Adenovirus carrying gag gene HIV vaccine Number:6,787,351 from the United States Patent and Trademark Office (PTO) owispatent An adenoviral vector is described which carries a codon-optimized gag gene, along with a heterologous promoter and transcription terminator. This viral vaccine can effectively prevent HIV infection when administered to humans either alone or as part of a prime and boost regime also with a vaccine plasmid.<H Adenovirus, carrying, Adenovirus, carr, 6787351.html, Patent, pto, 6787351

Title: Adenovirus carrying gag gene HIV vaccine

Abstract: An adenoviral vector is described which carries a codon-optimized gag gene, along with a heterologous promoter and transcription terminator. This viral vaccine can effectively prevent HIV infection when administered to humans either alone or as part of a prime and boost regime also with a vaccine plasmid.

Patent Number: 6,787,351 Issued on 09/07/2004 to Chen, et al.

Inventors: Chen; Ling (Blue Bell, PA), Shiver; John W. (Doylestown, PA), Bett; Andrew J. (Lansdale, PA), Casimiro; Danilo R. (Harleysville, PA), Caulfield; Michael J. (Fort Washington, PA), Chastain; Michael A. (Glenside, PA), Emini; Emilio A. (Strafford, PA)
Assignee: Merck & Co., Inc. (Rahway, NJ)

Appl. No.: 09/818,443

Filed: March 27, 2001

Current U.S. Class: 435/320.1 ; 424/93.2; 435/69.1; 435/69.2

Field of Search: 435/320.1,69.1,69.2 424/93.2

References Cited [Referenced By]

U.S. Patent Documents


5643579	July 1997	Hung et al.
5672508	September 1997	Gyuris et al.
5716613	February 1998	Guber et al.
5859193	January 1999	Devare et al.
6019978	February 2000	Ertl et al.
6033908	March 2000	Bout et al.
6287571	September 2001	Ertl et al.

Foreign Patent Documents


0 586 076	Jul., 1993	EP
0 638 316	Jul., 1994	EP
0 707 071	Aug., 1995	EP
WO96/21015	Jul., 1996	WO
WO96/39178	Dec., 1996	WO
WO97/00326	Jan., 1997	WO
WO97/31115	Aug., 1997	WO
WO97/39771	Oct., 1997	WO
WO97/48370	Dec., 1997	WO
WO98/34640	Aug., 1998	WO
WO98/56919	Dec., 1998	WO
WO01/21201	Sep., 2000	WO
WO01/02067	Jan., 2001	WO
WO01/43693	Jun., 2001	WO
WO01/45748	Jun., 2001	WO

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Primary Examiner: Park; Hankyel T.
Attorney, Agent or Firm: Cocuzzo; Anna L. Tribble; Jack L.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT International Application No. PCT/US00/18332, filed Jul. 3, 2000, which designates the U.S., which claims the benefit, under 35 U.S.C. .sctn.119(e), of U.S. Provisional Application Ser. No. 60/148,981, filed Aug. 13, 1999 and U.S. Provisional Application Ser. No. 60/142,631, filed Jul. 6, 1999.

Claims

What is claimed is:

1. A vaccine composition comprising a replication defective adenoviral vector comprising at least one gene encoding a HIV gag protein which is codon optimized for expression in a human, and the gene is operably linked to a heterologous promoter and transcription terminator.

2. An adenoviral vaccine vector comprising: a) a replication defective adenoviral vector, wherein the adenoviral vector does not have a functional E1 gene, and further comprising: b) a gene expression cassette comprising: i) a nucleic acid encoding a gag protein which is codon optimized for expression in a human host; ii) a heterologous promoter operatively linked to the nucleic acid encoding the gag protein; and iii) a transcription terminator.

3. A vector according to claim 2, wherein the E1 gene has been deleted from the adenoviral vector.

4. A vector according to claim 3, wherein the gene expression cassette has replaced the deleted E1 gene.

5. A vector according to claim 3, wherein the adenovirus vector does not have a functional E3 gene.

6. A vector according to claim 5 wherein the E3 gene has been deleted from the replication defective adenoviral vector.

7. A vector according to claim 6 comprising adenoviral 5 sequences deleted of E1 region base pairs (bp) 342-3523 and deleted of E3 region bp 28,133-30,818.

8. A vector according to claim 6 comprising adenoviral 2 sequences deleted of E1 region bp 559-3503 and E3 region bp 28,812-29,773.

9. A vector according to claim 8 comprising the sequence given in FIG. 6.

10. A vector according to claim 8 wherein the sequence is tPA-gag.

11. A vector according to claim 2 further comprising a physiologically acceptable carrier.

12. An adenoviral vaccine composition for producing an immune response against human immunodeficiency virus (HIV) in a human comprising: a) adenovirus serotype 5 sequences bp 1 to bp 341 and bp 3534 to 5798; and b) a gene expression cassette, located 3' to adenovirus sequence bp 341, comprising: i) a nucleic acid encoding gag which is codon-optimized and optionally has the tPA leader sequence at its 5' end; ii) a human CMV promoter plus intron A operatively linked to the nucleic acid encoding gag; and iii) a bovine growth hormone transcription terminator.

13. A plasmid vector comprising: a) an adenoviral portion comprising an adenoviral vector according to claim 2; and b) a plasmid portion.

14. A cell comprising an adenoviral vector of claim 2.

15. A method of producing the vector of claim 2 comprising introducing the adenoviral vector of claim 2 into a host cell which expresses adenoviral E1 protein, and harvesting the resultant adenoviral vectors.

16. A method according to claim 15 wherein the cell is a 293 cell or PER.C6 cell.

17. A method of generating a cellular immune response against an HIV protein in an individual comprising administering to the individual at least one adenovirus vaccine vector and a vaccine plasmid, wherein said adenovirus vaccine vector comprises: a) a replication defective adenoviral vector, wherein the adenoviral vector does not have a functional E1 gene, and b) a gene expression cassette comprising: i) a nucleic acid encoding gag protein optimized for expression in a human host; ii) a heterologous promoter operatively linked to the nucleic acid encoding the gag protein; and iii) a transcription terminator; and wherein said vaccine plasmid comprises: a) a gene expression cassette comprising: a nucleic acid encoding a gag protein, wherein the nucleic acid is codon optimized for expression in a human host; b) a promoter; and c) a transcription terminator

wherein the vaccine plasmid does not contain any adenoviral genes.

18. A method according to claim 17 comprising administering a vaccine plasmid to the individual, and after a predetermined minimum amount of time has passed, administering an adenovirus vaccine vector to the individual.

19. A method according to claim 17 comprising administering an adenovirus vaccine vector to the individual, and after a predetermined minimum amount of time has passed, administering a vaccine plasmid to the individual.

20. A method according to claim 17 comprising administering an adenovirus vaccine vector to the individual, and after a predetermined minimum amount of time has passed, re-administering an adenovirus vector to the individual.

Description

FIELD OF THE INVENTION

This invention relates to replication deficient adenovirus vectors comprising an optimized human immunodeficiency virus (HIV) gag gene under the control of a strong promoter, which are suitable for vaccines against HIV.

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus-1 (HIV-1) is the etiological agent of acquired human immune deficiency syndrome (AIDS) and related disorders.

Vaccination is an effective form of disease prevention and has proven successful against several types of viral infection. However, determining ways to present HIV-1 antigens to the human immune system in order to evoke protective humoral and cellular immunity is a difficult task. In AIDS patients, free virus is present in low levels only. Transmission of HIV-1 is enhanced by cell-to-cell interaction via fusion and syncytia formation. Hence, antibodies generated against free virus or viral subunits are generally ineffective in eliminating virus-infected cells.

European Patent Applications 0 638 316 (Published Feb. 15, 1995) and 0 586 076 (Published Mar. 9, 1994), (both assigned to American Home Products Corporation) describe replicating adenovirus vectors carrying an HIV gene, including env or gag. Various treatment regimens were used with chimpanzees and dogs, some of which included booster adenovirus or protein plus alum treatments.

Infection with HIV-1 is almost always fatal, and at present there are no cures for HIV-1 infection. Effective vaccines for prevention of HIV-1 infection are not yet available. Because of the danger of reversion or infection, live attenuated virus probably cannot be used as a vaccine, and. subunit vaccine approaches have not been successful at preventing HIV infection. Treatments for HIV-1 infection, while prolonging the lives of some infected persons, have serious side effects. There is thus a great need for effective treatments and vaccines to combat this lethal infection.

SUMMARY OF THE INVENTION

This invention relates to a vaccine composition comprising a replication-defective adenoviral vector comprising at least one gene encoding an HIV gag protein, wherein the gene comprises codons optimized for expression in a human, and the gene is operably linked to a heterologous promoter.

Another aspect of this invention relates to an adenoviral vaccine vector comprising: a replication defective adenoviral genome, wherein the adenoviral genome does not have a functional E1 gene, and the adenoviral genome further comprises a gene expression cassette comprising: i) a nucleic acid encoding a HIV gag protein, wherein the nucleic acid is codon optimized for expression in a human host; ii) a heterologous promoter is operatively linked to the nucleic acid encoding the gag protein; and iii) a transcription terminator.

In preferred embodiments, the E1 gene has been deleted from the adenoviral vector, and the HIV expression cassette has replaced the deleted E1 gene. In other preferred embodiments, the replication defective adenovirus genome does not have a functional E3 gene, and preferably the E3 gene has been deleted.

This invention also relates to a shuttle plasmid vector comprising: an adenoviral portion and a plasmid portion, wherein said adenovirus portion comprises: a) a replication defective adenovirus genome which does not have a functional E1 gene; and b) a gene expression cassette comprising: a nucleic acid encoding an HIV gag protein, wherein the nucleic acid is codon optimized for expression in a human host; a heterologous promoter operably linked to the nucleic acid encoding the gag protein; and a transcription terminator.

Other aspects of this invention include a host cell comprising the adenoviral vaccine vectors and/or the shuttle plasmid vectors, methods of producing the vectors comprising introducing the adenoviral vaccine vector into a host cell which expresses adenoviral E1 protein, and harvesting the resultant adenoviral vaccine vectors.

Another aspect of this invention is a method of generating a cellular immune response against an HIV protein in an individual comprising administering to the individual an adenovirus vaccine vector comprising:

a) a replication defective adenoviral vector, wherein the adenoviral vector does not have a functional E1 gene, and b) a gene expression cassette comprising: i) a nucleic acid encoding, an HIV gag protein, wherein the nucleic acid is codon optimized for expression in a human host; ii) a heterologous promoter operatively linked to the nucleic acid encoding the gag protein; and iii) a transcription terminator.

In some embodiments of this invention, the individual is given more than one administration of adenovirus vaccine vector, and it may be given in a regiment accompanied by the administration of a plasmid vaccine. The plasmid vaccine comprises a plasmid encoding a codon-optimized gag protein, a heterologous promoter operably linked to the gag protein nucleic acids, and a transcription terminator. There may be a predetermined minimum amount of time separating the, administrations. The individual can be given a first dose of plasmid vaccine, and then a second dose of plasmid vaccine. Alternatively, the individual may be given a first dose of adenovirus vaccine vector, and then a second dose of adenoviral vaccine vector. In other embodiments, the plasmid vaccine is administered first, followed after a time by administration of the adenovirus vector vaccine. Conversely, the adenovirus vaccine vector may be administered first, followed by administration of plasmid vaccine after a time. In these embodiments, an individual may be given multiple doses of the same adenovirus serotype in either viral vector or plasmid form, or the virus may be of differing serotypes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the number of gag peptide-specific interferon-gamma secreting splenocytes (.times.10.sup.6) from rats which were immunized with gag plasmid or Ad5FLgag.

FIG. 2 shows serum SEAP (secreted alkaline phosphatase) expression levels in rhesus monkeys following injection with FG Ad5-SEAP or SEAP DNA constructs.

FIGS. 3A, 3B and 3C show anti-HIV gag cytotoxic T lymphocyte responses in three rhesus monkeys vaccinated with FG Ad5 tPAgag. Each panel represents the specific killing response of a particular monkeys (denoted as numbers 92.times.024 in FIG. 3A, 94.times.012 in FIG. 3B, and 94.times.025 in FIG. 3C) at various time points following immunization at 0, 8, and 24 weeks. The abscissa axis shows the effector/target (E/T) ratios of cultured T cells and B cells employed in this assay, while the ordinate axis shows specific lysis values obtained for each sample. Specific lysis values of at least 10% difference between curves.+-.gag peptide antigen are generally considered significant. The square symbols represent target cells treated with an irrelevant influenza peptide antigen while the circles, triangles, and diamonds represent target cells treated with partial or complete gag peptide pools, respectively.

FIGS. 4A-H show anti-HIV gag cytotoxic T lymphocyte responses in rhesus monkeys vaccinated with FG Ad5FLgag. FIGS. 4A, B, and C are the first group of monkeys, D, E, and F are the second, and G, H, and I are the third group. Each represents specific killing responses of each monkey receiving the indicated treatment. The abscissa axis shows the effector/target (E/T) ratios of cultured T cells and B cells employed in this assay, while the ordinate axis shows specific lysis values obtained for each sample. Specific lysis values of at least 10% difference between curves.+-.gag peptide antigen are generally considered significant. The square symbols represent target cells treated with DMSO alone at the same concentration as samples containing peptides while the circles, triangles, and diamonds represent target cells treated with partial (F, G) or complete (H) gag peptide pools, respectively.

FIGS. 5A-H show anti-HIV gag cytotoxic T lymphocyte responses in rhesus monkeys vaccinated with Ad2Flgag priming, followed by either Ad2Flgag or Ad5Flgag boosting. Each panel (FIGS. 5A-G) represents specific killing responses of a group of three monkeys receiving the indicated treatment. The last panel (FIG. 5H) shows responses from two naive monkeys that were not vaccinated. The abscissa axis shows the effector/target (E/T) ratios of cultured T cells and B cells employed in this assay, while the ordinate axis shows specific lysis values obtained for each sample. Specific lysis values of at least 10% difference between curves +gag peptide antigen are generally considered significant.

FIG. 6 is the nucleic acid sequence (SEQ.ID.NO.1) of the optimized human HIV-1 gag open reading frame.

FIG. 7A shows construction of the adenovirus carrying codon-optimized gag. FIG. 7B shows construction of the adenovirus carrying codon-optimized tPA-gag.

FIG. 8 is the nucleic acid sequence of the optimized tPA-gag open reading frame.

FIG. 9 show the longevity (69 weeks after final boost) of cellular immune responses in rhesus monkeys immunized with Ad5FLgag

FIG. 10 shows CMI repsonses prior to and subsequent to a week 24 boost with Ad5-FLgag (SFC/10.sup.6 cells via ELIspot)

FIG. 11 shows long term CMI responses for an HIV gag DNA vaccine (0, 4 and 8 weeks) and Ad5FLgag (single prime at T=0) which were boosted. with 10.sup.7 particles of Ad5FLgag (SFC/10.sup.6 cells via ELIspot).

FIG. 12 shows a comparison of various single modality Ad5FLgag immunizations with DNA gag vaccines adjuvanted with various formulations of CRL1005. (D113:5 mg/ml DNA, 7.5 mg CRL1005 in PBS; D117:5 mg/ml DNA, 22.5 mg CRL1005 in PBS; D118:7.5 mg/ML CRL1005, 0.5 mM BAK and 5 mg/ml, DNA in PBS. DNA/POP-POE/BAK formulations are disclosed in U.S. Provisional Application Ser. Nos. 60/214,824 and 60/213622, filed Jun. 28, 2000 and Jun. 23, 2000, respectively; both of which are hereby incorporated by reference.) Four columns of data are presented for each animal. The far left column is a pre-immunization ELISPOT response; the column second from the left represents the ELISPOT after either the AdS or DNA priming, respectively; the column third from the left is taken the week of or prior to the boosting, and the fourth column measures a CMI response subsequent to the Ad5FLgag boost.

FIGS. 13A-E shows the longitudinal p11 C-specific tetramer staining results for all Mamu-A*01 monkeys up to one week before challenge. These data are presented as a percentage of the CD3.sup.+ CD8.sup.+ positive T cell population. Arrows indicate time of innoculations. (A) SIV gag DNA; (B) MVA-SIV gag; (C) SIV gag DNA+alum/MPL; (D) Ad5-SIV gag; and (E) SIV gag DNA+CRL1005.

FIGS. 14A-F show post challenge longitudinal results for peripheral p11C-specific tetramer staining for each group, as follows: (A) SIV gag DNA; (B) SIV gag DNA+CRL1005; (C) SIV gag DNA+alum/MPL; (D) MVA-SIV gag; (E) AdS-SIV gag; and, (F) naive animals.

FIGS. 15A-L shows post-challenge CD4 T cell counts (A, C, E, G, and I) and plasma viral load (B, D, F. H, J, L) for each group, as follows: (A, B) SIV gag DNA; (C,D) SIV gag DNA +CRL1005; (E, F) SIV gag DNA +alum/MPL; (G, H) MVA-SIV gag; (I, J) Ad5-SIV gag; and, (K, L) naive animals.

As used throughout the specification and claims, the following definitions and abbreviations are used:

In general, adenoviral constructs, gene constructs are named by reference to the genes contained therein, such as below:

"tPAgag" refers to a fusion between the leader sequence of the tissue plasminogen activator leader sequence and an optimized HIV gag gene. "Ad5-tPAgag" refers to an adenovirus serotype 5 replication deficient virus which carries an expression cassette which comprises a tissue plasminogen activator leader sequence fused to a codon-optimized gag gene which is under the control of the CMV promoter and contains Intron A.

"Fl" refers to a full length gene. "Flgag" refers to the full-length optimized gag gene. "Ad5-Flgag" refers to an adenovirus serotype 5 replication deficient virus which carries an expression cassette which comprises a full length optimized gag gene under the control of the CMV promoter and contains Intron A.

"FG Adenovirus" means a First Generation adenovirus, i.e. a replication deficient adenovirus which has either a non-functional or deleted E1 region, and optionally a non-functional or deleted E3 region.

"Promoter" means a recognition site on a DNA strand to which an RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences such as enhancers or inhibiting sequences such as silencers.

"Leader" means a DNA sequence at the 5' end of a structural gene which is transcribed along with the gene. This usually results a protein having an N-terminal peptide extension, often referred to as a pro-sequences.

"Intron" as used herein refers to a section of DNA occurring in the middle of a gene which does not code for an amino acid in the gene product. The precursor RNA of the intron is excised and is therefore not transcribed into mRNA not translated into protein.

"Cassette" refers to the a nucleic acid sequence which is to be expressed, along with its transcription and translational control sequences. By changing the cassette, a vector can express a different sequence.

It has been found according to this invention that first generation adenoviral vectors carrying a codon-optimized HIV gag gene regulated with a strong heterologous promoter can be used as human anti-HIV vaccines, and are capable of inducing immune responses.

The adenoviral vector which makes up the backbone of the vaccine construct of this invention is preferably a "first generation" adenoviral vector. This group of adenoviral vectors is known in the art, and these viruses are characterized by being replication-defective. They typically have a deleted or inactivated E1 gene region, and preferably additionally have a deleted or inactivated E3 gene region. In a preferred embodiment of this invention, the first generation replication incompetent adenovirus vector used is a serotype 5 adenovirus containing deletions in E1 (Ad5 base pairs 342-3523) and E3 (AdS base pairs 28133 to 30818). For adenovirus 2 serotype, the E1 deletions are preferably bp 559-3503 and the E3 deletions are preferably 28,812-29,773. (Genbank gb:J01917). Those of skill in the art can easily determine the equivalent sequences for other serotypes, such as serotypes 4, 12, 6, 17, 24, 33, 42, 31, 16.

Adenoviral serotypes 2 and 5, particularly 5 are preferred for use in this invention, since at this point in time, more is known about these serotypes generally than other serotypes, and their complete DNA sequences are known. The prototype serotype 5 adenovirus has been completely sequenced (Chroboczek et al, 1992 J. Virology 186:280, which is hereby incorporated by reference.) They also belong to the subgroup C adenoviruses, which are not associated with human or rodent malignancies. However, it is envisioned that any adenovirus serotype can be used in this invention, including non-human ones, as deletion of E1 genes should render all adenoviruses non-tumorogenic. Also it may be advantageous to use a serotype which has less prevalence in the wild, as patients are less likely to have previous exposure (and less pre-existing antibodies) to a rarer serotype.

The adenoviral vectors can be constructed using known techniques, such as those reviewed in Hitt et al, 1997 "Human Adenovirus Vectors for Gene Transfer into Mammalian Cells" Advances in Pharmacology 40:137-206, which is hereby incorporated by reference.

In constructing the adenoviral vectors of this invention, it is often convenient to insert them in to a plasmid or shuttle vector. These techniques are known and described in Hitt et al supra. This invention specifically includes both the adenovirus and the adenovirus when inserted into a shuttle plasmid.

Viral vectors can be propagated in various E1 complementing cell lines, including the known cell lines 293 and PER.C6. Both these cell lines express the adenoviral E1 gene product. PER.C6 is described in WO 97/00326, published Jan. 3, 1997, which is hereby incorporated by reference. It is a primary human retinoblast cell line transduced with an E1 gene segment that complements the production of replication deficient (FG) adenovirus, but is designed to prevent generation of replication competent adenovirus by homologous recombination. 293 cells are described in Graham et al 1977 J. Gen. Virol 36:59-72, which is hereby incorporated by reference.

The HIV gag gene selected to be expressed is of importance to the invention. Sequences for many genes of many HIV strains are publicly available in GENBANK and primary, field isolates of HIV are available from the National Institute of Allergy and Infectious Diseases (NIAID) which has contracted with Quality Biological (Gaithersburg, Md.) to make these strains available. Strains are also available from the World Health Organization (WHO), Geneva Switzerland. In a preferred embodiment of this invention, the gag gene is from an HIV-1 strain (CAM-1; Myers et al, eds. "Human Retroviruses and AIDS: 1995, IIA3-IIA19, which is incorporated by reference). This gene closely resembles the consensus amino acid sequence for the dade B (North American/European) sequence.

Regardless of the HIV gene chosen for expression, the sequence should be "optimized" for expression in a human cellular environment. A "triplet" codon of four possible nucleotide bases can exist in 64 variant forms. That these forms provide the message for only 20 different amino acids (as well as transcription initiation and termination) means that some amino acids can be coded for by more than one codon. Indeed, some amino acids have as many as six "redundant", alternative codons while some others have a single, required codon. For reasons not completely understood, alternative codons are not at all uniformly present in the endogenous DNA of differing types of cells and there appears to exist variable natural hierarchy or "preference" for certain codons in certain types of cells. As one example, the amino acid leucine is specified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA, and TTG (which correspond, respectively, to the MRNA codons, CUA, CUC, CUG, CUU, UUA and UUG). Exhaustive analysis of genome codon frequencies for microorganisms has revealed endogenous DNA of E. coli most commonly contains the CTG leucine-specifying codon, while the DNA of yeasts and slime molds most commonly includes a TTA leucine-specifying codon. In view of this hierarchy, it is generally held that the likelihood of obtaining high levels of expression of a leucine-rich polypeptide by an E. coli host will depend to some extent on the frequency of codon use. For example, a gene rich in TTA codons will in all probability be poorly expressed in E. coli, whereas a CTG rich gene will probably highly express the polypeptide. Similarly, when yeast cells are the projected transformation host cells for expression of a leucine-rich polypeptide, a preferred codon for use in an inserted DNA would be TTA.

The implications of codon preference phenomena on recombinant DNA techniques are manifest, and the phenomenon may serve to explain many prior failures to achieve high expression levels of exogenous genes in successfully transformed host organisms--a less "preferred" codon may be repeatedly present in the inserted gene and the host cell machinery for expression may not operate as efficiently. This phenomenon suggests that synthetic genes which have been designed to include a projected host cell's preferred codons provide a preferred form of foreign genetic material for practice of recombinant DNA techniques. Thus, one aspect of this invention is an adenovirus vector which specifically includes a gag gene which is codon optimized for expression in a human cellular environment.

The diversity of function that typifies eukaryotic cells depends upon the structural differentiation of their membrane boundaries. To generate and maintain these structures, proteins must be transported from their site of synthesis in the endoplasmic reticulum to predetermined destinations throughout the cell. This requires that the trafficking proteins display sorting signals that are recognized by the molecular machinery responsible for route selection located at the access points to the main trafficking pathways. Sorting decisions for most proteins need to be made only once as they traverse their biosynthetic pathways since their final destination, the cellular location at which they perform their function, becomes their permanent residence.

Maintenance of intracellular integrity depends in part on the selective sorting and accurate transport of proteins to their correct destinations. Over the past few years the dissection of the molecular machinery for targeting and localization of proteins has been studied vigorously. Defined sequence motifs have been identified on proteins which can act as "address labels". Leader or signal peptides such as that from the tissue-specific plasminogen activator protein, tPA, serve to transport a protein into the cellular secretory pathway through the endoplasmic reticulum and golgi apparatus. A number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins such as di-Leucine amino acid motifs or tyrosine-based sequences that can direct proteins to lysosomal compartments. For HIV, transport and extrusion from the cell of viral particles depend upon myristoylation of glycine residue number two at the amino terminus of gag. In some embodiments of the optimized gag gene, the tPA leader sequence has been attached 5' to the structural gene sequence.

The optimized gag gene is incorporated into an expression cassette. The cassette contains a transcriptional promoter recognized by an eukaryotic RNA polymerase; and a transcriptional terminator at the end of the gag gene coding sequence. In a preferred embodiment, the promoter is a "strong" or "efficient" promoter. An example of a strong promoter is the immediate early human cytomegalovirus promoter (Chapman et al, 1991 Nucl. Acids Res 19:3979-3986, which is incorporated by reference) with the intron A sequence (CMV-intA), although those skilled in the art will recognize that any of a number of other known promoters, such as the strong immunoglobulin, or other eukaryotic gene promoters may be used, including the EF1 alpha promoter, the murine CMV promoter, Rous sarcoma virus (RSV) promoter, SV40 early/late promoters and the beta-actin promoter. A preferred transcriptional terminator is the bovine growth hormone terminator. The combination of CMVintA-BGH terminator is particularly preferred although other promoter/terminator combinations in the context of FG adenovirus may also be used.

To assist in preparation of the polynucleotides in prokaryotic cells, a shuttle vector version of the adenovirus vector is often prepared. The shuttle vector contains an adenoviral portion and a plasmid portion. The adenoviral portion is essentially the same as the adenovirus vector discussed supra, containing adenoviral sequences (with non-functional or deleted E1 and E3 regions) and the gag expression cassette, flanked by convenient restriction sites. The plasmid portion of the shuttle vector often contains an antibiotic resistance marker under transcriptional control of a prokaryotic promoter so that expression of the antibiotic does not occur in eukaryotic cells. Ampicillin resistance genes, neomycin resistance genes and other pharmaceutically acceptable antibiotic resistance markers may be used. To aid in the high level production of the polynucleotide by fermentation in prokaryotic organisms, it is advantageous for the shuttle vector to contain a prokaryotic origin of replication and be of high copy number. A number of commercially available prokaryotic cloning vectors provide these benefits. It is desirable to remove non-essential DNA sequences. It is also desirable that the vectors not be able to replicate in eukaryotic cells. This minimizes the risk of integration of polynucleotide vaccine sequences into the recipients' genome. Tissue-specific promoters or enhancers may be used whenever it is desirable to limit expression of the polynucleotide to a particular tissue type.

In one embodiment of this invention, the shuttle plasmid used is pAD.CMVI-FLHIVgag, was made using homologous recombination techniques. For clinical use, the shuttle vector was rescued into virus in PER.C6 cells. To rescue, the shuttle plasmid was linearized by PacI restriction enzyme digestion and transfected into the PER.C6 cells using the calcium phosphate coprecipitate method. The plasmid in linear form is capable of replication after entering the PER.C6 cells and virus is produced. The infected cells and media were harvested after viral replication was complete.

Standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the DNA immunogens of this invention.

To ensure a clonal virus population a method of clonal purification was used for clinical material. The virus obtained from transfection of the PER.C6 cells was serially diluted to extinction using 2-fold dilutions. The dilutions were then used to infect PER.C6 cells in 96 well plates using 24 wells for each solution At the end of a 14-day incubation period the wells were scored positive or negative using adenovirus specific PCR and gag ELISA. Virus positive wells at the highest dilutions were selected for expansion. The selected well was the only positive well out of 24 wells plated at that dilution giving 98% assurance of clonality Verification of that endpoint had been reached in the dilution series, and that virus positive wells that had insufficient virus to be detected in the initial screening had not been missed, was obtained by subculturing the original 96 well plated two additional times and re-scoring them This confirmed the clonality of the selected well. The selected virus was designated AD5FLgag.

The adenoviral vaccine composition may contain physiologically acceptable components, such as buffer, normal saline or phosphate buffered saline, sucrose, other salts and polysorbate. One preferred formulation has: 2.5-10 mM TRIS buffer, preferably about 5 mM TRIS buffer; 25-100 mM NaCl, preferably about 75 mM NaCl; 2.5-10% sucrose, preferably about 5% sucrose; 0.01-2 mM MgCl.sub.2 ; and 0.001%-0.01% polysorbate 80 (plant derived). The pH should range from about 7.0-9.0, preferably about 8.0. One skilled in the art will appreciate that other conventional vaccine excipients may also be used it make the formulation. The preferred formulation contains 5 mM TRIS, 75 mM NaCl, 5% sucrose, 1 mM MgCl.sub.2, 0.005 % polysorbate 80 at pH 8.0 This has a pH and divalent cation composition which is near the optimum for AdS stability and minimizes the potential for adsorption of virus to a glass surface. It does not cause tissue irritation upon intramuscular injection. It is preferably frozen until use.

The amount of adenoviral particles in the vaccine composition to be introduced into a vaccine recipient will depend on the strength of the transcriptional and translational promoters used and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of 1.times.10.sup.7 to 1.times.10.sup.12 particles and preferably about 1.times.10.sup.10 to 1.times.10.sup.10 particles is administered directly into muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated. It is also contemplated that booster vaccinations are to be provided. Following vaccination with HIV adenoviral vector, boosting with a subsequent HIV adenoviral vector anchor plasmid may be desirable. Parenteral administration, such as intravenous, intramuscular, subcutaneous or other means of administration of interleukin-12 protein, concurrently with or subsequent to parenteral introduction of the vaccine compositions of this invention is also advantageous.

Another aspect of this invention is the administration of the adenoviral vector containing the optimized gag gene in a prime/boost regiment in conjunction with a plasmid DNA encoding gag. To distinguish this plasmid from the adenoviral-containing shuttle plasmids used in the construction of an adenovirus vector, this plasmid will be referred to as a "vaccine plasmid". The preferred vaccine plasmids to use in this administration protocol are disclosed in pending U.S. patent application Ser. No. 09/017,981, filed Feb. 3, 1998 and WO98/34640, published Aug. 13, 1998, both of which are hereby incorporated by reference. Briefly, the preferred vaccine plasmid is designated V1Jns-FL-gag, which expresses the same codon-optimized gag gene as the adenoviral vectors of this invention. The vaccine plasmid backbone, designated V1Jns contains the CMV immediate-early (IE) promoter and intron A, a bovine growth hormone-derived polyadenylation and transcriptional termination sequence as the gene expression regulatory elements, and a minimal pUC backbone (Montgomery et al, 1993 DNA Cell Biol. 12:777-783. The pUC sequence permits high levels of plasmid production in E. coli and has a neomycin resistance gene in place of an ampicillin resistance gene to provide selected growth in the presence of kanamycin. Those of skill in the art, however, will recognized that alternative vaccine plasmid vectors may be easily substituted for this specific construct, and this invention specifically envisions the use of alternative plasmid DNA vaccine vectors.

The adenoviral vector and/or vaccine plasmids of this invention polynucleotide may be unassociated with any proteins, adjuvants or other agents which impact on the recipients' immune system. In this case, it is desirable for the vector to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the vector may be associated with an adjuvant known in the art to boost immune responses, such as a protein or other carrier. Agents which assist in the cellular uptake of DNA, such as, but not limited to, calcium ions, may also be used to advantage. These agents are generally referred to herein as transfection facilitating reagents and pharmaceutically acceptable carriers. Techniques for coating microprojectiles coated with polynucleotide are known in the art and are also useful in connection with this invention.

The adenoviral vaccines of this invention may be administered alone, or may be part of a prime and boost administration regimen. A mixed modality priming and booster inoculation scheme will result in an enhanced immune response, particularly is pre-existing anti-vector immune responses are present. This one aspect of this invention is a method of priming a subject with the plasmid vaccine by administering the plasmid vaccine at least one time, allowing a predetermined length of time to pass, and then boosting by administering the adenoviral vaccine. Multiple primings typically, 1-4, are usually employed, although more may be used. The length of time between priming and boost may typically vary from about four months to a year, but other time frames may be used. In experiments with rhesus monkeys, the animals were primed four rimes with plasmid vaccines, then were boosted 4 months later with the adenoviral vaccine. Their cellular immune response was notably higher than that of animals which had only received adenoviral vaccine. The use of a priming regimen may be particularly preferred in situations where a person has a pre-existing anti-adenovirus immune response.

This invention also includes a prime and boost regimen wherein a first adenoviral vector is administered, then a booster dose is given. The booster dose may be repeated at selected time intervals.

A large body of human and animal data supports the importance of cellular immune responses, especially CTL in controlling (or eliminating) HIV infection. In humans, very high levels of CTL develop following primary infection and correlate with the control of viremia. Several small groups of individuals have been described who are repeatedly exposed to HIV by remain uninfected; CTL has been noted in several of these cohorts. In the SIV model of HIV infection, CTL similarly develops following primary infection, and it has been demonstrated that addition of anti-CD8 monoclonal antibody abrogated this control of infection and leads to disease progression. This invention uses adenoviral vaccines alone or in combination with plasmid vaccines to induce CTL. Cellular Immunity Assays for Pre-Clinical and Clinical Research Another aspect of this invention is an assay which measures the elicitation of HIV-1 protein, including gag-specific cellular immunity, particularly cytotoxic T-lymphocyte (CTL) responses. The "ELIspot" and cytotoxicity assays, discussed herein, measure HIV antigen-specific CD8+and CD4+T lymphocyte responses and can be used for a variety of mammals, such as humans, rhesus monkeys, mice, and rats.

The ELIspot assay provides a quantitative determination of HIV-specific T lymphocyte responses. PMBC cells are cultured in tissue culture microtiter plates. An HIV-1 gag peptide pool that encompasses the entire 500 amino acid open reading frame of gag (50 overlapping 20 mer peptides) is added to the cells and one day later the number of cells producing gamma interferon (or another selected interferon) is measured. Gamma interferon was selected as the cytokine visualized in this assay (using species specific anti-gamma interferon monoclonal antibodies) because it is the most common, and one of the most abundant cytokines synthesized and secreted by activated T lymphocytes. For this assay, the number of spot forming cells (SPC) per million PBMCs is determined for samples in the presence and absence (media control) of peptide antigens. This assay may be set up to determine overall T lymphocyte responses (both CD8+ and CD4+) or for specific cell populations by prior depletion of either CD8+ or CD4+ T cells. In addition, ELIspot assays, or variations of it, can be used to determine which peptide epitopes are recognized by particular individuals.

A distinguishing effector function of T lymphocytes is the ability of subsets of this cell population to directly lyse cells exhibiting appropriate MHC-associated antigenic peptides. This cytotoxic activity is most often associated with CD8+ T lymphocytes but may also be exhibited by CD4+ T lymphocytes. We have optimized bulk culture CTL assays in which PBMC samples are infected with recombinant vaccinia viruses expressing antigens (e.g., gag) in vitro for approximately 14 days to provide antigen restimulation and expansion of memory T cells that are then tested for cytoxicity against autologous B cell lines treated either with peptide antigen pools. Specific cytotoxicity is measured compared to irrelevant antigen or excipient-treated B cell lines. The phenotype of responding T lymphocytes is determined by appropriate depletion of either CD8+ or CD4+ populations prior to the cytotoxicity assay. This assay is semi-quantitative and is the preferred means for determining whether CTL responses were elicited by the vaccine.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLES

Example 1

Construction of Replication-Defective FG-Ad Expressing HIV Gag Antigen

Starting Vectors

Shuttle vector pHCMVIBGHpA1 contains Ad5 sequences from bp1 to bp 341 and bp 3534 to bp 5798 with a expression cassette containing human cytomegalovirus (HCMV) promoter plus intron A and bovine growth hormone polyadenylation signal.

The adenoviral backbone vector pAdE1-E3- (also named as phVad1) contains all Ad5 sequences except those nucleotides encompassing the E1 and E3 region.

Plasmid pV1JNStpaHIVgag contains tPA secretory signal sequence fused to the codon-optimized HIV gag nucleotides under the control of HCMV promoter plus intron A. It is described in pending U.S. patent application Ser. No. 09/017,981, filed Feb. 3, 1998 and WO98/34640, published Aug. 13, 1998, both of which are hereby incorporated by reference.

Plasmid pV1R-FLHIV gag (also named as pV1R-HIV gag-opt) contains codon-optimized full-length HIV gag under the control of the HCMV promoter plus intron A.

Construction of Ad5tpaHIV gag 1. Construction of adenoviral shuttle plasmid pA1-CMVI-tpaHIV gag containing tPAgag under the control of human CMV promoter and intron A.

The tPAgag insert was excised from pV1JNS-tPA gag by restriction enzymes PstI and XmaI, blunt-ended, and then cloned into EcoRV digested shuttle vector pHCMVIBGHpA1. The orientation of the transgene and the construct were verified by PCR using the insert specific primers hCMV5'-4 (5'TAG CGG CGG AGC TTC TAC ATC 3' SEQ.ID.NO. 2) and Gag3'-1 (5' ACT GGG AGG AGG GGT CGT TGC 3' SEQ.ID.NO.3), restriction enzyme analysis (RcaI, SspBI), and DNA sequencing spanning from CMV promoter to the initiation of the gag. 2. Homologous recombination to generate shuttle plasmid form of recombinant adenoviral vector pAd-CMVI-tpaHIV gag containing tpaHIV gag expression cassette.

Shuttle plasmid pA1-CMVI-tpaHIV gag was digested with restriction enzymes BstZ17 and SgrA1 and then co-transformed into E. coli strain BJ5183 with linearized (ClaI digested) adenoviral backbone plasmid pAdE1-E3-. One colony was verified by PCR analysis. The vector was transformed to competent E. coli HB 101 for large quantity production of the plasmid. 3. Generation of recombinant adenovirus Ad.CMVI-tpaHIV gag in 293 cells.

The shuttle plasmid was linearized by restriction enzyme PacI and transfected to 293 cells using CaPO.sub.4 method (InVitrogen kit). Ten days later, 10 plaques were picked and grown in 293 cells in 35-mm plates. PCR analysis of the adenoviral DNA indicated 10 out of 10 virus were positive for gag. 4. Evaluation of large scale recombinant adenovirus Ad.CMVI-tpaHIV gag

Clone No.9 was grown into large quantities through multiple rounds of amplification in 293 cells. One lot yielded of 1.7.times.10.sup.12 particles and a second lot yielded 6.7.times.10.sup.13 particles. The viral DNA was extracted by proteinase K digestion and confirmed by PCR and restriction enzyme (HindIII) analysis. The expression of tpaHIV gag was also verified by ELISA and Western blot analysis of the 293 or COS cells infected with the recombinant adenovirus. The recombinant adenovirus was used for evaluation in mice and rhesus monkeys.

Construction of Ad5.FHIV gag 1. Construction of adenoviral shuttle plasmid pA1-CMVI-FLHIV gag containing full length HIVgag under the control of human CMV promoter and intron A.

The FLHIV gag insert was excised from pV1R-FLHIV gag by restriction enzyme BglII and then cloned into BglII digested shuttle vector pHCMVIBGHpA1. The orientation and the construct were verified by PCR using the insert specific primers (hCMV5'-4 and Gag3'-1), restriction enzyme analysis, and DNA sequencing. 2. Homologous recombination to generate plasmid form of recombinant adenoviral vector pAd-CMVI-FLHIV gag containing FLHIV gag expression cassette.

Shuttle plasmid pA1-CMVI-FLHIV gag was digested with restriction enzymes BstZ17 and SgrA1 and then co-transformed into E. coli strain BJ5183 with linearized (ClaI digested) adenoviral backbone plasmid pAdE1-E3-. Colonies #6 and #7 were verified by PCR analysis. The vectors were transformed to competent E. coli HB101 for large quantity production of the plasmid. The plasmids were verified by HindIII digestion. 3. Generation of recombinant adenovirus Ad.CMVI-FLHIV gag in 293 cells.

The pAd plasmids were linearized by restriction enzyme PacI and transfected to 293 cells using Lipofectamine (BRL). Two weeks later, 6 viruses (#6-1.1, 6-1.2, 6-1.3, 7-1.1, 7-1.2, 7-1.3) were picked and grown in 293 cells in 35-mm plates. PCR analysis using the insert specific primers (hCMV5'-4 and Gag3'-1) of the adenoviral DNA verified the presence of HIV gag. 4. Evaluation of large scale recombinant adenovirus Ad. CMVI-FHIV gag

Virus clone #6-1 was grown into large quantities through multiple rounds of amplification in 293 cells. The viral DNA was extracted by proteinase K digestion and confirmed by PCR, restriction enzyme (HindIII, Bgl II, Bst E II, Xho I) analysis. A partial sequencing confirmed the junction between CMV promoter and the 5' end of HIV gag gene. The expression of FLHIV gag was also verified by ELISA and Western blot analysis of the 293 or COS cells infected with the recombinant adenovirus. The recombinant adenovirus was used for evaluation in mice and rhesus monkeys.

Construction of FG adenovirus FL gag.

The full-length (FL) humanized gag gene was ligated into an adenovirus-5 shuttle vector, pHCMVIBGHpA1, containing AdS sequences from bp 1 to bp 341 and bp 3534 to bp 5798 with a expression cassette containing human CMV promoter plus intron A and bovine growth hormone polyadenylation signal. The orientation was confirmed by restriction enzyme digestion analysis and DNA sequencing. Homologous recombination in E. coli was employed using the shuttle plasmid, pA1-CMVI-FLHIV gag, and adenoviral backbone plasmid, pAdE1-E3-, to generate a plasmid form of the recombinant adenovirus containing the expression regulatory elements and FL gag gene, pAd.CMVI-FHIV gag. Appropriate plasmid recombinants were confirmed by restriction enzyme digestion.

The pAd plasmid containing the gag expression cassette was linearized by restriction enzyme PacI and transfected to 293 cells (or PER.C6 cells for clinical development candidates) using Lipofectamine (BRL). Two weeks later, 6 viruses were picked and grown in 293 cells in 35-mm plates. PCR analysis using the insert specific primers (hCMV5'-4 and Gag3'-1) of the adenoviral DNA verified the presence of HIV gag. Virus clone #6-1 was grown into large quantities through multiple rounds of amplification in 293 cells. The viral DNA was extracted by proteinase K digestion and confirmed by PCR, restriction enzyme (HindIII, BglII, BstEII, XhoI) analysis. A partial sequencing confirmed the junction between CMV promoter and the 5' end of HIV gag gene. Restriction enzyme analysis demonstrated that the viral genome was stable over the course of these passages.

The expression of HIV gag was verified by ELISA and Western blot analysis of the 293 or COS cells infected with the recombinant adenovirus.

Example 2

Immunogenicity/Preclinical Efficacy

The "ELIspot" Assay

The ELIspot assay is a quantitative determination of IV-specific T lymphocyte responses by visualization of gamma interferon secreting cells in tissue culture microtiter plates one day following addition of an HIV-1 gag peptide pool that encompasses the entire 500 amino acid open reading frame of gag (50 overlapping 20 mer peptides) to PBMC samples. The number of spot forming cells (SPC) per million of PBMVs is determined for samples in the presence and absence (media control) of peptide antigens. The assay may be set up to determine overall T lymphocyte responses (both CD8+ and CD4+) or for specific cell populations by prior depletion of either CD8+ or CD4+ cells. In addition, the assay can be varied so as to determine which peptide epitopes are recognized by particular individuals.

Cytotoxic T Lymphocyte Assays

In this assay, PBMC samples are infected with recombinant vaccinia viruses expressing gag antigen in vitro for approximately 14 days to provide antigen restimulation and expansion of memory T cells. The cells are then tested for cytotoxicity against autologous B cell lines treated with peptide antigen pools. The phenotype of responding T lymphocytes is determined by appropriate depletion of either CD8+ or CD4+ cells.

A. Immune Responses to FG Adenovirus 5 FLgag Vaccine in Rodents

Adenovirus vectors coding for the gag antigen have consistently produced significantly stronger cellular immune responses than plasmid vectors in rodent species. Table 1 (below) shows ELIspot data from mice vaccinated with Ad5FLgag in comparison with plasmid DNA. Spleens from five mice were pooled and the number of gag peptide-specific interferon-gamma secreting cells was determined.

TABLE 1 Comparison of plasmid and adenovirus vaccination in mice SFC/10.sup.6 splenocytes Post 1st Post 2nd Vaccination Vaccination 10 .mu.g plasmid 68 324 10.sup.5 Ad5Flgag 18 170 10.sup.8 Ad5Flgag 530 5600

Similar enhancements in the cellular responses to gag were also seen in Fischer rats. FIG. 1 shows the ELIspot data from individual rats vaccinated with 10.sup.10 particles of adenovirus Ad5FLgag or with 0.4 mg FL gag plasmid. The mean response after one vaccination was 10-fold higher with adenovirus compared to plasmid. Both vaccines gave a boosted signal after a second vaccination, with the adenovirus vaccine signal 5-fold higher than the plasmid signal.

B. Immune Responses to FG Adenovirus 5 FL gag Vaccine in Rhesus Monkeys

Comparative in vivo expression of DNA vs. FGAd5 encoding a reporter gene.

Adenovirus and plasmid vectors expressing the secreted alkaline phosphatase (SEAP) as a reporter gene were injected into rhesus monkeys to compare the levels of antigen produced by the two forms of vaccination as shown below. FIG. 2 shows that at the highest possible plasmid dose (5 mg), the antigen levels are 1,000-fold lower than the levels achieved using 10.sup.10 particles of adenovirus, a dose which is ten fold lower than the maximum proposed clinical dose.

FG adenovirus-5 FLgag vaccinations of rhesus monkeys. Three Rhesus monkeys were vaccinated at 0, 8, and 24 weeks with 10.sup.11 p