Title:  Vaccines comprising synthetic genes

United States Patent:  6,534,312

Issued:  March 18, 2003

Inventors:  Shiver; John W. (Doylestown, PA); Davies; Mary Ellen (Norristown, PA); Freed; Daniel C. (King of Prussia, PA); Liu; Margaret A. (Rosemont, PA); Perry; Helen C. (Lansdale, PA)

Assignee:  Merck & Co., Inc. (Rahway, NJ)

Appl. No.:  340798

Filed:  June 28, 1999

Synthetic polynucleotides comprising a DNA sequence encoding a peptide or protein are provided. The DNA sequence of the synthetic polynucleotides comprise codons optimized for expression in a nonhomologous host. The invention is exemplified by synthetic DNA molecules encoding HIV env as well as modifications of HIV env. The codons of the synthetic molecules include the projected host cell's preferred codons. The synthetic molecules provide preferred forms of foreign genetic material. The synthetic molecules may be used as a polynucleotide vaccine which provides immunoprophylaxis against HIV infection through neutralizing antibody and cell-mediated immunity. This invention provides polynucleotides which, when directly introduced into a vertebrate in vivo, including mammals such as primates and humans, induces the expression of encoded proteins within the animal.

DETAILED DESCRIPTION OF THE INVENTION

Synthetic polynucleotides comprising a DNA sequence encoding a peptide or protein are provided. The DNA sequence of the synthetic polynucleotides comprise codons optimized for expression in a nonhomologous host. The invention is exemplified by synthetic DNA molecules encoding HIV env as well as modifications of HIV env are provided. The codons of the synthetic molecules include the projected host cell's preferred codons. The synthetic molecules provide preferred forms of foreign genetic material. The synthetic molecules may be used as a polynucleotide vaccine which provides immunoprophylaxis against HIV infection through neutralizing antibody and cell-mediated immunity. This invention provides polynucleotides which, when directly introduced into a vertebrate in vivo, including mammals such as primates and humans, induces the expression of encoded proteins within the animal.

Therefore, synthetic DNA molecules encoding HIV env and synthetic DNA molecules encoding modified forms of HIV env are provided. The codons of the synthetic molecules are designed so as to use the codons preferred by the projected host cell. As noted above, the synthetic molecules of this portion of the invention may be used as a polynucleotide vaccine which provides effective immunoprophylaxis against HIV infection through neutralizing antibody and cell-mediated immunity. The synthetic molecules may be used as an immunogenic composition. This portion of the invention also provides polynucleotides which, when directly introduced into a vertebrate in vivo, including mammals such as primates and humans, induces the expression of encoded proteins within the animal.

As used herein, a polynucleotide is a nucleic acid which contains essential regulatory elements such that upon introduction into a living, vertebrate cell, it is able to direct the cellular machinery to produce translation products encoded by the genes comprising the polynucleotide. In one embodiment of the invention, the polynucleotide is a polydeoxyribonucleic acid comprising at least one HIV gene operatively linked to a transcriptional promoter. In another embodiment of the invention, the polynucleotide vaccine (PNV) comprises polyribonucleic acid encoding at least one HIV gene which is amenable to translation by the eukaryotic cellular machinery (ribosomes, tRNAs, and other translation factors). Where the protein encoded by the polynucleotide is one which does not normally occur in that animal except in pathological conditions, (i.e., a heterologous protein) such as proteins associated with human immunodeficiency virus, (HIV), the etiologic agent of acquired immune deficiency syndrome, (AIDS), the animals' immune system is activated to launch a protective immune response. Because these exogenous proteins are produced by the animals' tissues, the expressed proteins are processed by the major histocompatibility system, MHC, in a fashion analogous to when an actual infection with the related organism (HIV) occurs. The result, as shown in this disclosure, is induction of immune responses against the cognate pathogen.

Accordingly, the instant inventors have prepared nucleic acids which, when introduced into the biological system induce the expression of HIV proteins and epitopes. The induced antibody response is both specific for the expressed HIV protein, and neutralizes HIV. In addition, cytotoxic T-lymphocytes which specifically recognize and destroy HIV infected cells are induced.

The instant invention provides a method for using a polynucleotide which, upon introduction into mammalian tissue, induces the expression in a single cell, in vivo, of discrete gene products. The instant invention provides a different solution which does not require multiple manipulations of rev dependent HIV genes to obtain rev-independent genes. The rev-independent expression system described herein is useful in its own right and is a system for demonstrating the expression in a single cell in vivo of a single desired gene-product.

Because many of the applications of the instant invention apply to anti-viral vaccination, the polynucleotides are frequently referred to as a polynucleotide vaccine, or PNV. This is not to say that additional utilities of these polynucleotides, in immune stimulation and in anti-tumor therapeutics, are considered to be outside the scope of the invention.

In one embodiment of this invention, a gene encoding an HIV gene product is incorporated in an expression vector. The vector contains a transcriptional promoter recognized by an eukaryotic RNA polymerase, and a transcriptional terminator at the end of the HIV gene coding sequence. In a preferred embodiment, the promoter is the cytomegalovirus promoter 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. A preferred transcriptional terminator is the bovine growth hormone terminator. The combination of CMVintA-BGH terminator is particularly preferred.

To assist in preparation of the polynucleotides in prokaryotic cells, an antibiotic resistance marker is also preferably included in the expression vector 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 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, the expression vector pnRSV is used, wherein the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) is used as the promoter. In another embodiment, V1, a mutated pBR322 vector into which the CMV promoter and the BGH transcriptional terminator were cloned is used. In another embodiment, the elements of V1 and pUC19 have been combined to produce an expression vector named V1J. Into V1J or another desirable expression vector is cloned an HIV gene, such as gp120, gp41, gp160, gag, pol, env, or any other HIV gene which can induce anti-HIV immune responses. In another embodiment, the ampicillin resistance gene is removed from V1J and replaced with a neomycin resistance gene, to generate V1J-neo into different HIV genes have been cloned for use according to this invention. In another embodiment, the vector is V1Jns, which is the same as V1Jneo except that a unique Sfi1 restriction site has been engineered into the single Kpn1 site at position 2114 of V1J-neo. The incidence of Sfi1 sites in human genomic DNA is very low (approximately 1 site per 100,000 bases). Thus, this vector allows careful monitoring for expression vector integration into host DNA, simply by Sfi1 digestion of extracted genomic DNA. In a further refinement, the vector is V1R. In this vector, as much non-essential DNA as possible was "trimmed" from the vector to produce a highly compact vector. This vector is a derivative of V1Jns. This vector allows larger inserts to be used, with less concern that undesirable sequences are encoded and optimizes uptake by cells.

One embodiment of this invention incorporates genes encoding HIV gp160, gp120, gag and other gene products from laboratory adapted strains of HIV such as SF2, IIIB or MN. Those skilled in the art will recognize that the use of genes from HIV-2 strains having analogous function to the genes from HIV-1 would be expected to generate immune responses analogous to those described herein for HIV-1 constructs. The cloning and manipulation methods for obtaining these genes are known to those skilled in the art.

It is recognized that elicitation of immune responses against laboratory adapted strains of HIV may not be adequate to provide neutralization of primary field isolates of HIV. Thus, in another embodiment of this invention, genes from virulent, primary field isolates of HIV are incorporated in the polynucleotide immunogen. This is accomplished by preparing cDNA copies of the viral genes and then subcloning the individual genes into the polynucleotide immunogen. Sequences for many genes of many HIV strains are now publicly available on GENBANK and such primary, field isolates of HIV are available from the National Institute of Allergy and Infectious Diseases (NIAID) which has contracted with Quality Biological, Inc., [7581 Lindbergh Drive, Gaithersburg, Md. 20879] to make these strains available. Such strains are also available from the World Health Organization (WHO) [Network for HIV Isolation and Characterization, Vaccine Development Unit, Office of Research, Global Programme on AIDS, CH-1211 Geneva 27, Switzerland]. From this work those skilled in the art will recognize that one of the utilities of the instant invention is to provide a system for in vivo as well as in vitro testing and analysis so that a correlation of HIV sequence diversity with serology of HIV neutralization, as well as other parameters can be made. Incorporation of genes from primary isolates of HIV strains provides an immunogen which induces immune responses against clinical isolates of the virus and thus meets a need as yet unmet in the field. Furthermore, as the virulent isolates change, the immunogen may be modified to reflect new sequences as necessary.

To keep the terminology consistent, the following convention is followed herein for describing polynucleotide immunogen constructs: "Vector name-HIV strain-gene-additional elements". Thus, a construct wherein the gp160 gene of the MN strain is cloned into the expression vector V1Jneo, the name it is given herein is: "V1Jneo-MN-gp160". The additional elements that are added to the construct are described in further detail below. As the etiologic strain of the virus changes, the precise gene which is optimal for incorporation in the pharmaceutical may be changed. However, as is demonstrated below, because CTL responses are induced which are capable of protecting against heterologous strains, the strain variability is less critical in the immunogen and vaccines of this invention, as compared with the whole virus or subunit polypeptide based vaccines. In addition, because the pharmaceutical is easily manipulated to insert a new gene, this is an adjustment which is easily made by the standard techniques of molecular biology.

The term "promoter" as used herein refers to a recognition site on a DNA strand to which the 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 termed "enhancers" or inhibiting sequences termed "silencers."

The term "leader" as used herein refers to a DNA sequence at the 5' end of a structural gene which is transcribed along with the gene. The leader usually results in the protein having an N-terminal peptide extension sometimes called a pro-sequence. For proteins destined for either secretion to the extracellular medium or a membrane, this signal sequence, which is generally hydrophobic, directs the protein into endoplasmic reticulum from which it is discharged to the appropriate destination.

The term "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 nor translated into protein.

The term "cassette" refers to the sequence of the present invention which contains the nucleic acid sequence which is to be expressed. The cassette is similar in concept to a cassette tape. Each cassette will have its own sequence. Thus by interchanging the cassette the vector will express a different sequence. Because of the restrictions sites at the 5' and 3' ends, the cassette can be easily inserted, removed or replaced with another cassette.

The term "3' untranslated region" or "3' UTR" refers to the sequence at the 3' end of a structural gene which is usually transcribed with the gene. This 3' UTR region usually contains the poly A sequence. Although the 3' UTR is transcribed from the DNA it is excised before translation into the protein.

The term "Non-Coding Region" or "NCR" refers to the region which is contiguous to the 3' UTR region of the structural gene. The NCR region contains a transcriptional termination signal.

The term "restriction site" refers to a sequence specific cleavage site of restriction endonucleases.

The term "vector" refers to some means by which DNA fragments can be introduced into a host organism or host tissue. There are various types of vectors including plasmid, bacteriophages and cosmids.

The term "effective amount" means sufficient PNV is injected to produce the adequate levels of the polypeptide. One skilled in the art recognizes that this level may vary.

To provide a description of the instant invention, the following background on HIV is provided. The human immunodeficiency virus has a ribonucleic acid (RNA) genome. This RNA genome must be reverse transcribed according to methods known in the art in order to produce a cDNA copy for cloning and manipulation according to the methods taught herein. At each end of the genome is a long terminal repeat which acts as a promoter. Between these termini, the genome encodes, in various reading frames, gag-pol-env as the major gene products: gag is the group specific antigen; pol is the reverse transcriptase, or polymerase; also encoded by this region, in an alternate reading frame, is the viral protease which is responsible for post-translational processing, for example, of gp160 into gp120 and gp41; env is the envelope protein; vif is the virion infectivity factor; rev is the regulator of virion protein expression; neg is the negative regulatory factor; vpu is the virion productivity factor "u"; tat is the trans-activator of transcription; vpr is the viral protein r. The function of each of these elements has been described.

In one embodiment of this invention, a gene encoding an HIV or SIV protein is directly linked to a transcriptional promoter. The env gene encodes a large, membrane bound protein, gp160, which is post-translationally modified to gp41 and gp120. The gp120 gene may be placed under the control of the cytomegalovirus promoter for expression. However, gp120 is not membrane bound and therefore, upon expression, it may be secreted from the cell. As HIV tends to remain dormant in infected cells, it is desirable that immune responses directed at cell-bound HIV epitopes also be generated. Additionally, it is desirable that a vaccine produce membrane bound, oligomeric ENV antigen similar in structure to that produced by viral infection in order to generate the most efficacious antibody responses for viral neutralization. This goal is accomplished herein by expression in vivo of a secreted gp140 epitope (gp140>gp120+ectodomain of gp41) or the cell-membrane associated epitope, gp160, to prime the immune system. However, expression of gp160 is repressed in the absence of rev due to non-export from the nucleus of non-spliced genes. For an understanding of this system, the life cycle of HIV must be described in further detail.

In the life cycle of HIV, upon infection of a host cell, HIV RNA genome is reverse-transcribed into a proviral DNA which integrates into host genomic DNA as a single transcriptional unit. The LTR provides the promoter which transcribes HIV genes from the 5' to 3' direction (gag, pol, env), to form an unspliced transcript of the entire genome. The unspliced transcript functions as the mRNA from which gag and pol are translated, while limited splicing must occur for translation of env encoded genes. For the regulatory gene product rev to be expressed, more than one splicing event must occur because in the genomic setting, rev and env overlap. In order for transcription of env to occur, rev transcription must stop, and vice versa. In addition, the presence of rev is required for export of unspliced RNA from the nucleus. For rev to function in this manner, however, a rev responsive element (RRE) must be present on the transcript [Malim et al., Nature 338:254-257 (1989)].

In the polynucleotide vaccine of this invention, the obligatory splicing of certain HIV genes is eliminated by providing fully spliced genes (i.e.: the provision of a complete open reading frame for the desired gene product without the need for switches in the reading frame or elimination of noncoding regions; those of ordinary skill in the art would recognize that when splicing a particular gene, there is some latitude in the precise sequence that results; however so long as a functional coding sequence is obtained, this is acceptable). Thus, in one embodiment, the entire coding sequence for gp160 is spliced such that no intermittent expression of each gene product is required.

The dual humoral and cellular immune responses generated according to this invention are particularly significant to inhibiting HIV infection, given the propensity of HIV to mutate within the population, as well as in infected individuals. In order to formulate an effective protective vaccine for HIV it is desirable to generate both a multivalent antibody response for example to gp160 (env is approximately 80% conserved across various HIV-1, lade B strains, which are the prevalent strains in US human populations), the principal neutralization target on HIV, as well as cytotoxic T cells reactive to the conserved portions of gp160 and, internal viral proteins encoded by gag. We have made an HIV vaccine comprising gp160 genes selected from common laboratory strains; from predominant, primary viral isolates found within the infected population; from mutated gp160s designed to unmask cross-strain, neutralizing antibody epitopes; and from other representative HIV genes such as the gag and pol genes (.about.95% conserved across HIV isolates.

Virtually all HIV seropositive patients who have not advanced towards an inimunodeficient state harbor anti-gag CTLs while about 60% of these patients show cross-strain, gp160-specific CTLs. The amount of HIV specific CTLs found in infected individuals that have progressed on to the disease state known as AIDS, however, is much lower, demonstrating the significance of our findings that we can induce cross-strain CTL responses.

Immune responses induced by our env and gag polynucleotide vaccine constructs are demonstrated in mice and primates. Monitoring antibody production to env in mice allows confirmation that a given construct is suitably immunogenic, i.e., a high proportion of vaccinated animals show an antibody response. Mice also provide the most facile animal model suitable for testing CTL induction by our constructs and are therefore used to evaluate whether a particular construct is able to generate such activity. Monkeys (African green, rhesus, chimpanzees) provide additional species including primates for antibody evaluation in larger, non-rodent animals. These species are also preferred to mice for antisera neutralization assays due to high levels of endogenous neutralizing activities against retroviruses observed in mouse sera. These data demonstrate that sufficient immunogenicity is engendered by our vaccines to achieve protection in experiments in a chimpanzee/HIV_IIIB challenge model based upon known protective levels of neutralizing antibodies for this system. However, the currently emerging and increasingly accepted definition of protection in the scientific community is moving away from so-called "sterilizing immunity", which indicates complete protection from HIV infection, to prevention of disease. A number of correlates of this goal include reduced blood viral titer, as measured either by HIV reverse transcriptase activity, by infectivity of samples of serum, by ELISA assay of p24 or other HIV antigen concentration in blood, increased CD4+ T-cell concentration, and by extended survival rates [see, for example, Cohen, J., Science 262:1820-1821, 1993, for a discussion of the evolving definition of anti-HIV vaccine efficacy]. The immunogens of the instant invention also generate neutralizing immune responses against infectious (clinical, primary field) isolates of HIV.

Immunology

A. Antibody Responses to env.

1. gp160 and gp120

An ELISA assay is used to determine whether vaccine vectors expressing either secreted gp120 or membrane-bound gp160 are efficacious for production of env-specific antibodies. Initial in vitro characterization of env expression by our vaccination vectors is provided by immunoblot analysis of gp160 transfected cell lysates. These data confirm and quantitate gp160 expression using anti-gp41 and anti-gp120 monoclonal antibodies to visualize transfectant cell gp160 expression. In one embodiment of this invention, gp160 is preferred to gp120 for the following reasons: (1) an initial gp120 vector gave inconsistent immunogenicity in mice and was very poorly or non-responsive in African green Monkeys; (2) gp160 contributes additional neutralizing antibody as well as CTL epitopes by providing the addition of approximately 190 amino acid residues due to the inclusion of gp41; (3) gp160 expression is more similar to viral env with respect to tetramer assembly and overall conformation, which may provide oligomer-dependent neutralization epitopes; and (4) we find that, like the success of membrane-bound, influenza HA constricts for producing neutralizing antibody responses in mice, ferrets, and nonhuman primates [see Ulmer et al., Science 259:1745-1749, 1993; Montgomery, D., et al., DNA and Cell Biol. 12:777-783, 1993] anti-gp160 antibody generation is superior to anti-gp120 antibody generation. Selection of which type of env, or whether a cocktail of env subfragments, is preferred is determined by the experiments outlined below.

2. Presence and Breadth of Neutralizing Activity

ELISA positive antisera from monkeys is tested and shown to neutralize both homologous and heterologous HIV strains.

3. V3 vs. Non-V3 Neutralizing Antibodies

A major goal for env PNVs is to generate broadly neutralizing antibodies. It has now been shown that antibodies directed against V3 loops are very strain specific, and the serology of this response has been used to define strains.

a. Non-V3 neutralizing antibodies appear to primarily recognize discontinuous, structural epitopes within gp120 which are responsible for CD4 binding. Antibodies to this domain are polyclonal and more broadly cross-neutralizing probably due to restraints on mutations imposed by the need for the virus to bind its cellular ligand. An in vitro assay is used to test for blocking gp120 binding to CD4 immobilized on 96 well plates by sera from immunized animals. A second in vitro assay detects direct antibody binding to synthetic peptides representing selected V3 domains immobilized on plastic. These assays are compatible for antisera from any of the animal types used in our studies and define the types of neutralizing antibodies our vaccines have generated as well as provide an in vitro correlate to virus neutralization.

b. gp41 harbors at least one major neutralization determinant, corresponding to the highly conserved linear epitope recognized by the broadly neutralizing 2F5 monoclonal antibody (commercially available from Viral Testing Systems Corp., Texas Commerce Tower, 600 Travis Street, Suite 4750, Houston, Tex. 77002-3005(USA), or Waldheim Pharmazeutika GmbH, Boltzmangasse 11, A1091 Wien, Austria), as well as other potential sites including the well-conserved "fusion peptide" domain located at the N-terminus of gp41. Besides the detection of antibodies directed against gp41 by immunoblot as described above, an in vitro assay test is used for antibodies which bind to synthetic peptides representing these domains immobilized on plastic.

4. Maturation of the Antibody Response

In HIV seropositive patients, the neutralizing antibody responses progress from chiefly anti-V3 to include more broadly neutralizing antibodies comprising the structural gp120 domain epitopes described above (#3), including gp41 epitopes. These types of antibody responses are monitored over the course of both time and subsequent vaccinations.

B. T Cell Reactivities Against env and gag.

1. Generation of CTL Responses

Viral proteins which are synthesized within cells give rise to MHC I-restricted CTL responses. Each of these proteins elicits CTL in seropositive patients. Our vaccines also are able to elicit CTL in mice. The immunogenetics of mouse strains are conducive to such studies, as demonstrated with influenza NP, [see Ulmer et al., Science 259:1745-1749, 1993]. Several epitopes have been defined for the HIV proteins env, rev, nef and gag in Balb/c mice, thus facilitating in vitro CTL culture and cytotoxicity assays. It is advantageous to use syngeneic tumor lines, such as the murine mastocytoma P815, transfected with these genes to provide targets for CTL as well as for in vitro antigen specific restimulation. Methods for defining immunogens capable of eliciting MHC class I-restricted cytotoxic T lymphocytes are known [see Calin-Laurens, et al., Vaccine 11(9):974-978, 1993; see particularly Eriksson, et al., Vaccine 11(8):859-865, 1993, wherein T-cell activating epitopes on the HIV gp120 were mapped in primates and several regions, including gp120 amino acids 142-192, 296-343, 367-400, and 410-453 were each found to induce lymphoproliferation; furthermore, discrete regions 248-269 and 270-295 were lymphoproliferative. A peptide encompassing amino acids 152-176 was also found to induce HIV neutralizing antibodies], and these methods may be used to identify immunogenic epitopes for inclusion in the PNV of this invention. Alternatively, the entire gene encoding gp160, gp120, protease, or gag could be used. For additional review on this subject, see for example, Shirai et al., J. Immunol 148:1657-1667, 1992; Choppin et al., J. Immunol 147:569-574, 1991; Choppin et al., J. Immunol 147:575-583, 1991; Berzofsky et al., J. Clin. Invest. 88:876-884, 1991. As used herein, T-cell effector function is associated with mature T-cell phenotype, for example, cytotoxicity, cytokine secretion for B-cell activation, and/or recruitment or stimulation of macrophages and neutrophils.

2. Measurement of T_H Activities

Spleen cell cultures derived from vaccinated animals are tested for recall to specific antigens by addition of either recombinant protein or peptide epitopes. Activation of T cells by such antigens, presented by accompanying splenic antigen presenting cells, APCs, is monitored by proliferation of these cultures or by cytokine production. The pattern of cytokine production also allows classification of T_H response as type 1 or type 2. Because dominant T_H 2 responses appear to correlate with the exclusion of cellular immunity in immunocompromised seropositive patients, it is possible to define the type of response engendered by a given PNV in patients, permitting manipulation of the resulting immune responses.

3. Delayed Type Hypersensitivity (DTH)

DTH to viral antigen after i.d. injection is indicative of cellular, primarily MHC II-restricted, immunity. Because of the commercial availability of recombinant HIV proteins and synthetic peptides for known epitopes, DTH responses are easily determined in vaccinated vertebrates using these reagents, thus providing an additional in vivo correlate for inducing cellular immunity.

Protection

Based upon the above immunologic studies, it is predictable that our vaccines are effective in vertebrates against challenge by virulent HIV. These studies are accomplished in an HIV_IIIB /chimpanzee challenge model after sufficient vaccination of these animals with a PNV construct, or a cocktail of PNV constructs comprised of gp160_IIIB, gag_IIIB, nef_IIIB and REV_IIIB. The IIIB strain is useful in this regard as the chimpanzee titer of lethal doses of this strain has been established. However, the same studies are envisioned using any strain of HIV and the epitopes specific to or heterologous to the given strain. A second vaccination/challenge model, in addition to chimpanzees, is the scid-hu PBL mouse. This model allows testing of the human lymphocyte immune system and our vaccine with subsequent HIV challenge in a mouse host. This system is advantageous as it is easily adapted to use with any HIV strain and it provides evidence of protection against multiple strains of primary field isolates of HIV. A third challenge model utilizes hybrid HIV/SIV viruses (SHIV), some of which have been shown to infect rhesus monkeys and lead to immunodeficiency disease resulting in death [see Li, J., et al., J. AIDS 5:639-646, 1992]. Vaccination of rhesus with our polynucleotide vaccine constructs is protective against subsequent challenge with lethal doses of SHIV.

PNV Construct Summary

HIV and other genes are ligated into an expression vector which has been optimized for polynucleotide vaccinations. Essentially all extraneous DNA is removed, leaving the essential elements of transcriptional promoter, immunogenic epitopes, transcriptional terminator, bacterial origin of replication and antibiotic resistance gene.

Expression of HIV late genes such as env and gag is rev-dependent and requires that the rev response element (RRE) be present on the viral gene transcript. A secreted form of gp120 can be generated in the absence of rev by substitution of the gp120 leader peptide with a heterologous leader such as from tPA (tissue-type plasminogen activator), and preferably by a leader peptide such as is found in highly expressed mammalian proteins such as immunoglobulin leader peptides. We have inserted a tPA-gp120 chimeric gene into V1Jns which efficiently expresses secreted gp120 in transfected cells (RD, a human rhabdomyosarcoma line). Monocistronic gp160 does not produce any protein upon transfection without the addition of a rev expression vector.

Representative Construct Components Include (but are not restricted to):

1. tPA-gp120_MN ;

2. gp160_IIIB ;

3. gag_IIIB : for anti-gag CTL;

4. tPA-gp120_IIIB ;

5. tPA-gp140

6. tPA-gp160 with structural mutations: V1, V2, and/or V3 loop deletions or substitutions

7. Genes encoding antigens expressed by pathogens other than HIV, such as, but not limited to, influenza virus nucleoprotein, hemagglutinin, matrix, neurarininidase, and other antigenic proteins; herpes simplex virus genes; human papillomavirus genes; tuberculosis antigens; hepatitis A, B, or C virus antigens.

The protective efficacy of polynucleotide HIV immunogens against subsequent viral challenge is demonstrated by immunization with the non-replicating plasmid DNA of this invention. This is advantageous since no infectious agent is involved, assembly of virus particles is not required, and determinant selection is permitted. Furthermore, because the sequence of gag and protease and several of the other viral gene products is conserved among various strains of HIV, protection against subsequent challenge by a virulent strain of HIV that is homologous to, as well as strains heterologous to the strain from which the cloned gene is obtained, is enabled.

The i.m. injection of a DNA expression vector encoding a gp160 results in the generation of significant protective immunity against subsequent viral challenge. In particular, gp160-specific antibodies and primary CTLs are produced. Immune responses directed against conserved proteins can be effective despite the antigenic shift and drift of the variable envelope proteins. Because each of the HIV gene products exhibit some degree of conservation, and because CTL are generated in response to intracellular expression and MHC processing, it is predictable that many virus genes give rise to responses analogous to that achieved for gp160. Thus, many of these genes have been cloned, as shown by the cloned and sequenced junctions in the expression vector (see below) such that these constructs are immunogenic agents in available form.

The invention offers a means to induce cross-strain protective immunity without the need for self-replicating agents or adjuvants. In addition, immunization with the instant polynucleotides offers a number of other advantages. This approach to vaccination should be applicable to tumors as well as infectious agents, since the CD8⁺ CTL response is important for both pathophysiological processes [K. Tanaka et al., Annu. Rev. Immunol. 6, 359 (1988)]. Therefore, eliciting an immune response against a protein crucial to the transformation process may be an effective means of cancer protection or immunotherapy. The generation of high titer antibodies against expressed proteins after injection of viral protein and human growth hormone DNA suggests that this is a facile and highly effective means of making antibody-based vaccines, either separately or in combination with cytotoxic T-lymphocyte vaccines targeted towards conserved antigens.

The ease of producing and purifying DNA constructs compares favorably with traditional methods of protein purification, thus facilitating the generation of combination vaccines. Accordingly, multiple constructs, for example encoding gp160, gp120, gp41, or any other HIV gene may be prepared, mixed and co-administered. Because protein expression is maintained following DNA injection, the persistence of B- and T-cell memory may be enhanced, thereby engendering long-lived humoral and cell-mediated immunity.

Standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the DNA immunogens of this invention. While standard techniques of molecular biology are therefore sufficient for the production of the products of this invention, the specific constructs disclosed herein provide novel polynucleotide immunogens which surprisingly produce cross-strain and primary HIV isolate neutralization, a result heretofore unattainable with standard inactivated whole virus or subunit protein vaccines.

The amount of expressible DNA or transcribed RNA 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 about 1 ng to 100 mg, and preferably about 10 .mu.g to 300 .mu.g 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 polynucleotide immunogen, boosting with HIV protein immunogens such as gp160, gp120, and gag gene products is also contemplated. Parenteral administration, such as intravenous, intramuscular, subcutaneous or other means of administration of interleukin-12 protein or GM-CSF or similar proteins alone or in combination, concurrently with or subsequent to parenteral introduction of the PNV of this invention is also advantageous.

The polynucleotide may be naked, that is, unassociated with any proteins, adjuvants or other agents which impact on the recipients' immune system. In this case, it is desirable for the polynucleotide to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, the DNA may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture, or the DNA 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.

Claim 1 of 12 Claims

What is claimed is:

1. A synthetic polynucleotide comprising a DNA sequence encoding HIV env protein or a fragment thereof, the DNA sequence comprising codons optimized for expression in a mammalian host, wherein said synthetic polynucleotide is selected from the group consisting of:

a) V1Jns-tPA-HIV_MN gp120, wherein the 5' end which is SEQ ID NO:4 and the 3' end which is SEQ ID NO:5;

b) V1Jns-tPA-HIV_IIIB gp120, wherein the 5' end which is SEQ ID NO:6 and the 3' end which is SEQ ID NO:7;

c) V1Jns-tPA-gp160/opt C1/opt41-A and V1Jns-tPA-gp160/opt C1/opt41-B, wherein the opt C1 comprises SEQ ID NO:30, and the gp120/41 proteolytic cleavage sites is retained in the "B" form (SEQ ID NO:33) and eliminated in the "A" form (SEQ ID NO:32);

d) V1Jns-tPA-gp160/opt all-A, V1Jns-tPA-gp160/opt all-B, V1Jns-tPA gp160/opt all-A (non_IIIB strains); V1Jns-tPA-gp160/opt all-B (non_IIIB strains), wherein the optimized codon usage is derived from opt C1 (SEQ ID NO:30), and wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32);

e) V1Jns-tPA-gp143, V1Jns-tPA-gp143/mutRRE-A, and V1Jns-tPA-gp143/mutRRE-B, wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32);

f) V1Jns-tPA-gp143/opt32-A and V1Jns-tPA-gp143/opt32-B, comprising a gp 32 opt sequence (SEQ ID NO:34), and wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32);

g) V1Jns-tPA-gp143/SRV-1 3'-UTR, wherein the SRV-1 3' UTR comprises SEQ ID NO:35;

h) V1Jns-tPA-gp143/opt C1/opt32A and V1Jns-tPA-gp143/opt C1/opt32B, wherein the optimized codon usage is derived from opt C1 (SEQ ID NO:30), and gp 32 opt (SEQ ID NO:34), and wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32);

i) V1Jns-tPA-gp143/opt all-A, V1Jns-tPA-gp143/opt all-B, V1Jns-tPA-gp143/opt all-A (non IIIB strains), and V1Jns-tPA-gp143/opt all-B (non IIIB strains), wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32); and,

j) V1Jns-tPA-gp143/opt32-A/glyB, V1Jns-tPA-gp143/opt32-B/glyB, V1Jns-tPA-gp143/opt C1/opt32-A/glyB, V1Jns-tPA-gp143/opt C1/opt32-B/glyB, V1Jns-tPA-gp143/opt all-A/glyB, V1Jns-tPA-gp143/opt all-B/glyB, V1Jns-tPA-gp143/opt all-A/glyB (non IIIB strains), V1Jns-tPA-gp143/opt all-B/glyB (non IIIB strains), which respectively contain gp 32 opt (SEQ ID NO:34) and/or opt C1 (SEQ ID NO:30), wherein the gp160 proteolytic cleavage site is retained in form "B" (SEQ ID NO:33) and is eliminated in form "A" (SEQ ID NO:32), and wherein the five carboxy-terminal amino acids of the expressed protein are NRLIKA (SEQ ID NO:27), and combinations thereof.
 

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