U.S. patent application number 12/377847 was filed with the patent office on 2010-06-10 for intergenic sites between conserved genes in the genome of modified vaccinia ankara (mva) vaccinia virus.
This patent application is currently assigned to THE USA, as represented by the Secretary, Departme. Invention is credited to Patricia Earl, Bernard Moss, Linda Wyatt.
Application Number | 20100143402 12/377847 |
Document ID | / |
Family ID | 40032225 |
Filed Date | 2010-06-10 |
United States Patent
Application |
20100143402 |
Kind Code |
A1 |
Moss; Bernard ; et
al. |
June 10, 2010 |
INTERGENIC SITES BETWEEN CONSERVED GENES IN THE GENOME OF MODIFIED
VACCINIA ANKARA (MVA) VACCINIA VIRUS
Abstract
The present invention relates to new insertion sites useful for
the integration of exogenous sequences into an intergenic region
(IGR) of a vaccinia virus genome, where the IGR is located between
or is flanked by two adjacent open reading frames (ORFs) of the
vaccinia virus genome, and where the ORFs correspond to conserved
genes, and to related plasmid vectors useful to insert exogenous
DNA into the genome of a vaccinia virus, and further to recombinant
vaccinia viruses comprising an exogenous sequence inserted into
said new insertion site as a medicine or vaccine.
Inventors: |
Moss; Bernard; (Bethesda,
MD) ; Wyatt; Linda; (Rockville, MD) ; Earl;
Patricia; (Chevy Chase, MD) |
Correspondence
Address: |
NIH-OTT
1560 Broadway, Suite 1200
Denver
CO
80238
US
|
Assignee: |
THE USA, as represented by the
Secretary, Departme
|
Family ID: |
40032225 |
Appl. No.: |
12/377847 |
Filed: |
August 24, 2007 |
PCT Filed: |
August 24, 2007 |
PCT NO: |
PCT/IB07/04575 |
371 Date: |
February 17, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60840093 |
Aug 25, 2006 |
|
|
|
60840755 |
Aug 28, 2006 |
|
|
|
Current U.S.
Class: |
424/199.1 ;
424/93.21; 435/235.1; 435/239; 435/320.1; 435/440; 435/69.1 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2710/24152 20130101; C12N 2710/24143 20130101; A61K 2039/5256
20130101; C12N 2710/24141 20130101; C12N 2710/24111 20130101; A61P
31/12 20180101; A61P 31/18 20180101; C12N 15/8636 20130101 |
Class at
Publication: |
424/199.1 ;
435/235.1; 435/69.1; 435/440; 424/93.21; 435/320.1; 435/239 |
International
Class: |
A61K 39/285 20060101
A61K039/285; A61P 37/00 20060101 A61P037/00; C12N 7/00 20060101
C12N007/00; C12P 21/00 20060101 C12P021/00; C12N 15/00 20060101
C12N015/00; A61K 35/12 20060101 A61K035/12; C12N 15/63 20060101
C12N015/63; C12N 7/02 20060101 C12N007/02 |
Claims
1. A recombinant modified vaccinia Ankara (MVA) virus comprising a
heterologous DNA sequence inserted into an intergenic region (IGR)
of the MVA genome, wherein the IGR is located between or is flanked
by two adjacent open reading frames (ORFs) of the MVA genome, and
wherein the ORFs correspond to conserved genes, and wherein the two
adjacent ORFs are selected from the group consisting of (using the
nomenclature according to CDC/Acambis): 044-045, 049-050, 050-051,
058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073,
073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082,
085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098,
100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115,
118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130,
130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and
142-143, in an exemplary manner or corresponding thereto in other
strains of vaccinia virus.
2. The MVA according to claim 1, wherein the two adjacent ORFs are
selected from the group consisting of 049-050, 071-072, 074-075,
078-079, 092-093, 100-101, 118-119, 121-122, and 132-133.
3. The MVA according to claim 2, wherein the two adjacent ORFs are
071-072.
4. The MVA according to any of claims 1 to 3, wherein the
heterologous DNA sequence comprises at least one coding sequence,
under the transcriptional control of a poxviral transcription
control element.
5. The MVA according to any of claims 1 to 4, wherein the
heterologous DNA sequence encodes one or more proteins,
polypeptides, peptides, foreign antigens or antigenic epitopes.
6. The MVA according to any of claims 1 to 5, wherein the
heterologous DNA sequence is derived from human immunodeficiency
virus (HIV).
7. The MVA according to claim 6, wherein the heterologous DNA
sequence derived from human immunodeficiency virus codes for HIV
env.
8. The MVA according to any of claims 1 to 7, wherein the MVA
genome is that of the MVA deposited at ATCC under accession number
PTA-5095.
9. The MVA according to any of claims 1 to 7, wherein the MVA
genome is that having the sequence of Genbank accession number
AY603355.
10. The MVA according to any of claims 1 to 7, wherein the MVA
genome is that having the sequence of Genbank accession number
U94848.
11. The MVA according to any of claims 1 to 10 for use as a
medicament and/or vaccine.
12. Use of the MVA according to any of claims 1 to 10 for the
preparation of a medicament and/or vaccine for the treatment and/or
prophylaxis of a viral infection and/or a proliferating
disease.
13. The use according to claim 12 for the treatment or prophylaxis
of human immunodeficiency virus (HIV) infection.
14. A vaccine or composition for inducing an immune response
comprising the MVA according to any of claims 1 to 10.
15. A pharmaceutical composition comprising the MVA according to
any of claims 1 to 10 and a pharmaceutically acceptable carrier,
additive, adjuvant, diluent and/or stabilizer.
16. A method for inducing an immune response in a living animal
body including a human comprising administering the MVA according
to any of claims 1 to 10 to the animal, including a human.
17. A method for producing a protein, polypeptide, peptide, antigen
or epitope in vitro comprising: infection of a host cell with the
recombinant MVA according to any of claims 1 to 10, cultivation of
the infected host cell under suitable conditions, and isolation
and/or enrichment of the polypeptide, protein, peptide, antigen,
epitope and/or virus produced by said host cell.
18. A method for introducing a DNA sequence into a cell in vitro,
said DNA sequence being homologous and/or heterologous to the
genome of said cell, wherein the cell is infected with the MVA
according to any of claims 1 to 10.
19. A method for introducing a DNA sequence into a cell ex vivo,
said DNA sequence being homologous and/or heterologous to the
genome of said cell, wherein the cell is infected with the MVA
according to any of claims 1 to 10 and wherein the infected cell
is, subsequently, administered to a living animal body, including a
human.
20. A method for introducing a DNA sequence into a living animal
body, including a human, said DNA sequence being homologous and/or
heterologous to the genome of said animal body, by administering
the MVA according to any of claims 1 to 10 to the animal, including
a human.
21. A plasmid vector comprising a DNA sequence derived from or
homologous to the genome of an MVA, wherein said DNA sequence
comprises a complete or partial fragment of an IGR sequence located
between or flanked by two adjacent ORFs of the MVA genome and
inserted into said IGR sequence a cloning site for the insertion of
a DNA sequence being heterologous to the genome of the MVA and,
optionally, of a reporter- and/or selection gene cassette, and
wherein the two adjacent ORFs are selected from the group
consisting of (using the nomenclature according to CDC/Acambis):
044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070,
070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077,
077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093,
093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105,
108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124,
127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134,
134-135, 135-136, 141-142, and 142-143, in an exemplary manner or
corresponding thereto in other strains of vaccinia virus.
22. A plasmid vector comprising a DNA sequence derived from or
homologous to the genome of an MVA, wherein said DNA sequence
comprises IGR flanking sequences of two adjacent ORFs and, inserted
into said IGR, a cloning site for the insertion of a DNA sequence
being heterologous to the genome of the MVA and, optionally, of a
reporter- and/or selection gene cassette, and wherein the two
adjacent ORFs are selected from the group consisting of (using the
nomenclature according to CDC/Acambis): 044-045, 049-050, 050-051,
058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073,
073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082,
085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098,
100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115,
118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130,
130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and
142-143, in an exemplary manner or corresponding thereto in other
strains of vaccinia virus.
23. The plasmid vector according to claim 21 or 22, wherein said
DNA sequence comprises a complete or partial fragment of an IGR
sequence, said IGR sequence including the cloning site for the
insertion of the heterologous DNA sequence and, optionally, of the
reporter- and/or selection gene cassette.
24. The plasmid vector according to claim 21 or 22, wherein the two
adjacent ORFs are selected from the group consisting of 049-050,
071-072, 074-075, 078-079, 092-093, 100-101, 118-119, 121-122, and
132-133.
25. The plasmid vector according to claim 24, wherein the two
adjacent ORFs are 071-072.
26. The plasmid vector according to any of claims 21 to 25, wherein
the DNA sequence is derived from or homologous to the genome of the
MVA deposited at ATCC under accession number PTA-5095.
27. The plasmid vector according to any of claims 21 to 25, wherein
the DNA sequence is derived from or homologous to the genome of the
MVA having the sequence of Genbank accession number AY603355.
28. The plasmid vector according to any of claims 21 to 25, wherein
the DNA sequence is derived from or homologous to the genome of the
MVA having the sequence of Genbank accession number U94848.
29. A method for producing the MVA according to claim 1 comprising
transfecting a cell with a plasmid vector according to any of
claims 21 to 39; infecting the transfected cell with an MVA; and
identifying, isolating and, optionally, purifying the MVA.
30. A method of stabilizing an insert in a recombinant vaccinia
virus comprising identifying a run of 4 or more G or C residues in
said insert, and making a silent mutation in said run so identified
by substitution so that the length of the run is reduced.
31. pLW-73.
32. MVA/UGD4d.
33. A recombinant vaccinia virus comprising a heterologous DNA
sequence inserted into an intergenic region (IGR) of the vaccinia
virus genome, wherein the IGR is located between or is flanked by
two adjacent open reading frames (ORFs) of the vaccinia virus
genome, and wherein the ORFs correspond to conserved genes, and
wherein the two adjacent ORFs are selected from the group
consisting of (using the nomenclature according to CDC/Acambis):
044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070,
070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077,
077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093,
093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105,
108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124,
127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134,
134-135, 135-136, 141-142, and 142-143, in an exemplary manner or
corresponding thereto in other strains of vaccinia virus.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/840,093 filed Aug. 25, 2006, and U.S.
Provisional Application No. 60/840,755 filed Aug. 28, 2006, both of
which are hereby expressly incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to insertion sites useful for
the stable integration of exogenous DNA sequences into the MVA
genome.
DESCRIPTION OF THE RELATED ART
[0003] The members of the poxvirus family have large
double-stranded DNA genomes encoding several hundred proteins
(Moss, B. 2007 "Poxyiridae: The Viruses and Their Replication" in
Fields Virology, 5.sup.th Ed. (D. M. Knipe, P. M. Howley, D. E.
Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus,
Eds), Lippincott Williams & Wilkins, Philadelphia, Pa.). The
genomic sequence of the highly attenuated vaccinia strain modified
vaccinia Ankara (MVA) (Mayr, A. et al. 1978 Zentralbl Bakteriol
167:375-390), which cannot grow in most mammalian cells and which
is a good candidate for a recombinant vaccine vector, is known
(Sutter, G. and Moss, B. 1992 Proc Natl Acad Sci USA
89:10847-10851; and Sutter, G. et al. 1994 Vaccine 12:1032-1040)
has been passaged over 570 times in chicken embryo fibroblasts,
during which six major deletions relative to the parental wild-type
strain Ankara, accompanied by a severe restriction in host range,
have occurred (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038).
SUMMARY OF THE INVENTION
[0004] The present invention relates to new insertion sites useful
for the integration of exogenous sequences into an intergenic
region (IGR) of a vaccinia virus genome, where the IGR is located
between or is flanked by two adjacent open reading frames (ORFs) of
the vaccinia virus genome, and where the ORFs correspond to
conserved genes, and to related plasmid vectors useful to insert
exogenous DNA into the genome of a vaccinia virus, and further to
recombinant vaccinia viruses comprising an exogenous sequence
inserted into said new insertion site as a medicine or vaccine.
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: 1) and two predicted helical domains
(helix-1 and -2) are shown. Amino acid numbers are indicated.
[0011] FIG. 7. pLW-73 transfer vector.
[0012] FIG. 8. Nucleotide sequence of the pLW-73 transfer vector
(top strand, SEQ ID NO: 2; bottom strand, SEQ ID NO: 3).
[0013] FIG. 9. Nucleotide sequence encoding Ugandan Glade D Env
protein (isolate AO7412) (SEQ ID NO: 4).
[0014] FIG. 10. Codon altered nucleotide sequence encoding Ugandan
Glade D gagpol protein (isolate AO3349) (SEQ ID NO: 5).
[0015] FIG. 11. Generation of recombinant MVAs and analysis of
stability of inserted genes. A) Schematic diagram of insertion of
env and gagpol into Del II and Del III sites, respectively. B)
Evaluation of stability by immunostaining.
[0016] FIG. 12. Types and frequency of env mutations in MVA/65A/G
env.
[0017] FIG. 13. Insertion of Env in I8R/G1L IGR and Gag Pol in Del
III.
[0018] FIG. 14. Modifications to A/G constructs to increase
stability.
[0019] FIG. 15. Env expression after plaque passages.
[0020] FIG. 16. PCR and Western blot analysis of individual
clones.
[0021] FIG. 17. Expression of A/G env by double recombinant
MVA.
[0022] FIG. 18. Recombinant viruses expressing env and gagpol from
Ugandan HIV-1 isolates.
[0023] FIG. 19. MVA/UGD4a--analysis of non-staining env
plaques.
[0024] FIG. 20. Modification of UGD env gene in recombinant
MVA.
[0025] FIG. 21. MVA/UGD4b--analysis of non-staining gag plaques. *,
location of runs of 4-6 G or C residues.
[0026] FIG. 22. Modification of UGD gagpol gene in recombinant
MVA.
[0027] FIG. 23. Construction of stable recombinant MVA expressing
UGD env and gagpol.
[0028] FIG. 24. Cellular responses elicited by MVA/UGD4d.
[0029] FIG. 25. Antibody responses elicited by MVA/UGD4d.
DEPOSIT OF MICROORGANISM
[0030] The following microorganism has been deposited in accordance
with the terms of the Budapest Treaty with the American Type
Culture Collection (ATCC), Manassas, Va., on the date
indicated:
TABLE-US-00001 Microorganism Accession No. Date MVA 1974/NIH Clone
1 PTA-5095 Mar. 27, 2003
[0031] MVA 1974/NIH Clone 1 was deposited as ATCC Accession No.:
PTA-5095 on Mar. 27, 2003 with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA. This
deposit was made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure and the Regulations thereunder
(Budapest Treaty). This assures maintenance of a viable culture of
the deposit for 30 years from date of deposit. The deposit will be
made available by ATCC under the terms of the Budapest Treaty, and
subject to an agreement between Applicant and ATCC which assures
permanent and unrestricted availability of the progeny of the
culture of the deposit to the public upon issuance of the pertinent
U.S. patent or upon laying open to the public of any U.S. or
foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 USC .sctn.122 and the Commissioner's rules pursuant
thereto (including 37 CFR .sctn.1.14). Availability of the
deposited strain is not to be construed as a license to practice
the invention in contravention of the rights granted under the
authority of any government in accordance with its patent laws.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0032] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g., Singleton P and Sainsbury D., Dictionary of Microbiology and
Molecular Biology, 3rd ed., J. Wiley & Sons, Chichester, N.Y.,
2001 and Fields Virology, 5.sup.th Ed. (D. M. Knipe, P. M. Howley,
D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E.
Straus, eds), Lippincott Williams & Wilkins, Philadelphia, Pa.,
2007.
[0033] The transitional term "comprising" is synonymous with
"including," "containing," or "characterized by," is inclusive or
open-ended and does not exclude additional, unrecited elements or
method steps.
[0034] The transitional phrase "consisting of" excludes any
element, step, or ingredient not specified in the claim, but does
not exclude additional components or steps that are unrelated to
the invention such as impurities ordinarily associated
therewith.
[0035] The transitional phrase "consisting essentially of" limits
the scope of a claim to the specified materials or steps and those
that do not materially affect the basic and novel characteristic(s)
of the claimed invention.
[0036] Poxviruses are divided into the subfamilies Chordopoxyirinae
and Entomopoxyirinae, based on vertebrate and insect host range.
The subfamily Chordopoxyirinae consists of eight genera:
Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus,
Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus.
The prototypal member of the genus Orthopoxvirus is vaccinia
virus.
[0037] Complete genome sequences have been reported for at least
one member of each chordopoxvirus genus and two entomopoxviruses.
Nearly 100 genes are conserved in all chordopoxviruses, and about
half of these are also present in entomopoxviruses. Based on the
above, several generalizations can be made: Genes are largely
nonoverlapping, tend to occur in blocks pointing toward the nearer
end of the genome, are usually located in the central region if
highly conserved and concerned with essential replication
functions, and are usually located in the end regions if variable
and concerned with host interactions. The arrangement of the
central genes is remarkably similar in all chordopoxviruses. A
convention for naming vaccinia virus genes or ORFs (open reading
frames), originating prior to sequencing the entire genome and
subsequently used for the complete sequence of the Copenhagen
strain of vaccinia virus, consists of using the HindIII restriction
endonuclease DNA fragment letter, followed by the ORF number (from
left to right) within the fragment, and L or R, depending on the
direction of the ORF. An exception to this rule was made for the
HindIII C fragment; the ORFs were numbered from the right in order
to avoid starting at the highly variable left end of the genome.
Polypeptide names correspond to gene names, except that L or R is
dropped. In most subsequent complete poxvirus genome sequences,
ORFs were numbered successively from one end of the genome to the
other. Nevertheless, the old letter designations have been retained
as common names to provide continuity in the literature. The ORF
number of the Western Reserve (WR) strain of vaccinia virus is
commonly shown in reference books because this strain has been used
for the great majority of biochemical and genetic studies.
[0038] The inventors of an embodiment of the present invention
identified new sites for the insertion of exogenous DNA sequences
into the genome of modified vaccinia Ankara (MVA) virus. The new
insertion sites are located in the intergenic regions (IGRs) of the
viral genome, wherein the IGRs are, in turn, located between or are
flanked by two adjacent open reading frames (ORFs) of the MVA
genome, and wherein the ORFs correspond to conserved genes.
[0039] Accordingly, an embodiment of the invention relates to a
recombinant MVA comprising a heterologous DNA sequence inserted
into an IGR of the viral genome. According to the present
embodiment, one or more exogenous DNA sequences may be inserted
into one or more IGRs.
[0040] It was surprisingly found that exogenous DNA sequences
remain stable inserted into IGRs of the MVA genome. The genome of
MVA is to be considered as being quite unstable. It seems that
genes or DNA sequences non-essential for propagation of the virus
are deleted or fragmented. Although it was--on the one hand--found
that stable recombinant MVAs are obtained when heterologous DNA
sequences are inserted into the naturally occurring deletion sites
of the MVA genome, it was--on the other hand--found that sometimes
these recombinant MVAs are unstable. Therefore, it could be
concluded that inserting heterologous DNA sequences non-essential
for viral propagation into spaces between ORFs would be expected to
be deleted by the virus as well.
[0041] While the nucleotide sequence of an ORF encodes an amino
acid sequence forming a peptide, polypeptide or protein, the IGRs
between two ORFs have no coding capacity, but may comprise
regulatory elements, binding sites, promoter and/or enhancer
sequences essential for or involved in the transcriptional control
of the viral gene expression. Thus, the IGR may be involved in the
regulatory control of the viral life cycle. However, the inventors
of the present embodiment have also shown that the new insertion
sites have the unexpected advantage that exogenous DNA sequences
can be stably inserted into the MVA genome without influencing or
changing the typical characteristics and gene expression of MVA.
The new insertion sites are especially useful, since no ORF or
coding sequence of MVA is altered.
[0042] The nucleotide sequence of an ORF regularly starts with a
start codon and ends with a stop codon. Depending on the
orientation of the two adjacent ORFs the IGR, the region in between
these ORFs, is flanked either by the two stop codons of the two
adjacent ORFs, or, by the two start codons of the two adjacent
ORFs, or, by the stop codon of the first ORF and the start codon of
the second ORF, or, by the start codon of the first ORF and the
stop codon of the second ORF.
[0043] Accordingly, the insertion site for the exogenous DNA
sequence into the IGR may be downstream or 3' of the stop codon of
a first ORF. In case the adjacent ORF, also termed second ORF, has
the same orientation as the first ORF, this insertion site
downstream of the stop codon of the first ORF lies upstream or 5'
of the start codon of the second ORF.
[0044] In case the second ORF has an opposite orientation relative
to the first ORF, which means the orientation of the two adjacent
ORFs points to each other, then the insertion site lies downstream
of the stop codons of both ORFs.
[0045] As a third alternative, in case the two adjacent ORFs read
in opposite directions, but the orientation of the two adjacent
ORFs points away from each other, which is synonymous with a
positioning that is characterized in that the start codons of the
two ORFs are adjacent to each other, then the exogenous DNA is
inserted upstream relative to both start codons.
[0046] ORFs in the MVA genome occur in two coding directions.
Consequently, mRNA synthesis activity occurs from left to right,
i.e., forward direction and, correspondingly, from right to left
(reverse direction). It is common practice in poxyirology and it
became a standard classification for vaccinia viruses to identify
ORFs by their orientation and their position on the different
HindIII restriction digest fragments of the genome. For the
nomenclature, the different HindIII fragments are named by
descending capital letters corresponding with their descending
size. The ORF are numbered from left to right on each HindIII
fragment and the orientation of the ORF is indicated by a capital L
(standing for transcription from right to Left) or R (standing for
transcription from left to Right). Additionally, there is a more
recent publication of the MVA genome structure, which uses a
different nomenclature, simply numbering the ORF from the left to
the right end of the genome and indicating their orientation with a
capital L or R (Antoine, G. et al. 1998 Virology 244:365-396). As
an example the I8R ORF, according to the old nomenclature,
corresponds to the 069R ORF according to Antoine et al.
[0047] In their efforts to make recombinants of modified vaccinia
virus Ankara (MVA) expressing HIV genes as candidate vaccines, the
inventors have observed instability in the expression of the HIV
genes. They have determined that one of the causes of instability
is due to deletions of the foreign gene and flanking MVA. To
overcome this problem they set out to insert foreign genes between
conserved genes in order to prevent viable deletions from occurring
in recombinant MVAs. Viruses with such deletions have a growth
advantage and will thus overgrow rMVA virus populations. If one
inserts foreign genes between conserved genes in the vaccinia
genome (these genes are considered to be required for vaccinia
virus replication and are therefore "essential genes"), any
deletion of an essential gene would inhibit virus replication, and,
therefore, not overgrow the recombinant MVAs. Thus, the stable
expression of the rMVA population is maintained. The strain of MVA
that the inventors have been using to make their recombinants was
provided by them to the Centers for Disease Control and Prevention
(CDC) and was subsequently sequenced by Acambis (Genbank Accession
number AY603355). The strain of MVA that Bavarian Nordic has based
their WO03/097845 publication on is vaccinia virus strain modified
vaccinia Ankara (Genbank Accession number U94848) sequenced by
Antoine, G. et al. 1998 Virology 244:365-396. (Please note that the
gene numbers in these two sequences for a given gene are
different.)
[0048] The inventors initially looked at genes conserved in the
Poxyiridae family as well as those genes conserved in subfamily
Chordopoxyirinae (the vertebrate poxviruses) (Upton, C. et al. 2003
Journal of Virology 77:7590-7600). These genes are listed in the
nomenclature of Copenhagen vaccinia virus (Genbank Accession number
M35027) given on the Poxvirus Bioinformatics Resource Center found
on the world wide web at poxvirus.org. These genes total 49
conserved genes in the Poxvirus family and 41 additional genes
conserved in chordopoxviruses, making a total of 90 conserved
genes. From these 90 conserved genes, the inventors listed
intergenic sites between conserved gene pairs. These gene pairs are
listed in Table 1. (Please note that genes are marked that have not
been included in the Bavarian Nordic WO03/097845 publication). The
orientations of these genes are variable, with some being
transcribed to the right, some to the left. This means that some of
the intergenic sites contain promoters that would have to be
preserved in the construction of the insertion vector. In addition,
for overlapping conserved genes, during vector construction the
genes would have to be reconstructed using alternative codons to
minimize the repeating sequences.
[0049] In a preferred embodiment, the inventors focused on
conserved genes whose orientation is "end to end" such that the 3'
stop codon of the genes are in close proximity to one another. The
construction of transfer vectors used in these sites are
facilitated by the fact that there would be no promoter in this
region between the stop codons. If there are intergenic nucleotides
separating the stop codons, then construction of the insertion
vector is straightforward. If the stop codon of one gene is within
the 3' end of the other gene, then during construction of the
plasmid transfer vector, the gene can be reconstructed using
alternative codons to minimize repeating sequences, or, depending
on the size of the overlap, simply corrected in the PCR of the
flanks so as not to overlap. Table 2 gives the intergenic sites
that meet the requirement of the orientation of the conserved genes
being "end to end". Those intergenic sites highlighted in gray have
no overlapping ends and therefore are simplest to construct.
[0050] The inventors specifically focused on the six intergenic
sites that have no overlapping ends. In a working example, of these
six, they chose the intergenic site, 071-072 (I8R-G1L), to insert
their foreign gene.
[0051] Besides using the requirement of conserved genes as listed
above in the Upton publication, for any gene that has been
experimentally deleted and virus replication is reduced by 10 fold
in the mutant, this gene could be considered as an "essential
gene". If this gene lies adjacent to another essential or conserved
gene, the intergenic site between the two genes could be considered
as a different site of insertion for a foreign gene.
TABLE-US-00002 TABLE 1 Intergenic Sites between Conserved Genes
Listed in Genes/ CDC/Acambis Antoine et WO03/097845 Copenhagen
Genes al. Genes publ ? N = No F9L-F10L 040-041 038L-039L F12L-F13L
044-045 042L-043L N F17R-E1L 049-050 047R-048L N E1L-E2L 050-051
048L-049L N E8R-E9L 057-058 055R-056L E9L-E10R 058-059 056L-057L N
I1L-I2L 064-065 062L-063L N I2L-I3L 065-066 063L-064L N I5L-I6L
068-069 066L-067L I6L-I7L 069-070 067L-068L N I7L-I8R 070-071
068L-069R N I8R-G1L 071-072 069R-070L N G1L-G3L 072-073 070L-071L N
G3L-G2R 073-074 071L-072R N G2R-G4L 074-075 072R-073L N G4L-G5R
075-076 073L-074R N G5R-G5.5R 076-077 074R-075R N G5.5R-G6R 077-078
075R-076R N G6R-G7L 078-079 076R-077L N G7L-G8R 079-080 077L-078R
G8R-G9R 080-081 078R-079R G9R-L1R 081-082 079R-080R N L1R-L2R
082-083 080R-081R L2R-L3L 083-084 081R-082L L3L-L4R 084-085
082L-083R L4R-L5R 085-086 083R-084R N L5R-J1R 086-087 084R-085R N
J3R-J4R 089-090 087R-088R N J4R-J5L 090-091 088R-089L J5L-J6R
091-092 089L-090R J6R-H1L 092-093 090R-091L N H1L-H2R 093-094
091L-092R N H2R-H3L 094-095 092R-093L H3L-H4L 095-096 093L-094L N
H4L-H5R 096-097 094L-095R H5R-H6R 097-098 095R-096R N H6R-H7R
098-099 096R-097R H7R-D1R 099-100 097R-098R D1R-D2L 100-101
098R-099L N D2L-D3R 101-102 099L-100R N D3R-D4R 102-103 100R-101R N
D4R-D5R 103-104 101R-102R D5R-D6R 104-105 102R-103R N D6R-D7R
105-106 103R-104R D9R-D10R 108-109 106R-107R N D10R-D11L 109-110
107R-108L D11L-D12L 110-111 108L-109L D12L-D13L 111-112 109L-110L
D13L-A1L 112-113 110L-111L A1L-A2L 113-114 111L-112L N A2L-A2.5L
114-115 112L-113L N A2.5L-A3L 115-116 113L-114L A3L-A4L 116-117
114L-115L A4L-A5R 117-118 115L-116R A5R-A6L 118-119 116R-117L N
A6L-A7L 119-120 117L-118L A7L-A8R 120-121 118L-119R A8R-A9L 121-122
119R-120L N A9L-A10L 122-123 120L-121L N A10L-A11R 123-124
121L-122R N A11R-A12L 124-125 122R-123L A12L-A13L 125-126 123L-124L
A13L-A14L 126-127 124L-125L A14L-A14.5L 127-128 .sup. 125L-125.5L N
A14.5L-A15L 128-129 125.5L-126L.sup. N A15L-A16L 129-130 126L-127L
N A16L-A17L 130-131 127L-128L N A17L-A18R 131-132 128L-129R N
A18R-A19L 132-133 129R-130L N A19L-A21L 133-134 130L-131L N
A21L-A20R 134-135 131L-132R N A20R-A22R 135-136 132R-133R N
A22R-A23R 136-137 133R-134R A23R-A24R 137-138 134R-135R A28L-A29L
141-142 139L-140L N A29L-A30L 142-143 140L-141L N
TABLE-US-00003 TABLE 2 Conserved genes with "end to end"
orientation ##STR00001## Gray highlighted genes have no
overlappping ends and thus are simplest to use as intergenic
sites.
[0052] According to the present invention, heterologous DNA
sequences can be inserted into one or more IGRs in between two
adjacent ORFs selected from the group consisting of (using the
nomenclature according to CDC/Acambis):
[0053] 044-045, 049-050, 050-051, 058-059, 064-065, 065-066,
069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076,
076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090,
092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103,
104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123,
123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133,
133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary
manner or corresponding thereto in other strains of vaccinia
virus.
[0054] In a preferred embodiment, the heterologous sequence is
inserted into an IGR flanked by two adjacent ORFs with "end to end"
orientation selected from the group consisting of 049-050, 071-072,
074-075, 078-079, 092-093, 100-101, 118-119, 121-122, and
132-133.
[0055] In a working example, the heterologous DNA sequence is
inserted into an IGR in which the conserved genes have no
overlapping ends 071-072.
[0056] Heterologous or exogenous DNA sequences are sequences which,
in nature, are not normally found associated with the poxvirus as
used according to the present invention. According to a further
embodiment of the present invention, the exogenous DNA sequence
comprises at least one coding sequence. The coding sequence is
operatively linked to a transcription control element, preferably
to a poxviral transcription control element. Additionally, also
combinations between poxviral transcription control element and,
e.g., internal ribosomal entry sites can be used.
[0057] According to a further embodiment, the exogenous DNA
sequence can also comprise two or more coding sequences linked to
one or several transcription control elements. Preferably, the
coding sequence encodes one or more proteins, polypeptides,
peptides, foreign antigens or antigenic epitopes, especially those
of therapeutically interesting genes.
[0058] Therapeutically interesting genes according to the present
invention may be genes derived from or homologous to genes of
pathogenous or infectious microorganisms which are disease causing.
Accordingly, in the context of the present invention such
therapeutically interesting genes are presented to the immune
system of an organism in order to affect, preferably induce a
specific immune response and, thereby, vaccinate or
prophylactically protect the organism against an infection with the
microorganism. In further preferred embodiments of the present
invention the therapeutically interesting genes are selected from
genes of infectious viruses, e.g., --but not limited to--dengue
virus, hepatitis virus B or C, or human immunodeficiency viruses
such as HIV.
[0059] According to a preferred embodiment of the present invention
the heterologous DNA sequence is derived from HIV and encodes HIV
env, wherein the HIV env gene is preferably inserted into the IGR
between the ORFs 071-072 (I8R-G1L).
[0060] Furthermore, therapeutically interesting genes according to
the present invention also comprise disease related genes, which
have a therapeutic effect on proliferative disorder, cancer or
metabolic diseases. For example, a therapeutically interesting gene
regarding cancer could be a cancer antigen that has the capacity to
induce a specific anti-cancer immune reaction.
[0061] According to a further embodiment of the present invention,
the coding sequence comprises at least one marker or selection
gene.
[0062] Selection genes transduce a particular resistance to a cell,
whereby a certain selection method becomes possible. The skilled
practitioner is familiar with a variety of selection genes, which
can be used in a poxviral system. Among these are, e.g., neomycin
resistance gene (NPT) or phosphoribosyl transferase gene (gpt).
[0063] Marker genes induce a color reaction in transduced cells,
which can be used to identify transduced cells. The skilled
practitioner is familiar with a variety of marker genes, which can
be used in a poxviral system. Among these are the gene encoding,
e.g., .beta.-galactosidase (.beta.-gal), .beta.-glucosidase
(.beta.-glu), green fluorescence protein (EGFP) or blue
fluorescence protein.
[0064] According to still a further embodiment of the present
invention the exogenous DNA sequence comprises a spacing sequence,
which separates poxyviral transcription control element and/or
coding sequence in the exogenous DNA sequence from the stop codon
and/or the start codon of the adjacent ORFs. This spacer sequence
between the stop/start codon of the adjacent ORF and the inserted
coding sequence in the exogenous DNA has the advantage to stabilize
the inserted exogenous DNA and, thus, any resulting recombinant
virus. The size of the spacer sequence is variable as long as the
sequence is without its own coding or regulatory function.
[0065] According to a further embodiment, the spacer sequence
separating the poxyviral transcription control element and/or the
coding sequence in the exogenous DNA sequence from the stop codon
of the adjacent ORF is at least one nucleotide long.
[0066] According to another embodiment of the present invention,
the spacing sequence separating the poxyviral transcription control
element and/or the coding sequence in the exogenous DNA sequence
from the start codon of the adjacent ORF is at least 30
nucleotides. Particularly, in cases where a typical vaccinia virus
promoter element is identified upstream of a start codon the
insertion of exogenous DNA may not separate the promoter element
from the start codon of the adjacent ORF. A typical vaccinia
promoter element can be identified by scanning for e.g., the
sequence "TAAAT" for late promoters (Davison & Moss 1989 J.
Mol. Biol.; 210:771-784) and an A/T rich domain for early
promoters. A spacing sequence of about 30 nucleotides is the
preferred distance to secure that a poxyviral promoter located
upstream of the start codon of the ORF is not influenced.
Additionally, according to a further preferred embodiment, the
distance between the inserted exogenous DNA and the start codon of
the adjacent ORF is around 50 nucleotides and more preferably
around 100 nucleotides.
[0067] According to a further preferred embodiment of the present
invention, the spacing sequence comprises an additional poxyviral
transcription control element which is capable to control the
transcription of the adjacent ORF.
[0068] A typical MVA strain which can be used according to the
present invention for generating a recombinant MVA is MVA 1974/NIH
Clone 1 that has been deposited as ATCC Accession No.: PTA-5095 on
Mar. 27, 2003 with the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, Va. 20110-2209, USA.
[0069] The term "derivatives" of a virus according to the present
invention refers to progeny viruses showing the same characteristic
features as the parent virus but showing differences in one or more
parts of its genome. The term "derivative of MVA" describes a
virus, which has the same functional characteristics compared to
MVA. For example, a derivative of MVA 1974/NIH Clone 1 has the
characteristic features of MVA 1974/NIH Clone 1. One of these
characteristics of MVA 1974/NIH Clone for derivatives thereof is
its attenuation and severe restriction in host range.
[0070] The recombinant MVA according to the present invention is
useful as a medicament or vaccine. It is, according to a further
embodiment, used for the introduction of the exogenous coding
sequence into a target cell, said sequence being either homologous
or heterologous to the genome of the target cell.
[0071] The introduction of an exogenous coding sequence into a
target cell may be done in vitro to produce proteins, polypeptides,
peptides, antigens or antigenic epitopes. This method comprises the
infection of a host cell with the recombinant MVA according to the
invention, cultivation of the infected host cell under suitable
conditions, and isolation and/or enrichment of the polypeptide,
peptide, protein, antigen, epitope and/or virus produced by said
host cell.
[0072] Furthermore, the method for introduction of one or more
homologous or one or more heterologous sequence into cells may be
applied for in vitro and in vivo therapy. For in vitro therapy,
isolated cells that have been previously (ex vivo) infected with
the recombinant MVA according to the invention are administered to
the living animal body for affecting, preferably inducing an immune
response. For in vivo therapy, the recombinant poxvirus according
to the invention is directly administered to the living animal body
for affecting, preferably inducing an immune response. In this
case, the cells surrounding the site of inoculation, but also cells
where the virus is transported to via, e.g., the blood stream, are
directly infected in vivo by the recombinant MVA according to the
invention. After infection, these cells synthesize the proteins,
peptides or antigenic epitopes of the therapeutic genes, which are
encoded by the exogenous coding sequences and, subsequently,
present them or parts thereof on the cellular surface. Specialized
cells of the immune system recognize the presentation of such
heterologous proteins, peptides or epitopes and launch a specific
immune response.
[0073] Since the MVA is highly growth restricted and, thus, highly
attenuated, it is useful for the treatment of a wide range of
mammals including humans, including immune-compromised animals or
humans. The present invention also provides pharmaceutical
compositions and vaccines for inducing an immune response in a
living animal body, including a human.
[0074] The pharmaceutical composition may generally include one or
more pharmaceutical acceptable and/or approved carriers, additives,
antibiotics, preservatives, adjuvants, diluents and/or stabilizers.
Such auxiliary substances can be water, saline, glycerol, ethanol,
wetting or emulsifying agents, pH buffering substances, or the
like. Suitable carriers are typically large, slowly metabolized
molecules such as proteins, polysaccharides, polylactic acids,
polyglycollic acids, polymeric amino acids, amino acid copolymers,
lipid aggregates, or the like.
[0075] For the preparation of vaccines, the recombinant poxvirus
according to the invention is converted into a physiologically
acceptable form. This can be done based on the experience in the
preparation of poxvirus vaccines used for vaccination against
smallpox (as described by Stickl, H. et al. 1974 Dtsch Med.
Wochenschr. 99:2386-2392). For example, the purified virus is
stored at -80.degree. C. with a titer of 5.times.10E8
TCID.sub.50/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.4.
For the preparation of vaccine shots, e.g., 10E2-10E8 particles of
the virus are lyophilized in 100 ml of phosphate-buffered saline
(PBS) in the presence of 2% peptone and 1% human albumin in an
ampoule, preferably a glass ampoule. Alternatively, the vaccine
shots can be produced by stepwise freeze-drying of the virus in a
formulation. This formulation can contain additional additives such
as mannitol, dextran, sugar, glycine, lactose or
polyvinylpyrrolidone or other aids such as antioxidants or inert
gas, stabilizers or recombinant proteins (e.g., human serum
albumin) suitable for in vivo administration. The glass ampoule is
then sealed and can be stored between 4.degree. C. and room
temperature for several months. However, as long as no need exists
the ampoule is stored preferably at temperatures below -20.degree.
C.
[0076] For vaccination or therapy the lyophilisate can be dissolved
in 0.1 to 0.5 ml of an aqueous solution, preferably physiological
saline or Tris buffer, and administered either systemically or
locally, i.e., parenterally, subcutaneous, intramuscularly, by
scarification or any other path of administration know to the
skilled practitioner. The mode of administration, the dose and the
number of administrations can be optimized by those skilled in the
art in a known manner. However, most commonly a patient is
vaccinated with a second shot about one month to six weeks after
the first vaccination shot.
[0077] The present invention further relates to plasmid vectors,
which can be used to generate recombinant MVA according to the
present invention, and also relates to certain DNA sequences.
[0078] Regularly, the IGR located between or flanked by two
adjacent ORFs comprises nucleotide sequences in which the exogenous
DNA sequence of interest can be inserted. Accordingly, the plasmid
vector according to the present invention comprises a DNA sequence
derived from or homologous to the genome of MVA, wherein said DNA
sequence comprises a complete or partial fragment of an IGR
sequence located between or flanked by two adjacent ORFs of the
viral genome. Preferably, the plasmid vector comprises inserted
into said IGR-derived sequence at least one cloning site for the
insertion of an exogenous DNA sequence of interest and, preferably,
for the insertion of a poxviral transcription control element
operatively linked to said heterologous DNA sequence. Optionally,
the plasmid vector comprises a reporter- and/or selection gene
cassette. The plasmid vector preferably also comprises sequences of
the two adjacent ORFs flanking said complete or partial fragment of
the IGR sequence.
[0079] Some IGRs have been identified which do not include
nucleotide sequences. In these cases, the plasmid vector comprises
DNA sequences of the IGR flanking sequences, i.e., DNA sequences of
the two adjacent ORFs. Preferably, the cloning site for the
insertion of the heterologous DNA sequence is inserted into the
IGR. The DNA of the IGR flanking sequences is used to direct the
insertion of exogenous DNA sequences into the corresponding IGR in
the MVA genome. Such a plasmid vector may additionally include a
complete or partial fragment of an IGR sequence which comprises the
cloning site for the insertion of the heterologous DNA sequence
and, optionally, of the, reporter- and/or selection gene
cassette.
[0080] IGR-DNA sequences as well as IGR flanking sequences of the
two adjacent ORFs are preferably selected from IGRs and ORFs,
respectively, selected from the group consisting of (using the
nomenclature according to CDC/Acambis):
[0081] 044-045, 049-050, 050-051, 058-059, 064-065, 065-066,
069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076,
076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090,
092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103,
104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123,
123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133,
133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary
manner or corresponding thereto in other strains of vaccinia
virus.
[0082] The sequences are, more preferably, selected from IGRs and
ORFs, respectively, selected from the group consisting of 049-050,
071-072, 074-075, 078-079, 092-093, 100-101, 118-119, 121-122, and
132-133.
[0083] In a working example, the IGR derived sequence is selected
as 071-072.
[0084] The DNA sequences are preferably derived from or homologous
to the genome of the MVA deposited as ATCC Accession No.: PTA-5095
on Mar. 27, 2003 with the American Type Culture Collection (ATCC),
10801 University Blvd., Manassas, Va. 20110-2209, USA.
[0085] To generate a plasmid vector according to the present
invention the sequences are isolated and cloned into a standard
cloning vector, such as pBluescript (Stratagene), wherein they
flank the exogenous DNA to be inserted into the MVA genome.
Optionally, such a plasmid vector comprises a selection- or
reporter gene cassette, which can be deleted from the final
recombinant virus, due to a repetitive sequence included into said
cassette.
[0086] Methods to introduce exogenous DNA sequences by a plasmid
vector into an MVA genome and methods to obtain recombinant MVA are
well known to the person skilled in the art and, additionally, can
be deduced can be deduced from Molecular Cloning, A Laboratory
Manual, Second Edition, J. Sambrook, E. F. Fritsch and T. Maniatis,
Cold Spring Harbor Laboratory Press, 1989 and Current Protocols in
Molecular Biology, John Wiley and Son Inc. 1998, Chapter 16,
section IV, "Expression of proteins in mammalian cells using
vaccinia viral vectors".
[0087] The MVA according to the present invention may be produced
by transfecting a cell with a plasmid vector according to the
present invention, infecting the transfected cell with an MVA and,
subsequently, identifying, isolating and, optionally, purifying the
MVA according to the invention.
[0088] The DNA sequences according to the invention can be used to
identify or isolate the MVA or its derivatives according to the
invention and cells or individuals infected with an MVA according
to the present invention. The DNA sequences are, e.g., used to
generate PCR-primers, hybridization probes or are used in array
technologies.
HIVs and their Replication
[0089] 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
SW.sub.cpz, are close genetic relatives of HIV-1.
[0090] 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-1 strains 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.
[0091] 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.
[0092] 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.
[0093] 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 contains 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 clade 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.
[0094] 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.
[0095] 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 Nee are the primary translation products of
spliced mRNAs.
Gag
[0096] 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).
[0097] 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.
Pol
[0098] 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-Pal. 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.
Protease
[0099] 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.
Reverse Transcriptase
[0100] 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).
[0101] 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.
[0102] 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.
Integrase
[0103] 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.
[0104] 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).
[0105] 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.
Env
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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 SW 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.
[0110] 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 SW 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 SW 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.
[0111] 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.
Use of Recombinant MVA Virus to Boost CD+8 Cell Immune Response
[0112] 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 prior 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.
[0113] 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.
[0114] The present invention employs MVA which, as prior
experiments 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.
[0115] Notably, prior experimental work demonstrates that use of
predecessors 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 may be found to induce a CD8.sup.+T cell response
after immunization. Recombinant MVA may also be shown to prime an
immune response that is boosted by one or more inoculations of
recombinant MVA.
[0116] Non-human primates immunized with plasmid DNA and boosted
with the MVA were effectively protected against intramucosal
challenge with live virus (Amara et al 2001 Science 292:69-74).
Advantageously, the inventors contemplate that a vaccination regime
using 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] An immune response to an HIV antigen may be primed by
immunization with plasmid DNA or by infection with an infectious
agent.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] Percent identity 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.
[0132] 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.
[0133] 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 on the world wide web at
hiv-web.lanl.gov/.
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.
[0134] 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 HN 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.
[0135] 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 HN Env, Gag, or Pol,
based on the teaching and guidance presented herein.
[0136] 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.
[0137] 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.
[0138] Within the MVA vector, regulatory sequences for expression
of the encoded antigen will include a 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). Poxyiridae: 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.
[0139] Promoters for use in aspects and embodiments of the present
invention may be compatible with poxvirus expression systems and
include natural, modified and synthetic sequences.
[0140] 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.
[0141] Administration of the boosting composition is generally
about 1 to 6 months after administration of the priming
composition, preferably about 1 to 3 months.
[0142] Preferably, administration of priming composition, boosting
composition, or both priming and boosting compositions, is
intradermal, intramuscular or mucosal immunization.
[0143] 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.
[0144] 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 ADDS which can be controlled by a CD8.sup.+T cell
response.
[0145] 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.
[0146] Most likely, administration will have prophylactic aim to
generate an immune response against HIV or AIDS before infection or
development of symptoms.
[0147] 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.
[0148] As noted, administration is preferably intradermal,
intramuscular or mucosal.
[0149] Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0150] 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.
[0151] A slow-release formulation may be employed.
[0152] 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.
[0153] 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.).
[0154] In one preferred regimen, DNA is administered at a dose of
300 .mu.g to 3 mg/injection, followed by MVA at a dose of 10.sup.6
to 10.sup.9 infectious virus particles/injection.
[0155] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0156] 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.
A Shuttle Plasmid, Recombinant MVA/HIV1 Clinical Vaccine Construct
and Mechanism for Retention of Intact Foreign Gene Inserts in
Recombinant MVA by Codon Alteration of the Foreign Gene and
Insertion of the Foreign Gene Between Two Vaccinia Virus Essential
Genes
[0157] The invention provides mechanisms for: [0158] retention of
intact foreign genes by inserting them between two vaccinia virus
genes that are essential for MVA replication. Deletion of the
foreign gene can provide a significant growth advantage for the
recombinant MVA allowing it to compete with MVA containing the
intact foreign gene upon repeated passage. However, most deletions
of a foreign gene include loss of some part of the flanking
vaccinia virus DNA. If that vaccinia virus DNA is essential, then
those viruses with deletions will not replicate and compete with
the MVA containing the intact foreign gene. This methodology will
be useful in production of recombinant vaccinia viruses that must
be amplified to large scale such as for use in clinical trials, and
[0159] stabilizing foreign gene inserts by alteration of specific
"hot spots" that otherwise readily undergo mutation after repeated
passage of the recombinant virus. This methodology is useful in
production of recombinant viruses that must be amplified to large
scale such as for use in clinical trials.
[0160] And describes: [0161] the shuttle plasmid, pLW-73, used for
insertion of a foreign gene between 2 essential vaccinia virus
genes; and [0162] the recombinant MVA/HIV-1 clinical vaccine
construct MVA/UGD4d, a material that embodies use of these two
mechanisms.
[0163] Novel Methods for Generation of Stable Recombinant MVA
Viruses
[0164] The inventors have made modified vaccinia virus Ankara (MVA)
recombinants expressing env and gagpol genes from HIV-1 isolates
from different geographical locations. The foreign genes were
inserted into 2 sites, Deletion II and Deletion III of MVA. The
stability of these genes after repeated passage of recombinant MVA
in tissue culture has proven to be variable. The inventors
demonstrated that the instability was due to either deletion of the
entire foreign gene and some flanking DNA or specific point
mutations resulting in propagation of progeny virions that have a
growth advantage because they do not express the foreign gene. Here
the inventors describe two novel methods of retaining the intact
foreign gene recombinant MVA. First, the inventors constructed a
transfer vector that directs insertion of a foreign gene between
two essential vaccinia virus genes in the conserved central region
of the genome. Use of this site for insertion of genes prevents the
outgrowth of variants containing large deletions that include the
essential vaccinia virus DNA. In addition, this plasmid can be used
for insertion of additional genes into recombinant viruses. Second,
analysis of isolates with point mutations revealed certain "hot
spots" with a propensity for insertion or deletion of a single base
that causes premature termination during translation. The inventors
showed that generation of silent mutations in these sites resulted
in stabilization of the inserted gene.
I. Novel Transfer Vector Construction and Application
[0165] Construction of Novel Transfer Vector, pLW-73
[0166] 1. The central region of the MVA genome, K7R-A24R, was
examined for 1) pairs of genes conserved in the poxvirus family or
chordopoxvirus subfamily and 2) genes that are in opposite
orientation such that their 3' ends are in close proximity, thereby
providing an insertion site that would not disrupt a vaccina
promoter. The site chosen as the new insertion site was between two
essential genes, I8R and G1L.
[0167] 2. The left flank of the new vector was constructed in the
following way: Plasmid LAS-1 was cut with restriction enzymes EcoRI
and XhoI to remove the del III MVA flank, GFP, and direct repeat of
MVA flank. This insert was cut with AscI and SacI and the GFP
fragment was isolated. Five hundred thirty one base pairs at the
end of the I8R gene (including the TAA stop codon) was PCR
amplified with EcoRI and AscI restriction sites on the ends of the
PCR product. PCR amplification of 229 base pairs of the direct
repeat (from the end of the I8R gene including the TAA stop codon)
was performed with oligonucleotides containing Sad and XhoI
restriction sites. All four pieces of DNA, 1) the vector backbone
with EcoRI and Xho I ends, 2) new left flank containing end of I8R
with EcoRI and AscI ends, 3) GFP with AcsI and Sad ends and the 4)
direct repeat of the I8R flank with Sad and XhoI ends were ligated
together to make plasmid pLW-72.
[0168] 3. The right flank was made as follows: pLW-72 was cut with
restriction enzymes PstI and HindIII to release del III flank of
the MVA in the plasmid. Seven hundred and two base pairs at the end
of the GIL gene was PCR amplified with PstI and HindIII restriction
enzyme sites on the ends and ligated into the pLW-72 vector to make
pLW-73 (FIG. 7). The sequence of pLW-73 is given in FIG. 8.
[0169] 4. The salient features of pLW-73 are: 1) the vector was
designed for insertion of foreign genes between essential genes in
MVA genome. The left flank consists of end of I8R gene and right
flank consists of end of G1L gene. 2) the GFP gene is included for
easy initial selection of recombinant virus 3) the GFP is flanked
by direct repeats of the I8R gene which allows for transient
expression of GFP as the GFP will be lost upon repeated passage of
the recombinant virus. Referring to WO 2004/087201, features 2 and
3 were also contained in earlier plasmids used for making MVA/HIV
recombinants, pLAS-1 and pLAS-2.
Application of pLW-73
[0170] 1. The env gene from the Glade B ADA isolate of HIV-1 was
cloned into pLW-73 and a recombinant MVA virus was made. DNA
sequencing confirmed the location and integrity of the env
gene.
[0171] 2. A recombinant MVA virus expressing the Ugandan Glade D
(isolate AO7412) env gene (FIG. 9) in the Deletion II site of MVA
proved to be unstable, i.e., after repeated serial passage in
culture, the gene was deleted from a significant portion of the
virus progeny. The same gene was then cloned into pLW-73 and a
recombinant MVA virus was made and characterized. The env gene
insert was stable after repeated serial passage (8.times.) in
culture i.e., no deletions of the inserted gene or the MVA flanking
region were found. In addition, no other mutations arose when the
gene was inserted into this site.
II. Point Mutation of "Hot Spots"
[0172] Analysis of Point Mutations
[0173] A recombinant MVA virus expressing the Ugandan Clade D
(isolate AO3349) gagpol gene in the Deletion III site of MVA proved
to be unstable. The major genetic alteration was the generation of
single point mutations in runs of 4-6 G or C residues (Table 3). In
addition, similar point mutations were found in non-staining
plaques from similar recombinant viruses expressing the gagpol
genes from a Kenyan Glade A isolate and a Tanzanian Glade C isolate
of HIV-1.
Mutagenesis of Hot Spots and Analysis of Stability in Recombinant
Virus
[0174] Using site-directed mutagenesis, silent mutations were made
in 6 such regions of the gag gene from the Ugandan HIV-1 isolate.
This altered gene, UGD 4d gagpol orf (FIG. 10), was cloned into
pLAS-1 and recombined into the same Deletion III site of MVA as was
done in construction of the unstable virus. After repeated serial
passage (8.times.) in culture, no non-expressing plaques were
found. DNA sequencing of the passage 8 virus stock verified that
the integrity of the gagpol gene was maintained.
III. Double Recombinant Construction
[0175] MVA/UGD4d Virus
[0176] MVA/UGD4d virus, a recombinant virus that expresses the
Ugandan subtype D AO7412 envelope and the AO3349 gagpol, was
constructed in the following way: The envelope and gagpol genes
were inserted into MVA 1974/NIH Clone 1 by homologous recombination
utilizing shuttle plasmids pLW-73 and pLAS-1, respectively.
MVA/UGD4d was isolated by 6 rounds of plaque purification in
chicken embryo fibroblast cells and subsequently amplified and
characterized.
SUMMARY
[0177] 1. A plasmid transfer vector was constructed that directs
recombination of a foreign gene between two essential genes, I8R
and G1L, in the conserved central region of the MVA genome. The use
of this site was shown to inhibit selection of mutant viruses with
deletions of inserted gene/MVA flanks.
[0178] 2. Highly mutable runs of G and C residues were altered by
site-directed mutagenesis and silent mutations in the coding
sequence were generated. This change was shown to stabilize the
gene when inserted into Deletion III of MVA.
[0179] 3. Utilizing these two methods above, UGD4d double MVA
recombinant that stably expresses both the env and gagpol of
Ugandan Clade D was constructed.
Example 1
[0180] Recombinant MVAs expressing HIV-1 env and gagpol genes from
many different isolates have been made. The stability of inserted
genes after repeated passage in tissue culture has proven to be
variable. Here the inventors (1) demonstrate that the instability
represents a combination of spontaneous mutation or deletion of the
inserted gene and selection for non-expressing mutants and (2)
describe novel methods for reducing instability.
Overview
[0181] Recombinant MVAs expressing env and gagpol from many
different isolates were constructed. Each virus was subjected to
repeated passages in chicken embryo fibroblast cells to mimic the
large-scale amplification required for production of virus for
clinical trials. Insert stability was monitored by env and gag
immunostaining of individual plaques. For some recombinant viruses,
env and/or gag expression was found to be rapidly lost in a
significant fraction of the virus population. To identify the
mechanism(s) of loss of expression, individual plaques were
isolated and the nature of the mutations was characterized. In some
cases, specific DNA sequences with propensity to mutate by addition
or deletion of a single nucleotide were identified. Generation of
such mutations could be avoided by altering codons without changing
the predicted translation product. In other cases, loss of
expression was caused by large deletions that frequently extended
into flanking non-essential MVA genes. To prevent this from
occurring, a new shuttle plasmid was constructed that was designed
to direct insertion of foreign genes between two essential MVA
genes. Recombination into this site reduced deletions of the
foreign DNA. In one case, however, the toxicity associated with
high-level HIV env expression was so severe that the selection of
rare mutants still resulted in an unstable population. In this
case, only truncation of the transmembrane domain of env allowed
the construction of a stable recombinant MVA.
Generation of Recombinant MVAs and Analysis of Stability of
Inserted Genes
[0182] Env and gagpol genes were cloned into MVA shuttle vectors.
Expression and function were analyzed by transient expression
assays. Gagpol was recombined into MVA 1974/NIH Clone 1.
Recombinant MVA were plaque purified with 6-8 rounds followed by
amplification of virus. Env was recombined into the MVA/gagpol
isolate and double-recombinant MVA (FIG. 11A) were plaque purified
with 6-8 rounds and were amplified. To assess the stability of
inserts, virus was serially passaged in CEF cells using a
multiplicity of infection (m.o.i.) of .about.1 pfu/cell to mimic
large-scale production. Stability was evaluated by determining the
percentage of cells expressing env or gag, as determined by
immunostaining with monoclonal antibodies (FIG. 11B).
Stability of Recombinant MVAs
[0183] Recombinant MVAs expressing genes from HIV-1 isolates from
different geographical locations were constructed. The env and
gagpol genes were inserted into deletions II and III of MVA,
respectively; both under control of the modified H5 promoter. The
stability of env and gagpol genes from seven recombinant MVAs is
shown in Table 4. Varying degrees of instability were observed in
the seven viruses. In MVA/65A/G, expression of env was rapidly lost
with only 25% of virions expressing env by passage 6. In MVA/UGD4a,
both env and gagpol expression were increasingly lost with
successive virus passages. Since at least 6-7 passages are required
for production of a lot of virus for a Phase I trial, these two
viruses were deemed unsuitable.
Analysis of Expression of MVA/65A/G
[0184] Referring to FIG. 12, thirteen plaques were randomly picked
from P3 and P5 of MVA/65A/G and analyzed by immunostaining with
T-24 mAb (binding site shown on a), Western blotting, PCR, and
sequencing. Five types of plaques were found and the number of
these plaques obtained for each type are given at right of FIG. 12.
Plaques a, b, and c stained, but b and c were truncated versions
due to base substitution (causing stop codon) (b) and deletion of
the end of the env gene and part of MVA flank (c). Nonstaining
plaques d and e resulted from addition of G to a 5G run causing a
frameshift (d) and large deletion of entire env gene and parts of
MVA flanks (e). Thus, base pair addition, substitution, and
deletions all contributed to unstable expression of the env gene in
MVA/65A/G. This A/G env, the most unstable example worked with, was
picked to study modifications that might enhance stability.
Modifications to A/G Constructs to Increase Stability
[0185] 1. Synthetic envelope was made by removing 4 and 5 G and C
runs by silent mutations to prevent point mutations.
[0186] 2. Vector I8/G1, i.e., pLW-73. was constructed with an
insertion site between essential genes I8R and G1L to prevent
deletions of genes and MVA flanks from being viable. The ends of
the I8R (500 bp) and G1L (750 bp) genes of MVA were amplified by
PCR and inserted into a vector containing vaccinia virus early/late
mH5 promoter controlling foreign gene expression. This I8/G1 vector
was used to insert foreign genes into MVA by homologous
recombination (FIG. 13). Deletions of inserted genes and MVA
flanking the inserted gene would not be viable because parts of
essential genes would be deleted. Therefore, viruses with these
mutations would not be able to overgrow the population with their
normal growth advantage.
[0187] 3. A/G gp140 envelope was mutated by deleting the
transmembrane domain and the cytoplasmic tail of gp41, resulting in
a secreted protein.
Testing Modifications to Increase Stability
[0188] Seven single recombinant viruses were made with env
modifications and/or use of new vector as shown in FIG. 14. Five
plaques of each virus were isolated and passaged independently in
CEF to determine if modifications enhanced envelope stable
expression. Passaged plaques were analyzed by immunostaining with
mAb T-43 (binding site mapped to 101-125aa of env), Western
blotting, PCR, and sequencing.
Env Expression after Plaque Passages
[0189] Referring to FIG. 15, five independently passaged plaque
isolates of each of the 7 recombinants listed above, were
characterized at passages 1, 3, 5, and 7 by immunostaining with mAb
T-43 (binds between 101-125a.a. in gp120). Four of 7 viruses (FIG.
15, a, b, c, e) had unstable protein expression in each of the 5
passaged plaques; two plaque passages of (FIG. 15f) also had
unstable env expression. These included viruses with the synthetic
env in both del II (FIG. 15c) and in the essential gene site (FIG.
15f) of MVA genome. Only recombinant viruses containing the
envelope as truncated, secreted gp140 remained stably expressing
envelope (FIG. 15, d and g).
Western Blotting, PCR and Sequence Analyses
[0190] From selected plaque passages, clones were picked to analyze
protein expression by Western blotting, PCR, and sequence analysis
(FIG. 16). For Western blot analysis, T-24 and T-32 binding at the
beginning and end of the clade A envelope, respectively, were used
in order to determine if only partial or full length envelope was
being made. Control viruses, marked c, are at the right of each
blot. For the three viruses made in deletion II of MVA (FIG. 16a,
b, and c), only in FIG. 16c (i.e., gp140 clones), were all the
clones expressing detectable protein in Western. This protein (as
measured by T-32) was not truncated. When envelope was inserted
into the essential gene site by vector I8/G1 (FIG. 16d, e and f),
again, only the gp140 envelope was being expressed in all clones
and was not truncated. Although use of I8/G1 vector did not prevent
mutations to the env sequence, it did prevent deletions which had
been seen in envelope inserted into del II. (Note positive PCR
products from all clones tested from I8/G1 vector, but negative PCR
products from clones tested using del II vector.)
Expression of Env in Clade A/G Double Recombinant
[0191] Based on previous results with single env analysis, double
recombinants expressing gagpol with either gp140 or the synthetic
gp160 gene were made and tested for stability of env expression
(FIG. 17). Five plaques were isolated from each as previously
described, and passaged 7 times to analyze stability of env
expression. At passage 7, the passaged plaques were immunostained
with both T-43 and T-32 mAbs (which bind to gp120 and gp41,
respectively). With T-43 mAb, one of five clones of recombinant
expressing synthetic envelope consisted of only non-staining
plaques. Subsequent T-32 staining of these plaques showed another
plaque had truncated envelope expression. All passaged plaques from
double recombinant containing gp140 envelope appeared stable by
both T-43 and T-32 immunostaining. Titers were also 2 logs higher
than with the other double recombinant. Thus a clade A/G double
recombinant stably expressing envelope could only be made with
gp140 envelope.
Recombinant Viruses Expressing env and gagpol from Ugandan HIV-1
Isolates
[0192] Recombinant MVA viruses expressing HIV-1 env and gagpol
genes from Ugandan isolates AO7412 and AO3349 were constructed as
shown in FIG. 18. Four to six independent isolates of each were
serially passaged and both genes were found to be unstable whether
expressed alone or in combination (Table 5). In contrast,
expression of gp140 instead of membrane bound gp160 resulted in
stability of the env gene after serial passage (FIG. 18 and Table
5).
MVA/UGD4a --Analysis of Non-Staining env Plaques
[0193] To determine the mechanism of instability, 24 individual
non-staining plaques (using Mab T-43) were isolated from passage 6
of MVA/UGD4a, amplified, and characterized. Two small deletions
(1.2 and 0.3 kb) were identified by PCR amplification and DNA
sequencing (FIG. 19). All other isolates contained very large
deletions that extended into the flanking MVA. The approximate
break-points for these deletions were identified using primer pairs
from within the env gene or flanking MVA regions.
Modification of UGD env Gene in Recombinant MVA
[0194] To ameliorate the problem of instability of the UGD env
gene, the AO7412 env gene was inserted into MVA using the new
vector, I8/G1, which directs recombination of a foreign gene
between 2 essential vaccinia virus genes, I8 and G1 and uses the
modified H5 promoter (FIG. 20). Four independent plaques were
serially passaged and analyzed for env expression by immunostaining
with Mabs T-43 and T-32 at passage 5. In all isolates, the gene was
stable (Table 6).
MVA/UGD4b--Analysis of Non-Staining gag Plaques
[0195] To determine the mechanism of instability of the gag gene, 8
individual non-staining plaques (using Mab 183-H12-5C--NIAID AIDS
Repository) were picked from passage 6 of MVA/UGD4b, amplified, and
the gagpol insert was sequenced (Table 7). In 7 isolates, an
insertion or deletion of a single G residue at position 564-569 was
found. In one isolate, a C residue was deleted from the sequence
CCCC at position 530-534. Furthermore, non-staining plaques from
high-passage stocks of MVA/KEA and MVA/TZC revealed a similar
hot-spot for mutation, i.e., position 564-569. Examination of the
full sequence of the UGD A07412 gagpol gene demonstrated 22 runs of
4 or more G or C residues (FIG. 21).
Modification of UGD gagpol Gene in Recombinant MVA
[0196] Since the mechanism of instability of the gagpol gene was
primarily insertion or deletion of a single nucleotide within a run
of 4-6 G or C residues, the strategy to improve the stability of
this gene was to generate silent mutations at such sites. Thus,
site-directed mutagenesis at 6 sites in p17 and p24 gag (Table 3)
was employed. The resulting codon altered (c.a.) gene inserted into
MVA at the same location, i.e., Deletion III, proved to be stable
upon serial passage (FIG. 22 and Table 8).
Construction of Stable, Recombinant MVA Expressing UGD env and
gagpol
[0197] A recombinant virus expressing the UGD env gene in the I8/G1
locus and the codon altered gagpol gene in Deletion III of MVA was
constructed (FIG. 23). Serial passage demonstrated no instability
of either gene. Furthermore, the level of protein expression and
DNA sequence were unaltered during passage (Table 9).
Conclusions
[0198] Instability of env and gagpol inserts is attributed to the
generation of point mutations and deletions and the growth
advantage of non-expressing MVA mutants. Instability can generally
be reduced by codon alteration and/or insertion into an essential
region of the MVA genome (MVA/UGD4d) but env had to be altered in
one case (MVA/65A/G).
Example 2
Immunogenicity of MVA/UGD4d in BALB/c Mice
[0199] Groups of 10 mice each were immunized by the intraperitoneal
route with either 10.sup.6 or 10.sup.7 infectious units of
MVA/UGD4d. Groups of 5 mice each were similarly immunized with
parental MVA-1974. Mice were immunized at weeks 0 and 3 and bled at
weeks 0, 3, and 5. Spleens were harvested at week 5.
[0200] Cellular responses were measured in fresh splenocytes by
intracellular cytokine staining. Splenocytes were separately
stimulated with the following: 1) immunodominant gag peptide
(AMQMLKETI (SEQ ID NO: 6)), 2) env peptides (DTEVHNVWATHACVP (SEQ
ID NO: 7) and QQQSNLLRAIEAQQH (SEQ ID NO: 8)), 3) pol peptides (8
peptides with single amino acid variants of ELRQHLLRWGLTT (SEQ ID
NO: 9) and HGVYYDPSKDLIAE (SEQ ID NO: 10)), and 4) MVA.
[0201] Cells were stained for surface expression of CD4 and CD8 and
then for intracellular expression of IFN-.gamma. and either IL2 or
TNF. As shown in FIG. 24, MVA/UGD4d elicited CD8/IFN-.gamma.
responses to the gag peptide, pol peptides, and MVA. The gag
peptide responses were multifunctional, expressing both IFN-.gamma.
and either IL2 or TNF. Also, CD4/IFN-.gamma. responses were
elicited to the pool of env peptides.
[0202] Humoral responses were measured by ELISA (FIG. 25). Strong
responses to UGD env were demonstrated at 3 weeks after one
immunization and were boosted by the second immunization. In
addition, strong vaccinia virus responses were elicited after one
and two immunizations.
TABLE-US-00004 TABLE 3 MVA/UGD Nucleotide Changes Made to Eliminate
Runs of G and C (HIV-1 isolate AO3349) Nucleotide # Original
Modified starting with ATG Sequence Sequence 28-32 GGGGG GGAGG
70-74 GGGGG GGAGG 408-411 GGGG GGGA 530-533 CCCC CACC 564-569
GGGGGG AGGAGG 686-689 GGGG GAGG
TABLE-US-00005 TABLE 4 Stability of Recombinant MVAs Percent
non-staining plaques LVD passage passage passage passage
Geographical seed 3/4 6/7 8/9 10-13 vaccine lot Virus Clade origin
env gag env gag env gag env gag env gag env gag KEA5b A Kenya <1
<1 0.13 0.33 0.34 0.36 0.54 2.4 0.64 0.77 65A/G A/G Ivory Coast
<2 <1 28 1 75 62B B US <1 <1 <1 <1 6 <1 10 1
TZCa C Tanzania <1 <1 <1 <1 1.7 2.8 3.6 3.7 71C C India
<1 <1 <1 1 <1 2 12 14 UGD4a D Uganda <1 <1 3 0.28
6.7 6 12.2 17.4 CMDR E/A Thailand <1 <1 <1 <1 <1
<1 <1 <1
TABLE-US-00006 TABLE 5 Recombinant Viruses Expressing env and
gagpol from Ugandan HIV-1 isolates % non-staining passage env gag
UGD4a 9 12.2 17.4 5 5.8 2.6 5 2.7 17.6 5 8.4 7.2 5 11.4 8.0 UGD4b 6
1.5 17.0 5 3.3 9.3 5 3.7 8.3 5 7.9 4.4 5 15.2 5.0 UGD1a 4 nd 18.8 4
nd 46.7 4 nd 64.9 4 nd 38.1 5 7.9 44.8 UGD gag3349 8 36.6 8 25.4 6
22.9 6 33.1 UGD env 8 9.0 8 2.9 8 13.3 8 12.5 8 14.3 UGDgag/gp140 5
1.2 18.9 5 2.3 17.6
TABLE-US-00007 TABLE 6 Modification of UGD env Gene in Recombinant
MVA % non-staining passage env gag UGD9 5 0.5 5 0.4 5 0.0 5 0.5
TABLE-US-00008 TABLE 7 MVA/UGD4b- Analysis of Non-Staining gag
Plaques # individual plagues with mutation gene base # sequence
MVA/UGD MVA/KEA MVA/TZC p17 28 GGGGG 70 GGGGG n = 1 p24 408 GGGG
530 CCCC n = 1 564 GGGGGG n = 7 n = 16 n = 21 686 GGGG 1050 GGGGGG
p7 1133 GGGG p1 1320 GGGG p6 1361 CCCC 1387 GGGG 1419 GGGG 1473
CCCC Protease 1494 GGGGG RT 1590 GGGGG 1599 GGGGG 2362 GGGG 2380
GGGG 2528 GGGGG 2596 GGGG 2893 GGGG 3001 CCCC
TABLE-US-00009 TABLE 8 Modification of UGD gagpol Gene in
Recombinant MVA % non-staining Passage env gag UGD gag (c.a.) 6 0.9
6 0.0 6 0.5
TABLE-US-00010 TABLE 9 Construction of Stable Recombinant MVA
Expressing UGD env and gagpol % non-staining Passage env gag UGD4d
11 0.0 0.7
[0203] 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
figures, tables, and appendices, as well as patents, applications,
and publications, referred to above, are hereby incorporated by
reference.
Sequence CWU 1
1
1014PRTHuman Immunodeficiency Virus Type 1VARIANT2, 3Xaa = Any
Amino Acid 1Tyr Xaa Xaa Leu125044DNAArtificial SequencepLW-73
Plasmid DNA, top strand 2gaattccctg ggacatacgt atatttctat
gatctgtctt atatgaagtc tatacagcga 60atagattcag aatttctaca taattatata
ttgtacgcta ataagtttaa tctaacactc 120cccgaagatt tgtttataat
ccctacaaat ttggatattc tatggcgtac aaaggaatat 180atagactcgt
tcgatattag tacagaaaca tggaataaat tattatccaa ttattatatg
240aagatgatag agtatgctaa actttatgta ctaagtccta ttctcgctga
ggagttggat 300aattttgaga ggacgggaga attaactagt attgtacaag
aagccatttt atctctaaat 360ttacgaatta agattttaaa ttttaaacat
aaagatgatg atacgtatat acacttttgt 420aaaatattat tcggtgtcta
taacggaaca aacgctacta tatattatca tagacctcta 480acgggatata
tgaatatgat ttcagatact atatttgttc ctgtagataa taactaaggc
540gcgcctttca ttttgttttt ttctatgcta taaatggtga gcaagggcga
ggagctgttc 600accggggtgg tgcccatcct ggtcgagctg gacggcgacg
taaacggcca caagttcagc 660gtgtccggcg agggcgaggg cgatgccacc
tacggcaagc tgaccctgaa gttcatctgc 720accaccggca agctgcccgt
gccctggccc accctcgtga ccaccctgac ctacggcgtg 780cagtgcttca
gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
840cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
ctacaagacc 900cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
gcatcgagct gaagggcatc 960gacttcaagg aggacggcaa catcctgggg
cacaagctgg agtacaacta caacagccac 1020aacgtctata tcatggccga
caagcagaag aacggcatca aggtgaactt caagatccgc 1080cacaacatcg
aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
1140ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
cgccctgagc 1200aaagacccca acgagaagcg cgatcacatg gtcctgctgg
agttcgtgac cgccgccggg 1260atcactctcg gcatgcacga gctgtacaag
taagagctcg aggacgggag aattaactag 1320tattgtacaa gaagccattt
tatctctaaa tttacgaatt aagattttaa attttaaaca 1380taaagatgat
gatacgtata tacacttttg taaaatatta ttcggtgtct ataacggaac
1440aaacgctact atatattatc atagacctct aacgggatat atgaatatga
tttcagatac 1500tatatttgtt cctgtagata ataactaact cgaggccgct
ggtacccaac ctaaaaattg 1560aaaataaata caaaggttct tgagggttgt
gttaaattga aagcgagaaa taatcataaa 1620taagcccggg gatcctctag
agtcgacctg cagtcaaact ctaatgacca catctttttt 1680tagagatgaa
aaattttcca catctccttt tgtagacacg actaaacatt ttgcagaaaa
1740aagtttatta gtgtttagat aatcgtatac ttcatcagtg tagatagtaa
atgtgaacag 1800ataaaaggta ttcttgctca atagattggt aaattccata
gaatatatta atcctttctt 1860cttgagatcc cacatcattt caaccagaga
cgttttatcc aatgatttac ctcgtactat 1920accacataca aaactagatt
ttgcagtgac gtcgtatctg gtattcctac caaacaaaat 1980tttactttta
gttcttttag aaaattctaa ggtagaatct ctatttgcca atatgtcatc
2040tatggaatta ccactagcaa aaaatgatag aaatatatat tgatacatcg
cagctggttt 2100tgatctacta tactttaaaa acgaatcaga ttccataatt
gcctgtatat catcagctga 2160aaaactatgt tttacacgta ttccttcggc
atttcttttt aatgatatat cttgtttaga 2220caatgataaa gttatcatgt
ccatgagaga cgcgtctccg tatcgtataa atatttcatt 2280agatgttaga
cgcttcatta ggggtatact tctataaggt ttcttaatca gtccatcatt
2340ggttgcgtca agaacaagct tgtctcccta tagtgagtcg tattagagct
tggcgtaatc 2400atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
acaattccac acaacatacg 2460agccggaagc ataaagtgta aagcctgggg
tgcctaatga gtgagctaac tcacattaat 2520tgcgttgcgc tcactgcccg
ctttcgagtc gggaaacctg tcgtgccagc tgcattaatg 2580aatcggccaa
cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
2640cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
actcaaaggc 2700ggtaatacgg ttatccacag aatcagggga taacgcagga
aagaacatgt gagcaaaagg 2760ccagcaaaag gccaggaacc gtaaaaaggc
cgcgttgctg gcgtttttcg ataggctccg 2820cccccctgac gagcatcaca
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 2880actataaaga
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
2940cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
cgctttctca 3000tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
cgctccaagc tgggctgtgt 3060gcacgaaccc cccgttcagc ccgaccgctg
cgccttatcc ggtaactatc gtcttgagtc 3120caacccggta agacacgact
tatcgccact ggcagcagcc actggtaaca ggattagcag 3180agcgaggtat
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
3240tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
gaaaaagagt 3300tggtagctct tgatccggca aacaaaccac cgctggtagc
ggtggttttt ttgtttgcaa 3360gcagcagatt acgcgcagaa aaaaaggatc
tcaagaagat cctttgatct tttctacggg 3420gtctgacgct cagtggaacg
aaaactcacg ttaagggatt ttggtcatga gattatcaaa 3480aaggatcttc
acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
3540atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
ctatctcagc 3600gatctgtcta tttcgttcat ccatagttgc ctgactcccc
gtcgtgtaga taactacgat 3660acgggagggc ttaccatctg gccccagtgc
tgcaatgata ccgcgagacc cacgctcacc 3720ggctccagat ttatcagcaa
taaaccagcc agccggaagg gccgagcgca gaagtggtcc 3780tgcaacttta
tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
3840ttcgccagtt aatagtttgc gcaacgttgt tggcattgct acaggcatcg
tggtgtcacg 3900ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
cgatcaaggc gagttacatg 3960atcccccatg ttgtgcaaaa aagcggttag
ctccttcggt cctccgatcg ttgtcagaag 4020taagttggcc gcagtgttat
cactcatggt tatggcagca ctgcataatt ctcttactgt 4080catgccatcc
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
4140atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
ataccgcgcc 4200acatagcaga actttaaaag tgctcatcat tggaaaacgt
tcttcggggc gaaaactctc 4260aaggatctta ccgctgttga gatccagttc
gatgtaaccc actcgtgcac ccaactgatc 4320ttcagcatct tttactttca
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 4380cgcaaaaaag
ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
4440atattattga agcatttatc agggttattg tctcatgagc ggatacatat
ttgaatgtat 4500ttagaaaaat aaacaaatag gggttccgcg cacatttccc
cgaaaagtgc cacctgacgt 4560ctaagaaacc attattatca tgacattaac
ctataaaaat aggcgtatca cgaggccctt 4620tcgtctcgcg cgtttcggtg
atgacggtga aaacctctga cacatgcagc tcccggagac 4680ggtcacagct
tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
4740gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
ttgtactgag 4800agtgcaccat atgcggtgtg aaataccgca cagatgcgta
aggagaaaat accgcatcag 4860gcgccattcg ccattcaggc tgcgcaactg
ttgggaaggg cgatcggtgc gggcctcttc 4920gctattacgc cagctggcga
aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 4980agggttttcc
cagtcacgac gttgtaaaac gacggccagt gaattggatt taggtgacac 5040tata
504435044DNAArtificial SequencepLW-73 Plasmid DNA, bottom strand,
5'-3' 3tatagtgtca cctaaatcca attcactggc cgtcgtttta caacgtcgtg
actgggaaaa 60ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
gctggcgtaa 120tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
cgcagcctga atggcgaatg 180gcgcctgatg cggtattttc tccttacgca
tctgtgcggt atttcacacc gcatatggtg 240cactctcagt acaatctgct
ctgatgccgc atagttaagc cagccccgac acccgccaac 300acccgctgac
gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
360gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
aacgcgcgag 420acgaaagggc ctcgtgatac gcctattttt ataggttaat
gtcatgataa taatggtttc 480ttagacgtca ggtggcactt ttcggggaaa
tgtgcgcgga acccctattt gtttattttt 540ctaaatacat tcaaatatgt
atccgctcat gagacaataa ccctgataaa tgcttcaata 600atattgaaaa
aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
660tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
taaaagatgc 720tgaagatcag ttgggtgcac gagtgggtta catcgaactg
gatctcaaca gcggtaagat 780ccttgagagt tttcgccccg aagaacgttt
tccaatgatg agcactttta aagttctgct 840atgtggcgcg gtattatccc
gtattgacgc cgggcaagag caactcggtc gccgcataca 900ctattctcag
aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
960catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
ctgcggccaa 1020cttacttctg acaacgatcg gaggaccgaa ggagctaacc
gcttttttgc acaacatggg 1080ggatcatgta actcgccttg atcgttggga
accggagctg aatgaagcca taccaaacga 1140cgagcgtgac accacgatgc
ctgtagcaat gccaacaacg ttgcgcaaac tattaactgg 1200cgaactactt
actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
1260tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
ataaatctgg 1320agccggtgag cgtgggtctc gcggtatcat tgcagcactg
gggccagatg gtaagccctc 1380ccgtatcgta gttatctaca cgacggggag
tcaggcaact atggatgaac gaaatagaca 1440gatcgctgag ataggtgcct
cactgattaa gcattggtaa ctgtcagacc aagtttactc 1500atatatactt
tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
1560cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
actgagcgtc 1620agaccccgta gaaaagatca aaggatcttc ttgagatcct
ttttttctgc gcgtaatctg 1680ctgcttgcaa acaaaaaaac caccgctacc
agcggtggtt tgtttgccgg atcaagagct 1740accaactctt tttccgaagg
taactggctt cagcagagcg cagataccaa atactgtcct 1800tctagtgtag
ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
1860cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
gtcttaccgg 1920gttggactca agacgatagt taccggataa ggcgcagcgg
tcgggctgaa cggggggttc 1980gtgcacacag cccagcttgg agcgaacgac
ctacaccgaa ctgagatacc tacagcgtga 2040gctatgagaa agcgccacgc
ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2100cagggtcgga
acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
2160tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
gctcgtcagg 2220ggggcggagc ctatcgaaaa acgccagcaa cgcggccttt
ttacggttcc tggccttttg 2280ctggcctttt gctcacatgt tctttcctgc
gttatcccct gattctgtgg ataaccgtat 2340taccgccttt gagtgagctg
ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 2400agtgagcgag
gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
2460gattcattaa tgcagctggc acgacaggtt tcccgactcg aaagcgggca
gtgagcgcaa 2520cgcaattaat gtgagttagc tcactcatta ggcaccccag
gctttacact ttatgcttcc 2580ggctcgtatg ttgtgtggaa ttgtgagcgg
ataacaattt cacacaggaa acagctatga 2640ccatgattac gccaagctct
aatacgactc actataggga gacaagcttg ttcttgacgc 2700aaccaatgat
ggactgatta agaaacctta tagaagtata cccctaatga agcgtctaac
2760atctaatgaa atatttatac gatacggaga cgcgtctctc atggacatga
taactttatc 2820attgtctaaa caagatatat cattaaaaag aaatgccgaa
ggaatacgtg taaaacatag 2880tttttcagct gatgatatac aggcaattat
ggaatctgat tcgtttttaa agtatagtag 2940atcaaaacca gctgcgatgt
atcaatatat atttctatca ttttttgcta gtggtaattc 3000catagatgac
atattggcaa atagagattc taccttagaa ttttctaaaa gaactaaaag
3060taaaattttg tttggtagga ataccagata cgacgtcact gcaaaatcta
gttttgtatg 3120tggtatagta cgaggtaaat cattggataa aacgtctctg
gttgaaatga tgtgggatct 3180caagaagaaa ggattaatat attctatgga
atttaccaat ctattgagca agaatacctt 3240ttatctgttc acatttacta
tctacactga tgaagtatac gattatctaa acactaataa 3300acttttttct
gcaaaatgtt tagtcgtgtc tacaaaagga gatgtggaaa atttttcatc
3360tctaaaaaaa gatgtggtca ttagagtttg actgcaggtc gactctagag
gatccccggg 3420cttatttatg attatttctc gctttcaatt taacacaacc
ctcaagaacc tttgtattta 3480ttttcaattt ttaggttggg taccagcggc
ctcgagttag ttattatcta caggaacaaa 3540tatagtatct gaaatcatat
tcatatatcc cgttagaggt ctatgataat atatagtagc 3600gtttgttccg
ttatagacac cgaataatat tttacaaaag tgtatatacg tatcatcatc
3660tttatgttta aaatttaaaa tcttaattcg taaatttaga gataaaatgg
cttcttgtac 3720aatactagtt aattctcccg tcctcgagct cttacttgta
cagctcgtgc atgccgagag 3780tgatcccggc ggcggtcacg aactccagca
ggaccatgtg atcgcgcttc tcgttggggt 3840ctttgctcag ggcggactgg
gtgctcaggt agtggttgtc gggcagcagc acggggccgt 3900cgccgatggg
ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
3960tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
atgatataga 4020cgttgtggct gttgtagttg tactccagct tgtgccccag
gatgttgccg tcctccttga 4080agtcgatgcc cttcagctcg atgcggttca
ccagggtgtc gccctcgaac ttcacctcgg 4140cgcgggtctt gtagttgccg
tcgtccttga agaagatggt gcgctcctgg acgtagcctt 4200cgggcatggc
ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc
4260actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
agcttgccgg 4320tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc
atcgccctcg ccctcgccgg 4380acacgctgaa cttgtggccg tttacgtcgc
cgtccagctc gaccaggatg ggcaccaccc 4440cggtgaacag ctcctcgccc
ttgctcacca tttatagcat agaaaaaaac aaaatgaaag 4500gcgcgcctta
gttattatct acaggaacaa atatagtatc tgaaatcata ttcatatatc
4560ccgttagagg tctatgataa tatatagtag cgtttgttcc gttatagaca
ccgaataata 4620ttttacaaaa gtgtatatac gtatcatcat ctttatgttt
aaaatttaaa atcttaattc 4680gtaaatttag agataaaatg gcttcttgta
caatactagt taattctccc gtcctctcaa 4740aattatccaa ctcctcagcg
agaataggac ttagtacata aagtttagca tactctatca 4800tcttcatata
ataattggat aataatttat tccatgtttc tgtactaata tcgaacgagt
4860ctatatattc ctttgtacgc catagaatat ccaaatttgt agggattata
aacaaatctt 4920cggggagtgt tagattaaac ttattagcgt acaatatata
attatgtaga aattctgaat 4980ctattcgctg tatagacttc atataagaca
gatcatagaa atatacgtat gtcccaggga 5040attc 504442214DNAHuman
Immunodeficiency Virus type 1, env 4atgagagtga gggagacagt
gaggaattat cagcacttgt ggagatgggg catcatgctc 60cttgggatgt taatgatatg
tagtgctgca gaccagctgt gggtcacagt gtattatggg 120gtacctgtgt
ggaaagaagc aaccactact ctattttgtg catcagatgc taaagcacat
180aaagcagagg cacataatat ctgggctaca catgcctgtg taccaacaga
ccccaatcca 240cgagaaataa tactaggaaa tgtcacagaa aactttaaca
tgtggaagaa taacatggta 300gagcagatgc atgaggatat aatcagttta
tgggatcaaa gtctaaaacc atgtgtaaaa 360ttaaccccac tctgtgttac
tttaaactgc actacatatt ggaatggaac tttacagggg 420aatgaaacta
aagggaagaa tagaagtgac ataatgacat gctctttcaa tataaccaca
480gaaataagag gtagaaagaa gcaagaaact gcacttttct ataaacttga
tgtggtacca 540ctagaggata aggatagtaa taagactacc aactatagca
gctatagatt aataaattgc 600aatacctcag tcgtgacaca ggcgtgtcca
aaagtaacct ttgagccaat tcccatacat 660tattgtgccc cagctggatt
tgcgattctg aaatgtaata ataagacgtt caatggaacg 720ggtccatgca
aaaatgtcag cacagtacag tgtacacatg gaattaggcc agtagtgtca
780actcaactgt tgttgaatgg cagtctagca gaagaagaga taataattag
atctgaaaat 840atcacaaata atgcaaaaac cataatagta cagcttaatg
agtctgtaac aattgattgc 900ataaggccca acaacaatac aagaaaaagt
atacgcatag gaccagggca agcactctat 960acaacagaca taatagggaa
tataagacaa gcacattgta atgttagtaa agtaaaatgg 1020ggaagaatgt
taaaaagggt agctgaaaaa ttaaaagacc ttcttaacca gacaaagaac
1080ataacttttg aaccatcctc aggaggggac ccagaaatta caacacacag
ctttaattgt 1140ggaggggaat tcttctactg caatacatca ggactattta
atgggagtct gcttaatgag 1200cagtttaatg agacatcaaa tgatactctc
acactccaat gcagaataaa acaaattata 1260aacatgtggc aaggagtagg
aaaagcaatg tatgcccctc ccattgcagg accaatcagc 1320tgttcatcaa
atattacagg actattgttg acaagagatg gtggtaatac tggtaatgat
1380tcagagatct tcagacctgg agggggagat atgagagaca attggagaag
tgaattatac 1440aaatataaag tagtaagaat tgaaccaatg ggtctagcac
ccaccagggc aaaaagaaga 1500gtggtggaaa gagaaaaaag agcaatagga
ctgggagcta tgttccttgg gttcttggga 1560gcggcaggaa gcacgatggg
cgcagcgtca ctgacgctga cggtacaggc cagacagtta 1620ttgtctggta
tagtgcaaca gcaaaacaat ttgctgagag ctatagaggc gcaacagcat
1680ctgttgcaac tcacagtctg gggcattaaa cagctccagg caagagtcct
ggctatggaa 1740agctacctaa aggatcaaca gctcctagga atttggggtt
gctctggaaa acacatttgc 1800accactactg tgccctggaa ctctacctgg
agtaatagat ctgtagagga gatttggaat 1860aatatgacct ggatgcagtg
ggaaagagaa attgagaatt acacaggttt aatatacacc 1920ttaattgaag
aatcgcaaac ccagcaagaa aagaatgaac aagaactatt gcaattggat
1980aaatgggcaa gtttgtggaa ttggtttagt ataacaaaat ggctgtggta
tataaaaata 2040ttcataatga tagtaggagg cttaataggt ttaagaatag
tttttgctgt gctttcttta 2100gtaaatagag ttaggcaggg atattcacct
ctgtcttttc agaccctcct cccagccccg 2160aggggacccg acaggcccga
aggaatagaa gaagaaggtg gagagcaagg ctaa 221453068DNAHuman
Immunodeficiency Virus, type 1, gagpol 5atgggtgcga gagcgtcagt
attaagcgga ggaaaattag atgaatggga aaaaattcgg 60ttacggccag gaggaaacaa
aaaatataga ttaaaacatt tagtatgggc aagcagggag 120ctagaacgat
ttgcacttaa tcctggtctt ttagaaacat cagaaggctg tagacaaata
180atagaacagc tacaaccatc tattcagaca ggatcagagg aacttaaatc
attacataat 240acagtagtaa ccctctattg tgtacatgaa aggataaagg
tagcagatac caaggaagct 300ttagataaga taaaggaaga acaaaccaaa
agtaagaaaa aagcacagca agcaacagct 360gacagcagcc aggtcagcca
aaattatcct atagtacaaa acctacaggg acaaatggta 420caccagtcct
tatcacctag gactttgaat gcatgggtaa aagtaataga agagaaggct
480ttcagcccag aagtaatacc catgttttca gcattatcag aaggagccac
accaacagat 540ttaaacacca tgctaaacac agtaggagga catcaagcag
ccatgcaaat gttaaaagag 600actatcaatg aggaagctgc agaatgggat
aggctacatc cagtgcctgc agggcctgtt 660gcaccaggcc aaatgagaga
accaagagga agtgatatag caggaactac cagtaccctt 720caggaacaaa
taggatggat gacaagcaat ccacctatcc cagtaggaga aatctataaa
780agatggataa tcctaggatt aaataaaata gtaagaatgt atagccctgt
cagcattttg 840gacataagac aaggaccaaa ggaacccttt agagactatg
tagatcggtt ctataaaact 900ctacgagccg agcaagcttc acaggatgta
aaaaattgga tgactgaaac cttgttagtc 960caaaatgcga atccagattg
taaaactatc ttaaaagcat tgggaccagc ggctacatta 1020gaagaaatga
tgacagcatg tcagggagtg gggggaccca gtcataaagc aagagttttg
1080gctgaggcaa tgagccaagc atcaaacaca aatgctgtta taatgatgca
gaggggcaat 1140ttcaagggca agaaaatcat taagtgtttc aactgtggca
aagaaggaca cctagcaaaa 1200aattgtaggg ctcctaggaa aagaggctgt
tggaaatgtg gaaaggaagg gcaccaaatg 1260aaagattgta atgaaagaca
ggctaatttt ttagggagaa tttggccttc ccacaagggg 1320aggccaggga
atttccttca gagcagacca gagccaacag ccccaccagc agagagcttc
1380gggtttgggg aagagataac accctcccag aaacaggagg ggaaagagga
gctgtatcct 1440tcagcctccc tcaaatcact ctttggcaac gacccctagt
cacaataaaa atagggggac 1500agctaaagga agctctatta gatacaggag
cagatgatac agtagtagaa gaaatgaatt 1560tgccaggaaa atggaaacca
aaaatgatag ggggaattgg gggctttatc aaagtaagac 1620agtatgatca
aatactcgta gaaatctatg gatataaggc tacaggtaca gtattagtag
1680gacctacacc tgtcaacata attggaagaa atttgttgac tcagattggt
tgcactttaa 1740attttccaat tagtcctatt gaaactgtac cagtaaaatt
aaagtcaggg atggatggtc 1800caagagttaa acaatggcca ttgacagaag
agaaaataaa agcactaata gaaatttgta 1860cagaaatgga aaaggaagga
aaactttcaa gaattggacc tgaaaatcca tacaatactc 1920caatatttgc
cataaagaaa aaagacagta ctaagtggag aaaattagta gatttcagag
1980aacttaataa gagaactcaa gatttctggg aagttcaact aggaatacca
catcctgcag 2040ggctaaaaaa gaaaaaatca gtaacagtac tggaggtggg
tgatgcatat ttttcagttc 2100ccttatatga agactttaga aaatacactg
cattcaccat acctagtata aacaatgaga 2160caccaggaat tagatatcag
tacaatgtgc ttccacaagg atggaaagga tcaccggcaa 2220tattccaaag
tagcatgaca aaaattttag aaccttttag aaaacaaaat ccagaagtgg
2280ttatctacca atacatgcac gatttgtatg taggatctga cttagaaata
gggcagcata 2340gaataaaaat
agaggaatta aggggacacc tattgaagtg gggatttacc acaccagaca
2400aaaatcatca gaaggaacct ccatttcttt ggatgggtta tgaactccat
cctgataaat 2460ggacagtaca gcctataaaa ctgccagaaa aagaaagctg
gactgtcaat gatctgcaga 2520agttagtggg gaaattaaat tgggcaagtc
aaatttattc aggaattaaa gtaagacaat 2580tatgcaaatg ccttagggga
accaaagcac tgacagaagt agtaccactg acagaagaag 2640cagaattaga
actggcagaa aacagggaac ttctaaaaga aacagtacat ggagtgtatt
2700atgacccatc aaaagactta atagcagaaa tacagaaaca agggcaagac
caatggacat 2760atcaaattta tcaagaacaa tataaaaatt tgaaaacagg
aaagtatgca aagaggagga 2820gtacccacac taatgatgta aaacaattaa
cagaggcagt gcaaaaaata gcccaagaat 2880gtatagtgat atggggaaag
actcctaaat tcagactacc catacaaaag gaaacatggg 2940aaacatggtg
gacagagtat tggcaggcca cctggattcc tgagtgggag tttgtcaata
3000cccctccctt ggttaaatta tggtaccagt tagagaagga acccatagta
ggagcagaaa 3060ccttctaa 306869PRTHuman Immunodeficiency Virus, type
1 6Ala Met Gln Met Leu Lys Glu Thr Ile1 5715PRTHuman
Immunodeficiency Virus, type 1 7Asp Thr Glu Val His Asn Val Trp Ala
Thr His Ala Cys Val Pro1 5 10 15815PRTHuman Immunodeficiency Virus,
type 1 8Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln
His1 5 10 15913PRTHuman Immunodeficiency Virus, type 1 9Glu Leu Arg
Gln His Leu Leu Arg Trp Gly Leu Thr Thr1 5 101014PRTHuman
Immunodeficiency Virus, type 1 10His Gly Val Tyr Tyr Asp Pro Ser
Lys Asp Leu Ile Ala Glu1 5 10
* * * * *