Title:  Adenovirus carrying gag gene HIV vaccine

United States Patent:  6,787,351

Issued:  September 7, 2004

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.:  818443

Filed:  March 27, 2001

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.

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.

Description of the Invention

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 MgCl₂ ; 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 MgCl₂, 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 1x10⁷ to 1x10¹² particles and preferably about 1x10¹⁰ to 1x10¹⁰ 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.

Claim 1 of 20 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.

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