U.S. patent number 5,741,492 [Application Number 08/590,288] was granted by the patent office on 1998-04-21 for preparation and use of viral vectors for mixed envelope protein vaccines against human immunodeficiency viruses.
This patent grant is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Julia L. Hurwitz, Randall J. Owens.
United States Patent |
5,741,492 |
Hurwitz , et al. |
April 21, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
Preparation and use of viral vectors for mixed envelope protein
vaccines against human immunodeficiency viruses
Abstract
Polyenv vaccines are provided that comprise mixtures of at least
4-40 to about 10,000 different recombinant vaccinia viruses that
each express a different HIV env variant or a portion thereof
containing both constant and variable regions, as well as methods
of making and using such polyenv vaccines and vaccinia viruses,
including the use of the polyenv vaccine, in live, attenuated or
inactivated form, for prophylaxis or treatment of HIV
infection.
Inventors: |
Hurwitz; Julia L. (Germantown,
TN), Owens; Randall J. (Millington, TN) |
Assignee: |
St. Jude Children's Research
Hospital (Memphis, TN)
|
Family
ID: |
24361647 |
Appl.
No.: |
08/590,288 |
Filed: |
January 23, 1996 |
Current U.S.
Class: |
424/208.1;
424/160.1; 536/23.2; 435/320.1; 424/199.1; 514/44R |
Current CPC
Class: |
A61K
39/12 (20130101); A61K 39/21 (20130101); A61P
37/00 (20180101); C12N 15/86 (20130101); A61P
31/00 (20180101); A61P 31/18 (20180101); A61P
37/02 (20180101); C07K 14/005 (20130101); A61K
2039/545 (20130101); C12N 2740/16134 (20130101); C12N
2710/24143 (20130101); A61K 2039/5256 (20130101); A61K
2039/53 (20130101); C12N 2740/16222 (20130101); C12N
2740/16122 (20130101); A61K 2039/54 (20130101); C12N
2710/24171 (20130101); A61K 39/00 (20130101); A61K
38/00 (20130101); A61K 2039/57 (20130101) |
Current International
Class: |
A61K
39/21 (20060101); C12N 15/863 (20060101); C07K
14/005 (20060101); C07K 14/16 (20060101); A61K
38/12 (20060101); A61K 39/00 (20060101); A61K
045/00 (); A61K 039/42 (); A61K 038/00 (); C12N
015/00 () |
Field of
Search: |
;424/160.1,199.1,208.1
;435/320.1 ;514/44 ;536/23.2 |
References Cited
[Referenced By]
U.S. Patent Documents
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5081226 |
January 1992 |
Berzofsky et al. |
5169763 |
December 1992 |
Kieny et al. |
|
Foreign Patent Documents
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2181435 |
|
Apr 1987 |
|
GB |
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WO 87/06262 |
|
Oct 1987 |
|
WO |
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WO 90/12880 |
|
Nov 1990 |
|
WO |
|
WO 92/22641 |
|
Dec 1992 |
|
WO |
|
Other References
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Progression," AIDS Res. Human Retroviruses 10(8):901-905 (1994).
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Conserved and Variable Regions in the Envelope Gene of
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after vaccination with recombinant glycoprotein gp120 but not
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Abstract, Jul. 1989..
|
Primary Examiner: Achutamurthy; Ponnathapura
Assistant Examiner: Park; Hankyel T.
Attorney, Agent or Firm: Klauber & Jackson
Government Interests
STATEMENT AS TO RIGHT TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED
RESEARCH AND DEVELOPMENT
Part of the work performed during development of this invention
utilized U.S. Government funds. This work was supported in part by
NCI grants R01-CA57419-03 and Cancer Center Support Core Grant
P30-CA21765,NIH-NIAID grants AI-32529 and P01-AI31596-04. The U.S.
Government has certain rights in this invention.
Claims
What is claimed is:
1. An immunogenic composition that can elicit an immune response to
more than one but not necessarily all of the env variants contained
in the composition, comprising at least 4 to about 10,000
recombinant vaccinia viruses, each comprising an env variant (EV)
nucleotide encoding a different envelope protein variant of a human
immunodeficiency virus (HIV), wherein
(i) said EV nucleotide encodes both variable and constant regions
of said envelope protein variant; and
(ii) said immunogenic composition is capable of eliciting at least
one of a cellular and a humoral immmune response in a mammal
against an HIV strain.
2. The immunogenic composition according to claim 1, wherein said
envelope protein variant comprises gp120 and an oligomerization
domain of gp41.
3. The immunogenic composition according to claim 2, wherein said
EV nucleotide is isolated from a patient infected with an HIV
virus.
4. The inmmunogenic composition according to claim 2, wherein said
EV nucleotide comprises a KpnI-BsmI restriction fragment of an HIV
envelope protein encoding nucleotide.
5. The immunogenic composition according to claim 1, wherein said
immunogenic composition further comprises envelope protein variants
expressed by said recombinant vaccinia viruscs.
6. The immunogenic composition according to claim 1, wherein said
immunogenic composition further comprises at least one of a
pharmaceutically acceptable carrier, an adjuvant and an antiviral
chemotherapeutic compound.
7. A method for making a immunogenic composition that can elicit an
immune response to more than one but not necessarily all of the env
variants contained in the composition, comprising combining in
admixture at least 4 to about 10,000 recombinant viruses to obtain
a immunogenic composition, wherein
(i) each of said recombinant viruses comprises an env variant (EV)
nucleotide encoding a different envelope protein variant of an HIV
envelope protein;
(ii) said EV nucleotide encodes both variable and constant regions
of said envelope protein variant; and
(iii) said immunogenic composition is capable of eliciting at least
one of a cellular and a humoral immune response in a mammal against
an HIV strain.
8. A method according to claim 7, wherein said immunogenic
composition further comprises envelope protein variants expressed
by said recombinant vaccinia viruses.
9. A method according to claim 7, wherein said envelope protein
variant comprises gp120 and an oligomerization domain of gp41.
10. A method according to claim 7, wherein said EV nucleotide is
isolated from a patient infected with an HIV virus.
11. A method according to claim 9, wherein said EV nucleotide
comprises a KpnI-BsmI restriction fragment of an HIV envelope
protein encoding nucleotide.
12. A method according to claim 7, wherein said combining step
further comprises adding at least one pharmaceutically acceptable
carrier, adjuvant and an antiviral chemotherapeutic compound.
13. A method for eliciting a humoral or cellular immune response,
or both, to a human immunodeficiency virus (HIV) in a mammal,
comprising
administering to said mammal an effective amount of a immunogenic
composition comprising at least 4 to about 10,000 different
recombinant viruses, wherein
(i) each of said recombinant viruses comprises an env variant (EV)
nucleotide encoding a different envelope protein variant of an HIV
envelope protein;
(ii) said EV nucleotide encodes both variable and constant regions
of said envelope protein variant; and
(iii) said amount of said immunogenic composition is effective to
elicit at least one of a cellular and a humoral immune response in
said mammal against an HIV strain infecting said mammal.
14. A method according to claim 13, wherein said immunogenic
composition further comprises variant envelope proteins expressed
by said recombinant vaccinia viruses.
15. A method according to claim 13, wherein said said envelope
protein variant comprises gp120 and an oligomerization domain of
gp41.
16. A method according to claim 13, wherein said EV nucleotide is
isolated from a patient infected with an HIV virus.
17. A method according to claim 15, wherein said EV nucleotide
comprises a KpnI-BsmI restriction fragment of an HIV envelope
protein encoding nucleotide.
18. A method according to claim 13, wherein said administering step
further comprises administering at least one pharmaceutically
acceptable carrier, adjuvant or an antiviral chemotherapeutic
compound.
Description
BACKGROUND OF THE INVENTION
1. Technical Field of the Invention
The present invention relates to polyenv vaccines for human
immunodeficiency virus (HIV), comprising a mixture of at least 4-40
and up to 10,000 recombinant vaccinia viruses that each express a
different variant of an HIV envelope protein. The vaccines are
suitable for the vaccination of mammals, including humans, in order
to provide unexpectedly enhanced cellular and/or humoral immune
responses to HIV infection. Additionally, the invention relates to
methods for making and using such recombinant vaccinia viruses and
polyenv vaccines.
2. Background Information
The AIDS virus is likely to claim tens of millions of lives by the
year 2,000, constituting a worldwide health concern of top priority
(see, DeVita, et al., AIDS, Etiology, Diagnosis, Treatment and
Prevention, 3rd edition, J. B. Lippincott Co., Philadelphia, Pa.
(1992); Wong-Staal, in Virology, pp 1529-1543; and Hirsch, et al.,
in Virology, pp. 1545-1570). The design of an effective HIV vaccine
poses a particular challenge to immunologists, as the reverse
transcriptase enzyme involved in the replication of HIV has a high
error rate. This results in many mutant HIV strains having outer
coat or envelope proteins with variant protein sequences. These
variant envelope proteins are often recognized as different
antigens by the mammalian immune system, which produces more than
10.sup.9 new lymphocytes per day for the sole purpose of countering
foreign antigens. B and T-cells constitute, respectively, the
humoral and cellular components of the immune response.
A good example of the qualitative strength of such immune responses
is shown in HIV-infected patients and in SIV-infected macaques. In
each case, successive rounds of infection, immunity, and
establishment of variant HIVs or SIVs occur (Wrin, et al., J.
Acquir. Immune Defic. Syndr. 7:211-219 (1994); Burns and
Desrosiers, Cur. Topics Microbiol. Immunol. 188:185-219 (1994)).
With each cycle, the diversity of HIV antigenic determinants (and
the corresponding immune responses) are increased, such that these
immune responses neutralize a broad range of SIV or HIV variants,
and superinfection is largely inhibited.
However, AIDS patients develop compromised immune responses that
become insufficient to prevent the HIV viral infection from
overcoming the patient's immune system. This may be due in part to
the establishment of HIV variants whose envelope variant proteins
are not recognized by the patient's immune system and thus escape
destruction (Sci. Amer. August 1995, pp). In such cases, even if
the immune response is capable of preventing de novo infection
(e.g., persistent mutation of the virus in privileged sequestered
sites), the HIV infection may ultimately overcome the patient's
immune response (Pantaleo et al., Nature 362:355-358 (1993);
Embretson. et al., Nature 362:359-362 (1993)).
The identification of B- and T-cell antigenic determinants among
HIV proteins remains incomplete. The HIV envelope protein has been
characterized as having variable (V1-V5) and constant (C1-C5)
regions. A peptide representative of the V3 region has been termed
the principal neutralizing determinant (PND)(Javaherian. et al.,
Proc. Natl. Acad Sci. (USA) 86:6768-6772 (1989)), although other
regions of the envelope protein may also be involved in eliciting
an immune response. The full length envelope protein from HIV
contains about 850 to 900 amino acids, with the variation in length
due to hypermutation (Starcich et al., Cell 45:637 (1986)).
The first vaccines against HIV evaluated in clinical trials were
designed to present single envelope proteins, or portions thereof,
to the immune system. However, neutralizing responses towards a
single or a few envelope proteins did not recognize diverse
isolates of HIV and the individuals were not protected from
infection (Belshe et al., J. Am. Med Assoc. 272:431-431 (1994);
U.S. Pat. No. 5,169,763; PCT publication WO 87/06262; Zagury et
al., Nature 332:728-731 (1988); Kieny et al., Int. Conf. AIDS 5:541
(1989); Eichberg, Int. Conf. AIDS 7:88 (1991); Cooney et al., Proc.
Natl. Acad Sci. USA 90:1882-1886 (1993); Graham et al., J. Infect.
Dis. 166:244-252 (1992); J. Infect. Dis. 167:533-537 (1993); Keefer
et al., AIDS Res. Hum. Retrovir. 10 (Suppl. 2):S139-143 (1994);
Gorse, AIDS Res. Hum. Retrovir. 10 (Suppl. 2): 141-143 (1994);
McElrath et al., J. Infect. Dis. 169:41-47 (1994); Fauci, Science
264:1072-1073 (May 1994)).
Accordingly, there is a long-felt and pressing need to discover
vaccines and methods that elicit an immune response that is
sufficient to treat or prevent HIV infections.
SUMMARY OF THE INVENTION
The present invention is intended to overcome one or more
deficiencies of the related arts.
To provide more effective HIV vaccines, the present invention
provides polyenv vaccines comprising mixtures of at least 4-40, and
up to 10,000 different recombinant vaccinia viruses, each
expressing a different HIV envelope protein variant (EPV) (or a
substantial portion thereof) that includes both constant and
variable regions of the envelope protein. Preferably, each of the
expressed envelope protein variants have a structure and/or
immunogenicity similar to that of a native HIV envelope protein
existing in an infected cell or HIV lipid bilayer, such as in an
oligomeric form. Also provided are methods of making and using such
recombinant vaccinia viruses and polyenv vaccines. In their use as
a vaccine, each of the variant envelope proteins preferably induces
a different subset of B and/or T cells, each subset responding to
different envelope proteins and, hence, to multiple HIV variants. A
mixture of this number, type and/or structure of envelope proteins
is a now-discovered method for eliciting a strong, durable
HIV-specific immune response with broad spectrum neutralizing
activity.
The present inventors have discovered that polyenv vaccines of the
present invention elicit unexpectedly enhanced immune responses by
the expression and/or presentation of multiple envelope protein
variants, each containing both constant and variable regions,
preferably having a structure that is substantially similar to that
of a native HIV envelope protein. The enhanced immune responses
recognize HIV strains in addition to those strains expressing the
envelope proteins provided in the polyenv vaccine. Thus, the aim of
such a vaccine is to provide enhanced immune responses to a wide
range of HIV strains, which immune responses are suitable for
treating or preventing infection (or continued infection due to
mutation) by different strains of the virus.
The present invention also provides env variant (EV) nucleic acid
encoding (or complementary to) at least one antigenic determinant
of an envelope protein variant (EPV). The EPV is preferably encoded
by a recombinant vaccinia virus, as further provided in a polyenv
vaccine of the present invention. The variant nucleic acid
comprises at least one mutation that confers differing antigenic
properties, or three dimensional structure, to the encoded EPV.
The present invention also provides a vaccine composition
comprising a polyenv vaccine of the present invention, and a
pharmaceutically acceptable carrier or diluent. The vaccine
composition can further comprise an adjuvant and/or cytokine which
enhances a polyenv vaccine immune response to at least one HIV
strain in a mammal administered the vaccine composition. A polyenv
vaccine of the present invention is capable of inducing an immune
response inclusive of at least one of a humoral immune response
(e.g., antibodies) and a cellular immune response (e.g., cytotoxic
T cells (CTLs)).
The present invention also provides a method for eliciting an
immune response to an HIV infection in a mammal which is
prophylactic for an HIV infection, the method comprising
administering to a mammal a vaccine composition comprising a
polyenv vaccine of the present invention, which is protective for
the mammal against a clinical HIV-related pathology caused by
infection of at least one HIV strain.
The present invention also provides a method for eliciting an
immune response to an HIV infection in a mammal for therapy of an
HIV infection. The method comprises administering to a mammal a
composition comprising an inactivated or attenuated polyenv vaccine
of the present invention, which composition elicits an enhanced
immune response, relative to controls, in the mammal against a
clinical virus pathology caused by infection with at least one HIV
strain.
Other objects, features, advantages, utilities and embodiments of
the present invention will be apparent to skilled practitioners
from the following detailed description and examples relating to
the present invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic representation of the orientation of the
HIV-1 gene in a vaccinia virus genome. The HIV-1 envelope gene is
positioned between right and left segments of the thymidine kinase
locus. A HindIII site exists at the C-terminus of the HIV-1
envelope gene. The appropriate insertion yields a HindIII fragment
of approximately 7 kb in size. Southern blots with this pattern
confirmed the position and correct orientation of the HIV-1
envelope gene.
FIG. 2 is a graphical representation of data showing that the
HIV-specific antibody response is long term in mammal models. The
results of representative mouse sera tested in the ELISA for
HIV-specific antibodies are shown. Each sample was diluted 1:100
(solid bars), 1:1,000 (hatched bars) and 1:10,000 (clear bars)
prior to assay on HIV-1-coated ELISA plates. Test mice were sampled
at various times (1 month, 4 months and 6 months) following the
injection of 10.sup.7 pfu of a vaccinia virus construct expressing
one envelope protein of HIV-1. The control mouse was immunized with
a vaccinia virus containing no envelope sequence. Standard error
bars are shown.
FIG. 3 is a graphical representation of data showing how the
vaccinia virus dose affects the induction of at least one immune
response, including HIV-specific antibody production.
Representative mouse serum samples were tested by the ELISA on
HIV-1-coated plates. Serum samples were taken from mice injected
with 10.sup.5, 10.sup.6, and 10.sup.7 pfu of one vaccinia virus
expressing the HIV-1-envelope protein. Serum samples were tested
approximately three weeks after injection. Each sample was diluted
1:100 (solid bars), 1:1,000 (hatched bars) and 1:10,000 (clear
bars) prior to assay on HIV-1-coated ELISA plates. Standard error
bars are shown.
FIG. 4 is a graphical representation of data showing that the
mixing of vaccinia virus constructs does not compromise the
elicitation of HIV-specific antibody in injected mammals.
Representative mouse serum samples were tested by the ELISA
approximately 2 months following the injection of 10.sup.7 pfu
vaccinia virus expressing HIV-1 envelope protein(s). "Single"
identifies a sample from a mouse that received a single vaccinia
virus. "Mix" represents a sample from a mouse that received a
mixture of vaccinia viruses expressing five distinct envelope
proteins. Each sample was diluted 1:100 (solid bars), 1:1,000
(hatched bars) and 1:10,000 (clear bars) prior to assay on
HIV-1-coated ELISA plates. Standard error bars are shown.
DETAILED DESCRIPTION OF THE DISCLOSURE
Discovery of Unexpectedly Enhanced Immune Responses to Mixed HIV
Polyenv Vaccines
Previous attempts to provide vaccines against different strains of
HIV have focused on one or more variable regions of gp 120 or gp
160. It was expected that such variable regions, provided in a
vaccine, would provide broad protection against HIV infection.
However, such vaccines have not been successful, where the
vaccine-induced immune response does not recognize many different
strains of HIV. Therefore, a critical need exists to provide
vaccines that elicit immune responses to multiple strains of HIV,
such that the vaccines are suitable for treatment and/or prevention
of HIV.
The present inventors have discovered that unexpectedly enhanced
immune responses can be induced against several or many different
HIV strains, by the use of polyenv vaccines that contain a mixture
of at least 4-40, up to as many as 10,000, recombinant vaccinia
viruses that each encode a different envelope protein variant
(EPV). The vaccine can also contain EPVs expressed by the vaccinia
viruses, e.g., as produced in the host cells used for the virus
production.
This immune response (as humoral and/or cellular) is found to be
effective for a broader range of strains of an infectious virus,
such as HIV, and is not limited to the virus strains expressing the
specific envelope protein variants (EPVs) provided by the polyenv
vaccine. The present invention thus provides multiple EPVs encoded
by a recombinant viral vaccine which give unexpectedly enhanced
immune responses to multiple strains of HIV.
The EPV encoding nucleic acid (envelope variant (EV) nucleic acid)
can be isolated from the same or different population (e.g.,
geographic) of humans infected with HIV. Alternatively, the
different EV nucleic acids can be obtained from any source and
selected based on screening of the sequences for differences in
coding sequence or in elicited humoral and/or cellular immune
responses to multiple HIV strains, in vitro or in vivo, according
to known methods.
Polyenv Vaccines and Envelope Protein Variants
The present invention thus provides, in one aspect, polyenv
vaccines using mixtures of at least 4-40, and up to 10,000,
different recombinant vaccinia viruses that each express a
different envelope protein variant, or an antigenic portion
thereof. Also provided are methods of making and using such polyenv
vaccines
A polyenv vaccine of the present invention induces at least one of
a humoral and a cellular immune response in a mammal who has been
administered the polyenv vaccine, but the response to the vaccine
is subclinical, or is effective in enhancing at least one immune
response to at least one strain of HIV, such that the vaccine
administration is suitable for vaccination purposes.
An EPV, encoded by a recombinant vaccinia virus alternatively
includes polypeptides having immunogenic activity elicited by an
amino acid sequence of an EPV amino acid sequence as at least one
epitope or antigenic determinant. This amino acid sequence
substantially corresponds to at least one 10-900 amino acid
fragment and/or consensus sequence of a known HIV EPV. Such an EPV
can have overall homology or identity of at least 50% to a known
envelope protein amino acid sequence, such as 50-99% homology, or
any range or value therein, while eliciting an immunogenic response
against at least one strain of an HIV.
Percent homology 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. 48:443 (1970), as revised by Smith and
Waterman (Adv. Appl. Math. 2:482 (1981)). Briefly, the GAP program
defines similarity as the number of aligned symbols (i.e.,
nucleotides or amino acids) which are similar, 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. 14:6745 (1986), 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.
In a preferred embodiment, an EPV of the present invention is a
variant form of at least one HIV envelope protein. Preferably, the
EPV includes gp120 and the oligomerization domain of gp41, as gp140
(Hallenberger, et al., Virology 193:510-514 (1993), entirely
incorporated herein by reference).
Known HIV envelope proteins contain about 750 to 900 aminio acids.
Examples of such 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). Substitutions or insertions
of an EPV to obtain an additional EPV, encoded by a nucleic acid
for use in a recombinant vaccinia virus or polyenv vaccine 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 EPV sequence. Preferably, such substitutions,
insertions or deletions are identified based on sequence
determination of envelope proteins obtained by nucleotide
sequencing of at least one EPV encoding nucleic acid from an
individual infected with HIV.
Non-limiting examples of such substitutions, insertions or
deletions preferably are made by the amplification of env DNA or
RNA sequences from HIV-1 infected patients, which can be determined
by routine experimentation to provide modified structural and
functional properties of an envelope protein or an EPV. The EPVs so
obtained preferably have different antigenic properties from the
original EPV. Such antigenic differences can be determined by
suitable assays, e.g., by testing with a panel of monoclonal
antibodies specific for HIV envelope proteins in an ELISA
assay.
Any substitution, insertion or deletion can be us&d as long as
the resulting EPV protein elicits antibodies which bind to HIV
envelope proteins, but which EPV has a different pattern than
antibodies elicited by a second EPV. Each of the above
substitutions, insertions or deletions can also include modified or
unusual amino acid, e.g., as provided in 37 C.F.R. .sctn.
1.822(p)(2), which is incorporated herein by reference.
The following Table 1 presents non-limiting examples of alternative
variants of envelope proteins of HIVs, that can be encoded by a
recombinant vaccinia virus according to present invention.
TABLE 1 - HIV Envelope Protein Variants 1 2 3 4 5 6 7 8 9 10 1 2 3
4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 K E Q K T V A M R V K E S Q M
K K Q H L W R W G W R W G T E K M K A M G T R R N C P N W L K I T K
G Y I T T M I K K S Y N C R K G K M L L M I R M G G E W R R K I T T
Y K E T D W Q S S I 1 2 3 4 5 6 7 8 9 40 1 2 3 4 5 6 7 8 9 50 1 2 3
4 5 6 7 8 9 60 31 M L L G L M I C S A T E K L W V T V Y Y G V P V W
K E A T L I F W I I T S L V V S Q Y A S I I E D E A M A I M T P L G
A Q D A H V I A M L T P C I E D N N T I A N K V A 1 2 3 4 5 6 7 8 9
70 1 2 3 4 5 6 7 8 9 80 1 2 3 4 5 6 7 8 9 90 61 T T L F C A S D A K
A Y D T E V H N V W A T H A C V P T D P P V E R R T H S R A K I C S
Y N N S T K A R K Q G L A K Q E P K 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5
6 7 8 9 110 1 2 3 4 5 5 7 8 9 120 91 N P Q E V V L V N V T E N F N
M W K N D M V E Q M H E D I I D H I L M G S G E D I R N I D Q T V S
R L Y E D K T S N T Y M D P D Y F S H 1 2 3 4 5 6 7 8 9 130 1 2 3 4
5 6 7 8 9 140 1 2 3 4 5 6 7 8 9 150 121 S L W D Q S L K P C V K L T
P L C V S L K C T D L K N D T N N E E E V M L C V T M N K H V T T A
S E Q N D I N Y G G M T Q S H Q W R I I G K F L S 1 2 3 4 S 6 7 8 9
160 1 2 3 4 5 6 7 8 9 170 1 2 3 4 5 6 7 8 9 180 151 T S N N V T S S
S W G R N I M E E G E I K N C S F N I S T S N K S S K T T K N W K R
E I D R E K M T K P Y K V T K G I E N V T I S K E K T G Q A G V R T
Y Q P N G S Q W V I G S R R Q E Q M I L G T V N K L T E 1 2 3 4 5 6
7 8 9 190 1 2 3 4 5 6 7 8 9 200 1 2 3 4 5 6 7 8 9 210 181 I R G K V
Q K E Y A F F Y K L D I I F I D K G N D S N D L G D R I K Q D N S L
L R N H V V Q V K D S D I N P K D A V K N Q M H R V R T Y H R T L A
K L G N T S R S E K E T A S T N T P M E E G S K T Q G H H V S S N N
1 2 3 4 5 6 7 8 9 220 1 2 3 4 5 6 7 8 9 230 1 2 3 4 5 6 7 8 9 240
211 T T S Y K F T L T S C N T S V I T Q A C P K V S F E S T T N A N
T W K R I I H S R T T V K S I T Q S N I R N Y I N D S A L T D S G K
T I M 1 2 3 4 5 6 7 8 9 250 1 2 3 4 5 6 7 8 9 260 1 2 3 4 5 6 7 8 9
270 241 P I P I H Y C A P A G F A I L K C N N K T F N G T G P C T N
F M F T G T Y V M F K D A K S K E Q K H L R S P E E S S H E C T S T
Q I R 1 2 3 4 5 6 7 8 9 280 1 2 3 4 5 6 7 8 9 290 1 2 3 4 5 6 7 8 9
300 271 V S T V Q C T H G I R P V V S T Q L L L N G S L A E E E V V
I T S R T K I T H I T S K G G I K V H S T S R K R D R R K G I D M 1
2 3 4 5 6 7 8 9 310 1 2 3 4 5 6 7 8 9 320 1 2 3 4 5 6 7 8 9 330 301
I R S A N F T D N A K T I I V Q L N Q S V E I N C T R P N N L M G D
D I S N S V R I W L A H K E P I A V V Y I E S I V A E L M E G T D N
V T T A T L Q T A A K M V S P A G V D A V M E E Y K K L H T T H H Q
1 2 3 4 5 6 7 8 9 340 1 2 3 4 5 6 7 8 9 350 1 2 3 4 5 6 7 8 9 360
331 N T R K S I R I Q R G F G R A F V T I G K I L G N M R Q A K V N
R R Y H R H I A P K Q V I H A T R R K I S D I G K Y K S G N Y K M P
S S R K T W Y V R K Q S R A N L L T R P Q T H L Y L M M S V F R L D
D G V F T S R S I V G P S W Y I N M E A V A N I T V 1 2 3 4 5 6 7 8
9 370 1 2 3 4 5 6 7 8 9 380 1 2 3 4 5 6 7 8 9 390 361 H C N I S R A
K W N N T L K Q I D S K L R E Q F G N N K T I Y K L A G E Q K A V I
E G V V K S Y K K K Y K D Q S V T V N K T D S K A V Q K L A T Q Q A
H L D H T Y E R N E R I S R T E H G V R S M A S A F D N L R I I D 1
2 3 4 5 6 7 8 9 400 1 2 3 4 5 6 7 8 9 410 1 2 3 4 5 6 7 8 9 420 391
I F K Q S S G G D P E I V T H S F N C G G E F F Y C N S T Q V S N H
H A C C L V T M Y N L I V R D I D T S G N L T S P I S L L T T V A A
N A A K G V H M W R P K S N T Q H E K 1 2 3 4 5 6 7 8 9 430 1 2 3 4
5 6 7 8 9 440 1 2 3 4 5 6 7 8 9 450 421 L F N S T W F N S T W S T K
G S N N T E G S D T I T L P C R M D S N I Y R L N K A G I E W N S G
M K E N N N L I H Q K I D T C N V G D D P I K D G D G G R E G P V V
I L T G F S D S K K N T C G T S N Q A R E L K D A G M G M L D I Q S
K R S 1 2 3 4 5 6 7 8 9 460 1 2 3 4 5 6 7 8 9 470 1 2 3 4 5 6 7 8 9
480 451 I K Q I I N M W Q E V G K A M Y A P P I S G Q I R C S S N I
E F V R I A G T R Q S T D L F G R V L S F I K R R A R L T Q E K E S
K I K T T V L V E L T 1 2 3 4 5 6 7 8 9 490 1 2 3 4 5 6 7 8 9 500 1
2 3 4 5 6 7 8 9 510 481 T G L L L T R D G G A N E N N E S E I F R P
G G G D M R D N T I V S S V T D Q T S D T V V I S L T N I K N I I E
E S K S A G E N T L A E D G T A K R N L V G E D K T I I 1 2 3 4 5 6
7 8 9 520 1 2 3 4 5 6 7 8 9 530 1 2 3 4 5 6 7 8 9 540 511 W R S E L
Y K Y K V V K I E P L G V A P T K A K R R V V Q R R I N K F N D I R
V K L I S I S R S R P I M E T T T F P S H I A Q M A W E I H 1 2 3 4
5 6 7 8 9 550 1 2 3 4 5 6 7 8 9 560 1 2 3 4 5 6 7 8 9 570 541 E K R
A V G E I G A L F L G F L G A A G S T M G A A S M T L K E I F I V V
M S I V S S A V A L A V Q A V T L M V L P R P I A M F I G V L I T T
1 2 3 4 5 6 7 8 9 580 1 2 3 4 5 6 7 8 9 590 1 2 3 4 5 6 7 8 9 600
571 T V Q A R Q L L S G I V Q Q Q N N L L R A I E A Q Q H L L Q A G
R T H H V M K D H S M K G Q M K P P L D R D E L K Q R S 1 2 3 4 5 6
7 8 9 610 1 2 3 4 5 6 7 8 9 620 1 2 3 4 5 6 7 8 9 630 601 L T V W G
I K Q L Q A R I L A V E R Y L K D Q Q L L G I W G S I V R R L V Q L
T F I R E R R M E F L W T L I S L Q N K I R M G S N N L 1 2 3 4 5 6
7 8 9 640 1 2 3 4 5 6 7 8 9 650 1 2 3 4 5 6 7 8 9 660 631 C S G K L
I C T T A V P W N A S W S N K S L E Q I W N N M T R K R T V P T K S
T G R R T M D D F D K T M H Y N F A S Y N Q N M G H L I F N G V S S
Q T N A S R K K W 1 2 3 4 5 6 7 8 9 670 1 2 3 4 5 6 7 8 9 680 1 2 3
4 5 6 7 8 9 690 661 W M E W D R E I N N Y T S L I H S L I E E S Q N
Q Q E K N E L Q E K L V D S V S N T Y T I L T D A A I G I Q I K Q H
E K I G I F N E Q Q T D Q V Q Q S D V N D R K E V 1 2 3 4 5 6 7 8 9
700 1 2 3 4 5 6 7 8 9 710 1 2 3 4 5 6 7 8 9 720 691 Q E L L E L D K
W A S L W N W F N I T N W L W Y I K L F I M L D G N E T N S S S S Q
S R I A V I R A A S K G Y G K K K K Q L D Q 1 2 3 4 5 6 7 8 9 730 1
2 3 4 5 6 7 8 9 740 1 2 3 4 5 6 7 8 9 750 721 I V G G L V G L R I V
F A V L S V V N R V R Q G Y S P L S F V I A A I I V K V I M S I F C
I I K S F S A Q L A T N L R N I N I 1 2 3 4 5 6 7 8 9 760 1 2 3 4 5
6 7 8 9 770 1 2 3 4 5 6 7 8 9 780 751 Q T H L P I P R G P D R P E G
I E E E G G E R D R D R S I R I R T H V Q E E L G Q L D R T D G G D
Q G K G T W V G L A N T T G A E T Q G E G P G G Q P P I A R Q E S K
N P F G S S A 1 2 3 4 5 6 7 8 9 790 1 2 3 4 5 6 7 8 9 800 1 2 3 4 5
6 7 8 9 810 781 L V N G S L A L I W D D L R S L C L F S Y H R L R D
L L L I A L D F S T Q F Y E C W T C F S S C R L T N F A S T S P H L
P L V G N I I I W L Q S S S C I C V T C Q G A G T Q S T H 1 2 3 4 5
6 7 8 9 820 1 2 3 4 5 6 7 8 9 830 1 2 3 4 5 6 7 8 9 840 811 V T R I
V E L L G R R G W E A L K Y W W N L L Q Y W S Q E L A A K T I D I K
H G L L D G I R L L G S V V L I K I V A L S T R L L I N I C I C A A
M I G R K L K Y V G C T T M V R L 1 2 3 4 5 6 7 8 9 850 1 2 3 4 5 6
7 8 9 860 1 2 3 4 5 6 7 8 9 870 841 K N S A V S L L N A T A I A V A
E G T D R V I E V V Q G A Y R I V I N W F D T I V V T G E G I L I A
R R I C Q S F S F V A V S N R K A A G A T L T L W A T V G F V 1 2 3
4 5 6 7 8 9 880 1 2 3 4 5 6 7 8 889 871 R A I R H I P R R I R Q G L
E R I L L Q G F L N V H T V F K G L Q T I V I A A V R S
Accordingly, based on the above examples of specific substitutions,
alternative substitutions can be made by routine experimentation,
to provide alternative EPVs of the present invention, e.g., by
making one or more substitutions, insertions or deletions in
envelope proteins or EPV's which give rise to differential immune
responses.
Amino acid sequence variations in an EPV of the present invention
can be prepared e.g., by mutations in the DNA. Such EPVs 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 EPV must not place the sequence out of reading frame
and preferably will not create complementary domains that could
produce secondary mRNA structures (see, e.g., Ausubel (1995 rev.),
infra; Sambrook (1989), infra).
EPV-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 envelope protein or an EPV,
and thereafter synthesizing or reverse transcribing the encoding
DNA to produce DNA or RNA encoding an EPV(see, e.g., Ausubel (1995
rev.), infra; Sambrook (1989), infra), based on the teaching and
guidance presented herein.
Recombinant vaccinia viruses expressing EPV's of the present
invention, or nucleic acid encoding therefor, include a finite set
of EPV-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., Principles of Protein
Structure, Springer-Verlag, New York, N.Y. (1978), and Creighton,
T.E., Proteins: Structure and Molecular Properties, W. H. Freeman
& Co., San Francisco, Calif. (1983), which are hereby
incorporated by reference. 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. (1987, 1988, 1989, 1990, 1991, 1992, 1993,
1994, 1995)(hereinafter, "Ausubel (1995 rev.)") at .sctn..sctn.
A.1.1-A.1.24, and Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989) at Appendices C and D.
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 env DNA or RNA to obtain
alternative EPVs, including substitutional, deletional or
insertional variants.
Screening Assays for HIV Activity
For screening HIV activity of an EPV encoding recombinant vaccinia
virus, any known and/or suitable screening assay can be used, as is
known in the art. For example, known HIV assays include viral
infectivity assays (see, e.g., Chesebro et al., J. Virol.
62:3779-3788 (1988); Aldovini et al., eds., Techniques in HIV
Research pp. 71-76 (1990)); neutralization assays (see, e.g.,
Golding et al., AIDS Res. Hum. Retrovir: 10:633-643 (1994);
Hanson., AIDS Res. Hum. Retrovir. 10:645-648 (1994); Laal et al.,
Res. Hum. Retrovir. 9:781-785 (1993); Hanson, J. Acquit. Immune
Defic. Syndr. 7:211-219 (1994)); peripheral mononuclear (PMN) cell
assays (see, e.g., Arduino et al., Antimicrob. Agents Chermother.
37:1095-1101 (1990)); and cytotoxic T-lymphocyte (CTL) assays (see,
e.g., Hammond et al., J. Exp. Med. 176:1531-1542 (1992); McElrath
et al., J. Virol. 68:5074-5083 (1994); Walker et al., Cell Immunol.
119:470-475 (1989); Weinhold et al., AIDS Res. Hum. Retrovir.
8:1373 (1992)). Other suitable activities, alone or in any
combination, include, but are not limited to, quantitative and/or
qualitative measurement of transcription, replication, translation,
virion incorporation, virulence, viral yield, and/or morphogenesis.
The above references are entirely incorporated herein by
reference.
Recombinant Vaccinia Virus Encoding EPV's, Polyenv Vaccines and
Methods of Making and Using Thereof
Overview. Recombinant vaccinia viruses (VV) expressing HIV envelope
proteins (e.g., gp41, gp120 and/or gp160, or a portion thereof)
provide materials useful for the production and testing of mixed
vaccines that induce at least one of a humoral or cellular immune
response against the virus, as well as for analyses of B-cell and
CTL determinants.
A polyenv vaccine of the present invention consists of a mixture of
n distinct recombinant vaccinia viruses, where n is a whole number
from about 4 to about 10,000 (or any range or value therein),
wherein each vaccinia vector construct expresses a variant of a
HIV-1 envelope protein (EPV) (e.g., gp41, gp 120 or gp160). The
recombinant vaccinia virus functionally encodes an EPV and is
prepared by recombination of wildtype VV with a plasmid. Multiple,
distinct plasmids encoding EPV can be prepared by substituting one
EPV encoding sequence with another, e.g., using a restriction
fragment or mutagenesis.
Preparation of Recombinant Vaccinia Viruses. Methods for the
preparation of individual plasmids (each expressing a unique HIV
protein sequence) can utilize DNA or RNA amplification for the
substitution of isolated envelope protein variant sequences into a
vector (e.g., pVenv4 or pVenv1(Hallenberger et at., Virology
193:510-514 (1993)), which vector encodes a known HIV envelope
protein sequence (e.g., available from the NIAID AIDS Research
& Reference Reagent Program, Rockville, Md.).
Methods of amplification of RNA or DNA are well known in the art
and can be used according to the present invention without undue
experimentation, based on the teaching and guidance presented
herein. Known methods of DNA or RNA amplification include, but are
not limited to polymerase chain reaction (PCR) and related
amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159, 4,965,188, to Mullis et al.; 4,795,699 and
4,921,794 to Tabor et al; 5,142,033 to Innis; 5,122,464 to Wilson
et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten et al;
4,889,818 to Gelfand et al; 4,994,370 to Silver et al; 4,766,067 to
Biswas; 4,656, 134 to Ringold) and RNA mediated amplification which
uses anti-sense RNA to the target sequence as a template for double
stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al,
with the trade name NASBA), the entire contents of which patents
are herein entirely incorporated by reference.
For example, recombinant vaccinia virus constructs prepared by this
route can be used for immunizations and elicitation of HIV-specific
T and/or B-cell responses. Primers utilize conserved HIV sequences
and thus successfully amplify env genes from many diverse HIV-1
patient samples. The basic techniques described here can similarly
be used with PCR or other types of amplification primers, in order
to substitute smaller or larger pieces of the env sequence from
field isolates for that found in vectors encoding an HIV envelope
protein. See, e.g., Ausubel; infra, Sambrook, infra.
EPV Encoding Nucleic Acids. The technique begins with the isolation
of DNA from HIV infected cells and the amplification of env
sequences by PCR. PCR or other amplification products provide the
simplest means for the isolation of HIV sequences, but any other
suitable and known methods can be used such as cloning and
isolation of EPV encoding nucleic acid or proteins (see Ausubel,
infra; Sambrook, infra). Enzyme restriction sites are preferably
incorporated into PCR or other amplification primer sequences to
facilitate gene cloning.
Isolated DNA for PCR can be prepared from multiple virus sources,
inclusive of fresh or frozen whole blood from HIV+ patients and
cells that have been infected in vitro with virus isolates.
In order to produce new HIV env constructs, the polymerase chain
reaction (PCR) is preferably used to amplify 100-2700 base pairs
(bp) of an env gene from each different HIV patient sample. The PCR
primers can represent well-conserved HIV sequences which are
suitable for amplifying env genes from known samples of env genes,
isolated HIVs or diverse HIV patient samples. The amplified DNA
preferably comprises a portion encoding 10-900 (such as 100-400,
400-600 or 600-900, or any range or value therein) amino acids of a
gp120and gp41 (both make up gp160). One or more of the envelope
variable regions (V1-V5) and constant regions (C1-C5) are
preferably included in the PCR products, more preferably most of
the V1, C1, V2, C2, V3, C3, V4, C4, and V5regions. In addition,
amplified sequences can encode 1-200 amino acids beyond the
cleavage site for gp120/gp41. Preferably, most or all of the entire
env gene is amplified. Optionally, the gp160 encoding sequence
amplified is missing part or all of sequences encoding the
transmembrane domain and/or the cytoplasmic tail domain (see,
e.g.,Hallenberger et al. (1993)).
The PCR primers can be designed so that restriction enzyme sites
flank the envelope gene sequence in vaccinia plasmid, such that
they are incorporated into the amplified DNA products. By using
well-known substitution cloning techniques, vaccinia plasmid
derivatives that express envelope protein variant sequences from
1-10,000 patients can be generated by substituting a portion of the
patient's EPV encoding sequence for corresponding portion of the
env sequence in the vaccinia plasmid such as by using restriction
fragments for the substitution. For example, the pVenv4 plasmid and
PCR products are treated with KpnI and BsmI to obtain a sequence
encoding a truncated gp160 of amino acids 1-639, which lacks both
the transmembrane domain and the cytoplasmic tail domain of gp41
(see, e.g., Hallenberger et al.(1993)).
Following ligation of the PCR product and the pVenv products,
bacterial host cells are transformed with the ligation mixture via
any of a number of methods well-known in the art, including, e.g.,
electroporation, and recombinant colonies are picked and examined
by sequencing.
Recombinant Vaccinia Virus Constructs Encoding HIV Envelope
Proteins. The EPV encoding vaccinia is then recombined with wild
type virus in a host cell and the EPV expressing virus plaques are
selected and virus stocks made. The virus stocks as VVenv's each
containing a different EPV encoding sequence are then mixed using
at least 4-40, and up to about 10,000 different recombinant
viruses, to form a polyenv vaccine of the present invention.
The recombinant vaccinia plasmids containing the EPV sequences are
then optionally sequenced or screened with HIV envelope
protein-specific antibodies to identify different EPVs. Sequencing
by the Sanger Method dideoxy-chain termination is preferred. This
involves the denaturation of DNA, annealing of primer, and
initiation of DNA synthesis in the presence of at least one
radio-labeled nucleotide, with plasmid DNA as the template. The
polymerization mix is then aliquoted in different tubes and DNA
synthesis is continued in the presence of one di-deoxynucleotide
(ddATP, ddCTP, ddGTP, ddTTP) per tube. Each chain of newly
synthesized DNA is terminated when ddNTP is incorporated. The
products of each reaction are run in parallel on an acrylamide gel,
which will resolve fragments differing by one nucleotide in size.
Thus, the sequence can be read by identifying the ddNTP responsible
for the termination of each fragment. The procedure is preferably
adapted from previously described methods (Sambrook et al. (1989),
infra; United States Biochemical, Sequenase Version 2.0--DNA
Sequencing Kit, Ninth Edition, Amersham Life Science, Inc., (1994))
and should read approximately 50-300 bp from the primer
position.
Methods for the production of VV expression vectors are well-known
in the art (see, e.g., Mackett, M. et al., Proc. Natl. Acad Sci.
(USA) 79:7415-7419 (1982); Panicali, D., and Paoletti, E., Proc.
Natl. Acad Sci. (USA) 79:4927-4931 (1982); U.S. Pat. No. 4,169,763;
Mazzara, G. P. et al., Methods in Enz. 217:557-581 (1993)), Ausubel
et al., infra, at .sctn..sctn. 16.15-16.19, each of which are
entirely incorporated herein by reference. The previously described
pSC11 vector (Chakrabarti, S. et al., Mol. Cell. Biol. 5:3403-3409
(1985)) can preferably be used to create an env-encoded plasmid,
such as pVenv4.
As a viral vector, vaccinia virus has a number of useful
characteristics, including capacity that permits cloning large
fragments of foreign DNA (greater than 20 Kb), retention of
infectivity after insertion of foreign DNA, a wide host range, a
relatively high level of protein synthesis, and suitable transport,
secretion, processing and post-translational modifications as
dictated by the primary structure of the expressed protein and the
host cell type use. For example, N-O-glycosylation,
phosphorylation, myristylation, and cleavage, as well as assembly
of expressed proteins, occur in a faithful manner.
Several variations of the vaccinia vector have been developed and
are suitable for use in the present invention (e.g., see Ausubel et
al., infra, .sctn..sctn. 16.15-16.19). Most commonly, after
obtaining the virus stock (Ausubel, infra at .sctn. 16.16), a
nucleic acid sequence encoding an EPV is placed under control of a
vaccinia virus promoter and integrated into the genome of vaccinia
so as to retain infectivity (Ausubel et al., infra at .sctn.
16.17). Alternatively, expression can be achieved by transfecting a
plasmid containing the vaccinia promoter-controlled gene encoding
an EPV into a cell that has been infected with wild-type
vaccinia.
Preferably, the host cell and vaccinia vector are suitable and
approved for use in vaccination of mammals and humans. These
recombinant viruses are then characterized using various known
methods (Ausubel et al., infra at .sctn. 16.18). In still another
variation, the bacteria phage T7 RNA polymerase chain can be
integrated into the genome of vaccinia so that the EPV encoding
sequences will be expressed under the control of a T7 promoter,
either in transfected plasma, plasmid or a recombinant vaccinia
virus, will be expressed.
The use of pox virus promoters is preferred because cellular and
other viral promoters are not usually recognized by the vaccinia
transcriptional apparatus. A compound early/late promoter is
preferably used in recombinant vaccinia for polyenv vaccines, as it
is desirable to express the EPV as an antigen that is presented in
recombinant vaccinia virus infected host cell in association with
major histocompatibility class (MHC) I or II. Such MHC associated
HIV envelope protein will then form cytotoxic T cell targets, and
prime vaccinated mammals for a cytotoxic T cell response and/or a
humoral response against the expressed HIV EPVs. This is because
the ability of vaccinia viral vectors to induce MHC presentation in
host cells for this type of antigen appears to diminish late in the
infection stage. Transcripts originating early will terminate after
the sequence TTTTTNT and lead to inadequate MHC presentation.
Alternatively, any such termination motifs within the coding
sequence of the gene can be altered by mutagenesis if an early pox
virus promoter is used, in order to enhance MHC presentation of
envelope protein antigens in host cells (Earl et al., infra, 1990).
To mimic vaccinia virus mRNAs, untranslated leader and 3'-terminal
sequences are usually kept short, if they are used in the vaccinia
plasmids incorporating HIV EPV encoding sequences.
Preferably, the plasmid used for making vaccinia constructs
according to the present invention has been designed with
restriction endonuclease sites for insertion of the env gene
downstream of the vaccinia promoter (Ausubel et al., infra, .sctn.
16.17). More preferably, the plasmid already contains an envelope
protein encoding sequence, wherein the restriction sites occur
uniquely near each of the beginning and ends of the envelope
protein coding sequence. The same restriction fragment of the EPV
encoding sequence can then replace the corresponding sequence in
the plasmid. In such cases, the major portion of the EPV encoding
sequence can be inserted after removing most or all of the envelope
protein encoding sequence from the plasmid.
Preferably, the resulting vaccinia construct (containing the EPV
encoding sequence and the vaccinia promoter) is flanked by vaccinia
DNA to permit homologous recombination when the plasmid is
transfected into cells that have been previously infected with
wild-type vaccinia virus. The flanking vaccinia virus DNA is chosen
so that the recombination will not interrupt an essential viral
gene.
Without selection, the ratio of recombinant to parental vaccinia
virus is usually about 1:1000. Although this frequency is high
enough to permit the use of plaque hybridization (see Ausubel et
al., infra at .sctn..sctn. 6.3 and 6.4) or immunoscreening (Ausubel
et al., infra at .sctn. 6.7) to pick recombinant viruses, a variety
of methods to facilitate recombinant-virus identification have been
employed. Nonlimiting examples of such selection or screening
techniques are known in the art (see Ausubel et al., infra at
.sctn. 16.17). Usually, the expression cassette is flanked by
segments of the vaccinia thymidine kinase (TK) genes so that
recombination results in inactivation of TK. Virus with a TK.sup.31
phenotype can then be distinguished from those with a TK.sup.+
phenotype by infecting a TK.sup.- cell line in the presence of
5-bromo-deoxyuridine (5-BrdU), which must be phosphorylated by TK
to be lethally incorporated into the virus genome.
Alternatively or additionally, recombinant viruses can be selected
by the co-expression of a bacterial antibiotic resistant gene such
as ampicillin (amp) or guanine phosphoribosyl transferase (gpt). As
a further example, co-expression of the Escherichia coli lac Z gene
allows coscreening of recombinant virus plaques with Xgal (Ausubel,
infra, .sctn. 16.17).
The recombinant vaccinia viruses expressing an EPV of the present
invention can be optionally attenuated or inactivated according to
known methods, such as by heat, paraformaldehyde treatment,
ultraviolet irradiation, proprioloactene treatment, hybrid or
chimera formation or by other known methods (see, e.g., Zagury et
al., Nature 332:728-731 (1988); Ito et al., Cancer Res.
50:6915-6918 (1990); Wellis et al., J. Immunol. 99:1134-9 (1967);
D'Honcht, Vaccine 10 Suppl. :548-52 (1992); Selenka et al., Arch.
Hyg. Bakteriol. 153:244-253 (1969); Grundwald-Bearch et al., J.
Cancer Res. Clin. Oncol. 117:561-567 (1991); the contents of which
are entirely incorporated here by reference. For example, heat
inactivation at 60.degree. C. will reduce virus titer considerably.
Such attenuation techniques are safety tested, as incomplete
inactivation might result in patient death (Dorozynski and
Anderson, Science 252:501-502 (1991)).
Such attenuated or inactivated recombinant vaccinia is to be used
where the patient may have a compromised immune system as
complications or death can occur when live vaccinia is
administered.
Pharmaceutical Compositions
Pharmaceutical preparations of the present invention, suitable for
inoculation or for parenteral or oral administration, include a
polyenv vaccine comprising of at least 4-40, and up to about
10,000, different recombinant vaccinia viruses, in the form of a
cell lysate, membrane-bound fraction, partially purified or
purified form. Preferably, the polyenv vaccine comprises
recombinant vaccinia virus containing cell lysate (or
membrane-bound fractions thereof) that further comprise EPV
proteins already expressed by the recombinant vaccinia viruses. The
inclusion of the expressed EPVs is now discovered to enhance the
primary antibody response.
The polyenv vaccine composition can be in the form of sterile
aqueous or non-aqueous solutions, suspensions, or emulsions, and
can also contain auxiliary agents or excipients which are known in
the art. Each of the at least about 4-40 to 10,000 different
vaccinia viruses encode and express a different EPV, as presented
herein. EPVs encoding DNA can be selected to represent EPVs
existing in a specific isolated community of AIDS patients. For
example, a vaccine could represent sequences from Memphis, Tenn.
and be targeted for use in Memphis, Tenn. Vaccines designed to
represent geographically restricted areas can also be useful for
use in communities outside of the targeted community.
Alternatively, EPVs encoding DNAs can be selected to represent
geographically distant communities, cities or countries, such as
clades. For example, multiple clones can be represented in one
polyenv vaccine. A polyenv vaccine composition can further comprise
immunomodulators such as cytokines which accentuate an immune
response to a vital infection.
See, e.g., Berkow et al., eds., The Merck Manual, Fifteenth
Edition, Merck and Co., Rahway, N.J. (1987); Goodman et al., eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
Eighth Edition, Pergamon Press, Inc., Elmsford, N.Y. (1990);
Avery's Drug Treatment: Principles and Practice of Clinical
Pharmacology and Therapeutics, Third Edition, ADIS Press, LTD.,
Williams and Wilkins, Baltimore, Md. (1987); and Katzung, ed. Basic
and Clinical Pharmacology, Fifth Edition, Appleton and Lange,
Norwalk, Conn. (1992), which references and references cited
therein, are entirely incorporated herein by reference as they show
the state of the art.
As would be understood by one of ordinary skill in the art, when a
polyenv vaccine of the present invention is provided to an
individual, it can be in a composition which can further comprise
at least one of salts, buffers, adjuvants, or other substances
which are desirable for improving the efficacy of the composition.
Adjuvants are substances that can be used to specifically augment
at least one immune response. Normally, the adjuvant and the
composition are mixed prior to presentation to the immune system,
or presented separately, but into the same site of the being
immunized. Adjuvants can be loosely divided into several groups
based upon their composition. These groups include oil adjuvants,
mineral salts (for example, AlK(SO.sub.4), .sub.2 AlNa(SO.sub.4),
.sub.2 AlNH.sub.4 (SO.sub.4), silica, kaolin, and carbon),
polynucleotides (for example, poly IC and poly AU nucleic acids),
and certain natural substances (for example, wax D from
Mycobacterium tuberculosis, substances found in Corynebacterium
parvum, or Bordetella pertussis, and members of the genus
Brucella). Among those substances particularly useful as adjuvants
are the saponins (e.g., Quil A., Superfos A/S, Denmark). Examples
of materials suitable for use in vaccine compositions are
disclosed, e.g., in Osol, A., ed., Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa. (1980), pp. 1324-1341,
which reference is entirely incorporated herein by reference).
A pharmaceutical polyenv vaccine composition of the present
invention can further or additionally comprise at least one
antiviral chemotherapeutic compound. Non-limiting examples can be
selected from at least one of the group consisting of gamma
globulin, amantadine, guanidine, hydroxy benzimidazole,
interferon-.alpha., interferon-.beta., interferon-.gamma.,
interleukin-16 (IL-16; Kurth, Nature, Dec. 8, 1995);
thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidine
analog (e.g., AZT and/or 3TC), a purine analog, foscarnet,
phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease
inhibitor (e.g., saquinavir (Hoffmann-La Roche); indinavir (Merck);
ritonavir (Abbott Labs); AG 1343 (Agovron Pharmaceuticals); VX-2/78
(Glaxo Wellcome)); chemokines, such as RANTES, MIP1.alpha. or
MIP1.beta. (Science 270:1560-1561 (1995)) or ganciclovir. See,
e.g., Richman: AIDs Res. Hum. Retroviruses 8:1065-1071(1992); Annu
Rev Pharmacol Toxico 33:149-164 (1993); Antimicrob Agents Chemother
37:1207-1213 (1993); AIDs Res. Hum. Retroviruses 10:901 (1994);
Katzung (1992), infra, and the references cited therein on pages
798-800 and 680-681, respectively, which references are herein
entirely incorporated by reference.
Pharmaceutical Uses
The administration of a polyenv vaccine (or the antisera which it
elicits) can be for either a "prophylactic" or "therapeutic"
purpose, and preferably for prophylactic purposes. When provided
prophylactically, the live polyenv vaccine composition is provided
in advance of any detection or symptom of HIV infection or AIDS
disease. The prophylactic administration of the compound(s) serves
to prevent or attenuate any subsequent HIV infection.
When provided therapeutically, the polyenv vaccine is provided upon
the detection of a symptom of actual infection. The administration
of a live polyenv vaccine after HIV infection is provided only
where the patient's immune system is determined to be capable of
responding to administration of the live polyenv vaccine without
substantive risk of unsuitable complications or death, where the
administration of a live vaccinia virus is provided in the required
dosage that serves to attenuate any actual HIV infection.
Alternatively, where the patients immune response is compromised,
therapeutic administration preferentially involves the use of an
attenuated or inactivated polyenv vaccine composition where the
recombinant vaccinia viruses are attenuated or inactivated, as
presented above. See, e.g., Berkow (1987), infra, Goodman (1990),
infra, Avery (1987), infra and Katzung (1992), infra, Dorozynski
and Anderson, Science 252:501-502 (1991) which are entirely
incorporated herein by reference, including all references cited
therein.
A composition is said to be "pharmacologically acceptable" if its
administration can be tolerated by a recipient patient. Such an
agent is said to be administered in a "therapeutically or
prophylactically effective amount" if the amount administered is
physiologically significant. A vaccine or composition of the
present invention is physiologically significant if its presence
results in a detectable change in the physiology of a recipient
patient, preferably by enhancing a humoral or cellular immune
response to an HIV.
The "protection" provided need not be absolute, i.e., the HIV
infection or AIDS disease need not be totally prevented or
eradicated, provided that there is a statistically significant
improvement relative to a control population. Protection can be
limited to mitigating the severity or rapidity of onset of symptoms
of the disease.
Pharmaceutical Administration
A vaccine of the present invention can confer resistance to one or
more strains of an HIV. The present invention thus concerns and
provides a means for preventing or attenuating infection by at
least one HIV strain. As used herein, a vaccine is said to prevent
or attenuate a disease if its administration to an individual
results either in the total or partial attenuation (i.e.
suppression) of a symptom or condition of the disease, or in the
total or partial immunity of the individual to the disease.
At least one polyenv vaccine of the present invention can be
administered by any means that achieve the intended purpose, using
a pharmaceutical composition as described herein.
For example, administration of such a composition can be by various
parenteral routes such as subcutaneous, intravenous, intradermal,
intramuscular, intraperitoneal, intranasal, transdermal, or buccal
routes. Subcutaneous administration is preferred. Parenteral
administration can be by bolus injection or by gradual perfusion
over time. See, e.g., Berkow (1987), infra, Goodman (1990), infra,
Avery (1987), infra, and Katzung (1992), infra, which are entirely
incorporated herein by reference, including all references cited
therein.
A typical regimen for preventing, suppressing, or treating a
disease or condition which can be alleviated by a cellular immune
response by active specific cellular immunotherapy, comprises
administration of an effective amount of a vaccine composition as
described above, administered as a single treatment, or repeated as
enhancing or booster dosages, over a period up to and including one
week to about 24 months.
According to the present invention, an "effective amount" of a
vaccine composition is one which is sufficient to achieve a desired
biological effect, in this case at least one of cellular or humoral
immune response to HIV. It is understood that the effective dosage
will be dependent upon the age, sex, health, and weight of the
recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. The ranges of
effective doses provided below are not intended to limit the
invention and represent preferred dose ranges. However, the most
preferred dosage will be tailored to the individual subject, as is
understood and determinable by one of skill in the art, without
undue experimentation. See, e.g., Berkow (1987), infra, Goodman
(1990), infra, Avery (1987), infra, Ebadi, Pharmacology, Little,
Brown and Co., Boston, Mass. (1985), and Katsung (1992), infra,
which references and references cited therein, are entirely
incorporated herein by reference.
Generally speaking, the dosage for a human adult will be from about
10.sup.5 -10.sup.9 plaque forming units (pfu)/kg or colony forming
units (CFU)/kg per dose, with 10.sup.6 -10.sup.8 preferred.
Whatever dosage is used, it should be a safe and effective amount
as determined by known methods, as also described herein.
Subjects
The recipients of the vaccines of the present invention can be any
mammal which can acquire specific immunity via a cellular or
humoral immune response to HIV, where the cellular response is
mediated by an MHC class I or class II protein. Among mammals, the
preferred recipients are mammals of the Orders Primata (including
humans, chimpanzees, apes and monkeys). The most preferred
recipients are humans. The subjects preferably are infected with
HIV or provide a model of HIV infection (e.g., Hu et al., Nature
328:721-723 (1987)), which reference is entirely incorporated
herein by reference.
Having now generally described the invention, the same will be more
readily understood through reference to the following example which
is provided by way of illustration, and is not intended to be
limiting of the present invention.
EXAMPLES
Example 1
Preparation of Vaccinia Virus Vectors for HIV Env Protein
Expression
Nomenclature
For purposes of reference, a recombinant vaccinia virus construct
is alternatively referred to herein as a VVenv construct, with
specific vaccinia virus constructs being designated according to a
patient, or to a depository (e.g., ATCC or the GenBank source of
the env DNA in the construct). For example, VVenv-Doe would refer
to a vaccinia virus vector construct having env sequences from
patient Doe, and VVenv-U28305 would refer to a vaccinia virus
vector having the env sequences found in GenBank accession No.
U28305.
The polyenv vaccine consists of 4-100 distinct recombinant vaccinia
viruses, each of which expresses a unique HIV-1 envelope protein.
For purposes of reference, each individual virus is designated as
VVenv, and the final virus mixture is referred to as polyenv.
The preparation of each VVenv uses the plasmid designated pVenv4
and a wildtype vaccinia virus designated NYCDH, described below.
For additional details, see Ryan et al., "Preparation and Use of
Vaccinia Virus Vectors for HIV Protein Expression and
Immunization," in Immunology Methods Manual, Lefkovits, ed.,
Academic Press (1996).
Vectors and Host Cells
The previously described pSC11 vector (Chakrabarti, S. et al., Mol.
Cell. Biol. 5:3403-3409 (1985)) can be used for the recombination
of multiple HIV genes into the VV genome. Elements of the pSC11
plasmid include the lacZ gene (a reporter gene by which transformed
bacteria and VV recombinants can be easily identified as those
having .beta.-galactosidase activity), a portion of the gene
encoding thymidine kinase (TK), and an ampicillin resistance gene
(amp). Genes cloned into pSC11 are inserted into the VV genome by
homologous recombination between the TK gene of the wildtype virus
and the portions of the TK gene contained in pSC11. Insertion of
plasmid DNA into the viral TK locus inactivates the viral gene so
that recombinant viruses can be readily selected from the
background of TK.sup.+ virus by growth in bromodeoxyuridine (BUdR).
In order for recombinant TK.sup.31 virus to survive this selection,
they must be grown in cells which do not supply an active TK
enzyme, such as the TK.sup.- 143 cell line, which is a TK-deficient
derivative of the human cell line R970-5, an osteosarcoma cell line
(Rhim, J.S. et al., Int. J. Cancer 15:23-29 (1975)) that supports
the growth of VV (Weir et al., infra (1982)). The production of HIV
gene segment expression can be by full gene insertion into the
Sinai site of the pSC11 vector. Full length genes can be expressed
under the control of the P7.5K promoter.
As an alternative to the cloning of complete HIV genes, one can
substitute partial gene sequences for HIV genes that have already
been cloned into pSC11. For example, a construct termed pVenv1 was
prepared from pSC11 and expresses the BH10 HIV envelope protein
(env) gene (Hallenberger et al., infra, (1993); Kilpatrick et al.
J. Biol. Chem. 262:116-121 (1987)). The construct can be used as a
parent vector to substitute and express variable envelope protein
regions from field HIV isolates. Similarly, a vector termed pVenv4
was constructed from pSC11 to express a BH10 env protein, truncated
to exclude the transmembrane and cytoplasmic tail domain encoding
gp41 sequences while retaining the oligomerization domain
(Hallenberger et al. (1993), infra). The pVenv4 vector encodes a
truncated gp160 (also: gp1601, gp140) that was discovered to form a
tertiary structure that is similar to that of the processed
gp41/gp120 oligomer (dimer, trimer or tetramer) as is present at
the cell surface of HIV infected cells. This tertiary structure is
maintained in both secreted and membrane associated form
(Hallenberger et al., (1993)). This vector is preferably used as a
parent vector for the substitution of alternative isolated env
sequences.
In this Example, the preparation of each VVenv construct involves
the use of a pVenv4 and a wildtype vaccinia virus NYCDH, and
appropriate host cells, as is described in detail below.
pVenv4: The pVenv4 vector was previously prepared by the insertion
of an HIV-1-envelope coding sequence into the pSC11 vaccinia virus
recombination vector (Hallenberger, et al., Virology 193:510-514
(1993); Chakrabarti et al., Mol. Cell Biology 5:3403-3409 (1985)).
The HIV-1 sequence was derived from a laboratory stock of live
virus. The sequence was named "BH10" (Ratner et al., Nature
313:277-284 (1985)). With PCR techniques unique envelope sequences
from HIV-1 infected patients may be amplified and substituted into
the BH10 env sequence to create new vectors. For example, the
following primers might be used for PCR.
(A) Sense, Position 5785 (SEQ ID NO:1):
AGCAGAAGACAGTGGCAATGAGAGTGA.
(B) Antisense, Position 7694 (SEQ ID NO:2):
CCACTCCATCCAGGTCATGTTATTCCAAAT.
(C) KpnI-Sense, position 5903 (SEQ ID NO:3):
GTGGGTCACAGTCTATTATGGGGTACCTGTGT.
(D) BsmI-Antisense, position 7659 (SEQ ID NO:4):
CCAGAGATTTATTACTCCAACTAGCATTCCAAGG.
(E) (optional) DraIII-Sense, position 6153 (SEQ ID NO:5):
CCATGTGTAAAATTAACCCCACTCTGTG.
(F) (optional) Bsu36I-Anti-sense, position 6917 (SEQ ID NO:6):
TACAATTTCTGGGTCCCCTCCTGAGG.
These primers are written 5' to 3'. Restriction sites are
underlined (numbered positions are based on the BH10 sequence
(Ratner et al., Nature 313:277-284 (1985)).
PCR Strategy: In order to produce new HIV-1 env constructs, the
polymerase chain reaction (PCR) is used to amplify 1800 base pairs
(bp) of envelope gene from forty different HIV-1 patient samples.
The PCR primers represent well-conserved HIV-1 sequences and thus
successfully amplified env genes from many diverse HIV-1 patient
samples. The amplified DNA encompasses the entire gp120 protein
except for approximately 10 highly conserved amino acids at the
protein's amino terminus. All envelope variable regions (V1-V5) are
included in the PCR products. In addition, amplified sequences
encode approximately 100 amino acids beyond the cleavage site for
gp120/gp41.
The PCR primers carrying the restriction enzyme sites for KpnI and
BsmI, which flank the BH10 envelope gene sequence in pVenv4, are
incorporated into the amplified DNA products.
First Round PCR: In a 500 .mu.l microcentrifuge tube, mix:
1 .mu.l Primer A (SEQ ID NO:1), 300 ng/.mu.l;
1 .mu.l primer B (SEQ ID NO:2), 300 ng/.mu.l;
2.5 .mu.l 10 mM of each of 4 dNTPs;
1 .mu.g DNA;
10 .mu.l 10X PCR buffer; and
HPLC H20 to 99 .mu.l
Vortex taq stock and dispense 1 .mu.l to PCR reaction. Mix well.
Overlay with mineral oil.
Run on a thermal-cycler as follows:
Incubate 95.degree. C., 3 minutes to melt DNA.
Run 40 cycles: 95.degree. C., 1 minute; 45.degree. C., 2 minutes;
72.degree. C., 3.5 minutes.
Second Round PCR: Prepare PCR reaction as above, but with primers C
and D (SEQ ID NOS:3 and 4, respectively) and without the DNA. Bring
the final solution to 95 .mu.l. Overlay with mineral oil. With a
plugged tip, remove 5 .mu.l from the first PCR reaction (from below
the oil). Mix the sample into the second reaction, below oil layer
and begin cycles as before. Thirty cycles is usually appropriate.
It can be desirable to monitor the product by removing 2 .mu.l for
gel analysis after each 10 cycles until a clear band is identified
of approximately 1800 bp. By using well-known substitution cloning
techniques, pVenv4. derivatives that express an env sequence from
one of the 40 patients, instead of the BH10 envelope sequence, were
generated. Briefly, the pVenv4 plasmid and PCR products are next
cut with KpnI and BsmI, and the cut pVenv4 was run on an agarose
gel and the large fragment isolated. The small fragment (1800 bp
fragment) of BH10 env was discarded. The cut PCR product was also
isolated and ligated to the large pVenv4 fragment to create a
chimetic envelope sequence, now containing 1800 bp of the variant
env from the patient DNA. Following ligation of the PCR product and
the pVenv products, bacterial host cells are transformed with the
ligation mixture via any of a number of methods well-known in the
art, including, e.g., electroporation, and recombinant colonies are
picked and examined by sequencing.
Plasmid pVenv4 or recombinants made with pVenv4 facilitates the
insertion of genes into the vaccinia virus genome by homologous
recombination between the tymidine kinase (Tk) gene of the wildtype
virus and the Tk sequences within the plasmid. Insertion of pVenv4
DNA into the viral Tk locus yields a vaccinia virus with the HIV-1
envelope gene expressed under the control of the P7.5K early/late
promoter. The virus is attenuated in growth activity due to the
disruption of the Tk locus. An additional element of pVenv4 is the
lacZ gene that encodes .beta.-galactosidase activity. lacZ activity
can be used to select vaccinia virus recombinants (see below).
The envelope gene expressed by pVenv4 is truncated to exclude the
transmembrane/C-terminal gp41 sequence. The vector is expressed as
an oligomeric structure that is found within cells and in secreted
form.
Vaccinia virus-NYCDH: Each new, substituted plasmid is individually
recombined with wildtype vaccinia virus NYCDH. This virus was
obtained from A.T.C.C. (Accession No. VR-325) and was
plaque-purified prior to use (Buck, C., and Paulino, M. S., eds.,
American Type Culture Collection Catalogue of Animal Viruses and
Antisera, Chlamydiae and Rickettsiae, 6th Ed., American Type
Culture Collection, Rockville, Md. (1990), p. 138).
Bacterial host cells: The plasmid may be grown on any suitable
host, as known in the art (see, e.g., Ausubel, infra (1995 rev),
.sctn..sctn. 16.15-16.19). A non-limiting example is DH5.alpha.
cells.
TK-deficient cells: The transformation and vaccinia virus
substitution is done on the human Tk.sup.- 143B cell line, which is
a TK-deficient derivative of the human cell line R970-5, an
osteosarcoma cell line (Rhim et al. (1975), infra) that supports
the growth of VV (Weir et al. (1982), infra). Each vaccinia virus
recombinant containing a unique HIV env gene sequence is selected
based on expression of the lacZ gene (Virus plaques are overlayed
with Bluo-gal and selected for .beta.-galactosidase activity as
judged by the development of a blue color).
Two rounds of PCR can be performed.
Example 2
Preparation of Polyenv Vaccine
Vero Cells: The final manufacturing step is to grow n VVenv
constructs on Vero cells newly purchased from the A.T.C.C.
(Accession No. CCL81 or X38) and cloned and expanded for virus
growth. The Vero cell line has been approved by the World Health
Organization for vaccine development (Hay, R., et al., eds.,
American Type Culture Collection Catalogue of Cell Lines and
Hybridomas, 7th Ed., American Type Culture Collection, Rockville,
Md. (1992), page 48).
Vero cells are grown with Dulbecco's Modified Eagles Medium
(Bio-Whittaker), a glutamine supplement (Bio-Whittaker) and
heat-inactivated fetal calf serum (Hyclone, Inc.). Alternatively,
serum-free media can be used. Each VVenv construct is inoculated
onto a separate confluent layer of Vero cells and harvested when
cells demonstrate cytopathic effects due to virus infection. Cell
extracts are washed extensively with PBS (Bio-Whittaker) after
harvest and before freezing. The cells are then broken open by
freeze-thawing, sonication or centrifuging at low speed in a
centrifuge (optional). Aliquots of supernatant are then stored at
-70.degree. C. Envelope protein is present in the lysate at
sufficient concentrations to elicit HIV envelope protein-specific
antibody (as detectable by ELISA) in mammal models, even if VV is
attenuated, e.g., prep is heated to 60.degree. C., 1 hr.
The Vaccine Product: Each virus (VVenv construct) stock from Vero
cells is individually frozen and subsequently titered and safety
tested. After tests have been completed, aliquots of each virus are
mixed to yield a stock vaccine of 10.sup.8 total pfu/ml ("pfu"
stands for plaque-forming units). If 40 VVenv constructs are
utilized, each VVenv is preferably equally represented, each VVenv
used at a titer of 2.5.times.10.sup.6 pfu/ml in the vaccine
product. This should yield 1.times.10.sup.8 total pfu.
Evaluation of Polyenv Vaccine
Mice: Mice can be infected with an intraperitoneal injection of
1.times.10.sup.7 pfu env-expressing VV. Antibody can be identified
by HIV ELISA or neutralization assays, as described above, three
weeks after VV injections.
Prior to manufacture of the polyenv vaccine for human use, a
similar group of viruses has been prepared for the purpose of
vaccine testing in mice. These viruses were administered to mice
either by the intraperitoneal or subcutaneous route. We then tested
serum HIV-1-specific antibody serum was tested for activity in an
enzyme-linked immunosorbant assay (ELISA). The assay involved the
plating of whole, disrupted HIV-1 (HTLV.sub.IIIB) on ELISA plates
and the blocking of plates with bovine serum albumin. Serum samples
were then added at dilutions of 1:100, 1:1,000 and 1:10,000 in
phosphate-buffered saline. The assay was developed with an
alkaline-phosphatase-conjugated goat-anti-mouse immunoglobulin
antibody and p-nitrophenyl phosphate. The color reaction was
stopped with a sodium hydroxide solution, and the optical density
reading was taken on an ELISA plate reader at 405 nm.
As shown in FIG. 2, a single inoculation with cell lysate
preparation of 10.sup.6 -10.sup.7 pfu vaccinia virus (containing a
single HIV-1/envelope protein encoding sequence and membrane bound
expressed envelope protein) elicited a strong antibody response
toward HIV-1 that was sustained throughout the experimental time
course of six months. Such an antibody response was significantly
higher than previously reported with other immunizations. This high
antibody response may be attributed to the presence of membrane
bound envelope protein in a vaccine preparation. As shown in FIG.
3, these responses were dose dependent. Lower responses were seen
in mammals given a dose of 10.sup.6 pfu than in mammals given a
dose of 10.sup.7 pfu.
Mixtures of vaccinia viruses expressing different HIV-1 envelope
proteins were also prepared. When mice received 10.sup.7 pfu of a
mixture of five viruses, their responses were essentially identical
in magnitude to responses generated against 10.sup.7 pfu of a
single vaccinia virus recombinant (FIG. 4). The mixing of numerous
env-expressing vaccinia viruses in high numbers has not been
reported, and is expected to provide broad spectrum of neutralizing
antibody.
Chimpanzees: The mixed vaccine is expected to neutralize a higher
number of challenge viruses. Two chimpanzees are injected, each
with a mixture of 10-40 VVenv, with approximately 3.times.10.sup.7
total pfu/mammal by the subcutaneous route. Two additional
chimpanzees receive 3.times.10.sup.7 total pfu/mammal of a single
VVenv. It is expected that the mammals will generate antibody
responses capable of neutralizing HIV-1 isolates in laboratory
tests, and that the neutralization will be superior with immune
sera from the mammals injected with 10-40 VVenv, as a polyenv
vaccine of the present invention, as compared to sera from mammals
immunized with 1-3 vaccinia viruses expressing: (1) one or several
variable regions of the HIV envelope protein, such as the V3
region; (2) one HIV envelope proteins; or (3) one or several
portions of the variable and constant regions from 1-3 HIV envelope
proteins.
Humans: Tests of the mixed virus stock are performed prior to
clinical trials, the first of which will be for the purpose of dose
escalation and safety testing.
The clinical trials will be a dose escalation study involving the
assembly of four volunteer groups. Each group receives one of the
following vaccine doses:
(1) 2.times.10.sup.4 pfu
(2) 2.times.10.sup.5 pfu
(3) 2.times.10.sup.6 pfu
(4) 2.times.10.sup.7 pfu
Each volunteer receives the mixed virus vaccine in 0.5 ml saline,
administered by a subcutaneous injection.
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