours after injection in one HDC recipient (37.8.degree. C.) and
in one vCP65 10.sup.5.5 TCID.sub.50 recipient (38.degree. C.). No
other systemic reaction attributable to vaccination was observed in
any participant.
[0243] Local reactions were noted in 9/10 recipients of HDC vaccine
injected subcutaneously and in 0/3, 1/3 and 9/9 recipients of vCP65
10.sup.3.5, 10.sup.4.5, 10.sup.5.5 TCID.sub.50, respectively.
[0244] Tenderness was the most common symptoms and was always mild.
Other local symptoms included redness and induration which were
also mild and transient. All symptoms usually subsided within 24
hours and never lasted more than 72 hours.
[0245] There was no significant change in blood cell counts, liver
enzymes or creatine kinase values.
[0246] Immune Responses; Neutralizing Antibodies to Rabies (Table
12). Twenty eight days after the first injection all the HDC
recipients had protective titers (.gtoreq.0.5 IU/ml). By contrast
none in groups A and B (10.sup.3.5 and 10.sup.4.5 TCID.sub.50) and
only 2/9 in group C (10.sup.5.5 TCID.sub.50) ALVAC-RG (vCP65)
recipients reached this protective titer.
[0247] At day 56 (i.e. 28 days after the second injection)
protective titers were achieved in 0/3 of Group A, 2/3 of Group B
and 9/9 of Group C recipients of ALVAC-RG (vCP65) vaccine and
persisted in all 10 HDC recipients.
[0248] At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and
11.5 IU/ml in groups A, B, C and HDC respectively.
[0249] At day 180, the rabies antibody titers had substantially
decreased in all subjects but remained above the minimum protective
titer of 0.5 IU/ml in 5/10 HCD recipients and in 5/9 ALVAC-RG
(vCP65) recipients; the geometric mean titers were 0.51 and 0.45
IU/ml in groups HCD and C, respectively.
[0250] Antibodies to the Canarypox virus (Table 13). The pre-immune
titers observed varied widely with titers varying from 0.22 to 1.23
O.D. units despite the absence of any previous contact with canary
birds in those subjects with the highest titers. When defined as a
greater than two-fold increase between preimmunization and post
second injection titers, a seroconversion was obtained in 1/3
subjects in group B and in 9/9 subjects in group C whereas no
subject seroconverted in groups A or HDC.
[0251] Booster Injection. The vaccine was similarly well tolerated
six months later, at the time of the booster injection: fever was
noted in 2/9 HDC booster recipients and in 1/10 ALVAC-RG (vCP65)
booster recipients. Local reactions were present in 5/9 recipients
of HDC booster and in 6/10 recipients of the ALVAC-RG (vCP65)
booster.
[0252] Observations. FIGS. 13A-13D show graphs of rabies
neutralizing antibody titers (Rapid Fluorescent Focus Inhibition
Test or RFFIT, IU/ml): Booster effect of HDC and vCP65 (10.sup.5.5
TCID.sub.50) in volunteers previously immunized with either the
same or the alternate vaccine. Vaccines were given at days 0, 28
and 180. Antibody titers were measured at days 0, 7, 28, 35, 56,
173, and 187 and 208.
[0253] As shown in FIGS. 13A to 13D, the booster dose given
resulted in a further increase in rabies antibody titers in every
subject whatever the immunization scheme. However, the ALVAC-RG
(vCP65) booster globally elicited lower immune responses than the
HDC booster and the ALVAC-RG (vCP65), ALVAC-RG (vCP65)-ALVAC-RG
(vCP65) group had significantly lower titers than the three other
groups. Similarly, the ALVAC-RG (vCP65) booster injection resulted
in an increase in canarypox antibody titers in 3/5 subjects who had
previously received the HDC vaccine and in all five subjects
previously immunized with ALVAC-RG (vCP65).
[0254] In general, none of the local side effects from
administration of vCP65 was indicative of a local replication of
the virus. In particular, lesions of the skin such as those
observed after injection of vaccine were absent. In spite of the
apparent absence of replication of the virus, the injection
resulted in the volunteers generating significant amounts of
antibodies to both the canarypox vector and to the expressed rabies
glycoprotein.
[0255] Rabies neutralizing antibodies were assayed with the Rapid
Fluorescent Focus Inhibition Test (RFFIT) which is known to
correlate well with the sero neutralization test in mice. Of 9
recipients of 10.sup.5.5TCID.sub.50, five had low level responses
after the first dose. Protective titers of rabies antibodies were
obtained after the second injection in all recipients of the
highest dose tested and even in 2 of the 3 recipients of the medium
dose. In this study, both vaccines were given subcutaneously as
usually recommended for live vaccines, but not for the inactivated
HDC vaccine. This route of injection was selected as it best
allowed a careful examination of the injection site, but this could
explain the late appearance of antibodies in HDC recipients:
indeed, none of the HDC recipients had an antibody increase at day
7, whereas, in most studies where HDC vaccine is give
intramuscularly a significant proportion of subjects do (Klietmann
et al., Int'l Green Cross--Geneva, 1981; Kuwert et al., Int'l Green
Cross--Geneva, 1981). However, this invention is not necessarily
limited to the subcutaneous route of administration.
[0256] The GMT (geometric mean titers) of rabies neutralizing
antibodies was lower with the investigational vaccine than with the
HDC control vaccine, but still well above the minimum titer
required for protection. The clear dose effect response obtained
with the three dosages used in this study suggest that a higher
dosage might induce a stronger response. Certainly from this
disclosure the skilled artisan can select an appropriate dosage for
a given patient.
[0257] The ability to boost the antibody response is another
important result of this Example; indeed, an increase in rabies
antibody titers was obtained in every subject after the 6 month
dose whatever the immunization scheme, showing that preexisting
immunity elicited by either the canarypox vector or the rabies
glycoprotein had no blocking effect on the booster with the
recombinant vaccine candidate or the conventional HDC rabies
vaccine. This contrasts findings of others with vaccinia
recombinants in humans that immune response may be blocked by
pre-existing immunity (Cooney et al.; Etinger et al.).
[0258] Thus, this Example clearly demonstrates that a
non-replicating poxvirus can serve as an immunizing vector in
humans, with all of the advantages that replicating agents confer
on the immune response, but without the safety problem created by a
fully permissive virus.
TABLE-US-00030 TABLE 11 Reactions in the 5 days following
vaccination vCP65 dosage H D C (TCID50) 10.sup.3.5 10.sup.4.5
10.sup.5.5 control Injection 1st 2nd 1st 2nd 1st 2nd 1st 2nd No.
vaccinees 3 3 3 3 9 9 10 10 temp >37.7.degree. C. 0 0 0 0 0 1 1
0 soreness 0 0 1 1 6 8 8 6 redness 0 0 0 0 0 4 5 4 induration 0 0 0
0 0 4 5 4
TABLE-US-00031 TABLE 12 Rabies neutralizing antibodies (REFIT;
IU/ml) Individual titers and geometric mean titers (GMT) Days No.
TCID50/dose 0 7 28 35 56 1 10.sup.3.5 <0.1 <0.1 <0.1
<0.1 0.2 3 10.sup.3.5 <0.1 <0.1 <0.1 <0.1 <0.1 4
10.sup.3.5 <0.1 <0.1 <0.1 <0.1 <0.1 G.M.T. <0.1
<0.1 <0.1 <0.1 <0.1 6 10.sup.4.5 <0.1 <0.1
<0.1 <0.1 <0.1 7 10.sup.4.5 <0.1 <0.1 <0.1 2.4
1.9 10 10.sup.4.5 <0.1 <0.1 <0.1 1.6 1.1 G.M.T. <0.1
<0.1 0.1 0.58 0.47 11 10.sup.5.5 <0.1 <0.1 1.0 3.2 4.3 13
10.sup.5.5 <0.1 <0.1 0.3 6.0 8.8 14 10.sup.5.5 <0.1
<0.1 0.2 2.1 9.4 17 10.sup.5.5 <0.1 <0.1 <0.1 1.2 2.5
18 10.sup.5.5 <0.1 <0.1 0.7 8.3 12.5 20 10.sup.5.5 <0.1
<0.1 <0.1 0.3 3.7 21 10.sup.5.5 <0.1 <0.1 0.2 2.6 3.9
23 10.sup.5.5 <0.1 <0.1 <0.1 1.7 4.2 25 10.sup.5.5 <0.1
<0.1 <0.1 0.6 0.9 G.M.T. <0.1 <0.1 0.16 1.9 4.4* 2 HDC
<0.1 <0.1 0.8 7.1 7.2 5 HDC <0.1 <0.1 9.9 12.8 18.7 8
HDC <0.1 <0.1 12.7 21.1 16.5 9 HDC <0.1 <0.1 6.0 9.9
14.3 12 HDC <0.1 <0.1 5.0 9.2 25.3 15 HDC <0.1 <0.1 2.2
5.2 8.6 16 HDC <0.1 <0.1 2.7 7.7 20.7 19 HDC <0.1 <0.1
2.6 9.9 9.1 22 HDC <0.1 <0.1 1.4 8.6 6.6 24 HDC <0.1
<0.1 0.8 5.8 4.7 G.M.T. <0.1 <0.1 2.96 9.0 11.5* *p =
0.007 student t test
TABLE-US-00032 TABLE 13 Canarypox antibodies: ELISA Geometric Mean
Titers* vCP65 dosage Days TCID50/dose 0 7 28 35 56 10.sup.3.5 0.69
ND 0.76 ND 0.68 10.sup.4.5 0.49 0.45 0.56 0.63 0.87 10.sup.5.5 0.38
0.38 0.77 1.42 1.63 HDC control 0.45 0.39 0.40 0.35 0.39 *optical
density at 1/25 dilution
Example 11
Comparison of the LD.sub.50 of ALVAC and NYVAC with Various
Vaccinia Virus Strains
[0259] Mice. Male outbred Swiss Webster mice were purchased from
Taconic Farms (Germantown, N.Y.) and maintained on mouse chow and
water ad libitum until use at 3 weeks of age ("normal" mice).
Newborn outbred Swiss Webster mice were of both sexes and were
obtained following timed pregnancies performed by Taconic Farms.
All newborn mice used were delivered within a two day period.
[0260] Viruses. ALVAC was derived by plaque purification of a
canarypox virus population and was prepared in primary chick embryo
fibroblast cells (CEF). Following purification by centrifugation
over sucrose density gradients, ALVAC was enumerated for plaque
forming units in CEF cells. The WR(L) variant of vaccinia virus was
derived by selection of large plaque phenotypes of WR (Panicali et
al., 1981). The Wyeth New York State Board of Health vaccine strain
of vaccinia virus was obtained from Pharmaceuticals Calf Lymph Type
vaccine Dryvax, control number 302001B. Copenhagen strain vaccinia
virus VC-2 was obtained from Institut Merieux, France. Vaccinia
virus strain NYVAC was derived from Copenhagen VC-2. All vaccinia
virus strains except the Wyeth strain were cultivated in Vero
African green monkey kidney cells, purified by sucrose gradient
density centrifugation and enumerated for plaque forming units on
Vero cells. The Wyeth strain was grown in CEF cells and enumerated
for plaque forming units in CEF cells.
[0261] Inoculations. Groups of 10 normal mice were inoculated
intracranially (ic) with 0.05 ml of one of several dilutions of
virus prepared by 10-fold serially diluting the stock preparations
in sterile phosphate-buffered saline. In some instances, undiluted
stock virus preparation was used for inoculation.
[0262] Groups of 10 newborn mice, 1 to 2 days old, were inoculated
ic similarly to the normal mice except that an injection volume of
0.03 ml was used.
[0263] All mice were observed daily for mortality for a period of
14 days (newborn mice) or 21 days (normal mice) after inoculation.
Mice found dead the morning following inoculation were excluded due
to potential death by trauma.
[0264] The lethal dose required to produce mortality for 50% of the
experimental population (LD.sub.50) was determined by the
proportional method of Reed and Muench.
[0265] Comparison of the LD.sub.50 of ALVAC and NYVAC with Various
Vaccinia Virus Strains for Normal, Young Outbred Mice by the ic
Route. In young, normal mice, the virulence of NYVAC and ALVAC were
several orders of magnitude lower than the other vaccinia virus
strains tested (Table 14). NYVAC and ALVAC were found to be over
3,000 times less virulent in normal mice than the Wyeth strain;
over 12,500 times less virulent than the parental VC-2 strain; and
over 63,000,000 times less virulent than the WR(L) variant. These
results would suggest that NYVAC is highly attenuated compared to
other vaccinia strains, and that ALVAC is generally nonvirulent for
young mice when administered intracranially, although both may
cause mortality in mice at extremely high doses
(3.85.times.10.sup.8 PFUs, ALVAC and 3.times.10.sup.8 PFUs, NYVAC)
by an undetermined mechanism by this route of inoculation.
[0266] Comparison of the LD.sub.50 of ALVAC and NYVAC with Various
Vaccinia Virus Strains for Newborn Outbred Mice by the ic Route.
The relative virulence of 5 poxvirus strains for normal, newborn
mice was tested by titration in an intracranial (ic) challenge
model system (Table 15). With mortality as the endpoint, LD.sub.50
values indicated that ALVAC is over 100,000 times less virulent
than the Wyeth vaccine strain of vaccinia virus; over 200,000 times
less virulent than the Copenhagen VC-2 strain of vaccinia virus;
and over 25,000,000 times less virulent than the WR-L variant of
vaccinia virus. Nonetheless, at the highest dose tested,
6.3.times.10.sup.7 PFUs, 100% mortality resulted. Mortality rates
of 33.3% were observed at 6.3.times.10.sup.6 PFUs. The cause of
death, while not actually determined, was not likely of
toxicological or traumatic nature since the mean survival time
(MST) of mice of the highest dosage group (approximately 6.3
LD.sub.50) was 6.7.+-.1.5 days. When compared to WR(L) at a
challenge dose of 5 LD.sub.50, wherein MST is 4.8.+-.0.6 days, the
MST of ALVAC challenged mice was significantly longer
(P=0.001).
[0267] Relative to NYVAC, Wyeth was found to be over 15,000 times
more virulent; VC-2, greater than 35,000 times more virulent; and
WR(L), over 3,000,000 times more virulent. Similar to ALVAC, the
two highest doses of NYVAC, 6.times.10.sup.8 and 6.times.10.sup.7
PFUs, caused 100% mortality. However, the MST of mice challenged
with the highest dose, corresponding to 380 LD.sub.50, was only 2
days (9 deaths on day 2 and 1 on day 4). In contrast, all mice
challenged with the highest dose of WR-L, equivalent to 500
LD.sub.50, survived to day 4.
TABLE-US-00033 TABLE 14 Calculated 50% Lethal Dose for mice by
various vaccinia virus strains and for canarypox virus (ALVAC) by
the ic route. POXVIRUS CALCULATED STRAIN LD.sub.50 (PFUs) WR(L) 2.5
VC-2 1.26 .times. 10.sup.4 WYETH 5.00 .times. 10.sup.4 NYVAC 1.58
.times. 10.sup.8 ALVAC 1.58 .times. 10.sup.8
TABLE-US-00034 TABLE 15 Calculated 50% Lethal Dose for newborn mice
by various vaccinia virus strains and for canarypox virus (ALVAC)
by the ic route. POXVIRUS CALCULATED STRAIN LD.sub.50 (PFUs) WR(L)
0.4 VC-2 0.1 WYETH 1.6 NYVAC 1.58 .times. 10.sup.6 ALVAC 1.00
.times. 10.sup.7
Example 12
Evaluation of NYVAC (vP866) and NYVAC-RG (vP879)
[0268] Immunoprecipitations. Preformed monolayers of avian or
non-avian cells were inoculated with 10 pfu per cell of parental
NYVAC (vP866) or NYVAC-RG (vP879) virus. The inoculation was
performed in EMEM free of methionine and supplemented with 2%
dialyzed fetal bovine serum. After a one hour incubation, the
inoculum was removed and the medium replaced with EMEM (methionine
free) containing 20 .mu.Ci/ml of .sup.35S-methionine. After an
overnight incubation of approximately 16 hours, cells were lysed by
the addition of Buffer A (1% Nonidet P-40, 10 mM Tris pH7.4, 150 mM
NaCl, 1 mM EDTA, 0.01% sodium azide, 500 units per ml of aprotinin,
and 0.02% phenyl methyl sulfonyl fluoride). Immunoprecipitation was
performed using a rabies glycoprotein specific monoclonal antibody
designated 24-3F10 supplied by Dr. C. Trinarchi, Griffith
Laboratories, New York State Department of Health, Albany, N.Y.,
and a rat anti-mouse conjugate obtained from Boehringer Mannheim
Corporation (Cat. #605-500). Protein A Sepharose CL-48 obtained
from Pharmacia LKB Biotechnology Inc., Piscataway, N.J., was used
as a support matrix. Immunoprecipitates were fractionated on 10%
polyacrylamide gels according to the method of Dreyfuss et. al.
(1984). Gels were fixed, treated for fluorography with 1M
Na-salicylate for one hour, and exposed to Kodak XAR-2 film to
visualize the immunoprecipitated protein species.
[0269] Sources of Animals. New Zealand White rabbits were obtained
from Hare-Marland (Hewitt, N.J.). Three week old male Swiss Webster
outbred mice, timed pregnant female Swiss Webster outbred mice, and
four week old Swiss Webster nude (nu.sup.+nu.sup.+) mice were
obtained from Taconic Farms, Inc. (Germantown, N.Y.). All animals
were maintained according to NIH guidelines. All animal protocols
were approved by the institutional IACUC. When deemed necessary,
mice which were obviously terminally ill were euthanized.
[0270] Evaluation of Lesions in Rabbits. Each of two rabbits was
inoculated intradermally at multiple sites with 0.1 ml of PBS
containing 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, or 10.sup.8 pfu
of each test virus or with PBS alone. The rabbits were observed
daily from day 4 until lesion resolution. Indurations and
ulcerations were measured and recorded.
[0271] Virus Recovery from Inoculation Sites. A single rabbit was
inoculated intradermally at multiple sites of 0/1 ml of PBS
containing 10.sup.6, 10.sup.7, or 10.sup.8 pfu of each test virus
or with PBS alone. After 11 days, the rabbit was euthanized and
skin biopsy specimens taken from each of the inoculation sites were
aseptically prepared by mechanical disruption and indirect
sonication for virus recovery. Infectious virus was assayed by
plaque titration on CEF monolayers.
[0272] Virulence in Mice. Groups of ten mice, or five in the nude
mice experiment, were inoculated ip with one of several dilutions
of virus in 0.5 ml of sterile PBS. Reference is also made to
Example 11.
[0273] Cyclophosphamide (CY) Treatment. Mice were injected by the
ip route with 4 mg (0.02 ml) of CY (SIGMA) on day -2, followed by
virus injection on day 0. On the following days post infection,
mice were injected ip with CY: 4 mg on day 1; 2 mg on days 4, 7 and
11; 3 mg on days 14, 18, 21, 25 and 28. Immunosuppression was
indirectly monitored by enumerating white blood cells with a
Coulter Counter on day 11. The average white blood cell count was
13,500 cells per .mu.l for untreated mice (n=4) and 4,220 cells per
.mu.l for CY-treated control mice (n=5).
[0274] Calculation of LD.sub.50. The lethal dose required to
produce 50% mortality (LD.sub.50) was determined by the
proportional method of Reed and Muench (Reed and Muench 1938).
[0275] Potency Testing of NYVAC-RG in Mice. Four to six week old
mice were inoculated in the footpad with 50 to 100 .mu.l of a range
of dilutions (2.0-8.0 log.sub.10 tissue culture infective dose 50%
(TCID.sub.50)) of either VV-RG (Kieny et al., 1984), ALVAC-RG
(Taylor et al., 1991b), or the NYVAC-RG. Each group consisted of
eight mice. At 14 days post-vaccination, the mice were challenged
by intracranial inoculation with 15 LD.sub.50 of the rabies virus
CVS strain (0.03 ml). On day 28, surviving mice were counted and
protective does 50% (PD.sub.50) calculated.
[0276] Derivation of NYVAC (vP866). The NYVAC strain of vaccinia
virus was generated from VC-2, a plaque cloned isolate of the
COPENHAGEN vaccine strain. To generate NYVAC from VC-2, eighteen
vaccinia ORFs, including a number of viral functions associated
with virulence, were precisely deleted in a series of sequential
manipulations as described earlier in this disclosure. These
deletions were constructed in a manner designed to prevent the
appearance of novel unwanted open reading frames. FIG. 10
schematically depicts the ORFs deleted to generate NYVAC. At the
top of FIG. 10 is depicted the HindIII restriction map of the
vaccinia virus genome (VC-2 plaque isolate, COPENHAGEN strain).
Expanded are the six regions of VC-2 that were sequentially deleted
in the generation of NYVAC. The deletions were described earlier in
this disclosure (Examples 1 through 6). Below such deletion locus
is listed the ORFs which were deleted from that locus, along with
the functions or homologies and molecular weight of their gene
products.
[0277] Replication Studies of NYVAC and ALVAC on Human Tissue Cell
Lines. In order to determine the level of replication of NYVAC
strain of vaccinia virus (vP866) in cells of human origin, six cell
lines were inoculated at an input multiplicity of 0.1 pfu per cell
under liquid culture and incubated for 72 hours. The COPENHAGEN
parental clone (VC-2) was inoculated in parallel. Primary chick
embryo fibroblast (CEF) cells (obtained from 10-11 day old
embryonated eggs of SPF origin, Spafas, Inc., Storrs, Conn.) were
included to represent a permissive cell substrate for all viruses.
Cultures were analyzed on the basis of two criteria: the occurrence
of productive viral replication and expression of an extrinsic
antigen.
[0278] The replication potential of NYVAC in a number of human
derived cells are shown in Table 16. Both VC-2 and NYVAC are
capable of productive replication in CEF cells, although NYVAC with
slightly reduced yields. VC-2 is also capable of productive
replication in the six human derived cell lines tested with
comparable yields except in the EBV transformed lymphoblastoid cell
line JT-1 (human lymphoblastoid cell line transformed with
Epstein-Barr virus, see Rickinson et al., 1984). In contrast, NYVAC
is highly attenuated in its ability to productively replicate in
any of the human derived cell lines tested. Small increases of
infectious virus above residual virus levels were obtained from
NYVAC-infected MRC-5 (ATCC #CCL171, human embryonic lung origin),
DETROIT 532 (ATCC #CCL54, human foreskin, Downs Syndrome), HEL 299
(ATCC #CCL137, human embryonic lung cells) and HNK (human neonatal
kidney cells, Whittiker Bioproducts, Inc. Walkersville, Md., Cat
#70-151) cells. Replication on these cell lines was significantly
reduced when compared to virus yields obtained from NYVAC-infected
CEF cells or with parental VC-2 (Table 16). It should be noted that
the yields at 24 hours in CEF cells for both NYVAC and VC-2 is
equivalent to the 72-hour yield. Allowing the human cell line
cultures to incubate an additional 48 hours (another two viral
growth cycles) may, therefore, have amplified the relative virus
yield obtained.
[0279] Consistent with the low levels of virus yields obtained in
the human-derived cell lines, MRC-5 and DETROIT 532, detectable but
reduced levels of NYVAC-specific DNA accumulation were noted. The
level of DNA accumulation in the MRC-5 and DETROIT 532
NYVAC-infected cell lines relative to that observed in
NYVAC-infected CEF cells paralleled the relative virus yields.
NYVAC-specific viral DNA accumulation was not observed in any of
the other human-derived cells.
[0280] An equivalent experiment was also performed using the avipox
virus, ALVAC. The results of virus replication are also shown in
Table 16. No progeny virus was detectable in any of the human cell
lines consistent with the host range restriction of canarypox virus
to avian species. Also consistent with a lack of productive
replication of ALVAC in these human-derived cells is the
observation that no ALVAC-specific DNA accumulation was detectable
in any of the human-derived cell lines.
[0281] Expression of Rabies Glycoprotein by NYVAC-RG (vP879) in
Human Cells. In order to determine whether efficient expression of
a foreign gene could be obtained in the absence of significant
levels of productive viral replication, the same cell lines were
inoculated with the NYVAC recombinant expressing the rabies virus
glycoprotein (vP879, Example 7) in the presence of
.sup.35S-methionine. Immunoprecipitation of the rabies glycoprotein
was performed from the radiolabelled culture lysate using a
monoclonal antibody specific for the rabies glycoprotein.
Immunoprecipitation of a 67 kDa protein was detected consistent
with a fully glycosylated form of the rabies glycoprotein. No
serologically crossreactive product was detected in uninfected or
parental NYVAC infected cell lysates. Equivalent results were
obtained with all other human cells analyzed.
[0282] Inoculations on the Rabbit Skin. The induction and nature of
skin lesions on rabbits following intradermal (id) inoculations has
been previously used as a measure of pathogenicity of vaccinia
virus strains (Buller et al., 1988; Child et al., 1990; Fenner,
1958, Flexner et al., 1987; Ghendon and Chemos 1964). Therefore,
the nature of lesions associated with id inoculations with the
vaccinia strains WR (ATCC #VR119 plaque purified on CV-1 cells,
ATCC #CCL70, and a plaque isolate designated L variant, ATCC
#VR2035 selected, as described in Panicali et al., 1981)), WYETH
(ATCC #VR325 marketed as DRYVAC by Wyeth Laboratories, Marietta,
Pa.), COPENHAGEN (VC-2), and NYVAC was evaluated by inoculation of
two rabbits (A069 and A128). The two rabbits displayed different
overall sensitivities to the viruses, with rabbit A128 displaying
less severe reactions than rabbit A069. In rabbit A128, lesions
were relatively small and resolved by 27 days post-inoculation. On
rabbit A069, lesions were intense, especially for the WR
inoculation sites, and resolved only after 49 days. Intensity of
the lesions was also dependent on the location of the inoculation
sites relative to the lymph drainage network. In particular, all
sites located above the backspine displayed more intense lesions
and required longer times to resolve the lesions located on the
flanks. All lesions were measured daily from day 4 to the
disappearance of the last lesion, and the means of maximum lesion
size and days to resolution were calculated (Table 17). No local
reactions were observed from sites injected with the control PBS.
Ulcerative lesions were observed at sites injected with WR, VC-2
and WYETH vaccinia virus strains. Significantly, no induration or
ulcerative lesions were observed at sites of inoculation with
NYVAC.
[0283] Persistence of Infectious Virus at the Site of Inoculation.
To assess the relative persistence of these viruses at the site of
inoculation, a rabbit was inoculated intradermally at multiple
sites with 0.1 ml PBS containing 10.sup.6, 10.sup.7 or 10.sup.8 pfu
of VC-2, WR, WYETH or NYVAC. For each virus, the 10.sup.7 pfu dose
was located above the backspine, flanked by the 10.sup.6 and
10.sup.8 doses. Sites of inoculation were observed daily for 11
days. WR elicited the most intense response, followed by VC-2 and
WYETH (Table 18). Ulceration was first observed at day 9 for WR and
WYETH and day 10 for VC-2. Sites inoculated with NYVAC or control
PBS displayed no induration or ulceration. At day 11 after
inoculation, skin samples from the sites of inoculation were
excised, mechanically disrupted, and virus was titrated on CEF
cells. The results are shown in Table 18. In no case was more virus
recovered at this timepoint than was administered. Recovery of
vaccinia strain, WR, was approximately 10.sup.6 pfu of virus at
each site irrespective of amount of virus administered. Recovery of
vaccinia strains WYETH and VC-2 was 10.sup.3 to 10.sup.4 pfu
regardless of amount administered. No infectious virus was
recovered from sites inoculated with NYVAC.
[0284] Inoculation of Genetically or Chemically Immune Deficient
Mice. Intraperitoneal inoculation of high doses of NYVAC
(5.times.10.sup.8 pfu) or ALVAC (10.sup.9 pfu) into nude mice
caused no deaths, no lesions, and no apparent disease through the
100 day observation period. In contrast, mice inoculated with WR
(10.sup.3 to 10.sup.4 pfu), WYETH (5.times.10.sup.7 or
5.times.10.sup.8 pfu) or VC-2 (10.sup.4 to 10.sup.9 pfu) displayed
disseminated lesions typical of poxviruses first on the toes, then
on the tail, followed by severe orchitis in some animals. In mice
infected with WR or WYETH, the appearance of disseminated lesions
generally led to eventual death, whereas most mice infected with
VC-2 eventually recovered. Calculated LD.sub.50 values are given in
Table 19.
[0285] In particular, mice inoculated with VC-2 began to display
lesions on their toes (red papules) and 1 to 2 days later on the
tail. These lesions occurred between 11 and 13 days
post-inoculation (pi) in mice given the highest doses (10.sup.9,
10.sup.8, 10.sup.7 and 10.sup.6 pfu), on day 16 pi in mice given
10.sup.5 pfu and on day 21 pi in mice given 10.sup.4 pfu. No
lesions were observed in mice inoculated with 10.sup.3 and 10.sup.2
pfu during the 100 day observation period. Orchitis was noticed on
day 23 pi in mice given 10.sup.9 and 10.sup.8 pfu, and
approximately 7 days later in the other groups (10.sup.7 to
10.sup.4 pfu). Orchitis was especially intense in the 10.sup.9 and
10.sup.8 pfu groups and, although receding, was observed until the
end of the 100 day observation period. Some pox-like lesions were
noticed on the skin of a few mice, occurring around 30-35 days pi.
Most pox lesions healed normally between 60-90 days pi. Only one
mouse died in the group inoculated with 10.sup.9 pfu (Day 34 pi)
and one mouse died in the group inoculated with 10.sup.8 pfu (Day
94 pi). No other deaths were observed in the VC-2 inoculated
mice.
[0286] Mice inoculated with 10.sup.4 pfu of the WR strain of
vaccinia started to display pox lesions on Day 17 pi. These lesions
appeared identical to the lesions displayed by the VC-2 injected
mice (swollen toes, tail). Mice inoculated with 10.sup.3 pfu of the
WR strain did not develop lesions until 34 days pi. Orchitis was
noticed only in the mice inoculated with the highest dose of WR
(10.sup.4pfu). During the latter stages of the observation period,
lesions appeared around the mouth and the mice stopped eating. All
mice inoculated with 10.sup.4 pfu of WR died or were euthanized
when deemed necessary between 21 days and 31 days pi. Four out of
the 5 mice injected with 10.sup.3 pfu of WR died or were euthanized
when deemed necessary between 35 days and 57 days pi. No deaths
were observed in mice inoculated with lower doses of WR (1 to 100
pfu).
[0287] Mice inoculated with the WYETH strain of vaccinia virus at
higher doses 5.times.10.sup.7 and 5.times.10.sup.8 pfu) showed
lesions on toes and tails, developed orchitis, and died. Mice
injected with 5.times.10.sup.6 pfu or less of WYETH showed no signs
of disease or lesions.
[0288] As shown in Table 19, CY-treated mice provided a more
sensitive model for assaying poxvirus virulence than did nude mice.
LD.sub.50 values for the WR, WYETH, and VC-2 vaccinia virus strains
were significantly lower in this model system than in the nude
mouse model. Additionally, lesions developed in mice injected with
WYETH, WR and VC-2 vaccinia viruses, as noted below, with higher
doses of each virus resulting in more rapid formation of lesions.
As was seen with nude mice, CY-treated mice injected with NYVAC or
ALVAC did not develop lesions. However, unlike nude mice, some
deaths were observed in CY-treated mice challenged with NYVAC or
ALVAC, regardless of the dose. These random incidences are suspect
as to the cause of death.
[0289] Mice injected with all doses of WYETH (9.5.times.10.sup.4 to
9.5.times.10.sup.9 pfu) displayed pox lesions on their tail and/or
on their toes between 7 and 15 days pi. In addition, the tails and
toes were swollen. Evolution of lesions on the tail was typical of
pox lesions with formation of a papule, ulceration and finally
formation of a scab. Mice inoculated with all doses of VC-2
(1.65.times.10.sup.5 to 1.65.times.10.sup.9) also developed pox
lesions on their tails and/or their toes analogous to those of
WYETH injected mice. These lesions were observed between 7-12 days
post inoculation. No lesions were observed on mice injected with
lower doses of WR virus, although deaths occurred in these
groups.
[0290] Potency Testing of NYVAC-RG. In order to determine that
attenuation of the COPENHAGEN strain of vaccinia virus had been
effected without significantly altering the ability of the
resulting NYVAC strain to be a useful vector, comparative potency
tests were performed. In order to monitor the immunogenic potential
of the vector during the sequential genetic manipulations performed
to attenuate the virus, a rabiesvirus glycoprotein was used as a
reporter extrinsic antigen. The protective efficacy of the vectors
expressing the rabies glycoprotein gene was evaluated in the
standard NIH mouse potency test for rabies (Seligmann, 1973). Table
20 demonstrates that the PD.sub.50 values obtained with the highly
attenuated NYVAC vector are identical to those obtained using a
COPENHAGEN-based recombinant containing the rabies glycoprotein
gene in the tk locus (Kieny et al., 1984) and similar to PD.sub.50
values obtained with ALVAC-RG, a canarypox based vector restricted
to replication to avian species.
[0291] Observations. NYVAC, deleted of known virulence genes and
having restricted in vitro growth characteristics, was analyzed in
animal model systems to assess its attenuation characteristics.
These studies were performed in comparison with the neurovirulent
vaccinia virus laboratory strain, WR, two vaccinia virus vaccine
strains, WYETH (New York City Board of Health) and COPENHAGEN
(VC-2), as well as with a canarypox virus strain, ALVAC (See also
Example 11). Together, these viruses provided a spectrum of
relative pathogenic potentials in the mouse challenge model and the
rabbit skin model, with WR being the most virulent strain, WYETH
and COPENHAGEN (VC-2) providing previously utilized attenuated
vaccine strains with documented characteristics, and ALVAC
providing an example of a poxvirus whose replication is restricted
to avian species. Results from these in vivo analyses clearly
demonstrate the highly attenuated properties of NYVAC relative to
the vaccinia virus strains, WR, WYETH and COPENHAGEN (VC-2) (Tables
14-20). Significantly, the LD.sub.50 values for NYVAC were
comparable to those observed with the avian host restricted
avipoxvirus, ALVAC. Deaths due to NYVAC, as well as ALVAC, were
observed only when extremely high doses of virus were administered
via the intracranial route (Example 11, Tables 14, 15, 19). It has
not yet been established whether these deaths were due to
nonspecific consequences of inoculation of a high protein mass.
Results from analyses in immunocompromised mouse models (nude and
CY-treated) also demonstrate the relatively high attenuation
characteristics of NYVAC, as compared to WR, WYETH and COPENHAGEN
strains (Tables 17 and 18). Significantly, no evidence of
disseminated vaccinia infection or vaccinial disease was observed
in NYVAC-inoculated animals or ALVAC-inoculated animals over the
observation period. The deletion of multiple virulence-associated
genes in NYVAC shows a synergistic effect with respect to
pathogenicity. Another measure of the inocuity of NYVAC was
provided by the intradermal administration on rabbit skin (Tables
17 and 18). Considering the results with ALVAC, a virus unable to
replicate in nonavian species, the ability to replicate at the site
of inoculation is not the sole correlate with reactivity, since
intradermal inoculation of ALVAC caused areas of induration in a
dose dependent manner. Therefore, it is likely that factors other
than the replicative capacity of the virus contribute to the
formation of the lesions. Deletion of specific virulence-associated
genes in NYVAC prevents lesion occurrence.
[0292] Together, the results in this Example and in foregoing
Examples, including Example 11, demonstrate the highly attenuated
nature of NYVAC relative to WR, and the previously utilized
vaccinia virus vaccine strains, WYETH and COPENHAGEN. In fact, the
pathogenic profile of NYVAC, in the animal model systems tested,
was similar to that of ALVAC, a poxvirus known to productively
replicate only in avian species. The apparently restricted capacity
of NYVAC to productively replicate on cells derived from humans
(Table 16) and other species, including the mouse, swine, dog and
horse, provides a considerable barrier that limits or prevents
potential transmission to unvaccinated contacts or to the general
environment in addition to providing a vector with reduced
probability of dissemination within the vaccinated individual.
[0293] Significantly, NYVAC-based vaccine candidates have been
shown to be efficacious. NYVAC recombinants expressing foreign gene
products from a number of pathogens have elicited immunological
responses towards the foreign gene products in several animal
species, including primates. In particular, a NYVAC-based
recombinant expressing the rabies glycoprotein was able to protect
mice against a lethal rabies challenge. The potency of the
NYVAC-based rabies glycoprotein recombinant was comparable to the
PD.sub.50 value for a COPENHAGEN-based recombinant containing the
rabies glycoprotein in the tk locus (Table 20). NYVAC-based
recombinants have also been shown to elicit measles virus
neutralizing antibodies in rabbits and protection against
pseudorabies virus and Japanese encephalitis virus challenge in
swine. The highly attenuated NYVAC strain confers safety advantages
with human and veterinary applications (Tartaglia et al., 1992).
Furthermore, the use of NYVAC as a general laboratory expression
vector system may greatly reduce the biological hazards associated
with using vaccinia virus.
[0294] By the following criteria, the results of this Example and
the Examples herein, including Example 11, show NYVAC to be highly
attenuated: a) no detectable induration or ulceration at site of
inoculation (rabbit skin); b) rapid clearance of infectious virus
from intradermal site of inoculation (rabbit skin); c) absence of
testicular inflammation (nude mice); d) greatly reduced virulence
(intracranial challenge, both three-week old and newborn mice); e)
greatly reduced pathogenicity and failure to disseminate in
immunodeficient subjects (nude and cyclophosphamide treated mice);
and f) dramatically reduced ability to replicate on a variety of
human tissue culture cells. Yet, in spite of being highly
attenuated, NYVAC, as a vector, retains the ability to induce
strong immune responses to extrinsic antigens.
TABLE-US-00035 TABLE 16 Replication of COPENHAGEN (VC-2), NYVAC and
ALVAC in avian or human derived cell lines Hours post- Yield.sup.a
Cells infection VC-2 NYVAC ALVAC % Yield CEF 0 3.8.sup.b 3.7 4.5 24
8.3 7.8 6.6 48 8.6 7.9 7.7 72 8.3 7.7 7.5 25 72A.sup.c <1.4 1.8
3.1 MRC-5 0 3.8 3.8 4.7 72 7.2 4.6 3.8 0.25 72A 2.2 2.2 3.7 WISH* 0
3.4 3.4 4.3 72 7.6 2.2 3.1 0.0004 72A --.sup.d 1.9 2.9 DETROIT 0
3.8 3.7 4.4 72 7.2 5.4 3.4 1.6 72A 1.7 1.7 2.9 HEL 0 3.8 3.5 4.3 72
7.5 4.6 3.3 0.125 72A 2.5 2.1 3.6 JT-1 0 3.1 3.1 4.1 72 6.5 3.1 4.2
0.039 72A 2.4 2.1 4.4 HNK 0 3.8 3.7 4.7 72 7.6 4.5 3.6 0.079 72A
3.1 2.7 3.7 .sup.aYield of NYVAC at 72 hours post-infection
expressed as a percentage of yield of VAC-2 after 72 hours on the
same cell line. .sup.bTiter expressed as LOG.sub.50 pfu per ml.
.sup.cSample was incubated in the presence of 40 g/ml of cytosine
arabinoside. .sup.dNot determined. *ATCC #CCL25 Human amnionic
cells.
TABLE-US-00036 TABLE 17 Induration and ulceration at the site of
intradermal inoculation of the rabbit skin VIRUS INDURATION
ULCERATION STRAIN DOSE.sup.a Size.sup.b Days.sup.c Size Days WR
10.sup.4 386 30 88 30 10.sup.5 622 35 149 32 10.sup.6 1057 34 271
34 10.sup.7 877 35 204 35 10.sup.8 581 25 88 26 WYETH 10.sup.4 32 5
.sup. --.sup.d -- 10.sup.5 116 15 -- -- 10.sup.6 267 17 3 15
10.sup.7 202 17 3 24 10.sup.8 240 29 12 31 VC-2 10.sup.4 64 7 -- --
10.sup.5 86 8 -- -- 10.sup.6 136 17 -- -- 10.sup.7 167 21 6 10
10.sup.8 155 32 6 8 NYVAC 10.sup.4 -- -- -- -- 10.sup.5 -- -- -- --
10.sup.6 -- -- -- -- 10.sup.7 -- -- -- -- 10.sup.8 -- -- -- --
.sup.apfu of indicated vaccinia virus in 0.1 ml PBS inoculated
intradermally into one site. .sup.bmean maximum size of lesions
(mm.sup.2) .sup.cmean time after inoculation for complete healing
of lesion. .sup.dno lesions discernable.
TABLE-US-00037 TABLE 18 Persistence of poxviruses at the site of
intradermal inoculation Total Virus Virus Inoculum Dose Recovered
WR .sup. 8.0.sup.a 6.14 7.0 6.26 6.0 6.21 WYETH 8.0 3.66 7.0 4.10
6.0 3.59 VC-2 8.0 4.47 7.0 4.74 6.0 3.97 NYVAC 8.0 0 7.0 0 6.0 0
.sup.aexpressed as log.sub.10 pfu.
TABLE-US-00038 TABLE 19 Virulence studies in immunocompromised mice
LD.sub.50.sup.a Poxvirus Cyclophosphamide Strain Nude mice treated
mice WR 422.sup. 42 VC-2 >10.sup.9 <1.65 .times. 10.sup.5
WYETH 1.58 .times. 10.sup.7 1.83 .times. 10.sup.6 NY VAC >5.50
.times. 10.sup.8 7.23 .times. 10.sup.8 ALVAC >10.sup.9
.gtoreq.5.00 .times. 10.sup.8b .sup. .sup.aCalculated 50% lethal
dose (pfu) for nude or cyclophosphamide treated mice by the
indicated vaccinia viruses and for ALVAC by intraperitoneal route.
.sup.b5 out of 10 mice died at the highest dose of 5 .times.
10.sup.8 pfu.
TABLE-US-00039 TABLE 20 Comparative efficacy of NYVAC-RG and
ALVAC-RG in mice Recombinant PD.sub.50.sup.a VV-RG 3.74 ALVAC-RG
3.86 NYVAC-RG 3.70 .sup.aFour to six week old mice were inoculated
in the footpad with 50-100 .mu.l of a range of dilutions (2.0-8.0
log.sub.10 tissue culture infection dose 50% (TCID.sub.50) of
either the VV-RG (Kieny et al., 1984), ALVAC-RG (vCP65) or NYVAC-RG
(vP879). At day 14, mice of each group were challenged by
intracranial inoculation of 30 .mu.l of a live CVS strain rabies
virus corresponding to 15 lethal dose 50% (LD.sub.50) per mouse. At
day 28, surviving mice were counted and a protective dose 50%
(PD.sub.50) was calculated.
Example 13
Construction of TROVAC Recombinants Expressing the Hemagglutinin
Glycoproteins of Avian Influenza Viruses
[0295] This Example describes the development of fowlpox virus
recombinants expressing the hemagglutinin genes of three serotypes
of avian influenza virus.
[0296] Cells and Viruses. Plasmids containing cDNA clones of the
H4, H5 and H7 hemagglutinin genes were obtained from Dr. Robert
Webster, St. Jude Children's Research Hospital, Memphis, Tenn. The
strain of FPV designated FP-1 has been described previously (Taylor
et al., 1988a, b). It is a vaccine strain useful in vaccination of
day old chickens. The parental virus strain Duvette was obtained in
France as a fowlpox scab from a chicken. The virus was attenuated
by approximately 50 serial passages in chicken embryonated eggs
followed by 25 passages on chick embryo fibroblast (CEF) cells.
This virus was obtained in September 1980 by Rhone Merieux, Lyon,
France, and a master viral seed established. The virus was received
by Virogenetics in September 1989, where it was subjected to four
successive plaque purifications. One plaque isolate was further
amplified in primary CEF cells and a stock virus, designated as
TROVAC, was established. The stock virus used in the in vitro
recombination test to produce TROVAC-AIH5 (vFP89) and TROVAC-AIH4
(vFP92) had been further amplified though 8 passages in primary CEF
cells. The stock virus used to produce TROVAC-AIH7 (vFP100) had
been further amplified through 12 passages in primary CEF
cells.
[0297] Construction of Fowlpox Insertion Plasmid at F8 Locus.
Plasmid pRW731.15 contains a 10 kbp PvuII-PvuII fragment cloned
from TROVAC genomic DNA. The nucleotide sequence was determined on
both strands for a 3659 bp PvuII-EcoRV fragment. This sequence is
shown in FIG. 11 (SEQ ID NO:67). The limits of an open reading
frame designated in this laboratory as F8 were determined within
this sequence. The open reading frame is initiated at position 495
and terminates at position 1887. A deletion was made from position
779 to position 1926, as described below.
[0298] Plasmid pRW761 is a sub-clone of pRW731.15 containing a 2430
bp EcoRV-EcoRV fragment. Plasmid pRW761 was completely digested
with XbaI and partially digested with SspI. A 3700 bp XbaI-SspI
band was isolated and ligated with the annealed double-stranded
oligonucleotides JCA017 (SEQ ID NO:37) and JCA018 (SEQ ID
NO:38).
TABLE-US-00040 JCA017 (SEQ ID NO: 37) 5'
CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGG
GATCCTTATACGGGGAATAAT 3' JCA018 (SEQ ID NO: 38) 5'
ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATA TTAAAAAACATAAAGTGT
3'
[0299] The plasmid resulting from this ligation was designated
pJCA002. Plasmid pJCA004 contains a non-pertinent gene linked to
the vaccinia virus H6 promoter in plasmid pJCA002. The sequence of
the vaccinia virus H6 promoter has been previously described
(Taylor et al., 1988a, b; Guo et al. 1989; Perkus et al., 1989).
Plasmid pJCA004 was digested with EcoRV and BamHI which deletes the
non-pertinent gene and a portion of the 3' end of the H6 promoter.
Annealed oligonucleotides RW178 (SEQ ID NO:48) and RW179 (SEQ ID
NO:49) were cut with EcoRV and BamHI and inserted between the EcoRV
and BamHI sites of JCA004 to form pRW846.
TABLE-US-00041 RW178 (SEQ ID NO: 48): 5'
TCATTATCGCGATATCCGTGTTAACTAGCTAGCTAATTTTTATTCCC GGGATCCTTATCA 3'
RW179 (SEQ ID NO: 49): 5'
GTATAAGGATCCCGGGAATAAAAATTAGCTAGCTAGTTAACACGGAT ATCGCGATAATGA
3'
Plasmid pRW846 therefore contains the H6 promoter 5' of EcoRV in
the de-ORFed F8 locus. The HincII site 3' of the H6 promoter in
pRW846 is followed by translation stop codons, a transcriptional
stop sequence recognized by vaccinia virus early promoters (Yuen et
al., 1987) and a SmaI site.
[0300] Construction of Fowlpox Insertion Plasmid at F7 Locus. The
original F7 non-de-ORFed insertion plasmid, pRW731.13, contained a
5.5 kb FP genomic PvuII fragment in the PvuII site of pUC9. The
insertion site was a unique HincII site within these sequences. The
nucleotide sequence shown in FIG. 12 (SEQ ID NO:68) was determined
for a 2356 bp region encompassing the unique HincII site. Analysis
of this sequence revealed that the unique HincII site (FIG. 12,
underlined) was situated within an ORF encoding a polypeptide of 90
amino acids. The ORF begins with an ATG at position 1531 and
terminates at position 898 (positions marked by arrows in FIG.
12).
[0301] The arms for the de-ORFed insertion plasmid were derived by
PCR using pRW731.13 as template. A 596 bp arm (designated as HB)
corresponding to the region upstream from the ORF was amplified
with oligonucleotides F73PH2 (SEQ ID NO:50)
(5'-GACAATCTAAGTCCTATATTAGAC-3') and F73PB (SEQ ID NO:51)
(5'-GGATTTTTAGGTAGACAC-3'). A 270 bp arm (designated as EH)
corresponding to the region downstream from the ORF was amplified
using oligonucleotides F75PE (SEQ ID NO:52)
(5'-TCATCGTCTTCATCATCG-3') and F73PH1 (SEQ ID NO:53)
(5'-GTCTTAAACTTATTGTAAGGGTATACCTG-3').
[0302] Fragment EH was digested with EcoRV to generate a 126 bp
fragment. The EcoRV site is at the 3'-end and the 5'-end was
formed, by PCR, to contain the 3' end of a HincII site. This
fragment was inserted into pBS-SK (Stratagene, La Jolla, Calif.)
digested with HincII to form plasmid pF7D1. The sequence was
confirmed by dideoxynucleotide sequence analysis. The plasmid pF7D1
was linearized with ApaI, blunt-ended using T4 DNA polymerase, and
ligated to the 596 bp HB fragment. The resultant plasmid was
designated as pF7D2. The entire sequence and orientation were
confirmed by nucleotide sequence analysis.
[0303] The plasmid pF7D2 was digested with EcoRV and BglII to
generate a 600 bp fragment. This fragment was inserted into pBS-SK
that was digested with ApaI, blunt-ended with T4 DNA polymerase,
and subsequently digested with BamHI. The resultant plasmid was
designated as pF7D3. This plasmid contains an HB arm of 404 bp and
a EH arm of 126 bp.
[0304] The plasmid pF7D3 was linearized with XhoI and blunt-ended
with the Klenow fragment of the E. coli DNA polymerase in the
presence of 2 mM dNTPs. This linearized plasmid was ligated with
annealed oligonucleotides F7MCSB (SEQ ID NO:54)
(5'-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTTTTTATTAGTTTAGTT
AGTC-3') and F7MCSA (SEQ ID NO:55) (5'-GACTAACTAACTAATAAAAAA
CCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAATCGTT-3'). This was performed to
insert a multiple cloning region containing the restriction sites
for HindIII, PstI and SmaI between the EH and HB arms. The
resultant plasmid was designated as pF7DO.
[0305] Construction of Insertion Plasmid for the H4 Hemagglutinin
at the F8 Locus. A cDNA copy encoding the avian influenza H4
derived from A/Ty/Min/833/80 was obtained from Dr. R. Webster in
plasmid pTM4H833. The plasmid was digested with HindIII and NruI
and blunt-ended using the Klenow fragment of DNA polymerase in the
presence of dNTPs. The blunt-ended 2.5 kbp HindIII-NruI fragment
containing the H4 coding region was inserted into the HincII site
of pIBI25 (International Biotechnologies, Inc., New Haven, Conn.).
The resulting plasmid pRW828 was partially cut with BanII, the
linear product isolated and recut with HindIII. Plasmid pRW828 now
with a 100 bp HindIII-BanII deletion was used as a vector for the
synthetic oligonucleotides RW152 (SEQ ID NO:56) and RW153 (SEQ ID
NO:57). These oligonucleotides represent the 3' portion of the H6
promoter from the EcoRV site and align the ATG of the promoter with
the ATG of the H4 cDNA.
TABLE-US-00042 RW152 (SEQ ID NO: 56): 5'
GCACGGAACAAAGCTTATCGCGATATCCGTTAAGTTTGTATCGTAAT
GCTATCAATCACGATTCTGTTCCTGCTCATAGCAGAGGGCTCATCTCAGA AT 3' RW153 (SEQ
ID NO: 57): 5' ATTCTGAGATGAGCCCTCTGCTATGAGCAGGAACAGAATCGTGATTG
ATAGCATTACGATACAAACTTAACGGATATCGCGATAAGCTTTGTTCCGT GC 3'
[0306] The oligonucleotides were annealed, cut with BanII and
HindIII and inserted into the HindIII-BanII deleted pRW828 vector
described above. The resulting plasmid pRW844 was cut with EcoRV
and DraI and the 1.7 kbp fragment containing the 3' H6 promoted H4
coding sequence was inserted between the EcoRV and HincII sites of
pRW846 (described previously) forming plasmid pRW848. Plasmid
pRW848 therefore contains the H4 coding sequence linked to the
vaccinia virus H6 promoter in the de-ORFed F8 locus of fowlpox
virus.
[0307] Construction of Insertion Plasmid for H5 Hemagglutinin at
the F8 Locus. A cDNA clone of avian influenza H5 derived from
A/Turkey/Ireland/1378/83 was received in plasmid pTH29 from Dr. R.
Webster. Synthetic oligonucleotides RW10 (SEQ ID NO:58) through
RW13 (SEQ ID NO:61) were designed to overlap the translation
initiation codon of the previously described vaccinia virus H6
promoter with the ATG of the H5 gene. The sequence continues
through the 5' SalI site of the H5 gene and begins again at the 3'
H5 DraI site containing the H5 stop codon.
TABLE-US-00043 RW10 (SEQ ID NO: 58): 5'
GAAAAATTTAAAGTCGACCTGTTTTGTTGAGTTGTTTGCGTGGTAAC CAATGCAAATCTGGTCACT
3' RW11 (SEQ ID NO: 59): 5'
TCTAGCAAGACTGACTATTGCAAAAAGAAGCACTATTTCCTCCATTA CGATACAAACTTAACGGAT
3' RW12 (SEQ ID NO: 60): 5'
ATCCGTTAAGTTTGTATCGTAATGGAGGAAATAGTGCTTCTTTTTGC
AATAGTCAGTCTTGCTAGAAGTGACCAGATTTGCATTGGT 3' RW13 (SEQ ID NO:61): 5'
TACCACGCAAACAACTCAACAAAACAGGTCGACTTTAAATTTTTCTG CA 3'
[0308] The oligonucleotides were annealed at 95.degree. C. for
three minutes followed by slow cooling at room temperature. This
results in the following double strand structure with the indicated
ends.
##STR00002##
[0309] Cloning of oligonucleotides between the EcoRV and PstI sites
of pRW742B resulted in pRW744. Plasmid pRW742B contains the
vaccinia virus H6 promoter linked to a non-pertinent gene inserted
at the HincII site of pRW731.15 described previously. Digestion
with PstI and EcoRV eliminates the non-pertinent gene and the
3'-end of the H6 promoter. Plasmid pRW744 now contains the 3'
portion of the H6 promoter overlapping the ATG of avian influenza
H5. The plasmid also contains the H5 sequence through the 5' SalI
site and the 3' sequence from the H5 stop codon (containing a DraI
site). Use of the DraI site removes the H5 3' non-coding end. The
oligonucleotides add a transcription termination signal recognized
by early vaccinia virus RNA polymerase (Yuen et al., 1987). To
complete the H6 promoted H5 construct, the H5 coding region was
isolated as a 1.6 kpb SalI-DraI fragment from pTH29. Plasmid pRW744
was partially digested with DraI, the linear fragment isolated,
recut with SalI and the plasmid now with eight bases deleted
between SalI and DraI was used as a vector for the 1.6 kpb pTH29
SalI and DraI fragment. The resulting plasmid pRW759 was cut with
EcoRV and DraI. The 1.7 kbp PRW759 EcoRV-DraI fragment containing
the 3' H6 promoter and the H5 gene was inserted between the EcoRV
and HincII sites of pRW846 (previously described). The resulting
plasmid pRW849 contains the H6 promoted avian influenza virus H5
gene in the de-ORFed F8 locus.
[0310] Construction of Insertion Vector for H7 Hemagglutinin at the
F7 Locus. Plasmid pCVH71 containing the H7 hemagglutinin from
A/CK/VIC/1/85 was received from Dr. R. Webster. An EcoRI-BamHI
fragment containing the H7 gene was blunt-ended with the Klenow
fragment of DNA polymerase and inserted into the HincII site of
pIBI25 as PRW827. Synthetic oligonucleotides RW165 (SEQ ID NO:62)
and RW166 (SEQ ID NO:63) were annealed, cut with HincII and StyI
and inserted between the EcoRV and StyI sites of pRW827 to generate
pRW845.
TABLE-US-00044 RW165 (SEQ ID NO: 62): 5'
GTACAGGTCGACAAGCTTCCCGGGTATCGCGATATCCGTTAAGTTTG
TATCGTAATGAATACTCAAATTCTAATACTCACTCTTGTGGCAGCCATTC
ACACAAATGCAGACAAAATCTGCCTTGGACATCAT 3' RW166 (SEQ ID NO: 63): 5'
ATGATGTCCAAGGCAGATTTTGTCTGCATTTGTGTGAATGGCTGCCA
CAAGAGTGAGTATTAGAATTTGAGTATTCATTACGATACAAACTTAACGG
ATATCGCGATACCCGGGAAGCTTGTCGACCTGTAC 3'
[0311] Oligonucleotides RW165 (SEQ ID NO:62) and RW166 (SEQ ID
NO:63) link the 3' portion of the H6 promoter to the H7 gene. The
3' non-coding end of the H7 gene was removed by isolating the
linear product of an ApaLI digestion of pRW845, recutting it with
EcoRI, isolating the largest fragment and annealing with synthetic
oligonucleotides RW227 (SEQ ID NO:64) and RW228 (SEQ ID NO:65). The
resulting plasmid was pRW854.
TABLE-US-00045 RW227 (SEQ ID NO: 64): 5'
ATAACATGCGGTGCACCATTTGTATATAAGTTAACGAATTCCAAGTC AAGC 3' RW228 (SEQ
ID NO: 65): 5' GCTTGACTTGGAATTCGTTAACTTATATACAAATGGTGCACCGCATG TTAT
3'
The stop codon of H7 in PRW854 is followed by an HpaI site. The
intermediate H6 promoted H7 construct in the de-ORFed F7 locus
(described below) was generated by moving the pRW854 EcoRV-HpaI
fragment into pRW858 which had been cut with EcoRV and blunt-ended
at its PstI site. Plasmid pRW858 (described below) contains the H6
promoter in an F7 de-ORFed insertion plasmid.
[0312] The plasmid pRW858 was constructed by insertion of an 850 bp
SmaI/HpaI fragment, containing the H6 promoter linked to a
non-pertinent gene, into the SmaI site of pF7DO described
previously. The non-pertinent sequences were excised by digestion
of pRW858 with EcoRV (site 24 bp upstream of the 3'-end of the H6
promoter) and PstI. The 3.5 kb resultant fragment was isolated and
blunt-ended using the Klenow fragment of the E. coli DNA polymerase
in the presence of 2 mM dNTPs. This blunt-ended fragment was
ligated to a 1700 bp EcoRV/HpaI fragment derived from pRW854
(described previously). This EcoRV/HpaI fragment contains the
entire AIV HA (H7) gene juxtaposed 3' to the 3'-most 24 bp of the
VV H6 promoter. The resultant plasmid was designated pRW861.
[0313] The 126 bp EH arm (defined previously) was lengthened in
pRW861 to increase the recombination frequency with genomic TROVAC
DNA. To accomplish this, a 575 bp AccI/SnaBI fragment was derived
from pRW 731.13 (defined previously). The fragment was isolated and
inserted between the AccI and NaeI sites of pRW861. The resultant
plasmid, containing an EH arm of 725 bp and a HB arm of 404 bp
flanking the AIV H7 gene, was designated as pRW869. Plasmid pRW869
therefore consists of the H7 coding sequence linked at its 5' end
to the vaccinia virus H6 promoter. The left flanking arm consists
of 404 bp of TROVAC sequence and the right flanking arm of 725 bp
of TROVAC sequence which directs insertion to the de-ORFed F7
locus.
[0314] Development of TROVAC-Avian Influenza Virus Recombinants.
Insertion plasmids containing the avian influenza virus HA coding
sequences were individually transfected into TROVAC infected
primary CEF cells by using the calcium phosphate precipitation
method previously described (Panicali et al., 1982; Piccini et al.,
1987). Positive plaques were selected on the basis of hybridization
to HA specific radiolabelled probes and subjected to sequential
rounds of plaque purification until a pure population was achieved.
One representative plaque was then amplified to produce a stock
virus. Plasmid pRW849 was used in an in vitro recombination test to
produce recombinant TROVAC-AIH5 (vFP89) expressing the H5
hemagglutinin. Plasmid pRW848 was used to produce recombinant
TROVAC-AIH4 (vFP92) expressing the H4 hemagglutinin. Plasmid pRW869
was used to produce recombinant TROVAC-AIH7 (vFP100) expressing the
H7 hemagglutinin.
[0315] Immunofluorescence. In influenza virus infected cells, the
HA molecule is synthesized and glycosylated as a precursor molecule
at the rough endoplasmic reticulum. During passage to the plasma
membrane it undergoes extensive post-translational modification
culminating in proteolytic cleavage into the disulphide linked
HA.sub.1 and HA.sub.2 subunits and insertion into the host cell
membrane where it is subsequently incorporated into mature viral
envelopes. To determine whether the HA molecules produced in cells
infected with the TROVAC-AIV recombinant viruses were expressed on
the cell surface, immunofluorescence studies were performed.
Indirect immunofluorescence was performed as described (Taylor et
al., 1990). Surface expression of the H5 hemagglutinin in
TROVAC-AIH5, H4 hemagglutinin in TROVAC-AIH4 and H7 hemagglutinin
in TROVAC-AIH7 was confirmed by indirect immunofluorescence.
Expression of the H5 hemagglutinin was detected using a pool of
monoclonal antibodies specific for the H5HA. Expression of the H4HA
was analyzed using a goat monospecific anti-H4 serum. Expression of
the H7HA was analyzed using a H7 specific monoclonal antibody
preparation.
[0316] Immunoprecipitation. It has been determined that the
sequence at and around the cleavage site of the hemagglutinin
molecule plays an important role in determining viral virulence
since cleavage of the hemagglutinin polypeptide is necessary for
virus particles to be infectious. The hemagglutinin proteins of the
virulent H5 and H7 viruses possess more than one basic amino acid
at the carboxy terminus of HA1. It is thought that this allows
cellular proteases which recognize a series of basic amino acids to
cleave the hemagglutinin and allow the infectious virus to spread
both in vitro and in vivo. The hemagglutinin molecules of H4
avirulent strains are not cleaved in tissue culture unless
exogenous trypsin is added.
[0317] In order to determine that the hemagglutinin molecules
expressed by the TROVAC recombinants were authentically processed,
immunoprecipitation experiments were performed as described (Taylor
et al., 1990) using the specific reagents described above.
[0318] Immunoprecipitation analysis of the H5 hemagglutinin
expressed by TROVAC-AIH5 (vFP89) showed that the glycoprotein is
evident as the two cleavage products HA.sub.1 and HA.sub.2 with
approximate molecular weights of 44 and 23 kDa, respectively. No
such proteins were precipitated from uninfected cells or cells
infected with parental TROVAC. Similarly immunoprecipitation
analysis of the hemagglutinin expressed by TROVAC-AIH7 (vFP100)
showed specific precipitation of the HA.sub.2 cleavage product. The
HA.sub.1 cleavage product was not recognized. No proteins were
specifically precipitated from uninfected CEF cells or TROVAC
infected CEF cells. In contrast, immunoprecipitation analysis of
the expression product of TROVAC-AIH4 (vFP92) showed expression of
only the precursor protein HA.sub.0. This is in agreement with the
lack of cleavage of the hemagglutinins of avirulent subtypes in
tissue culture. No H4 specific proteins were detected in uninfected
CEF cells or cells infected with TROVAC. Generation of recombinant
virus by recombination, in situ hybridization of nitrocellulose
filters and screening for B-galactosidase activity are as
previously described (Panicali et al., 1982; Perkus et al.,
1989).
Example 14
Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)
(IIIB), gp120 (MN) (+transmembrane) Epitopes
[0319] A plasmid, pHXB2D, containing HIV1 (IIIB) cDNA sequence
(Ratner et al, 1985), was obtained from Robert Gallo (NCI, NIH).
The sequence encoding the 5'-end of the gag gene was cloned between
vaccinia virus tk flanking arms. This was accomplished by cloning
the 1,625 bp BglII fragment of pHXB2D, containing the 5'-end of the
gag gene, into the 4,075 bp BglII fragment of pSD542VCVQ. The
plasmid generated by this manipulation is called pHIVG2.
[0320] The 3'-end of the gag gene was then cloned into pHIVG2. This
was accomplished by cloning a 280 bp ApaI-BamHI PCR fragment,
containing the 3'-end of the gag gene, into the 5,620 bp ApaI-BamHI
fragment of pHIVG2. (This PCR fragment was generated from the
plasmid, pHXB2D, with the oligonucleotide primers, HIVP5 (SEQ ID
NO:69; 5'-TGTGGCAAAGAAGGGC-3') and HIVP6 (SEQ ID NO:70;
5'-TTGGATCCTTATTGTGACGAGGGGTC-3').) The plasmid generated by this
manipulation is called pHIVG3.
[0321] The I3L promoter was then cloned upstream of the gag gene.
This was accomplished by cloning the oligonucleotides, HIVL17 (SEQ
ID NO:71;
5'-GATCTTGAGATAAAGTGAAAATATATATCATTATATTACAAAGTACAATTATTTA
GGTTTAATCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3') and HIVL18
(SEQ ID NO:72; 5'-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCT
CGCACCCATGATTAAACCTAAATAATTGTACTTTGTAATATAATGATATATATTTTCACTTT
ATCTCAA-3'), encoding the vaccinia virus I3L promoter and the
5'-end of the gag gene, into the 5,540 bp partial BglII-ClaI
fragment of pHIVG3. The plasmid generated by this manipulation is
called pHIVG4.
[0322] The portion of the gag gene encoding p24, p2, p7 and p6 was
then eliminated. This was accomplished by cloning the
oligonucleotides, HIVL19 (SEQ ID NO:73; 5'-CTGACACAGG
ACACAGCAATCAGGTCAGCCAAAATTACTAATTTTTATCTCGAGGTCGACAGGACCCG-3') and
HIVL20 (SEQ ID NO:74; 5'-GATCCGGGTCCTGTCGACCTCGAGATAAAAA
TTAGTAATTTTGGCTGACCTGATTGCTGTGTCCTGTGTCAG-3'), into the 4,450 bp
partial PvuII-BamHI fragment of pHIVG4. The plasmid generated by
this manipulation is called pHIVG5.
[0323] The remainder of the gag gene, as well as the pol gene, was
then cloned downstream of the p17 "gene". This was accomplished by
cloning the 4,955 bp ClaI-SalI fragment of pHXB2D, containing most
of the gag gene and all of the pol gene, into the 4,150 bp
ClaI-SalI fragment of pHIVG5. The plasmid generated by this
manipulation is called pHIVG6.
[0324] Extraneous 3'-noncoding sequence was then eliminated. This
was accomplished by cloning a 360 bp AflII-BamHI PCR fragment,
containing the 3'-end of the pol gene, into the 8,030 bp
AflII-BamHI fragment of pHIVG6. (This PCR fragment was generated
from the plasmid, pHXB2D, with the oligonucleotide primers, HIVP7
(SEQ ID NO:75; 5'-AAG AAAATTATAGGAC-3') and HIVP8 (SEQ ID NO:76;
5'-TTGGATCCC TAATCCTCATCCTGT-3').) The plasmid generated by this
manipulation is called pHIVG7.
[0325] The I3L-promoted gag and pol genes were then cloned between
canary pox C3 flanking arms. This was accomplished by cloning the
4,360 bp partial BglII-BamHI fragment of pHIVG7, containing the
I3L-promoted gag and pol genes, into the BamHI site of pVQH6CP3L.
The plasmid generated by this manipulation is called pHIVGE14.
[0326] The H6-promoted HIV1gp120(MN) (+transmembrane) "gene" (Gurgo
et al, 1988) was then cloned into pHIVGE14. This was accomplished
by cloning the 1,700 bp NruI-SmaI fragment of pC5HIVMN120T,
containing the gp120(+transmembrane) "gene", into the 11,400 bp
NruI-SmaI fragment of pHIVGE14. The plasmid generated by this
manipulation is called pHIVGE14T.
[0327] Most of the pol gene was then removed. This was accomplished
by cloning a 540 bp ApaI-BamHI PCR fragment, containing the 3'-end
of the HIV1 protease "gene", into the 10,000 bp ApaI-BamHI fragment
of pHIVGE14T. (This PCR fragment was generated from the plasmid,
pHIVG7, with the oligonucleotide primers, HIVP5 and HIVP37 (SEQ ID
NO:77; 5'-AAAGGATCCCCCGGGTTAAAAATTTAAAGTGCAACC-3').) This
manipulation removes most of the pol gene, but leaves the protease
"gene" intact. The plasmid generated by this manipulation is called
pHIV32. The DNA sequence of pHIV32 (SEQ ID NOS: 78 and 79) is shown
in FIGS. 14A-14C which shows the nucleotide sequence of the
H6-promoted HIV1gp120(+transmembrane) gene and the I3L-promoted
HIV1gag(+pro) gene contained in pHIV32:
TABLE-US-00046 gag (+pro) and gp120 (+transmembrane) FEATURES From
To/Span Description frag 1 56 C3 flanking arm frag 162 76 (C) HIV1
(IIIB) env transmembrane region frag 1728 163 (C) HIV1 (MM) gp120
gene frag 1853 1729 (C) vaccinia H6 promoter frag 1925 1983
vaccinia I3L promoter frag 1984 3746 HIV1 (IIB) gag/pro gene frag
3753 3808 C3 flanking arm
[0328] The DNA sequence of the ALVAC C3 flanking arm (SEQ ID NOS:
80 and 81) is shown in FIGS. 15A-F. FIGS. 15A to 15F show the
nucleotide sequence of the C3 locus in pVQH6CP3L:
TABLE-US-00047 C3 LOCUS pVQH6CP3L FEATURES From To/Span Description
frag 1 1460 C3 flanking arm frag 1461 1501 Cloning sites frag 1630
1502 H6 promoter frag 1717 4291 C3 flanking arm
[0329] pHIV32 was used in vitro recombination with ALVAC as the
rescuing virus to yield vCP205.
[0330] vCP205 (ALVAC-MN120TMG) was deposited on Mar. 6, 1997 under
the terms of the Budapest Treaty with the American Type Culture
Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA under
ATCC accession number VR-2557.
Example 15
Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)
(IIIB), gp120 (MN) (+transmembrane) and 2 nef (BRU) Epitopes
[0331] Expression cassettes encoding two nef CTL epitopes, CTL1
(amino acids 66-147) and CTL2 (amino acids 182-206) (Wain-Hobson et
al, 1985; Nixon and McMichael, 1991), were then inserted into
vCP205. The insertion plasmid, p2-60-HIV.3, containing the nef CTL
epitopes, was generated by the following procedure. The
I3L-promoted CTL2 epitope was cloned into pBSH6. This was
accomplished by cloning a 255 bp PCR HindIII-XhoI fragment,
containing the I3L-promoted CTL2 epitope, into the 3,100 bp
HindIII-XhoI fragment of pBSH6. (The 255 bp PCR fragment was
generated by the following procedure. A 216 bp PCR fragment,
containing the I3L-promoter and the 5'-end of the CTL2 epitope, was
generated from the plasmid, pMPI3H, with the oligonucleotide
primers, VQPCRI3 (SEQ ID NO: 82;
5'-ATCATCAAGCTTAATTAATTAGTTATTAGACAAGGTGAAAACGAAACTATTTGTAGCTT
AATTATTAGACATCATGCAGTGGTTAAAC-3') and I3PCRCTL (SEQ ID NO: 83;
5'-CTAGCTACGTGATGAAATGCTAATCTAGAATCAAATCTCCACTCCATGATTAAACCTAA
ATAATTGTAC-3'). This 216 bp PCR fragment was then used as a
template in a second PCR reaction with the oligonucleotide primers,
VQPCRI3 and CTLPCR (SEQ ID NO: 84;
5'-GAATTCCTCGAGGATCCTCTAGATTAACAATTTTTAAAATATTCAGGATGTAATTCTCT
AGCTACGTGATGAAATGC-3'), to generate the PCR fragment, containing
the I3L-promoted CTL2 epitope, that was digested with HindIII and
XhoI and cloned into pBSH6). The plasmid generated by this
manipulation is called p2-60-HIV.1.
[0332] The H6-promoted CTL1 epitope was then cloned into
p2-60-HIV.1. This was accomplished by cloning a 290 bp NruI-EcoRI
fragment, containing the H6-promoted CTL1 epitope, into the 3,300
bp NruI-EcoRI fragment of p2-60-HIV.1. (The 290 bp NruI-EcoRI
fragment was generated by the following procedure. A 195 bp PCR
fragment, containing the H6-promoter and the 5'-end of the CTL1
epitope, was generated from the plasmid, pH6T2, with the
oligonucleotide primers, H6PCR1 (SEQ ID NO: 85;
5'-ACTACTAAGCTTCTTTATTCTATACTTAAAAAGTG-3') and NCCPCR1 (SEQ ID NO:
86; 5'-CAGCTGCTTTGTAAGTCATTGGTCTTAAAGGTACTTGAGGTGTTACTGGAAAACCTACC
ATTACGATACAAACTTAACGGATATCGCG-3'). This 195 bp PCR fragment and the
oligonucleotides, NCC174A (SEQ ID NO: 87;
5'-ACTTACAAAGCAGCTGTAGATCTTTCTCACTTTTTAAAAGAAAAAGGAGGTTTAGAAGG
GCTAATTCATTCTCAACGAAGACAAGATATTCTTGATTTGTGG-3') and NCC174B (SEQ ID
NO: 88;
5'-CCACAAATCAAGAATATCTTGTCTTCGTTGAGAATGAATTAGCCCTTCTAAACCTCCTT
TTTCTTTTAAAAAGTGAGAAAGATCTACAGCTGCTTTGTAAGT-3'), were then used as
templates in a second PCR reaction with the oligonucleotide
primers, H6PCR1 and NCCPCR2 (SEQ ID NO: 89;
5'-CTGCCAATCAGGAAAATATCCTTGTGTATGATAAATCCACAAATCAAGAATATC-3'). The
resulting 317 bp PCR fragment, containing the H6-promoter and most
of the CTL1 epitope, and the oligonucleotides, NCC291A (SEQ ID NO:
90; 5'-GGATATTTTCCTGATTGGCAGAATTACACACCAGGACCAGGAGTCAGATACCCATTAAC
CTTTGGTTGGTGCTACAAGC-3') and NCC291B (SEQ ID NO: 91;
5'-GCTTGTAGCACCAACCAAAGGTTAATGGGTATCTGACTCCTGGTCCTGGTGTGTAATTC
TGCCAATCAGGAAAATATCC-3'), were then used as templates in a third
PCR reaction with the oligonucleotide primers, H6PCR1 and NCCPCR3
(SEQ ID NO: 92;
5'-ACTACTGAATTCTCGAGAAAAATTATGGTACTAGCTTGTAGCACCAACC-3'), to
generate the PCR fragment, containing the H6-promoted CTL1 epitope,
that was digested with NruI and EcoRI and cloned into p2-60-HIV.1)
The plasmid generated by this manipulation is called
p2-60-HIV.2.
[0333] The I3L-promoted CTL2 and H6-promoted CTL1 epitopes were
then cloned between canarypox C6 flanking arms. This was
accomplished by cloning the 640 bp XhoI fragment of p2-60-HIV.2,
containing the two (2) nef CTL epitopes, into the XhoI site of
pC6L. The plasmid generated by this manipulation is called
p2-60-HIV.3. The DNA sequence of p2-60-HIV.3 (SEQ ID NOS: 93-96) is
shown in FIG. 16. FIG. 16 shows the nucleotide sequence of the
I3L-promoted nef CTL2 epitope and H6-promoted nef CTL1 epitope
contained in p2-60-HIV.3:
TABLE-US-00048 NEF CTL epitopes FEATURES From To/Span Description
frag 1 51 C6 Left Arm pept 175 98 (C) nef CTL2 frag 275 176 (C) I3L
promoter frag 337 460 H6 promoter pept 461 709 1 nef CTL1 frag 751
801 C6 Right Arm
[0334] The DNA sequence of the ALVAC C6 flanking arm (SEQ ID NOS:
97 and 98) is shown in FIGS. 17A-C. FIG. 17A to 17C show the
nucleotide sequence of the C6 locus in pC6L:
TABLE-US-00049 C6 LOCUS pC6L FEATURES From To/Span Description frag
1 381 C6 flanking arm frag 382 447 Cloning sites frag 448 1615 C6
flanking arm
[0335] p2-60HIV.3 was used in in vitro recombination experiments
with vCP205 as the rescuing virus to yield vCP264.
Example 16
Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)
(IIIB), gp120 (MN) (+transmembrane) and 2 nef (BRU) and 3 pol
(IIIB) CTL Epitope Containing Regions
[0336] Expression cassettes encoding three (3) pol CTL epitopes,
poll (amino acids 172-219), pol2 (amino acids 325-383) and pol3
(amino acids 461-519) (Ratner et al, 1985; Nixon and McMichael,
1991), were then inserted into vCP264. The insertion plasmid,
pC5POLT5A, containing the three (3) pol CTL epitopes, was generated
by cloning a 948 bp XhoI-BamHI fragment, containing the H6-promoted
poll epitope, the I3L-promoted pol2 epitope and the 42K-promoted
pol3 epitope, into the 2,940 bp XhoI-BamHI fragment of PBSK.sup.+.
(The 948 bp XhoI-BamHI fragment was generated by the following
procedure. A 183 bp PCR fragment, containing the poll epitope, was
generated from the plasmid, pHXB2D, with the oligonucleotide
primers, P1A (SEQ ID NO: 99;
5'-TTTGTATCGTAATGATTGAGACTGTACCAGTAAAATTAAAGCC-3') and P1B (SEQ ID
NO: 100;
5'-GGGCTGCAGGAATTCTAATCAATTAAGGCCCAATTTTTGAAATTTTCCCTTCCTTTTCC
ATCTCTG-3'). A 224 bp PCR fragment, containing the pol2 epitope,
was generated from the plasmid, pHXB2D, with the oligonucleotide
primers, P2A (SEQ ID NO: 101;
5'-ACAAAGTACAATTATTTAGGTTTAATCATGGCAATATTCCAAAGTAGCATGAC-3') and
P2B (SEQ ID NO: 102;
5'-ATCATCCTCGAGAAAAATTAGGTAAGTCCCCACCTCAACAGATG-3'). A 236 bp PCR
fragment, containing the pol3 epitope, was generated from the
plasmid, pHXB2D, with the oligonucleotide primers, P3A (SEQ ID NO:
103;
5'-AAAATATATAATTACAATATAAAATGCCACTAACAGAAGAAGCAGAGCTAGAACTGGC-3')
and P3B (SEQ ID NO: 104;
5'-ATCATCTCTAGACTCGAGGATCCATAAAAATTATCCTGTTTTCAGATTTTTAAATGGCT
C-3'). A 340 bp PCR fragment, containing the I3L and H6 promoters
(in a head-to-head configuration) was generated from the plasmid,
p2-60-HIV.2, with the oligonucleotide primers, P2IVH (SEQ ID NO:
105; 5'-GTCATGCTACTTTTGAATATTGCCATGATTAAACCTAAATAATTGTACTTTG-3,)
and IVHP1 (SEQ ID NO: 106;
5'-TTTAATTTTACTGGTACAGTCTCAATCATTACGATACAAACTTAACGGATATCGCG-3'). A
168 bp PCR fragment, containing the 42K promoter, was generated
from the plasmid, pVQ42KTh4.1, with the oligonucleotide primers,
EPS42K (SEQ ID NO: 107;
5'-AATTGATTAGAATTCCTGCAGCCCGGGTCAAAAAAATATAAATG-3') and 42 KP3B
(SEQ ID NO: 108;
5'-CCAGTTCTAGCTCTGCTTCTTCTGTTAGTGGCATTTTATATTGTAATTATATATTTTC-3').
A 511 bp PCR fragment, containing the H6 promoter and I3L-promoted
pol2 epitope, was generated by using the 224 bp PCR fragment,
containing the pol2 epitope, and the 340 bp PCR fragment,
containing the I3L and H6 promoters, as templates in a PCR reaction
with the oligonucleotide primers, P2B and IVHP1. A 347 bp PCR
fragment, containing the 42K-promoted pol3 epitope, was generated
by using the 168 bp PCR fragment, containing the 42K promoter, and
the 236 bp PCR fragment, containing the pol3 epitope, as templates
in a PCR reaction with the oligonucleotide primers, IPS42K and P3B.
A 506 bp PCR fragment, containing the poll epitope and the
42K-promoted pol3 epitope, was generated by using the 183 bp PCR
fragment, containing the poll epitope, and the 347 bp PCR fragment,
containing the 42K-promoted pol3 epitope, as templates in a PCR
reaction with the oligonucleotide primers, P1A and P3B. A 977 bp
PCR fragment, containing the H6-promoted poll epitope, the
I3L-promoted pol2 epitope and the 42K-promoted pol3 epitope, was
generated by using the 511 bp PCR fragment, containing the H6
promoter and I3L-promoted pol2 epitope, and the 506 bp PCR
fragment, containing the poll epitope and 42K-promoted pol3
epitope, as templates in a PCR reaction with the oligonucleotide
primers, P2B and P3B. The 977 bp PCR fragment was then digested
with XhoI and BamHI and cloned into the 2,940 bp XhoI-BamHI
fragment of pBSK.sup.+.) The plasmid generated by this manipulation
is called pBSPOLT5.
[0337] Nucleotide sequence analysis of pBSPOLT5 indicated that
there was an error in the pol2 epitope. In order to correct this
mistake, the 948 bp XhoI-BamHI fragment, containing the H6-promoted
poll epitope, the I3L-promoted pol2 epitope and the 42K-promoted
pol3 epitope, was used as a template in a PCR reaction with the
oligonucleotide primers, 13PCR1 (SEQ ID NO: 109;
5'-ATCATCGGATCCAAGCTTACATCATGCAGTGG-3') and FIXPOL2 (SEQ ID NO:
110; 5'-ATCATCCTCGAGCTATTCAATTAGGTTGTAAGTCCCCACCTCAAC-3'). The
resulting PCR fragment, containing the corrected I3L-promoted pol2
epitope, was digested with HindIII and XhoI and cloned into the
3,650 bp HindIII-XhoI fragment of pBSPOLT5. The plasmid generated
by this manipulation is called pBSPOLT5A.
[0338] The H6-promoted poll epitope, I3L-promoted pol2 epitope and
42K-promoted pol3 epitope was then cloned between canary pox C5
flanking arms. This was accomplished by cloning the 897 bp
BamHI-XhoI fragment of pBSPOLT5A, containing the H6-promoted poll
epitope, the I3L-promoted pol2 epitope and the 42K-promoted pol3
epitope, into the 4,675 bp BamHI-XhoI fragment of pNC5L-SP5. The
plasmid generated by this manipulation is called pC5POLT5A. The DNA
sequence of pC5POLT5A (SEQ ID NOS: 111-115) is shown in FIGS.
18A-B. FIGS. 18A to 18B shows the nucleotide sequence of the
I3L-promoted pol2 epitope, H6-promoted poll epitope and
42K-promoted pol3 epitope contained in pC5POLT5a:
TABLE-US-00050 POL CTL epitopes FEATURES From To/Span Description
frag 1 50 C5 Left Arm pept 272 92 (C) POL 2 frag 372 273 (C) I3L
promoter frag 377 500 H6 promoter pept 501 647 1 POL1 frag 676 782
_42K promoter pept 783 962 1 POL 3 frag 986 1035 C5 Right Arm
[0339] The DNA sequence of the ALVAC C5 flanking arm (SEQ ID NOS:
116 and 117) is shown in FIGS. 19A-C. FIGS. 19A to 19C show the
nucleotide sequence of the C5 locus in pNC5L-SP5:
TABLE-US-00051 C5 LOCUS pNC5L-SP5 FEATURES From To/Span Description
frag 1 1549 C5 flanking arm frag 1550 1637 Cloning sites frag 1638
2049 C5 flanking arm
[0340] pC5POLT5A was used in in vitro recombination experiments
with vCP264 as the rescuing virus to yield vCP300.
Example 17
Restriction and Immunoprecipitation Analyses
[0341] Restriction enzyme analysis was performed to confirm that
the HIV1 sequences in vCP300 are in the proper loci. ALVAC, vCP205,
vCP264 and vCP300 DNA were digested with HindIII, PstI or XhoI and
the resultant fragments fractionated on an agarose gel. When the
sizes of the resulting fragments were compared, it was determined
that, as expected, the gag(+pro) and gp120(+transmembrane) genes
were inserted into the C3 locus, the nef epitopes were inserted
into the C6 locus and the pol epitopes were inserted into the C5
locus.
[0342] Immunoprecipitation analysis was performed to determine
whether vCP300 expresses authentic HIV1gag and
gp120(+transmembrane) gene products. HeLa cell monolayers were
either mock infected or infected at an m.o.i. of 10 pfu/cell with
ALVAC or vCP300. Following an hour adsorption period, the inoculum
was aspirated and the cells were overlayed with 2 mls of modified
Eagle's medium (minus methionine) containing 2% fetal bovine serum
and [.sup.35S]-methionine (20 .mu.Ci/ml). Cells were harvested at
18 hrs post-infection by the addition of 1 ml 3.times. buffer A (3%
NP-40, 30 mM Tris (pH7.4), 3 mM EDTA, 0.03% Na Azide and 0.6 mg/ml
PMSF) and 50 ul aprotinin, with subsequent scraping of the cell
monolayers. Lysates from the infected cells were analyzed for
HIV1gag and gp120(+transmembrane) gene expression using serum from
HIV1-seropositive individuals (obtained from New York State
Department of Health). The sera was bound to Protein A-sepharose in
an overnight incubation at 4.degree. C. Following this incubation
period, the material was washed 4.times. with 1.times. buffer A.
Lysates, precleared with normal human sera and protein A-sepharose,
were then incubated overnight at 4.degree. C. with the
HIV1-seropositive human sera bound to protein A-sepharose. After
the overnight incubation period, the samples were washed 4.times.
with 1.times. buffer A and 2.times. with a LiCl.sub.2/urea buffer.
Precipitated proteins were dissociated from the immune complexes by
the addition of 2.times. Laemmli's buffer (125 mM Tris (pH6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min.
Proteins were fractionated on a 10% Dreyfuss gel system (Dreyfuss
et al., 1984), fixed and treated with 1M Na-salicylate for
fluorography. This analysis indicated that HIV1gag and
gp120(+transmembrane) gene products were precipitated from
vCP300-infected cells, but were not precipitated from mock infected
or ALVAC-infected cells.
[0343] Expression of the nef and pol epitopes in vCP300-infected
cells has not been confirmed because no epitope specific
serological reagents are yet available. Nucleotide sequence
analysis, however, has confirmed that the nef and pol sequences
cloned into vCP300 are correct. PCR fragments, containing the nef
and pol expression cassettes, were generated from vCP300 DNA.
Nucleotide sequence analysis of these fragments indicated that the
nef and pol sequences are correct. vCP205 expresses the same cell
surface-associated form of HIV1 gp120 as expressed by vCP300. The
immunogenicity of this gene product, as expressed by vCP205, has
been assayed in small laboratory animals.
Example 18
Immunogenicity Studies
[0344] Groups of two rabbits or guinea pigs were inoculated
intramuscularly (im) with 10.sup.8 pfu of ALVAC, vCP205, or with
0.1 mg of peptide CLTB-36 (GPKEPFRDYVDRFYKNKRKRIHIGPGRAFYTTKN) (SEQ
ID NOS: 118) adjuvanted with 0.05 mg of QS-21 according to the
schedule below (Table 21).
TABLE-US-00052 TABLE 21 Immunization schedule for rabbits and
guinea pigs inoculated with ALVAC, vCP205, or with peptide CLTB-36
in QS-21. INOCULATION GROUP WEEK 0 WEEK 4 WEEK 8 1 ALVAC ALVAC
CLTB-36/QS-21 2 vCP205 vCP205 vCP205 3 vCP205 vCP205 CLTB-36/QS-21
4 CLTB-36/QS-21 CLTB-36/QS-21 CLTB-36/QS-21
[0345] Each rabbit and guinea pig was bled prior to the first
inoculation and at 2-week intervals following the first inoculation
through week 14. Serum was prepared from each blood sample and
stored at -70.degree. C. until use. Each serum was tested for
antibody responses to recombinant HIV MN/BRU hybrid gp160 or to
25-mer synthetic HIV MN gp120 V3 loop (American Bio-Technologies,
Inc. Cambridge, Mass., product # 686010) by kinetics enzyme-linked
immunosorbant assay (KELISA).
[0346] Rabbits immunized with vCP205 (Group 2) produced the highest
levels of anti-gp160 antibodies (FIG. 20). Rabbits were immunized
according to the schedule in Table 21 (arrows in FIG. 20) and bled
at 2 week intervals. Each serum was diluted 1:100 in dilution
buffer and tested for reactivity with purified recombinant HIV
MN/BRU gp160 using a kinetics ELISA. Both rabbits in this group
began producing gp160 reactive antibodies after a single
inoculation. Boosting with subsequent inoculations produced only
minor increases in antibody levels. Rabbits inoculated twice with
vCP205 and boosted with peptide CLTB-36 in QS-21 adjuvant (Group 3)
apparently failed to make anti-gp160 antibodies when compared to
control rabbits (Group 1). Of the rabbits immunized three times
with peptide CLTB-36 in QS-21, only one responded by generating
gp160-specific antibodies. The one responsive rabbit (A353) began
producing gp160 antibodies only after the third immunization.
[0347] Rabbits immunized only with peptide CLTB-36 would not be
expected to generate broadly reactive gp160-specific antibodies
since the peptide contains only a small portion of the envelope
glycoprotein, the V3 loop. Thus, sera were tested for reactivity to
a peptide containing 25 amino acids of the HIV MN V3 loop (FIG.
21). Rabbits were immunized according to the schedule in Table 21
(arrows in FIG. 22) and bled at 2 week intervals. Each serum was
diluted 1:100 in dilution buffer and tested for reactivity with a
25-mer synthetic peptide representing the HIV MN gp120 V3 loop
(CNKRKRIHIGPGRAFYTTKNIIGTIC; (SEQ ID NO: 119) American
Bio-Technologies, Inc. Cambridge, Mass., product # 686010) using a
kinetics ELISA. As before, the highest V3 antibody responses were
found in the sera of rabbits inoculated three times with vCP205.
Two inoculations with vCP205 followed by peptide CLTB-36 in QS-21
produced anti-V3 antibody responses, but not as high as Group 2
rabbits. Also, as before, only one rabbit responded to three
inoculations with peptide CLTB-36 in QS-21.
[0348] Guinea pigs in all groups, including one animal in Group 1,
produced antibodies that reacted with HIV gp160 (FIG. 22). Guinea
pigs were immunized according to the schedule in Table 21 (arrows
in FIG. 22) and bled at 2 week intervals. Each serum was diluted
1:100 in dilution buffer and tested for reactivity with purified
recombinant HIV MN/BRU gp160 using a kinetics ELISA. The single
animal in Group 1 that responded did so only after inoculation with
peptide CLTB-36 in QS-21 adjuvant. Antibody levels in the sera of
all guinea pigs in Groups 2, 3, and 4 were similar. Most of the
guinea pigs responded to a single inoculation by producing gp160
antibodies.
[0349] Similar results were seen using a HIV MN V3 25-mer peptide
as the KELISA antigen (FIG. 23). Guinea pigs were immunized
according to the schedule in Table 21 (arrows in FIG. 23) and bled
at 2 week intervals. Each serum was diluted 1:100 in dilution
buffer and tested for reactivity with a 25-mer synthetic peptide
representing the HIV MN gp120 V3 loop (CNKRKRIHIGPGRAFYTTKNIIGTIC
(SEQ ID NO: 120) American Bio-Technologies, Inc. Cambridge, Mass.,
product # 686010) using a kinetics ELISA. A single inoculation of
peptide CLTB-36 in QS-21 elicited V3 antibody responses which were
boosted by second and third inoculations. Two inoculations with
vCP205 were necessary to induce V3 antibody responses which was
boosted to higher levels by the third inoculation of vCP205 or
CLTB-36 in QS-21.
[0350] Expression and immunogenicity of the Nef and Pol CTL
epitopes expressed by vCP300 is demonstrated by the following in
vitro assays. Fresh PBMC samples were derived from HIV-seropositive
individuals. Twenty-percent of these cells were inoculated with
vCP300 at an m.o.i. of 10. Two hours post-infection the cells were
washed and mixed with autologous, uninoculated PBMCs at a ratio of
uninoculated/inoculated of 10:1. (Seeding density equaled
1.5.times.10.sup.6 cells/ml). On day 0, exogenous IL-7 was added at
a final concentration of 1000 U/ml. On day three, the addition of
an exogenous source of IL-7 and IL-2 was added at a final
concentration of 1000 U/ml and 220 U/ml, respectively. After 12
days in culture, the in vitro stimulated cell population was used
in a standard .sup.51Cr-release assay using autologous Epstein-Barr
virus-transformed B cells infected with vaccinia virus (WR)
recombinants expressing HIV-1 proteins as targets. The results from
assays obtained using an Effector/Target (E/T) cell ratio of 20:1
are shown in FIGS. 24 and 25 and expressed as percent specific
lysis. The combined results demonstrate the ability of
vCP300-infected PBMCs to stimulate HIV-1, Env-, Gag-, Pol-, and
Nef-specific cytolytic activity. Further, abrogation of the
cytolytic activities by anti-CD8 monoclonal antibodies demonstrates
that the nature of the cell mediating the cytolytic activities are
classical CD8.sup.+ CTLs.
[0351] In summary, the inoculation of rabbits and guinea pigs with
the HIV ALVAC recombinant canarypox virus, vCP205, elicited
antibodies to the HIV envelope glycoprotein and a region of the HIV
envelope glycoprotein associated with neutralization of HIV, the
gp120 V3 loop region. The expression and immunogenicity of the
vCP300 expressed Env, Gag, Pol and Nef encoded products is
demonstrated by the in vitro stimulation of CD8.sup.+ CTLs from
seropositive individuals. Thus, vCP205 and vCP300 and precursors to
these recombinants and expression products and DNA from these
recombinants are useful, as described above.
Example 19
Generation of vCP1307; an ALVAC Recombinant Expressing a Form of
HIV1 gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3
Loop
[0352] vCP1307, an ALVAC recombinant expressing HIV1 gp120+TM with
2 ELDKWA epitopes from HIV1 gp41 inserted into the gp120 V3 loop
region, was generated by the following procedure. The sequence
encoding part of the ELDKWA elements and V3 loop was cloned into
pBSK+ (Stratagene, LaJolla, Calif.). This was accomplished by
cloning a 225 bp EcoRI-SacI-digested PCR fragment, containing part
of the ELDKWA-V3 loop sequence, into the 2,900 bp EcoRI-SacI
fragment of pBSK+. (This PCR fragment was generated from the
plasmid, pBSHIVMN120T, with the primers, HIVP72 (SEQ ID NO: 121;
5'-TTATTACCATTCCAAGTACTATT-3') and HIVP74 (SEQ ID NO: 122)
5'-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGAC- C
AGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3').) The plasmid
generated by this manipulation is called pHIV55.
[0353] pBSHIVMN120T, a plasmid containing the H6-promoted HIV1
gp120+TM gene, was generated by the following procedure. A plasmid,
pMN1.8-9, containing a cDNA copy of the HIV1 (MN) env gene, was
obtained from Marvin Reitz (NCI, NIH). An early transcription
termination signal sequence, T.sub.5NT, in the env gene was
modified. This was accomplished by cloning a 1,100 bp
KpnI-EcoRI-digested PCR fragment, containing the T.sub.5NT-modified
5'-end of the env gene, into the 2,900 bp KpnI-EcoRI fragment of
pBSK+. (This PCR fragment was generated from the plasmid, pMN1.8-9,
with the oligonucleotides, HIVMN6 (SEQ ID NO: 123;
5'-GGGTTATTAATGATCTGTAG-3') and HIV3B2 (SEQ ID NO: 124;
5'-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3').) The plasmid
generated by this manipulation is called pBSMIDMN.
[0354] The T.sub.5NT-modified 5'-end of the env gene was then
cloned upstream to the rest of the env gene. This was accomplished
by cloning the 1,025 bp KpnI-EcoRI fragment of pBSMIDMN, containing
the T.sub.5NT-modified 5'-end of the env gene, into the 4,300 bp
KpnI-EcoRI fragment of pBS3MN. (pBS3MN was generated by cloning a
430 bp EcoRI-SacI-digested PCR fragment, containing a central
portion of the env gene, and a 1,050 bp SacI-XbaI-digested PCR
fragment, containing the 3'-end of the env gene, into the 2,900 bp
EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragment was generated
from the plasmid, pMN1.8-9, with the oligonucleotides, HIV3B1 (SEQ
ID NO: 125; 5'-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3') and HIVMN4
(SEQ ID NO: 126; 5'-ATCATCGAGCTCCTATCGCTGCTC-3'). The 1,050 bp PCR
fragment was generated from the plasmid, pMN1.8-9, with the
oligonucleotides, HIVMN5 (SEQ ID NO: 127;
5'-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3') and HIVMN3P (SEQ ID NO: 128;
5'-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3').) The plasmid
generated by this manipulation is called pBSMID3MN.
[0355] The H6 promoter (Perkus et al., 1989) was then cloned
upstream to the env gene. This was accomplished by cloning the 320
bp KpnI fragment of pH6IIIBE, containing the H6 promoter linked to
the 5'-end of the HIV1 (IIIB) env gene, and the 2,450 bp KpnI-XbaI
fragment of pBSMID3MN, containing the bulk of the HIV1 (MN) env
gene, into the 2,900 bp KpnI-XbaI fragment of pBSK+. The plasmid
generated by this manipulation is called pH6HMNE.
[0356] The sequence encoding gp41 was then replaced with the
sequence encoding the HIV1 env transmembrane (TM) region. This was
accomplished by cloning a 480 bp EcoRI-XbaI-digested PCR fragment,
containing the 3'-end of the gp120 gene and the HIV1 env
transmembrane region, into the 4,200 bp EcoRI-XbaI fragment of
pH6HMNE. (This PCR fragment was generated from the PCR fragment,
PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO: 129;
5'-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGT
AGTGAATAGAGTTAGGCAGGGATAA-3') and HIVTM2 (SEQ ID NO: 130;
5'-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCA
AGCCTCCTACTATCATTATGAATAA-3'), with the oligonucleotides, HIV3B1
(SEQ ID NO: 125) and HIVTM3 (SEQ ID NO: 131;
5'-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3'). PCR-MN11 was
generated from the plasmid, pH6HMNE, with the oligonucleotides,
HIV3B1 (SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;
5'-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3').) The plasmid
generated by this manipulation is called pBSHIVMN120T.]
[0357] Another part of the sequence encoding the ELDKWA epitopes
and V3 loop was then cloned into PBSK+. This was accomplished by
cloning a 300 bp HindIII-SacI-digested PCR fragment, containing
part of the ELDKWA-V3 loop sequence, into the 2,900 bp HindIII-SacI
fragment of PBSK+. (This PCR fragment was generated from the
plasmid, pBSHIVMN120T, with the primers, HIVP69 (SEQ ID NO: 133;
5'-TGATAGTACCAGCTATAGGTTGAT-3') and HIVP75 (SEQ ID NO: 134;
5'-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3').) The plasmid generated by
this manipulation is called pHIV56.
[0358] The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were
then cloned into the H6-promoted gp120+TM gene. This was
accomplished by cloning the 225 bp EcoRI-SacI fragment of pHIV55
and the 300 bp HindIII-SacI fragment of pHIV56, containing the
ELDKWA-V3 loop sequences, into the 4,300 bp EcoRI-HindIII fragment
of pBSHIVMN120T. The plasmid generated by this manipulation is
called pHIV57.
[0359] The H6-promoted gp120+TM construct containing the ELDKWA
epitopes was then cloned between C5 flanking arms. This was
accomplished by cloning the 1,700 bp NruI-XbaI fragment of pHIV57,
containing the H6-promoted gp120+TM (with ELDKWA epitopes) gene,
into the 4,700 bp NruI-XbaI fragment of pSIVGC15. (pSIVGC15
contains the H6-promoted SIV env gene cloned between C5 flanking
arms.) The plasmid generated by this manipulation is called pHIV59.
The DNA sequence of the H6-promoted gp120+TM (with ELDKWA epitopes)
gene in pHIV59 is shown in FIG. 26. pHIV59 was used in in vitro
recombination experiments with ALVAC as the rescuing virus to yield
vCP1307.
[0360] FACS analysis was performed to determine whether HIV1
gp120+TM (with ELDKWA epitopes) was expressed on the surface of
vCP1307-infected cells. 5.times.10.sup.6 HeLa-S3 cells in S-MEM
(Sigma M-8028 Joklik suspension media) were infected at an m.o.i.
of 5 pfu/cell with ALVAC, vP1286 (a WR recombinant expressing HIV1
gp120+TM) or vCP1307. Following a 60 minute adsorption period at
37.degree. C., the cells were washed with 10 mls of S-MEM and
centrifuged at 1,000 RPM for 5 minutes. The samples were then
resuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes
and placed on a rotator at 37.degree. C. After 18 hours, 200 ul
aliquots (1.times.10.sup.6 cells) were placed in polypropylene
tubes and washed with 3 mls of PBS-CMF (with 0.2% NaN.sub.3+0.2%
BSA). The supernatant was then decanted and the pellet was
resuspended in 100 ul of a 1:100 dilution of sera from
HIV1-seropositive humans (obtained from the New York State Dept. of
Health) or a 1:100 dilution of a human monoclonal antibody specific
for the ELDKWA epitope, IAM41-2F5 (obtained from Viral Testing
Systems Corp., Houston, Tex.). The samples were incubated at
4.degree. C. for 60 minutes, washed two times with PBS-CMF (with
0.2% NaN.sub.3+0.2% BSA) and centrifuged at 1,000 RPM for 5
minutes. The supernatant was decanted and the pellet was
resuspended in a 1:100 dilution of goat anti-human FITC (obtained
from Boehringer Mannheim). The samples were incubated at 4.degree.
C. for 30 minutes, washed twice with PBS-CMF (with 0.2%
NaN.sub.3+0.2% BSA) and analyzed on a Facscan flow cytometer. A
gene product containing the ELDKWA epitope was expressed at low
levels on the surface of vCP1307-infected cells, but was not
expressed on the surface of ALVAC-infected or vP1286-infected cells
(FIG. 27, lower panel). A gene product reactive with the
HIV1-seropositive sera was expressed on the surface of
vP1286-infected cells and vCP1307-infected cells, but was not
expressed on the surface of ALVAC-infected cells (FIG. 27, upper
panel). These results indicate that the ELDKWA epitope of the HIV1
gp120+TM (with ELDKWA epitopes) gene product is expressed on the
surface of vCP1307-infected cells, consistent with the fact that a
portion of the V3 loop of this gene product has been replaced with
ELDKWA epitopes.
[0361] Immunoprecipitation analysis was performed to determine
whether vCP1307 expresses a form of gp120+TM which contains an
immunogenic ELDKWA epitope. HeLa cell monolayers were infected at
an m.o.i. of 10 pfu/cell with ALVAC (the parental virus), vP1286 (a
WR recombinant expressing HIV1 gp120+TM) or vCP1307. Following an
hour adsorption period, the inoculum was removed and the cells were
overlayed with 2 mls of modified Eagle's medium (minus cysteine and
methionine) containing 2% dialyzed fetal bovine serum and
[.sup.35S]-TRANS label (30 .mu.Ci/ml). The lysates were harvested
at 18 hrs post-infection by addition of 1 ml 3.times. buffer A (450
mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide
and 0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA
epitope, using a 1:100 dilution of a human monoclonal antibody
specific for the ELDKWA epitope, IAM41-2F5 (obtained from Viral
Testing Systems Corp., Houston, Tex.) and 2) HIV1 gene products,
using a 1:100 dilution of sera from HIV1-seropositive humans
(obtained from the New York State Dept. of Health). Lysates,
precleared with normal human sera and a protein A-sepharose
complex, were incubated overnight at 4.degree. C. with an
IAM41-2F5-protein A-sepharose complex or an HIV1-seropositive
sera-protein A-sepharose complex. The samples were washed 4.times.
with 1.times. buffer A and 2.times. with a LiCl.sub.2/urea buffer.
Precipitated proteins were dissociated from the immune complexes by
the addition of 2.times. Laemmli's buffer (125 mM Tris (pH=6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min.
Proteins were fractionated on an SDS-polyacrylamide gel, fixed and
treated with 1 M Na-salicylate for fluorography. Proteins of the
appropriate size were precipitated with the monoclonal antibody to
the ELDKWA epitope from vCP1307-infected cells, but were not
precipitated from ALVAC-infected cells or vP1286-infected cells.
Furthermore, proteins of the appropriate size were precipitated
with the human HIV1-seropositive sera from vP1286-infected cells
and vCP1307-infected cells, but were not precipitated from
ALVAC-infected cells. These results indicate that vCP1307 expresses
a form of gp120+TM which contains an antigenic ELDKWA epitope.
Example 20
Generation of vP1313; a NYVAC Recombinant Expressing a Form of HIV1
gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3 Loop
[0362] vP1313, a NYVAC recombinant expressing HIV1 gp120+TM with 2
ELDKWA epitopes from HIV1 gp41 inserted into the gp120 V3 loop, was
generated by the following procedure. The sequence encoding part of
the ELDKWA epitopes and V3 loop was cloned into pBSK+. This was
accomplished by cloning a 225 bp EcoRI-SacI-digested PCR fragment,
containing part of the ELDKWA-V3 loop sequence, into the 2,900 bp
EcoRI-SacI fragment of pBSK+. (This PCR fragment was generated from
the plasmid, pBSHIVMN120T, with the primers, HIVP72 (SEQ ID NO:
144; 5'-TTATTACCATTCCAAGTACTATT-3') and HIVP74 (SEQ ID NO: 145;
5'-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGACC
AGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3').) The plasmid
generated by this manipulation is called pHIV55.
[0363] pBSHIVMN120T, a plasmid containing the H6-promoted HIV1
gp120+TM gene, was generated by the following procedure. A plasmid,
pMN1.8-9, containing a cDNA copy of the HIV1 (MN) env gene, was
obtained from Marvin Reitz (NCI, NIH). An early transcription
termination signal sequence, T.sub.5NT, in the env gene was
modified. This was accomplished by cloning a 1,100 bp
KpnI-EcoRI-digested PCR fragment, containing the T.sub.5NT-modified
5'-end of the env gene, into the 2,900 bp KpnI-EcoRI fragment of
pBSK+. (This PCR fragment was generated from the plasmid, pMN1.8-9,
with the oligonucleotides, HIVMN6 (SEQ ID NO: 146;
5'-GGGTTATTAATGATCTGTAG-3') and HIV3B2 (SEQ ID NO: 124;
5'-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3').) The plasmid
generated by this manipulation is called pBSMIDMN.
[0364] The T.sub.5NT-modified 5%-end of the env gene was then
cloned upstream to the rest of the env gene. This was accomplished
by cloning the 1,025 bp KpnI-EcoRI fragment of pBSMIDMN, containing
the T.sub.5NT-modified 5'-end of the env gene, into the 4,300 bp
KpnI-EcoRI fragment of pBS3MN. (pBS3MN was generated by cloning a
430 bp EcoRI-SacI-digested PCR fragment, containing a central
portion of the env gene, and a 1,050 bp SacI-XbaI-digested PCR
fragment, containing the 3'-end of the env gene, into the 2,900 bp
EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragment was generated
from the plasmid, pMN1.8-9, with the oligonucleotides, HIV3B1 (SEQ
ID NO: 125; 5'-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3') and HIVMN4
(SEQ ID NO: 126; 5'-ATCATCGAGCTCCTATCGCTGCTC-3'). The 1,050 bp PCR
fragment was generated from the plasmid, pMN1.8-9, with the
oligonucleotides, HIVMN5 (SEQ ID NO: 127;
5'-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3') and HIVMN3P (SEQ ID NO: 128;
5'-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3').) The plasmid
generated by this manipulation is called pBSMID3MN.
[0365] The H6 promoter was then cloned upstream to the env gene.
This was accomplished by cloning the 320 bp KpnI fragment of
pH6IIIBE, containing the H6 promoter linked to the 5'-end of the
HIV1 (IIIB) env gene, and the 2,450 bp KpnI-XbaI fragment of
pBSMID3MN, containing the bulk of the HIV1 (MN) env gene, into the
2,900 bp KpnI-XbaI fragment of pBSK+. The plasmid generated by this
manipulation is called pH6HMNE.
[0366] The sequence encoding gp41 was then replaced with the
sequence encoding the HIV1 env transmembrane (TM) region. This was
accomplished by cloning a 480 bp EcoRI-XbaI-digested PCR fragment,
containing the 3'-end of the gp120 gene and the HIV1 env
transmembrane region, into the 4,200 bp EcoRI-XbaI fragment of
pH6HMNE. (This PCR fragment was generated from the PCR fragment,
PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO: 129;
5'-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGT
AGTGAATAGAGTTAGGCAGGGATAA-3') and HIVTM2 (SEQ ID NO: 130;
5'-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCA
AGCCTCCTACTATCATTATGAATAA-3'), with the oligonucleotides, HIV3B1
(SEQ ID NO: 125) and HIVTM3 (SEQ ID NO: 131;
5'-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3'). PCR-MN11 was
generated from the plasmid, pH6HMNE, with the oligonucleotides,
HIV3B1 (SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;
5'-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3').) The plasmid
generated by this manipulation is called pBSHIVMN120T.]
[0367] Another part of the sequence encoding the ELDKWA epitopes
and V3 loop was then cloned into pBSK+. This was accomplished by
cloning a 300 bp HindIII-SacI-digested PCR fragment, containing
part of the ELDKWA-V3 loop sequence, into the 2,900 bp HindIII-SacI
fragment of pBSK+. (This PCR fragment was generated from the
plasmid, pBSHIVMN120T, with the primers, HIVP69 (SEQ ID NO: 133;
5'-TGATAGTACCAGCTATAGGTTGAT-3') and HIVP75 (SEQ ID NO: 134;
5'-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3').) The plasmid generated by
this manipulation is called pHIV56.
[0368] The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were
then cloned into the H6-promoted gp120+TM gene. This was
accomplished by cloning the 225 bp EcoRI-SacI fragment of pHIV55
and the 300 bp HindIII-SacI fragment of pHIV56, containing the
ELDKWA-V3 loop sequences, into the 4,300 bp EcoRI-HindIII fragment
of pBSHIVMN120T. The plasmid generated by this manipulation is
called pHIV57.
[0369] The H6-promoted gp120+TM construct containing the ELDKWA
epitopes was then cloned between C5 flanking arms. This was
accomplished by cloning the 1,700 bp NruI-XbaI fragment of pHIV57,
containing the H6-promoted gp120+TM (with ELDKWA epitopes) gene,
into the 4,700 bp NruI-XbaI fragment of pSIVGC15. (pSIVGC15
contains the H6-promoted SIV env gene cloned between C5 flanking
arms) The plasmid generated by this manipulation is called
pHIV59.
[0370] The H6-promoted gp120+TM (with ELDKWA epitopes) gene was
then cloned between I4L flanking arms. This was accomplished by
cloning the 1,850 bp BamHI-SmaI fragment of pHIV59, containing the
H6-promoted gp120+TM (with ELDKWA epitopes) gene, into the 3,600 bp
BamHI-SmaI fragment of pSD550VC. The plasmid generated by this
manipulation is called pHIV60. The DNA sequence of the H6-promoted
gp120+TM (with ELDKWA epitopes) gene in pHIV60 is shown in FIG.
28.
[0371] pHIV60 was used in in vitro recombination experiments with
NYVAC as the rescuing virus to yield vP1313.
[0372] FACS analysis was performed to determine whether HIV1
gp120+TM (with ELDKWA epitopes) was expressed on the surface of
vP1313-infected cells. 5.times.10.sup.6 HeLa-S3 cells in S-MEM
(Sigma M-8028 Joklik suspension media) were infected at an m.o.i.
of 5 pfu/cell with NYVAC, vP1286 (a WR recombinant expressing HIV1
gp120+TM) or vP1313. Following a 60 minute adsorption period at
37.degree. C., the cells were washed with 10 mls of S-MEM and
centrifuged at 1,000 RPM for 5 minutes. The samples were then
resuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes
and placed on a rotator at 37.degree. C. After 18 hours, 200 ul
aliquots (1.times.10.sup.6 cells) were placed in polypropylene
tubes and washed with 3 mls of PBS-CMF (with 0.2% NaN.sub.3+0.2%
BSA). The supernatant was then decanted and the pellet was
resuspended in 100 ul of a 1:100 dilution of sera from
HIV1-seropositive humans (obtained from the New York State Dept. of
Health), or a 1:100 dilution of a human monoclonal antibody
specific for the ELDKWA epitope, IAM41-2F5 (obtained from Viral
Testing Systems Corp. Houston, Tex.). The samples were incubated at
4.degree. C. for 60 minutes, washed two times with PBS-CMF (with
0.2% NaN.sub.3+0.2% BSA) and centrifuged at 1,000 RPM for 5
minutes. The supernatant was decanted and the pellet was
resuspended in a 1:100 dilution of goat anti-human FITC (obtained
from Boehringer Mannheim). The samples were incubated at 4.degree.
C. for 30 minutes, washed twice with PBS-CMF (with 0.2%
NaN.sub.3+0.2% BSA) and analyzed on a Facscan flow cytometer. A
gene product containing the ELDKWA epitope was expressed on the
surface of vP1313-infected cells, but was not expressed on the
surface of NYVAC-infected or vP1286-infected cells (FIG. 29, lower
panel). A gene product reactive with the HIV1-seropositive sera was
expressed on the surface of vP1286-infected cells and
vP1313-infected cells, but was not expressed on the surface of
NYVAC-infected cells (FIG. 29, upper panel). These results indicate
that the ELDKWA epitope of the HIV1 gp120+TM (with ELDKWA epitopes)
gene product is expressed on the surface of vP1313-infected cells,
consistent with the fact that a portion of the V3 loop of this gene
product has been replaced with ELDKWA epitopes.
[0373] Immunoprecipitation analysis was performed to determine:
whether vP1313 expresses a form of gp120+TM which contains an
immunogenic ELDKWA epitope. HeLa cell monolayers were infected at
an m.o.i. of 10 pfu/cell with NYVAC (the parental virus), vP1286 (a
WR recombinant expressing HIV1 gp120+TM) or vP1313. Following an
hour adsorption period, the inoculum was removed and the cells were
overlayed with 2 mls of modified Eagle's medium (minus cysteine and
methionine) containing 2% dialyzed fetal bovine serum and
[.sup.35S]-TRANS label (30 .mu.Ci/ml). The lysates were harvested
at 18 hrs post-infection by addition of 1 ml 3.times. buffer A (450
mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide
and 0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA
epitope, using a 1:100 dilution of a human monoclonal antibody
specific for the ELDKWA epitope, IAM41-2F5 (obtained from Viral
Testing Systems Corp., Houston, Tex.) and 2) HIV1 gene products,
using a 1:100 dilution of sera from HIV1-seropositive humans
(obtained from the New York State Dept. of Health). Lysates,
precleared with normal human sera and a protein A-sepharose
complex, were incubated overnight at 4.degree. C. with an
IAM41-2F5-protein A-sepharose complex or an HIV1-seropositive
sera-protein A-sepharose complex. The samples were washed 4.times.
with 1.times. buffer A and 2.times. with a LiCl.sub.2/urea buffer.
Precipitated proteins were dissociated from the immune complexes by
the addition of 2.times. Laemmli's buffer (125 mM Tris (pH=6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min.
Proteins were fractionated on an SDS-polyacrylamide gel, fixed and
treated with 1 M Na-salicylate for fluorography. Proteins of the
appropriate size were precipitated with the monoclonal antibody to
the ELDKWA epitope from vP1313-infected cells, but were not
precipitated from NYVAC-infected cells or vP1286-infected cells.
Furthermore, proteins of the appropriate size were precipitated
with the human HIV1-seropositive sera from vP1286-infected cells
and vP1313-infected cells, but were not precipitated from
NYVAC-infected cells. These results indicate that vP1313 expresses
a form of gp120+TM which contains an immunogenic ELDKWA
epitope.
Example 21
Generation of vP1319; a COPAK Recombinant Expressing a Form of HIV1
gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3 Loop
[0374] vP1319, a COPAK recombinant expressing HIV1 gp120+TM with 2
ELDKWA epitopes from HIV1 gp41 inserted into the gp120 V3 loop, was
generated by the following procedure. The sequence encoding part of
the ELDKWA epitopes and V3 loop was cloned into pBSK+. This was
accomplished by cloning a 225 bp EcoRI-SacI-digested PCR fragment,
containing part of the ELDKWA-V3 loop sequence, into the 2,900 bp
EcoRI-SacI fragment of pBSK+. (This PCR fragment was generated from
the plasmid, pBSHIVMN120T, with the primers, HIVP72 (SEQ ID NO:
121; 5'-TTATTACCATTCCAAGTACTATT-3') and HIVP74 (SEQ ID NO: 122;
5'-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGACC
AGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3').) The plasmid
generated by this manipulation is called pHIV55. pBSHIVMN120T, a
plasmid containing the H6-promoted HIV1 gp120+TM gene, was
generated by the following procedure. A plasmid, pMN1.8-9,
containing a cDNA copy of the HIV1 (MN) env gene, was obtained from
Marvin Reitz (NCI, NIH). An early transcription termination signal
sequence, T.sub.5NT, in the env gene was modified. This was
accomplished by cloning a 1,100 bp KpnI-EcoRI-digested PCR
fragment, containing the T.sub.5NT-modified 5'-end of the env gene,
into the 2,900 bp KpnI-EcoRI fragment of pBSK+. (This PCR fragment
was generated from the plasmid, pMN1.8-9, with the
oligonucleotides, HIVMN6 (SEQ ID NO: 123;
5'-GGGTTATTAATGATCTGTAG-3') and HIV3B2 (SEQ ID NO: 124;
5'-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3').) The plasmid
generated by this manipulation is called pBSMIDMN.
[0375] The T.sub.5NT-modified 5'-end of the env gene was then
cloned upstream to the rest of the env gene. This was accomplished
by cloning the 1,025 bp KpnI-EcoRI fragment of pBSMIDMN, containing
the T.sub.5NT-modified 5'-end of the env gene, into the 4,300 bp
KpnI-EcoRI fragment of pBS3MN. (pBS3MN was generated by cloning a
430 bp EcoRI-SacI-digested PCR fragment, containing a central
portion of the env gene, and a 1,050 bp SacI-XbaI-digested PCR
fragment, containing the 3'-end of the env gene, into the 2,900 bp
EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragment was generated
from the plasmid, pMN1.8-9, with the oligonucleotides, HIV3B1 (SEQ
ID NO: 125; 5'-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3') and HIVMN4
(SEQ ID NO: 126; 5'-ATCATCGAGCTCCTATCGCTGCTC-3'). The 1,050 bp PCR
fragment was generated from the plasmid, pMN1.8-9, with the
oligonucleotides, HIVMN5 (SEQ ID NO: 127;
5'-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3') and HIVMN3P (SEQ ID NO: 128;
5'-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3').) The plasmid
generated by this manipulation is called pBSMID3MN.
[0376] The H6 promoter was then cloned upstream to the env gene.
This was accomplished by cloning the 320 bp KpnI fragment of
pH6IIIBE, containing the H6 promoter linked to the 5'-end of the
HIV1 (IIIB) env gene, and the 2,450 bp KpnI-XbaI fragment of
pBSMID3MN, containing the bulk of the HIV1 (MN) env gene, into the
2,900 bp KpnI-XbaI fragment of pBSK+. The plasmid generated by this
manipulation is called pH6HMNE.
[0377] The sequence encoding gp41 was then replaced with the
sequence encoding the HIV1 env transmembrane (TM) region. This was
accomplished by cloning a 480 bp EcoRI-XbaI-digested PCR fragment,
containing the 3'-end of the gp120 gene and the HIV1 env
transmembrane region, into the 4,200 bp EcoRI-XbaI fragment of
pH6HMNE. (This PCR fragment was generated from the PCR fragment,
PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO: 129;
5'-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGT
AGTGAATAGAGTTAGGCAGGGATAA-3') and HIVTM2 (SEQ ID NO: 130;
5'-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCA
AGCCTCCTACTATCATTATGAATAA-3'), with the oligonucleotides, HIV3B1
(SEQ ID NO: 125) and HIVTM3 (SEQ ID NO: 131;
5'-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3'). PCR-MN11 was
generated from the plasmid, pH6HMNE, with the oligonucleotides,
HIV3B1 (SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;
5'-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3').) The plasmid
generated by this manipulation is called pBSHIVMN120T.
[0378] Another part of the sequence encoding the ELDKWA epitopes
and V3 loop was then cloned into pBSK+. This was accomplished by
cloning a 300 bp HindIII-SacI-digested PCR fragment, containing
part of the ELDKWA-V3 loop sequence, into the 2,900 bp HindIII-SacI
fragment of pBSK+. (This PCR fragment was generated from the
plasmid, pBSHIVMN120T, with the primers, HIVP69 (SEQ ID NO: 133;
5'-TGATAGTACCAGCTATAGGTTGAT-3') and HIVP75 (SEQ ID NO: 134;
5'-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3').) The plasmid generated by
this manipulation is called pHIV56.
[0379] The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were
then cloned into the H6-promoted gp120+TM gene. This was
accomplished by cloning the 225 bp EcoRI-SacI fragment of pHIV55
and the 300 bp HindIII-SacI fragment of pHIV56, containing the
ELDKWA-V3 loop sequences, into the 4,300 bp EcoRI-HindIII fragment
of pBSHIVMN120T. The plasmid generated by this manipulation is
called pHIV57.
[0380] The H6-promoted gp120+TM construct containing the ELDKWA
epitopes was then cloned between C5 flanking arms. This was
accomplished by cloning the 1,700 bp NruI-XbaI fragment of pHIV57,
containing the H6-promoted gp120+TM (with ELDKWA epitopes) gene,
into the 4,700 bp NruI-XbaI fragment of pSIVGC15. (pSIVGC15
contains the H6-promoted SIV env gene cloned between C5 flanking
arms.) The plasmid generated by this manipulation is called
pHIV59.
[0381] The H6-promoted gp120+TM (with ELDKWA epitopes) gene was
then cloned between COPAK flanking arms. This was accomplished by
cloning the 1,850 bp BamHI-SmaI fragment of pHIV59, containing the
H6-promoted gp120+TM (with ELDKWA epitopes) gene, into the 4,600 bp
BamHI-SmaI fragment of pSD553VC. The plasmid generated by this
manipulation is called pHIV61. The DNA sequence of the H6-promoted
gp120+TM (with ELDKWA epitopes) gene in pHIV61 is shown in FIG.
30.
[0382] pHIV61 was used in in vitro recombination experiments with
COPAK as the rescuing virus to yield vP1319.
[0383] FACS analysis was performed to determine whether HIV1
gp120+TM (with ELDKWA epitopes) was expressed on the surface of
vP1319-infected cells. 5.times.10.sup.6 HeLa-S3 cells in S-MEM
(Sigma M-8028 Joklik suspension media) were infected at an m.o.i.
of 5 pfu/cell with WR, vP1286 (a WR recombinant expressing HIV1
gp120+TM) or vP1319. Following a 60 minute adsorption period at
37.degree. C., the cells were washed with 10 mls of S-MEM and
centrifuged at 1,000 RPM for 5 minutes. The samples were then
resuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes
and placed on a rotator at 37.degree. C. After 18 hours, 200 ul
aliquots (1.times.10.sup.6 cells) were placed in polypropylene
tubes and washed with 3 mls of PBS-CMF (with 0.2% NaN.sub.3+0.2%
BSA). The supernatant was then decanted and the pellet was
resuspended in 100 ul of a 1:100 dilution of sera from
HIV1-seropositive humans (obtained from the New York State Dept. of
Health), a 1:500 dilution of a mouse monoclonal antibody specific
for the HIV1 (MN) V3 loop, 50.1 (obtained from M. Robert-Guroff,
NCI, NIH.) or a 1:100 dilution of a human monoclonal antibody
specific for the ELDKWA epitope, IAM41-2F5 (obtained from Viral
Testing Systems Corp., Houston, Tex.). The samples were incubated
at 4.degree. C. for 60 minutes, washed two times with PBS-CMF (with
0.2% NaN.sub.3+0.2% BSA) and centrifuged at 1,000 RPM for 5
minutes. The supernatant was decanted and the pellet was
resuspended in a 1:100 dilution of goat anti-human FITC or goat
anti-mouse FITC (obtained from Boehringer Mannheim). The samples
were incubated at 4.degree. C. for 30 minutes, washed twice with
PBS-CMF (with 0.2% NaN.sub.3+0.2% BSA) and analyzed on a Facscan
flow cytometer. A gene product containing the ELDKWA epitope was
expressed on the surface of vP1319-infected cells, but was not
expressed on the surface of WR-infected cells or vP1286-infected
cells (FIG. 31, middle panel). A gene product containing the V3
loop was expressed on the surface of vP1286-infected cells, but was
not expressed on the surface of WR-infected cells or
vP1319-infected cells (FIG. 31, lower panel). A gene product
reactive with the HIV1-seropositive sera was expressed on the
surface of vP1286-infected cells and vP1319-infected cells, but was
not expressed on the surface of WR-infected cells (FIG. 31, upper
panel). This analysis indicated that 1) the ELDKWA epitope of the
HIV1 gp120+TM (with ELDKWA epitopes) gene product is expressed on
the surface of vP1319-infected cells and 2) the gene product
expressed on the surface of vP1319-infected cells does not contain
a wild-type V3 loop, consistent with the fact that the V3 loop of
this gene product has been replaced with ELDKWA epitopes.
[0384] Immunoprecipitation analysis was performed to determine
whether vP1319 expresses a form of gp120+TM which contains an
immunogenic ELDKWA epitope. HeLa cell monolayers were infected at
an m.o.i. of 10 pfu/cell with NYVAC (the parental virus), vP1286 (a
WR recombinant expressing HIV1 gp120+TM) or vP1319. Following an
hour adsorption period, the inoculum was removed and the cells were
overlayed with 2 mls of modified Eagle's medium (minus cysteine and
methionine) containing 2% dialyzed fetal bovine serum and
[.sup.35S]-TRANS label (30 .mu.Ci/ml). The lysates were harvested
at 18 hrs post-infection by addition of 1 ml 3.times. buffer A (450
mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide
and 0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA
epitope, using a 1:100 dilution of a human monoclonal antibody
specific for the ELDKWA epitope, IAM41-2F5 (obtained from Viral
Testing Systems Corp., Houston, Tex.) and 2) HIV1 gene products,
using a 1:100 dilution of sera from HIV1-seropositive humans
(obtained from the New York State Dept. of Health). Lysates,
precleared with normal human sera and a protein A-sepharose
complex, were incubated overnight at 4.degree. C. with an
IAM41-2F5-protein A-sepharose complex or an HIV1-seropositive
sera-protein A-sepharose complex. The samples were washed 4.times.
with 1.times. buffer A and 2.times. with a LiCl.sub.2/urea buffer.
Precipitated proteins were dissociated from the immune complexes by
the addition of 2.times. Laemmli's buffer (125 mM Tris (pH=6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min.
Proteins were fractionated on an SDS-polyacrylamide gel, fixed and
treated with 1 M Na-salicylate for fluorography. Proteins of the
appropriate size were precipitated with the monoclonal antibody to
the ELDKWA epitope from vP1319-infected cells, but were not
precipitated from NYVAC-infected cells or vP1286-infected cells.
Furthermore, proteins of the appropriate size were precipitated
with the human HIV1-seropositive sera from vP1286-infected cells
and vP1319-infected cells, but were not precipitated from
NYVAC-infected cells. These results indicate that vP1319 expresses
a form of gp120+TM which contains an immunogenic ELDKWA
epitope.
[0385] Since vCP1307, vP1313 and vP1319 each express the ELDKWA
epitope in an immunogenic configuration, these recombinants have
numerous utilities, as do the expression products, antibodies
elicited thereby, and DNA from these recombinants, as discussed
above.
Example 22
Inocuity and the Immunogenicity of vCP205 in Macaques Inoculated by
Intramuscular Route
Experimental Animals:
[0386] Species: Macaca fascicularis (adult, wild caught)
Number: 8
Sex: Males
[0387] Origin: Mauritius Island considered free of Herpes B,
Filovirus and Tuberculosis. Previous history: polio experiments.
Diet and drinking water: Commercial diet and fruits; tap water ad
libitum.
[0388] Four male Cynomolgus macaques were inoculated five times, at
one month intervals, with one dose of ALVAC-HIV (vCP205)
(containing 10.sup.5.8 TCID.sub.50/dose) by intramuscular route.
The injections caused neither symptoms nor lesions. Body weight of
monkeys was not altered by the injections. Four control animals
were injected with placebo. (Mixture of DMEM-F12 medium (25%),
Lactoglutamate (25%) and freeze-drying substrate (50%)). General
condition was monitored daily and injection sites were observed on
days 1, 2, 3, 4 and 7 after each inoculation. Body weights were
recorded once a week.
[0389] Blood samples were collected in EDTA blood collection
Vacutainer.RTM. tubes (Becton-Dickinson, Meyland, France) for
hematological analyses, in lithium-heparin microvette CB 100 tubes
(Sarstedt, Numbrecht, Germany) for biochemical analysis and in
tubes with serum separator SST Vacutainer.RTM. tubes
(Becton-Dickinson, Meylan, France) for serological analyses
(MON:TSA.034.00). Samplings were done on days 0, 3, 7, 14, 28, 31,
35, 42, 56, 59, 63, 70, 84, 87, 91, 98, 112, 115, 119, 126 and 140.
Anesthesia (Ketamine, 20 mg/kg, intramuscular) was used for
clinical examinations and blood sampling.
[0390] Hematological and biochemical analysis were carried out with
the VET TEST 8008 Apparatus. Anti-HIV gp160, p24 proteins and V3
peptide antibodies were titered according to an ELISA technique.
Maxisorp F96 NUNC plates wells were coated for 1 hour at 37.degree.
C., with one of the following antigens diluted in 0.1 M carbonate
buffer, pH 9.6: [0391] 130 ng per well of purified gp160 MN/BRU
(from recombinant vaccinia containing 30% of cleaved gp160 and no
alpha.sub.2 macroglobulin), [0392] 200 ng of V3MN peptide, [0393]
130 ng of purified "p24"HIV (E. coli, P25 LAI isolate, batch 672Cl,
Transgene).
[0394] All incubations were carried out in a final volume of 100
.mu.l, followed by 3 washings performed with phosphate buffered
saline, pH 7.1-0.1% Tween 20. Plates were blocked for 1 hour at
37.degree. C. with 150 .mu.l of phosphate buffered saline pH
7.1-0.05% Tween 20-5% (W/V) powdered skim milk (Gloria).
[0395] Serial threefold dilutions of the sera, ranging from 1/50 to
1/12150, in phosphate buffered saline--0.05% Tween 20-5% (W/V)
powdered skim milk, were added to the wells and incubated for 90
min at 37.degree. C.
[0396] After washing, anti-monkey IgG peroxidase conjugate (Cappel,
goat IgG fraction), diluted 1/3000 in phosphate buffered
saline--0.5% Tween 20-5% powdered skim milk, was added and the
plates incubated for another 90 min at 37.degree. C. The plates,
washed four times, were incubated in the dark for 30 min at room
temperature with the substrate O-phenylenediamine dihydrochloride
(Sigma tablets). The substrate was used at the concentration of 1.5
mg/ml in 0.05 M phosphate citrate buffer, pH 5.0 containing 0.03
containing 0.03% sodium perborate (Sigma capsules).
[0397] The reactions were stopped with 50 .mu.l of 4N
H.sub.2SO.sub.4. The optical density was measured at 490-650 nm
with an automatic plate reader (Vmax, Molecular Devices). The
antibody titers were calculated according to the formula:
Titer = log OD 490 - 650 .times. 10 1 / dilution ( OD value range :
0.2 to 1 ) ##EQU00001##
[0398] Neutralizing antibody assay determines the dilution of serum
that prevents the development of syncytia in 50% of microwells
infected with 10 TCID.sub.50 of HIV MN. The MN strain was obtained
from F. Barre-Sinoussi and propagated in CEM clone 166 cells.
[0399] Sera were decomplemented and twofold serial dilutions in
RPMI beginning 1/10 were prepared. Equal volumes of serum dilution
and HIV suspension (500 ul each) were mixed and incubated for 2 h
at 37.degree. C. The HIV suspension had been adjusted to contain
10.sup.2 to 10.sup.2.5 TCID.sub.50 per ml.
[0400] Prior to use, indicator CEMss cells were plated in
microwells coated with poly-L-lysine, and incubated for 1 h at
37.degree. C. Culture medium was removed and replaced with the
virus/serum mixtures (100 .mu.l/well, 6 wells per dilution). After
1 h incubation at 37.degree. C. culture medium was added to each
well and the plates were incubated at 37.degree. C. All incubations
were in a CO.sub.2 5% incubator.
[0401] Seven and 14 days later, the cultures were examined under
the microscope and wells showing syncytia were recorded.
Neutralizing 50% titer was computed according to SPEARMAN and
KARBER and expressed as the log.sub.10 of the end-point. As a
confirmation, supernatants of the cultures were collected on day
seven, pooled for each dilution and assayed for RT activity. Each
assay includes an infectivity titration of the virus suspension,
titration of antibody in a reference serum and a set of uninfected
microwells as negative controls.
[0402] Results:
[0403] A majority of the parameters selected to monitor
hematological and biochemical status varied within normal limits,
including: erythrocyte number, mean corpuscle volume, hematocrite,
creatinin and alanine aminotransferase.
[0404] Some variations were seen, which cannot be attributed to the
repeated immunizations with ALVAC-HIV (vCP205): i) unexplained
lymphocytosis on weeks 8 and 9 in both the test and the control
groups; ii) a decreased level of hemoglobin in both groups on weeks
17 and 20 and in control groups; ii) a decreased level of
hemoglobin in both groups on weeks 17 and 20 and in controls on
week 9; iii) erratic high values of the aspartate aminotransferase
in two control monkeys and last; iv) irregular trombocytes counts
caused by microcoagulation of some specimens.
[0405] The anti-HIV immune response induced by vCP205 was assessed
by ELISA tests using purified gp160 MN/BRU (from recombinant
vaccinia), V3MN synthetic peptide and p24 (from E. coli, LAI
isolate). All the animals developed antibodies against gp160 and
V3, and 2/4 against p24. The positive anti-HIV immune response
usually appeared after 2, or maximum, 3 injections. Subsequent
vCP205 inoculations mainly maintained (sometimes slightly
increased) the antibody levels and improved homogeneity of the
response between macaques. Highest antibody titers were usually
observed two weeks after each inoculation, followed by a decrease
until the next booster. Neutralizing antibodies against HIV/MN were
detectable in all immunized monkeys.
[0406] Clinical observation: neither erythema nor edema were
reported at the site of inoculation. Body weights were stable in
control monkeys and in ALVAC-HIV (vCP205) recipients (FIG. 32).
[0407] Hematological analyses: Leucocyte counts were greatly
modified in both control and tested animals on weeks 8 and 9 with
formula inversion (FIG. 33a). This fact, noted in both groups, is
without correlation with the viral injections.
[0408] Erythrocyte number, corpuscle mean volume and hematocrite
varied within normal limits but hemoglobin showed some discrepancy
on weeks 9, 17 and 20 in controls and on weeks 17 and 20 in animals
inoculated with ALVAC-HIV (vCP205) (FIGS. 33b and c). Thrombocytes
values varied depending on the sampling quality (microcoagulation)
(FIG. 33c).
[0409] Biochemical analyses: Creatinin and ALAT (SGPTransaminase)
did not vary significantly after the reiterated inoculations FIGS.
34 and 36). The AST (SGOTransaminase) values presented variations
particularly important in control monkeys #3 and 2 respectively on
week 5 and 8-9 (FIG. 35).
[0410] Serological analyses (considering the ELISA titers of the
negative control group of macaques sera, the negative detection
threshold of the serological response was considered to be, in
log.sub.10: 1.56.+-.0.24, 1.92.+-.0.12 and 2.18.+-.0.34, for gp160,
V3 and p24 respectively): gp160 and V3 specific response (FIGS.
37a-37b, 38a-38b): the kinetics of antibody of gp160 was similar to
that to V3. The magnitude of the latter was slightly weaker (mean
titer at week 20 of 4.37 versus 8.84).
[0411] Two injections were necessary to induce a detectable immune
response. Three monkeys showed a maximum response after the second
injection; the fourth one required three (gp160) or four (V3)
injections to do so. During the four week interval between
injections, antibody titers consistently increased then faded, to
be boosted by the next inoculation. While there were significant
individual differences in the responses to the initial injections,
the responses leveled out as the experiment progressed.
[0412] p24 a specific response (FIGS. 37c, 38c): a response was
observed for 2 macaques (macaques 5 and 7) out of 4 after
respectively 2 and 3 injections of vCP205. This is in contrast with
a guinea-pig test in which no anti-p24 antibodies were detected in
any inoculated animal groups. As with anti-gp160 and anti-V3
antibodies, titers fluctuated up and down between injections and
individual differences progressively disappeared. Unlike that of
gp160 and V3, the anti-p24 antibody profile did not reach a
plateau.
[0413] Neutralizing antibody: as shown in FIG. 39, all four monkeys
with vCP205 developed detectable levels of neutralizing antibody
against HIV (MN). One of them tested positive after one injection,
two after the second injection and the last one required five
administrations. It is noteworthy that the latter animal also
showed the slowest kinetics of ELISA antibody. The levels of
neutralizing activity are relatively modest (1/10 to 1/30 in
arithmetical expression) and, likewise ELISA measurements, went up
and down in the intervals between injections.
[0414] Effect of a late boost with the proteins gp160 and p24: The
four monkeys were boosted 7 weeks post the last injection of vCP205
with 200 .mu.g of gp160 and 200 .mu.g of p24 proteins in incomplete
Freund's adjuvant (Montanide ISA51) to raise hyperimmune reference
sera; this caused a pronounced increase (at least 10-fold) in
antibody titers, suggesting that the plateau seen in the ELISA
analysis were not the limit of response.
[0415] The immunization regimen induced high levels of binding
antibody to gp160 MN/BRU and V3 MN peptide, and low but definite
neutralizing antibody. Serological results showed higher antibody
levels than those observed in macaques inoculated with ALVAC-HIV
(vCP125) and one booster (instead of two) was sufficient to obtain
a good anamnestic type response. This Example shows that vCP205 and
expression products thereof, antibodies therefrom, and DNA from
vCP205, can be used as described above.
Example 23
Inocuity and Immunogenicity of vCP300 In Macaques
Experimental Animals:
[0416] Species: Macaca fascicularis
Number: 8
Sex: Males
[0417] Origin: Mauritius Island considered without Herpes B,
Filovirus and tuberculosis.
[0418] Four male Cynomolgus macaques were immunized five times, at
one month intervals, with one dose of ALVAC-HIV (vCP300)
(containing 10.sup.6.16 TCID.sub.50/dose) by intramuscular route.
Four control animals were injected with placebo. As a sixth
injection, all the animals received ALVAC-HIV (vCP300) by
intravenous route. The regimen was as follows:
Group #1
Product: Placebo
[0419] Route: Intramuscular alternately in the left or right
deltoid muscle
Volume: 1 ml
[0420] Number of injections: 5 (on weeks 0, 4, 8, 12 and 16).
Group #2
Product: ALVAC-HIV
[0421] Route: Intramuscular alternately in the left or right
deltoid muscle.
Volume: 1 ml.
Dose: 10.sup.6.16 TCID.sub.50.
[0422] Number of injections: 5 (on weeks 0, 4, 8, 12 and 16).
Groups #1 and #2
[0423] On week 20 Product: ALVAC-HIV (vCP300)
Route: Intravenous
Volume: 1 ml
Dose: 106.16 TCID.sub.50.
[0424] Number of injections: 1.
[0425] Clinical observations: Injection site was observed on days
1, 2, 3, 4 and 7 following each inoculation. Animals were weighed
once a week. Samplings: Blood samples were taken under ketamin
anesthesia from the femoral vein. Blood was collected in the
following order in Vacutainer tubes (Becton Dickinson, Meylan,
France):
1) 1.8 ml in 0.129M buffered sodium citrated tube (prothrombine).
2) 1 ml in 5 mg sodium fluoride and 4 mg potassium oxalate tube
(glucose). 3) 2 ml in 0.17M EDTA K.sub.3 tube (hematological
analyses). 4) 2 to 3 ml in tubes for serum separation with inert
barrier material and clot activator (biochemical and serological
analyses).
[0426] Samplings were done on days 0, 3, 7, 14, 28, 31, 35, 42, 56,
59, 63, 70, 84, 87, 91, 98, 112, 119, 126, 140 and 143.
[0427] Dosages:
[0428] Hematological analyses included: blood counts and
differential leucocyte counts, hemoglobin, thrombocytes,
prothrombin, reticulocytes and sedimentation rate.
[0429] Biochemical analyses included: sodium, potassium, glucose,
alkalin phosphatases, cholesterol, total proteins and
electrophoresis, transaminases SGOT and SGPT.
[0430] Serological analyses: Anti-HIV gp160 glycoprotein, p24
protein, V3 peptide and nef protein antibodies were titrated
according to a modification of the ELISA technique:
[0431] Maxisorp F96 NUNC plates wells were coated for 1 hour at
37.degree. C., then overnight at 4.degree. C., with one of the
following antigens diluted in 0.1 M carbonate buffer, pH 9.6:
[0432] 130 ng per well of purified gp160 MN/BRU (from recombinant
vaccinia VVTG 5156), [0433] 200 ng of V3MN peptide, [0434] 130 ng
of purified p24 HIV (E. coli, p25 LAI isolate, batch 672Cl,
Transgene).
[0435] All incubations were carried out in a final volume of 100
.mu.l, followed by 3 washings performed with phosphate buffered
saline, pH 7.1-0.1% Tween 20. Plates were blocked for 1 hour at
37.degree. C. with 150 .mu.l of phosphate buffered saline pH
7.1-0.05% Tween 20-5% (W/V) powdered skim milk (Gloria). Serial
threefold dilutions of the sera, ranging from 1/50 to 1/12150 or
1/500 to 1/121500, in phosphate buffered saline--0.05% Tween 20-5%
(W/V) powdered skim milk, were added to the wells and incubated for
90 min. at 37.degree. C.
[0436] After washing, anti-monkey IgG peroxidase conjugate (Cappel,
goat IgG fraction), diluted 1/3000 in phosphate buffered
saline--0.05% Tween 20-5% powdered skim milk, was added and the
plates incubated for another 90 min. at 37.degree. C. The plates,
washed four times, were incubated in the dark for 30 min. at room
temperature with the substrate O-phenylenediamine dihydrochloride
(Sigma tablets), the substrate was used at the concentration of 1.5
mg/ml in 0.05 M phosphate citrate buffer, pH 5.0 containing 0.03%
sodium perborate (Sigma capsules). The reactions were stopped with
50 .mu.l of 4N H.sub.2SO.sub.4.
[0437] The optical density was measured at 490-650 nm with an
automatic plate reader (Vmax, Molecular Devices). The blanks were
subtracted and the values of the duplicates averaged. The antibody
titers were calculated for the OD value range of 0.2 to 1.2, from
the regression curve of a standard anti-gp160 and anti-p24
hyperimmune serum of guinea-pig which was present on each ELISA
plate. The titer of the standard serum had been previously
determined according to the formula:
Titer = log OD 490 - 650 .times. 10 1 / dilution ( OD value range :
0.2 to 1.2 ) ##EQU00002##
[0438] Since no standard monkey anti-nef serum was available, the
determination of anti-nef antibodies was performed by including in
the test a reference monoclonal mouse antibody (anti-nef HIV1 ALI,
MATG0020, Transgene) as an internal positive control. Anti-mouse
IgG peroxidase conjugate (Amersham) diluted 1/5000, was then used
and sera titers were calculated using the formula mentioned
above.
[0439] Results:
[0440] The injections caused neither symptoms nor lesions. Body
weight of monkeys was not altered by the injections. Hematological
parameters did not vary significantly and biochemical analyses
showed no alteration of kidneys and liver functions.
[0441] Hematological results: Variations, when present, were
similar in pattern and in range in the placebo and in the vaccinee
groups.
[0442] Individual WBC counts varied within normal limits.
Differential WBC counts often showed a diminution of lyphocytes 3
days after the blood samplings (FIG. 40). Erythrocyte counts,
hematocrite and hemoglobin transiently decreased after each blood
sampling contrary to reticulocyte counts which increased regularly
after the puncture (FIGS. 41a, 41b, 41c). Mean corpuscle volume was
stable in all the animals except an increase on day 140.
Thrombocyte counts showed some variations (FIG. 41) but prothrombin
level was stable (FIG. 42). There was no sign of anemia in either
group. Sedimentation rate was 1 mm after one hour in all the
samples.
[0443] Biochemical results: For a better interpretation of the
variations observed, a standard serum (pool of 18 macaque sera) was
analyzed at the beginning (Std a) and at the end (Std p) of each
series of samples to be tested.
[0444] Cholesterol values varied in the same way in controls and in
test group (FIGS. 43a and 43b).
[0445] Sodium and potassium were stable after all the injections
but, as for total proteins and glucose, there was a great rising on
day 140. On that day, blood specimens were drawn prior to the
intravenous injection without anesthesia (so that clinical
reactions could be monitored without interference); the change
observed in several biological parameters was likely due to the
stress associated with handling and sampling in the absence of
anesthesia (FIGS. 43a and 43b). Electrophoresis profiles (data not
shown) were very similar in all the samples. Creatinine, bilirubin,
glutamic oxaloacetic and glutamic pyruvic transaminases and alkalin
phosphatases varied within normal limits and always in the same
direction in both control and test groups (FIGS. 43c and 43d).
Kidneys and liver functions were therefore not affected by the
inoculations of ALVAV-HIV (vCP300).
[0446] Serological results: Two (macaques # 6, 7, 8) or three
(macaque #5) injections were necessary to induce detectable
anti-gp160 and anti-V3 responses (FIG. 44a, 44b, 45a, 45b). Two
weeks after the second injection, these responses were variable
between animals (anti-gp160 titers fluctuating between 2 to 4.6
logs). This response heterogeneity was smoothed out by the third
injection. The subsequent intramuscular injections mainly
maintained or increased the titers which reached around 4.3 and 3.6
for respectively anti-gp160 and anti-V3 antibodies after the fourth
to fifth injection. Detectable anti-p24 antibodies (FIGS. 46a, 46b)
were observed after three (macaques # 6, 8) to four (macaque #7) or
five (macaque 5) vCP300 intramuscular injections. The animals with
the highest anti-gp160/V3 titers exhibited the highest anti-p24
(.apprxeq.4.3 on week 18 post-primoimmunization). None of the
animals raised anti-nef antibodies (FIG. 47a, 47b).
[0447] The immune response induced by vCP300 was assessed by
analyzing in ELISA the anti-HIV-1 gp160, V3, p24 and nef sera
antibodies.
[0448] All the animals developed antibodies against gp160, V3 and
p24: significant anti-gp160 or V3 responses were obtained after 2
or at most 3 intramuscular injections. Subsequent inoculations
maintained or increased the antibody levels. Anti-p24 responses
were detected after 3 to 5 injections and each inoculation of
vCP300 increased the levels. No anti-nef antibodies could be
detected in any of the animals.
[0449] The highest antibody titers were usually observed two weeks
after each intramuscular injection, followed by a decrease until
the next boost.
[0450] These serological results are very close to those obtained
with ALVAC-HIV (vCP205) which expressed the same proteins but no
CTL epitopes. Two minor differences can be pointed out in this
example with vCP300: slightly lower anti-gp160/V3 antibody titers
(.apprxeq.0.2 to 0.5 log), and higher anti-p24 responses (all
macaques positive, higher sera titers). Because of the individual
variations between animals, these differences were not deemed
significant. No indication of hypersensitivity was seen following
intravenous inoculation. No side effects were recorded. This
regimen induced high levels of binding antibodies to gp160 (though
lower than with vCP205), V3 and p24 antigens. This Example shows
that vCP300 and expression products thereof, antibodies therefrom,
and DNA from vCP300 can be used as described above.
[0451] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited by particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope thereof.
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Sequence CWU 1
1
149120DNAArtificial SequenceSynthetic oligonucleotide referred to
as MPSYN43 1taattaacta gctacccggg 20228DNAArtificial
SequenceSynthetic oligonucleotide referred to as MPSYN44
2gtacattaat tgatcgatgg gcccttaa 28373DNAArtificial
SequenceSynthetic oligonucleotide referred to as MPSYN45
3agcttcccgg gtaagtaata cgtcaaggag aaaacgaaac gatctgtagt tagcggccgc
60ctaattaact aat 73469DNAArtificial SequenceSynthetic nucleotide
referred to as MPSYN46 4agggcccatt cattatgcag ttcctctttt gctttgctag
acatcaatcg ccggcggatt 60aattgatta 69520DNAArtificial
SequenceComplementary 20mer oligonucleotide referred to as MPSYN47
5ttagttaatt aggcggccgc 20622DNAArtificial SequenceSynthetic
oligonucleotide referred to as SD22mer 6cgattactat gaaggatccg tt
22720DNAArtificial SequenceSynthetic oligonucleotide referred to as
SD20mer 7taatgatact tcctaggcaa 20841DNAArtificial SequenceSynthetic
oligonucleotide referred to as SD42mer 8cgattactag atctgagctc
cccgggctcg agggatccgt t 41939DNAArtificial SequenceSynthetic
oligonucleotide referred to as SD40mer 9taatgatcta gactcgaggg
gcccgagctc cctaggcaa 391016DNAArtificial SequenceSynthetic
oligonucleotide referred to as HEM5 10gatccgaatt ctagct
161112DNAArtificial SequenceSynthetic oligonucleotide referred to
as HEM6 11gcttaagatc ga 121275DNAArtificial SequenceSynthetic
oligonucleotide referred to as ATI3 12tatgagtaac ttaactcttt
tgttaattaa aagtatattc aaaaaataag ttatataaat 60agatctgaat tcgtt
751373DNAArtificial SequenceSynthetic oligonucleotide referred to
as ATI4 13actcattgaa ttgagaaaac aattaatttt catataagtt ttttattcaa
tatatttatc 60tagacttaag caa 731449DNAArtificial SequenceSynthetic
oligonucleotide referred to as MPSYN177 14aaaatgggcg tggattgtta
actttatata acttattttt tgaatatac 491567DNAArtificial
SequenceSynthetic oligonucleotide referred to as MPSYN59
15acacgaatga ttttctaaag tatttggaaa gttttatagg tagttgatag aacaaaatac
60ataattt 671651DNAArtificial SequenceSynthetic oligonucleotide
referred to as MPSYN62 16tgtgcttact aaaagatttc ataaaccttt
caaaatatcc atcaactatc t 511746DNAArtificial SequenceSynthetic
oligonucleotide referred to as MPSYN60 17tgtaaaaata aatcactttt
tatactaaga tctcccgggc tgcagc 461866DNAArtificial SequenceSynthetic
oligonucleotide referred to as MPSYN61 18tgttttatgt attaaaacat
ttttatttag tgaaaaatat gattctagag ggcccgacgt 60cgccgg
661950DNAArtificial SequenceSynthetic oligonucleotide referred to
as MPSYN82 19tttctgtata tttgcaccaa tttagatctt actcaaaata tgtaacaata
502044DNAArtificial SequenceSynthetic oligonucleotide referred to
as MPSYN233 20tgtcatttaa cactatactc atattaataa aaataatatt tatt
442172DNAArtificial SequenceSynthetic oligonucleotide referred to
as 518A1 21gatcctgagt actttgtaat ataatgatat atattttcac tttatctcat
ttgagaataa 60aaagatctta gg 722272DNAArtificial SequenceSynthetic
oligonucleotide referred to as 518A2 22gactcatgaa acattatatt
actatatata aaagtgaaat agagtaaact cttatttttc 60tagaatcctt aa
722372DNAArtificial SequenceSynthetic oligonucleotide referred to
as 518B1 23gatccagatc tcccgggaaa aaaattattt aacttttcat taatagggat
ttgacgtatg 60tagcgtacta gg 722472DNAArtificial SequenceSynthetic
oligonucleotide referred to as 518B2 24gtctagaggg cccttttttt
aataaattga aaagtaatta tccctaaact gcatactacg 60catgatcctt aa
722540DNAArtificial SequenceSynthetic oligonucleotide referred to
as VQ1A 25gggagatctc tcgagctgca gggcgccgga tcctttttct
402640DNAArtificial SequenceSynthetic oligonucleotide referred to
as VQ1B 26ccctctagag agctcgacgt cccgcggcct aggaaaaaga
402759DNAArtificial SequenceOligonucleotide referred to as CE75
27cgatatccgt taagtttgta tcgtaatggg ctccagatct tctaccagga tcccggtac
592855DNAArtificial SequenceOligonucleotide referred to as CE76
28cgggatcctg gtagaagatc tggagcccat tacgatacaa acttaacgga tatcg
552917DNAArtificial SequenceOligonucleotide referred to as CE42
29aattcgagct ccccggg 173013DNAArtificial SequenceOligonucleotide
referred to as CE43 30cccggggagc tcg 133126DNAArtificial
SequenceOligonucleotide referred to as CE166 31ctttttataa
aaagttaact acgtag 263234DNAArtificial SequenceOligonucleotide
referred to as CE167 32gatcctacgt agttaacttt ttataaaaag agct
343320DNAArtificial SequenceOligonucleotide referred to as CE182
33cttaactcag ctgactatcc 203444DNAArtificial SequenceOligonucleotide
referred to as CE183 34tacgtagtta actttttata aaaatcatat ttttgtagtg
gctc 443567DNAArtificial SequenceOligonucleotide referred to as
CE162 35aattcaggat cgttccttta ctagttgaga ttctcaagga tgatgggatt
taatttttat 60aagcttg 673667DNAArtificial SequenceOligonucleotide
referred to as CE163 36aattcaagct tataaaaatt aaatcccatc atccttgaga
atctcaacta gtaaaggaac 60gatcctg 673768DNAArtificial
SequenceDouble-stranded oligonucleotide referred to as JCA017
37ctagacactt tatgtttttt aatatccggt cttaaaagct tcccggggat ccttatacgg
60ggaataat 683865DNAArtificial SequenceDouble-stranded
oligonucleotide referred to as JCA018 38attattcccc gtataaggat
cccccgggaa gcttttaaga ccggatatta aaaaacataa 60agtgt
653929DNAArtificial SequenceReplacement sequence for bases from 167
through position 455 of 880 bp canarypox PvuII fragment
39gcttcccggg aattctagct agctagttt 294046DNAArtificial
SequenceSynthetic oligonucleotide referred to as RW145 40actctcaaaa
gcttcccggg aattctagct agctagtttt tataaa 464150DNAArtificial
SequenceSynthetic oligonucleotide referred to as RW146 41gatctttata
aaaactagct agctagaatt cccgggaagc ttttgagagt 504271DNAArtificial
SequenceOligonucleotide referred to as Oligonucleotide A
42ctgaaattat ttcattatcg cgatatccgt taagtttgta tcgtaatggt tcctcaggct
60ctcctgtttg t 714348DNAArtificial SequenceOligonucleotide referred
to as Oligonucleotide B 43cattacgata caaacttaac ggatatcgcg
ataatgaaat aatttcag 484473DNAArtificial SequenceOligonucleotide
referred to as Oligonucleotide C 44accccttctg gtttttccgt tgtgttttgg
gaaattccct atttacacga tcccagacaa 60gcttagatct cag
734551DNAArtificial SequenceOligonucleotide referred to as
Oligonucleotide D 45ctgagatcta agcttgtctg ggatcgtgta aatagggaat
ttcccaaaac a 514645DNAArtificial SequenceOligonucleotide referred
to as Oligonucleotide E 46caacggaaaa accagaaggg gtacaaacag
gagagcctga ggaac 454711DNAArtificial SequencepRW824 sequence of
BamHI followed by SmaI 47ggatccccgg g 114860DNAArtificial
SequenceOligonucleotide referred to as RW178 48tcattatcgc
gatatccgtg ttaactagct agctaatttt tattcccggg atccttatca
604960DNAArtificial SequenceOligonucleotide referred to as RW179
49gtataaggat cccgggaata aaaattagct agctagttaa cacggatatc gcgataatga
605024DNAArtificial SequenceOligonucleotide referred to as F73PH2
50gacaatctaa gtcctatatt agac 245118DNAArtificial
SequenceOligonucleotide referred to as F73PB 51ggatttttag gtagacac
185218DNAArtificial SequenceOligonucleotide referred to as F75PE
52tcatcgtctt catcatcg 185329DNAArtificial SequenceOligonucleotide
referred to as F73PH1 53gtcttaaact tattgtaagg gtatacctg
295461DNAArtificial SequenceOligonucleotide referred to as F7MCSB
54aacgattagt tagttactaa aagcttgctg cagcccgggt tttttattag tttagttagt
60c 615560DNAArtificial SequenceOligonucleotide referred to as
F7MCSA 55gactaactaa ctaataaaaa acccgggctg cagcaagctt tttgtaacta
actaatcgtt 605699DNAArtificial SequenceSynthetic oligonucleotide
referred to as RW152 56gcacggaaca aagcttatcg cgatatccgt taagtttgta
tcgtaatgct atcaatcacg 60attctgttcc tgctcatagc agagggctca tctcagaat
995799DNAArtificial SequenceSynthetic oligonucleotide referred to
as RW153 57attctgagat gagccctctg ctatgagcag gaacagaatc gtgattgata
gcattacgat 60acaaacttaa cggatatcgc gataagcttt gttccgtgc
995866DNAArtificial SequenceSynthetic oligonucleotide referred to
as RW10 58gaaaaattta aagtcgacct gttttgttga gttgtttgcg tggtaaccaa
tgcaaatctg 60gtcact 665966DNAArtificial SequenceSynthetic
oligonucleotide referred to as RW11 59tctagcaaga ctgactattg
caaaaagaag cactatttcc tccattacga tacaaactta 60acggat
666087DNAArtificial SequenceSynthetic oligonucleotide referred to
as RW12 60atccgttaag tttgtatcgt aatggaggaa atagtgcttc tttttgcaat
agtcagtctt 60gctagaagtg accagatttg cattggt 876149DNAArtificial
SequenceSynthetic oligonucleotide referred to as RW13 61taccacgcaa
acaactcaac aaaacaggtc gactttaaat ttttctgca 4962132DNAArtificial
SequenceSynthetic oligonucleotide referred to as RW165 62gtacaggtcg
acaagcttcc cgggtatcgc gatatccgtt aagtttgtat cgtaatgaat 60actcaaattc
taatactcac tcttgtggca gccattcaca caaatgcaga caaaatctgc
120cttggacatc at 13263132DNAArtificial SequenceSynthetic
oligonucleotide referred to as RW166 63atgatgtcca aggcagattt
tgtctgcatt tgtgtgaatg gctgccacaa gagtgagtat 60tagaatttga gtattcatta
cgatacaaac ttaacggata tcgcgatacc cgggaagctt 120gtcgacctgt ac
1326451DNAArtificial SequenceSynthetic oligonucleotide referred to
as RW227 64ataacatgcg gtgcaccatt tgtatataag ttaacgaatt ccaagtcaag c
516551DNAArtificial SequenceSynthetic oligonucleotide referred to
as RW228 65gcttgacttg gaattcgtta acttatatac aaatggtgca ccgcatgtta t
51663209DNACanarypox virus 66tgaatgttaa atgttatact ttggatgaag
ctataaatat gcattggaaa aataatccat 60ttaaagaaag gattcaaata ctacaaaacc
taagcgataa tatgttaact aagcttattc 120ttaacgacgc tttaaatata
cacaaataaa cataattttt gtataaccta acaaataact 180aaaacataaa
aataataaaa ggaaatgtaa tatcgtaatt attttactca ggaatggggt
240taaatattta tatcacgtgt atatctatac tgttatcgta tactctttac
aattactatt 300acgaatatgc aagagataat aagattacgt atttaagaga
atcttgtcat gataattggg 360tacgacatag tgataaatgc tatttcgcat
cgttacataa agtcagttgg aaagatggat 420ttgacagatg taacttaata
ggtgcaaaaa tgttaaataa cagcattcta tcggaagata 480ggataccagt
tatattatac aaaaatcact ggttggataa aacagattct gcaatattcg
540taaaagatga agattactgc gaatttgtaa actatgacaa taaaaagcca
tttatctcaa 600cgacatcgtg taattcttcc atgttttatg tatgtgtttc
agatattatg agattactat 660aaactttttg tatacttata ttccgtaaac
tatattaatc atgaagaaaa tgaaaaagta 720tagaagctgt tcacgagcgg
ttgttgaaaa caacaaaatt atacattcaa gatggcttac 780atatacgtct
gtgaggctat catggataat gacaatgcat ctctaaatag gtttttggac
840aatggattcg accctaacac ggaatatggt actctacaat ctcctcttga
aatggctgta 900atgttcaaga ataccgaggc tataaaaatc ttgatgaggt
atggagctaa acctgtagtt 960actgaatgca caacttcttg tctgcatgat
gcggtgttga gagacgacta caaaatagtg 1020aaagatctgt tgaagaataa
ctatgtaaac aatgttcttt acagcggagg ctttactcct 1080ttgtgtttgg
cagcttacct taacaaagtt aatttggtta aacttctatt ggctcattcg
1140gcggatgtag atatttcaaa cacggatcgg ttaactcctc tacatatagc
cgtatcaaat 1200aaaaatttaa caatggttaa acttctattg aacaaaggtg
ctgatactga cttgctggat 1260aacatgggac gtactccttt aatgatcgct
gtacaatctg gaaatattga aatatgtagc 1320acactactta aaaaaaataa
aatgtccaga actgggaaaa attgatcttg ccagctgtaa 1380ttcatggtag
aaaagaagtg ctcaggctac ttttcaacaa aggagcagat gtaaactaca
1440tctttgaaag aaatggaaaa tcatatactg ttttggaatt gattaaagaa
agttactctg 1500agacacaaaa gaggtagctg aagtggtact ctcaaaatgc
agaacgatga ctgcgaagca 1560agaagtagag aaataacact ttatgacttt
cttagttgta gaaaagatag agatataatg 1620atggtcataa ataactctga
tattgcaagt aaatgcaata ataagttaga tttatttaaa 1680aggatagtta
aaaatagaaa aaaagagtta atttgtaggg ttaaaataat acataagatc
1740ttaaaattta taaatacgca taataataaa aatagattat acttattacc
ttcagagata 1800aaatttaaga tatttactta tttaacttat aaagatctaa
aatgcataat ttctaaataa 1860tgaaaaaaaa gtacatcatg agcaacgcgt
tagtatattt tacaatggag attaacgctc 1920tataccgttc tatgtttatt
gattcagatg atgttttaga aaagaaagtt attgaatatg 1980aaaactttaa
tgaagatgaa gatgacgacg atgattattg ttgtaaatct gttttagatg
2040aagaagatga cgcgctaaag tatactatgg ttacaaagta taagtctata
ctactaatgg 2100cgacttgtgc aagaaggtat agtatagtga aaatgttgtt
agattatgat tatgaaaaac 2160caaataaatc agatccatat ctaaaggtat
ctcctttgca cataatttca tctattccta 2220gtttagaata cttttcatta
tatttgttta cagctgaaga cgaaaaaaat atatcgataa 2280tagaagatta
tgttaactct gctaataaga tgaaattgaa tgagtctgtg ataatagcta
2340taatcagaga agttctaaaa ggaaataaaa atctaactga tcaggatata
aaaacattgg 2400ctgatgaaat caacaaggag gaactgaata tagctaaact
attgttagat agaggggcca 2460aagtaaatta caaggatgtt tacggttctt
cagctctcca tagagctgct attggtagga 2520aacaggatat gataaagctg
ttaatcgatc atggagctga tgtaaactct ttaactattg 2580ctaaagataa
tcttattaaa aaaaaataat atcacgttta gtaatattaa aatatattaa
2640taactctatt actaataact ccagtggata tgaacataat acgaagttta
tacattctca 2700tcaaaatctt attgacatca agttagattg tgaaaatgag
attatgaaat taaggaatac 2760aaaaatagga tgtaagaact tactagaatg
ttttatcaat aatgatatga atacagtatc 2820tagggctata aacaatgaaa
cgattaaaaa ttataaaaat catttcccta tatataatac 2880gctcatagaa
aaattcattt ctgaaagtat actaagacac gaattattgg atggagttat
2940aaattctttt caaggattca ataataaatt gccttacgag attcagtaca
ttatactgga 3000gaatcttaat aaccatgaac taaaaaaaat tttagataat
atacattaaa aaggtaaata 3060gatcatctgt tattataagc aaagatgctt
gttgccaata atatacaaca ggtatttgtt 3120tttattttta actacatatt
tgatgttcat tctctttata tagtatacac agaaaattca 3180taatccactt
agaatttcta gttatctag 3209673659DNAFowlpox virus 67gatatctgtg
gtctatatat actacaccct accgatatta accaacgagt ttctcacaag 60aaaacttgtt
tagtagatag agattctttg attgtgttta aaagaagtac cagtaaaaag
120tgtggcatat gcatagaaga aataaacaaa aaacatattt ccgaacagta
ttttggaatt 180ctcccaagtt gtaaacatat tttttgccta tcatgtataa
gacgttgggc agatactacc 240agaaatacag atactgaaaa tacgtgtcct
gaatgtagaa tagtttttcc tttcataata 300cccagtaggt attggataga
taataaatat gataaaaaaa tattatataa tagatataag 360aaaatgattt
ttacaaaaat acctataaga acaataaaaa tataattaca tttacggaaa
420atagctggtt ttagtttacc aacttagagt aattatcata ttgaatctat
attgtttttt 480agttatataa aaacatgatt agcccccaat cggatgaaaa
tataaaagat gttgagaatt 540tcgaatacaa caaaaagagg aatcgtacgt
tgtccatatc caaacatata aataaaaatt 600caaaagtagt attatactgg
atgtttagag atcaacgtgt acaagataat tgggctttaa 660tttacgcaca
acgattagcg ttaaaactca aaatacctct aagaatatgc ttttgtgtcg
720tgccaaaatt tcacactact acttctagac actttatgtt tttaatatcc
ggtcttaaag 780aagtcgcgga agaatgtaaa agactatgta tagggttttc
attgatatat ggcgtaccaa 840aagtaataat tccgtgtata gtaaaaaaat
acagagtcgg agtaatcata acggatttct 900ttccattacg tgttcccgaa
agattaatga aacagactgt aatatctctt ccagataaca 960taccttttat
acaagtagac gctcataata tagtaccttg ttgggaagct tctgataaag
1020aagaatacgg tgcacgaact ttaagaaaaa agatatttga taaattatat
gaatatatga 1080cagaatttcc tgttgttcgt aaacatccat acggtccatt
ttctatatct attgcaaaac 1140ccaaaaatat atcattagac aagacggtat
tacccgtaaa atgggcaacg cctggaacaa 1200aagctggaat aattgtttta
aaagaattta taaaaaacag attaccgtca tacgacgcgg 1260atcataacaa
tcctacgtgt gacgctttga gtaacttatc tccgtggcta cattttggtc
1320atgtatccgc acaacgtgtt gccttagaag tattaaaatg tatacgagaa
agcaaaaaaa 1380acgttgaaac gtttatagat gaaataattg taagaagaga
actatcggat aatttttgtt 1440actataacaa acattatgat agtatccagt
ctactcattc atgggttaga aaaacattag 1500aagatcacat taatgatcct
agaaagtata tatattccat taaacaactc gaaaaagcgg 1560aaactcatga
tcctctatgg aacgcgtcac aaatgcagat ggtgagagaa ggaaaaatgc
1620atagtttttt acgaatgtat tgggctaaga agatacttga atggactaga
acacctgaag 1680acgctttgag ttatagtatc tatttgaaca acaagtacga
actagacggc acggatccta 1740acggatacgt aggttgtatg tggtctattt
gcggattaca cgatagagcg tggaaagcaa 1800gaccgatatt tggaaagata
agatatatga attatgagag ttctaagaag aaatttgatg 1860ttgctgtatt
tatacagaaa tacaattaag ataaataata tacagcattg taaccatcgt
1920catccgttat acggggaata atattaccat acagtattat taaattttct
tacgaagaat 1980atagatcggt atttatcgtt agtttatttt acatttatta
attaaacatg tctactatta 2040cctgttatgg
aaatgacaaa tttagttata taatttatga taaaattaag ataataataa
2100tgaaatcaaa taattatgta aatgctacta gattatgtga attacgagga
agaaagttta 2160cgaactggaa aaaattaagt gaatctaaaa tattagtcga
taatgtaaaa aaaataaatg 2220ataaaactaa ccagttaaaa acggatatga
ttatatacgt taaggatatt gatcataaag 2280gaagagatac ttgcggttac
tatgtacacc aagatctggt atcttctata tcaaattgga 2340tatctccgtt
attcgccgtt aaggtaaata aaattattaa ctattatata tgtaatgaat
2400atgatatacg acttagcgaa atggaatctg atatgacaga agtaatagat
gtagttgata 2460aattagtagg aggatacaat gatgaaatag cagaaataat
atatttgttt aataaattta 2520tagaaaaata tattgctaac atatcgttat
caactgaatt atctagtata ttaaataatt 2580ttataaattt tataaatttt
aataaaaaat acaataacga cataaagata tttaatcttt 2640aattcttgat
ctgaaaaaca catctataaa actagataaa aagttattcg ataaagataa
2700taatgaatcg aacgatgaaa aattggaaac agaagttgat aagctaattt
ttttcatcta 2760aatagtatta ttttattgaa gtacgaagtt ttacgttaga
taaataataa aggtcgattt 2820ttactttgtt aaatatcaaa tatgtcatta
tctgataaag atacaaaaac acacggtgat 2880tatcaaccat ctaacgaaca
gatattacaa aaaatacgtc ggactatgga aaacgaagct 2940gatagcctca
atagaagaag cattaaagaa attgttgtag atgttatgaa gaattgggat
3000catcctcaac gaagaaatag ataaagttct aaactggaaa aatgatacat
taaacgattt 3060agatcatcta aatacagatg ataatattaa ggaaatcata
caatgtctga ttagagaatt 3120tgcgtttaaa aagatcaatt ctattatgta
tagttatgct atggtaaaac tcaattcaga 3180taacgaacat tgaaagataa
aattaaggat tattttatag aaactattct taaagacaaa 3240cgtggttata
aacaaaagcc attacccgga ttggaaacta aaatactaga tagtattata
3300agattttaaa aacataaaat taataggttt ttatagattg acttattata
tacaatatgg 3360ataaaagata tatatcaact agaaagttga atgacggatt
cttaatttta tattatgatt 3420caatagaaat tattgtcatg tcgtgtaatc
attttataaa tatatcagcg ttactagcta 3480agaaaaacaa ggactttaat
gaatggctaa agatagaatc atttagagaa ataatagata 3540ctttagataa
aattaattac gatctaggac aacgatattg tgaagaactt acggcgcatc
3600acattccagt gtaattattg aggtcaaagc tagtaactta atagatgaca
ggacagctg 3659682356DNAFowlpox virus 68tgtctggact aactgatttc
atggaacaat tttcatcaaa aatatcagtt atacctagtt 60ctacaaagac agaactttga
tgttatgttt gtgtttgtat agaaaatttt gggatactaa 120ctgatatttc
tgaatatttc tgaatatttc atgttactta cttactccta tcttagacga
180taataaaatt cgaggcgtaa tatgtttttc caaatatttg aaattcttat
acgtatcggc 240gaagaaaagt aacatactat aagtgttatg caagtaaggt
atgttaatga tattggattt 300aatttcattg acaatacata tgtccaaaca
ttccactcgt aattatgtac ggaacgactt 360tagttaaata cttagtcaca
aaaaacttat gactgtcatt atctgaaaac ggtgattccc 420ataaatcaga
atacttaata ttaaatagaa tgctcgcttc tggaggtttc cggatactag
480ataacatatc ttctgtatta tagtttaatt cactcatttt attacataat
acagtaacat 540ctcccgaaac caatgatgtt atattagatt tacttacata
cttcttgtaa ctatcatgaa 600tacgtttgtt atgatctata aagaagatgg
atgtatattc tgttctagat agcaagttct 660ttaagttatt ctttgtctgt
attactatca tcgtcttcat catcgtctaa aggtagcatt 720atataataaa
tctaatagtt gatttctcga tctatcagta ctcgctttca ataacatttt
780tactataagc ataatagaag gcggtgatat cactatattt ttatcgggta
ttcttttagt 840aattagttag ttcgtagaat ttcgtagaga taaaagccaa
tttgttgttg atactgctta 900cgttactcat gtttcttgtt tctgttaatt
aacaggtata cccttacaat aagtttaatt 960aacttttagg tttttgtgaa
gaacttttag cttctagttc ccttatccat aattgggtct 1020tagatctaga
ttcttcccat gtataaaggg ggacataccc aaaatcttta aatgctttgt
1080ccgtttctat agtaaatgtc gtacattcct taatcaaagt ataaggattt
agtaaaggcg 1140tgtaagaaca aataggtgat agtaatactc ttaaaccttt
attaatatta gcgataaacc 1200ttaaacacca taaaggaaga catgtattcc
gtagatccat ccctaattga ttaaagaaat 1260gcatgttaaa atcatgataa
tgttcagtag gagaggtatc gtaacagtaa tacacgttat 1320tgcagagagg
actatgttga ccattttcta tcatatttct tgctgctaaa atatgcatcc
1380aagctacgtt tcctgcatag actctgctat gaaatacttt atcatccgca
tatttataca 1440ttttcctgct tttatacgat cttctgtata aagtttctag
tactggacag tattctccga 1500aaacacctaa tgggcgtagc gacaagtgca
taatctaagt cctatattag acatagtacc 1560gttagcttct agtatatatt
tctcagataa cttgtttact aagaggataa gcctctttat 1620ggttagattg
ataatacgta ttctcgtttc ctcttatcat cgcatctccg gagaaagtta
1680ggacctaccg cagaataact actcgtatat actaagactc ttacgccgtt
atacagacaa 1740gaatctacta cgttcttcgt tccgttgata ttaacgtcca
ttatagagtc gttagtaaac 1800ttacccgcta catcatttat cgaagcaata
tgaatgacca catctgctga tctaagcgct 1860tcgtccaaag tacttttatt
tctaacatct ccaatcacgg gaactatctt tattatatta 1920catttttcta
caagatctag taaccattgg tcgattctaa tatcgtaaac acgaacttct
1980ttttaaagag gattcgaaca agataagatt atttataatg tgtctaccta
aaaatccaca 2040ccctccggtt accacgtata ctagtgtacg cattttgagt
attaactata taagaccaaa 2100attatatttt cattttctgt tatattatac
tatataataa aaacaaataa atatacgaat 2160attataagaa atttagaaca
cgttattaaa gtattgcctt ttttattaac ggcgtgttct 2220tgtaattgcc
gtttagaata gtctttattt actttagata actcttctat cataaccgtc
2280tccttattcc aatcttcttc agaagtacat gagtacttac cgaagtttat
catcatagag 2340attatatatg aagaaa 23566916DNAArtificial
SequenceOligonucleotide primer referred to as HIVP5 69tgtggcaaag
aagggc 167026DNAArtificial SequenceOligonucleotide primer referred
to as HIVP6 70ttggatcctt attgtgacga ggggtc 2671106DNAArtificial
SequenceOligonucleotide referred to as HIVL17 71gatcttgaga
taaagtgaaa atatatatca ttatattaca aagtacaatt atttaggttt 60aatcatgggt
gcgagagcgt cagtattaag cgggggagaa ttagat 10672104DNAArtificial
SequenceOligonucleotide referred to HIVL18 72cgatctaatt ctcccccgct
taatactgac gctctcgcac ccatgattaa acctaaataa 60ttgtactttg taatataatg
atatatattt tcactttatc tcaa 1047368DNAArtificial
SequenceOligonucleotide referred to as HIVL19 73ctgacacagg
acacagcaat caggtcagcc aaaattacta atttttatct cgaggtcgac 60aggacccg
687472DNAArtificial SequenceOligonucleotide referred to as HIVL20
74gatccgggtc ctgtcgacct cgagataaaa attagtaatt ttggctgacc tgattgctgt
60gtcctgtgtc ag 727516DNAArtificial SequenceOligonucleotide primer
referred to as HIVP7 75aagaaaatta taggac 167624DNAArtificial
SequenceOligonucleotide primer referred to as HIVP8 76ttggatccct
aatcctcatc ctgt 247736DNAArtificial SequenceOligonucleotide primer
referred to as HIVP37 77aaaggatccc ccgggttaaa aatttaaagt gcaacc
36783807DNAArtificial SequenceDNA sequence of plasmid referred to
as pHIV32, coding strand 78taatgtagta tactaatatt aactcacatt
tgactaatta gctataaaaa cccgggatcg 60attctagaat aaaaattatc cctgcctaac
tctattcact acagagagta cagcaaaaac 120tattcttaaa cctaccaagc
ctcctactat cattatgaat aatctttttt ctctctgcac 180cactcttctc
tttgccttgg tgggtgctac tcctaatggt tcaattgtta ctactttata
240tttatataat tcacttctcc aattgtccct catatctcct cctccaggtc
tgaagatctc 300ggtgtcgttc gtgtccgtgt ccttaccacc atctcttgtt
aatagtagcc ctgtaatatt 360tgatgaacat ctaatttgtc cttcaatggg
aggggcatat attgcttttc ctacttcctg 420ccacatgttt ataatttgtt
ttattttgca ttgaagtgtg atattgttat ttgaccctgt 480agtattattc
caagtattat taccattcca agtactatta aacagtggtg atgaattaca
540gtagaagaat tcccctccac aattaaaact gtgcattaca atttctgggt
cccctcctga 600ggattgatta aagactattg ttttattctt aaattgttct
tttaatttgc taactatctg 660tcttaaagtg tcattccatt ttgctctact
aatgttacaa tgtgcttgtc ttatagttcc 720tattatattt tttgttgtat
aaaatgctct ccctggtcct atatgtatcc tttttctttt 780attgtagttg
ggtcttgtac aattaatttg tacagattca ttcagatgta ctatgatggt
840tttagcatta tcattgaaat tctcagatct aattactacc tcttcttctg
ctagactgcc 900atttaacagc agttgagttg atactactgg cctaattcca
tgtgtacatt gtactgtgct 960gacattttta catgatcctt ttccactgaa
ctttttatcg ttacacttta gaatcgcaaa 1020accagccggg gcacaatagt
gtatgggaat tggctcaaag gatatctttg gacaagcttg 1080tgtaatgact
gaggtattac aacttatcaa cctatagctg gtactatcat tatttattga
1140tactatatca agtttataaa gaagtgcata ttctttctgc atcttatctc
ttatgcttgt 1200ggtgatattg aaagagcagt ttttcatttc tcctcccttt
attgttccct cgctattact 1260attgttatta gcagtactat tattggtatt
agtagtattc ctcaaatcag tgcaatttaa 1320agtaacacag agtggggtta
attttacaca tggctttagg ctttgatccc ataaactgat 1380tatatcctca
tgcatctgtt ctaccatgtt atttttccac atgttaaaat tttctgtcac
1440atttaccaat tctacttctt gtgggttggg gtctgtgggt acacaggcat
gtgtggccca 1500aacattatgt acctctgtat catatgcttt agcatctgat
gcacaaaata gagtggtggt 1560tgcttctttc cacacaggta ccccataata
gactgtgacc cacaattttt ctgtagcact 1620acagatcatc aacatcccaa
ggagcatggt gccccatctc cacccccatc tccacaagtg 1680ctgatatttc
tccttcactc tcattgccac tgtcttctgc tctttcatta cgatacaaac
1740ttaacgcata tcgcgataat gaaataattt atgattattt ctcgctttca
atttaacaca 1800accctcaaga acctttgtat ttattttcac tttttaagta
tagaataaag aagctctaat 1860taattaagct acaaatagtt tcgttttcac
cttgtctaat aactaattaa ttaacccgga 1920tcttgagata aagtgaaaat
atatatcatt atattacaaa gtacaattat ttaggtttaa 1980tcatgggtgc
gagagcgtca gtattaagcg ggggagaatt agatcgatgg gaaaaaattc
2040ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
gcaagcaggg 2100agctagaacg attcgcagtt aatcctggcc tgttagaaac
atcagaaggc tgtagacaaa 2160tactgggaca gctacaacca tcccttcaga
caggatcaga agaacttaga tcattatata 2220atacagtagc aaccctctat
tgtgtgcatc aaaggataga gataaaagac accaaggaag 2280ctttagacaa
gatagaggaa gagcaaaaca aaagtaagaa aaaagcacag caagcagcag
2340ctgacacagg acacagcaat caggtcagcc aaaattaccc tatagtgcag
aacatccagg 2400ggcaaatggt acatcaggcc atatcaccta gaactttaaa
tgcatgggta aaagtagtag 2460aagagaaggc tttcagccca gaagtgatac
ccatgttttc agcattatca gaaggagcca 2520ccccacaaga tttaaacacc
atgctaaaca cagtgggggg acatcaagca gccatgcaaa 2580tgttaaaaga
gaccatcaat gaggaagctg cagaatggga tagagtgcat ccagtgcatg
2640cagggcctat tgcaccaggc cagatgagag aaccaagggg aagtgacata
gcaggaacta 2700ctagtaccct tcaggaacaa ataggatgga tgacaaataa
tccacctatc ccagtaggag 2760aaatttataa aagatggata atcctgggat
taaataaaat agtaagaatg tatagcccta 2820ccagcattct ggacataaga
caaggaccaa aagaaccctt tagagactat gtagaccggt 2880tctataaaac
tctaagagcc gagcaagctt cacaggaggt aaaaaattgg atgacagaaa
2940ccttgttggt ccaaaatgcg aacccagatt gtaagactat tttaaaagca
ttgggaccag 3000cggctacact agaagaaatg atgacagcat gtcagggagt
aggaggaccc ggccataagg 3060caagagtttt ggctgaagca atgagccaag
taacaaattc agctaccata atgatgcaga 3120gaggcaattt taggaaccaa
agaaagattg ttaagtgttt caattgtggc aaagaagggc 3180acacagccag
aaattgcagg gcccctagga aaaagggctg ttggaaatgt ggaaaggaag
3240gacaccaaat gaaagattgt actgagagac aggctaattt tttagggaag
atctggcctt 3300cctacaaggg aaggccaggg aattttcttc agagcagacc
agagccaaca gccccaccag 3360aagagagctt caggtctggg gtagagacaa
caactccccc tcagaagcag gagccgatag 3420acaaggaact gtatccttta
acttccctca gatcactctt tggcaacgac ccctcgtcac 3480aataaagata
ggggggcaac taaaggaagc tctattagat acaggagcag atgatacagt
3540attagaagaa atgagtttgc caggaagatg gaaaccaaaa atgatagggg
gaattggagg 3600ttttatcaaa gtaagacagt atgatcagat actcatagaa
atctgtggac ataaagctat 3660aggtacagta ttagtaggac ctacacctgt
caacataatt ggaagaaatc tgttgactca 3720gattggttgc actttaaatt
tttaacccgg gggatcccga tttttatgac tagttaatca 3780aataaaaagc
atacaagcta ttgcttc 3807793808DNAArtificial SequenceDNA sequence of
plasmid referred to as pHIV32, template strand 79attacatcat
atgattataa ttgagtgtaa actgattaat cgatattttt gggccctagc 60taagatctta
tttttaatag ggacggattg agataagtga tgtctctcat gtcgtttttg
120ataagaattt ggatggttcg gaggatgata gtaatactta ttagaaaaaa
gagagacgtg 180gtgagaagag aaacggaacc acccacgatg aggattacca
agttaacaat gatgaaatat 240aaatatatta agtgaagagg ttaacaggga
gtatagagga ggaggtccag acttctagag 300ccacagcaag cacaggcaca
ggaatggtgg tagagaacaa ttatcatcgg gacattataa 360actacttgta
gattaaacag gaagttaccc tccccgtata taacgaaaag gatgaaggac
420ggtgtacaaa tattaaacaa aataaaacgt aacttcacac tataacaata
aactgggaca 480tcataataag gttcataata atggtaaggt tcatgataat
ttgtcaccac tacttaatgt 540catcttctta aggggaggtg ttaattttga
cacgtaatgt taaagaccca ggggaggact 600cctaactaat ttctgataac
aaaataagaa tttaacaaga aaattaaacg attgatagac 660agaatttcac
agtaaggtaa aacgagatga ttacaatgtt acacgaacag aatatcaagg
720ataatataaa aaacaacata ttttacgaga gggaccagga tatacatagg
aaaaagaaaa 780taacatcaac ccagaacatg ttaattaaac atgtctaagt
aagtctacat gatactacca 840aaatcgtaat agtaacttta agagtctaga
ttaatgatgg agaagaagac gatctgacgg 900taaattgtcg tcaactcaac
tatgatgacc ggattaaggt acacatgtaa catgacacga 960ctgtaaaaat
gtactaggaa aaggtgactt gaaaaatagc aatgtgaaat cttagcgttt
1020tggtcggccc cgtgttatca cataccctta accgagtttc ctatagaaac
ctgttcgaac 1080acattactga ctccataatg ttgaatagtt ggatatcgac
catgatagta ataaataact 1140atgatatagt tcaaatattt cttcacgtat
aagaaagacg tagaatagag aatacgaaca 1200ccactataac tttctcgtca
aaaagtaaag aggagggaaa taacaaggga gcgataatga 1260taacaataat
cgtcatgata ataaccataa tcatcataag gagtttagtc acgttaaatt
1320tcattgtgtc tcaccccaat taaaatgtgt accgaaatcc gaaactaggg
tatttgacta 1380atataggagt acgtagacaa gatggtacaa taaaaaggtg
tacaatttta aaagacagtg 1440taaatggtta agatgaagaa cacccaaccc
cagacaccca tgtgtccgta cacaccgggt 1500ttgtaataca tggagacata
gtatacgaaa tcgtagacta cgtgttttat ctcaccacca 1560acgaagaaag
gtgtgtccat ggggtattat ctgacactgg gtgttaaaaa gacatcgtga
1620tgtctagtag ttgtagggtt cctcgtacca cggggtagag gtgggggtag
aggtgttcac 1680gactataaag aggaagtgag agtaacggtg acagaagacg
agaaagtata tgctatgttt 1740gaattgcgta tagcgctatt actttattaa
atactaataa agagcgaaag ttaaattgtg 1800ttgggagttc ttggaaacat
aaataaaagt gaaaaattca tatcttattt cttcgagatt 1860aattaattcg
atgtttatca aagcaaaagt ggaacagatt attgattaat taattgggcc
1920tagaactcta tttcactttt atatatagta atataatgtt tcatgttaat
aaatccaaat 1980tagtacccac gctctcgcag tcataattcg ccccctctta
atctagctac ccttttttaa 2040gccaattccg gtcccccttt cttttttata
tttaattttg tatatcatac ccgttcgtcc 2100ctcgatcttg ctaagcgtca
attaggaccg gacaatcttt gtagtcttcc gacatctgtt 2160tatgaccctg
tcgatgttgg tagggaagtc tgtcctagtc ttcttgaatc tagtaatata
2220ttatgtcatc gttgggagat aacacacgta gtttcctatc tctattttct
gtggttcctt 2280cgaaatctgt tctatctcct tctcgttttg ttttcattct
tttttcgtgt cgttcgtcgt 2340cgactgtgtc ctgtgtcgtt agtccagtcg
gttttaatgg gatatcacgt cttgtaggtc 2400cccgtttacc atgtagtccg
gtatagtgga tcttgaaatt tacgtaccca ttttcatcat 2460cttctcttcc
gaaagtcggg tcttcactat gggtacaaaa gtcgtaatag tcttcctcgg
2520tggggtgttc taaatttgtg gtacgatttg tgtcaccccc ctgtagttcg
tcggtacgtt 2580tacaattttc tctggtagtt actccttcga cgtcttaccc
tatctcacgt aggtcacgta 2640cgtcccggat aacgtggtcc ggtctactct
cttggttccc cttcactgta tcgtccttga 2700tgatcatggg aagtccttgt
ttatcctacc tactgtttat taggtggata gggtcatcct 2760ctttaaatat
tttctaccta ttaggaccct aatttatttt atcattctta catatcggga
2820tggtcgtaag acctgtattc tgttcctggt tttcttggga aatctctgat
acatctggcc 2880aagatatttt gagattctcg gctcgttcga agtgtcctcc
attttttaac ctactgtctt 2940tggaacaacc aggttttacg cttgggtcta
acattctgat aaaattttcg taaccctggt 3000cgccgatgtg atcttcttta
ctactgtcgt acagtccctc atcctcctgg gccggtattc 3060cgttctcaaa
accgacttcg ttactcggtt cattgtttaa gtcgatggta ttactacgtc
3120tctccgttaa aatccttggt ttctttctaa caattcacaa agttaacacc
gtttcttccc 3180gtgtgtcggt ctttaacgtc ccggggatcc tttttcccga
caacctttac acctttcctt 3240cctgtggttt actttctaac atgactctct
gtccgattaa aaaatccctt ctagaccgga 3300aggatgttcc cttccggtcc
cttaaaagaa gtctcgtctg gtctcggttg tcggggtggt 3360cttctctcga
agtccagacc ccatctctgt tgttgagggg gagtcttcgt cctcggctat
3420ctgttccttg acataggaaa ttgaagggag tctagtgaga aaccgttgct
ggggagcagt 3480gttatttcta tccccccgtt gatttccttc gagataatct
atgtcctcgt ctactatgtc 3540ataatcttct ttactcaaac ggtccttcta
cctttggttt ttactatccc ccttaacctc 3600caaaatagtt tcattctgtc
atactagtct atgagtatct ttagacacct gtatttcgat 3660atccatgtca
taatcatcct ggatgtggac agttgtatta accttcttta gacaactgag
3720tctaaccaac gtgaaattta aaaattgggc cccctagggc taaaaatact
gatcaattag 3780tttatttttc gtatgttcga taacgaag
3808804291DNAArtificial SequenceNucleotide sequence of C3 locus of
ALVAC in the plasmid, pVQH6CP3L, coding strand 80agatatttgt
tagcttctgc cggagatacc gtgaaaatct attttctgga aggaaaggga 60ggtcttatct
attctgtcag cagagtaggt tcctctaatg acgaagacaa tagtgaatac
120ttgcatgaag gtcactgtgt agagttcaaa actgatcatc agtgtttgat
aactctagcg 180tgtacgagtc cttctaacac tgtggtttat tggctggaat
aaaaggataa agacacctat 240actgattcat tttcatctgt caacgtttct
ctaagagatt cataggtatt attattacat 300cgatctagaa gtctaataac
tgctaagtat attattggat ttaacgcgct ataaacgcat 360ccaaaaccta
caaatatagg agaagcttct cttatgaaac ttcttaaagc tttactctta
420ctattactac tcaaaagaga tattacatta attatgtgat gaggcatcca
acatataaag 480aagactaaag ctgtagaagc tgttatgaag aatatcttat
cagatatatt agatgcattg 540ttagttctgt agatcagtaa cgtatagcat
acgagtataa ttatcgtagg tagtaggtat 600cctaaaataa atctgataca
gataataact ttgtaaatca attcagcaat ttctctatta 660tcatgataat
gattaataca cagcgtgtcg ttattttttg ttacgatagt atttctaaag
720taaagagcag gaatccctag tataatagaa ataatccata tgaaaaatat
agtaatgtac 780atatttctaa tgttaacata tttataggta aatccaggaa
gggtaatttt tacatatcta 840tatacgctta ttacagttat taaaaatata
cttgcaaaca tgttagaagt aaaaaagaaa 900gaactaattt tacaaagtgc
tttaccaaaa tgccaatgga aattacttag tatgtatata 960atgtataaag
gtatgaatat cacaaacagc aaatcggcta ttcccaagtt gagaaacggt
1020ataatagata tatttctaga taccattaat aaccttataa gcttgacgtt
tcctataatg 1080cctactaaga aaactagaag atacatacat actaacgcca
tacgagagta actactcatc 1140gtataactac tgttgctaac agtgacactg
atgttataac tcatctttga tgtggtataa 1200atgtataata actatattac
actggtattt tatttcagtt atatactata tagtattaaa 1260aattatattt
gtataattat attattatat tcagtgtaga aagtaaaata ctataaatat
1320gtatctctta tttataactt attagtaaag tatgtactat tcagttatat
tgttttataa 1380aagctaaatg ctactagatt gatataaatg aatatgtaat
aaattagtaa tgtagtatac 1440taatattaac tcacatttga ctaattagct
ataaaaaccc gggctgcagg aattcctcga 1500gtacgataca aacttaacgg
atatcgcgat aatgaaataa tttatgatta tttctcgctt 1560tcaatttaac
acaaccctca agaacctttg tatttatttt cactttttaa gtatagaata
1620aagaagctct aattaattaa gctacaaata gtttcgtttt caccttgtct
aataactaat 1680taattaaccc ggatcccgat ttttatgact agttaatcaa
ataaaaagca tacaagctat 1740tgcttcgcta tcgttacaaa atggcaggaa
ttttgtgtaa actaagccac atacttgcca 1800atgaaaaaaa tagtagaaag
gatactattt taatgggatt agatgttaag gttccttggg 1860attatagtaa
ctgggcatct gttaactttt acgacgttag gttagatact gatgttacag
1920attataataa tgttacaata aaatacatga caggatgtga tatttttcct
catataactc 1980ttggaatagc aaatatggat caatgtgata gatttgaaaa
tttcaaaaag caaataactg 2040atcaagattt acagactatt tctatagtct
gtaaagaaga gatgtgtttt cctcagagta 2100acgcctctaa acagttggga
gcgaaaggat gcgctgtagt tatgaaactg gaggtatctg 2160atgaacttag
agccctaaga aatgttctgc tgaatgcggt accctgttcg aaggacgtgt
2220ttggtgatat cacagtagat aatccgtgga atcctcacat aacagtagga
tatgttaagg 2280aggacgatgt cgaaaacaag aaacgcctaa tggagtgcat
gtccaagttt agggggcaag 2340aaatacaagt tctaggatgg tattaataag
tatctaagta tttggtataa tttattaaat 2400agtataatta taacaaataa
taaataacat gataacggtt tttattagaa taaaatagag 2460ataatatcat
aatgatatat aatacttcat taccagaaat gagtaatgga agacttataa
2520atgaactgca taaagctata aggtatagag atataaattt agtaaggtat
atacttaaaa 2580aatgcaaata caataacgta aatatactat caacgtcttt
gtatttagcc gtaagtattt 2640ctgatataga aatggtaaaa ttattactag
aacacggtgc cgatatttta aaatgtaaaa 2700atcctcctct tcataaagct
gctagtttag ataatacaga aattgctaaa ctactaatag 2760attctggcgc
tgacatagaa cagatacatt ctggaaatag tccgttatat atttctgtat
2820atagaaacaa taagtcatta actagatatt tattaaaaaa aggtgttaat
tgtaatagat 2880tctttctaaa ttattacgat gtactgtatg ataagatatc
tgatgatatg tataaaatat 2940ttatagattt taatattgat cttaatatac
aaactagaaa ttttgaaact ccgttacatt 3000acgctataaa gtataagaat
atagatttaa ttaggatatt gttagataat agtattaaaa 3060tagataaaag
tttatttttg cataaacagt atctcataaa ggcacttaaa aataattgta
3120gttacgatat aatagcgtta cttataaatc acggagtgcc tataaacgaa
caagatgatt 3180taggtaaaac cccattacat cattcggtaa ttaatagaag
aaaagatgta acagcacttc 3240tgttaaatct aggagctgat ataaacgtaa
tagatgactg tatgggcagt cccttacatt 3300acgctgtttc acgtaacgat
atcgaaacaa caaagacact tttagaaaga ggatctaatg 3360ttaatgtggt
taataatcat atagataccg ttctaaatat agctgttgca tctaaaaaca
3420aaactatagt aaacttatta ctgaagtacg gtactgatac aaagttggta
ggattagata 3480aacatgttat tcacatagct atagaaatga aagatattaa
tatactgaat gcgatcttat 3540tatatggttg ctatgtaaac gtctataatc
ataaaggttt cactcctcta tacatggcag 3600ttagttctat gaaaacagaa
tttgttaaac tcttacttga ccacggtgct tacgtaaatg 3660ctaaagctaa
gttatctgga aatactcctt tacataaagc tatgttatct aatagtttta
3720ataatataaa attactttta tcttataacg ccgactataa ttctctaaat
aatcacggta 3780atacgcctct aacttgtgtt agctttttag atgacaagat
agctattatg ataatatcta 3840aaatgatgtt agaaatatct aaaaatcctg
aaatagctaa ttcagaaggt tttatagtaa 3900acatggaaca tataaacagt
aataaaagac tactatctat aaaagaatca tgcgaaaaag 3960aactagatgt
tataacacat ataaagttaa attctatata ttcttttaat atctttcttg
4020acaataacat agatcttatg gtaaagttcg taactaatcc tagagttaat
aagatacctg 4080catgtatacg tatatatagg gaattaatac ggaaaaataa
atcattagct tttcatagac 4140atcagctaat agttaaagct gtaaaagaga
gtaagaatct aggaataata ggtaggttac 4200ctatagatat caaacatata
ataatggaac tattaagtaa taatgattta cattctgtta 4260tcaccagctg
ttgtaaccca gtagtataaa g 4291814291DNAArtificial SequenceNucleotide
sequence of C3 locus of ALVAC in the plasmid, pVQH6CP3L, template
strand 81tctataaaca atcgaagacg gcctctatgg cacttttaga taaaagacct
tcctttccct 60ccagaataga taagacagtc gtctcatcca aggagattac tgcttctgtt
atcacttatg 120aacgtacttc cagtgacaca tctcaagttt tgactagtag
tcacaaacta ttgagatcgc 180acatgctcag gaagattgtg acaccaaata
accgacctta ttttcctatt tctgtggata 240tgactaagta aaagtagaca
gttgcaaaga gattctctaa gtatccataa taataatgta 300gctagatctt
cagattattg acgattcata taataaccta aattgcgcga tatttgcgta
360ggttttggat gtttatatcc tcttcgaaga gaatactttg aagaatttcg
aaatgagaat 420gataatgatg agttttctct ataatgtaat taatacacta
ctccgtaggt tgtatatttc 480ttctgatttc gacatcttcg acaatacttc
ttatagaata gtctatataa tctacgtaac 540aatcaagaca tctagtcatt
gcatatcgta tgctcatatt aatagcatcc atcatccata 600ggattttatt
tagactatgt ctattattga aacatttagt taagtcgtta aagagataat
660agtactatta ctaattatgt gtcgcacagc aataaaaaac aatgctatca
taaagatttc 720atttctcgtc cttagggatc atattatctt tattaggtat
actttttata tcattacatg 780tataaagatt acaattgtat aaatatccat
ttaggtcctt cccattaaaa atgtatagat 840atatgcgaat aatgtcaata
atttttatat gaacgtttgt acaatcttca ttttttcttt 900cttgattaaa
atgtttcacg aaatggtttt acggttacct ttaatgaatc atacatatat
960tacatatttc catacttata gtgtttgtcg tttagccgat aagggttcaa
ctctttgcca 1020tattatctat ataaagatct atggtaatta ttggaatatt
cgaactgcaa aggatattac 1080ggatgattct tttgatcttc tatgtatgta
tgattgcggt atgctctcat tgatgagtag 1140catattgatg acaacgattg
tcactgtgac tacaatattg agtagaaact acaccatatt 1200tacatattat
tgatataatg tgaccataaa ataaagtcaa tatatgatat atcataattt
1260ttaatataaa catattaata taataatata agtcacatct ttcattttat
gatatttata 1320catagagaat aaatattgaa taatcatttc atacatgata
agtcaatata acaaaatatt 1380ttcgatttac gatgatctaa ctatatttac
ttatacatta tttaatcatt acatcatatg 1440attataattg agtgtaaact
gattaatcga tatttttggg cccgacgtcc ttaaggagct 1500catgctatgt
ttgaattgcc tatagcgcta ttactttatt aaatactaat aaagagcgaa
1560agttaaattg tgttgggagt tcttggaaac ataaataaaa gtgaaaaatt
catatcttat 1620ttcttcgaga ttaattaatt cgatgtttat caaagcaaaa
gtggaacaga ttattgatta 1680attaattggg cctagggcta aaaatactga
tcaattagtt tatttttcgt atgttcgata 1740acgaagcgat agcaatgttt
taccgtcctt aaaacacatt tgattcggtg tatgaacggt 1800tacttttttt
atcatctttc ctatgataaa attaccctaa tctacaattc caaggaaccc
1860taatatcatt gacccgtaga caattgaaaa tgctgcaatc caatctatga
ctacaatgtc 1920taatattatt acaatgttat tttatgtact gtcctacact
ataaaaagga gtatattgag 1980aaccttatcg tttataccta gttacactat
ctaaactttt aaagtttttc gtttattgac 2040tagttctaaa tgtctgataa
agatatcaga catttcttct ctacacaaaa ggagtctcat 2100tgcggagatt
tgtcaaccct cgctttccta cgcgacatca atactttgac ctccatagac
2160tacttgaatc tcgggattct ttacaagacg acttacgcca tgggacaagc
ttcctgcaca 2220aaccactata gtgtcatcta ttaggcacct taggagtgta
ttgtcatcct atacaattcc 2280tcctgctaca gcttttgttc tttgcggatt
acctcacgta caggttcaaa tcccccgttc 2340tttatgttca agatcctacc
ataattattc atagattcat aaaccatatt aaataattta 2400tcatattaat
attgtttatt atttattgta ctattgccaa aaataatctt attttatctc
2460tattatagta ttactatata ttatgaagta atggtcttta ctcattacct
tctgaatatt 2520tacttgacgt atttcgatat tccatatctc tatatttaaa
tcattccata tatgaatttt 2580ttacgtttat gttattgcat ttatatgata
gttgcagaaa cataaatcgg cattcataaa 2640gactatatct ttaccatttt
aataatgatc ttgtgccacg gctataaaat tttacatttt 2700taggaggaga
agtatttcga cgatcaaatc tattatgtct ttaacgattt gatgattatc
2760taagaccgcg actgtatctt gtctatgtaa gacctttatc aggcaatata
taaagacata 2820tatctttgtt attcagtaat tgatctataa ataatttttt
tccacaatta acattatcta 2880agaaagattt aataatgcta catgacatac
tattctatag actactatac atattttata 2940aatatctaaa attataacta
gaattatatg tttgatcttt aaaactttga ggcaatgtaa 3000tgcgatattt
catattctta tatctaaatt aatcctataa caatctatta tcataatttt
3060atctattttc aaataaaaac gtatttgtca tagagtattt ccgtgaattt
ttattaacat 3120caatgctata ttatcgcaat gaatatttag tgcctcacgg
atatttgctt gttctactaa 3180atccattttg gggtaatgta gtaagccatt
aattatcttc ttttctacat tgtcgtgaag 3240acaatttaga tcctcgacta
tatttgcatt atctactgac atacccgtca gggaatgtaa 3300tgcgacaaag
tgcattgcta tagctttgtt gtttctgtga aaatctttct cctagattac
3360aattacacca attattagta tatctatggc aagatttata tcgacaacgt
agatttttgt 3420tttgatatca tttgaataat gacttcatgc catgactatg
tttcaaccat cctaatctat 3480ttgtacaata agtgtatcga tatctttact
ttctataatt atatgactta cgctagaata 3540atataccaac gatacatttg
cagatattag tatttccaaa gtgaggagat atgtaccgtc 3600aatcaagata
cttttgtctt aaacaatttg agaatgaact ggtgccacga atgcatttac
3660gatttcgatt caatagacct ttatgaggaa atgtatttcg atacaataga
ttatcaaaat 3720tattatattt taatgaaaat agaatattgc ggctgatatt
aagagattta ttagtgccat 3780tatgcggaga ttgaacacaa tcgaaaaatc
tactgttcta tcgataatac tattatagat 3840tttactacaa tctttataga
tttttaggac tttatcgatt aagtcttcca aaatatcatt 3900tgtaccttgt
atatttgtca ttattttctg atgatagata ttttcttagt acgctttttc
3960ttgatctaca atattgtgta tatttcaatt taagatatat aagaaaatta
tagaaagaac 4020tgttattgta tctagaatac catttcaagc attgattagg
atctcaatta ttctatggac 4080gtacatatgc atatatatcc cttaattatg
cctttttatt tagtaatcga aaagtatctg 4140tagtcgatta tcaatttcga
cattttctct cattcttaga tccttattat ccatccaatg 4200gatatctata
gtttgtatat tattaccttg ataattcatt attactaaat gtaagacaat
4260agtggtcgac aacattgggt catcatattt c 42918288DNAArtificial
SequenceOligonucleotide primer referred to as VQPCR13 82atcatcaagc
ttaattaatt agttattaga caaggtgaaa acgaaactat ttgtagctta 60attattagac
atcatgcagt ggttaaac 888369DNAArtificial SequenceOligonucleotide
primer referred to as I3PCRCTL 83ctagctacgt gatgaaatgc taatctagaa
tcaaatctcc actccatgat taaacctaaa 60taattgtac 698477DNAArtificial
SequenceOligonucleotide primer referred to as CTLPCR 84gaattcctcg
aggatcctct agattaacaa tttttaaaat attcaggatg taattctcta 60gctacgtgat
gaaatgc 778535DNAArtificial SequenceOligonucleotide primer referred
to as H6PCR1 85actactaagc ttctttattc tatacttaaa aagtg
358688DNAArtificial SequenceOligonucleotide primer referred to as
NCCPCR1 86cagctgcttt gtaagtcatt ggtcttaaag gtacttgagg tgttactgga
aaacctacca 60ttacgataca aacttaacgg atatcgcg 8887102DNAArtificial
SequenceOligonucleotide referred to as NCC174A 87acttacaaag
cagctgtaga tctttctcac tttttaaaag aaaaaggagg tttagaaggg 60ctaattcatt
ctcaacgaag acaagatatt cttgatttgt gg 10288102DNAArtificial
SequenceOligonucleotide referred to as NCC174B 88ccacaaatca
agaatatctt gtcttcgttg agaatgaatt agcccttcta aacctccttt 60ttcttttaaa
aagtgagaaa gatctacagc tgctttgtaa gt 1028954DNAArtificial
SequenceOligonucleotide primer referred to as NCCPCR2 89ctgccaatca
ggaaaatatc cttgtgtatg ataaatccac aaatcaagaa tatc
549079DNAArtificial SequenceOligonucleotide referred to as NCC291A
90ggatattttc ctgattggca gaattacaca ccaggaccag gagtcagata cccattaacc
60tttggttggt gctacaagc 799179DNAArtificial SequenceOligonucleotide
referred to as NCC291B 91gcttgtagca ccaaccaaag gttaatgggt
atctgactcc tggtcctggt gtgtaattct 60gccaatcagg aaaatatcc
799249DNAArtificial SequenceOligonucleotide primer referred to as
NCCPCR3 92actactgaat tctcgagaaa aattatggta ctagcttgta gcaccaacc
4993801DNAArtificial SequenceDNA sequence of the plasmid,
p2-60-HIV.3, coding strand 93agtacaataa aaagtattaa ataaaaatac
ttacttacga aaaaatgact aattagctat 60aaaaacccgg gctgcagctc gaggatcctc
tagattaaca atttttaaaa tattcaggat 120gtaattctct agctacgtga
tgaaatgcta atctagaatc aaatctccac tccatgatta 180aacctaaata
attgtacttt gtaatataat gatatatatt ttcactttat ctcatttgag
240aataaaaatg tttttgttta accactgcat gatgtctaat taattaagct
acaaatagtt 300tcgttttcac cttgtctaat aactaattaa ttaagcttct
ttattctata cttaaaaagt 360gaaaataaat acaaaggttc ttgagggttg
tgttaaattg aaagcgagaa ataatcataa 420attatttcat tatcgcgata
tccgttaagt ttgtatcgta atggtaggtt ttccagtaac 480acctcaagta
cctttaagac caatgactta caaagcagct gtagatcttt ctcacttttt
540aaaagaaaaa ggaggtttag aagggctaat tcattctcaa cgaagacaag
atattcttga 600tttgtggatt tatcatacac aaggatattt tcctgattgg
cagaattaca caccaggacc 660aggagtcaga tacccattaa cctttggttg
gtgctacaag ctagtaccat aatttttctc 720gaggaattct ttttattgat
taactagtca aatgagtata tataattgaa aaagtaaaat 780ataaatcata
taataatgaa a 80194801DNAArtificial SequenceDNA sequence of the
plasmid, p2-60-HIV.3, template strand 94tcatgttatt tttcataatt
tatttttatg aatgaatgct tttttactga ttaatcgata 60tttttgggcc cgacgtcgag
ctcctaggag atctaattgt taaaaatttt ataagtccta 120cattaagaga
tcgatgcact actttacgat tagatcttag tttagaggtg aggtactaat
180ttggatttat taacatgaaa cattatatta ctatatataa aagtgaaata
gagtaaactc 240ttatttttac aaaaacaaat tggtgacgta ctacagatta
attaattcga tgtttatcaa 300agcaaaagtg gaacagatta ttgattaatt
aattcgaaga aataagatat gaatttttca 360cttttattta tgtttccaag
aactcccaac acaatttaac tttcgctctt tattagtatt 420taataaagta
atagcgctat aggcaattca aacatagcat taccatccaa aaggtcattg
480tggagttcat ggaaattctg gttactgaat gtttcgtcga catctagaaa
gagtgaaaaa 540ttttcttttt cctccaaatc ttcccgatta agtaagagtt
gcttctgttc tataagaact 600aaacacctaa atagtatgtg ttcctataaa
aggactaacc gtcttaatgt gtggtcctgg 660tcctcagtct atgggtaatt
ggaaaccaac cacgatgttc gatcatggta ttaaaaagag 720ctccttaaga
aaaataacta attgatcagt ttactcatat atattaactt tttcatttta
780tatttagtat attattactt t 8019527PRTHuman immunodeficiency virus
type 1 95Cys Asn Lys Phe Tyr Glu Pro His Leu Glu Arg Ala Val His
His Phe1 5 10 15Ala Leu Arg Ser Asp Phe Arg Trp Glu Met Pro 20
259683PRTHuman immunodeficiency virus type 1 96Met Val Gly Phe Pro
Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr1 5 10 15Tyr Lys Ala Ala
Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 20 25 30Leu Glu Gly
Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu 35 40 45Trp Ile
Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr 50 55 60Pro
Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys65 70 75
80Leu Val Pro971615DNAArtificial SequenceNucleotide sequence of the
ALVAC C6 locus in the plasmid, pC6L, coding strand 97gagctcgcgg
ccgcctatca aaagtcttaa tgagttaggt gtagatagta tagatattac 60tacaaaggta
ttcatatttc ctatcaattc taaagtagat gatattaata actcaaagat
120gatgatagta gataatagat acgctcatat aatgactgca aatttggacg
gttcacattt 180taatcatcac gcgttcataa gtttcaactg catagatcaa
aatctcacta aaaagatagc 240cgatgtattt gagagagatt ggacatctaa
ctacgctaaa gaaattacag ttataaataa 300tacataatgg attttgttat
catcagttat atttaacata agtacaataa aaagtattaa 360ataaaaatac
ttacttacga aaaaatgact aattagctat aaaaacccgg gctgcagctc
420gaggaattct ttttattgat taactagtca aatgagtata tataattgaa
aaagtaaaat 480ataaatcata taataatgaa acgaaatatc agtaatagac
aggaactggc agattcttct 540tctaatgaag taagtactgc taaatctcca
aaattagata aaaatgatac agcaaataca 600gcttcattca acgaattacc
ttttaatttt ttcagacaca ccttattaca aactaactaa 660gtcagatgat
gagaaagtaa atataaattt aacttatggg tataatataa taaagattca
720tgatattaat aatttactta acgatgttaa tagacttatt ccatcaaccc
cttcaaacct 780ttctggatat tataaaatac cagttaatga tattaaaata
gattgtttaa gagatgtaaa 840taattatttg gaggtaaagg atataaaatt
agtctatctt tcacatggaa atgaattacc 900taatattaat aattatgata
ggaatttttt aggatttaca gctgttatat gtatcaacaa 960tacaggcaga
tctatggtta tggtaaaaca ctgtaacggg aagcagcatt ctatggtaac
1020tggcctatgt ttaatagcca gatcatttta ctctataaac attttaccac
aaataatagg 1080atcctctaga tatttaatat tatatctaac aacaacaaaa
aaatttaacg atgtatggcc 1140agaagtattt tctactaata aagataaaga
tagtctatct tatctacaag atatgaaaga 1200agataatcat ttagtagtag
ctactaatat ggaaagaaat gtatacaaaa acgtggaagc 1260ttttatatta
aatagcatat tactagaaga tttaaaatct agacttagta taacaaaaca
1320gttaaatgcc aatatcgatt ctatatttca tcataacagt agtacattaa
tcagtgatat 1380actgaaacga tctacagact caactatgca aggaataagc
aatatgccaa ttatgtctaa 1440tattttaact ttagaactaa aacgttctac
caatactaaa aataggatac gtgataggct 1500gttaaaagct gcaataaata
gtaaggatgt agaagaaata ctttgttcta taccttcgga 1560ggaaagaact
ttagaacaac ttaagtttaa tcaaacttgt atttatgaag gtacc
1615981615DNAArtificial SequenceNucleotide sequence of the ALVAC C6
locus in the plasmid, pC6L, template strand 98ctcgagcgcc ggcggatagt
tttcagaatt actcaatcca catctatcat atctataatg 60atgtttccat aagtataaag
gatagttaag atttcatcta ctataattat tgagtttcta 120ctactatcat
ctattatcta tgcgagtata ttactgacgt ttaaacctgc caagtgtaaa
180attagtagtg cgcaagtatt caaagttgac gtatctagtt ttagagtgat
ttttctatcg 240gctacataaa ctctctctaa cctgtagatt gatgcgattt
ctttaatgtc aatatttatt 300atgtattacc taaaacaata gtagtcaata
taaattgtat tcatgttatt tttcataatt 360tatttttatg aatgaatgct
tttttactga ttaatcgata tttttgggcc cgacgtcgag 420ctccttaaga
aaaataacta attgatcagt ttactcatat atattaactt tttcatttta
480tatttagtat attattactt tgctttatag tcattatctg tccttgaccg
tctaagaaga 540agattacttc attcatgacg atttagaggt tttaatctat
ttttactatg tcgtttatgt 600cgaagtaagt tgcttaatgg aaaattaaaa
aagtctgtgt ggaataatgt ttgattgatt 660cagtctacta ctctttcatt
tatatttaaa ttgaataccc atattatatt atttctaagt 720actataatta
ttaaatgaat tgctacaatt atctgaataa ggtagttggg gaagtttgga
780aagacctata atattttatg gtcaattact ataattttat ctaacaaatt
ctctacattt 840attaataaac ctccatttcc tatattttaa tcagatagaa
agtgtacctt tacttaatgg 900attataatta ttaatactat ccttaaaaaa
tcctaaatgt cgacaatata catagttgtt 960atgtccgtct agataccaat
accattttgt gacattgccc ttcgtcgtaa gataccattg 1020accggataca
aattatcggt ctagtaaaat gagatatttg taaaatggtg tttattatcc
1080taggagatct ataaattata atatagattg ttgttgtttt tttaaattgc
tacataccgg 1140tcttcataaa agatgattat ttctatttct atcagataga
atagatgttc tatactttct 1200tctattagta aatcatcatc gatgattata
cctttcttta catatgtttt tgcaccttcg 1260aaaatataat ttatcgtata
atgatcttct aaattttaga tctgaatcat attgttttgt 1320caatttacgg
ttatagctaa gatataaagt agtattgtca tcatgtaatt agtcactata
1380tgactttgct agatgtctga gttgatacgt tccttattcg ttatacggtt
aatacagatt 1440ataaaattga aatcttgatt ttgcaagatg gttatgattt
ttatcctatg cactatccga 1500caattttcga cgttatttat cattcctaca
tcttctttat gaaacaagat atggaagcct 1560cctttcttga aatcttgttg
aattcaaatt agtttgaaca taaatacttc catgg 16159943DNAArtificial
SequenceOligonucleotide primer referred to as P1A 99tttgtatcgt
aatgattgag actgtaccag taaaattaaa gcc 4310066DNAArtificial
SequenceOligonucleotide primer referred to as P1B 100gggctgcagg
aattctaatc aattaaggcc caatttttga
aattttccct tccttttcca 60tctctg 6610153DNAArtificial
SequenceOligonucleotide primer referred to as P2A 101acaaagtaca
attatttagg tttaatcatg gcaatattcc aaagtagcat gac
5310244DNAArtificial SequenceOligonucleotide primer referred to as
P2B 102atcatcctcg agaaaaatta ggtaagtccc cacctcaaca gatg
4410358DNAArtificial SequenceOligonucleotide primer referred to as
P3A 103aaaatatata attacaatat aaaatgccac taacagaaga agcagagcta
gaactggc 5810460DNAArtificial SequenceOligonucleotide primer
referred to as P3B 104atcatctcta gactcgagga tccataaaaa ttatcctgtt
ttcagatttt taaatggctc 6010552DNAArtificial SequenceOligonucleotide
primer referred to as P2IVH 105gtcatgctac ttttgaatat tgccatgatt
aaacctaaat aattgtactt tg 5210656DNAArtificial
SequenceOligonucleotide primer referred to as IVHP1 106tttaatttta
ctggtacagt ctcaatcatt acgatacaaa cttaacggat atcgcg
5610744DNAArtificial SequenceOligonucleotide primer referred to as
EPS42K 107aattgattag aattcctgca gcccgggtca aaaaaatata aatg
4410858DNAArtificial SequenceOligonucleotide primer referred to as
42KP3B 108ccagttctag ctctgcttct tctgttagtg gcattttata ttgtaattat
atattttc 5810932DNAArtificial SequenceOligonucleotide primer
referred to as I3PCR1 109atcatcggat ccaagcttac atcatgcagt gg
3211045DNAArtificial SequenceOligonucleotide primer referred to as
FIXPOL2 110atcatcctcg agctattcaa ttaggttgta agtccccacc tcaac
451111035DNAArtificial SequenceDNA sequence of the plasmid,
pC5POLT5A, coding strand 111ttagaaatta tgcattttag atctttataa
gcggccgtga ttaactagtc ataaaaaccc 60gggatcgatt ctagactcga gctattcaat
taggttgtaa gtccccacct caacagatgt 120tgtctcagct cctctatttt
tgttctatgc tgccctattt ctaagtcaga tcctacatac 180aaatcatcca
tgtattgata gataactatg tctggatttt gttttctaaa aggctctaag
240atttttgtca tgctactttg gaatattgcc atgattaaac ctaaataatt
gtactttgta 300atataatgat atatattttc actttatctc atttgagaat
aaaaatgttt ttgtttaacc 360actgcatgat gtaagcttct ttattctata
cttaaaaagt gaaaataaat acaaaggttc 420ttgagggttg tgttaaattg
aaagcgagaa ataatcataa attatttcat tatcgcgata 480tccgttaagt
ttgtatcgta atgattgaga ctgtaccagt aaaattaaag ccaggaatgg
540atggcccaaa agttaaacaa tggccattga cagaagaaaa aataaaagca
ttagtagaaa 600tttgtacaga gatggaaaag gaagggaaaa tttcaaaaat
tgggccttaa ttgattagaa 660ttcctgcagc ccaggtcaaa aaaatataaa
tgattcacca tctgatagaa aaaaaattta 720ttgggaagaa tatgataata
ttttgggatt tcaaaattga aaatatataa ttacaatata 780aaatgccact
aacagaagaa gcagagctag aactggcaga aaacagagag attctaaaag
840aaccagtaca tggagtgtat tatgacccat caaaagactt aatagcagaa
atacagaagc 900aggggcaagg ccaatggaca tatcaaattt atcaagagcc
atttaaaaat ctgaaaacag 960gataattttt atggatcctt tttatagcta
attagtcacg tacctttgag agtaccactt 1020cagctacctc ctttg
10351121035DNAArtificial SequenceDNA sequence of the plasmid,
pC5POLT5A, template strand 112aatctttaat acgtaaaatc tagaaatatt
cgccggcact aattgatcag tatttttggg 60ccctagctaa gatctgagct cgataagtta
atccaacatt caggggtgga gttgtctaca 120acagagtcga ggagataaaa
acaagatacg acgggataaa gattcagtct aggatgtatg 180tttagtaggt
acataactat ctattgatac agacctaaaa caaaagattt tccgagattc
240taaaaacagt acgatgaaac cttataacgg tactaatttg gatttattaa
catgaaacat 300tatattacta tatataaaag tgaaatagag taaactctta
tttttacaaa aacaaattgg 360tgacgtacta cattcgaaga aataagatat
gaatttttca cttttattta tgtttccaag 420aactcccaac acaatttaac
tttcgctctt tattagtatt taataaagta atagcgctat 480aggcaattca
aacatagcat tactaactct gacatggtca ttttaatttc ggtccttacc
540taccgggttt tcaatttgtt accggtaact gtcttctttt ttattttcgt
aatcatcttt 600aaacatgtct ctaccttttc cttccctttt aaagttttta
acccggaatt aactaatctt 660aaggacgtcg ggtccagttt ttttatattt
actaagtggt agactatctt ttttttaaat 720aacccttctt atactattat
aaaaccctaa agttttaact tttatatatt aatgttatat 780tttacggtga
ttgtcttctt cgtctcgatc ttgaccgtct tttgtctctc taagattttc
840ttggtcatgt acctcacata atactgggta gttttctgaa ttatcgtctt
tatgtcttcg 900tccccgttcc ggttacctgt atagtttaaa tagttctcgg
taaattttta gacttttgtc 960ctattaaaaa tacctaggaa aaatatcgat
taatcagtgc atggaaactc tcatggtgaa 1020gtcgatggag gaaac
103511360PRTHuman immunodeficiency virus type 1 113Thr Thr Leu Gly
Trp Arg Leu Leu His Gln Arg Leu Glu Glu Ile Lys1 5 10 15Thr Arg His
Gln Gly Ile Glu Leu Asp Ser Gly Val Tyr Leu Asp Asp 20 25 30Met Tyr
Gln Tyr Ile Val Ile Asp Pro Asn Gln Lys Arg Phe Pro Glu 35 40 45Leu
Ile Lys Thr Met Ser Ser Gln Phe Ile Ala Met 50 55 6011449PRTHuman
immunodeficiency virus type 1 114Met Ile Glu Thr Val Pro Val Lys
Leu Lys Pro Gly Met Asp Gly Pro1 5 10 15Lys Val Lys Gln Trp Pro Leu
Thr Glu Glu Lys Ile Lys Ala Leu Val 20 25 30Glu Ile Cys Thr Glu Met
Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly 35 40 45Pro11559PRTHuman
immunodeficiency virus type 1 115Met Pro Leu Thr Glu Glu Ala Glu
Leu Glu Leu Ala Glu Asn Arg Glu1 5 10 15Ile Leu Lys Glu Pro Val His
Gly Val Tyr Tyr Asp Pro Ser Lys Asp 20 25 30Leu Ile Ala Glu Ile Gln
Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln 35 40 45Ile Tyr Gln Glu Pro
Phe Lys Asn Leu Lys Thr 50 551162049DNAArtificial
SequenceNucleotide sequence of the ALVAC C5 locus of the plasmid,
pNC5L-SP5, coding strand 116gaattgcggc cgctgaatgt taaatgttat
actttggatg aagctataaa tatgcattgg 60aaaaataatc catttaaaga aaggattcaa
atactacaaa acctaagcga taatatgtta 120actaagctta ttcttaacga
cgctttaaat atacacaaat aaacataatt tttgtataac 180ctaacaaata
actaaaacat aaaaataata aaaggaaatg taatatcgta attattttac
240tcaggaatgg ggttaaatat ttatatcacg tgtatatcta tactgttatc
gtatactctt 300tacaattact attacgaata tgcaagagat aataagatta
cgtatttaag agaatcttgt 360catgataatt gggtacgaca tagtgataaa
tgctatttcg catcgttaca taaagtcagt 420tggaaagatg gatttgacag
atgtaactta ataggtgcaa aaatgttaaa taacagcatt 480ctatcggaag
ataggatacc agttatatta tacaaaaatc actggttgga taaaacagat
540tctgcaatat tcgtaaaaga tgaagattac tgcgaatttg taaactatga
caataaaaag 600ccatttatct caacgacatc gtgtaattct tccatgtttt
atgtatgtgt ttcagatatt 660atgagattac tataaacttt ttgtatactt
atattccgta aactatatta atcatgaaga 720aaatgaaaaa gtatagaagc
tgttcacgag cggttgttga aaacaacaaa attatacatt 780caagatggct
tacatatacg tctgtgaggc tatcatggat aatgacaatg catctctaaa
840taggtttttg gacaatggat tcgaccctaa cacggaatat ggtactctac
aatctcctct 900tgaaatggct gtaatgttca agaataccga ggctataaaa
atcttgatga ggtatggagc 960taaacctgta gttactgaat gcacaacttc
ttgtctgcat gatgcggtgt tgagagacga 1020ctacaaaata gtgaaagatc
tgttgaagaa taactatgta aacaatgttc tttacagcgg 1080aggctttact
cctttgtgtt tggcagctta ccttaacaaa gttaatttgg ttaaacttct
1140attggctcat tcggcggatg tagatatttc aaacacggat cggttaactc
ctctacatat 1200agccgtatca aataaaaatt taacaatggt taaacttcta
ttgaacaaag gtgctgatac 1260tgacttgctg gataacatgg gacgtactcc
tttaatgatc gctgtacaat ctggaaatat 1320tgaaatatgt agcacactac
ttaaaaaaaa taaaatgtcc agaactggga aaaattgatc 1380ttgccagctg
taattcatgg tagaaaagaa gtgctcaggc tacttttcaa caaaggagca
1440gatgtaaact acatctttga aagaaatgga aaatcatata ctgttttgga
attgattaaa 1500gaaagttact ctgagacaca aaagaggtag ctgaagtggt
actctcaaag gtacgtgact 1560aattagctat aaaaaggatc cggtaccctc
gagtctagaa tcgatcccgg gtttttatga 1620ctagttaatc acggccgctt
ataaagatct aaaatgcata atttctaaat aatgaaaaaa 1680aagtacatca
tgagcaacgc gttagtatat tttacaatgg agattaacgc tctataccgt
1740tctatgttta ttgattcaga tgatgtttta gaaaagaaag ttattgaata
tgaaaacttt 1800aatgaagatg aagatgacga cgatgattat tgttgtaaat
ctgttttaga tgaagaagat 1860gacgcgctaa agtatactat ggttacaaag
tataagtcta tactactaat ggcgacttgt 1920gcaagaaggt atagtatagt
gaaaatgttg ttagattatg attatgaaaa accaaataaa 1980tcagatccat
atctaaaggt atctcctttg cacataattt catctattcc tagtttagaa
2040tacctgcag 20491172049DNAArtificial SequenceDNA sequence of the
ALVAC C5 locus in the plasmid, pCN5L-SP5, template strand
117cttaacgccg gcgacttaca atttacaata tgaaacctac ttcgatattt
atacgtaacc 60tttttattag gtaaatttct ttcctaagtt tatgatgttt tggattcgct
attatacaat 120tgattcgaat aagaattgct gcgaaattta tatgtgttta
tttgtattaa aaacatattg 180gattgtttat tgattttgta tttttattat
tttcctttac attatagcat taataaaatg 240agtccttacc ccaatttata
aatatagtgc acatatagat atgacaatag catatgagaa 300atgttaatga
taatgcttat acgttctcta ttattctaat gcataaattc tcttagaaca
360gtactattaa cccatgctgt atcactattt acgataaagc gtagcaatgt
atttcagtca 420acctttctac ctaaactgtc tacattgaat tatccacgtt
tttacaattt attgtcgtaa 480gatagccttc tatcctatgg tcaatataat
atgtttttag tgaccaacct attttgtcta 540agacgttata agcattttct
acttctaatg acgcttaaac atttgatact gttatttttc 600ggtaaataga
gttgctgtag cacattaaga aggtacaaaa tacatacaca aagtctataa
660tactctaatg atatttgaaa aacatatgaa tataaggcat ttgatataat
tagtacttct 720tttacttttt catatcttcg acaagtgctc gccaacaact
tttgttgttt taatatgtaa 780gttctaccga atgtatatgc agacactccg
atagtaccta ttactgttac gtagagattt 840atccaaaaac ctgttaccta
agctgggatt gtgccttata ccatgagatg ttagaggaga 900actttaccga
cattacaagt tcttatggct ccgatatttt tagaactact ccatacctcg
960atttggacat caatgactta cgtgttgaag aacagacgta ctacgccaca
actctctgct 1020gatgttttat cactttctag acaacttctt attgatacat
ttgttacaag aaatgtcgcc 1080tccgaaatga ggaaacacaa accgtcgaat
ggaattgttt caattaaacc aatttgaaga 1140taaccgagta agccgcctac
atctataaag tttgtgccta gccaattgag gagatgtata 1200tcggcatagt
ttatttttaa attgttacca atttgaagat aacttgtttc cacgactatg
1260actgaacgac ctattgtacc ctgcatgagg aaattactag cgacatgtta
gacctttata 1320actttataca tcgtgtgatg aatttttttt attttacagg
tcttgaccct ttttaactag 1380aacggtcgac attaagtacc atcttttctt
cacgagtccg atgaaaagtt gtttcctcgt 1440ctacatttga tgtagaaact
ttctttacct tttagtatat gacaaaacct taactaattt 1500ctttcaatga
gactctgtgt tttctccatc gacttcacca tgagagtttc catgcactga
1560ttaatcgata tttttcctag gccatgggag ctcagatctt agctagggcc
caaaaatact 1620gatcaattag tgccggcgaa tatttctaga ttttacgtat
taaagattta ttactttttt 1680ttcatgtagt actcgttgcg caatcatata
aaatgttacc tctaattgcg agatatggca 1740agatacaaat aactaagtct
actacaaaat cttttctttc aataacttat acttttgaaa 1800ttacttctac
ttctactgct gctactaata acaacattta gacaaaatct acttcttcta
1860ctgcgcgatt tcatatgata ccaatgtttc atattcagat atgatgatta
ccgctgaaca 1920cgttcttcca tatcatatca cttttacaac aatctaatac
taatactttt tggtttattt 1980agtctaggta tagatttcca tagaggaaac
gtgtattaaa gtagataagg atcaaatctt 2040atggacgtc
204911834PRTArtificial SequencePeptide referred to as CLTB-36,
which is a small portion of the HIV1 gp120 envelope glycoprotein,
the V3 loop. 118Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe
Tyr Lys Asn1 5 10 15Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala
Phe Tyr Thr Thr 20 25 30Lys Asn11926PRTArtificial SequenceSynthetic
peptide representing the HIV MN gp120 V3 loop 119Cys Asn Lys Arg
Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr1 5 10 15Thr Thr Lys
Asn Ile Ile Gly Thr Ile Cys 20 2512026PRTArtificial
SequenceSynthetic peptide representing HIV MN gp120 V3 loop 120Cys
Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr1 5 10
15Thr Thr Lys Asn Ile Ile Gly Thr Ile Cys 20 2512123DNAArtificial
SequencePrimer referred to as HIVP72 121ttattaccat tccaagtact att
23122109DNAArtificial SequencePrimer referred to as HIVP74
122tctgtacaaa ttaattgtac aagacccaac tacgagctcg acaaatgggc
ccatatagga 60ccagggagag aattggataa gtgggcgaat ataataggaa ctataagac
10912320DNAArtificial SequenceOligonucleotide referred to as HIVMN6
123gggttattaa tgatctgtag 2012437DNAArtificial
SequenceOligonucleotide referred to as HIV3B2 124gaattacagt
agaagaattc ccctccacaa ttaaaac 3712537DNAArtificial
SequenceOligonucleotide referred to as HIV3B1 125gttttaattg
tggaggggaa ttcttctact gtaattc 3712624DNAArtificial
SequenceOligonucleotide referred to as HIVMN4 126atcatcgagc
tcctatcgct gctc 2412730DNAArtificial SequenceOligonucleotide
referred to as HIVMN5 127atcatcgagc tctgttcctt gggttcttag
3012843DNAArtificial SequenceOligonucleotide referred to as HIVMN3P
128atcatctcta gaataaaaat tatagcaaag ccctttccaa gcc
4312987DNAArtificial SequenceOligonucleotide referred to as HIVTM1
129ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
tgtactctct 60gtagtgaata gagttaggca gggataa 8713087DNAArtificial
SequenceOligonucleotide referred to as HIVTM2 130ttatccctgc
ctaactctat tcactacaga gagtacagca aaaactattc ttaaacctac 60caagcctcct
actatcatta tgaataa 8713143DNAArtificial SequenceOligonucleotide
referred to as HIVTM3 131atcatctcta gaataaaaat tatccctgcc
taactctatt cac 4313239DNAArtificial SequenceOligonucleotide
referred to as HIVMN18 132gcctcctact atcattatga ataatctttt
ttctctctg 3913324DNAArtificial SequencePrimer referred to as HIVP69
133tgatagtacc agctataggt tgat 2413433DNAArtificial SequencePrimer
referred to as HIVP75 134tttgtcgagc tcgtagttgg gtcttgtaca att
331352020DNAArtificial SequenceDNA sequence of the H6-promoted HIV1
gp120 + TM (with ELDKWA epitopes) gene between C5 flanking arms,
coding strand 135agaaagttac tctgagacac aaaagaggta gctgaagtgg
tactctcaaa ggtacgtgac 60taattagcta taaaaaggat ccgggttaat taattagtca
tcaggcaggg cgagaacgag 120actatctgct cgttaattaa ttagagcttc
tttattctat acttaaaaag tgaaaataaa 180tacaaaggtt cttgagggtt
gtgttaaatt gaaagcgaga aataatcata aattatttca 240ttatcgcgat
atgcgttaag tttgtatcgt atatgaaaga gcagaagaca gtggcaatga
300gagtgaagga gaaatatcag cacttgtgga gatgggggtg gagatggggc
accatgctcc 360ttgggatgtt gatgatctgt agtgctacag aaaaattgtg
ggtcacagtc tattatgggg 420tacctgtgtg gaaagaagca accaccactc
tattttgtgc atcagatgct aaagcatatg 480atacagaggt acataatgtt
tgggccacac atgcctgtgt acccacagac cccaacccac 540aagaagtaga
attggtaaat gtgacagaaa attttaacat gtggaaaaat aacatggtag
600aacagatgca tgaggatata atcagtttat gggatcaaag cctaaagcca
tgtgtaaaat 660taaccccact ctgtgttact ttaaattgca ctgatttgag
gaatactact aataccaata 720atagtactgc taataacaat agtaatagcg
agggaacaat aaagggagga gaaatgaaaa 780actgctcttt caatatcacc
acaagcataa gagataagat gcagaaagaa tatgcacttc 840tttataaact
tgatatagta tcaataaata atgatagtac cagctatagg ttgataagtt
900gtaatacctc agtcattaca caagcttgtc caaagatatc ctttgagcca
attcccatac 960actattgtgc cccggctggt tttgcgattc taaagtgtaa
cgataaaaag ttcagtggaa 1020aaggatcatg taaaaatgtc agcacagtac
aatgtacaca tggaattagg ccagtagtat 1080caactcaact gctgttaaat
ggcagtctag cagaagaaga ggtagtaatt agatctgaga 1140atttcaatga
taatgctaaa accatcatag tacatctgaa tgaatctgta caaattaatt
1200gtacaagacc caactacgag ctcgacaaat gggcccatat aggaccaggg
agagaattgg 1260ataagtgggc gaatataata ggaactataa gacaagcaca
ttgtaacatt agtagagcaa 1320aatggaatga cactttaaga cagatagtta
gcaaattaaa agaacaattt aagaataaaa 1380caatagtctt taatcaatcc
tcaggagggg acccagaaat tgtaatgcac agttttaatt 1440gtggagggga
attcttctac tgtaattcat caccactgtt taatagtact tggaatggta
1500ataatacttg gaataatact acagggtcaa ataacaatat cacacttcaa
tgcaaaataa 1560aacaaattat aaacatgtgg caggaagtag gaaaagcaat
atatgcccct cccattgaag 1620gacaaattag atgttcatca aatattacag
ggctactatt aacaagagat ggtggtaagg 1680acacggacac gaacgacacc
gagatcttca gacctggagg aggagatatg agggacaatt 1740ggagaagtga
attatataaa tataaagtag taacaattga accattagga gtagcaccca
1800ccaaggcaaa gagaagagtg gtgcagagag aaaaaagatt attcataatg
atagtaggag 1860gcttggtagg tttaagaata gtttttgctg tactctctgt
agtgaataga gttaggcagg 1920gataattttt attctagaat cgatcccggg
tttttatgac tagttaatca cggccgctta 1980taaagatcta aaatgcataa
tttctaaata atgaaaaaaa 20201362020DNAArtificial SequenceDNA sequence
of the H6-promoted HIV1 gp120 + TM (with ELDKWA epitopes) gene
between C5 flanking arms, template strand 136tctttcaatg agactctgtg
ttttctccat cgacttcacc atgagagttt ccatgcactg 60attaatcgat atttttccta
ggcccaatta attaatcagt agtccgtccc gctcttgctc 120tgatagacga
gcaattaatt aatctcgaag aaataagata tgaatttttc acttttattt
180atgtttccaa gaactcccaa cacaatttaa ctttcgctct ttattagtat
ttaataaagt 240aatagcgcta tacgcaattc aaacatagca tatactttct
cgtcttctgt caccgttact 300ctcacttcct ctttatagtc gtgaacacct
ctacccccac ctctaccccg tggtacgagg 360aaccctacaa ctactagaca
tcacgatgtc tttttaacac ccagtgtcag ataatacccc 420atggacacac
ctttcttcgt tggtggtgag ataaaacacg tagtctacga tttcgtatac
480tatgtctcca tgtattacaa acccggtgtg tacggacaca tgggtgtctg
gggttgggtg 540ttcttcatct taaccattta cactgtcttt taaaattgta
caccttttta ttgtaccatc 600ttgtctacgt actcctatat tagtcaaata
ccctagtttc ggatttcggt acacatttta 660attggggtga gacacaatga
aatttaacgt gactaaactc cttatgatga ttatggttat 720tatcatgacg
attattgtta tcattatcgc tcccttgtta tttccctcct ctttactttt
780tgacgagaaa gttatagtgg tgttcgtatt ctctattcta cgtctttctt
atacgtgaag 840aaatatttga actatatcat agttatttat
tactatcatg gtcgatatcc aactattcaa 900cattatggag tcagtaatgt
gttcgaacag gtttctatag gaaactcggt taagggtatg 960tgataacacg
gggccgacca aaacgctaag atttcacatt gctatttttc aagtcacctt
1020ttcctagtac atttttacag tcgtgtcatg ttacatgtgt accttaatcc
ggtcatcata 1080gttgagttga cgacaattta ccgtcagatc gtcttcttct
ccatcattaa tctagactct 1140taaagttact attacgattt tggtagtatc
atgtagactt acttagacat gtttaattaa 1200catgttctgg gttgatgctc
gagctgttta cccgggtata tcctggtccc tctcttaacc 1260tattcacccg
cttatattat ccttgatatt ctgttcgtgt aacattgtaa tcatctcgtt
1320ttaccttact gtgaaattct gtctatcaat cgtttaattt tcttgttaaa
ttcttatttt 1380gttatcagaa attagttagg agtcctcccc tgggtcttta
acattacgtg tcaaaattaa 1440cacctcccct taagaagatg acattaagta
gtggtgacaa attatcatga accttaccat 1500tattatgaac cttattatga
tgtcccagtt tattgttata gtgtgaagtt acgttttatt 1560ttgtttaata
tttgtacacc gtccttcatc cttttcgtta tatacgggga gggtaacttc
1620ctgtttaatc tacaagtagt ttataatgtc ccgatgataa ttgttctcta
ccaccattcc 1680tgtgcctgtg cttgctgtgg ctctagaagt ctggacctcc
tcctctatac tccctgttaa 1740cctcttcact taatatattt atatttcatc
attgttaact tggtaatcct catcgtgggt 1800ggttccgttt ctcttctcac
cacgtctctc ttttttctaa taagtattac tatcatcctc 1860cgaaccatcc
aaattcttat caaaaacgac atgagagaca tcacttatct caatccgtcc
1920ctattaaaaa taagatctta gctagggccc aaaaatactg atcaattagt
gccggcgaat 1980atttctagat tttacgtatt aaagatttat tacttttttt
2020137551PRTArtificial SequenceHIV1 gp120 + TM (with ELDKWA
epitopes) expressed by vCP1307-infected cells 137Met Lys Glu Gln
Lys Thr Val Ala Met Arg Val Lys Glu Lys Tyr Gln1 5 10 15His Leu Trp
Arg Trp Gly Trp Arg Trp Gly Thr Met Leu Leu Gly Met 20 25 30Leu Met
Ile Cys Ser Ala Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr 35 40 45Gly
Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser 50 55
60Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala Thr His65
70 75 80Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Val Glu Leu Val
Asn 85 90 95Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu
Gln Met 100 105 110His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu
Lys Pro Cys Val 115 120 125Lys Leu Thr Pro Leu Cys Val Thr Leu Asn
Cys Thr Asp Leu Arg Asn 130 135 140Thr Thr Asn Thr Asn Asn Ser Thr
Ala Asn Asn Asn Ser Asn Ser Glu145 150 155 160Gly Thr Ile Lys Gly
Gly Glu Met Lys Asn Cys Ser Phe Asn Ile Thr 165 170 175Thr Ser Ile
Arg Asp Lys Met Gln Lys Glu Tyr Ala Leu Leu Tyr Lys 180 185 190Leu
Asp Ile Val Ser Ile Asn Asn Asp Ser Thr Ser Tyr Arg Leu Ile 195 200
205Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe
210 215 220Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala
Ile Leu225 230 235 240Lys Cys Asn Asp Lys Lys Phe Ser Gly Lys Gly
Ser Cys Lys Asn Val 245 250 255Ser Thr Val Gln Cys Thr His Gly Ile
Arg Pro Val Val Ser Thr Gln 260 265 270Leu Leu Leu Asn Gly Ser Leu
Ala Glu Glu Glu Val Val Ile Arg Ser 275 280 285Glu Asn Phe Asn Asp
Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu 290 295 300Ser Val Gln
Ile Asn Cys Thr Arg Pro Asn Tyr Glu Leu Asp Lys Trp305 310 315
320Ala His Ile Gly Pro Gly Arg Glu Leu Asp Lys Trp Ala Asn Ile Ile
325 330 335Gly Thr Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys
Trp Asn 340 345 350Asp Thr Leu Arg Gln Ile Val Ser Lys Leu Lys Glu
Gln Phe Lys Asn 355 360 365Lys Thr Ile Val Phe Asn Gln Ser Ser Gly
Gly Asp Pro Glu Ile Val 370 375 380Met His Ser Phe Asn Cys Gly Gly
Glu Phe Phe Tyr Cys Asn Ser Ser385 390 395 400Pro Leu Phe Asn Ser
Thr Trp Asn Gly Asn Asn Thr Trp Asn Asn Thr 405 410 415Thr Gly Ser
Asn Asn Asn Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile 420 425 430Ile
Asn Met Trp Gln Glu Val Gly Lys Ala Ile Tyr Ala Pro Pro Ile 435 440
445Glu Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr
450 455 460Arg Asp Gly Gly Lys Asp Thr Asp Thr Asn Asp Thr Glu Ile
Phe Arg465 470 475 480Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg
Ser Glu Leu Tyr Lys 485 490 495Tyr Lys Val Val Thr Ile Glu Pro Leu
Gly Val Ala Pro Thr Lys Ala 500 505 510Lys Arg Arg Val Val Gln Arg
Glu Lys Arg Leu Phe Ile Met Ile Val 515 520 525Gly Gly Leu Val Gly
Leu Arg Ile Val Phe Ala Val Leu Ser Val Val 530 535 540Asn Arg Val
Arg Gln Gly Asn545 5501382028DNAArtificial SequenceNucleotide
sequence of H6-promoted HIV1 gp120 + TM (with ELDKWA epitopes) gene
between I4L flanking arms, coding strand. 138tactttgtaa tataatgata
tatattttca ctttatctca tttgagaata aaaagatcac 60aaaaattaac taatcaggat
ccgggttaat taattagtca tcaggcaggg cgagaacgag 120actatctgct
cgttaattaa ttagagcttc tttattctat acttaaaaag tgaaaataaa
180tacaaaggtt cttgagggtt gtgttaaatt gaaagcgaga aataatcata
aattatttca 240ttatcgcgat atgcgttaag tttgtatcgt atatgaaaga
gcagaagaca gtggcaatga 300gagtgaagga gaaatatcag cacttgtgga
gatgggggtg gagatggggc accatgctcc 360ttgggatgtt gatgatctgt
agtgctacag aaaaattgtg ggtcacagtc tattatgggg 420tacctgtgtg
gaaagaagca accaccactc tattttgtgc atcagatgct aaagcatatg
480atacagaggt acataatgtt tgggccacac atgcctgtgt acccacagac
cccaacccac 540aagaagtaga attggtaaat gtgacagaaa attttaacat
gtggaaaaat aacatggtag 600aacagatgca tgaggatata atcagtttat
gggatcaaag cctaaagcca tgtgtaaaat 660taaccccact ctgtgttact
ttaaattgca ctgatttgag gaatactact aataccaata 720atagtactgc
taataacaat agtaatagcg agggaacaat aaagggagga gaaatgaaaa
780actgctcttt caatatcacc acaagcataa gagataagat gcagaaagaa
tatgcacttc 840tttataaact tgatatagta tcaataaata atgatagtac
cagctatagg ttgataagtt 900gtaatacctc agtcattaca caagcttgtc
caaagatatc ctttgagcca attcccatac 960actattgtgc cccggctggt
tttgcgattc taaagtgtaa cgataaaaag ttcagtggaa 1020aaggatcatg
taaaaatgtc agcacagtac aatgtacaca tggaattagg ccagtagtat
1080caactcaact gctgttaaat ggcagtctag cagaagaaga ggtagtaatt
agatctgaga 1140atttcaatga taatgctaaa accatcatag tacatctgaa
tgaatctgta caaattaatt 1200gtacaagacc caactacgag ctcgacaaat
gggcccatat aggaccaggg agagaattgg 1260ataagtgggc gaatataata
ggaactataa gacaagcaca ttgtaacatt agtagagcaa 1320aatggaatga
cactttaaga cagatagtta gcaaattaaa agaacaattt aagaataaaa
1380caatagtctt taatcaatcc tcaggagggg acccagaaat tgtaatgcac
agttttaatt 1440gtggagggga attcttctac tgtaattcat caccactgtt
taatagtact tggaatggta 1500ataatacttg gaataatact acagggtcaa
ataacaatat cacacttcaa tgcaaaataa 1560aacaaattat aaacatgtgg
caggaagtag gaaaagcaat atatgcccct cccattgaag 1620gacaaattag
atgttcatca aatattacag ggctactatt aacaagagat ggtggtaagg
1680acacggacac gaacgacacc gagatcttca gacctggagg aggagatatg
agggacaatt 1740ggagaagtga attatataaa tataaagtag taacaattga
accattagga gtagcaccca 1800ccaaggcaaa gagaagagtg gtgcagagag
aaaaaagatt attcataatg atagtaggag 1860gcttggtagg tttaagaata
gtttttgctg tactctctgt agtgaataga gttaggcagg 1920gataattttt
attctagaat cgatcccggg agatcttagc taactgattt ttctgggaaa
1980aaaattattt aacttttcat taatagggat ttgacgtatg tagcgtac
20281392028DNAArtificial SequenceNucleotide sequence of H6-promoted
HIV1 gp120 + TM (with ELDKWA epitopes) gene between I4L flanking
arms, template strand 139atgaaacatt atattactat atataaaagt
gaaatagagt aaactcttat ttttctagtg 60tttttaattg attagtccta ggcccaatta
attaatcagt agtccgtccc gctcttgctc 120tgatagacga gcaattaatt
aatctcgaag aaataagata tgaatttttc acttttattt 180atgtttccaa
gaactcccaa cacaatttaa ctttcgctct ttattagtat ttaataaagt
240aatagcgcta tacgcaattc aaacatagca tatactttct cgtcttctgt
caccgttact 300ctcacttcct ctttatagtc gtgaacacct ctacccccac
ctctaccccg tggtacgagg 360aaccctacaa ctactagaca tcacgatgtc
tttttaacac ccagtgtcag ataatacccc 420atggacacac ctttcttcgt
tggtggtgag ataaaacacg tagtctacga tttcgtatac 480tatgtctcca
tgtattacaa acccggtgtg tacggacaca tgggtgtctg gggttgggtg
540ttcttcatct taaccattta cactgtcttt taaaattgta caccttttta
ttgtaccatc 600ttgtctacgt actcctatat tagtcaaata ccctagtttc
ggatttcggt acacatttta 660attggggtga gacacaatga aatttaacgt
gactaaactc cttatgatga ttatggttat 720tatcatgacg attattgtta
tcattatcgc tcccttgtta tttccctcct ctttactttt 780tgacgagaaa
gttatagtgg tgttcgtatt ctctattcta cgtctttctt atacgtgaag
840aaatatttga actatatcat agttatttat tactatcatg gtcgatatcc
aactattcaa 900cattatggag tcagtaatgt gttcgaacag gtttctatag
gaaactcggt taagggtatg 960tgataacacg gggccgacca aaacgctaag
atttcacatt gctatttttc aagtcacctt 1020ttcctagtac atttttacag
tcgtgtcatg ttacatgtgt accttaatcc ggtcatcata 1080gttgagttga
cgacaattta ccgtcagatc gtcttcttct ccatcattaa tctagactct
1140taaagttact attacgattt tggtagtatc atgtagactt acttagacat
gtttaattaa 1200catgttctgg gttgatgctc gagctgttta cccgggtata
tcctggtccc tctcttaacc 1260tattcacccg cttatattat ccttgatatt
ctgttcgtgt aacattgtaa tcatctcgtt 1320ttaccttact gtgaaattct
gtctatcaat cgtttaattt tcttgttaaa ttcttatttt 1380gttatcagaa
attagttagg agtcctcccc tgggtcttta acattacgtg tcaaaattaa
1440cacctcccct taagaagatg acattaagta gtggtgacaa attatcatga
accttaccat 1500tattatgaac cttattatga tgtcccagtt tattgttata
gtgtgaagtt acgttttatt 1560ttgtttaata tttgtacacc gtccttcatc
cttttcgtta tatacgggga gggtaacttc 1620ctgtttaatc tacaagtagt
ttataatgtc ccgatgataa ttgttctcta ccaccattcc 1680tgtgcctgtg
cttgctgtgg ctctagaagt ctggacctcc tcctctatac tccctgttaa
1740cctcttcact taatatattt atatttcatc attgttaact tggtaatcct
catcgtgggt 1800ggttccgttt ctcttctcac cacgtctctc ttttttctaa
taagtattac tatcatcctc 1860cgaaccatcc aaattcttat caaaaacgac
atgagagaca tcacttatct caatccgtcc 1920ctattaaaaa taagatctta
gctagggccc tctagaatcg attgactaaa aagacccttt 1980ttttaataaa
ttgaaaagta attatcccta aactgcatac atcgcatg 2028140550PRTArtificial
SequenceHIV1 gp120 + TM (with ELDKWA epitopes) expressed by
vP1313-infected cells 140Met Lys Glu Gln Lys Thr Val Ala Met Arg
Val Lys Glu Lys Tyr Gln1 5 10 15His Leu Trp Arg Trp Gly Trp Arg Trp
Gly Thr Met Leu Leu Gly Met 20 25 30Leu Met Ile Cys Ser Ala Thr Glu
Lys Leu Trp Val Thr Val Tyr Tyr 35 40 45Gly Val Pro Val Trp Lys Glu
Ala Thr Thr Thr Leu Phe Cys Ala Ser 50 55 60Asp Ala Lys Ala Tyr Asp
Thr Glu Val His Asn Val Trp Ala Thr His65 70 75 80Ala Cys Val Pro
Thr Asp Pro Asn Pro Gln Glu Val Glu Leu Val Asn 85 90 95Val Thr Glu
Asn Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met 100 105 110His
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val 115 120
125Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Thr Asp Leu Arg Asn
130 135 140Thr Thr Asn Thr Asn Asn Ser Thr Ala Asn Asn Asn Ser Asn
Ser Glu145 150 155 160Gly Thr Ile Lys Gly Gly Glu Met Lys Asn Cys
Ser Phe Asn Ile Thr 165 170 175Thr Ser Ile Arg Asp Lys Met Gln Lys
Glu Tyr Ala Leu Leu Tyr Lys 180 185 190Leu Asp Ile Val Ser Ile Asn
Asn Asp Ser Thr Ser Tyr Arg Leu Ile 195 200 205Ser Cys Asn Thr Ser
Val Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe 210 215 220Glu Pro Ile
Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu225 230 235
240Lys Cys Asn Asp Lys Lys Phe Ser Gly Lys Gly Ser Cys Lys Asn Val
245 250 255Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser
Thr Gln 260 265 270Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val
Val Ile Arg Ser 275 280 285Glu Asn Phe Asn Asp Asn Ala Lys Thr Ile
Ile Val His Leu Asn Glu 290 295 300Ser Val Gln Ile Asn Cys Thr Arg
Pro Asn Tyr Glu Leu Asp Lys Trp305 310 315 320Ala His Ile Gly Pro
Gly Arg Glu Leu Asp Lys Trp Ala Asn Ile Ile 325 330 335Gly Thr Ile
Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys Trp Asn 340 345 350Asp
Thr Leu Arg Gln Ile Val Ser Lys Leu Lys Glu Gln Phe Lys Asn 355 360
365Lys Thr Ile Val Phe Asn Gln Ser Ser Gly Gly Asp Pro Glu Ile Val
370 375 380Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn
Ser Ser385 390 395 400Pro Leu Phe Asn Ser Thr Trp Asn Gly Asn Asn
Thr Trp Asn Asn Thr 405 410 415Thr Gly Ser Asn Asn Asn Ile Thr Leu
Gln Cys Lys Ile Lys Gln Ile 420 425 430Ile Asn Met Trp Gln Glu Val
Gly Lys Ala Ile Tyr Ala Pro Pro Ile 435 440 445Glu Gly Gln Ile Arg
Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr 450 455 460Arg Asp Gly
Gly Lys Asp Thr Asp Thr Asn Asp Thr Glu Ile Phe Arg465 470 475
480Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys
485 490 495Tyr Lys Val Val Thr Ile Glu Pro Leu Gly Val Ala Pro Thr
Lys Ala 500 505 510Lys Arg Arg Val Val Gln Arg Glu Lys Arg Leu Phe
Ile Met Ile Val 515 520 525Gly Gly Leu Val Gly Leu Arg Ile Val Phe
Ala Val Leu Ser Val Val 530 535 540Asn Arg Val Arg Gln Gly545
5501412060DNAArtificial SequenceH6-promoted HIV1 gp120 + TM (with
ELDKWA epitopes) gene between A24R and K1L flanking arms, coding
sequence 141ataaaccatt agataaagtt gatctcaagc gctcttttct ggtgtaataa
aaattaatta 60attactcgag ggtaccggat ccgggttaat taattagtca tcaggcaggg
cgagaacgag 120actatctgct cgttaattaa ttagagcttc tttattctat
acttaaaaag tgaaaataaa 180tacaaaggtt cttgagggtt gtgttaaatt
gaaagcgaga aataatcata aattatttca 240ttatcgcgat atgcgttaag
tttgtatcgt atatgaaaga gcagaagaca gtggcaatga 300gagtgaagga
gaaatatcag cacttgtgga gatgggggtg gagatggggc accatgctcc
360ttgggatgtt gatgatctgt agtgctacag aaaaattgtg ggtcacagtc
tattatgggg 420tacctgtgtg gaaagaagca accaccactc tattttgtgc
atcagatgct aaagcatatg 480atacagaggt acataatgtt tgggccacac
atgcctgtgt acccacagac cccaacccac 540aagaagtaga attggtaaat
gtgacagaaa attttaacat gtggaaaaat aacatggtag 600aacagatgca
tgaggatata atcagtttat gggatcaaag cctaaagcca tgtgtaaaat
660taaccccact ctgtgttact ttaaattgca ctgatttgag gaatactact
aataccaata 720atagtactgc taataacaat agtaatagcg agggaacaat
aaagggagga gaaatgaaaa 780actgctcttt caatatcacc acaagcataa
gagataagat gcagaaagaa tatgcacttc 840tttataaact tgatatagta
tcaataaata atgatagtac cagctatagg ttgataagtt 900gtaatacctc
agtcattaca caagcttgtc caaagatatc ctttgagcca attcccatac
960actattgtgc cccggctggt tttgcgattc taaagtgtaa cgataaaaag
ttcagtggaa 1020aaggatcatg taaaaatgtc agcacagtac aatgtacaca
tggaattagg ccagtagtat 1080caactcaact gctgttaaat ggcagtctag
cagaagaaga ggtagtaatt agatctgaga 1140atttcaatga taatgctaaa
accatcatag tacatctgaa tgaatctgta caaattaatt 1200gtacaagacc
caactacgag ctcgacaaat gggcccatat aggaccaggg agagaattgg
1260ataagtgggc gaatataata ggaactataa gacaagcaca ttgtaacatt
agtagagcaa 1320aatggaatga cactttaaga cagatagtta gcaaattaaa
agaacaattt aagaataaaa 1380caatagtctt taatcaatcc tcaggagggg
acccagaaat tgtaatgcac agttttaatt 1440gtggagggga attcttctac
tgtaattcat caccactgtt taatagtact tggaatggta 1500ataatacttg
gaataatact acagggtcaa ataacaatat cacacttcaa tgcaaaataa
1560aacaaattat aaacatgtgg caggaagtag gaaaagcaat atatgcccct
cccattgaag 1620gacaaattag atgttcatca aatattacag ggctactatt
aacaagagat ggtggtaagg 1680acacggacac gaacgacacc gagatcttca
gacctggagg aggagatatg agggacaatt 1740ggagaagtga attatataaa
tataaagtag taacaattga accattagga gtagcaccca 1800ccaaggcaaa
gagaagagtg gtgcagagag aaaaaagatt attcataatg atagtaggag
1860gcttggtagg tttaagaata gtttttgctg tactctctgt agtgaataga
gttaggcagg 1920gataattttt attcgagaat cgatcccggg aatcgattcg
cgatagctga ttagtttttg 1980ttaacaaaaa tgtgggagaa tctaattagt
ttttctttac acaattgacg tacatgagtc 2040tgagttcctt gtttttgcta
20601422060DNAArtificial SequenceH6-promoted HIV1 gp120 + TM (with
ELDKWA epitopes) gene between A24R and K1L flanking arms, template
strand 142tatttggtaa tctatttcaa ctagagttcg cgagaaaaga ccacattatt
tttaattaat 60taatgagctc ccatggccta ggcccaatta attaatcagt agtccgtccc
gctcttgctc 120tgatagacga gcaattaatt aatctcgaag aaataagata
tgaatttttc acttttattt 180atgtttccaa gaactcccaa cacaatttaa
ctttcgctct ttattagtat ttaataaagt 240aatagcgcta tacgcaattc
aaacatagca tatactttct cgtcttctgt caccgttact
300ctcacttcct ctttatagtc gtgaacacct ctacccccac ctctaccccg
tggtacgagg 360aaccctacaa ctactagaca tcacgatgtc tttttaacac
ccagtgtcag ataatacccc 420atggacacac ctttcttcgt tggtggtgag
ataaaacacg tagtctacga tttcgtatac 480tatgtctcca tgtattacaa
acccggtgtg tacggacaca tgggtgtctg gggttgggtg 540ttcttcatct
taaccattta cactgtcttt taaaattgta caccttttta ttgtaccatc
600ttgtctacgt actcctatat tagtcaaata ccctagtttc ggatttcggt
acacatttta 660attggggtga gacacaatga aatttaacgt gactaaactc
cttatgatga ttatggttat 720tatcatgacg attattgtta tcattatcgc
tcccttgtta tttccctcct ctttactttt 780tgacgagaaa gttatagtgg
tgttcgtatt ctctattcta cgtctttctt atacgtgaag 840aaatatttga
actatatcat agttatttat tactatcatg gtcgatatcc aactattcaa
900cattatggag tcagtaatgt gttcgaacag gtttctatag gaaactcggt
taagggtatg 960tgataacacg gggccgacca aaacgctaag atttcacatt
gctatttttc aagtcacctt 1020ttcctagtac atttttacag tcgtgtcatg
ttacatgtgt accttaatcc ggtcatcata 1080gttgagttga cgacaattta
ccgtcagatc gtcttcttct ccatcattaa tctagactct 1140taaagttact
attacgattt tggtagtatc atgtagactt acttagacat gtttaattaa
1200catgttctgg gttgatgctc gagctgttta cccgggtata tcctggtccc
tctcttaacc 1260tattcacccg cttatattat ccttgatatt ctgttcgtgt
aacattgtaa tcatctcgtt 1320ttaccttact gtgaaattct gtctatcaat
cgtttaattt tcttgttaaa ttcttatttt 1380gttatcagaa attagttagg
agtcctcccc tgggtcttta acattacgtg tcaaaattaa 1440cacctcccct
taagaagatg acattaagta gtggtgacaa attatcatga accttaccat
1500tattatgaac cttattatga tgtcccagtt tattgttata gtgtgaagtt
acgttttatt 1560ttgtttaata tttgtacacc gtccttcatc cttttcgtta
tatacgggga gggtaacttc 1620ctgtttaatc tacaagtagt ttataatgtc
ccgatgataa ttgttctcta ccaccattcc 1680tgtgcctgtg cttgctgtgg
ctctagaagt ctggacctcc tcctctatac tccctgttaa 1740cctcttcact
taatatattt atatttcatc attgttaact tggtaatcct catcgtgggt
1800ggttccgttt ctcttctcac cacgtctctc ttttttctaa taagtattac
tatcatcctc 1860cgaaccatcc aaattcttat caaaaacgac atgagagaca
tcacttatct caatccgtcc 1920ctattaaaaa taagatctta gctagggccc
ttagctaagc gctatcgact aatcaaaaac 1980aattgttttt acaccctctt
agattaatca aaaagaaatg tgttaactgc atgtactcag 2040actcaaggaa
caaaaacgat 2060143551PRTArtificial SequenceHIV1 gp120 + TM (with
ELDKWA epitopes) expressed by vP1319 infected cells 143Met Lys Glu
Gln Lys Thr Val Ala Met Arg Val Lys Glu Lys Tyr Gln1 5 10 15His Leu
Trp Arg Trp Gly Trp Arg Trp Gly Thr Met Leu Leu Gly Met 20 25 30Leu
Met Ile Cys Ser Ala Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr 35 40
45Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser
50 55 60Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala Thr
His65 70 75 80Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Val Glu
Leu Val Asn 85 90 95Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asn Met
Val Glu Gln Met 100 105 110His Glu Asp Ile Ile Ser Leu Trp Asp Gln
Ser Leu Lys Pro Cys Val 115 120 125Lys Leu Thr Pro Leu Cys Val Thr
Leu Asn Cys Thr Asp Leu Arg Asn 130 135 140Thr Thr Asn Thr Asn Asn
Ser Thr Ala Asn Asn Asn Ser Asn Ser Glu145 150 155 160Gly Thr Ile
Lys Gly Gly Glu Met Lys Asn Cys Ser Phe Asn Ile Thr 165 170 175Thr
Ser Ile Arg Asp Lys Met Gln Lys Glu Tyr Ala Leu Leu Tyr Lys 180 185
190Leu Asp Ile Val Ser Ile Asn Asn Asp Ser Thr Ser Tyr Arg Leu Ile
195 200 205Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile
Ser Phe 210 215 220Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly
Phe Ala Ile Leu225 230 235 240Lys Cys Asn Asp Lys Lys Phe Ser Gly
Lys Gly Ser Cys Lys Asn Val 245 250 255Ser Thr Val Gln Cys Thr His
Gly Ile Arg Pro Val Val Ser Thr Gln 260 265 270Leu Leu Leu Asn Gly
Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser 275 280 285Glu Asn Phe
Asn Asp Asn Ala Lys Thr Ile Ile Val His Leu Asn Glu 290 295 300Ser
Val Gln Ile Asn Cys Thr Arg Pro Asn Tyr Glu Leu Asp Lys Trp305 310
315 320Ala His Ile Gly Pro Gly Arg Glu Leu Asp Lys Trp Ala Asn Ile
Ile 325 330 335Gly Thr Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala
Lys Trp Asn 340 345 350Asp Thr Leu Arg Gln Ile Val Ser Lys Leu Lys
Glu Gln Phe Lys Asn 355 360 365Lys Thr Ile Val Phe Asn Gln Ser Ser
Gly Gly Asp Pro Glu Ile Val 370 375 380Met His Ser Phe Asn Cys Gly
Gly Glu Phe Phe Tyr Cys Asn Ser Ser385 390 395 400Pro Leu Phe Asn
Ser Thr Trp Asn Gly Asn Asn Thr Trp Asn Asn Thr 405 410 415Thr Gly
Ser Asn Asn Asn Ile Thr Leu Gln Cys Lys Ile Lys Gln Ile 420 425
430Ile Asn Met Trp Gln Glu Val Gly Lys Ala Ile Tyr Ala Pro Pro Ile
435 440 445Glu Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu
Leu Thr 450 455 460Arg Asp Gly Gly Lys Asp Thr Asp Thr Asn Asp Thr
Glu Ile Phe Arg465 470 475 480Pro Gly Gly Gly Asp Met Arg Asp Asn
Trp Arg Ser Glu Leu Tyr Lys 485 490 495Tyr Lys Val Val Thr Ile Glu
Pro Leu Gly Val Ala Pro Thr Lys Ala 500 505 510Lys Arg Arg Val Val
Gln Arg Glu Lys Arg Leu Phe Ile Met Ile Val 515 520 525Gly Gly Leu
Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Val Val 530 535 540Asn
Arg Val Arg Gln Gly Asn545 55014423DNAArtificial SequencePrimer
referred to as HIVP72 144ttattaccat tccaagtact att
23145109DNAArtificial SequencePrimer referred to as HIVP74
145tctgtacaaa ttaattgtac aagacccaac tacgagctcg acaaatgggc
ccatatagga 60ccagggagag aattggataa gtgggcgaat ataataggaa ctataagac
10914620DNAArtificial SequenceOligonucleotide referred to as HIVMN6
146gggttattaa tgatctgtag 201476PRTHuman immunodeficiency virus type
1 147Glu Leu Asp Lys Trp Ala1 51484PRTHuman immunodeficiency virus
type 1 148Leu Asp Lys Trp11495PRTNewcastle disease virus 149Arg Arg
Gln Arg Arg1 5
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