U.S. patent application number 10/646628 was filed with the patent office on 2004-07-29 for mva expressing modified hiv envelope, gag, and pol genes.
Invention is credited to Earl, Patricia, Moss, Bernard, Robinson, Harriet L., Wyatt, Linda.
Application Number | 20040146528 10/646628 |
Document ID | / |
Family ID | 23048178 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146528 |
Kind Code |
A1 |
Moss, Bernard ; et
al. |
July 29, 2004 |
MVA expressing modified HIV envelope, gag, and pol genes
Abstract
The invention provides modified virus Ankara (MVA), a
replication-deficient strain of vaccinia virus, expressing human
immunodeficiency virus (HIV) env, gag, and pol genes.
Inventors: |
Moss, Bernard; (Bethesda,
MD) ; Wyatt, Linda; (Rockville, MD) ; Earl,
Patricia; (Chevy Chase, MD) ; Robinson, Harriet
L.; (Atlanta, GA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
23048178 |
Appl. No.: |
10/646628 |
Filed: |
August 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646628 |
Aug 22, 2003 |
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PCT/US02/06713 |
Mar 1, 2002 |
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60274434 |
Mar 8, 2001 |
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Current U.S.
Class: |
424/199.1 ;
435/235.1 |
Current CPC
Class: |
A61K 39/21 20130101;
C12N 15/86 20130101; C12N 2740/15022 20130101; C12N 2740/15034
20130101; A61K 2039/525 20130101; A61K 2039/57 20130101; C12N
2740/16034 20130101; C12N 2830/00 20130101; C12N 2710/24171
20130101; C12N 2740/16122 20130101; A61P 31/18 20180101; C12N
2830/15 20130101; C12N 2740/16234 20130101; A61K 2039/545 20130101;
A61K 2039/70 20130101; C12N 2740/16134 20130101; C12N 2740/16222
20130101; C12N 2830/60 20130101; C12N 2710/24143 20130101; A61K
39/12 20130101; A61P 43/00 20180101; C07K 14/005 20130101; A61K
2039/53 20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/199.1 ;
435/235.1 |
International
Class: |
A61K 039/12; C12N
007/00 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising a recombinant MVA virus
expressing an HIV env, gag, and pol gene or modified gene thereof
for production of an HIV Env, Gag, and Pol antigen by expression
from said recombinant MVA virus, wherein said HIV env gene is
modified to encode an HIV Env protein composed of gp120 and the
membrane-spanning and ectodomain of gp41 but lacking part or all of
the cytoplasmic domain of gp41, and a pharmaceutically acceptable
carrier.
2. The pharmaceutical composition of claim 1, wherein said HIV pol
gene or modified gene thereof is modified to inactivate reverse
transcriptase and integrase.
3. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
A.
4. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
B.
5. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
C.
6. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
D.
7. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
E.
8. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
F.
9. The pharmaceutical composition of claim 1, wherein said HIV env,
gag, or pol gene or modified gene thereof is taken from clade
G.
10. The pharmaceutical composition of claim 1, wherein said HIV
env, gag, or pol gene or modified gene thereof is taken from clade
H.
11. The pharmaceutical composition of claim 1, wherein said HIV
env, gag, or pol gene or modified gene thereof is taken from clade
J.
12. The pharmaceutical composition of claim 1 wherein said HIV env,
gag, or pol gene or modified gene thereof is inserted at the site
of deletion III within the MVA genome.
13. The pharmaceutical composition of claim 1 wherein said HIV env,
gag, or pol gene or modified gene thereof is under transcriptional
initiation regulation of a H5-like early/late vaccinia virus
promoter.
14. The pharmaceutical composition of claim 1 wherein recombinant
MVA virus additionally expresses an additional HIV gene or modified
gene thereof for production of an HIV antigen by expression from
said recombinant MVA virus, wherein said additional HIV gene is a
member selected from the group consisting of vif, vpr, tat, rev,
vpu, and nef.
15. MVA/HIV48 comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and
SEQ ID NO:5.
16. pLW-48 having SEQ ID NO:1.
17. A plasmid transfer vector having the sequence of pLW-48 (SEQ ID
NO:1) excluding the HIV env, gag, and pol genes.
18. pLW-48 (SEQ ID NO:1) wherein the HIV env, gag, and pol genes
have a sequence taken from another clade.
19. A poxvirus comprising a promoter selected from the group
consisting of m7.5 promoter having SEQ ID NO:10, Psyn II promoter
having SEQ ID NO:2, Psyn III promoter having SEQ ID NO:11, Psyn IV
promoter having SEQ ID NO:12, and Psyn V promoter having SEQ ID
NO:13.
20. A method of boosting a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of any of claims 1-15,
whereby a CD8.sup.+ T cell immune response to the antigen
previously primed in the primate is boosted.
21. The method of claim 20, wherein the primate is a human.
22. The method of claim 20, wherein administration of the
recombinant MVA virus is by needleless injection.
23. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a composition of any of claims 1-15,
whereby a CD8.sup.+ T cell immune response to the antigen in the
primate is induced.
24. The method of claim 23, wherein the primate is a human.
25. The method of claim 23, wherein administration of the
recombinant MVA virus is by needleless injection.
26. A method of inducing a CD8.sup.+ T cell immune response to an
HIV Env, Gag, or Pol antigen in a primate, the method comprising
provision in the primate of a priming composition comprising
nucleic acid encoding said antigen and then provision in the
primate of a boosting composition which comprises any of claims
1-15, whereby a CD8.sup.+ T cell immune response to the antigen is
induced.
27. The method of claim 26, wherein the primate is a human.
28. The method of claim 26, wherein administration of the
recombinant MVA virus is by needleless injection.
29. The method of claim 26, wherein the priming composition
comprises plasmid DNA encoding said antigen.
30. A method of making a composition of any of claims 1-15
comprising preparing a plasmid transfer vector encoding an HIV env,
gag, and pol gene or modified gene thereof, wherein said HIV env
gene is modified to encode an HIV Env protein composed of gp120 and
the membrane-spanning and ectodomain of gp41 but lacking part or
all of the cytoplasmic domain of gp41, and recombining said plasmid
transfer vector with a MVA virus to produce a composition of any of
claims 1-15.
Description
RELATED APPLICATIONS
[0001] This application is a continuation and claims the benefit of
priority of International Application No. PCT/US02/06713 filed Mar.
1, 2002, designating the United States of America and published in
English, which claims the benefit of priority of U.S. Provisional
Application No. 60/274,434 filed Mar. 8, 2001, both of which are
hereby expressly incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention provides modified vaccinia Ankara (MVA), a
replication-deficient strain of vaccinia virus, expressing human
immunodeficiency virus (HIV) env, gag, and pol genes.
BACKGROUND OF THE INVENTION
[0003] Cellular immunity plays an important role in the control of
immunodeficiency virus infections (P. J. Goulder et al. 1999 AIDS
13:S121). Recently, a DNA vaccine designed to enhance cellular
immunity by cytokine augmentation successfully contained a highly
virulent immunodeficiency virus challenge (D. H. Barouch et al.
2000 Science 290:486). Another promising approach to raising
cellular immunity is DNA priming followed by recombinant poxvirus
boosters (H. L. Robinson et al. 2000 AIDS Rev 2:105). This
heterologous prime/boost regimen induces 10- to 100-fold higher
frequencies of T cells than priming and boosting with DNA or
recombinant poxvirus vaccines alone. Previously, investigators
showed that boosting a DNA-primed response with a poxvirus was
superior to boosting with DNA or protein for the control of a
non-pathogenic immunodeficiency virus (H. L. Robinson et al. 1999
Nat Med 5:526). There is a need for the control of a pathogenic
immnunodeficiency virus.
SUMMARY OF THE INVENTION
[0004] Here we report that DNA priming followed by a recombinant
modified vaccinia Ankara (rMVA) booster has controlled a highly
pathogenic immunodeficiency virus challenge in a rhesus macaque
model. Both the DNA and rMVA components of the vaccine expressed
multiple immnunodeficiency virus proteins. Two DNA inoculations at
0 and 8 weeks and a single rMVA booster at 24 weeks effectively
controlled an intrarectal challenge administered seven months after
the booster. These findings are envisioned as indicating that a
relatively simple multiprotein DNA/MVA vaccine can help to control
the acquired immune deficiency syndrome (AIDS) epidemic. We also
report that inoculations of rMVA induce good immune responses even
without DNA priming.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1. Phylogenetic relationships of HIV-1 and HIV-2 based
on identity of pol gene sequences. SIV.sub.cpz and SIV.sub.smm are
subhuman primate lentiviruses recovered from a chimpanzee and sooty
mangabey monkey, respectively.
[0006] FIG. 2. Phylogenetic relationships of HIV-1 groups M, N and
O with four different SIV.sub.cpz isolates based on full-length pol
gene sequences. The bar indicates a genetic distance of 0.1 (10%
nucleotide divergence) and the asterisk positions group N HIV-1
isolates based on env sequences.
[0007] FIG. 3. Tropic and biologic properties of HIV-1
isolates.
[0008] FIG. 4. HIV-encoded proteins. The location of the HIV genes,
the sizes of primary translation products (in some cases
polyproteins), and the processed mature viral proteins are
indicated.
[0009] FIG. 5. Schematic representation of a mature HIV-1
virion.
[0010] FIG. 6. Linear representation of the HIV-1 Env glycoprotein.
The arrow indicates the site of gp160 cleavage to gp120 and gp41.
In gp120, cross-hatched areas represent variable domains (V.sub.1
to V.sub.5) and open boxes depict conserved sequences (C.sub.1 to
C.sub.5). In the gp41 ectodomain, several domains are indicated:
the N-terminal fusion peptide, and the two ectodomain helices (N-
and C-helix). The membrane-spanning domain is represented by a
black box. In the gp41 cytoplasmic domain, the Tyr-X-X-Leu (YXXL)
endocytosis motif (SEQ ID NO: 9) and two predicted helical domains
(helix-1 and -2) are shown. Amino acid numbers are indicated.
[0011] FIG. 7. Temporal frequencies of Gag-specific T cells. (A)
Gag-specific CD8 T cell responses raised by DNA priming and rMVA
booster immunizations. The schematic presents mean Gag-CM9-tetramer
data generated in the high-dose i.d. DNA-immunized animals. (B)
Gag-specific IFN-.gamma. ELISPOTs in A*01 (open bars) and non-A*01
(filled bars) macaques at various times before challenge and at two
weeks after challenge. Three pools of 10 to 13 Gag peptides
(22-mers overlapping by 12) were used for the analyses. The numbers
above data bars represent the arithmetic mean.+-.SD for the
ELISPOTs within each group. The numbers at the top of the graphs
designate individual animals. *, data not available; #, <20
ELISPOTs per 1.times.10.sup.6 peripheral blood mononuclear cells
(PBMC). Temporal data for Gag-CM9-Mamu-A*01 tetramer-specific T
cells can be found in FIG. 12.
[0012] FIG. 8. Temporal viral loads, CD4 counts, and survival after
challenge of vaccinated and control animals. (A) Geometric mean
viral loads and (B) geometric mean CD4 counts. (C) Survival curve
for vaccinated and control animals. The dotted line represents all
24 vaccinated animals. (D) Viral loads and (E) CD4 counts for
individual animals in the vaccine and control groups. The key to
animal numbers is presented in (E). Assays for the first 12 weeks
after challenge had a detection level of 1000 copies of RNA per
milliliter of plasma. Animals with loads below 1000 were scored
with a load of 500. For weeks 16 and 20, the detection level was
300 copies of RNA per milliliter. Animals with levels of virus
below 300 were scored at 300.
[0013] FIG. 9. Postchallenge T cell responses in vaccine and
control groups. (A) Temporal tetramer.sup.+ cells (dashed line) and
viral loads (solid line). (B) Intracellular cytokine assays for
IFN-.gamma. production in response to stimulation with the Gag-CM9
peptide at two weeks after challenge. This ex vivo assay allows
evaluation of the functional status of the peak postchallenge
tetramer.sup.+ cells displayed in FIG. 7A. (C) Proliferation assay
at 12 weeks after challenge. Gag-Pol-Env (open bars) and Gag-Pol
(hatched bars) produced by transient transfections were used for
stimulation. Supernatants from mock-transfected cultures served as
control antigen. Stimulation indices are the growth of cultures in
the presence of viral antigens divided by the growth of cultures in
the presence of mock antigen.
[0014] FIG. 10. Lymph node histomorphology at 12 weeks after
challenge. (A) Typical lymph node from a vaccinated macaque showing
evidence of follicular hyperplasia characterized by the presence of
numerous secondary follicles with expanded germinal centers and
discrete dark and light zones. (B) Typical lymph node from an
infected control animal showing follicular depletion and
paracortical lymphocellular atrophy. (C) A representative lymph
node from an age-matched, uninfected macaque displaying nonreactive
germinal centers. (D) The percentage of the total lymph node area
occupied by germinal centers was measured to give a non-specific
indicator of follicular hyperplasia. Data for uninfected controls
are for four age-matched rhesus macaques.
[0015] FIG. 11. Temporal antibody responses. Micrograms of total
Gag (A) or Env (B) antibody were determined with ELISAs. The titers
of neutralizing antibody for SHIV-89.6 (C) and SHIV-89.6P (D) were
determined with MT-2 cell killing and neutral red staining (D. C.
Montefiori et al. 1988 J Clin Microbiol 26:231). Titers are the
reciprocal of the serum dilution giving 50% neutralization of the
indicated viruses grown in human PBMC. Symbols for animals are the
same as in FIG. 8.
[0016] FIG. 12. Gag-CM9-Mamu-A*01 tetramer-specific T cells in
Mamu-A*01 vaccinated and control macaques at various times before
challenge and at two weeks after challenge. The number at the upper
right corner of each plot represents the frequency of
tetramer-specific CD8 T cells as a % of total CD8 T cells. The
numbers above each column of FACS data designate individual
animals.
[0017] FIG. 13. Map of plasmid transfer vector pLW-48.
[0018] FIG. 14. Sequence of plasmid transfer vector pLW-48.
[0019] FIG. 15. Sequences of plasmid transfer vector pLW-48, Psy II
promoter (which controls ADA envelope expression), ADA envelope
truncated, PmH5 promoter (which controls HXB2 gag pol expression),
and HXB2 gag pol (with safety mutations, .DELTA. integrase).
[0020] FIG. 16. Plasmid transfer vector pLW-48 and making MVA
recombinant virus MVA/HIV 48.
[0021] FIG. 17. A clade B gag pol.
[0022] FIG. 18. Sequence of new Psyn II promoter.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] Recombinant MVA Virus
[0024] Vaccinia virus, a member of the genus Orthopoxvirus in the
family of Poxviridae, was used as live vaccine to immunize against
the human smallpox disease. Successful worldwide vaccination with
vaccinia virus culminated in the eradication of variola virus, the
causative agent of the smallpox (The global eradication of
smallpox. Final report of the global commission for the
certification of smallpox eradication. History of Public Health,
No. 4, Geneva: World Health Organization, 1980). Since that WHO
declaration, vaccination has been universally discontinued except
for people at high risk of poxvirus infections (e.g. laboratory
workers).
[0025] More recently, vaccinia viruses have also been used to
engineer viral vectors for recombinant gene expression and for the
potential use as recombinant live vaccines (Mackett, M. et al. 1982
PNAS USA 79:7415-7419; Smith, G. L. et al. 1984 Biotech Genet Engin
Rev 2:383-407). This entails DNA sequences (genes) which code for
foreign antigens being introduced, with the aid of DNA
recombination techniques, into the genome of the vaccinia viruses.
If the gene is integrated at a site in the viral DNA which is
non-essential for the life cycle of the virus, it is possible for
the newly produced recombinant vaccinia virus to be infectious,
that is to say able to infect foreign cells and thus to express the
integrated DNA sequence (EP Patent Applications No. 83,286 and No.
110,385). The recombinant vaccinia viruses prepared in this way can
be used, on the one hand, as live vaccines for the prophylaxis of
infectious diseases, on the other hand, for the preparation of
heterologous proteins in eukaryotic cells.
[0026] For vector applications health risks would be lessened by
the use of a highly attenuated vaccinia virus strain. Several such
strains of vaccinia virus were especially developed to avoid
undesired side effects of smallpox vaccination. Thus, the modified
vaccinia Ankara (MVA) has been generated by long-term serial
passages of the Ankara strain of vaccinia virus (CVA) on chicken
embryo fibroblasts (for review see Mayr, A. et al. 1975 Infection
3:6-14; Swiss Patent No. 568,392). The MVA virus is publicly
available from American Type Culture Collection as ATCC No.
VR-1508. MVA is distinguished by its great attenuation, that is to
say by diminished virulence and ability to replicate in primate
cells while maintaining good immunogenicity. The MVA virus has been
analyzed to determine alterations in the genome relative to the
parental CVA strain. Six major deletions of genomic DNA (deletion
I, II, III, IV, V, and VI) totaling 31,000 base pairs have been
identified (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038). The
resulting MVA virus became severely host cell restricted to avian
cells.
[0027] Furthermore, MVA is characterized by its extreme
attenuation. When tested in a variety of animal models, MVA was
proven to be avirulent even in immunosuppressed animals. More
importantly, the excellent properties of the MVA strain have been
demonstrated in extensive clinical trials (Mayr A. et al. 1978
Zentralbl Bakteriol [B] 167:375-390; Stickl et al. 1974 Dtsch Med
Wschr 99:2386-2392). During these studies in over 120,000 humans,
including high-risk patients, no side effects were associated with
the use of MVA vaccine.
[0028] MVA replication in human cells was found to be blocked late
in infection preventing the assembly to mature infectious virions.
Nevertheless, MVA was able to express viral and recombinant genes
at high levels even in non-permissive cells and was proposed to
serve as an efficient and exceptionally safe gene expression vector
(Sutter, G. and Moss, B. 1992 PNAS USA 89:10847-10851).
Additionally, novel vaccinia vector vaccines were established on
the basis of MVA having foreign DNA sequences inserted at the site
of deletion III within the MVA genome (Sutter, G. et al. 1994
Vaccine 12:1032-1040).
[0029] The recombinant MVA vaccinia viruses can be prepared as set
out hereinafter. A DNA-construct which contains a DNA-sequence
which codes for a foreign polypeptide flanked by MVA DNA sequences
adjacent to a naturally occurring deletion, e.g. deletion III, or
other non-essential sites, within the MVA genome, is introduced
into cells infected with MVA, to allow homologous recombination.
Once the DNA-construct has been introduced into the eukaryotic cell
and the foreign DNA has recombined with the viral DNA, it is
possible to isolate the desired recombinant vaccinia virus in a
manner known per se, preferably with the aid of a marker. The
DNA-construct to be inserted can be linear or circular. A plasmid
or polymerase chain reaction product is preferred. The
DNA-construct contains sequences flanking the left and the right
side of a naturally occurring deletion, e.g. deletion III, within
the MVA genome. The foreign DNA sequence is inserted between the
sequences flanking the naturally occurring deletion. For the
expression of a DNA sequence or gene, it is necessary for
regulatory sequences, which are required for the transcription of
the gene, to be present on the DNA. Such regulatory sequences
(called promoters) are known to those skilled in the art, and
include for example those of the vaccinia 11 kDa gene as are
described in EP-A-198,328, and those of the 7.5 kDa gene
(EP-A-110,385). The DNA-construct can be introduced into the MVA
infected cells by transfection, for example by means of calcium
phosphate precipitation (Graham et al. 1973 Virol 52:456-467;
Wigler et al. 1979 Cell 16:777-785), by means of electroporation
(Neumann et al. 1982 EMBO J 1:841-845), by microinjection
(Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of
liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by
means of spheroplasts (Schaffner 1980 PNAS USA 77:2163-2167) or by
other methods known to those skilled in the art.
[0030] HIVs and Their Replication
[0031] The etiological agent of acquired immune deficiency syndrome
(AIDS) is recognized to be a retrovirus exhibiting characteristics
typical of the lentivirus genus, referred to as human
immunodeficiency virus (HIV). The phylogenetic relationships of the
human lentiviruses are shown in FIG. 1. HIV-2 is more closely
related to SIV.sub.smm, a virus isolated from sooty mangabey
monkeys in the wild, than to HIV-1. It is currently believed that
HIV-2 represents a zoonotic transmission of SIV.sub.smm to man. A
series of lentiviral isolates from captive chimpanzees, designated
SIV.sub.cpz, are close genetic relatives of HIV-1.
[0032] The earliest phylogenetic analyses of HIV-1 isolates focused
on samples from Europe/North America and Africa; discrete clusters
of viruses were identified from these two areas of the world.
Distinct genetic subtypes or clades of HIV-1 were subsequently
defined and classified into three groups: M (major); O (outlier);
and N (non-M or O) (FIG. 2). The M group of HIV-1, which includes
over 95% of the global virus isolates, consists of at least eight
discrete clades (A, B, C, D, F, G, H, and J), based on the sequence
of complete viral genomes. Members of HIV-1 group O have been
recovered from individuals living in Cameroon, Gabon, and
Equatorial Guinea; their genomes share less than 50% identity in
nucleotide sequence with group M viruses. The more recently
discovered group N HIV-1strains have been identified in infected
Cameroonians, fail to react serologically in standard whole-virus
enzyme-linked immunosorbent assay (ELISA), yet are readily
detectable by conventional Western blot analysis.
[0033] Most current knowledge about HIV-1 genetic variation comes
from studies of group M viruses of diverse geographic origin. Data
collected during the past decade indicate that the HIV-1 population
present within an infected individual can vary from 6% to 10% in
nucleotide sequence. HIV-1 isolates within a clade may exhibit
nucleotide distances of 15% in gag and up to 30% in gp120 coding
sequences. Interclade genetic variation may range between 30% and
40% depending on the gene analyzed.
[0034] All of the HIV-1 group M subtypes can be found in Africa.
Clade A viruses are genetically the most divergent and were the
most common HIV-1 subtype in Africa early in the epidemic. With the
rapid spread of HIV-1 to southern Africa during the mid to late
1990s, clade C viruses have become the dominant subtype and now
account for 48% of HIV-1 infections worldwide. Clade B viruses, the
most intensively studied HIV-1 subtype, remain the most prevalent
isolates in Europe and North America.
[0035] High rates of genetic recombination are a hallmark of
retroviruses. It was initially believed that simultaneous
infections by genetically diverse virus strains were not likely to
be established in individuals at risk for HIV-1. By 1995, however,
it became apparent that a significant fraction of the HIV-1 group M
global diversity included interclade viral recombinants. It is now
appreciated that HIV-1 recombinants will be found in geographic
areas such as Africa, South America, and Southeast Asia, where
multiple HIV-1 subtypes coexist and may account for more than 10%
of circulating HIV-1 strains. Molecularly, the genomes of these
recombinant viruses resemble patchwork mosaics, with juxtaposed
diverse HIV-1 subtype segments, reflecting the multiple crossover
events contributing to their generation. Most HIV-1 recombinants
have arisen in Africa and a majority contain segments originally
derived from clade A viruses. In Thailand, for example, the
composition of the predominant circulating strain consists of a
clade A gag plus pol gene segment and a clade E env gene. Because
the clade E env gene in Thai HIV-1 strains is closely related to
the lade E env present in virus isolates from the Central African
Republic, it is believed that the original recombination event
occurred in Africa, with the subsequent introduction of a
descendent virus into Thailand. Interestingly, no full-length HIV-1
subtype E isolate (i.e., with subtype E gag, pol, and env genes)
has been reported to date.
[0036] The discovery that .alpha. and .beta. chemokine receptors
function as coreceptors for virus fusion and entry into susceptible
CD4.sup.+ cells has led to a revised classification scheme for
HIV-1 (FIG. 3). Isolates can now be grouped on the basis of
chemokine receptor utilization in fusion assays in which HIV-1
gp120 and CD4.sup.+ coreceptor proteins are expressed in separate
cells. As indicated in FIG. 3, HIV-1 isolates using the CXCR4
receptor (now designated X4 viruses) are usually T cell line
(TCL)-tropic syncytium inducing (SI) strains, whereas those
exclusively utilizing the CCR5 receptor (R5 viruses) are
predominantly macrophage (M)-tropic and non-syncytium inducing
(NSI). The dual-tropic R5/X4 strains, which may comprise the
majority of patient isolates and exhibit a continuum of tropic
phenotypes, are frequently SI.
[0037] As is the case for all replication-competent retroviruses,
the three primary HIV-1 translation products, all encoding
structural proteins, are initially synthesized as polyprotein
precursors, which are subsequently processed by viral or cellular
proteases into mature particle-associated proteins (FIG. 4). The
55-kd Gag precursor Pr55.sup.Gag is cleaved into the matrix (MA),
capsid (CA), nucleocapsid (NC), and p6 proteins. Autocatalysis of
the 160-kd Gag-Pol polyprotein, Pr160.sup.Gag-Pol, gives rise to
the protease (PR), the heterodimeric reverse transcriptase (RT),
and the integrase (IN) proteins, whereas proteolytic digestion by a
cellular enzyme(s) converts the glycosylated 160-kd Env precursor
gp160 to the gp120 surface (SU) and gp41 transmembrane (TM)
cleavage products. The remaining six HIV-1-encoded proteins (Vif,
Vpr, Tat, Rev, Vpu, and Nef) are the primary translation products
of spliced mRNAs.
[0038] Gag
[0039] The Gag proteins of HIV, like those of other retroviruses,
are necessary and sufficient for the formation of noninfectious,
virus-like particles. Retroviral Gag proteins are generally
synthesized as polyprotein precursors; the HIV-1 Gag precursor has
been named, based on its apparent molecular mass, Pr55.sup.Gag. As
noted previously, the mRNA for Pr55.sup.Gag is the unspliced 9.2-kb
transcript (FIG. 4) that requires Rev for its expression in the
cytoplasm. When the pol ORF is present, the viral protease (PR)
cleaves Pr55.sup.Gag during or shortly after budding from the cell
to generate the mature Gag proteins p17 (MA), p24 (CA), p7 (NC),
and p6 (see FIG. 4). In the virion, MA is localized immediately
inside the lipid bilayer of the viral envelope, CA forms the outer
portion of the cone-shaped core structure in the center of the
particle, and NC is present in the core in a ribonucleoprotein
complex with the viral RNA genome (FIG. 5).
[0040] The HIV Pr55.sup.Gag precursor oligomerizes following its
translation and is targeted to the plasma membrane, where particles
of sufficient size and density to be visible by EM are assembled.
Formation of virus-like particles by Pr55.sup.Gag is a
self-assembly process, with critical Gag-Gag interactions taking
place between multiple domains along the Gag precursor. The
assembly of virus-like particles does not require the participation
of genomic RNA (although the presence of nucleic acid appears to be
essential), pol-encoded enzymes, or Env glycoproteins, but the
production of infectious virions requires the encapsidation of the
viral RNA genome and the incorporation of the Env glycoproteins and
the Gag-Pol polyprotein precursor Pr160.sup.Gag-Pol.
[0041] Pol
[0042] Downstream of gag lies the most highly conserved region of
the HIV genome, the pol gene, which encodes three enzymes: PR, RT,
and IN (see FIG. 4). RT and IN are required, respectively, for
reverse transcription of the viral RNA genome to a double-stranded
DNA copy, and for the integration of the viral DNA into the host
cell chromosome. PR plays a critical role late in the life cycle by
mediating the production of mature, infectious virions. The pol
gene products are derived by enzymatic cleavage of a 160-kd Gag-Pol
fusion protein, referred to as Pr160.sup.Gag-Pol. This fusion
protein is produced by ribosomal frameshifting during translation
of Pr55.sup.Gag (see FIG. 4). The frame-shifting mechanism for
Gag-Pol expression, also utilized by many other retroviruses,
ensures that the pol-derived proteins are expressed at a low level,
approximately 5% to 10% that of Gag. Like Pr55.sup.Gag, the
N-terminus of Pr160.sup.Gag-Pol is myristylated and targeted to the
plasma membrane.
[0043] Protease
[0044] Early pulse-chase studies performed with avian retroviruses
clearly indicated that retroviral Gag proteins are initially
synthesized as polyprotein precursors that are cleaved to generate
smaller products. Subsequent studies demonstrated that the
processing function is provided by a viral rather than a cellular
enzyme, and that proteolytic digestion of the Gag and Gag-Pol
precursors is essential for virus infectivity. Sequence analysis of
retroviral PRs indicated that they are related to cellular
"aspartic" proteases such as pepsin and renin. Like these cellular
enzymes, retroviral PRs use two apposed Asp residues at the active
site to coordinate a water molecule that catalyzes the hydrolysis
of a peptide bond in the target protein. Unlike the cellular
aspartic proteases, which function as pseudodimers (using two folds
within the same molecule to generate the active site), retroviral
PRs function as true dimers. X-ray crystallographic data from HIV-1
PR indicate that the two monomers are held together in part by a
four-stranded antiparallel .beta.-sheet derived from both N- and
C-terminal ends of each monomer. The substrate-binding site is
located within a cleft formed between the two monomers. Like their
cellular homologs, the HIV PR dimer contains flexible "flaps" that
overhang the binding site and may stabilize the substrate within
the cleft; the active-site Asp residues lie in the center of the
dimer. Interestingly, although some limited amino acid homology is
observed surrounding active-site residues, the primary sequences of
retroviral PRs are highly divergent, yet their structures are
remarkably similar.
[0045] Reverse Transcriptase
[0046] By definition, retroviruses possess the ability to convert
their single-stranded RNA genomes into double-stranded DNA during
the early stages of the infection process. The enzyme that
catalyzes this reaction is RT, in conjunction with its associated
RNaseH activity. Retroviral RTs have three enzymatic activities:
(a) RNA-directed DNA polymerization (for minus-strand DNA
synthesis), (b) RNaseH activity (for the degradation of the tRNA
primer and genomic RNA present in DNA-RNA hybrid intermediates),
and (c) DNA-directed DNA polymerization (for second- or plus-strand
DNA synthesis).
[0047] The mature HIV-1 RT holoenzyme is a heterodimer of 66 and 51
kd subunits. The 51-kd subunit (p51) is derived from the 66-kd
(p66) subunit by proteolytic removal of the C-terminal 15-kd RNaseH
domain of p66 by PR (see FIG. 4). The crystal structure of HIV-1 RT
reveals a highly asymmetric folding in which the orientations of
the p66 and p51 subunits differ substantially. The p66 subunit can
be visualized as a right hand, with the polymerase active site
within the palm, and a deep template-binding cleft formed by the
palm, fingers, and thumb subdomains. The polymerase domain is
linked to RNaseH by the connection subdomain. The active site,
located in the palm, contains three critical Asp residues (110,
185, and 186) in close proximity, and two coordinated Mg.sup.2+
ions. Mutation of these Asp residues abolishes RT polymerizing
activity. The orientation of the three active-site Asp residues is
similar to that observed in other DNA polymerases (e.g., the Klenow
fragment of E. coli DNA poll). The p51 subunit appears to be rigid
and does not form a polymerizing cleft; Asp 110, 185, and 186 of
this subunit are buried within the molecule. Approximately 18 base
pairs of the primer-template duplex lie in the nucleic acid binding
cleft, stretching from the polymerase active site to the RNaseH
domain.
[0048] In the RT-primer-template-dNTP structure, the presence of a
dideoxynucleotide at the 3' end of the primer allows visualization
of the catalytic complex trapped just prior to attack on the
incoming dNTP. Comparison with previously obtained structures
suggests a model whereby the fingers close in to trap the template
and dNTP prior to nucleophilic attack of the 3'-OH of the primer on
the incoming dNTP. After the addition of the incoming dNTP to the
growing chain, it has been proposed that the fingers adopt a more
open configuration, thereby releasing the pyrophosphate and
enabling RT to bind the next dNTP. The structure of the HIV-1
RNaseH has also been determined by x-ray crystallography; this
domain displays a global folding similar to that of E. coli
RNaseH.
[0049] Integrase
[0050] A distinguishing feature of retrovirus replication is the
insertion of a DNA copy of the viral genome into the host cell
chromosome following reverse transcription. The integrated viral
DNA (the provirus) serves as the template for the synthesis of
viral RNAs and is maintained as part of the host cell genome for
the lifetime of the infected cell. Retroviral mutants deficient in
the ability to integrate generally fail to establish a productive
infection.
[0051] The integration of viral DNA is catalyzed by integrase, a
32-kd protein generated by PR-mediated cleavage of the C-terminal
portion of the HIV-1 Gag-Pol polyprotein (see FIG. 4).
[0052] Retroviral IN proteins are composed of three structurally
and functionally distinct domains: an N-terminal,
zinc-finger-containing domain, a core domain, and a relatively
nonconserved C-terminal domain. Because of its low solubility, it
has not yet been possible to crystallize the entire 288-amino-acid
HIV-1 IN protein. However, the structure of all three domains has
been solved independently by x-ray crystallography or NMR methods.
The crystal structure of the core domain of the avian sarcoma virus
IN has also been determined. The N-terminal domain (residues 1 to
55), whose structure was solved by NMR spectroscopy, is composed of
four helices with a zinc coordinated by amino acids His-12, His-16,
Cys-40, and Cys-43. The structure of the N-terminal domain is
reminiscent of helical DNA binding proteins that contain a
so-called helix-turn-helix motif; however, in the HIV-1 structure
this motif contributes to dimer formation. Initially, poor
solubility hampered efforts to solve the structure of the core
domain. However, attempts at crystallography were successful when
it was observed that a Phe-to-Lys change at IN residue 185 greatly
increased solubility without disrupting in vitro catalytic
activity. Each monomer of the HIV-1 IN core domain (IN residues 50
to 212) is composed of a five-stranded .beta.-sheet flanked by
helices; this structure bears striking resemblance to other
polynucleotidyl transferases including RNaseH and the bacteriophage
MuA transposase. Three highly conserved residues are found in
analogous positions in other polynucleotidyl transferases; in HIV-1
IN these are Asp-64, Asp-116 and Glu-152, the so-called D,D-35-E
motif. Mutations at these positions block HIV IN function both in
vivo and in vitro. The close proximity of these three amino acids
in the crystal structure of both avian sarcoma virus and HIV-1 core
domains supports the hypothesis that these residues play a central
role in catalysis of the polynucleotidyl transfer reaction that is
at the heart of the integration process. The C-terminal domain,
whose structure has been solved by NMR methods, adopts a
five-stranded .beta.-barrel folding topology reminiscent of a Src
homology 3 (SH3) domain. Recently, the x-ray structures of SIV and
Rous sarcoma virus IN protein fragments encompassing both the core
and C-terminal domains have been solved.
[0053] Env
[0054] The HIV Env glycoproteins play a major role in the virus
life cycle. They contain the determinants that interact with the
CD4 receptor and coreceptor, and they catalyze the fusion reaction
between the lipid bilayer of the viral envelope and the host cell
plasma membrane. In addition, the HIV Env glycoproteins contain
epitopes that elicit immune responses that are important from both
diagnostic and vaccine development perspectives.
[0055] The HIV Env glycoprotein is synthesized from the singly
spliced 4.3-kb Vpu/Env bicistronic mRNA (see FIG. 4); translation
occurs on ribosomes associated with the rough endoplasmic reticulum
(ER). The 160-kd polyprotein precursor (gp160) is an integral
membrane protein that is anchored to cell membranes by a
hydrophobic stop-transfer signal in the domain destined to be the
mature TM Env glycoprotein, gp41 (FIG. 6). The gp160 is
cotranslationally glycosylated, forms disulfide bonds, and
undergoes oligomerization in the ER. The predominant oligomeric
form appears to be a trimer, although dimers and tetramers are also
observed. The gp160 is transported to the Golgi, where, like other
retroviral envelope precursor proteins, it is proteolytically
cleaved by cellular enzymes to the mature SU glycoprotein gp120 and
TM glycoprotein gp41 (see FIG. 6). The cellular enzyme responsible
for cleavage of retroviral Env precursors following a highly
conserved Lys/Arg-X-Lys/Arg-Arg motif is furin or a furin-like
protease, although other enzymes may also catalyze gp160
processing. Cleavage of gp160 is required for Env-induced fusion
activity and virus infectivity. Subsequent to gp160 cleavage, gp120
and gp41 form a noncovalent association that is critical for
transport of the Env complex from the Golgi to the cell surface.
The gp120-gp41 interaction is fairly weak, and a substantial amount
of gp120 is shed from the surface of Env-expressing cells.
[0056] The HIV Env glycoprotein complex, in particular the SU
(gp120) domain, is very heavily glycosylated; approximately half
the molecular mass of gp160 is composed of oligosaccharide side
chains. During transport of Env from its site of synthesis in the
ER to the plasma membrane, many of the side chains are modified by
the addition of complex sugars. The numerous oligosaccharide side
chains form what could be imagined as a sugar cloud obscuring much
of gp120 from host immune recognition. As shown in FIG. 6, gp120
contains interspersed conserved (C.sub.1 to C.sub.5) and variable
(V.sub.1 to V.sub.5) domains. The Cys residues present in the
gp120s of different isolates are highly conserved and form
disulfide bonds that link the first four variable regions in large
loops.
[0057] A primary function of viral Env glycoproteins is to promote
a membrane fusion reaction between the lipid bilayers of the viral
envelope and host cell membranes. This membrane fusion event
enables the viral core to gain entry into the host cell cytoplasm.
A number of regions in both gp120 and gp41 have been implicated,
directly or indirectly, in Env-mediated membrane fusion. Studies of
the HA.sub.2 hemagglutinin protein of the orthomyxoviruses and the
F protein of the paramyxoviruses indicated that a highly
hydrophobic domain at the N-terminus of these proteins, referred to
as the fusion peptide, plays a critical role in membrane fusion.
Mutational analyses demonstrated that an analogous domain was
located at the N-terminus of the HIV-1, HIV-2, and SIV TM
glycoproteins (see FIG. 6). Nonhydrophobic substitutions within
this region of gp41 greatly reduced or blocked syncytium formation
and resulted in the production of noninfectious progeny
virions.
[0058] C-terminal to the gp41 fusion peptide are two amphipathic
helical domains (see FIG. 6) which play a central role in membrane
fusion. Mutations in the N-terminal helix (referred to as the
N-helix), which contains a Leu zipper-like heptad repeat motif,
impair infectivity and membrane fusion activity, and peptides
derived from these sequences exhibit potent antiviral activity in
culture. The structure of the ectodomain of HIV-1 and SIV gp41, the
two helical motifs in particular, has been the focus of structural
analyses in recent years. Structures were determined by x-ray
crystallography or NMR spectroscopy either for fusion proteins
containing the helical domains, a mixture of peptides derived from
the N- and C-helices, or in the case of the SIV structure, the
intact gp41 ectodomain sequence from residue 27 to 149. These
studies obtained fundamentally similar trimeric structures, in
which the two helical domains pack in an antiparallel fashion to
generate a six-helix bundle. The N-helices form a coiled-coil in
the center of the bundle, with the C-helices packing into
hydrophobic grooves on the outside.
[0059] In the steps leading to membrane fusion CD4 binding induces
conformation changes in Env that facilitate coreceptor binding.
Following the formation of a ternary gp120/CD4/coreceptor complex,
gp41 adopts a hypothetical conformation that allows the fusion
peptide to insert into the target lipid bilayer. The formation of
the gp41 six-helix bundle (which involves antiparallel interactions
between the gp41 N- and C-helices) brings the viral and cellular
membranes together and membrane fusion takes place.
[0060] Use of Recombinant MVA Virus To Boost CD+8 Cell Immune
Response
[0061] The present invention relates to generation of a CD8.sup.+ T
cell immune response against an antigen and also eliciting an
antibody response. More particularly, the present invention relates
to "prime and boost" immunization regimes in which the immune
response induced by administration of a priming composition is
boosted by administration of a boosting composition. The present
invention is based on inventors' experimental demonstration that
effective boosting can be achieved using modified vaccinia Ankara
(MVA) vectors, following priming with any of a variety of different
types of priming compositions including recombinant MVA itself.
[0062] A major protective component of the immune response against
a number of pathogens is mediated by T lymphocytes of the CD8.sup.+
type, also known as cytotoxic T lymphocytes (CTL). An important
function of CD8.sup.+ cells is secretion of gamma interferon
(IFN.gamma.), and this provides a measure of CD8.sup.+ T cell
immune response. A second component of the immune response is
antibody directed to the proteins of the pathogen.
[0063] The present invention employs MVA which, as the experiments
described below show, has been found to be an effective means for
providing a boost to a CD8.sup.+ T cell immune response primed to
antigen using any of a variety of different priming compositions
and also eliciting an antibody response.
[0064] Remarkably, the experimental work described below
demonstrates that use of embodiments of the present invention
allows for recombinant MVA virus expressing an HIV antigen to boost
a CD8.sup.+ T cell immune response primed by a DNA vaccine and also
eliciting an antibody response. The MVA was found to induce a
CD8.sup.+ T cell response after intradermal, intramuscular or
mucosal immunization. Recombinant MVA has also been shown to prime
an immune response that is boosted by one or more inoculations of
recombinant MVA.
[0065] Non-human primates immunized with plasmid DNA and boosted
with the MVA were effectively protected against intramucosal
challenge with live virus. Advantageously, the inventors found that
a vaccination regime used intradermal, intramuscular or mucosal
immunization for both prime and boost can be employed, constituting
a general immunization regime suitable for inducing CD8.sup.+ T
cells and also eliciting an antibody response, e.g. in humans.
[0066] The present invention in various aspects and embodiments
employs an MVA vector encoding an HIV antigen for boosting a
CD8.sup.+ T cell immune response to the antigen primed by previous
administration of nucleic acid encoding the antigen and also
eliciting an antibody response.
[0067] A general aspect of the present invention provides for the
use of an MVA vector for boosting a CD8.sup.+ T cell immune
response to an HIV antigen and also eliciting an antibody
response.
[0068] One aspect of the present invention provides a method of
boosting a CD8.sup.+ T cell immune response to an HIV antigen in an
individual, and also eliciting an antibody response, the method
including provision in the individual of an MVA vector including
nucleic acid encoding the antigen operably linked to regulatory
sequences for production of antigen in the individual by expression
from the nucleic acid, whereby a CD8.sup.+ T cell immune response
to the antigen previously primed in the individual is boosted.
[0069] An immune response to an HIV antigen may be primed by
immunization with plasmid DNA or by infection with an infectious
agent.
[0070] A further aspect of the invention provides a method of
inducing a CD8.sup.+ T cell immune response to an HIV antigen in an
individual, and also eliciting an antibody response, the method
comprising administering to the individual a priming composition
comprising nucleic acid encoding the antigen and then administering
a boosting composition which comprises an MVA vector including
nucleic acid encoding the antigen operably linked to regulatory
sequences for production of antigen in the individual by expression
from the nucleic acid.
[0071] A further aspect provides for use of an MVA vector, as
disclosed, in the manufacture of a medicament for administration to
a mammal to boost a CD8.sup.+ T cell immune response to an HIV
antigen, and also eliciting an antibody response. Such a medicament
is generally for administration following prior administration of a
priming composition comprising nucleic acid encoding the
antigen.
[0072] The priming composition may comprise any viral vector, such
as a vaccinia virus vector such as a replication-deficient strain
such as modified vaccinia Ankara (MVA) or NYVAC (Tartaglia et al.
1992 Virology 118:217-232), an avipox vector such as fowlpox or
canarypox, e.g. the strain known as ALVAC (Paoletti et al. 1994 Dev
Biol Stand 82:65-69), or an adenovirus vector or a vesicular
stomatitis virus vector or an alphavirus vector.
[0073] The priming composition may comprise DNA encoding the
antigen, such DNA preferably being in the form of a circular
plasmid that is not capable of replicating in mammalian cells. Any
selectable marker should not be resistance to an antibiotic used
clinically, so for example Kanamycin resistance is preferred to
Ampicillin resistance. Antigen expression should be driven by a
promoter which is active in mammalian cells, for instance the
cytomegalovirus immediate early (CMV IE) promoter.
[0074] In particular embodiments of the various aspects of the
present invention, administration of a priming composition is
followed by boosting with a boosting composition, or first and
second boosting compositions, the first and second boosting
compositions being the same or different from one another. Still
further boosting compositions may be employed without departing
from the present invention. In one embodiment, a triple
immunization regime employs DNA, then adenovirus as a first
boosting composition, then MVA as a second boosting composition,
optionally followed by a further (third) boosting composition or
subsequent boosting administration of one or other or both of the
same or different vectors. Another option is DNA then MVA then
adenovirus, optionally followed by subsequent boosting
administration of one or other or both of the same or different
vectors.
[0075] The antigen to be encoded in respective priming and boosting
compositions (however many boosting compositions are employed) need
not be identical, but should share at least one CD8.sup.+ T cell
epitope. The antigen may correspond to a complete antigen, or a
fragment thereof. Peptide epitopes or artificial strings of
epitopes may be employed, more efficiently cutting out unnecessary
protein sequence in the antigen and encoding sequence in the vector
or vectors. One or more additional epitopes may be included, for
instance epitopes which are recognized by T helper cells,
especially epitopes recognized in individuals of different HLA
types.
[0076] An HIV antigen of the invention to be encoded by a
recombinant MVA virus includes polypeptides having immunogenic
activity elicited by an amino acid sequence of an HIV Env, Gag,
Pol, Vif, Vpr, Tat, Rev, Vpu, or Nef amino acid sequence as at
least one CD8.sup.+ T cell epitope. This amino acid sequence
substantially corresponds to at least one 10-900 amino acid
fragment and/or consensus sequence of a known HIV Env or Pol; or at
least one 10-450 amino acid fragment and/or consensus sequence of a
known HIV Gag; or at least one 10-100 amino acid fragment and/or
consensus sequence of a known HIV Vif, Vpr, Tat, Rev, Vpu, or
Nef.
[0077] Although a full length Env precursor sequence is presented
for use in the present invention, Env is optionally deleted of
subsequences. For example, regions of the gp120 surface and gp41
transmembrane cleavage products can be deleted.
[0078] Although a full length Gag precursor sequence is presented
for use in the present invention, Gag is optionally deleted of
subsequences. For example, regions of the matrix protein (p17),
regions of the capsid protein (p24), regions of the nucleocapsid
protein (p7), and regions of p6 (the C-terminal peptide of the Gag
polyprotein) can be deleted.
[0079] Although a full length Pol precursor sequence is presented
for use in the present invention, Pol is optionally deleted of
subsequences. For example, regions of the protease protein (p10),
regions of the reverse transcriptase protein (p66/p51), and regions
of the integrase protein (p32) can be deleted.
[0080] Such an HIV Env, Gag, or Pol can have overall identity of at
least 50% to a known Env, Gag, or Pol protein amino acid sequence,
such as 50-99% identity, or any range or value therein, while
eliciting an immunogenic response against at least one strain of an
HIV.
[0081] Percent identify can be determined, for example, by
comparing sequence information using the GAP computer program,
version 6.0, available from the University of Wisconsin Genetics
Computer Group (UWGCG). The GAP program utilizes the alignment
method of Needleman and Wunsch (J Mol Biol 1970 48:443), as revised
by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP
program defines identity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are identical, divided by the
total number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unitary comparison matrix (containing a value of 1 for identities
and 0 for non-identities) and the weighted comparison matrix of
Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by
Schwartz and Dayhoff (eds., Atlas of Protein Sequence and
Structure, National Biomedical Research Foundation, Washington,
D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an
additional 0.10 penalty for each symbol in each gap; and (3) no
penalty for end gaps.
[0082] In a preferred embodiment, an Env of the present invention
is a variant form of at least one HIV envelope protein. Preferably,
the Env is composed of gp120 and the membrane-spanning and
ectodomain of gp41 but lacks part or all of the cytoplasmic domain
of gp41.
[0083] Known HIV sequences are readily available from commercial
and institutional HIV sequence databases, such as GENBANK, or as
published compilations, such as Myers et al. eds., Human
Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid
and Amino Acid Sequences, Vol. I and II, Theoretical Biology and
Biophysics, Los Alamos, N. Mex. (1993), or
http://hiv-web.lanl.gov/.
[0084] Substitutions or insertions of an HIV Env, Gag, or Pol to
obtain an additional HIV Env, Gag, or Pol, encoded by a nucleic
acid for use in a recombinant MVA virus of the present invention,
can include substitutions or insertions of at least one amino acid
residue (e.g., 1-25 amino acids). Alternatively, at least one amino
acid (e.g., 1-25 amino acids) can be deleted from an HIV Env, Gag,
or Pol sequence. Preferably, such substitutions, insertions or
deletions are identified based on safety features, expression
levels, immunogenicity and compatibility with high replication
rates of MVA.
[0085] Amino acid sequence variations in an HIV Env, Gag, or Pol of
the present invention can be prepared e.g., by mutations in the
DNA. Such HIV Env, Gag, or Pol include, for example, deletions,
insertions or substitutions of nucleotides coding for different
amino acid residues within the amino acid sequence. Obviously,
mutations that will be made in nucleic acid encoding an HIV Env,
Gag, or Pol must not place the sequence out of reading frame and
preferably will not create complementary domains that could produce
secondary mRNA structures.
[0086] HIV Env, Gag, or Pol-encoding nucleic acid of the present
invention can also be prepared by amplification or site-directed
mutagenesis of nucleotides in DNA or RNA encoding an HIV Env, Gag,
or Pol and thereafter synthesizing or reverse transcribing the
encoding DNA to produce DNA or RNA encoding an HIV Env, Gag, or
Pol, based on the teaching and guidance presented herein.
[0087] Recombinant MVA viruses expressing HIV Env, Gag, or Pol of
the present invention, include a finite set of HIV Env, Gag, or
Pol-encoding sequences as substitution nucleotides that can be
routinely obtained by one of ordinary skill in the art, without
undue experimentation, based on the teachings and guidance
presented herein. For a detailed description of protein chemistry
and structure, see Schulz, G. E. et al., 1978 Principles of Protein
Structure, Springer-Verlag, New York, N.Y., and Creighton, T. E.,
1983 Proteins: Structure and Molecular Properties, W. H. Freeman
& Co., San Francisco, Calif. For a presentation of nucleotide
sequence substitutions, such as codon preferences, see Ausubel et
al. eds. Current Protocols in Molecular Biology, Greene Publishing
Assoc., New York, N.Y. 1994 at .sctn..sctn. A.1.1-A.1.24, and
Sambrook, J. et al. 1989 Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. at Appendices C and D.
[0088] Thus, one of ordinary skill in the art, given the teachings
and guidance presented herein, will know how to substitute other
amino acid residues in other positions of an HIV env, gag, or pol
DNA or RNA to obtain alternative HIV Env, Gag, or Pol, including
substitutional, deletional or insertional variants.
[0089] Within the MVA vector, regulatory sequences for expression
of the encoded antigen will include a natural, modified or
synthetic poxvirus promoter. By "promoter" is meant a sequence of
nucleotides from which transcription may be initiated of DNA
operably linked downstream (i.e. in the 3' direction on the sense
strand of double-stranded DNA). "Operably linked" means joined as
part of the same nucleic acid molecule, suitably positioned and
oriented for transcription to be initiated from the promoter. DNA
operably linked to a promoter is "under transcriptional initiation
regulation" of the promoter. Other regulatory sequences including
terminator fragments, polyadenylation sequences, marker genes and
other sequences may be included as appropriate, in accordance with
the knowledge and practice of the ordinary person skilled in the
art: see, for example, Moss, B. (2001). Poxviridae: the viruses and
their replication. In Fields Virology, D. M. Knipe, and P. M.
Howley, eds. (Philadelphia, Lippincott Williams & Wilkins), pp.
2849-2883. Many known techniques and protocols for manipulation of
nucleic acid, for example in preparation of nucleic acid
constructs, mutagenesis, sequencing, introduction of DNA into cells
and gene expression, and analysis of proteins, are described in
detail in Current Protocols in Molecular Biology, 1998 Ausubel et
al. eds., John Wiley & Sons.
[0090] Promoters for use in aspects and embodiments of the present
invention must be compatible with poxvirus expression systems and
include natural, modified and synthetic sequences.
[0091] Either or both of the priming and boosting compositions may
include an adjuvant, such as granulocyte macrophage-colony
stimulating factor (GM-CSF) or encoding nucleic acid therefor.
[0092] Administration of the boosting composition is generally
about 1 to 6 months after administration of the priming
composition, preferably about 1 to 3 months.
[0093] Preferably, administration of priming composition, boosting
composition, or both priming and boosting compositions, is
intradermal, intramuscular or mucosal immunization.
[0094] Administration of MVA vaccines may be achieved by using a
needle to inject a suspension of the virus. An alternative is the
use of a needleless injection device to administer a virus
suspension (using, e.g., Biojector.TM. needleless injector) or a
resuspended freeze-dried powder containing the vaccine, providing
for manufacturing individually prepared doses that do not need cold
storage. This would be a great advantage for a vaccine that is
needed in rural areas of Africa.
[0095] MVA is a virus with an excellent safety record in human
immunizations. The generation of recombinant viruses can be
accomplished simply, and they can be manufactured reproducibly in
large quantities. Intradermal, intramuscular or mucosal
administration of recombinant MVA virus is therefore highly
suitable for prophylactic or therapeutic vaccination of humans
against AIDS which can be controlled by a CD8.sup.+ T cell
response.
[0096] The individual may have AIDS such that delivery of the
antigen and generation of a CD8.sup.+ T cell immune response to the
antigen is of benefit or has a therapeutically beneficial
effect.
[0097] Most likely, administration will have prophylactic aim to
generate an immune response against HIV or AIDS before infection or
development of symptoms.
[0098] Components to be administered in accordance with the present
invention may be formulated in pharmaceutical compositions. These
compositions may comprise a pharmaceutically acceptable excipient,
carrier, buffer, stabilizer or other materials well known to those
skilled in the art. Such materials should be non-toxic and should
not interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material may depend on the
route of administration, e.g. intravenous, cutaneous or
subcutaneous, nasal, intramuscular, intraperitoneal routes.
[0099] As noted, administration is preferably intradermal,
intramuscular or mucosal.
[0100] Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0101] For intravenous, cutaneous, subcutaneous, intramuscular or
mucosal injection, or injection at the site of affliction, the
active ingredient will be in the form of a parenterally acceptable
aqueous solution which is pyrogen-free and has suitable pH,
isotonicity and stability. Those of relevant skill in the art are
well able to prepare suitable solutions using, for example,
isotonic vehicles such as Sodium Chloride Injection, Ringer's
Injection, Lactated Ringer's Injection. Preservatives, stabilizers,
buffers, antioxidants and/or other additives may be included as
required.
[0102] A slow-release formulation may be employed.
[0103] Following production of MVA particles and optional
formulation of such particles into compositions, the particles may
be administered to an individual, particularly human or other
primate. Administration may be to another mammal, e.g. rodent such
as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig,
horse, cow, donkey, dog or cat.
[0104] Administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the
case may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual. The actual
amount administered, and rate and time-course of administration,
will depend on the nature and severity of what is being treated.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors, or in a veterinary context a veterinarian, and typically
takes account of the disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners. Examples
of the techniques and protocols mentioned above can be found in
Remington's Pharmaceutical Sciences, 16th edition, 1980, Osol, A.
(ed.).
[0105] In one preferred regimen, DNA is administered at a dose of
250 .mu.g to 2.5 mg/injection, followed by MVA at a dose of
10.sup.6 to 10.sup.9 infectious virus particles/injection.
[0106] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0107] Delivery to a non-human mammal need not be for a therapeutic
purpose, but may be for use in an experimental context, for
instance in investigation of mechanisms of immune responses to an
antigen of interest, e.g. protection against HIV or AIDS.
[0108] Further aspects and embodiments of the present invention
will be apparent to those of ordinary skill in the art, in view of
the above disclosure and following experimental exemplification,
included by way of illustration and not limitation, and with
reference to the attached figures.
EXAMPLE 1
Control of a Mucosal Challenge and Prevention of AIDS by a
Multiprotein DNA/MVA Vaccine
[0109] Here we tested DNA priming and poxvirus boosting for the
ability to protect against a highly pathogenic mucosal challenge.
The 89.6 chimera of simian and human immunodeficiency viruses
(SHIV-89.6) was used for the construction of immunogens and its
highly pathogenic derivative, SHIV-89.6P, for challenge (G. B.
Karlsson et al. 1997 J Virol 71:4218). SHIV-89.6 and SHIV-89.6P do
not generate cross-neutralizing antibody (D. C . . . Montefiori et
al. 1998 J Virol 72:3427) and allowed us to address the ability of
vaccine-raised T cells and non-neutralizing antibodies to control
an immunodeficiency virus challenge. Modified vaccinia Ankara (MVA)
was used for the construction of the recombinant poxvirus. MVA has
been highly effective at boosting DNA-primed CD8 T cells and enjoys
the safety feature of not replicating efficiently in human or
monkey cells (H. L. Robinson et al. 2000 AIDS Reviews 2:105).
[0110] To ensure a broad immune response both the DNA and
recombinant MVA (rMVA) components of the vaccine expressed multiple
immunodeficiency virus proteins. The DNA prime (DNA/89.6) expressed
simian immunodeficiency virus (SIV) Gag, Pol, Vif, Vpx, and Vpr and
human immunodeficiency virus-1 (HIV-1) Env, Tat, and Rev from a
single transcript (R. J. Gorelick et al. 1999 Virology 253:259; M.
M. Sauter et al. 1996 J Cell Biol 132:795).
[0111] Molecularly cloned SHIV-89.6 sequences were cloned into the
vector pGA2 using ClaI and RsrII sites. This cloning deleted both
long terminal repeats (LTRs) and nef. The SHIV-89.6 sequences also
were internally mutated for a 12-base pair region encoding the
first four amino acids of the second zinc finger in nucleocapsid.
This mutation renders SHIV viruses noninfectious (R. J. Gorelick et
al. 1999 Virology 253:259). A mutation in gp41 converted the
tyrosine at position 710 to cysteine to achieve better expression
of Env on the plasma membrane of DNA-expressing cells (M. M. Sauter
et al. 1996 J Cell Biol 132:795). pGA2 uses the CMV immediate early
promoter without intron A and the bovine growth hormone
polyadenylation sequence to express vaccine inserts. Vaccine DNA
was produced by Althea (San Diego, Calif.). In transient
transfections of 293T cells, DNA/89.6 produced about 300 ng of Gag
and 85 ng of Env per 1.times.10.sup.6 cells.
[0112] The rMVA booster (MVA/89.6) expressed SIV Gag, Pol, and
HIV-1 Env under the control of vaccinia virus early/late
promoters.
[0113] The MVA double recombinant virus expressed both the HIV 89.6
Env and the SIV 239 Gag-Pol, which were inserted into deletion II
and deletion III of MVA, respectively. The 89.6 Env protein was
truncated for the COOH-terminal 115 amino acids of gp41. The
modified H5 promoter controlled the expression of both foreign
genes.
[0114] Vaccination was accomplished by priming with DNA at 0 and 8
weeks and boosting with rMVA at 24 weeks (FIG. 7A).
[0115] I.d. and i.m. DNA immunizations were delivered in
phosphate-buffered saline (PBS) with a needleless jet injector
(Bioject, Portland, Oreg.) to deliver five i.d. 100-.mu.l
injections to each outer thigh for the 2.5-mg dose of DNA or one
i.d. 100-.mu.l injection to the right outer thigh for the 250-.mu.g
dose of plasmid. I.m. deliveries of DNA were done with one 0.5-ml
injection of DNA in PBS to each outer thigh for the 2.5-mg dose and
one 100-.mu.l injection to the right outer thigh for the 250-.mu.g
dose. 1.times.10.sup.8 pfu of MVA/89.6 was administered both i.d.
and i.m. with a needle. One 100-.mu.l dose was delivered to each
outer thigh for the i.d. dose and one 500-.mu.l dose to each outer
thigh for the i.m dose. Control animals received 2.5 mg of the pGA2
vector without vaccine insert with the Bioject device to deliver
five 100-.mu.l doses i.d. to each outer thigh. The control MVA
booster immunization consisted of 2.times.10.sup.8 pfu of MVA
without an insert delivered i.d. and i.m. as described for
MVA/89.6.
[0116] Four groups of six rhesus macaques each were primed with
either 2.5 mg (high-dose) or 250 .mu.g (low-dose) of DNA by
intradermal (i.d.) or intramuscular (i.m.) routes using a
needleless jet injection device (Bioject, Portland, Oreg.) (T. M.
Allen et al. 2000 J Immunol 164:4968).
[0117] Young adult rhesus macaques from the Yerkes breeding colony
were cared for under guidelines established by the Animal Welfare
Act and the NIH "Guide for the Care and Use of Laboratory Animals"
with protocols approved by the Emory University Institutional
Animal Care and Use Committee. Macaques were typed for the
Mamu-A*01 allele with polymerase chain reaction (PCR) analyses (M.
A. Egan et al. 2000 J Virol 74:7485; I. Ourmanov et al. 2000 J
Virol 74:2740). Two or more animals containing at least one
Mamu-A*01 allele were assigned to each group. Animal numbers are as
follows: 1, RBr-5*; 2, RIm-5*; 3, RQf-5*; 4, RZe-5; 5, ROm-5; 6,
RDm-5; 7, RAj-5*; 8, RJi-5*; 9, RAl-5*; 10, RDe-5*; 11, RAi-5; 12,
RPr-5; 13, RKw-4*; 14, RWz-5*; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18,
RAt-5; 19, RPb-5*; 20, RIi-5*; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24,
RGd-6; 25, RMb-5*; 26, RGy-5*; 27, RUs-4; and 28, RPm-5. Animals
with the A*01 allele are indicated with asterisks.
[0118] Gene gun deliveries of DNA were not used because these had
primed non-protective immune responses in a 1996-98 trial (H. L.
Robinson et al. 1999 Nat Med 5:526). The MVA/89.6 booster
immunization (2.times.10.sup.8 plaque-forming units, pfu) was
injected with a needle both i.d. and i.m. A control group included
two mock immunized animals and two naive animals. The challenge was
given at 7 months after the rMVA booster to test for the generation
of long-term immunity. Because most HIV-1 infections are
transmitted across mucosal surfaces, an intrarectal challenge was
administered.
[0119] DNA priming followed by rMVA boosting generated high
frequencies of virus-specific T cells that peaked at one week
following the rMVA booster (FIG. 7). The frequencies of T cells
recognizing the Gag-CM9 epitope were assessed by means of Mamu-A*01
tetramers, and the frequencies of T cells recognizing epitopes
throughout Gag were assessed with pools of overlapping peptides and
an enzyme-linked immunospot (ELISPOT) assay (C. A. Power et al.
1999 J Immunol Methods 227:99).
[0120] For tetramer analyses, about 1.times.10.sup.6 peripheral
blood mononuclear cells (PBMC) were surface-stained with antibodies
to CD3 conjugated to fluorescein isothiocyanate (FITC) (FN-18;
Biosource International, Camarillo, Calif.), CD8 conjugated to
peridinin chlorophyl protein (PerCP) (SK1; Becton Dickinson, San
Jose, Calif.), and Gag-CM9 (CTPYDINQM)-Mamu-A*01 tetramer (SEQ ID
NO: 6) conjugated to allophycocyanin (APC), in a volume of 100
.mu.l at 8.degree. to 10.degree. C. for 30 min. Cells were washed
twice with cold PBS containing 2% fetal bovine serum (FBS), fixed
with 1% paraformaldehyde in PBS, and analyzed within 24 hrs on a
FACScaliber (Becton Dickinson, San Jose, Calif.). Cells were
initially gated on lymphocyte populations with forward scatter and
side scatter and then on CD3 cells. The CD3 cells were then
analyzed for CD8 and tetramer-binding cells. About 150,000
lymphocytes were acquired for each sample. Data were analyzed using
FloJo software (Tree Star, San Carlos, Calif.).
[0121] For interferon-.gamma. (IFN-.gamma.) ELISPOTs, MULTISCREEN
96 well filtration plates (Millipore Inc. Bedford, Mass.) were
coated overnight with antibody to human IFN-.gamma. (Clone B27,
Pharmingen, San Diego, Calif.) at a concentration of 2 .mu.g/ml in
sodium bicarbonate buffer (pH 9.6) at 8.degree. to 10.degree. C.
Plates were washed two times with RPMI medium and then blocked for
1 hour with complete medium (RPMI containing 10% FBS) at 37.degree.
C. Plates were washed five more times with plain RPMI medium, and
cells were seeded in duplicate in 100 .mu.l complete medium at
numbers ranging from 2.times.10.sup.4 to 5.times.10.sup.5 cells per
well. Peptide pools were added to each well to a final
concentration of 2 .mu.g/ml of each peptide in a volume of 100
.mu.l in complete medium. Cells were cultured at 37.degree. C. for
about 36 hrs under 5% CO.sub.2. Plates were washed six times with
wash buffer (PBS with 0.05% Tween-20) and then incubated with 1
.mu.g of biotinylated antibody to human IFN-.gamma. per milliliter
(clone 7-86-1; Diapharma Group, West Chester, Ohio) diluted in wash
buffer containing 2% FBS. Plates were incubated for 2 hrs at
37.degree. C. and washed six times with wash buffer.
Avidin-horseradish peroxidase (Vector Laboratories, Burlingame,
Calif.) was added to each well and incubated for 30 to 60 min at
37.degree. C. Plates were washed six times with wash buffer and
spots were developed using stable DAB as substrate (Research
Genetics, Huntsville, Ala.). Spots were counted with a stereo
dissecting microscope. An ovalbumin peptide (SIINFEKL) (SEQ ID NO:
7) was included as a control in each analysis. Background spots for
the ovalbumin peptide were generally <5 for 5.times.10.sup.5
PBMCs. This background when normalized for 1.times.10.sup.6 PBMC
was <10. Only ELISPOT counts of twice the background
(.gtoreq.20) were considered significant. The frequencies of
ELISPOTs are approximate because different dilutions of cells have
different efficiencies of spot formation in the absence of feeder
cells (C. A. Power et al. 1999 J Immunol Methods 227: 99). The same
dilution of cells was used for all animals at a given time point,
but different dilutions were used to detect memory and acute
responses.
[0122] Gag-CM9 tetramer analyses were restricted to macaques that
expressed the Mamu-A*01 histocompatibility type, whereas ELISPOT
responses did not depend on a specific histocompatibility type. As
expected, the DNA immunizations raised low levels of memory cells
that expanded to high frequencies within 1 week of the rMVA booster
(FIGS. 7 and 12). In Mamu-A*01 macaques, CD8 cells specific to the
Gag-CM9 epitope expanded to frequencies as high as 19% of total CD8
T cells (FIG. 12). This peak of specific cells underwent a 10- to
100-fold contraction into the DNA/MVA memory pool (FIGS. 7A and
12). ELISPOTs for three pools of Gag peptides also underwent a
major expansion (frequencies up to 4000 spots for 1.times.10.sup.6
PBMC) before contracting from 5- to 20-fold into the DNA/MVA memory
response (FIG. 7B). The frequencies of ELISPOTs were the same in
macaques with and without the A*01 histocompatibility type
(P>0.2).
[0123] Simple linear regression was used to estimate correlations
between postbooster and postchallenge ELISPOT responses, between
memory and postchallenge ELISPOT responses, and between
logarithmically transformed viral loads and ELISPOT frequencies.
Comparisons between vaccine and control groups and A*01 and non
A*01 macaques were performed by means of two-sample t tests with
logarithmically transformed viral load and ELISPOT responses.
Two-way analyses of variance were used to examine the effects of
dose and route of administration on peak DNA/MVA ELISPOTs, on
memory DNA/MVA ELISPOTs, and on logarithmically transformed Gag
antibody data.
[0124] At both peak and memory phases of the vaccine response, the
rank order for the height of the ELISPOTs in the vaccine groups was
2.5 mg i.d.>2.5 mg i.m.>250 .mu.g i.d.>250 .mu.g i.m.
(FIG. 7B). The IFN-.gamma. ELISPOTs included both CD4 and CD8
cells. Gag-CM9-specific CD8 cells had good lytic activity after
restimulation with peptide.
[0125] The highly pathogenic SHIV-89.6P challenge was administered
intrarectally at 7 months after the rMVA booster, when
vaccine-raised T cells were in memory (FIG. 7).
[0126] The challenge stock (5.7.times.10.sup.9 copies of viral RNA
per milliliter) was produced by one intravenous followed by one
intrarectal passage in rhesus macaques of the original SHIV-89.6P
stock (G. B. Karlsson et al. 1997 J Virol 71:4218). Lymphoid cells
were harvested from the intrarectally infected animal at peak
viremia, CD8-depleted, and mitogen-stimulated for stock production.
Before intrarectal challenge, fasted animals were anesthetized
(ketamine, 10 mg/kg) and placed on their stomach with the pelvic
region slightly elevated. A feeding tube (8Fr (2.7 mm).times.16
inches (41 cm); Sherwood Medical, St. Louis, Mo.) was inserted into
the rectum for a distance of 15 to 20 cm. Following insertion of
the feeding tube, a syringe containing 20 intrarectal infectious
doses in 2 ml of RPMI-1640 plus 10% FBS was attached to the tube
and the inoculum was slowly injected into the rectum. After
delivery of the inoculum, the feeding tube was flushed with 3.0 ml
of RPMI without FBS and then slowly withdrawn. Animals were left in
place, with pelvic regions slightly elevated, for a period of ten
minutes after the challenge.
[0127] The challenge infected all of the vaccinated and control
animals (FIG. 8). However, by 2 weeks after challenge, titers of
plasma viral RNA were at least 10-fold lower in the vaccine groups
(geometric means of 1.times.10.sup.7 to 5.times.10.sup.7) than in
the control animals (geometric mean of 4.times.10.sup.8) (FIG. 8A)
(S. Staprans et al. in: Viral Genome Methods K. Adolph, ed. CRC
Press, Boca Raton, Fla., 1996 pp. 167-184; R. Hofmann-Lehmann et
al. 2000 AIDS Res Hum Retroviruses 16:1247).
[0128] For the determination of SHIV copy number, viral RNA from
150 .mu.l of ACD anticoagulated plasma was directly extracted with
the QIAamp Viral RNA kit (Qiagen), eluted in 60 .mu.l of AVE
buffer, and frozen at -80.degree. C. until SHIV RNA quantitation
was performed. Five microliters of purified plasma RNA was
reverse-transcribed in a final 20-.mu.l volume containing 50 mM
KCl, 10 mM Tris-HCl (pH 8.3), 4 mM MgCl.sub.2, 1 mM each
deoxynucleotide triphosphate (dNTP), 2.5 .mu.M random hexamers, 20
units MultiScribe RT, and 8 units ribonuclease inhibitor. Reactions
were incubated at 25.degree. C. for 10 min, followed by incubation
at 42.degree. C. for 20 min, and inactivation of reverse
transcriptase at 99.degree. C. for 5 min. The reaction mix was
adjusted to a final volume of 50 .mu.l containing 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 4 mM MgCl.sub.2, 0.4 mM each dNTP, 0.2 .mu.M
forward primer, 0.2 .mu.M reverse primer, 0.1 .mu.M probe, and 5
units AmpliTaq Gold DNA polymerase (all reagents from PerkinElmer
Applied Biosystems, Foster City, Calif.). The primer sequences
within a conserved portion of the SIV gag gene are the same as
those described previously (S. Staprans et al. in: Viral Genome
Methods K. Adolph, ed. CRC Press, Boca Raton, Fla., 1996 pp.
167-184). A PerkinElmer Applied Biosystems 7700 Sequence Detection
System was used with the PCR profile: 95.degree. C. for 10 min,
followed by 40 cycles at 93.degree. C. for 30 s, and 59.5.degree.
C. for 1 min. PCR product accumulation was monitored with the 7700
sequence detector and a probe to an internal conserved gag gene
sequence: 6FAM-CTGTCTGCGTCATTTGGTGC-Tamra (SEQ ID NO: 8), where FAM
and Tamra denote the reporter and quencher dyes. SHIV RNA copy
number was determined by comparison with an external standard curve
consisting of virion-derived SIVmac239 RNA quantified by the SIV
bDNA method (Bayer Diagnostics, Emeryville, Calif.). All specimens
were extracted and amplified in duplicate, with the mean result
reported. With a 0.15-ml plasma input, the assay has a sensitivity
of 10.sup.3 RNA copies per milliliter of plasma and a linear
dynamic range of 10.sup.3 to 10.sup.8 RNA copies (R.sup.2=0.995).
The intraassay coefficient of variation was <20% for samples
containing >10.sup.4 SHIV RNA copies per milliliter, and <25%
for samples containing 10.sup.3 to 10.sup.4 SHIV RNA copies per
milliliter. To more accurately quantitate low SHIV RNA copy number
in vaccinated animals at weeks 16 and 20, we made the following
modifications to increase the sensitivity of the SHIV RNA assay:
(i) Virions from .ltoreq.1 ml of plasma were concentrated by
centrifugation at 23,000 g at 10.degree. C. for 150 min before
viral RNA extraction, and (ii) a one-step reverse transcriptase PCR
method was used (R. Hofmann-Lehmann et al. 2000 AIDS Res Hum
Retroviruses 16:1247). These changes provided a reliable
quantification limit of 300 SHIV RNA copies per milliliter, and
gave SHIV RNA values that were highly correlated to those obtained
by the first method used (r=0.91, P<0.0001).
[0129] By 8 weeks after challenge, both high-dose DNA-primed groups
and the low-dose i.d. DNA-primed group had reduced their geometric
mean loads to about 1000 copies of viral RNA per milliliter. At
this time, the low-dose i.m. DNA-primed group had a geometric mean
of 6.times.10.sup.3 copies of viral RNA and the nonvaccinated
controls had a geometric mean of 2.times.10.sup.6. By 20 weeks
after challenge, even the low-dose i.m. group had reduced its
geometric mean copies of viral RNA to 1000. Among the 24 vaccinated
animals, only one animal, animal number 22 in the low-dose i.m.
group, had intermittent viral loads above 1.times.10.sup.4 copies
per milliliter (FIG. 8D).
[0130] By 5 weeks after challenge, all of the nonvaccinated
controls had undergone a profound depletion of CD4 cells (FIG. 8B).
All of the vaccinated animals maintained their CD4 cells, with the
exception of animal 22 in the low dose i.m. group (see above),
which underwent a slow CD4 decline (FIG. 8E). By 23 weeks after
challenge, three of the four control animals had succumbed to AIDS
(FIG. 8C). These animals had variable degrees of enterocolitis with
diarrhea, cryptosporidiosis, colicystitis, enteric campylobacter
infection, splenomegaly, lymphadenopathy, and SIV-associated giant
cell pneumonia. In contrast, all 24 vaccinated animals maintained
their health.
[0131] Containment of the viral challenge was associated with a
burst of antiviral T cells (FIGS. 7 and 9A). At one week after
challenge, the frequency of tetramer.sup.+ cells in the peripheral
blood had decreased, potentially reflecting the recruitment of
specific T cells to the site of infection (FIG. 9A). However, by
two weeks after challenge, tetramer.sup.+ cells in the peripheral
blood had expanded to frequencies as high as, or higher than, after
the rMVA booster (FIG. 7 and 9A). The majority of the
tetramer.sup.+ cells produced IFN-.gamma. in response to a 6-hour
peptide stimulation (FIG. 9B) (S. L. Waldrop et al. 1997 J Clin
Invest 99:1739) and did not have the "stunned" IFN-.gamma. negative
phenotype sometimes observed in viral infections (F. Lechner et al.
2000 J Exp Med 191:1499).
[0132] For intracellular cytokine assays, about 1.times.10.sup.6
PBMC were stimulated for 1 hour at 37.degree. C. in 5 ml
polypropylene tubes with 100 .mu.g of Gag-CM9 peptide (CTPYDINQM)
(SEQ ID NO: 6) per milliliter in a volume of 100 .mu.l RPMI
containing 0.1% bovine serum albumin (BSA) and 1 .mu.g of antibody
to human CD28 and 1 .mu.g of antibody to human CD49d (Pharmingen,
San Diego, Calif.) per milliliter. Then, 900 .mu.l of RPMI
containing 10% FBS and monensin (10 .mu.g/ml) was added, and the
cells were cultured for an additional 5 hrs at 37.degree. C. at an
angle of 5.degree. under 5% CO.sub.2. Cells were surface stained
with antibodies to CD8 conjugated to PerCP (clone SK1, Becton
Dickinson) at 8.degree. to 10.degree. C. for 30 min, washed twice
with cold PBS containing 2% FBS, and fixed and permeabilized with
Cytofix/Cytoperm solution (Pharmingen). Cells were then incubated
with antibodies to human CD3 (clone FN-18; Biosource International,
Camarillo, Calif.) and IFN-.gamma. (Clone B27; Pharmingen)
conjugated to FITC and phycoerythrin, respectively, in Perm wash
solution (Pharmingen) for 30 min at 4.degree. C. Cells were washed
twice with Perm wash, once with plain PBS, and resuspended in 1%
paraformaldehyde in PBS. About 150,000 lymphocytes were acquired on
the FACScaliber and analyzed with FloJo software.
[0133] The postchallenge burst of T cells contracted concomitant
with the decline of the viral load. By 12 weeks after challenge,
virus-specific T cells were present at about one-tenth of their
peak height (FIGS. 7A and 9A). In contrast to the vigorous
secondary response in the vaccinated animals, the naive animals
mounted a modest primary response (FIGS. 7B and 9A). Tetramer.sup.+
cells peaked at less than 1% of total CD8 cells (FIG. 9A), and
IFN-.gamma.-producing ELISPOTs were present at a mean frequency of
about 300 as opposed to the much higher frequencies of 1000 to 6000
in the vaccine groups (FIG. 7B) (P<0.05).
[0134] The tetramer.sup.+ cells in the control group, like those in
the vaccine group, produced IFN-.gamma. after peptide stimulation
(FIG. 9B). By 12 weeks after challenge, three of the four controls
had undetectable levels of IFN-.gamma.-producing ELISPOTs. This
rapid loss of antiviral T cells in the presence of high viral loads
may reflect the lack of CD4 help.
[0135] T cell proliferative responses demonstrated that
virus-specific CD4 cells had survived the challenge and were
available to support the antiviral immune response (FIG. 9C).
[0136] About 0.2 million PBMC were stimulated in triplicate for 5
days with the indicated antigen in 200 .mu.l of RPMI at 37.degree.
C. under 5% CO.sub.2. Supernatants from 293T cells transfected with
DNA expressing either SHIV-89.6 Gag and Pol or SHIV-89.6 Gag, Pol
and Env were used directly as antigens (final concentration of
.about.0.5 .mu.g of p27 Gag per milliliter). Supernatants from mock
DNA (vector alone)-transfected cells served as negative controls.
On day six, cells were pulsed with 1 .mu.Ci of tritiated thymidine
per well for 16 to 20 hours. Cells were harvested with an automated
cell harvester (TOMTEC, Harvester 96, Model 1010, Hamden, Conn.)
and counted with a Wallac 1450 MICROBETA Scintillation counter
(Gaithersburg, Md.). Stimulation indices are the counts of
tritiated thymidine incorporated in PBMC stimulated with 89.6
antigens divided by the counts of tritiated thymidine incorporated
by the same PBMC stimulated with mock antigen.
[0137] At 12 weeks after challenge, mean stimulation indices for
Gag-Pol-Env or Gag-Pol proteins ranged from 35 to 14 in the vaccine
groups but were undetectable in the control group. Consistent with
the proliferation assays, intracellular cytokine assays
demonstrated the presence of virus-specific CD4 cells in vaccinated
but not control animals. The overall rank order of the vaccine
groups for the magnitude of the proliferative response was 2.5 mg
i.d.>2.5 mg i.m.>250 .mu.g i.d.>250 .mu.g i.m.
[0138] At 12 weeks after challenge, lymph nodes from the vaccinated
animals were morphologically intact and responding to the
infection, whereas those from the infected controls had been
functionally destroyed (FIG. 10). Nodes from vaccinated animals
contained large numbers of reactive secondary follicles with
expanded germinal centers and discrete dark and light zones (FIG.
10A). By contrast, lymph nodes from the non-vaccinated control
animals showed follicular and paracortical depletion (FIG. 10B),
while those from unvaccinated and unchallenged animals displayed
normal numbers of minimally reactive germinal centers (FIG. 10C).
Germinal centers occupied <0.05% of total lymph node area in the
infected controls, 2% of the lymph node area in the uninfected
controls, and up to 18% of the lymph node area in the vaccinated
groups (FIG. 10D). More vigorous immune reactivity in the low-dose
than the high-dose DNA-primed animals was suggested by more
extensive germinal centers in the low dose group (FIG. 10D). At 12
weeks after challenge, in situ hybridization for viral RNA revealed
rare virus-expressing cells in lymph nodes from 3 of the 24
vaccinated macaques, whereas virus-expressing cells were readily
detected in lymph nodes from each of the infected control animals.
In the controls, which had undergone a profound depletion in CD4 T
cells, the cytomorphology of infected lymph node cells was
consistent with a macrophage phenotype.
[0139] The prime/boost strategy raised low levels of antibody to
Gag and undetectable levels of antibody to Env (FIG. 11).
Postchallenge, antibodies to both Env and Gag underwent anamnestic
responses with total Gag antibody reaching heights approaching 1
mg/ml and total Env antibody reaching heights of up to 100
.mu.g/ml.
[0140] Enzyme-linked immunosorbent assays (ELISAs) for total
antibody to Gag used bacterially produced SIV gag p27 to coat wells
(2 .mu.g per milliliter in bicarbonate buffer). ELISAs for antibody
to Env antibody used 89.6 Env produced in transiently transfected
293T cells and captured with sheep antibody against Env (catalog
number 6205; International Enzymes, Fairbrook Calif.). Standard
curves for Gag and Env ELISAs were produced with serum from a
SHIV-89.6-infected macaque with known amounts of immunoglobulin G
(IgG) specific for Gag or Env. Bound antibody was detected with
peroxidase-conjugated goat antibody to macaque IgG (catalog #
YNGMOIGGFCP; Accurate Chemical, Westbury, N.Y.) and TMB substrate
(Catalog # T3405; Sigma, St. Louis, Mo.). Sera were assayed at
threefold dilutions in duplicate wells. Dilutions of test sera were
performed in whey buffer (4% whey and 0.1% tween 20 in 1.times.
PBS). Blocking buffer consisted of whey buffer plus 0.5% nonfat dry
milk. Reactions were stopped with 2M H.sub.2SO.sub.4 and the
optical density read at 450 nm. Standard curves were fitted and
sample concentrations were interpolated as .mu.g of antibody per ml
of serum using SOFTmax 2.3 software (Molecular Devices, Sunnyvale,
Calif.).
[0141] By 2 weeks after challenge, neutralizing antibodies for the
89.6 immunogen, but not the SHIV-89.6P challenge, were present in
the high-dose DNA-primed groups (geometric mean titers of 352 in
the i.d. and 303 in the i.m. groups) (FIG. 11C) (D. C. Montefiori
et al. 1988 J Clin Microbiol 26:231). By 5 weeks after challenge,
neutralizing antibody to 89.6P had been generated (geometric mean
titers of 200 in the high-dose i.d. and 126 in the high-dose i.m.
group) (FIG. 11D) and neutralizing antibody to 89.6 had started to
decline. By 16 to 20 weeks after challenge, antibodies to Gag and
Env had fallen in most animals.
[0142] Our results demonstrate that a multiprotein DNA/MVA vaccine
can raise a memory immune response capable of controlling a highly
virulent mucosal immunodeficiency virus challenge. Our levels of
viral control were more favorable than have been achieved using
only DNA (M. A. Egan et al. 2000 J Virol 74:7485) or rMVA vaccines
(I. Ourmanov et al. 2000 J Virol 74:2740) and were comparable to
those obtained for DNA immunizations adjuvanted with interleukin-2
(D. H. Barouch et al. 2000 Science 290:486). All of these previous
studies have used more than three vaccine inoculations, none have
used mucosal challenges, and most have challenged at peak effector
responses and not allowed a prolonged post vaccination period to
test for "long term" efficacy.
[0143] The dose of DNA had statistically significant effects on
both cellular and humoral responses (P<0.05), whereas the route
of DNA administration affected only humoral responses. Intradermal
DNA delivery was about 10 times more effective than i.m.
inoculations for generating antibody to Gag (P=0.02). Neither route
nor dose of DNA appeared to have a significant effect on
protection. At 20 weeks after challenge, the high-dose DNA-primed
animals had slightly lower geometric mean levels of viral RNA
(7.times.10.sup.2 and 5.times.10.sup.2) than the low-dose
DNA-primed animals (9.times.10.sup.2 and 1.times.10.sup.3).
[0144] The DNA/MVA vaccine controlled the infection, rapidly
reducing viral loads to near or below 1000 copies of viral RNA per
milliliter of blood. Containment, rather than prevention of
infection, affords the opportunity to establish a chronic infection
(H. L. Robinson et al. 1999 Nat Med 5:526). By rapidly reducing
viral loads, a multiprotein DNA/MVA vaccine will extend the
prospect for long-term non-progression and limit HIV transmission.
(J. W. Mellors et al. 1996 Science 272:1167; T. C. Quinn et al.
2000 N Engl J Med 342:921).
EXAMPLE 2
MVA Expressing Modified HIV Env, Gag, and Pol Genes
[0145] This disclosure describes the construction of a modified
vaccinia Ankara (MVA) recombinant virus, MVA/HIV clade B
recombinant virus expressing the HIV strain ADA env and the HXB2
gag pol (MVA/HIV ADA env+HXB2 gag pol). For amplification, the lab
name of MVA/HIV 48 will be used, which denotes the plasmid from
which the construct comes.
[0146] The HIV gag-pol genes were derived from the Clade B
infectious HXB2 virus. The gag-pol gene was truncated so that most
of the integrase coding sequences were removed and amino acids 185,
266, and 478 were mutated to inactivate reverse transcriptase,
inhibit strand transfer activity, and inhibit the RNaseH activity,
respectively. The Clade B CCR5 tropic envelope gene was derived
from the primary ADA isolate; TTTTTNT sequences were mutated
without changing coding capacity to prevent premature transcription
termination and the cytoplasmic tail was truncated in order to
improve surface expression, immunogenicity, and stability of the
MVA vector. The HIV genes were inserted into a plasmid transfer
vector so that gag-pol gene was regulated by the modified H5
early/late vaccinia virus promoter and the env gene was regulated
by the newly designed early/late Psyn II promoter to provide
similar high levels of expression. A self-deleting GUS reporter
gene was included to allow detection and isolation of the
recombinant virus. The HIV genes were flanked by MVA sequences to
allow homologous recombination into the deletion 3 site so that the
recombinant MVA would remain TK positive for stability and high
expression in resting cells. The recombinant MVA was isolated and
shown to express abundant amounts of gag-pol-env and to process
gag. Production of HIV-like particles was demonstrated by
centrifugation and by electron microscopy. The presence of env in
the HIV-like particles was demonstrated by immunoelectron
microscopy.
1 Table of Sequences Description SEQ ID NO FIG. NO pLW-48 1 14
pLW-48 1 15 Psyn II promoter 2 15 ADA envelope truncated 3 15 PmH5
promoter 4 15 HXB2 gag pol 5 15
[0147] Plasmid Transfer Vector
[0148] The plasmid transfer vector used to make the MVA recombinant
virus, pLW-48, (FIG. 16) by homologous recombination was
constructed as follows:
[0149] 1. From the commercially obtained plasmid, pGem-4Z
(Promega), flanking areas on either side of deletion III,
designated flank 1 and flank 2, containing 926 and 520 base pairs
respectively, were amplified by PCR from the MVA stains of vaccinia
virus. Within these flanks, a promoter, the mH5, which had been
modified from the originally published sequence by changing two
bases that had been shown by previously published work to increase
the expression of the cloned gene, was added.
[0150] 2. A clade B gag pol (FIG. 17) was truncated so that the
integrase was removed and was cloned into the plasmid so that it
was controlled by the mH5 promoter. This gene contained the
complete HXB2 sequence of the gag. The pol gene has reverse
transcriptase safety mutations in amino acid 185 within the active
site of RT, in amino acid 266 which inhibits strand transfer
activity, and at amino acid 478 which inhibits the RNaseH activity.
In addition, the integrase gene was deleted past EcoRI site.
[0151] 3. A direct repeat of 280 basepairs, corresponding to the
last 280 base pairs of MVA flank 1, was added after flank 1.
[0152] 4. The p11 promoter and GUS reporter gene were added between
the two direct repeats of flank 1 so that this screening marker
could initially be used for obtaining the recombinant virus, yet
deleted out in the final recombinant virus (Scheiflinger, F. et al.
1998 Arch Virol 143:467-474; Carroll, M. W. and B. Moss 1995
BioTechniques 19:352-355).
[0153] 5. A new promoter, Psyn II, was designed to allow for
increased expression of the ADA env. The sequence of this new
early/late promoter is given in FIG. 18.
[0154] 6. A truncated version of the ADA envelope with a silent
5TNT mutation was obtained by PCR and inserted in the plasmid under
the control of the Psyn II promoter. The envelope was truncated in
the cytoplasmic tail of the gp41 gene, deleting 115 amino acids of
the cytoplasmic tail. This truncation was shown to increase the
amount of envelope protein on the surface of infected cells and
enhance immunogenicity of the envelope protein in mice, and
stability of the recombinant virus in tissue culture.
[0155] Recombinant MVA Construction
[0156] 1. MVA virus, which may be obtained from ATCC Number
VR-1508, was plaque purified three times by terminal dilutions in
chicken embryo fibroblasts (CEF), which were made from 9 day old
SPF Premium SPAFAS fertile chicken eggs, distributed by B and E
Eggs, Stevens, Pa.
[0157] Secondary CEF cells were infected at an MOI of 0.05 of MVA
and transfected with 2 .mu.g of pLW-48, the plasmid described
above. Following a two day incubation at 37.degree. C., the virus
was harvested, frozen and thawed 3.times., and plated out on CEF
plates.
[0158] 3. At 4 days, those foci of infection that stained blue
after addition of X-gluc substrate, indicating that recombination
had occurred between the plasmid and the infecting virus, were
picked and inoculated on CEF plates. Again, those foci that stained
blue were picked.
[0159] 4. These GUS containing foci were plated out in triplicate
and analyzed for GUS staining (which we wanted to now delete) and
ADA envelope expression. Individual foci were picked from the 3rd
replicate plates of those samples that had about equal numbers of
mixed populations of GUS staining and nonstaining foci as well as
mostly envelope staining foci.
[0160] 5. These foci were again plated out in triplicate, and
analyzed the same way. After 5 passages, a virus was derived which
expressed the envelope protein but which had deleted the GUS gene
because of the double repeat. By immunostaining, this virus also
expressed the gag pol protein.
[0161] Characterization of MVA Recombinant Virus, MVA/HIV 48
[0162] 1. Aliquots of MVA/HIV 48 infected cell lysates were
analyzed by radioimmunoprecipitation and immunostaining with
monoclonal antibodies for expression of both the envelope and gag
pol protein. In both of these tests, each of these proteins was
detected.
[0163] 2. The recombinant virus was shown to produce gag particles
in the supernatant of infected cells by pelleting the
.sup.35S-labeled particles on a 20% sucrose cushion.
[0164] 3. Gag particles were also visualized both outside and
budding from cells as well as within vacuoles of cells in the
electron microscope in thin sections. These gag particles had
envelope protein on their surface.
[0165] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer, and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
SUMMARY
[0166] In summary, we have made a recombinant MVA virus, MVA/HIV
48, which has high expression of the ADA truncated envelope and the
HXB2 gag pol. The MVA recombinant virus is made using a transiently
expressed GUS marker that is deleted in the final virus. High
expression of the ADA envelope is possible because of a new hybrid
early/late promoter, Psyn II. In addition, the envelope has been
truncated because we have shown truncation of the envelope enhances
the amount of protein on the surface of the infected cells, and
hence enhances immunogenicity; stability of the recombinant is also
enhanced. The MVA recombinant makes gag particles which has been
shown by pelleting the particles through sucrose and analyzing by
PAGE. Gag particles with envelope protein on the surface have also
been visualized in the electron microscope.
EXAMPLE 3
Additional Modified or Synthetic Promoters Designed for Gene
Expression in MVA or Other Poxviruses
[0167] Additional modified or synthetic promoters were designed for
gene expression in MVA or other poxviruses. Promoters were modified
to allow expression at early and late times after infection and to
reduce possibility of homologous recombination between identical
sequences when multiple promoters are used in same MVA vector.
Promoters are placed upstream of protein coding sequence.
2 m7.5 promoter: CGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACA-
ATAATTAATTTC (SEQ ID NO:10) TCGTAAAAATTGAAAAACTATTCTAATTT-
ATTGCACGGT Psyn II promoter:
TAAAAAATGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTC (SEQ ID
NO:2) TATGCTATAAATAATAAATA Psyn III promoter:
TAAAAATTGAAAAAATATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTC (SEQ ID
NO:11) TATACTATAAATAATAAATA Psyn IV promoter:
TAAAAATTGAAAAACTATTCTAATTTATAGGACGGTTTTGATTTTCTTTTTTTC (SEQ ID
NO:12) TATACTATAAATAATAAATA Psyn V promoter:
AAAAAATGATAAAGTAGGTTCAGTTTTATTGCTGGTTTAAAATCACGCTTTCG (SEQ ID
NO:13) AGTAAAAACTACGAATATAAAT
EXAMPLE 4
Tables A-F
[0168] Table A: MVA/48 immunization--guinea pigs.
[0169] Groups of guinea pigs were immunized at days 0 and 30 with
1.times.10.sup.8 infectious units of MVA/48 by either the
intramuscular (IM) or intradermal (ID) route. As a control another
group was immunized IM with the same dose of non-recombinant MVA.
Sera taken before as well as after each immunization was analyzed
for neutralizing activity against HIV-1-MN. Titers are the
reciprocal serum dilution at which 50% of MT-2 cells were protected
from virus-induced killing. Significant neutralizing activity was
observed in all animals after the second immunization with MVA/48
(day 49).
[0170] Table B: Frequencies of HIV-1 gag-specific T cells following
immunization of mice with MVA/48.
[0171] Groups of BalbC mice were immunized at days 0 and 21 with
1.times.10.sup.7 infectious units of MVA/48 by one of three routes:
intraperitoneal (IP), intradermal (ID), or intramuscular (IM). A
control group was immunized with non-recombinant MVA. At 5 weeks
after the last immunization, splenocytes were prepared and
stimulated in vitro with an immunodominant peptide from HIV-1 p24
for 7 days. The cells were then mixed either with peptide-pulsed
P815 cells or with soluble peptide. Gamma interferon-producing
cells were enumerated in an ELISPOT assay. A value of >500 was
assigned to wells containing too many spots to count. Strong T cell
responses have been reported in mice immunized IP with other
viruses. In this experiment, IP immunization of mice with MVA/48
elicited very strong HIV-1 gag-specific T cell responses.
[0172] Table C: DNA prime and MVA/48 boost--total ELISPOTS per
animal.
[0173] Ten rhesus macaques were primed (weeks 0 and 8) with a DNA
vaccine expressing HIV-1 antigens including Ada envelope and HXB2
gagpol. At week 24 the animals were boosted intramuscularly with
1.times.10.sup.8 infectious units of MVA/48. Fresh peripheral blood
mononuclear cells (PBMC) were analyzed for production of gamma
interferon in an ELISPOT assay as follows: PBMC were incubated for
30-36 hours in the presence of pools of overlapping peptides
corresponding to the individual HIV-1 antigens in the vaccines. The
total number of gamma interferon-producing cells from each animal
is shown in the table. T cell responses to DNA vaccination were
limited (weeks 2-20). However, boosting with MVA/48 resulted in
very strong HIV-1-specific T cell responses in all animals (week
25).
[0174] Table D: Antibody response following immunization of
macaques with MVA/SHIV KB9.
[0175] Groups of rhesus macaques were immunized with
2.times.10.sup.8 infectious units of MVA/SHIV-KB9 at weeks 0 and 4
by one of several routes: Tonsilar, intradermal (ID), or
intramuscular (IM). Another group was immunized with
non-recombinant MVA using the same routes. Serum samples from 2
weeks after the second immunization were analyzed for binding to
KB9 envelope protein by ELISA and for neutralization of SHIV-89.6P
and SHIV-89.6. In the ELISA assay, soluble KB9 envelope protein was
captured in 96 well plates using an antibody to the C-terminus of
gp120. Serial dilutions of sera were analyzed and used to determine
the endpoint titers. Neutralization of SHIV-89.6P and SHIV-89.6 was
determined in an MT-2 cell assay. Titers are the reciprocal serum
dilution at which 50% of the cells were protected from
virus-induced killing. In in vitro neutralization assays,
SHIV-89.6P and SHIV-89.6 are heterologous, i.e. sera from animals
infected with one of the viruses does not neutralize the other
virus. Thus, two immunizations with MVA/SHIV-KB9 elicited good
ELISA binding antibodies in all animals and neutralizing antibodies
to the homologous virus (SHIV-89.6P) in some animals. In addition,
heterologous neutralizing antibodies were observed in a subset of
animals.
[0176] Table E: Frequencies of gag CM-9-specific CD3/CD8 T cells
following immunization of macaques with MVA/SHIV-KB9.
[0177] Groups of MamuA*01 positive rhesus macaques were immunized
with 2.times.10.sup.8 infectious units of MVA/SHIV-KB9 at weeks 0
and 4 by one of several routes: tonsilar, intradermal (ID), or
intramuscular IM). Another group was immunized with non-recombinant
MVA. The frequencies of CD3+/CD8+ T cells that bound tetrameric
complex containing the SIV gag-specific peptide CM9 were determined
by flow cytometry at various times after each immunization. Time
intervals were as follows: 1a, 1b, and 1d were one, two, and four
weeks after the first immunization, respectively; 2a, 2b, 2c, and
2d were one, two, three, and twelve weeks after the second
immunization, respectively. Values above background are shown in
bold face. Strong SIV gag-specific responses were observed after a
single immunization with MVA/SHIV-KB9 in all immunized animals.
Boosting was observed in most animals following the second
immunization. In addition, measurable tetramer binding was still
found twelve weeks after the second immunization.
[0178] Table F: Frequencies of specific T cells following
immunization of macaques with MVA/SHIV KB9.
[0179] Groups of macaques were immunized with MVA/SHIV-KB9 as
described above. MVA/SHIV-KB9 expresses 5 genes from the chimeric
virus, SHIV-89.6P: envelope, gag, polymerase, tat, and nef. Thus,
the frequencies of T cells specific for each of the 5 antigens was
analyzed using pools of peptides corresponding to each individual
protein. Fresh PBMC were stimulated with pools of peptides for
30-36 hours in vitro. Gamma interferon-producing cells were
enumerated in an ELISPOT assay. The total number of cells specific
for each antigen is given as "total # spots". In addition, the
number of responding animals and average # of spots per group is
shown. PBMC were analyzed at one week after the first immunization
(1a) and one week after the second immunization (2a). Another group
of 7 animals was immunized with non-recombinant MVA. In these
animals, no spots above background levels were detected. Thus, a
single immunization with MVA/SHIV-KB9 elicited strong SHIV-specific
T cell responses in all animals. Gag and envelope responses were
the strongest; most animals had responses to gag, all animals had
responses to envelope. The Elispot responses were also observed
after the second immunization with MVA/SHIV-KB9, albeit at lower
levels. At both times, the rank order of responses was:
tonsilar>ID>IM. We show good immune response to nef and some
immune response to tat.
3TABLE A MVA/48 immunization - guinea pigs HIV-MN neutralizing
antibody - reciprocal titer Animal Day 4 day day 33 # Group Route
day 0 MVA #1 30 MVA#2 day 49 885 MVA I.M. <20 I.M. 31 I.M. 24
891 " " <20 " 85 " <20 882 MVA/48 I.M. <20 I.M. <20
I.M. 5,524 883 " " <20 " 68 " 691 886 " " <20 " <20 "
4,249 890 " " <20 " 180 " 89 879 MVA/48 I.D. <20 I.D. <20
I.D. 817 881 " " <20 " <20 " 234 888 " " <20 " 24 " 112
889 " " <20 " 22 " 376
[0180]
4TABLE B Frequencies of HIV-gag-specific T cells following
immunization of mice with MVA/48 Group P815 cells + gag peptide gag
peptide no stimulation MVA control 0 2 0 4 1 2 MVA/48 (IP) >500
>500 >500 >500 8 8 MVA/48 (ID) 12 5 49 33 4 2 MVA/48 (IM)
22 18 66 49 12 8
[0181]
5TABLE C DNA prime and MVA/48 boost Total ELISPOTS per Animal WEEKS
Animal # -2 2 6 10.sup.2 14.sup.2 20.sup.2 25.sup.2 RLw 4 731* <
47 43 50 3905 RVI 5 997* < < < 8 205 Roa .sup. <.sup.1
< 1 < < < 245 RHc < < < < < < 535 Ryl
< < < < < < 4130 RQk < 46 < < < <
630 RDr < < < 14 < < 1965 RZc < 5 < 58 <
< 925 RSf < 118 < < < 20 5570 Ras < 69 < <
< < 1435 Total 9 1966 1 119 43 78 19545 Geo Mean 4.5 105.3
1.0 33.7 43.0 20.0 1147.7 DNA primes were at 0 and 8 weeks and
MVA/48 boost was at 24 weeks .sup.1< = Background (2 .times. the
number of ELISPOTs in the unstimulated control + 10)
.sup.2Costimulatory antibodies were added to the ELISPOT
incubations *Animals from this bleed date exhibited higher than
usual ELISPOTs.
[0182]
6TABLE D Antibody response following immunization of macaques with
MVA/SHIV KB9 KB9 SHIV- SHIV- env SHIV- SHIV- 89.6 89.6P ELISA KB9
env elisa 89.6 89.6P # pos # pos Animal # Route titer average std
dev. Nab titer Nab titer animals animals 598 tonsil 25,600 31,086
20,383 <20 <20 3 2 601 " 51,200 <20 <20 606 " 25,600
<20 <20 642 " 51,200 75 31 646 " 51,200 61 48 653 " 6,400
<20 <20 654 " 6,400 22 <20 602 i.d. 25,600 18,800 15,341
38 <20 2 4 604 " 12,800 <20 262 608 " 3,200 20 66 637 "
12,800 <20 35 638 " 51,200 <20 <20 645 " 25,600 <20
<20 647 " 12,800 32 162 650 " 6,400 <20 <20 599 i.m. 6,400
17,000 16,516 <20 <20 0 3 600 " 6,400 <20 29 609 " 6,400
<20 <20 639 " 51,200 <20 85 640 " 12,800 <20 <20 641
" 25,600 <20 41 649 " 1,600 <20 <20 651 " 25,600 20 <20
603 Control <100 <100 <20 <20 0 0 605 " <100 <20
<20 607 " <100 <20 <20 643 " <100 <20 <20 644
" <100 <20 <20 648 " <100 <20 <20 652 " <100
<20 <20
[0183]
7TABLE E Frequencies of gag CM9-specific CD3/CD8 T cells following
immunization of macaques with MVA/SHIV KB9 pre- Animal # Route
Virus bleed 1a 1b 1d 2a 2b 2c 2d 598 Tonsil MVA/KB9 0.018 0.41 0.79
0.25 2.64 1.13 0.51 0.21 601 " " 0.071 0.34 0.38 0.27 0.83 0.7 0.36
0.039 646 " " 0.022 0.68 0.76 0.43 1.12 0.91 0.53 0.15 653 " "
0.041 0.69 0.85 0.53 0.68 0.49 0.47 0.3 648 " MVA 0.033 0.039 0.022
0.058 0.033 0.013 602 i.d. MVA/KB9 0.019 0.17 0.92 0.5 0.95 0.59
0.5 0.2 604 " " 0.013 0.11 0.38 0.32 0.44 0.38 0.19 0.25 650 " "
0.095 0.17 0.6 0.23 2.87 1.12 0.9 0.16 647 " " 0.032 0.22 0.38 0.14
0.84 0.91 0.34 0.17 652 " MVA 0.041 0.038 0.059 0.025 0.022 0.026
0.055 599 i.m. MVA/KB9 0.081 0.31 0.082 0.12 0.054 0.11 600 " "
0.034 0.15 0.41 0.17 0.29 0.27 0.16 0.049 649 " " 0.00486 0.35 1.34
0.56 2.42 0.77 0.69 0.22 651 " " 0.049 0.12 0.69 0.25 1.01 0.32
0.24 0.22 603 " MVA 0.024 0.087 0.073 0.082 0.027 0.17
[0184]
8TABLE F Frequencies of specific T cells following immunization of
macaques with MVA/SHIV KB9 Gag specific Tat specific Nef specific
Env specific Total # Total average # total average # total average
# total Average # Study responding # # responding # # responding #
# responding # # responding groups animals spots spots animals
spots spots animals spots spots animals spots spots animals tonsil
1a 4/6 1325 221 0/6 0 0 3/6 195 33 6/6 8760 1460 6/6 tonsil 2a 5/6
1405 234 0/6 0 0 1/6 560 93 6/6 4485 748 6/6 i.d. 1a 7/7 1335 191
0/7 0 0 2/7 215 31 7/7 7320 1046 7/7 i.d. 2a 4/7 755 108 0/7 0 0
1/7 55 8 7/7 2700 386 7/7 i.m. 1a 7/7 925 132 1/7 60 9 3/7 180 26
7/7 5490 784 7/7 i.m. 2a 4/7 250 36 0/7 0 0 0/7 0 0 6/7 2205 315
6/7
[0185] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
patents, patent applications and publications referred to above are
hereby incorporated by reference.
Sequence CWU 1
1
13 1 12225 DNA Artificial Sequence Plasmid pLW-48 1 gaattcgttg
gtggtcgcca tggatggtgt tattgtatac tgtctaaacg cgttagtaaa 60
acatggcgag gaaataaatc atataaaaaa tgatttcatg attaaaccat gttgtgaaaa
120 agtcaagaac gttcacattg gcggacaatc taaaaacaat acagtgattg
cagatttgcc 180 atatatggat aatgcggtat ccgatgtatg caattcactg
tataaaaaga atgtatcaag 240 aatatccaga tttgctaatt tgataaagat
agatgacgat gacaagactc ctactggtgt 300 atataattat tttaaaccta
aagatgccat tcctgttatt atatccatag gaaaggatag 360 agatgtttgt
gaactattaa tctcatctga taaagcgtgt gcgtgtatag agttaaattc 420
atataaagta gccattcttc ccatggatgt ttcctttttt accaaaggaa atgcatcatt
480 gattattctc ctgtttgatt tctctatcga tgcggcacct ctcttaagaa
gtgtaaccga 540 taataatgtt attatatcta gacaccagcg tctacatgac
gagcttccga gttccaattg 600 gttcaagttt tacataagta taaagtccga
ctattgttct atattatata tggttgttga 660 tggatctgtg atgcatgcaa
tagctgataa tagaacttac gcaaatatta gcaaaaatat 720 attagacaat
actacaatta acgatgagtg tagatgctgt tattttgaac cacagattag 780
gattcttgat agagatgaga tgctcaatgg atcatcgtgt gatatgaaca gacattgtat
840 tatgatgaat ttacctgatg taggcgaatt tggatctagt atgttgggga
aatatgaacc 900 tgacatgatt aagattgctc tttcggtggc tgggtaccag
gcgcgccttt cattttgttt 960 ttttctatgc tataaatggt acgtcctgta
gaaaccccaa cccgtgaaat caaaaaactc 1020 gacggcctgt gggcattcag
tctggatcgc gaaaactgtg gaattgatca gcgttggtgg 1080 gaaagcgcgt
tacaagaaag ccgggcaatt gctgtgccag gcagttttaa cgatcagttc 1140
gccgatgcag atattcgtaa ttatgcgggc aacgtctggt atcagcgcga agtctttata
1200 ccgaaaggtt gggcaggcca gcgtatcgtg ctgcgtttcg atgcggtcac
tcattacggc 1260 aaagtgtggg tcaataatca ggaagtgatg gagcatcagg
gcggctatac gccatttgaa 1320 gccgatgtca cgccgtatgt tattgccggg
aaaagtgtac gtatcaccgt ttgtgtgaac 1380 aacgaactga actggcagac
tatcccgccg ggaatggtga ttaccgacga aaacggcaag 1440 aaaaagcagt
cttacttcca tgatttcttt aactatgccg gaatccatcg cagcgtaatg 1500
ctctacacca cgccgaacac ctgggtggac gatatcaccg tggtgacgca tgtcgcgcaa
1560 gactgtaacc acgcgtctgt tgactggcag gtggtggcca atggtgatgt
cagcgttgaa 1620 ctgcgtgatg cggatcaaca ggtggttgca actggacaag
gcactagcgg gactttgcaa 1680 gtggtgaatc cgcacctctg gcaaccgggt
gaaggttatc tctatgaact gtgcgtcaca 1740 gccaaaagcc agacagagtg
tgatatctac ccgcttcgcg tcggcatccg gtcagtggca 1800 gtgaagggcg
aacagttcct gattaaccac aaaccgttct actttactgg ctttggtcgt 1860
catgaagatg cggacttgcg tggcaaagga ttcgataacg tgctgatggt gcacgaccac
1920 gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc
ttacgctgaa 1980 gagatgctcg actgggcaga tgaacatggc atcgtggtga
ttgatgaaac tgctgctgtc 2040 ggctttaacc tctctttagg cattggtttc
gaagcgggca acaagccgaa agaactgtac 2100 agcgaagagg cagtcaacgg
ggaaactcag caagcgcact tacaggcgat taaagagctg 2160 atagcgcgtg
acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 2220
acccgtccgc aaggtgcacg ggaatatttc gcgccactgg cggaagcaac gcgtaaactc
2280 gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca
caccgatacc 2340 atcagcgatc tctttgatgt gctgtgcctg aaccgttatt
acggatggta tgtccaaagc 2400 ggcgatttgg aaacggcaga gaaggtactg
gaaaaagaac ttctggcctg gcaggagaaa 2460 ctgcatcagc cgattatcat
caccgaatac ggcgtggata cgttagccgg gctgcactca 2520 atgtacaccg
acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc 2580
gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc cgattttgcg
2640 acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac
tcgcgaccgc 2700 aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga
ctggcatgaa cttcggtgaa 2760 aaaccgcagc agggaggcaa acaatgagag
ctcggttgtt gatggatctg tgatgcatgc 2820 aatagctgat aatagaactt
acgcaaatat tagcaaaaat atattagaca atactacaat 2880 taacgatgag
tgtagatgct gttattttga accacagatt aggattcttg atagagatga 2940
gatgctcaat ggatcatcgt gtgatatgaa cagacattgt attatgatga atttacctga
3000 tgtaggcgaa tttggatcta gtatgttggg gaaatatgaa cctgacatga
ttaagattgc 3060 tctttcggtg gctggcggcc cgctcgagta aaaaatgaaa
aaatattcta atttatagga 3120 cggttttgat tttctttttt tctatgctat
aaataataaa tagcggccgc accatgaaag 3180 tgaaggggat caggaagaat
tatcagcact tgtggaaatg gggcatcatg ctccttggga 3240 tgttgatgat
ctgtagtgct gtagaaaatt tgtgggtcac agtttattat ggggtacctg 3300
tgtggaaaga agcaaccacc actctatttt gtgcatcaga tgctaaagca tatgatacag
3360 aggtacataa tgtttgggcc acacatgcct gtgtacccac agaccccaac
ccacaagaag 3420 tagtattgga aaatgtgaca gaaaatttta acatgtggaa
aaataacatg gtagaacaga 3480 tgcatgagga tataatcagt ttatgggatc
aaagcctaaa gccatgtgta aaattaaccc 3540 cactctgtgt tactttaaat
tgcactgatt tgaggaatgt tactaatatc aataatagta 3600 gtgagggaat
gagaggagaa ataaaaaact gctctttcaa tatcaccaca agcataagag 3660
ataaggtgaa gaaagactat gcacttttct atagacttga tgtagtacca atagataatg
3720 ataatactag ctataggttg ataaattgta atacctcaac cattacacag
gcctgtccaa 3780 aggtatcctt tgagccaatt cccatacatt attgtacccc
ggctggtttt gcgattctaa 3840 agtgtaaaga caagaagttc aatggaacag
ggccatgtaa aaatgtcagc acagtacaat 3900 gtacacatgg aattaggcca
gtagtgtcaa ctcaactgct gttaaatggc agtctagcag 3960 aagaagaggt
agtaattaga tctagtaatt tcacagacaa tgcaaaaaac ataatagtac 4020
agttgaaaga atctgtagaa attaattgta caagacccaa caacaataca aggaaaagta
4080 tacatatagg accaggaaga gcattttata caacaggaga aataatagga
gatataagac 4140 aagcacattg caacattagt agaacaaaat ggaataacac
tttaaatcaa atagctacaa 4200 aattaaaaga acaatttggg aataataaaa
caatagtctt taatcaatcc tcaggagggg 4260 acccagaaat tgtaatgcac
agttttaatt gtggagggga attcttctac tgtaattcaa 4320 cacaactgtt
taatagtact tggaatttta atggtacttg gaatttaaca caatcgaatg 4380
gtactgaagg aaatgacact atcacactcc catgtagaat aaaacaaatt ataaatatgt
4440 ggcaggaagt aggaaaagca atgtatgccc ctcccatcag aggacaaatt
agatgctcat 4500 caaatattac agggctaata ttaacaagag atggtggaac
taacagtagt gggtccgaga 4560 tcttcagacc tgggggagga gatatgaggg
acaattggag aagtgaatta tataaatata 4620 aagtagtaaa aattgaacca
ttaggagtag cacccaccaa ggcaaaaaga agagtggtgc 4680 agagagaaaa
aagagcagtg ggaacgatag gagctatgtt ccttgggttc ttgggagcag 4740
caggaagcac tatgggcgca gcgtcaataa cgctgacggt acaggccaga ctattattgt
4800 ctggtatagt gcaacagcag aacaatttgc tgagggctat tgaggcgcaa
cagcatctgt 4860 tgcaactcac agtctggggc atcaagcagc tccaggcaag
agtcctggct gtggaaagat 4920 acctaaggga tcaacagctc ctagggattt
ggggttgctc tggaaaactc atctgcacca 4980 ctgctgtgcc ttggaatgct
agttggagta ataaaactct ggatatgatt tgggataaca 5040 tgacctggat
ggagtgggaa agagaaatcg aaaattacac aggcttaata tacaccttaa 5100
ttgaggaatc gcagaaccaa caagaaaaga atgaacaaga cttattagca ttagataagt
5160 gggcaagttt gtggaattgg tttgacatat caaattggct gtggtatgta
aaaatcttca 5220 taatgatagt aggaggcttg ataggtttaa gaatagtttt
tactgtactt tctatagtaa 5280 atagagttag gcagggatac tcaccattgt
catttcagac ccacctccca gccccgaggg 5340 gacccgacag gcccgaagga
atcgaagaag aaggtggaga cagagactaa tttttatgcg 5400 gccgctggta
cccaacctaa aaattgaaaa taaatacaaa ggttcttgag ggttgtgtta 5460
aattgaaagc gagaaataat cataaataag cccggggatc ctctagagtc gacaccatgg
5520 gtgcgagagc gtcagtatta agcgggggag aattagatcg atgggaaaaa
attcggttaa 5580 ggccaggggg aaagaaaaaa tataaattaa aacatatagt
atgggcaagc agggagctag 5640 aacgattcgc agttaatcct ggcctgttag
aaacatcaga aggctgtaga caaatactgg 5700 gacagctaca accatccctt
cagacaggat cagaagaact tagatcatta tataatacag 5760 tagcaaccct
ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag 5820
acaagataga ggaagagcaa aacaaaagta agaaaaaagc acagcaagca gcagctgaca
5880 caggacacag caatcaggtc agccaaaatt accctatagt gcagaacatc
caggggcaaa 5940 tggtacatca ggccatatca cctagaactt taaatgcatg
ggtaaaagta gtagaagaga 6000 aggctttcag cccagaagtg atacccatgt
tttcagcatt atcagaagga gccaccccac 6060 aagatttaaa caccatgcta
aacacagtgg ggggacatca agcagccatg caaatgttaa 6120 aagagaccat
caatgaggaa gctgcagaat gggatagagt gcatccagtg catgcagggc 6180
ctattgcacc aggccagatg agagaaccaa ggggaagtga catagcagga actactagta
6240 cccttcagga acaaatagga tggatgacaa ataatccacc tatcccagta
ggagaaattt 6300 ataaaagatg gataatcctg ggattaaata aaatagtaag
aatgtatagc cctaccagca 6360 ttctggacat aagacaagga ccaaaagaac
cctttagaga ctatgtagac cggttctata 6420 aaactctaag agccgagcaa
gcttcacagg aggtaaaaaa ttggatgaca gaaaccttgt 6480 tggtccaaaa
tgcgaaccca gattgtaaga ctattttaaa agcattggga ccagcggcta 6540
cactagaaga aatgatgaca gcatgtcagg gagtaggagg acccggccat aaggcaagag
6600 ttttggctga agcaatgagc caagtaacaa attcagctac cataatgatg
cagagaggca 6660 attttaggaa ccaaagaaag attgttaagt gtttcaattg
tggcaaagaa gggcacacag 6720 ccagaaattg cagggcccct aggaaaaagg
gctgttggaa atgtggaaag gaaggacacc 6780 aaatgaaaga ttgtactgag
agacaggcta attttttagg gaagatctgg ccttcctaca 6840 agggaaggcc
agggaatttt cttcagagca gaccagagcc aacagcccca ccagaagaga 6900
gcttcaggtc tggggtagag acaacaactc cccctcagaa gcaggagccg atagacaagg
6960 aactgtatcc tttaacttcc ctcagatcac tctttggcaa cgacccctcg
tcacaataaa 7020 gatagggggg caactaaagg aagctctatt agatacagga
gcagatgata cagtattaga 7080 agaaatgagt ttgccaggaa gatggaaacc
aaaaatgata gggggaattg gaggttttat 7140 caaagtaaga cagtatgatc
agatactcat agaaatctgt ggacataaag ctataggtac 7200 agtattagta
ggacctacac ctgtcaacat aattggaaga aatctgttga ctcagattgg 7260
ttgcacttta aattttccca ttagccctat tgagactgta ccagtaaaat taaagccagg
7320 aatggatggc ccaaaagtta aacaatggcc attgacagaa gaaaaaataa
aagcattagt 7380 agaaatttgt acagaaatgg aaaaggaagg gaaaatttca
aaaattgggc ctgagaatcc 7440 atacaatact ccagtatttg ccataaagaa
aaaagacagt actaaatgga ggaaattagt 7500 agatttcaga gaacttaata
agagaactca agacttctgg gaagttcaat taggaatacc 7560 acatcccgca
gggttaaaaa agaaaaaatc agtaacagta ctggatgtgg gtgatgcata 7620
tttttcagtt cccttagatg aagacttcag gaagtatact gcatttacca tacctagtat
7680 aaacaatgag acaccaggga ttagatatca gtacaatgtg cttccacagg
gatggaaagg 7740 atcaccagca atattccaaa gtagcatgac aaaaatctta
gagcctttta aaaaacaaaa 7800 tccagacata gttatctatc aatacatgaa
cgatttgtat gtaggatctg acttagaaat 7860 agggcagcat agaacaaaaa
tagaggagct gagacaacat ctgttgaggt ggggacttac 7920 cacaccagac
aaaaaacatc agaaagaacc tccattcctt tggatgggtt atgaactcca 7980
tcctgataaa tggacagtac agcctatagt gctgccagaa aaagacagct ggactgtcaa
8040 tgacatacag aagttagtgg ggaaattgaa taccgcaagt cagatttacc
cagggattaa 8100 agtaaggcaa ttatgtaaac tccttagagg aaccaaagca
ctaacagaag taataccact 8160 aacagaagaa gcagagctag aactggcaga
aaacagagag attctaaaag aaccagtaca 8220 tggagtgtat tatgacccat
caaaagactt aatagcagaa atacagaagc aggggcaagg 8280 ccaatggaca
tatcaaattt atcaagagcc atttaaaaat ctgaaaacag gaaaatatgc 8340
aagaatgagg ggtgcccaca ctaatgatgt aaaacaatta acagaggcag tgcaaaaaat
8400 aaccacagaa agcatagtaa tatggggaaa gactcctaaa tttaaactac
ccatacaaaa 8460 ggaaacatgg gaaacatggt ggacagagta ttggcaagcc
acctggattc ctgagtggga 8520 gtttgttaat acccctcctt tagtgaaatt
atggtaccag ttagagaaag aacccatagt 8580 aggagcagaa accttctatg
tagatggggc agctaacagg gagactaaat taggaaaagc 8640 aggatatgtt
actaacaaag gaagacaaaa ggttgtcccc ctaactaaca caacaaatca 8700
gaaaactcag ttacaagcaa tttatctagc tttgcaggat tcaggattag aagtaaacat
8760 agtaacagac tcacaatatg cattaggaat cattcaagca caaccagata
aaagtgaatc 8820 agagttagtc aatcaaataa tagagcagtt aataaaaaag
gaaaaggtct atctggcatg 8880 ggtaccagca cacaaaggaa ttggaggaaa
tgaacaagta gataaattag tcagtgctgg 8940 aatcaggaaa atactatttt
tagatggaat agataaggcc caagatgaac attagttttt 9000 atgtcgacct
gcagggaaag ttttataggt agttgataga acaaaataca taattttgta 9060
aaaataaatc actttttata ctaatatgac acgattacca atacttttgt tactaatatc
9120 attagtatac gctacacctt ttcctcagac atctaaaaaa ataggtgatg
atgcaacttt 9180 atcatgtaat cgaaataata caaatgacta cgttgttatg
agtgcttggt ataaggagcc 9240 caattccatt attcttttag ctgctaaaag
cgacgtcttg tattttgata attataccaa 9300 ggataaaata tcttacgact
ctccatacga tgatctagtt acaactatca caattaaatc 9360 attgactgct
agagatgccg gtacttatgt atgtgcattc tttatgacat cgcctacaaa 9420
tgacactgat aaagtagatt atgaagaata ctccacagag ttgattgtaa atacagatag
9480 tgaatcgact atagacataa tactatctgg atctacacat tcaccagaaa
ctagttaagc 9540 ttgtctccct atagtgagtc gtattagagc ttggcgtaat
catggtcata gctgtttcct 9600 gtgtgaaatt gttatccgct cacaattcca
cacaacatac gagccggaag cataaagtgt 9660 aaagcctggg gtgcctaatg
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 9720 gctttcgagt
cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 9780
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
9840 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
gttatccaca 9900 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
gccagcaaaa ggccaggaac 9960 cgtaaaaagg ccgcgttgct ggcgtttttc
gataggctcc gcccccctga cgagcatcac 10020 aaaaatcgac gctcaagtca
gaggtggcga aacccgacag gactataaag ataccaggcg 10080 tttccccctg
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 10140
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
10200 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
ccccgttcag 10260 cccgaccgct gcgccttatc cggtaactat cgtcttgagt
ccaacccggt aagacacgac 10320 ttatcgccac tggcagcagc cactggtaac
aggattagca gagcgaggta tgtaggcggt 10380 gctacagagt tcttgaagtg
gtggcctaac tacggctaca ctagaaggac agtatttggt 10440 atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 10500
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
10560 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac 10620 gaaaactcac gttaagggat tttggtcatg agattatcaa
aaaggatctt cacctagatc 10680 cttttaaatt aaaaatgaag ttttaaatca
atctaaagta tatatgagta aacttggtct 10740 gacagttacc aatgcttaat
cagtgaggca cctatctcag cgatctgtct atttcgttca 10800 tccatagttg
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 10860
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
10920 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
atccgcctcc 10980 atccagtcta ttaattgttg ccgggaagct agagtaagta
gttcgccagt taatagtttg 11040 cgcaacgttg ttggcattgc tacaggcatc
gtggtgtcac gctcgtcgtt tggtatggct 11100 tcattcagct ccggttccca
acgatcaagg cgagttacat gatcccccat gttgtgcaaa 11160 aaagcggtta
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 11220
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
11280 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg 11340 agttgctctt gcccggcgtc aatacgggat aataccgcgc
cacatagcag aactttaaaa 11400 gtgctcatca ttggaaaacg ttcttcgggg
cgaaaactct caaggatctt accgctgttg 11460 agatccagtt cgatgtaacc
cactcgtgca cccaactgat cttcagcatc ttttactttc 11520 accagcgttt
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 11580
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
11640 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
taaacaaata 11700 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
tctaagaaac cattattatc 11760 atgacattaa cctataaaaa taggcgtatc
acgaggccct ttcgtctcgc gcgtttcggt 11820 gatgacggtg aaaacctctg
acacatgcag ctcccggaga cggtcacagc ttgtctgtaa 11880 gcggatgccg
ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 11940
ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
12000 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
gccattcagg 12060 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
cgctattacg ccagctggcg 12120 aaagggggat gtgctgcaag gcgattaagt
tgggtaacgc cagggttttc ccagtcacga 12180 cgttgtaaaa cgacggccag
tgaattggat ttaggtgaca ctata 12225 2 74 DNA Artificial Sequence Psyn
II promoter 2 taaaaaatga aaaaatattc taatttatag gacggttttg
attttctttt tttctatgct 60 ataaataata aata 74 3 2214 DNA Artificial
Sequence HIV env gene 3 atgaaagtga aggggatcag gaagaattat cagcacttgt
ggaaatgggg catcatgctc 60 cttgggatgt tgatgatctg tagtgctgta
gaaaatttgt gggtcacagt ttattatggg 120 gtacctgtgt ggaaagaagc
aaccaccact ctattttgtg catcagatgc taaagcatat 180 gatacagagg
tacataatgt ttgggccaca catgcctgtg tacccacaga ccccaaccca 240
caagaagtag tattggaaaa tgtgacagaa aattttaaca tgtggaaaaa taacatggta
300 gaacagatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc
atgtgtaaaa 360 ttaaccccac tctgtgttac tttaaattgc actgatttga
ggaatgttac taatatcaat 420 aatagtagtg agggaatgag aggagaaata
aaaaactgct ctttcaatat caccacaagc 480 ataagagata aggtgaagaa
agactatgca cttttctata gacttgatgt agtaccaata 540 gataatgata
atactagcta taggttgata aattgtaata cctcaaccat tacacaggcc 600
tgtccaaagg tatcctttga gccaattccc atacattatt gtaccccggc tggttttgcg
660 attctaaagt gtaaagacaa gaagttcaat ggaacagggc catgtaaaaa
tgtcagcaca 720 gtacaatgta cacatggaat taggccagta gtgtcaactc
aactgctgtt aaatggcagt 780 ctagcagaag aagaggtagt aattagatct
agtaatttca cagacaatgc aaaaaacata 840 atagtacagt tgaaagaatc
tgtagaaatt aattgtacaa gacccaacaa caatacaagg 900 aaaagtatac
atataggacc aggaagagca ttttatacaa caggagaaat aataggagat 960
ataagacaag cacattgcaa cattagtaga acaaaatgga ataacacttt aaatcaaata
1020 gctacaaaat taaaagaaca atttgggaat aataaaacaa tagtctttaa
tcaatcctca 1080 ggaggggacc cagaaattgt aatgcacagt tttaattgtg
gaggggaatt cttctactgt 1140 aattcaacac aactgtttaa tagtacttgg
aattttaatg gtacttggaa tttaacacaa 1200 tcgaatggta ctgaaggaaa
tgacactatc acactcccat gtagaataaa acaaattata 1260 aatatgtggc
aggaagtagg aaaagcaatg tatgcccctc ccatcagagg acaaattaga 1320
tgctcatcaa atattacagg gctaatatta acaagagatg gtggaactaa cagtagtggg
1380 tccgagatct tcagacctgg gggaggagat atgagggaca attggagaag
tgaattatat 1440 aaatataaag tagtaaaaat tgaaccatta ggagtagcac
ccaccaaggc aaaaagaaga 1500 gtggtgcaga gagaaaaaag agcagtggga
acgataggag ctatgttcct tgggttcttg 1560 ggagcagcag gaagcactat
gggcgcagcg tcaataacgc tgacggtaca ggccagacta 1620 ttattgtctg
gtatagtgca acagcagaac aatttgctga gggctattga ggcgcaacag 1680
catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagagt cctggctgtg
1740 gaaagatacc taagggatca acagctccta gggatttggg gttgctctgg
aaaactcatc 1800 tgcaccactg ctgtgccttg gaatgctagt tggagtaata
aaactctgga tatgatttgg 1860 gataacatga cctggatgga gtgggaaaga
gaaatcgaaa attacacagg cttaatatac 1920 accttaattg aggaatcgca
gaaccaacaa gaaaagaatg aacaagactt attagcatta 1980 gataagtggg
caagtttgtg gaattggttt gacatatcaa attggctgtg gtatgtaaaa 2040
atcttcataa tgatagtagg aggcttgata ggtttaagaa tagtttttac tgtactttct
2100 atagtaaata gagttaggca gggatactca ccattgtcat ttcagaccca
cctcccagcc 2160 ccgaggggac ccgacaggcc cgaaggaatc gaagaagaag
gtggagacag agac 2214 4 70 DNA Artificial Sequence PmH5 promoter 4
aaaaattgaa aataaataca aaggttcttg agggttgtgt taaattgaaa gcgagaaata
60 atcataaata 70 5 3479 DNA Artificial Sequence HIV genes 5
atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgatggga aaaaattcgg
60 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
aagcagggag 120 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
cagaaggctg tagacaaata 180 ctgggacagc tacaaccatc ccttcagaca
ggatcagaag aacttagatc attatataat 240 acagtagcaa ccctctattg
tgtgcatcaa aggatagaga taaaagacac caaggaagct 300 ttagacaaga
tagaggaaga gcaaaacaaa agtaagaaaa aagcacagca agcagcagct 360
gacacaggac acagcaatca ggtcagccaa aattacccta tagtgcagaa catccagggg
420 caaatggtac atcaggccat atcacctaga actttaaatg catgggtaaa
agtagtagaa 480 gagaaggctt tcagcccaga agtgataccc atgttttcag
cattatcaga aggagccacc 540 ccacaagatt taaacaccat gctaaacaca
gtggggggac atcaagcagc catgcaaatg 600 ttaaaagaga ccatcaatga
ggaagctgca gaatgggata gagtgcatcc agtgcatgca 660 gggcctattg
caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact 720
agtacccttc aggaacaaat aggatggatg acaaataatc cacctatccc agtaggagaa
780 atttataaaa gatggataat cctgggatta aataaaatag taagaatgta
tagccctacc 840 agcattctgg acataagaca aggaccaaaa gaacccttta
gagactatgt agaccggttc 900 tataaaactc taagagccga gcaagcttca
caggaggtaa aaaattggat gacagaaacc 960 ttgttggtcc aaaatgcgaa
cccagattgt aagactattt taaaagcatt gggaccagcg 1020 gctacactag
aagaaatgat gacagcatgt cagggagtag gaggacccgg ccataaggca 1080
agagttttgg ctgaagcaat gagccaagta acaaattcag ctaccataat gatgcagaga
1140 ggcaatttta ggaaccaaag aaagattgtt aagtgtttca attgtggcaa
agaagggcac 1200 acagccagaa attgcagggc ccctaggaaa aagggctgtt
ggaaatgtgg aaaggaagga 1260 caccaaatga aagattgtac tgagagacag
gctaattttt tagggaagat ctggccttcc 1320 tacaagggaa ggccagggaa
ttttcttcag agcagaccag agccaacagc cccaccagaa 1380 gagagcttca
ggtctggggt agagacaaca actccccctc agaagcagga gccgatagac 1440
aaggaactgt atcctttaac ttccctcaga tcactctttg gcaacgaccc ctcgtcacaa
1500 taaagatagg ggggcaacta aaggaagctc tattagatac aggagcagat
gatacagtat 1560 tagaagaaat gagtttgcca ggaagatgga aaccaaaaat
gataggggga attggaggtt 1620 ttatcaaagt aagacagtat gatcagatac
tcatagaaat ctgtggacat aaagctatag 1680 gtacagtatt agtaggacct
acacctgtca acataattgg aagaaatctg ttgactcaga 1740 ttggttgcac
tttaaatttt cccattagcc ctattgagac tgtaccagta aaattaaagc 1800
caggaatgga tggcccaaaa gttaaacaat ggccattgac agaagaaaaa ataaaagcat
1860 tagtagaaat ttgtacagaa atggaaaagg aagggaaaat ttcaaaaatt
gggcctgaga 1920 atccatacaa tactccagta tttgccataa agaaaaaaga
cagtactaaa tggaggaaat 1980 tagtagattt cagagaactt aataagagaa
ctcaagactt ctgggaagtt caattaggaa 2040 taccacatcc cgcagggtta
aaaaagaaaa aatcagtaac agtactggat gtgggtgatg 2100 catatttttc
agttccctta gatgaagact tcaggaagta tactgcattt accataccta 2160
gtataaacaa tgagacacca gggattagat atcagtacaa tgtgcttcca cagggatgga
2220 aaggatcacc agcaatattc caaagtagca tgacaaaaat cttagagcct
tttaaaaaac 2280 aaaatccaga catagttatc tatcaataca tgaacgattt
gtatgtagga tctgacttag 2340 aaatagggca gcatagaaca aaaatagagg
agctgagaca acatctgttg aggtggggac 2400 ttaccacacc agacaaaaaa
catcagaaag aacctccatt cctttggatg ggttatgaac 2460 tccatcctga
taaatggaca gtacagccta tagtgctgcc agaaaaagac agctggactg 2520
tcaatgacat acagaagtta gtggggaaat tgaataccgc aagtcagatt tacccaggga
2580 ttaaagtaag gcaattatgt aaactcctta gaggaaccaa agcactaaca
gaagtaatac 2640 cactaacaga agaagcagag ctagaactgg cagaaaacag
agagattcta aaagaaccag 2700 tacatggagt gtattatgac ccatcaaaag
acttaatagc agaaatacag aagcaggggc 2760 aaggccaatg gacatatcaa
atttatcaag agccatttaa aaatctgaaa acaggaaaat 2820 atgcaagaat
gaggggtgcc cacactaatg atgtaaaaca attaacagag gcagtgcaaa 2880
aaataaccac agaaagcata gtaatatggg gaaagactcc taaatttaaa ctacccatac
2940 aaaaggaaac atgggaaaca tggtggacag agtattggca agccacctgg
attcctgagt 3000 gggagtttgt taatacccct cctttagtga aattatggta
ccagttagag aaagaaccca 3060 tagtaggagc agaaaccttc tatgtagatg
gggcagctaa cagggagact aaattaggaa 3120 aagcaggata tgttactaac
aaaggaagac aaaaggttgt ccccctaact aacacaacaa 3180 atcagaaaac
tcagttacaa gcaatttatc tagctttgca ggattcagga ttagaagtaa 3240
acatagtaac agactcacaa tatgcattag gaatcattca agcacaacca gataaaagtg
3300 aatcagagtt agtcaatcaa ataatagagc agttaataaa aaaggaaaag
gtctatctgg 3360 catgggtacc agcacacaaa ggaattggag gaaatgaaca
agtagataaa ttagtcagtg 3420 ctggaatcag gaaaatacta tttttagatg
gaatagataa ggcccaagat gaacattag 3479 6 9 PRT Simian
Immunodeficiency virus 6 Cys Thr Pro Tyr Asp Ile Asn Gln Met 1 5 7
8 PRT Chicken 7 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 8 20 DNA
Artificial Sequence probe 8 ctgtctgcgt catttggtgc 20 9 4 PRT Human
immunodeficiency Virus VARIANT (1)...(4) Xaa = Any Amino Acid 9 Tyr
Xaa Xaa Leu 1 10 93 DNA Artificial Sequence m7.5 promoter 10
cgctttttat agtaagtttt tcacccataa ataataaata caataattaa tttctcgtaa
60 aaattgaaaa actattctaa tttattgcac ggt 93 11 74 DNA Artificial
Sequence Psyn III promoter 11 taaaaattga aaaaatattc taatttatag
gacggttttg attttctttt tttctatact 60 ataaataata aata 74 12 74 DNA
Artificial Sequence Psyn IV promoter 12 taaaaattga aaaactattc
taatttatag gacggttttg attttctttt tttctatact 60 ataaataata aata 74
13 75 DNA Artificial Sequence Psyn V promoter 13 aaaaaatgat
aaagtaggtt cagttttatt gctggtttaa aatcacgctt tcgagtaaaa 60
actacgaata taaat 75
* * * * *
References