CA2258568A1 - Vaccines comprising synthetic genes - Google Patents

Abstract

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

Description

CA 022~8~68 1998-12-18 TITLE OF THE INVENTION
VACCINES COMPRISING SYNTHETIC GENES

BACKGROI~NO OF THE INVENTION
5 1. HIV Infection:
~ Iuman Imrnunodeficiency Virus-1 (HIV-1) is the etiological agent of acquired human immune deficiency syndrome (AIDS) and related disorders. HIV-1 is an RNA virus of the Retroviridae family and exhibits the 5'LTR-gag-pol-env-LTR3' 10 org~ni7~ion of all retroviruses. In addition, H~V-1 comprises a handful of genes with regulatory or unknown functions, including the tat and rev genes. The env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gpl60) and then cleaved by a cellular protease to yield the external 120-kDa envelope 15 glycoprotein (gpl20) and the transmembrane 41-kDa envelope glycoprotein (gp41). Gpl20 and gp41 remain associated and are displayed on the viral particles and the surface of HIV-infected cells.
Gpl20 binds to the CD4 receptor present on the surface of helper T-lymphocytes, macrophages and other target cells. After gpl20 binds to 20 CD4, gp4 l mediates the fusion event responsible for virus entry.
Infection begins when gp 120 on the viral particle binds to the CD4 receptor on the surface of T4 Iymphocytes or other target cells.
The bound virus merges with the target cell and reverse transcribes its RNA genome into the double-stranded DNA of the cell. The viral DNA
25 is incorporated into the genetic material in the cell's nucleus, where the viral DNA directs the production of new viral RNA, viral proteins, and new virus particles. The new particles bud from the target cell membrane and infect other cells.
Destruction of T4 Iymphocytes, which are critical to 30 immllne defense, is a major cause of the progressive immune dysfunction that is the h~llm~rk of HIV infection. The loss of target cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.

.. ....

CA 022~8~68 1998-12-18 HIV-1 kills the cells it infects by replicating, budding from them and ~l~m~ging the cell membrane. HIV-1 may kill target cells indirectly by means of the viral gpl20 that is displayed on an infected cell's surface. Since the CD4 receptor on T cells has a strong affinity for 5 gpl20, healthy cells expressing CD4 receptor can bind to gpl20 and fuse with infected cells to form a syncytium. A syncytium cannot survlve.
HIV-1 can also elicit normal cellular immune defenses against infected cells. With or without the help of antibodies, cytotoxic 10 defensive cells can destroy an infected cell that displays viral proteins on its surface. Finally, free gpl20 may circulate in the blood of individuals infected with HIV-1. The free protein may bind to the CD4 receptor of uninfected cells, making them appear to be infected and evoking an immune response.
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. Most subunit vaccine approaches have not been 20 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.

25 2. Vaccines Vaccination is an effective form of disease prevention and has proven successful against several types of viral infection.
Determining ways to present HIV-1 antigens to the human immune system in order to evoke protective humoral and cellular immunity, is a 30 difficult task. To date, attempts to generate an effective HIV vaccine have not been successful. In AIDS patients, free virus is present in low levels only. Tr~n~mi.~sion of HIV-l is enhanced by cell-to-cell interaction via fusion and syncytia formation. Hence, antibodies CA 022~8~68 1998-12-18 generated against free virus or viral subunits are generally ineffective in elimin~ting virus-infected cells.
Vaccines exploit the body's ability to "remember" an antigen. After first encounters with a given antigen the immlme system S generates cells that retain an immunological memory of the antigen for an individual's lifetime. Subsequent exposure to the antigen stim~ tes the immune response and results in elimin~tion or inactivation of the pathogen.
The immune system deals with pathogens in two ways: by 10 humoral and by cell-mediated responses. In the humoral response Iymphocytes generate specific antibodies that bind to the antigen thus inactivating the pathogen. The cell-mediated response involves cytotoxic and helper T Iymphocytes that specifically attack and destroy infected cells.
Vaccine development with HIV-1 virus presents problems because HIV-1 infects some of the same cells the vaccine needs to activate in the immune system (i.e., T4 lymphocytes). It would be advantageous to develop a vaccine which inactivates HIV before impairment of the immune system occurs. A particularly suitable type 20 of HIV vaccine would generate an anti-H~V immune response which recognizes HIV variants and which works in HIV-positive individuals who are at the beginning of their infection.
A major challenge to the development of vaccines against viruses, particularly those with a high rate of mutation such as the 25 human immunodeficiency virus, against which elicitation of neutralizing and protective immllne responses is desirable, is the diversity of the viral envelope proteins among different viral isolates or strains.
Because cytotoxic T-lymphocytes (CTLs) in both mice and humans are capable of recognizing epitopes derived from conserved internal viral 30 proteins, and are thought to be important in the immune response against viruses, efforts have been directed towards the development of CTL vaccines capable of providing heterologous protection against different viral strains.

CA 022~8~68 1998-12-18 WO 97/48370 PCT/USg7/lO!jl7 It is known that CD8+ CTLs kill virally-infected cells when their T cell receptors recognize viral peptides associated with MHC class I molecules. The viral peptides are derived from endogenously synthesized viral proteins, regardless of the protein's location or function within the virus. Thus, by recognition of epitopes from conserved viral proteins, CTLs may provide cross-strain protection.
Peptides capable of associating with MHC class I for CTL recognition originate from proteins that are present in or pass through the cytoplasm or endoplasmic reticulum. In general, exogenous proteins, which enter the endosomal processing pathway (as in the case of antigens presented by MHC class Il molecules), are not effective at generating CD8+ CTL responses.
Most efforts to generate CTL responses have used replicating vectors to produce the protein antigen within the cell or they have focused upon the introduction of peptides into the cytosol. These approaches have limi~tions that may reduce their utility as vaccines.
Retroviral vectors have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while m~int~ininp;
the ability of the recombinant virus to replicate, and the effectiveness of vectors such as vaccinia for subsequent immlmi7~tions may be compromised by immune responses against the vectors themselves.
Also, viral vectors and modified pathogens have inherent risks that may hinder their use in hllm~n~. Furthermore, the selection of peptide epitopes to be presented is dependent upon the structure of an individual's MHC antigens and, therefore, peptide vaccines may have limited effectiveness due to the diversity of MHC haplotypes in outbred populations.

3. DNA Vaccines Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555, (1986)] showed that CaPO4 precipitated DNA introduced into mice intraperitoneally (i.p.), intravenously (i.v.) or intramuscularly (i.m.) could be expressed. The i.m. injection of DNA expression vectors without CaCl2 treatment in mice resulted in the uptake of DNA by the CA 022~8~68 1998-12-18 muscle cells and expression of the protein encoded by the DNA . The plasmids were m:lint~ined episomally and did not replicate.
Subsequently, persistent expression has been observed after i.m.
injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats. The technique of using nucleic acids as therapeutic agents was reported in WO90/11092 (4 October 1990), in which naked polynucleotides were used to vaccinate vertebrates.
It is not necessary for the success of the method that immunization be intramuscular. The introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH
antibodies in the mice. A jet injector has been used to transfect skin, muscle, fat, and m~mm~ry tissues of living ~nim~l~. Various methods for introducing nucleic have been reviewed. Intravenous injection of a DNA:cationic liposome complex in mice was shown by Zhu et al., [Science 261:209-211 (9 July 1993) to result in systemic expression of a cloned transgene. ULrner et al., [Science 259:1745-1749, (1993)]
reported on the heterologous protection against influenza virus infection by intramuscular injection of DNA encoding influenza virus proteins.
The need for specific therapeutic and prophylactic agents capable of eliciting desired immune responses against pathogens and tumor antigens is met by the instant invention. Of particular importance in this therapeutic approach is the ability to induce T-cell immune responses which can prevent infections or disease caused even by virus strains which are heterologous to the strain from which the antigen gene was obtained. This is of particular concern when dealing with HIV as this virus has been recognized to mutate rapidly and many virulent isolates have been identified [see, for example, LaRosa et al., Science 249:932-935 (1990), identifying 245 separate HIV isolates]. In response to this recognized diversity, researchers have attempted to generate CTLs based on peptide immllni7~tion. Thus, T~k~h~hi et al., [Science 255:333-336 (1992)] reported on the induction of broadly cross-reactive cytotoxic T cells recogni7ing an HIV envelope (gpl60) determinant. However, those workers recognized the difficulty in CA 022~8~68 1998-12-18 WO 97/48370 PCT/~JS97/lOS17 achieving a truly cross-reactive CTL response and suggested that there is a dichotomy between the priming or restimulation of T cells, which is very stringent, and the elicitation of effector function, including cytotoxicity, from already stimulated CTLs.
S Wang et al. reported on elicitation of immllne responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. However, the level of immune responses achieved in these studies was very low. In addition, the Wang et al., DNA construct utilized an essentially genomic piece of HIV encoding contiguous Tat/rev-gpl60-Tat/rev coding sequences. As is described in detail below, this is a suboptimal system for obtaining high-level expression of the gpl60. It also is potentially dangerous because expression of Tat contributes to the progression of Kaposi's Sarcoma.
WO 93/17706 describes a method for vaccinating an ~nim~l against a virus, wherein carrier particles were coated with a gene construct and the coated particles are accelerated into cells of an ~nim~l.
ln regard to HIV, essentially the entire genome, minus the long terminal repeats, was proposed to be used. That method represents substantial risks for recipients. It is generally believed that constructs of HIV
should contain less than about 50% of the HIV genome to ensure safety of the vaccine; thi.s ensures that enzymatic moieties and viral regulatory proteins, many of which have unknown or poorly understood functions have been elimin~ted. Thus, a number of problems remain if a useful human HIV vaccine is to emerge from the gene-delivery technology.
The instant invention contemplates any of the known methods for introducing polynucleotides into living tissue to induce expression of proteins. However, this invention provides a novel immunogen for introducing HIV and other proteins into the antigen processing pathway to efficiently generate HIV-specific CTLs and antibodies. The ph~rm~ceutical is effective as a vaccine to induce both cellular and humoral anti-HIV and HIV neutralizing immune responses.
In the instant invention, the problems noted above are addressed and solved by the provision of polynucleotide immlmogens which, when introduced into an ~nim~l, direct the efficient expression of HIV

CA 022~8~68 1998-12-18 WO 97/48370 PCT/US97/lOS17 proteins and epitopes without the attendant risks associated with those methods. The immune responses thus generated are effective at recogni7ing HIV, at inhibiting replication of HIV, at identifying and killing cells infected with HIV, and are cross-reactive against many HIV
5 strains.

4. Codon Usage and Codon Context The codon pairings of org~ni~m~ are highly nonrandom, and differ from organism to organism. This information is used to 10 construct and express altered or synthetic genes having desired levels of translational efficiency, to determine which regions in a genome are protein coding regions, to introduce translational pause sites into heterologous genes, and to ascertain relationship or ancestral origin of nucleotide sequences.
The expression of foreign heterologous genes in transformed org~ni~ms is now commonplace. A large number of m~mm~ n genes, including, for example, murine and human genes, have been successfully inserted into single celled org~ni~m~. Standard techniques in this regard include introduction of the foreign gene to be expressed into a vector such as a plasmid or a phage and l-tili7ing that vector to insert the gene into an org~ni~m. The native promoters for such genes are commonly replaced with strong promoters compatible with the host into which the gene is inserted. Protein sequencing machinery permits elucidation of the amino acid sequences of even minute quantities of native protein. From these amino acid sequences, DNA sequences coding for those proteins can be inferred. DNA
synthesis is also a rapidly developing art, and synthetic genes corresponding to those inferred DNA sequences can be readily constructed.
Despite the burgeoning knowledge of expression systems and recombinant DNA, significant obstacles remain when one attempts to express a foreign or synthetic gene in an organism. Many native, active proteins, for example, are glycosylated in a manner different from that which occurs when they are expressed in a foreign host. For CA 022~8~68 l998- l2- l8 this reason, eukaryotic hosts such as yeast may be preferred to bacterial hosts for expressing many m~mm~ n genes. The glycosylation problem is the subject of continlling research.
Another problem is more poorIy understood. Often translation of a synthetic gene, even when coupled with a strong promoter, proceeds much less efficiently than would be expected. The same is frequently true of exogenous genes foreign to the expression organism. Even when the gene is transcribed in a sufficiently efficient manner that recoverable quantities of the translation product are produced, the protein is often inactive or otherwise different in properties from the native protein.
It is recognized that the latter problem is commonly due to differences in protein folding in various org~ni.cm~. The solution to this problem has been elusive, and the mech~nisms controlling protein folding are poorly understood.
- The problems related to translational efficiency are believed to be related to codon context effects. The protein coding regions of genes in all organisms are subject to a wide variety of functional constraints, some of which depend on the requirement for encoding a properly functioning protein, as well as a~ro~liate translational start and stop signals. However, several features of protein coding regions have been discerned which are not readily understood in terms of these constraints. Two important classes of such features are those involving codon usage and codon context.
It is known that codon utilization is highly biased and varies considerably between different org~ni~ms. Codon usage patterns have been shown to be related to the relative abundance of tRNA
isoacceptors. Genes encoding proteins of high versus low abundance show differences in their codon preferences. The possibility that biases 30 in codon usage alter peptide elongation rates has been widely discussed.
While differences in codon use are associated with differences in translation rates, direct effects of codon choice on translation have been difficult to demonstrate. Other proposed constraints on codon usage CA 022~8~68 1998-12-18 Wo 97148370 PCT/US97/10517 patterns include ma~imi7.in~ the fidelity of translation and optimi7.ing the kinetic efficiency of protein synthesis.
Apart from the non-random use of codons, considerable evidence has accumulated that codon/anticodon recognition is influenced 5 by sequences outside the codon itself, a phenomenon termed "codon context." There exists a strong influence of nearby nucleotides on the efficiency of suppression of nonsense codons as well as missense codons.
Clearly, the abundance of suppressor activity in natural bacterial populations, as well as the use of "termination" codons to encode 10 selenocysteine and phosphoserine require that termination be context-dependent. Similar context effects have been shown to influence the fidelity of translation, as well as the efficiency of translation initiation.
Statistical analyses of protein coding regions of E. coli have demonstrate another manifestation of "codon context." The presence of 15 a particular codon at one position strongly influences the frequency of occurrence of certain nucleotides in neighboring codons, and these context constraints differ markedly for genes expressed at high versus low levels. Although the context effect has been recognized, the predictive value of the statistical rules relating to preferred nucleotides 20 adjacent to codons is relatively low. This has limited the utility of such nucleotide preference data for selecting codons to effect desired levels of translational efficiency.
The advent of automated nucleotide sequencing equipment has made available large quantities of sequence data for a wide variety 25 of or~ni~m~. Understanding those data presents substantial difficulties.
For example, it is important to identify the coding regions of the genome in order to relate the genetic sequence data to protein sequences.
In addition~ the ancestry of the genome of certain org~ni~m~ is of substantial interest. It is known that genomes of some org~ni~m~ are of 30 mixed ancestry. Some sequences that are viral in origin are now stably incorporated into the genome of eukaryotic organisms. The viral sequences themselves may have originated in another substantially unrelated species. An understanding of the ancestry of a gene can be CA 022~8~68 1998-12-18 important in drawing proper analogies between related genes and their translation products in other org~nism~.
There is a need for a better underst~nclin~; of codon context effects on translation, and for a method for determining the appropriate codons for any desired translational effect. There is also a need for a method for identifying coding regions of the genome from nucleotide sequence data. There is also a need for a method for controlling protein folding and for insuring that a foreign gene will fold appropriately when expressed in a host. Genes altered or constructed in accordance with desired translational efficiencies would be of significant worth.
Another aspect of the practice of recombinant DNA
techniques for the expression by microorg~ni~m~ of proteins of industrial and pharmaceutical interest is the phenomenon of "codon preference". While it was earlier noted that the existing machinery for gene expression is genetically transformed host cel}s will "operate" to construct a given desired product, levels of expression attained in a microorganism can be subject to wide variation, depending in part on specific alternative forms of the amino acid-specifying genetic code present in an inserted exogenous gene. 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 a 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 microorg~nism~ has revealed endogenous DNA of E.

CA 022~8~68 1998-12-18 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 obt~ining high levels of expression of a leucine-5 rich polypeptide by an E. coli host will depend to some extent on thei~requency 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 e~press the polypeptide. Similarly, when yeast cells are the projected transformation host cells for expression of a 10 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 15 exogenous genes in successfully transformed host org~nism~-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 20 preferred form of foreign genetic material for practice of recombinant DNA techniques.

5. Protein Trafficking The diversity of function that typifies eukaryote cells 25 depends upon the structural differentiation of their membrane boundaries. To generate and m~int~in 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 30 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 CA 022~8~68 1998-12-18 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 S destinations. Over the past few years the dissection of the molecular machinery for targeting and loc~li7~tion of proteins has been studied vigorously. Defined sequence motifs have been identified on proteins which can act as 'address labels'. A number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins.
SUMMARY 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. 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 effective 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 m~mm~ls such as primates and hllm~ns, induces the expression of encoded proteins within the ~nim~l.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows HIV env cassette-based expression strategies.
Figure 2 shows DNA vaccine mediated anti-gpl20 responses.
Figure 3 shows anti-gpl20 ELISA titers of murine DNA
vaccinee sera.

CA 022~8~68 1998-12-18 WO 97/48370 PCTtUS97/10517 Figure 4 shows the relative expression of gpl20 after HIV
env PNV cell culture transfection.
Figure 5 shows the mean anti-gpl20 ELISA responses following tPA-gpl43/optA vs. opt~ DNA vaccination.
5Figure 6 shows the neutralization of HIV by murine DNA
vaccinee sera.
Figure 7 shows HIV neutralization by sera from murine HIV env DNA vaccinees.
Figure 8 is an immunoblot analysis of optimized HIV env DNA constructs.
Figure 9 shows anti-gpl20 ELISA responses in rhesus monkeys following final vaccination with gpl40 DNA and o-gpl60 protein.
Figure 10 shows SHIV neutralizing antibody responses of rhesus monkeys following final vaccination.
DETAILED DESCRIPTION OF THE INVENTION
Synthetic polynucleotides comprising a DNA sequence encoding a peptide or protein are provided. The DNA se~uence 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 m~mm~l~ such as - 30 primates and h~lm~ns, induces the expression of encoded proteins within the ~nim~l.
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 ~ . . .

CA 022~8~68 1998-12-18 synthetic molecules of this portion of the invention may be used as a polynucleotide vaccine which provides effective immunoprophylaxis against HIV infection through neutr~li7.in~ antibody and cell-mediated immlmity. The synthetic molecules may be used as an immunogenic 5 composition. This portion of the invention also provides polynucleotides which, when directly introduced into a vertebrate in vivo, including m~mm~ such as primates and humans, induces the expression of encoded proteins within the ~nim~l.
As used herein, a polynucleotide is a nucleic acid which 10 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 15 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 20 encoded by the polynucleotide is one which does not normally occur in that ~nim~l except in pathological conditions, (i.e., a heterologous protein) such as proteins associated with hllm~n immunodeficiency virus, (HIV), the etiologic agent of acquired immune deficiency syndrome, (AIDS), the ~nim~l~' immune system is activated to launch a 25 protective immune response. Because these exogenous proteins are produced by the ~nim~l~' tissues, the expressed proteins are processed by the major histocompatibility system, M HC, in a fashion analogous to when an actual infection with the related org~ni~m (HIV) occurs. The result, as shown in this disclosure, is induction of immune responses 30 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 CA 022~8~68 1998-12-18 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 m~mm~ n tissue, induces 5 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 10 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 15 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
20 polymerase, and a transcriptional termin~or 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 25 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 polynuc}eotides in prokaryotic cells, an antibiotic resistance marker is also preferably 30 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 CA 022~8~68 l998- l2- l8 polynucleotide by fermentation in prokaryotic or~ni~ms, 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 5 desirable to remove non-essential DNA sequences. It is also desirable that the vectors not be able to replicate in eukaryotic cells. This minimi7.es 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 10 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 15 terminator were cloned is used. In another embodiment, the elements of V1 and pUC19 have been combined to produce an expression vector named VlJ. Into VlJ or another desirable expression vector is cloned an HIV gene, such as gpl20, gp41, gpl60, gag, pol, env, or any other HIV gene which can induce anti-HlV immllne responses. In another 20 embodiment, the ampicillin resistance gene is removed from VlJ and replaced with a neomycin resistance gene, to generate VlJ-neo into different HIV genes have been cloned for use according to this invention. In another embodiment, the vector is VlJns, which is the same as VlJneo except that a unique Sfil restriction site has been 25 engineered into the single Kpnl site at position 2114 of VlJ-neo. The incidence of Sfil sites in hllm~n 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 Sfil digestion of extracted genomic DNA. In a further 30 refinement, the vector is VlR. 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 VlJns. This vector allows larger inserts to be used, with less concern that undesirable sequences are encoded and optimizes uptake by cells.

..

CA 022~8~68 l998- l2- l8 One embodiment of this invention incorporates genes encoding HIV gpl60, gpl20, gag and other gene products from ~ Iaboratory 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 5 having analogous function to the genes from HIV-l would be expected to generate immllne 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 10 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 15 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, lnc., [7581 20 Lindbergh Drive, Gaithersburg, Maryland 20879] to make these strains available. Such strains are also available from the World Health Organization (W HO) [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 25 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 30 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.

CA 022~8~68 l998- l2- l8 To keep the terminology consistent, the following convention is followed herein for describing polynucleotide immllnogen constructs: "Vector name-HIV strain-gene-additional elements". Thus, a construct wherein the gp~60 gene of the MN strain is cloned into the S expression vector VlJneo, the name it is given herein is: "VlJneo-MN-gpl60". 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 ph~ ceutical may be changed. However, as is demonstrated below, 10 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 ph~ ceutical is easily manipulated to insert a new gene, this is an 15 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 20 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 25 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 30 the ~yprol~riate 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 CA 022~8~68 l998- l2- l8 - 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 o~,vn sequence. Thus by interch~nging 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-CA 022~8~68 l998- l2- l8 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 gpl60 into gpl20 and 5 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, gpl60, which is post-translationally modified to gp41 and gpl20. The gpl20 gene may be placed under the control of the cytomegalovirus promoter for 15 expression. However, gpl20 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
20 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 gpl40 epitope (gpl40 > gpl20 + ectodomain of gp41) or the cell-membrane associated epitope, gpl60, to prime the immllne 25 system. However, expression of gpl60 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
30 RNA genome is reverse-transcribed into a proviral DNA which integrates into host genomic DNA as a single transcriptional unit. The LTE~ 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 CA 022~8~68 1998-12-18 gag and pol are tr~n~l~ted, 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 5 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 elimin~ted 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 elimin~tion 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 gpl60 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 form~ te an effective protective vaccine for HIV it is desirable to generate both a multivalent antibody response for example to gpl60 (env is approximately 80%
conserved across various HIV-1, clade B strains, which are the prevalent strains in US hllm~n populations), the principal neutralization target on HIV, as well as cytotoxic T cells reactive to the conserved portions of gpl60 and, internal viral proteins encoded by gag. We have made an HIV vaccine comprising gpl60 genes selected from common laboratory strains; from predomin~nt, primary viral isolates found within the infected population; from mutated gpl60s designed to nnm~.~k cross-strain, neutralizing antibody epitopes; and from other representative CA 022~8~68 1998-12-18 HIV genes such as the gag and pol genes (~95% conserved across HIV
isolates.
Virtually all HIV seropositive patients who have not advanced towards an immllnodeficient state harbor anti-gag CTLs while 5 about 60% of these patients show cross-strain, gpl60-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 andgag 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 ~nim~ show an antibody response. Mice also 15 provide the most facile ~nim~l 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 ~nim~l~. These 20 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 immllnogenicity is engendered by our vaccines to achieve protection in experiments in a chimpanzee/HIVIIIg challenge model based upon 25 known protective levels of neutralizing antibodies for this system.
However, the currently emerging and increasingly accepted definition of protection in the scientific commllnity 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 30 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 CA 022~8~68 1998-12-18 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.

5 Immunolo~y A. Antibody Responses to env.
1. ~pl60 and gpl20. An ELISA assay is used to determine whether vaccine vectors expressing either secreted gpl20 or membrane-bound gpl60 are efficacious for production of env-specific 10 antibodies. Initial in vitro characterization of env expression by our vaccination vectors is provided by immunoblot analysis of gpl60 transfected cell lysates. These data confirm and quantitate gpl60 expression using anti-gp41 and anti-gpl20 monoclonal antibodies to visualize transfectant cell gpl60 expression. In one embodiment of this 15 invention, gpl60 is preferred to gpl20 for the following reasons: (1) an initial gpl20 vector gave inconsistent immllnogenicity in mice and was very poorly or non-responsive in African green Monkeys; (2) gpl60 contributes additional neutralizing antibody as well as CTL
epitopes by providing the addition of approximately 190 amino acid 20 residues due to the inclusion of gp41; (3) gpl60 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 constructs for producing neutr~li7ing antibody responses 25 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-gpl60 antibody generation is superior to anti-gpl20 antibody generation. Selection of which type of env, or whether a cocktail of env subfragments, is preferred is determined by the 30 experiments outlined below.

2. Presence and Breadth of Neutralizin~ Activity.
ELISA positive antisera from monkeys is tested and shown to neutralize both homologous and heterologous HIV strains.

CA 022~8~68 1998-12-18 3. V3 vs. non-V3 Neutr~1i7in~ Antibodies. A major goal for env PNVs is to generate broadly neutr~li7ing 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 5 strains.
a. Non-V3 neutr~li7.in~ antibodies appear to primarily recognize discontinuous, structural epitopes within gpl20 which are responsible for CD4 binding. Antibodies to this domain are polyclonal and more broadly cross-neutralizing probably due to 10 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 gp l 20 binding to CD4 immobilized on 96 well p}ates by sera from immlmi7ed ~nim~ls. A second in vitro assay detects direct antibody binding to synthetic peptides representing selected V3 domains immobilized on 15 plastic. These assays are compatible for antisera from any of the ~nim~l 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 20 determin~nt, 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, TX 77002-3005(USA), or Waldheim Ph~rm~7eutika GmbH, Boltzmangasse 11, A-25 1091 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 im~nunoblot as described above, an in vitro assay test is used for antibodies which bind to synthetic peptides representing these domains immobilized on 30 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 gpl20 domain epitopes described above (#3), CA 022~8~68 l998- l2- l8 WO 97/48370 PCTtUS97/10517 including gp41 epitopes. These types of antibody responses are monitored over the course of both time and subsequent vaccinations.

B. T Cell Reactivities A~ainst env and gaB.
1. Generation of CTL Responses. Viralproteins 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 immlmogens capable of eliciting MHC class I-restricted cytotoxic T Iymphocytes 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
gpl20 were mapped in primates and several regions, including gpl20 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 immllnogenic epitopes for inclusion in the PNV of this invention. Alternatively, the entire gene encoding gpl60, gpl20, 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 stim~ tion of macrophages and neutrophils.

CA 022~8~68 1998-12-18 WO 97/48370 PCT/US97/lOS17 2. Measurement of T~ Activities. Spleen cell cultures derived from vaccinated ~nim~ are tested for recall to specific antigens by addition of either recombinant protein or peptide epitopes.
Act*ation of T cells by such antigens, presented by accompanying 5 splenic antigen presenting cells, APCs, is monitored by proliferation of these cultures or by cytol~ine production. The pattern of cytokine production also allows classification of TH response as type 1 or type 2.
Because domin~nt TH2 responses appear to correlate with the exclusion of cellular immunity in immunocompromised seropositive patients, it is 10 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 15 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.

20 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 HIVIIIg/chimpanzee challenge model after sufficient vaccination of 25 these ~nim~ls with a PNV construct, or a cocktail of PNV constructs comprised of gpl60IIIg, gagIIIg~ nefIIIB and REVIIIg. 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 30 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 hllm~n 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 CA 022~8~68 1998-12-18 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 immnnodeficiency disease resulting in death [see S Li, J., et al., J. AIDS 5:639-646, 1992]. Vaccination of rhesus with our polynucleotide vaccine constructs is protective against subse~uent 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, immllnogenic 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 gpl20 can be generated in the absence of rev by substitution of the gpl20 leader peptide with a heterologous leader such as from tPA (tissue-type plasminogen 20 activator), and preferably by a leader peptide such as is found in highly expressed m~mm~ n proteins such as immunoglobulin leader peptides.
We have inserted a tPA-gpl20 chimeric gene into VlJns which efficiently expresses secreted gpl20 in transfected cells (RD, a human rhabdomyosarcoma line). Monocistronic gpl60 does not produce any 25 protein upon transfection without the addition of a rev expression vector.

Representative Construct Components Include (but are not restricted to):
1 . tPA-gp 1 20MN;
- 30 2. gpl6oIIIB;
3. gagIIIB: for anti-gag CTL;
4 . tPA-gp 1 2oIIIB;
5. tPA-gpl40 .. . ., . ~ , , CA 022~8~68 1998-12-18 6. tPA-gpl60 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, neuraminidase, 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 immllni7.~tion 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 determin~nt selection is permitted.
lS 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 gpl60 results in the generation of significant protective immunity against subsequent viral challenge. In particular, gpl60-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 gpl60. 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 CA 022~8~68 1998-12-18 WO 97148370 PCT/US97tlO517 adjuvants. In addition, immllni7~tion 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. Imml-nol. 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 gpl60, gpl20, gp41, or any other HIV gene may be prepared, mixed and co-~(lmini~tered. Because protein expression is m~int~ined 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
immllnogens 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 lln~tt~in~ble with standard inactivated whole virus or subunit protein vaccines.
- 30 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 CA 022~8~68 1998-12-18 mg, and preferably about 10 ~lg to 300 ~lg is ~tlrnini~tered directly into muscle tissue. Subcutaneous injection, intradermal introduction, impression through the skin, and other modes of ~lmini~tration such as intraperitoneal, intravenous, or inh~l~tion delivery are also S 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 gpl60, gpl20, and gag gene products is also contemplated. Parenteral ~lmini~tration, such as intravenous, intramuscular, subcutaneous or other means of 10 ~mini~tration 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' 15 immllne 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 20 associated with an adjuvant known in the art to boost immllne 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 25 carriers. Techniques for coating microprojectiles coated with polynucleotide are known in the art and are also useful in connection with this invention.
The following examples are offered by way of illustration and are not intended to limit the invention in any manner.

CA 022~8~68 1998-12-18 Materials descriptions Vectors pF411 and pF412: These vectors were subcloned from vector pSP62 which was constructed in R. Gallo's lab. pSP62 is an S available reagent from Biotech Research Laboratories, Inc. pSP62 has a 12.5 kb XbaI fragment of the HXB2 genome subcloned from lambda HXB2. SalI and Xba I digestion of pSP62 yields to HXB2 fragments:
S'-XbaI/SalI, 6.5 kb and 3'- SalI/XbaI, 6 kb. These inserts were subcloned into pUC 18 at SmaI and SalI sites yielding pF411 (5'-10 XbaVSalI) and pF412 (3'-XbaI/SalI). pF411 contains gag/pol and pF412 contains tat/rev/env/nef.

Repligen reagents:
recombinant rev (IIIB), ~tRPl024-10 15 rec. gpl20 (IIIB), #RP1001-10 anti-rev monoclonal antibody, #RP1029-10 anti-gpl20 mAB, #lC1, #RPlO10-10 AIDS Research and Reference Reagent Program:
20 anti-gp41 mAB hybridoma, Chessie 8, #526 The strategies are designed to induce both cytotoxic T
lymphocyte (CTL) and neutralizing antibody responses to HIV, principally directed at the HIV gag (~95% conserved) and env (gpl60 or gpl20; 70-80% conserved) gene products. gpl60 contains the only 2~ kno~vn neutr~li7ing antibody epitopes on the HIV particle while the importance of anti-env and anti-gag CTL responses are highlighted by the known association of the onset of these cellular immunities with clearance of primary viremia following infection, which occurs prior to the appearance of neutralizing antibodies, as well as a role for CTL in 30 m;lint~ining disease-free status. Because HIV is notorious for its genetic diversity, we hope to obtain greater breadth of neutralizing antibodies by including several representative env genes derived from clinical isolates and gp4l (~90% conserved), while the highly conserved gag gene should generate broad cross-strain CTL responses.

CA 022~8~68 1998-12-18 Heterolo~ous Expression of HIV Late Gene Products HIV structural genes such as env and gag require expression of the HIV regulatory gene, rev, in order to efficiently S produce full-length proteins. We have found that rev-dependent expression of gag yielded low levels of protein and that rev itself may be toxic to cells. Although we achieved relatively high levels of rev-dependent expression of gpl60 in vitro this vaccine elicited low levels of antibodies to gpl60 following in vivo immllni7~tion with rev/gpl60 10 DNA. This may result from known cytotoxic effects of rev as well as increased difficulty in obt~ining rev function in myotubules cont~ining hundreds of nuclei (rev protein needs to be in the same nucleus as a rev-dependent transcript for gag or env protein expression to occur).
However, it has been possible to obtain rev-independent expression 15 using selected modifications of the env gene. Evaluation of these plasmids for vaccine purposes is under~vay.
In general, our vaccines have utilized primarily HIV (IIIB) env and gag genes for optimization of expression within our generalized vaccination vector, VlJns, which is comprised of a CMV immediate-20 early (IE) promoter, BGH polyadenylation site, and a pUC backbone.Varying efficiencies, depending upon how large a gene segment is used (e.g., gpl20 vs. gpl60), of rev-independent expression may be achieved for env by replacing its native secretory leader peptide with that from the tissue-specific pl~minogen activator (tPA) gene and expressing the 25 resulting chimeric gene behind the CMVIE promoter with the CMV
intron A. tPA-gpl20 is an example of a secreted gpl20 vector constructed in this fashion which functions well enough to elicit anti-gpl20 immune responses in vaccinated mice and monkeys.
Because of reports that membrane-anchored proteins may 30 induce much more substantial (and perhaps more specific for HIV
neutralization) antibody responses compared to secreted proteins as well as to gain additional immllne epitopes, we prepared VlJns-tPA-gpl60 and V13ns-rev/gpl60. The tPA-gpl60 vector produced detectable CA 022~8~68 1998-12-18 quantities of gpl60 and gpl20, without the addition of rev, as shown by immunoblot analysis of transfected cells, although levels of expression were much lower than that obtained for rev/gpl60, a rev-dependent gpl60-expressing plasmid. This is probably because inhibitory regions 5 (designated INS), which confer rev dependence upon the gpl60 transcript, occur at multiple sites within gpl60 including at the COOH-terminus of gp41 (see Figure 1 for schematic view of gpl43 construct strategies). A vector was prepared for a COOH-terminally truncated form of tPA-gpl60, tPA-gpl43, which was designed to increase the 10 overall expression levels of env by elimin~tion of these inhibitory sequences. The gpl43 vector also elimin~tes intracellular gp41 regions cont~ining peptide motifs (such as leu-leu) known to cause diversion of membrane proteins to the lysosomes rather than the cell surface. Thus, gpl43 may be expected to increase both expression of the enV protein 15 (by decreasing rev-dependence) and the efficiency of transport of protein to the cell surface compared to full-length gpl60 where these proteins may be better able to elicit anti-gpl60 antibodies following DNA vaccination. tPA-gpl43 was further modified by extensive silent mutagenesis of the rev response element (RRE) sequence (350 bp) to 20 elimin~te additional inhibitory sequences for expression. This construct, gpl43/mutRRE, was prepared in two forrns: either elimin~ting (form A) or retaining (form B) proteolytic cleavage sites for gpl20/41. Both forrns were prepared because of literature reports that vaccination of mice using uncleavable gpl60 expressed in vaccinia elicited much higher 25 levels of antibodies to gpl60 than did cleavable folms.
A quantitative ELISA for gpl60/gpl20 expression in cell transfectants was developed to determine the relative expression capabilities for these vectors. In vitro transfection of 293 cells followed by quantification of cell-associated vs. secreted/released gpl20 yielded 30 the following results: (1) tPA-gpl60 expressed 5-lOX less gpl20 than rev/gpl60 with similar proportions retained intracellularly vs.
trafficked to the cell surface; (2) tPA-gpl43 gave 3-6X greater secretion of gpl20 than rev/gpl60 with only low levels of cell-associated gpl~3, CA 022~8~68 1998-12-18 confirming that the cytoplasmic tail of gpl60 causes intracellular retention of gpl60 which can be overcome by partial deletion of this sequence; and, (3) tPA-gpl43/mutRRE A and B gave ~lOX greater expression levels of protein than did parental tPA-gpl43 while S elimin~tion of proteolytic processing was confirmed for forrn A.
Figure 4 presents representative data supporting points (1) - (3).
Thus, our strategy to increase rev-independent expression has yielded stepwise increases in overall expression as well as redirecting membrane-anchored gpl43 to the cell surface away from 10 lysosomes. It is important to note that it should be possible to insert gpl20 sequences derived from various viral isolates within a vector cassette containing these modifications which reside either at the NH2-terminus (tPA leader) or COOH-terminus (gp41), where few antigenic differences exist between different viral strains. In other words, this is 15 a generic construct which can easily be modified by inserting gpl20 derived from various primary viral isolates to obtain clinically relevant vaccmes.
To apply these expression strategies to viruses that are relevant for vaccine purposes and confirm the generality of our 20 approaches, we also prepared a tPA-gpl20 vector derived from a primary HIV isolate (cont~ining the North American consensus V3 peptide loop; macrophage-tropic and nonsyncytia-inducing phenotypes).
This vector gave high expression/secretion of gpl20 with transfected 293 cells and elicited anti-gpl20 antibodies in mice demonstrating that it 25 was cloned in a functional form. Primary isolate gpl60 genes will also be used for expression in the same way as for gpl60 derived from laboratory strains.

30 Immune Responses to HIV-l env Polynucleotide Vaccines:
African green (AGM) and Rhesus (RHM) monkeys which received gpl20 DNA vaccines showed low levels of neutralizing antibodies following 2-3 vaccinations, which could not be increased by additional vaccination. These results, as well as increasing awareness CA 022~8~68 1998-12-18 within the HIV vaccine field that oligomeric gpl60 is probably a more relevant target antigen for eliciting neutr~li7.in~ antibodies than gpl20 monomers (Moore and Ho, J. Virol. 67: 863 (1993)), have led us to focus upon obtaining effective expression of gpl60-based vectors (see 5 above). Mice and AGM were also vaccinated with the primary isolate derived tPA-gpl20 vaccine. These ~nim~ exhibited anti-V3 peptide (using homologous sequence) reciprocal endpoint antibody titers ranging from 500-5000, demonstrating that this vaccine design is functional for clinically relevant viral isolates.
The gpl60-based vaccines, rev-gpl60 and tPA-gpl60, failed to consistently elicit antibody responses in mice and nonhllm~n primates or yielded low antibody titers. Our initial results with the tPA-gpl43 plasmid yielded geometric mean titers > 103 in mice and AGM following two vaccinations. These data indicate that we have 15 significantly improved the immunogenicity of gpl60-like vaccines by increasing expression levels. This construct, as well as the tPA-gpl43/mutRRE A and B vectors, will continue to be characterized for antibody responses, especially for virus neutralization.
Significantly, gpl20 DNA vaccination produced potent 20 helper T cell responses in all lymphatic compartments tested (spleen, blood, inguinal, mesenteric, and iliac nodes) with TH 1-like cytokine secretion profiles (i.e., g-interferon and IL-2 production with little or no IL-4). These cytokines generally promote strong cellular immunity and have been associated with m~intenance of a disease-free state for 25 HIV-seropositive patients. Lymph nodes have been shown to be primary sites for HIV replication, harboring large reservoirs of virus even when virus cannot be readily detected in the blood. A vaccine which can elicit anti-HIV immune responses at a variety of Iymph sites, such as we have shown with our DNA vaccine, may help prevent 30 successful colonization of the lymphatics following initial infection.
As stated previously, we consider realization of the following objectives to be essential to maximize our chances for success with this program: (1) env-based vectors capable of generating stronger neutralizing antibody responses in primates; (2) gag and env vectors CA 022~8~68 1998-12-18 which elicit strong T-lymphocyte responses as characterized by CTL
and helper effector functions in primates; (3) use of env and gag genes from clinically relevant HIV-l strains in our vaccines and characterization of the immunologic responses, especially neutralization 5 of primary isolates, they elicit; (4) demonstration of protection in an ~nim~l challenge model such as chimpanzee/HIV (IIIB) or rhesus/SHIV
using appropriate optimized vaccines; and, (5) determination of the duration of immune responses appropriate to clinical use. Significant progress has been made on the first three of these objectives and 10 experiments are in progress to determine whether our recent vaccination constructs for gpl60 and gag will improve upon these initial results.

15 Vectors For Vaccine Production A. VlJneo EXPRESSION VECTOR~ SEO. ID 1:
It was necessary to remove the ampr gene used for antibiotic selection of bacteria harboring VlJ because ampicillin may not be used in large-scale fermenters. The ampr gene from the pUC
20 backbone of VlJ was removed by digestion with SspI and Eaml 105I
restriction enzymes. The rem~ining plasmid was purified by agarose gel electrophoresis, blunt-ended with T4 DNA polymerase, and then treated with calf intestinal :~lk~line phosphatase. The commercially available kanr gene, derived from transposon 903 and contained within 25 the pUC4K plasmid, was excised using the PstI restriction enzyme, purified by agarose gel electrophoresis, and blunt-ended with T4 DNA
polymerase. This fragment was ligated with the VlJ backbone and plasmids with the kanr gene in either orientation were derived which were designated as VlJneo #'s 1 and 3. Each of these plasmids was 30 confirmed by restriction enzyme digestion analysis, DNA sequencing of the junction regions, and was shown to produce similar quantities of plasmid as VlJ. Expression of heterologous gene products was also comparable to VlJ for these VlJneo vectors. We arbitrarily selected VlJneo#3, referred to as VlJneo hereafter (SEQ ID:1), which contains CA 022~8~68 1998-12-18 the kanr gene in the same orientation as the ampr gene in VlJ as the expression construct.

B. V13ns Expression Vector:
An Sfi I site was added to VlJneo to facilitate integration studies. A commercially available 13 base pair Sfi I linker (New England BioLabs) was added at the Kpn I site within the BGH sequence of the vector. VlJneo was linearized with Kpn I, gel purified, blunted by T4 DNA polymerase, and ligated to the blunt Sfi I linker. Clonal isolates were chosen by restriction mapping and verified by sequencing through the linker. The new vector was designated VlJns. Expression of heterologous genes in VlJns (with Sfi I) was comparable to expression of the same genes in VlJneo (with Kpn I).

1~ C. VlJns-tPA:
In order to provide an heterologous leader peptide sequence to secreted and/or membrane proteins, VlJn was modified to include the human tissue-specific pl~minogen activator (tPA) leader. Two synthetic complementary oligomers were annealed and then ligated into VlJn which had been BglII digested. The sense and antisense oligomers were 5'-GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC
TGT GTG CTG CTG CTG TGT GGA GCA GTC l~C GTT TCG CCC
AGC GA-3' (SEQ.ID:2), and 5'-GAT CTC GCT GGG CGA AAC GAA
GAC TGC TCC ACA CAG CAG CAG CAC ACA GCA GAG CCC
TCT CTT CAT TGC ATC CAT GGT-3' (SEQ. ID:3). The Kozak sequence is underlined in the sense oligomer. These oligomers have overhanging bases compatible for ligation to BglII-cleaved sequences.
After ligation the upstream BglII site is destroyed while the downstream BglII is retained for subsequent ligations. Both the junction sites as well as the entire tPA leader sequence were verified by DNA sequencing.
Additionally, in order to conform with our consensus optimized vector VlJns (=VlJneo with an SfiI site), an SfiI restriction site was placed at the KpnI site within the BGH terminator region of VlJn-tPA by blunting the KpnI site with T4 DNA polymerase followed by ligation CA 022~8~68 1998-12-18 with an SfiI linker (catalogue #1138, New F.n~l~n~l Biolabs). This modification was verified by restriction digestion and agarose gel electrophoresis .

I. HIV env Vaccine Constructs:
Vaccines Producing Secreted env-derived Anti~en (gp120 and gp140):
Expression of the rev -dependent env gene as gpl20 was conducted as follows: gpl20 was PCR-cloned from the MN strain of HIV with either the native leader peptide sequence (VlJns-gpl20), or as a fusion with the tissue-plasminogen activator (tPA) leader peptide replacing the native leader peptide (VlJns-tPA-gpl20). tPA-gpl20 expression has been shown to be rev-independent [B.S. Chapman et al., Nuc. Acids Res. 19, 3979 (1991); it should be noted that other leader sequences would provide a similar function in rendering the gpl20 gene rev independent]. This was accomplished by preparing the following gpl20 constructs lltili7ing the above described vectors.

gp 120 Vaccine Constructs:
A. VlJns-tPA-HIV~N ~pl20:
HIVMN gpl20 gene (~edimmune) was PCR amplified using oligomers designed to remove the first 30 amino acids of the peptide leader sequence and to facilitate cloning into VlJns-tPA creating a chimeric protein consisting of the tPA leader peptide followed by the rem~ining gpl20 sequence following amino acid residue 30. This design allows for rev -independent gpl20 expression and secretion of soluble gpl20 from cells harboring this plasmid. The sense and antisense PCR oligomers used were 5'-CCC CGG ATC CTG ATC ACA
GAA AAA TTG TGGGTC ACA GTC-3' (SEQ. ID:4), and 5'-C CCC
AGG AATC CAC CTG TTA GCG CTT TTC TCT CTG CAC CAC
TCT TCT C-3' (SEQ. ID:5). The translation stop codon is underlined.
These oligomers contain BamHI restriction enzyme sites at either end of the translation open reading frame with a BclI site located 3' to the CA 022~8~68 1998-12-18 BamHI of the sense oligomer. The PCR product was sequentially digested with BclI followed by BamHI and ligated into VlJns-tPA which had been BglII digested followed by calf intestinal ~Ik~line phosphatase treatrnent. The resulting vector was sequenced to confirm in-frame 5 fusion between the tPA leader and gpl20 coding sequence, and gpl20 expression and secretion was verified by immllnoblot analysis of transfected RB cells.

B. VlJns-tpA-HIvnIR ~pl20:
This vector is analogous to I.A. except that the HIV IIIB
strain was used for gpl20 sequence. The sense and antisense PCR
oligomers used were: 5'-GGT ACA TGA TCA CA GAA AAA TTG
TGG GTC ACA GTC-3' (SEQ.ID:6), and 5'-CCA CAT TGA TCA
GAT ATC TTA TCT TTT TTC TCT CTG CAC CAC TCT TC-3' 15 (SEQ.ID:7), respectively. These oligomers provide BclI sites at either end of the insert as well as an EcoRV just upstream of the BclI site at the 3'-end. The 5'-terminal BclI site allows ligation into the BglII site of VlJns-tPA to create a chimeric tPA-gpl20 gene encoding the tPA
leader sequence and gpl20 without its native leader sequence. Ligation 20 products were verified by restriction digestion and DNA sequencing.

gp l 40 Vaccine Constructs:
These constructs was prepared by PCR similarly as tPA-25 gpl20 with the tPA leader in place of the native leader, but designed toproduce secreted antigen by termin~ting the gene immediately NH2-terminal of the transmembrane peptide (projected carboxyterminal amino acid sequence = NH2-.. TNWLWYIK-COOH) [SEQ.ID:8].
Unlike the gpl20-producing constructs, gpl40 constructs should 30 produce oligomeric antigen and retain known gp4 1 -contained antibody neutralization epitopes such as ELDKWA (SEQ.ID:53) defined by the 2F5 monoclonal antibody.
Constructs were prepared in two forms (A or B) depending upon whether the gpl60 proteolytic cleavage sites at the junction of W O 97/48370 PCTrUS97/10517 gpl20 and gp41 were retained (B) or elimin~ted (A) by appropriate amino acid substitutions as described by Kieny et al., (Prot. Eng. 2:
219-255 (1988)) (wild type sequence = NH2-...KAKRRVVQREKR...COOH (SEQ.ID:9) and the mutated sequence =
S NH2-...KAQNHvvQNEHo...cooH (sEQ~ o) withmllt~tedamino acids underlined).

A. V lJns-tPA-gp 1 40/mutRRE-A/SRV- 1 3'-UTR (based on HIV-l ~IIB~:
This construct was obtained by PCR using the following sense and antisense PCR oligomers: S'-CT GAA AGA CCA GCA ACT
CCT AGG GAAT l~G GGG l~G CTC TGG-3' (SEQ.ID: 11 ) :, and 5'-CGC AGG GGA GGT GGT CTA GAT ATC l~A TTA TTT TAT
ATA CCA CAG CCA ATT TGT TAT G-3' (SEQ ID:12) to obtain an AvrII/EcoRV segment from vector IVB (con~ining the optimized RRE-A segment). The 3'-UTR, prepared as a synthetic gene segment, that is derived from the Simian Retrovirus-l (SRV-1, see below) was inserted into an SrfT restriction enzyme site introduced immediately 3'- of the gpl40 open reading frame. This UTR sequence has been described previously as facilitating rev-independent expression of HIV env and gag.

B . V 1 Jns-tPA-gp l 40/mutRRE-B/SRV- 1 3'-UTR (based on HIV-~IIIB):
This construct is similar to TlA except that the env proteolytic cleavage sites have been retained by using construct IVC as starting material.

C. VlJns-tPA-gpl40/opt30-A (based on HTV-lnn~):
This construct was derived from IVB by AvrTI and Srfl restriction enzyme digestion followed by ligation of a synthetic DNA
segment corresponding to gp30 but comprised of optimal codons for translation (see gp32-opt below). The gp30-opt DNA was obtained from gp32-opt by PCR amplification using the following sense and anti-CA 022~8~68 1998-12-18 WO 97/48370 PCT/US97tlO517 sense oligomers: 5'-GGT ACA CCT AGG CAT CTG GGG CTG CTC
TGG-3', (SEQ ID:13) and, 5'-CCA CAT GAT ATC G CCC GGG C
TTA TTA l l l GAT GTA CCA CAG CCA GTT GGT GAT G-3', (SEQ ID:14), respectively. This DNA segment was digested with AvrII
5 and EcoRV restriction enzymes and ligated into VlJns-tPA-gpl43/opt32-A (IVD) that had been digested with AvrII and SrfI to remove the corresponding DNA segment. The resulting products were verified by DNA sequencing of ligation junctions and immunoblot analysis.
D. VlJns-tPA-g;pl40/opt30-B (based on HIV~
This construct is similar to IIC except that the env proteolytic cleavage sites have been retained.

15 E. VlJns-tPA-~pl40/opt all-A:
The env gene of this construct is comprised completely of optimal codons. The constant regions (C1, C5, gp32) are those described in IVB,D,H with an additional synthetic DNA segment corresponding to variable regions 1-5 is inserted using a synthetic DNA
20 segment comprised of optimal codons for translation (see example below based on HIV-1 MN V1-V5).

F. VlJns-tPA-gpl40/opt all-B:
This construct is similar to IE except that the env 25 proteolytic cleavage sites have been retained.

G. VlJns-tPA-gpl40/opt all-A (non-IIIB strains):
This construct is similar to IIE above except that env amino acid sequences from strains other than IIIB are used to determine 30 optimum codon usage throughout the variable (V1-V5) regions.

H. VlJns-tPA-gpl40/opt all-B (non-IIIB strains):
This construct is similar to IIG except that the env proteolytic cleavage sites have been retained.

, . . ~.

CA 022~8~68 1998-12-18 ~p l 60 Vaccine Constructs:
Constructs were prepared in two forms (A or B) depending upon whether the gpl60 proteolytic cleavage sites as described above.

A. V lJns-rev/env:
This vector is a variation of the one described in section D
above except that the entire tat coding region in exon l is deleted up to the beginning of the rev open reading frame. VlJns-gpl60IIIg (see section A. above) was digested with Pstl and KpnI restriction enzymes to remove the 5'-region of the gpl60 gene. PCR amplification was used to obtain a DNA segment encoding the first REV exon up to the KpnI
site in gpl60 from the HXB2 genomic clone. The sense and antisense PCR oligomers were 5'-GGT ACA CTG CAG TCA CCG TCC T ATG
GCA GGA AGA AGC GGA GAC-3' (SEQ.ID:15) and 5'-CCA CAT
CA GGT ACC CCA TAA TAG ACT GTG ACC-3'(SEQ.ID:16) respectively. These oligomers provide PstI and KpnI restriction enzyme sites at the 5'- and 3'- termini of the DNA fragment, respectively. The resulting DNA was digested with PstI and KpnI, purified from an agarose electrophoretic gel, and ligated with VlJns-gpl60(PstI/KpnI). The resulting plasmid was verified by restriction enzyme digestion.

B. VlJns-~160:
HIVIIIb gpl60 was cloned by PCR amplification from plasmid pF412 which contains the 3'-terminal half of the ~IVIIIb genome derived from HIVIIIb clone HXB2. The PCR sense and antisense oligomers were 5'-GGT ACA TGA TCA ACC ATG AGA
GTG AAG GAG AAA TAT CAG C-3'(SEQ.ID:17), and 5'-CCA CAT
TGA TCA GAT ATC CCC ATC TTA TAG CAA AAT CCT TTC C-3' (SEQ. ID: 18), respectively. The Kozak sequence and translation stop codon are underlined. These oligomers provide BclI restriction enzyme sites outside of the translation open reading frame at both ends of the env gene. (BclI-digested sites are compatible for ligation with BglII-CA 022~8~68 1998-12-18 WO 97/48370 PCT/US97/lOS17 digested sites with subsequent loss of sensitivity to both restriction enzymes. BclI was chosen for PCR-cloning gpl60 because this gene contains internal Bglrl and as well as BamHI sites). The antisense oligomer also inserts an EcoRV site just prior to the BclI site as described above for other PCR-derived genes. The amplified gpl60 gene was agarose gel-purified, digested with BclI, and ligated to VlJns which had been digested with BglII and treated with calf intestinal ~lk~line phosphatase. The cloned gene was about 2.6 kb in size and each junction of gpl60 with VlJns was confirmed by DNA sequencing.
C. VlJns-tPA-gpl60 (based on HIv-lTnR):
This vector is similar to Example l(C) above, except that the full-length gpl60, without the native leader se~uence, was obtained by PCR. The sense oligomer was the sarne as used in I.C. and the antisense oligomer was 5'-CCA CAT TGA TCA GAT ATC CCC ATC
TTA TAG CAA AAT CCT TTC C-3' (SEQ.ID:l9). These oligomers provide BclI sites at either end of the insert as well as an EcoRV just upstream of the BclI site at the 3'-end. The 5'-te~ninal BclI site allows ligation into the BglII site of VlJns-tPA to create a chimeric tPA-gpl60 gene encoding the tPA leader sequence and gpl60 without its native leader sequence. Ligation products were verified by restriction digestion and DNA sequencing.

D. VlJns-tPA-gpl60/opt C1/opt41 -A (~oased on HIV-1 lR~:
This construct was based on IVH, having a complete optimized codon segment for C5 and gp41, rather than gp32, with an additional optimized codon segment (see below) replacing C1 at the amino terminus of gpl20 following the tPA leader. The new C1 segment was joined to the rem~ining gpl43 segment via SOE PCR using the following oligomers for PCR to synthesize the joined C1/143 segment: 5'-CCT GTG TGT GAG TTT AAA C TGC ACT GAT TTG
AAG AAT GAT ACT AAT AC-3' (SEQ ID:20). The resulting gpl43 gene contains optimal codon usage except for Vl-V5 regions and has a .. ... .

CA 022~8~68 l998- l2- l8 unique PmeI restriction enzyme site placed at the junction of Cl and V1 for insertion of variable regions from other HIV genes.

E. VlJns-tPA-gp160/opt C1/opt41-B (based on HIV-lmp~:
S This construct is similar to IIID except that the env proteolytic cleavage sites have been retained.

F VlJns-tPA-~pl60/opt all-A (based on HIV-lmp~):
The env gene of this construct is comprised completely of optimal codons as described above. The constant regions (Cl, CS, gp32) are those described in IIID,E which is used as a cassette (employed for all completely optimized gpl60s) while the variable regions, V l-V5, are derived from a synthetic DNA segment comprised of optimal codons.
G. VlJns-tPA-gpl60/opt all-B:
This construct is similar to IIIF except that the env proteolytic cleavage sites have been retained.

H. VlJns-tPA-gpl60/opt all-A (non-IIIB strains):
This construct is similar to IIIF above except that env amino acid sequences from strains other than IIIB were used to determine optimum codon useage throughout the variable (Vl-VS) regions.
I. VlJns-tPA-gpl60/opt all-B (non-IIIB strains):
This construct is similar to IIIH except that the env proteolytic cleavage sites have been retained.

CA 022~8~68 1998-12-18 gpl43 Vaccine Constructs:
These constructs were prepared by PCR similarly as other tPA-cont~ining constructs described above (tPA-gpl20, tPA-gpl40, and 5 tPA-gpl60), with the tPA leader in place of the native leader, but designed to produce COOH-termin~ted, membrane-bound env (projected intracellular amino acid sequence= NH2-NRVRQGYSP-COOH). This construct was designed with the purpose of combining the increased expression of env accompanying tPA introduction and 10 minimi7.ing the possibility that a transcript or peptide region corresponding to the intracellular portion of env might negatively impact expression or protein stability/transport to the cell surface.
Constructs were prepared in two forms (A or B) depending upon whether the gpl60 proteo}ytic cleavage sites were removed or retained 15 as described above. The residual gp41 fragment resulting from truncation to gpl43 is referred to as gp32.

A. VlJns-tPA-~pl43:
This construct was prepared by PCR using plasmid pF412 20 with the following sense and antisense PCR oligomers: 5'-GGT ACA
TGA TCA CA GAA AAA TTG TGG GTC ACA GTC-3' (SEQ.ID:21):, and 5'- CCA CAT TGA TCA G CCC GGG C TTA GGG TGA ATA
GCC CTG CCT CAC TCT GTT CAC-3' (SEQ.ID:22). The resulting DNA segment contains BclI restriction sites at either end for cloning 25 into VlJns-tPA/BglII-digested with an Sr~ site located immediately 3'-to the env open reading frame. Constructs were verified by DNA
sequencing of ligation junctions and immunoblot analysis of transfected cells (Figure 8).

~ 30 B. VlJns-tPA-gpl43/mutRRE-A:
This construct was based on IVA by excising the DNA
~ segment using the unique MunI restriction enzyme site and the downstream SrfI site described above. This segment corresponds to a portion of the gpl20 C5 domain and the entirety of gp32. A synthetic - 4~ -,. . _ ~

CA 022~8~68 1998-12-18 DNA segment corresponding to ~350 bp of the rev response element (RRE A) of gpl60, comprised of optimal codons for translation, was joined to the rem~ining gp32 segment by splice overlap extension (SOE) PCR creating an AvrII restriction enzyme site at the junction of the two 5 segments (but no changes in amino acid sequence). These PCR reactions were performed using the following sense and antisense PCR oligomers for generating the gp32-cont~ining domain: 5'-CT GAA AGA CCA
GCA ACT CCT AGG GAT TTG GGG TTG CTG TGG-3' (SEQ ID:23) and 5'-CCA CAT TGA TCA G CCC GGG C TTA GGG TGA ATA
10 GCC CTG CCT CAC TCT GTT CAC-3' [SEQ ID:24] (which was used as the antisense oligomer for IVA), respectively. The mutated RRE
(mutRRE-A) segment was joined to the wild type sequence of gp32 by SOE PCR using the following sense oligomer, 5'-GGT ACA CAA TTG
GAG GAG CGA GTT ATA TAA ATA TAA G-3' (SEQ ID:25), and 15 the antisense oligomer used to make the gp32 segment. The resulting joined DNA segment was digested with MunI and SrfI restriction enzymes and ligated into the parent gpl43/Munl/SrfI digested plasmid.
The resulting construct was verified by DNA sequencing of ligation and SOE PCR junctions and immunoblot analysis of transfected cells (Figure 20 8).

C. VlJns-tPA-gp143/mutRRE-B:
This construct is similar to IVB except that the env proteolytic cleavage sites have been retained by using the mutRRE-B
25 synthetic gene segment in place of mutRRE-A.

D. V lJns-tPA-gpl43/opt32-A:
This construct was derived from IVB by AvrII and SrfI
restriction enzyme digestion followed by ligation of a synthetic DNA
30 segment corresponding to gp32 but comprised of optimal codons for translation (see gp32 opt below). The resulting products were verified by DNA sequencing of ligation junctions and immllnoblot analysis.

CA 022~8~68 1998-12-18 E. VlJns-tPA-gp143/opt32-B:
This construct is similar to IVD except that the env proteolytic cleavage sites have been retained by using IVC as the initial plasmid.
s F. VlJns-tPA-gpl43/SRV-1 3'-UTR:
This construct is similar to IVA except that the 3'-UTR
derived from the Simian Retrovirus-l (SRV-l, see below) was inserted into the SrfI restriction enzyme site introduced immediately 3'- of the 10 gpl43 open reading frame. This UTR sequence has been described previously as facilit~ting rev-independent expression of HIV env and gag.

G. VlJns-tPA-~pl43/opt C1/opt32A:
This construct was based on IVD, having a complete optimized codon segment for C5 and gp32 with an additional optimized codon segment (see below) replacing C1 at the amino terminus of gpl20 following the tPA leader. The new Cl segment was joined to the rem~ining gpl43 segment via SOE PCR using the following oligomers 20 for PCR to synthesize the joined C1/143 segment: 5'-CCT GTG TGT
GAG TTT AAA C TGC ACT GAT TTG AAG AAT GAT ACT AAT
AC-3' (SEQ ID:26). The resulting gpl43 gene contains optimal codon useage except for V1-V5 regions and has a unique PmeI restriction enzyme site placed at the junction of Cl and V1 for insertion of variable 25 regions from other HIV genes.

H. VlJns-tPA-~pl43/opt C1/opt32B:
This construct is similar to IVH except that the env proteolytic cleavage sites have been retained.

. . . ~ . .

CA 022~8~68 1998-12-18 I. VlJns-tPA-gpl43/opt all-A:
The env gene of this construct is comprised completely of optimal codons. The constant regions (Cl, C5, gp32) are those described in 4B,D,H with an additional synthetic DNA segment 5 corresponding to variable regions V1-V5 is inserted using a synthetic DNA segment comprised of optimal codons for translation.

J. VlJns-tPA-~pl43/opt all-B:
This construct is similar to IVJ except that the env 10 proteolytic cleavage sites have been retained.

K. VlJns-tPA-gpl43/opt all-A (non-IIIB strains):
This construct is similar to IIIG above except that env amino acid sequences from strains other than IIIB were used to 15 determine optimum codon useage throughout the variable (Vl-V5) reglons.

L. VlJns-tPA-gpl43/opt all-B (non-IIIB strains):
This construct is similar to IIIG above except that env 20 amino acid sequences from strains other than IIIB were used to determine optimum codon useage throughout the variable (Vl-V5) reglons.

25 gpl43/~elyB Vaccine Constructs:
These constructs were prepared by PCR similarly as other tPA-cont~ining constructs described above (tPA-gpl20, tPA-gpl40, tPA-gpl43 and tPA-gpl60), with the tPA leader in place of the native leader, but designed to produce COOH-termin~ted, membrane-bound 30 env as with gpl43. However, gpl43/glyB constructs differ from gpl43 in that of the six amino acids projected to comprise the intracellular peptide domain, the last 4 are the same those at the carboxyl terminus of human glycophorin B (glyB) protein (projected intracellular amino acid sequence= NH2-NRLIKA-COOH (SEQ.ID:27) with the underlined 35 residues corresponding to glyB and "R" common to both env and glyB).

CA 022~8~68 1998-12-18 This construct was designed with the purpose gaining additional env expression and directed targeting to the cell surface by completely elimin~ting any transcript or peptide region corresponding to the intracellular portion of env that might negatively impact expression or protein stability/transport to the cell surface by replacing this region with a peptide sequence from an abundantly expressed protein (glyB) having a short cytoplasmic domain (intracellular amino acid sequence=
NH2-RRLIKA-COOH). Constructs were prepared in two forms (A or B) depending upon whether the gpl60 proteolytic cleavage sites were removed or retained as described above.

A . V 1 Jns-tPA-gp 1 43/opt32-A/glyB:
This construct is the same as IVD except that the following antisense PCR oligomer was used to replace the intracellular peptide domain of gpl43 with that of glycophorin B as described above: 5'-CCA CAT GAT ATC G CCC GGG C TTA TTA GGC CTT GAT CAG
CCG GTT CAC AAT GGA CAG CAC AGC-3' (SEQ ID:28).

B. VlJns-tPA-gpl43/opt32-B/glyB:
This construct is similar to VA except that the env proteolytic cleavage sites have been retained.

C. VlJns-tPA-gpl43/opt C1/opt32-A/glyB:
This constmct is the same as VA except that the first constant region (C1) of gpl20 is replaced by optimal codons for translation as with IVH.

D. VlJns-tPA-gpl43/opt C1/opt32-B/glyB:
This construct is similar to VC except that the env proteolytic cleavage sites have been retained.

E. VlJns-tPA-gpl43/opt all-A/glyB:
The env gene of this construct is comprised completely of optimal codons as described above.

CA 022~8~68 1998-12-18 F. VlJns-tPA-~pl43/opt all-B/glyB:
This construct is similar to VE except that the env proteolytic cleavage sites have been retained.

5 G. VlJns-tPA-gpl43/opt all-A/glyB (non-IIIB strains):
This construct is similar to IIIG above except that env amino acid sequences from strains other than IIIB were used to determine optimum codon useage throughout the variable (V1-V5) reglons.
H. VlJns-tPA-~pl43/opt all-B/glyB (non-IIIB strains):
This construct is similar to VG except that the env proteolytic cleavage sites have been retained.

15 HIV env Vaccine Constructs with Variable Loop Deletions:
These constructs may include all env forms listed above (gpl20, gpl40, gpl43, gpl60, gpl43/glyB) but have had variable loops within the gpl20 region deleted during preparation (e.g., V1, V2, and/or V3). The purpose of these modifications is to elimin~te peptide 20 segments which may occlude exposure of conserved neutralization epitopes such as the CD4 binding site. For exarnple, the following oligomer was used in a PCR reaction to create a V1/V2 deletion resulting in adjoining THE C1 and C2 segments: 5'-CTG ACC CCC
CTG TGT GTG GGG GCT GGC AGT TGT AAC ACC TCA GTC
25 ATT ACA CAG-3' (SEQ ID:29).

EXAMPLE 1 l Design of Synthetic Gene Segments for Increased env Gene Expression:
Gene segments were converted to sequences having 30 identical translated sequences (except where noted) but with alternative codon usage as defined by R. Lathe in a research article from J. Molec.
Biol. Vol. 183, pp. 1-12 (1985) entitled "Synthetic Oligonucleotide Probes Deduced from Arnino Acid Sequence Data: Theoretical and Practical Considerations". The methodology described below to CA 022~8~68 1998-12-18 increase rev-independent expression of HIV env gene segments was based on our hypothesis that the known inability to express this gene - efficiently in m~mm~ n cells is a consequence of the overall transcript composition. Thus, using alternative codons encoding the same protein 5 sequence may remove the constraints on env expression in the absence of rev. Inspection of the codon usage within env revealed that a high percentage of codons were among those infrequently used by highly expressed human genes. The specific codon replacement method employed may be described as follows employing data from Lathe et al.:
1. Identify placement of codons for proper open reading frame.
2. Compare wild type codon for observed frequency of use by human genes (refer to Table 3 in Lathe et al.).
3. If codon is not the most commonly employed, replace it with an optimal codon for high expression based on data in Table 5.
4. Inspect the third nucleotide of the new codon and the first nucleotide of the adjacent codon immediately 3'- of the first. If a 5'-CG-3' pairing has been created by the new codon selection, replace it 20 with the choice indicated in Table 5.
5. Repeat this procedure until the entire gene segment has been replaced.
6. Inspect new gene sequence for undesired sequences generated by these codon replacements (e.g., "ATTTA" sequences, 25 inadvertent creation of intron splice recognition sites, unwanted restriction enzyme sites, etc.) and substitute codons that elimin~te these sequences.
7. Assemble synthetic gene segments and test for improved expression.

CA 022~8~68 1998-12-18 These me~ods were used to create the following synthetic gene segments for HIV env creating a gene comprised entirely of optimal codon usage for expression: (i) gpl20-C1 (opt); (ii) V1-V5 (opt); (iii) RRE-A/B (mut or opt); and (iv) gp30 (opt) with percentages of codon replacements/nucleotide substitutions of 56/19, 73/26, 78/28, and 61/25 obtained for each segment, respectively. Each of these segments has been described in detail above with actual sequences listed below.

~p120-Cl (o~t) This is a gpl20 constant region 1 (Cl) gene segment from the mature N-terminus to the beginning of Vl designed to have optimal codon usage for expression.
l TGATCACAGA GAAGCTGTGG GTGACAGTGT ATTATGGCGT GCCAGTCTGG
51 AAGGAGGCCA CCACCACCCT(i~ irGCC TCTGATGCCA AGGCCTATGA

301 TAAAC(SEQ ID:30) MN Vl-V5 (opt) This is a gene segment corresponding to the derived protein sequence for HIV MN V1-V5 (1066BP) having optimal codon usage for expression.

51 AGCG4ACAACMCTCCMCTCCGAGGGCAC CATCAAGGGG G(~3GAGATGA
101 AGMCTGCTC C; l I CMCATC ACCACCTCCATCAGGGACM GATGCAGAAG
151 GAGTATGCCC l (i~ l ci I ACM GCTGGACATT GTGTCCATTG ACMTGACTC
201 CACCTCCTAC AGGCTGATCT CCTGCMCAC C 1~ i l C;ATC ACCCAGGCCT
251 GCCCCAAAAT C I (;C I I I GAG CCCATCCCCA TCCACTACTG TGCCCCTGCT

~ 301 GG~i I I I dCCATCCTGAAGTG CAATGACAAG M~ ; 1 (i I G GCMGGG~
351 CTGCAAGAAT~ CACAGTGCAGTGCACACATGGCATCAGG~i I ~ iG
401 TGTCCACCCA~; I G(; I (~i I G MT~ I ~;CC l (~;1 GAGGAGGAG(~i I ~i 1~
451 ATCAGGTCTG AGAACTTCAC AGACAATGCC MGACCATCA l (~i l GCACCT

551 AGAGGATCCA CA I I (3GcccT GGCAGGG~; I l t~ l ACACCAC CMGMCATC
601 ATTG(3CACCATCAGGCAGGC CCACTGCMC ATCTCCAGGG CCMGTGGAA
l 5 651 TGACACCCTGAGGCAGATTGTGTCCMGCTGMGGAGCAGTTCMGMCA
701 AGACCATTGT GTTCMCCAG TC~ GGG GG,GACCCTGA GATTGTGATG
751 CACTCCTTCAACi l ~ iGGGGGGA(i I I (; I IC TACTGCMCACCTCCCCCCT

901 T(~CA~G TG~C4AGGC CATGTATGCC CCCO~ TTG Ar~r~l~GAT
951 CA~i I Gl; I (~C TCCAACATCA CAG~CC I (i~; l GCTGACCAGG GAT~'~;~
1001 AGGACACAGA CACCAACGAC ACCGr4MTCT TCAGGCC; 1~ ('~fi{-~C
1051 ATGAGGGACAATTGG (SEQ ID:31) RRF.Mut (~

This is a DNA segment corresponding to the rev response element (RRE) of HIV-1 comprised of optimal codon usage for expression. The "A" form also has removed the known proteolytic cleavage sites at the gpl20/gp41 junction by using the nucleotides indicated in boldface.
1 GACMTTG,GA GGAGCGAGTT ATATMMTAT MG,GTGGTGA AGATTGAGCC
51 CC I GGGGGTG GCCCCAACM MGCTC~ i I ~i I G CAG~Q~GAGC
101 A~:AGGC~ I G~GCATTG~;G GCCC; I (i I I I ~T(~GC; I I l ~i l t'~; I G(i I
151 G~;CTCCACMTr~C TAGCATGACC CTCACCGTGC AAGCTCGCCA
201 GCTG,CTGAGTGGCATCGTCC AGCAGCAG~A CMC(i l ~; l (~ CGCGCCATCG

251 AAGCCCAGCA GCAC;C 1~1~; CAGCTGACTG TGTGG,GGGAT CAAACAGC~T
301 CA(~ GGC~il CGA;3CGCrATCTGAAAGACCAGCAACTCCT
351 AGGC (SEQ ID:32) RRE.Mut (8) This is a DNA segment corresponding to the rev response element (RRE) of HIV-1 comprised of optimal codon usage for expression. The "B" form retains the known proteolytic cleavage sites at the gpl20/gp41 Junctlon.
GACMTTGGA GGAGCGAGTT ATATAAATAT AAGGTGGTGA AGATTGAGCC
51 CCTGGGGGTG GCCCCAACM AAGCTA~ '~r~AGA(i I G~ I G CAG~GAG~
101 AGAGAGCCGTGGGCATTGt3GGC~I(il I IC l~;l I l ~;l t'~r~( I(i~;
151 GGC I CCACAA I ~CGCCGCTAGCATGACC CTCACCGTGC AAG~ I CGCCA
201 G,CTG,CTGAGT GG,CA I (li I (;C AGCAGCAGAA CAAC;CTGCTC CGCGCCATCG
251 MG~CCAG,CA GCAC~ x; l C CAGCTGACTG TGTGGGGGAT CAAACAGC~T
25 301 CAr{~ GCC(il CGAGCGCrATCTGAAAGACCAGCAACTCCT
351 AGGC (SEQ ID:33) gp32 (opt) This is a gp32 gene segment from the AvrII site (starting immediately at the end of the RRE) to the end of gpl43 comprised of optimal codons for expression.

35 1 CCTAG,GCATCTG,G~GCTG CTCTGGCAAG CTGATCTGCACCACAGCTGT
51 G,CCCTGGAAT GCCTCCTGGT CCAACAAGAG CCTG~;AGCM ATCTGSMCA

201 GGAGC; 1~;1 a GAGCTGGACAAGTGGGCCTC CC; I ~:i l ~iMC I ~i I I ~MCA
45 251 TCACCMCTG GC; l ~ i I AC ATCMMTCT TCATCATGAT TGT('~r~

~ 3û1 CTGGTGGGGCTGCGGATTGTC; I I 1~1 (j I GC;I ~ ;CATTGTGAACOGGGT
351 GAGACAGGGC TACTCCCCCT MTMGCCCG GGCGATATC (SEQ ID~4) SRV-1 CTE (A) This is a synthetic gene segment corresponding to a 3'-UTR from the Simian Retrovirus-1 genome. This DNA is placed in the following orientation at the 3'-terminus of HIV genes to increase rev-independent 10 expression.
S rf I Ec oRV
5~ ~t~ TATC TA GACCACCTCC CCTGCGAGCT MGCTGGACA
GCCAATGACGGGTA~GAGAGTGACAI ~ CACTAACCTAAGACAGGAGG
GCOt~u (iAGAG CTA~ AATCCAAAGAC GGGTAAAAGT GATAAAAATG
TATCACTCCAACCTA~GACAGGCGCAGCTTCCGAGGGA I I ~ w I
I I IATATATATTTAAAAGGGTGACCTGTCCGGAGCCGTGCTGCCGGGATG
ATGTCTTGGt'-~TATC t~CC~ C-3' (SEQID:35) E~RV S rf l CA 02258568 l998- l2- l8 WO 97/48370 PCT/US97tlO517 SRV-1 CTE (~) This synthetic gene segment is identical to SRV-1 CTE (A) shown above except that a single nucleotide mutation was used (indicated by boldface) 5 to elimin~te an Al-rTA sequence. This sequence has been associated with increased mRNA turnover.
S rf l EmRV
5~ t~r~ ~r~ t~TATC TA GACCACCTCC CCTGCGAGCT AAGCTGGACA

GCC~ATGACGGGTA~GAGAGTGACATmTcACTA~CCTAA~AGG
G~i I ~iAGAG CTA~; 1~; l A AT~A~ G~;GTAAAAGT GATAAMATG
TATCACTCCAACCTA~GACA(~TCCGAGGG~ l~TCTGT
mATATATA TTL4AAA(3GG TGAC(i ~ C GGa!~rGC ll~CCGGATG
A I G I C; I I GG ~I~ CCC l~r~C -3' (SEQ ID:36) ~oRV Srfl EXAMPLE l l In Vitro ~pl20 Vaccine Expression:
In vitro expression was tested in transfected hllm~n rhabdomyosarcoma (RD) cells for these constructs. Quantitation of secreted tPA-gpl20 from transfected RD cells showed that VlJns-tPA-gp 120 vector produced secreted gp 120.

In Vivo gpl20 Vaccination:
VlJns-tPA-gpl20~N PNV-induced Class II MHC-restricted T Iymphocyte gpl20 specific antigen reactivities. Balb/c mice which had been vaccinated two times with 200 )lg VlJns-tPA-gpl20MN
were sacrificed and their spleens extracted for in vitro determin~tions of helper T lymphocyte reactivities to recombinant gpl20. T cell proliferation assays were performed with PBMC (peripheral blood mononuclear cells) using recombinant gpl20IIIg (Repligen, catalogue #RP1016-20) at 5 ~lg/ml with 4 x 105 cells/ml. Basal levels of 3H-thymidine uptake by these cells were obtained by culturing the cells in media alone, while maximum proliferation was induced using ConA

CA 022~8~68 1998-12-18 stimulation at 2 ~lg/ml. ConA-induced reactivities peak at ~3 days and were harvested at that time point with media control samples while antigen-treated samples were harvested at 5 days with an additional media control. Vaccinated mice responses were compared with naive, 5 age-matched syngeneic mice. ConA positive controls gave very high proliferation for both naive and immunized mice as expected. Very strong helper T cell memory responses were obtained by gpl20 treatment in vaccinated mice while the naive mice did not respond (the threshold for specific reactivity is an stimulation index (SI) of >3-4; SI
lO is calculated as the ratio of sample cpm/media cpm). SI's of 65 and 14 were obtained for the vaccinated mice which compares with anti-gpl20 ELISA titers of 5643 and 11,900, respectively, for these mice.
Interestingly, for these two mice the higher responder for antibody gave significantly lower T cell reactivity than the mouse having the lower 15 antibody titer. This experiment demonstrates that the secreted gpl20 vector efficiently activates helper T cells in vivo as well as generates strong antibody responses. In addition, each of these immune responses was determined using antigen which was heterologous compared to that encoded by the inoculation PNV (IIIB vs. MN):

gpl60 Vaccines In addition to secreted gpl20 constructs, we have prepared expression constructs for full-length, membrane-bound gpl60. The 25 rationales for a gpl60 construct, in addition to gpl20, are (1) more epitopes are available both for both CTL stimulation as well as neutralizing antibody production including gp41, against which a potent HIV neutralizing monoclonal antibody (2F5, see above) is directed; (2) a more native protein structure may be obtained relative to virus-30 produced gpl60; and, (3) the success of membrane-bound influenza HA
constructs for immllnogenicity [Ulmer et al., Science 259:1745-1749, 1993; Montgomery, D., et al., DNA and Cell Biol.. 12:777 783, 1993].

. . ,.~. i .. .. .. .

CA 022~8~68 1998-12-18 gpl60 retains substantial rev dependence even with a heterologous leader peptide sequence so that further constructs were made to increase expression in the absence of rev.

Assay For HIV Cytotoxic T-Lymphocytes:
The methods described in this section illustrate the assay as used for vaccinated mice. An essentially similar assay can be used with primates except that autologous B cell lines must be established for use as target cells for each ~nim~l. This can be accomplished for humans using the Epstein-Barr virus and for rhesus monkey using the herpes B
virus.
Peripheral blood mononuclear cells (PBMC) are derived from either freshly drawn blood or spleen using Ficoll-Hypaque centrifugation to separate erythrocytes from white blood cells. For mice, lymph nodes may be used as well. Effecter CTLs may be prepared from the PBMC either by in vitro culture in IL-2 (20 U/ml) and concanavalin A (2,ug/ml) for 6-12 days or by using specific antigen using an e~ual number of irradiated antigen presenting cells. Specific antigen can consist of either synthetic peptides (9-15 amino acids usually) that are known epitopes for CTL recognition for the MHC
haplotype of the ~nim~ls used, or vaccinia virus constructs engineered to express appropriate antigen. Target cells may be either syngeneic or MHC haplotype-matched cell lines which have been treated to present appropriate antigen as described for in vitro stimulation of the CTLs.
For Balb/c mice the P18 peptide (ArgIleHisIleGlyProGlyArgAlaPheTyrThrThrLysAsn [SEQ.ID:37~, for HIV MN strain) can be used at 10 ,uM concentration to restimulate CTL
in vitro using irradiated syngeneic splenocytes and can be used to sensitize target cells during the cytotoxicity assay at 1-10 }lM by incubation at 37~C for about two hours prior to the assay. For these H-2d MHC haplotype mice, the murine mastocytoma cell line, P815, provides good target cells. Antigen-sensitized target cells are loaded with Na5 l CrO4, which is released from the interior of the target cells CA 022~8~68 1998-12-18 WO 97/48370 PCT/US97/lOS17 upon killing by CTL, by incubation of targets for 1-2 hours at 37~C
(0.2 mCi for ~5 x 106 cells) followed by several washings of the target cells. CTL populations are mixed with target cells at varying ratios of effectors to targets such as 100:1, 50:1, 25:1, etc., pelleted together, and S incubated 4-6 hours at 37~C before harvest of the supern~t~nt~ which are then assayed for release of radioactivity using a gamma counter.
Cytotoxicity is calculated as a percentage of total releasable counts from the target cells (obtained using 0.2% Triton X-100 treatment) from which spontaneous release from target cells has been subtracted.

Assay For HIV Specific Antibodies:
ELISA were designed to detect antibodies generated against HIV using either specific recombinant protein or synthetic peptides as 15 substrate antigens. 96 well microtiter plates were coated at 4~C
overnight with recombinant antigen at 2 ,ug/ml in PBS (phosphate buffered saline) solution using 50 ,ul/well on a rocking platform.
Antigens consisted of either recombinant protein (gpl20, rev: Repligen ~orp.; gpl60, gp41: American Bio-Technologies, Inc.) or synthetic 20 peptide (V3 peptide corresponding to virus isolate sequences from IIIB, etc.: American Bio-Technologies, lnc.; gp41 epitope for monoclonal antibody 2F5). Plates were rinsed four times using wash buffer (PBS/0.05% Tween 20) followed by addition of 200111/well of blocking buffer (1% Carnation milk solution in PBS/0.05% Tween-20) for 1 hr 25 at room temperature with rocking. Pre-sera and immune sera were diluted in blocking buffer at the desired range of dilutions and 100 ,ul added per well. Plates were incubated for 1 hr at room temperature with rocking and then washed four times with wash buffer. Secondary antibodies conjugated with horse radish peroxidase, (anti-rhesus Ig, 30 Southern Biotechnology Associates; anti- mouse and anti-rabbit Igs, Jackson Immuno Research) diluted 1:2000 in blocking buffer, were then added to each sample at 100 Ill/well and incubated 1 hr at room temperature with rocking. Plates were washed 4 times with wash buffer and then developed by addition of 100 ~l/well of an o-phenylenediamine .

CA 022~8~68 1998-12-18 (o-PD, Calbiochem) solution at 1 mg/ml in 100 mM citrate buffer at pH
4.5. Plates were read for absorbance at 450 nm both kinetically (first ten min~ltes of reaction) and at 10 and 30 minute endpoints (Thermo-max microplate reader, Molecular Devices).

Assay For HIV Neutralizing Antibodies:
In vitro neutralization of HIV isolates assays using sera derived from vaccinated ~nim~l~ was performed as follows. Test sera 10 and pre-immune sera were heat inactivated at 56~c for 60 min before use. A titrated amount of HIV-l was added in 1:2 serial dilutions of test sera and incubated 60 min at room temperature before addition to 105 MT-4 hllm~n lymphoid cells in 96 well microtiter plates. The virus/cell mixtures were incubated for 7 days at 37~C and assayed for virus-15 mediated killing of cells by staining cultures with tetrazolium dye.Neutralization of virus is observed by prevention of virus-mediated cell death.

20 Isolation Of Genes From Clinical HIV Isolates:
HIV viral genes were cloned from infected PBMC's which had been activated by ConA treatment. The preferred method for obtaining the viral genes was by PCR amplification from infected cellular genome using specific oligomers fl~nking the desired genes. A
25 second method for obtaining viral genes was by purification of viral RNA from the supern~t~nts of infected cells and preparing cDNA from this material with subsequent PCR. This method was very analogous to that described above for cloning of the murine B7 gene except for the PCR oligomers used and random hexamers used to make cDNA rather 30 than specific ~lilll-llg oligomers.
Genomic DNA was purified from infected cell pellets by Iysis in STE solution (10 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 8.0) to which Proteinase K and SDS were added to 0.1 mg/ml and 0.5% final concentrations, respectively. This mixture was incubated CA 022~8~68 1998-12-18 overnight at 56~C and extracted with 0.5 volumes of phenol:chloroform:isoarnyl alcohol (25:24:1). The aqueous phase was then precipitated by addition of sodium acetate to 0.3 M final concentration and two volumes of cold ethanol. After pelleting the S DNA from solution the DNA was resuspended in O.lX TE solution (lX
TE = 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). At this point SDS was added to 0.1% with 2 U of RNAse A with incubation for 30 minutes at 37~C. This solution was extracted with phenol/chloroform/isoamyl alcohol and then precipitated with ethanol as before. DNA was suspended in 0.1 X TE and quantitated by measuring its ultraviolet absorbance at 260 nm. Samples were stored at -20~C until used for PCR.
PCR was performed using the Perkin-Elmer Cetus kit and procedure using the following sense and antisense oligomers for gpl60:
5'-GA AAG AGC AGA AGA CAG TGG CAA TGA -3' (SEQ.ID:38) and 5'-GGG Cl-r TGC TAA ATG GGT GGC AAG TGG CCC GGG C
ATG TGG-3' (SEQ.ID:39), respectively. These oligomers add an SrfI
site at the 3'-terminus of the resulting DNA fragment. PCR-derived segments are cloned into either the VlJns or VlR vaccination vectors and V3 regions as well as ligation junction sites confirmed by DNA
sequencmg.

T Cell Proliferation Assays:
PBMCs are obtained and tested for recall responses to specific antigen as determined by proliferation within the PBMC
population. Proliferation is monitored using 3H-thymidine which is added to the cell cultures for the last 18-24 hours of incubation ~efore harvest. Cell harvesters retain isotope-cont~ining DNA on filters if proliferation has occurred while quiescent cells do not incorporate the isotope which is not retained on the filter in free form. For either rodent or primate species 4 X 105 cells are plated in 96 well microtiter plates in a total of 200 ~1 of complete media (RPMI/10% fetal calf serum). Background proliferation responses are determined using CA 022~8~68 1998-12-18 PBMCs and media alone while nonspecific responses are generated by using lectins such as phytohaemagglutin (PHA) or concanavalin A
(ConA) at 1- 5 ,ug/ml concentrations to serve as a positive control.
Specific antigen consists of either known peptide epitopes, purified 5 protein, or inactivated virus. Antigen concentrations range from 1- 10 ~lM for peptides and 1-10 ,ug/ml for protein. Lectin-induced proliferation peaks at 3-5 days of cell culture incubation while antigen-specific responses peak at 5-7 days. Specific proliferation occurs when radiation counts are obtained which are at least three-fold over the 10 media background and is often given as a ratio to background, or Stimulation Index (SI). HIV gpl60 is known to contain several peptides known to cause T cell proliferation of gpl60/gpl20 immunized or HIV-infected individuals. The most commonly used of these are:
Tl (LysGlnIleIleAsnMetTrpGlnGluValGlyLysAlaMetTyrAla 15 [SEQ .ID :40] ) ; T2 (HisGluAspIleIleSerLeuTrpAspGlnSerLeuLys [SEQ.ID:411; and, TH4 (AspArgValIleGluValValGlnGlyAlaTyrArgAla IleArg [SEQ.ID:42]). These peptides have been demonstrated to stim~ te proliferation of PBMC from antigen-sensitized mice, nonhuman primates, and humans.

Vector V 1 R Preparation:
In an effort to continue to optimize our basic vaccination vector, we prepared a derivative of VlJns which was designated as VlR
25 The purpose for this vector construction was to obtain a minimum-sized vaccine vector, i.e., without unnecessary DNA sequences, which still retained the overall optimized heterologous gene expression characteristics and high plasmid yields that VlJ and VlJns afford. We determined from the literature as well as by experiment that (1) regions 30 within the pUC backbone comprising the E. coli origin of replication could be removed without affecting plasmid yield from bacteria; (2) the 3'-region of the kanr gene following the k~n~mycin open reading frame could be removed if a bacterial terminator was inserted in its stead; and, (3) ~300 bp from the 3'- half of the BGH terminator could be removed CA 022~8~68 1998-12-18 without affecting its regulatory function (following the original KpnI
restriction enzyme site within the BGH element).
- VlR was constructed by using PCR to synthesize three segments of DNA from VlJns representing the CMVintA
S promoter/BGH terminator, origin of replication, and k~n~mycin resistance elements, respectively. Restriction enzymes unique for each segment were added to each segment end using the PCR oligomers: SspI
and XhoI for CMVintA/BGH; EcoRV and BamHI for the kan r gene;
and, BclI and SalI for the ori r. These enzyme sites were chosen 10 because they allow directional ligation of each of the PCR-derived DNA
segments with subsequent loss of each site: EcoRV and SspI leave blunt-ended DNAs which are compatible for ligation while BamHI and BclI
leave complementary overhangs as do SalI and XhoI. After obtaining these segments by PCR each segment was digested with the appropriate 15 restriction enzymes indicated above and then ligated together in a single reaction mixture cont~ining all three DNA segments. The 5'-end of the ori r was designed to include the T2 rho independent termin~tor sequence that is normally found in this region so that it could provide termination information for the k7~n~mycin resistance gene. The ligated 20 product was confirmed by restriction enzyme digestion (>8 enzymes) as well as by DNA sequencing of the ligation junctions. DNA plasmid yields and heterologous expression using viral genes within VlR appear similar to V lJns. The net reduction in vector size achieved was 1346 bp (VlJns = 4.86 kb; VlR = 3.52 kb), [SEQ.ID:43 of this specification;
25 also see Figure 11 and SEQ ID:lO0 of WO95/24485; PCT International Application No. PCT/US95/02633].

PCR oligomer sequences used to synthesize VlR
(restriction enzyme sites are underlined and identified in brackets 30 following sequence):

- (1) 5'-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3' [SspI], (SEQ.ID:44):, CA 022~8~68 l998- l2- l8 (2) 5'-CCA CAT CTC GAG GAA CCG GGT CAA l~C lY~C AGC
ACC-3' [XhoI], (SEQ.ID:45):
(for CMVintA/BGH segment) 5 (3) 5'-GGT ACA GAT ATC GGA AAG CCA CGT TGT GTC TCA
AAA TC-3'[EcoRV], (SEQ.ID:46):
(4) 5'-CCA CAT GGA TCC G TAA TGC TCT GCC AGT G~T ACA
ACC-3' [BamHI], (SEQ.ID:47):
(for k~n~mycin resistance gene segment) (5) 5'-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC
~YC TTG-3'[BclI], (SEQ.ID:48):, (6) 5'-CCA CAT GTC GAC CC GTA AAA AGG CCG CGT TGC
TGG-3' [SalI], (SEQ.ID:49):
(for E. coli origin of replication) Ligation junctions were sequenced for VlR using the following oligomers:
5'-GAG CCA ATA TAA ATG TAC-3' (SEQ.ID:50):
20 [CMVintA/kanr junction]
5'-CAA TAG CAG GCA TGC-3' (SEQ.ID:Sl): [BGH/ori junction]
5'-G CAA GCA GCA GAT TAC-3' (SEQ.ID:52): [ori/kanr junction]
EXAMPLE l9 Heterologous Expression of HIV Late Gene Products HIV structural genes such as env and gag require expression of the HIV regulatory gene, rev, in order to efficiently 30 produce full-length proteins. We have found that rev-dependent expression of gag yielded low levels of protein and that rev itself may be toxic to cells. Although we achieved relatively high levels of rev-dependent expression of gpl60 in vitro this vaccine elicited low levels of antibodies to gpl60 following in vivo imml-ni7.~tion with rev/gpl60 CA 022~8~68 1998-12-18 DNA. This may result from known cytotoxic effects of rev as well as increased difficulty in obt~ining rev function in myotubules cont~ining hundreds of nuclei (rev protein needs to be in the same nucleus as a rev-dependent transcript in order for gag or env protein expression to 5 occur). However, it has been possible to obtain rev-independent expression using selected modifications of the env gene.

1. rev-independent expression of env:
In general, our vaccines have utilized primarily HIV (IIIB) 10 env and gag genes for optimi7~tion of expression within our generalized vaccination vector, VlJns, which is comprised of a CMV immediate-early (IE) promoter, a BGH-derived polyadenylation and transcriptional termination sequence, and a pUC backbone. Varying efficiencies, depending upon how large a gene segment is used (e.g., gpl20 vs.
15 gpl60), of rev-independent expression may be achieved for env by replacing its native secretory leader peptide with that from the tissue-specific pl~minogen activator (tPA) gene and expressing the resulting chimeric gene behind the CMVIE promoter with the CMV intron A.
tPA-gpl20 is an example of a secreted gpl20 vector constructed in this 20 fashion which functions well enough to elicit anti-gpl20 immune responses in vaccinated mice and monkeys.
Because of reports that membrane-anchored proteins may induce much more substantial (and perhaps more specific for HIV
neutralization) antibody responses compared to secreted proteins as well 25 as to gain additional epitopes, we prepared VlJns-tPA-gpl60 and VlJns-rev/gpl60. The tPA-gpl60 vector produced detectable quantities of gpl60 and gpl20, without the addition of rev, as shown by immunoblot analysis of transfected cells, although levels of expression were much lower than that obtained for rev/gpl60, a rev-dependent gpl60-30 expressing plasmid. This is probably because inhibitory regions, whichconfer rev dependence upon the gpl60 transcript, occur at multiple sites within gpl60 including at the COOH-terminus of gp41. A vector was prepared for a COOH-terminally truncated form of tPA-gpl60 (tPA-CA 022~8~68 1998-12-18 gpl43) which was designed to increase the overall expression levels of env by elimin~tion of these inhibitory sequences. The gpl43 vector also elimin~tes intracellular gp41 regions cont~ining peptide motifs (such as Leu-Leu) known to cause diversion of membrane proteins to the 5 lysosomes rather than the cell surface. Thus, gpl43 may be expected to have increased levels of expression of the env protein (by decreasing rev-dependence) and greater efficiency of transport of protein to the cell surface compared to full-length gpl60 where these proteins may be better able to elicit anti-gpl60 antibodies following DNA vaccination.
10 tPA-gpl43 was further modified by extensive silent mutagenesis of the rev response element (RRE) sequence (350 bp) to eliminate additional inhibitory sequences for expression. This construct, gpl43/mutRRE, was prepared in two forms: either elimin~ling (form A) or re~ining (form B) proteolytic cleavage sites for gpl20/41. Both forms were 15 prepared because of literature reports that vaccination of mice using uncleavable gpl60 expressed in vaccinia elicited much higher levels of antibodies to gpl60 than did cleavable forms.
A quantitative El,ISA for gpl60/gpl20 expression in cell transfectants was developed to determine the relative expression 20 capabilities for these vectors. In vitro transfection of 293 cells followed by quantification of cell-associated vs. secreted/released gpl20 yielded the following results: (l) tPA-gpl60 expressed 5-lOX less gpl20 than rev/gpl60 with similar proportions retained intracellularly vs. released from the cell surface; (2) tPA-gpl43 gave 3-6X greater secretion of 25 gpl20 than rev/gpl60 with only low levels of cell-associated gpl43, confirming that the cytoplasmic tail of gpl60 causes intracellular retention of gpl60 which can be overcome by partial deletion of this sequence; and, (3) tPA-gpl43/mutRR~ A and B gave ~lOX greater expression levels of protein than did parental tPA-gpl43 while 30 elimin~tion of proteolytic processing was confirmed for form A.
Thus, our strategy to increase rev-independent expression has yielded stepwise increases in overall expression levels as well as redirecting membrane-anchored gpl43 to the cell surface away from lysosomes. It is important to note that this is a generic constmct into CA 022~8~68 1998-12-18 which it should be possible to insert gpl20 sequences derived from various primary viral isolates within a vector cassette cont~ining these modifications which reside either at the NH2-terminus (tPA leader) or COOH-terminus (gp41), where few antigenic differences exist between 5 different viral strains.
Figures 2-7 present data supporting the use of various constructs, including but not limited to a gpl43-based construct, and preferably a tPA-gpl43 based construct, as a DNA vaccine against HIV
infection. Figure 2 shows that tPA-143 (opt41) elicits an anti-gpl20 10 antibody response in the in the range of GMT=103. Figure 3 measures and compares anti-gpl20 antibody titers for several DNA vaccines, including gpl43-based constructs. Figure 4 shows the relative expression of tPA-gpl43 and tPA-143/mutRRE in comparison to the tPA-gpl60 construct. Figure 5 measures generation of anti-gpl20 15 antibodies for both the optA and optB forrns of tPA-gpl43 constructs.
Figure 6 shows the ability of several DNA vaccines, including tPA-gpl43-optA and tPA-gpl43-optB, to promote generation of neutralizing antibodies against HIV strains subsequent to murine DNA vaccination.
Figure 7 also shows HIV neutralization data for various DNA vaccine 20 constructions, including tPA-gpl43-optA, tPA-gpl43-optB, tPA-gpl43-optA-glyB and tPA-gpl43-optB-glyB.
2. Expression of ~pl20 derived from a clinical isolate:
To apply these expression strategies to viruses that are 25 relevant for vaccine purposes and confirm the generality of our approaches, we also prepared a tPA-gpl20 vector derived from a primary HIV isolate (cont~ining the North American concensus V3 peptide loop; macrophage-tropic and nonsyncytia-inducing phenotypes).
This vector gave high expression/secretion of gpl20 with transfected 30 293 cells and elicited anti-gpl20 antibodies in mice thus demonstrating that it was cloned in a functional form. Primary isolate gpl60 genes will also be used for expression in the same way as for gpl60 derived from laboratory strains.

... . . .

CA 022~8~68 1998-12-18 3. ~mmlme Responses to HIV-1 env Polynucleotide Vaccines Effect of vaccination route on immune responses in mice:
While efforts to improve expression of gpl60 are ongoing, we have utilized the tPA-gpl20 DNA construct to assess immlme responses and S ways to augment them. Intramuscular (i.m.) and intradermal (i.d.) vaccination routes were compared for this vector at 100, 10, and 1 ,ug doses in mice. Vaccination by either route elicited antibody responses (GMTs = 103-104) in all recipients following 2-3 vaccinations at all three dosage levels. Each route elicited similar anti-gpl20 antibody 10 titers with clear dose-dependent responses. However, we observed greater variability of responses for i.d. vaccination, particularly at the lower doses following the initial inoculation. Moreover, helper T-cell responses, as determined by antigen-specific in vitro proliferation and cytokine secretion, were higher following i.m. vaccination than i.d. We 15 concluded that i.d. vaccination did not offer any advantages compared to i.m. for this vaccine.
4. ~pl20 DNA vaccine-mediated helper T cell immunity in mice:
gp 120 DNA vaccination produced potent helper T-cell 20 responses in all lymphatic compartments tested (spleen, blood, inguinal, mesenteric, and iliac nodes) with TH1-like cytokine secretion profiles (i.e., g-interferon and IL-2 production with little or no IL-4). These cytokines generally promote strong cellular immunity and have been associated with maintenance of a disease-free state for HIV-seropositive 25 patients. Lymph nodes have been shown to be primary sites for HIV
replication, harboring large reservoirs of virus even when virus cannot be readily detected in the blood. A vaccine which can elicit anti-HIV
immllne responses at a variety of Iymph sites, such as we have shown with our DNA vaccine, may help prevent successful colonization of the 30 lymphatics following initial infection.

5. env DNA vaccine-mediated antibody responses:
African green (AGM) and Rhesus (RHM) monkeys which received gpl20 DNA vaccines showed low levels of neutralizing 35 antibodies following 2-3 vaccinations, which could not be increased by - 6~ -CA 022~8~68 1998-12-18 additional vaccination. These results, as well as increasing awareness within the HIV vaccine field that oligomeric gpl60 is probably a more relevant target antigen for eliciting neutr~li7.in~ antibodies than gpl20 monomers, have led us to focus upon obt~ining effective expression of 5 gpl60-based vectors (see above). Mice and AGM were also vaccinated with the primary isolate derived tPA-gpl20 vaccine. These ~nim~l~
exhibited anti-V3 peptide (using homologous sequence) reciprocal endpoint antibody titers ranging 500-5000, demonstrating that this vaccine design is functional for clinically relevant viral isolates.
The gpl60-based vaccines, rev-gpl60 and tPA-gpl60, failed to consistently elicit antibody responses in mice and nonhuman primates or yielded low antibody titers. Our initial results with the tPA-gpl43 plasmid yielded geometric mean titers (GMT) > 103 in mice and AGM following two vaccinations. These data indicate that we have 15 signficantly improved the immunogenicity of gpl60-like vaccines by increasing expression levels and more efficient intracellular trafficking of env to the cell surface. This construct, as well as the tPA-gpl43/mutRRE A and B vectors, will continue to be characterized for antibody responses, especially for virus neutralization.
6. env DNA vaccine-mediated CTL responses in monkeys:
We continued to characterize CTL responses of RHM that had been vaccinated with gpl20 and gpl60/IRES/rev DNA. All four monkeys that received this vaccine showed significant MHC Class I-25 restricted CTL activities (20-35% specific killing at an effector/target =
20) following two vaccinations. Following a fourth vaccination these activities increased to 50-60% killing under similar test conditions, indicating that additional vaccination boosted responses significantly.
The CTL activities have persisted for at least seven months subsequent 30 to the final vaccination at about 50% of their peak levels indicating that long-term memory had been established.

CA 022~8~68 1998-12-18 SIV/HIV (SHIV) Chimeras:
A major obstacle for testing the protective efficacy of candidate HrV-1 vaccines has been the lack of a suitable ~nim~l 5 challenge model for this virus. Although the simian immunodeficiency virus (SIV), which is closely related to HIV, is infectious and causes AIDS in rhesus monkeys, the only ~nim~l species which can be infected with HIV- 1 viral isolates is the chimpanzee. However, the resulting viremia from this infection is low-level, transient, and no pathogenic 10 effects (e.g., Iymphopenia, immunodeficiency-related opportunistic infections, etc.) develop. Recently, hybrid viruses comprised of SIV
and HIV genomes have been developed which are also infectious to rhesus monkeys and which can cause infection-related AIDS. An example of this type of virus is SHIV-4 (IIIB) (Li et al., J. of Acquired 15 Immune Deficiency Syndrome, Vol. 5, 639-646 (1992)). This virus contains the SIV (MAC239) genome except for the regulatory genes, tat and rev, and the structural gene, env. Because the principle component of candidate HIV vaccines is based upon env this virus allows testing vaccines developed for human clinical purposes for protective efficacy 20 against infection in an ~nim~l model.

Plasmid DNA and Recombinant Protein Combination Vaccines.
Vaccines having both a plasmid DNA HIV env component 25 and a recombinant HIV env protein component were tested for their abilities to induce antibody responses in rhesus monkeys. Figure 9 and Figure 10 show the resulting anti-gpl20 ELISA antibody and SHIV-4 (IIIB) virus neutralizing antibody titers, respectively, following vaccination of rhesus with Hrv env gene-cont~ining DNA vaccines and 30 recombinant protein (formulated in an appropriate adjuvant). These monkeys developed high titers of env-specific antibodies and neutr~li7ing antibodies. Control monkeys, vaccinated with "blank"
DNA that did not contain a gene and ovalbumin did not develop any detectable env-specific responses while monkeys vaccinated only with the protein component of this vaccine showed low levels of antigen-specific antibodies detected by ELISA and no neutr~li7in~ antibodies.
When these monkeys were challenged with SHIV-4 (IIIB) virus all control and protein only monkeys became infected while those receiving 5 both env DNA and protein did not develop a detectable SHIV viremia.
These monkeys are currently being tested periodically for possible delayed onset of infection.

, . ...... . .. ... . .

CA 022~8~68 1998-12-18 W 097/48370 PCT~Us97/10517 SEQUENCE LISTING

(l) GENERAL INFORMATION:
~i) APPLICANTS: MERCK & CO., INC.

(ii) TITLE OF INVENTION: VACCINES COMPRISING SYNTHETIC GENES

(iii) NUMBER OF SEQUENCES: 53 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: J. MARK HAND - MERCK ~ CO., INC.
~B) STREET: 126 E. LINCOLN AVE., P.O. BOX 2000 (C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: US
(F) ZIP: 07065-0907 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HAND, J. MARK
(B) REGISTRATION NUMBER: 36,545 (C) REFERENCE/DOCKET NUMBER: l9729Y PCT

(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3905 (B) TELEFAX: 908-594-4720 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4864 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both SUeS~ (RUlE~

CA 022~8~68 1998-12-18 W 097/48370 PCT~US97110517 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAG~ 'l' GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120 CGTCAATGGG AGlll~llll GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA 840 CTATTGGTGA CGATACTTTC CATTACTAAT CCATAACATG G~'l'C'l"l"l'GCC ACAACTCTCT 1260 SU~ Sn~

CA 022~8~68 l998-l2-l8 W O 97148370 PCT~US97/10517 GGCAGAAGAA GATGCAGGCA GCTGAGTTGT 'l~l~ll~lGA TAAGAGTCAG AGGTAACTCC 1740 GCGCGCCACC AGACATAATA GCTGACAGAC TAACAGACTG TTCCTTTCCA TGG~l~llll 1860 CTGCAGTCAC CGTCCTTAGA TCTGCTGTGC CTTCTAGTTG CCAGCCATCT ~ll~lllGCC 1920 AGGCACATCC C~ l~l~l GACACACCCT GTCCACGCCC CTGGTTCTTA GTTCCAGCCC 2220 S~ (~ULE2i) CA 022~8~68 l998-l2-l8 W O 97/48370 PCT~US97/10517 TCCGGCAAAC AAACCACCGC TGGTAGCGGT G~lllll~llG TTTGCAAGCA GCAGATTACG 3180 GCCACGGTTG ATGAGAGCTT 'l'~l"l'~'l'AGGT GGACCAGTTG GTGATTTTGA ACTTTTGCTT 3600 TTATTCATAT CAGGATTATC AATACCATAT TTTTGAAAAA GCCGl'll~"l'~ TAATGAAGGA 3840 AAACCGTTAT TCATTCGTGA TTGCGCCTGA GCGAGACGAA ATACGCGATC GCTGTTAAAA 4~40 ATATTTTCAC CTGAATCAGG ATA'll~ l' AATACCTGGA ATG~'l'~llll CCCGGGGATC 4260 SUBS~ S~ (NUIE26) ... ..

CA 022~8~68 1998-12-18 W 097/48370 PCTrUS97/10S17 TTA~ l'G CAATGTAACA TCAGAGATTT TGAGACACAA CGTGGCTTTC CCCCCCCCCC 4680 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATCACCATG GATGCAATGA AGAGAGGGCT CTG~ ~ CTGCTGCTGT GTGGAGCAGT 60 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs SU~~ 26) CA 022~8~68 1998-12-18 W O 97/4B370 PCTrUS97/10517 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSIllul~Sh~l (RULE26) CA 022~8~68 1998-12-18 W O 97/48370 PCT~US97/10517 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCACATTGAT CAGATATCTT A~ lC TCTCTGCACC ACTCTTC 47 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Thr Asn Trp Leu Trp Tyr Ile Lys l 5 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg (2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SUBSllJU~tSh~l (RULE26) CA 022~8~68 l998-l2-l8 W 097/48370 PCTrUS97/10517 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Lys Ala Gln Asn His Val Val Gln Asn Glu His Gln (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ''oligonucleotideU
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

SUBSIIIUI~ SIIEr (RUlE26) CA 022~8~68 l998-l2-l8 W 097/48370 PCTrUS97/10517 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs Ul~ S~l (RULE 2G) CA 022~8~68 l998-l2-l8 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSIIlul~: ShEE~ ~RUL~~) CA 022~8~68 l998-l2-l8 W O 97/48370 rCT~US97/10517 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
C~ '~'l'~'l'G AGTTTAAACT GCACTGATTT GAAGAATGAT ACTAATAC 48 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCACATTGAT CAGCCCGGGC TTAGGGTGAA TAGCCCTGCC TCA~~ CAC 53 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"

SUI~ SH~ ULE2ii) CA 022~8~68 1998-12-18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTGAAAGACC AGCAACTCCT AGGGATTTGG GGTTGCTGTG G 4l (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCACATTGAT CAGCCCGGGC TTAGGGTGAA TAGCCCTGCC TCA~ l CAC 53 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

SUlBS~ Sh~tl (RUIE~;) CA 022~8~68 1998-12-18 W O 97/48370 PCTrUS97/10517 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Asn Arg Leu Ile Lys Ala (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CTGACCCCCC ~ GGG GGCTGGCAGT TGTAACACCT CAGTCATTAC ACAG 54 (2) INFORMATION FOR SEQ ID NO:30:

u~Sh~ll (RUIE26~

CA 022~8~68 l998-l2-l8 (i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 305 base pairs - (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CCACCACCCT ~ ~lGCC TCTGATGCCA AGGCCTATGA CACAGAGGTG CACAATGTGT 120 (2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

SUBSI~ Sh~l (RULE21i) .. . . .

CA 022~8~68 1998-12-18 TCCTCTGGGG GGGACCCTGA GATTGTGATG CACTCCTTCA ACTGTGGGGG GGA~ll~ 780 ACCGAAATCT TCAGGCCTGG GGGGGGGGAC ATGAGGGACA ATTGG l065 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

GCCCTGTTTC TGGGCTTTCT GGGGGCTGCT GGCTCCACAA TGGGCGCCGC TAGCATGACC l80 (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SU~ Sh~ UIE26) CA 022~8~68 l998-l2-l8 W O 97/48370 PCTrUS97/10S17 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SU~ SM~E~(RULE26) CA 022~8~68 l998-l2-l8 W 097/48370 PCT~US97/10517 (ii) MOLECULE TYPE: DNA (genomic~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

GGGATTTGTC GT~'l'~'l"l"l"l'A TATATATTTA AAAGGGTGAC CTGTCCGGAG CCGTGCTGCC 240 (2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

AAAGACGGGT AAAAGTGATA AAAATGTATC ACTCCAACCT AAGACAGGCG CAGCTTCCGA l80 GGGATTTGTC GT~l~llllA TATATATTAA AAAGGGTGAC CTGTCCGGAG CCGTGCTGCC 240 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sing-le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn Su~ sht~ (XUlE2~i) CA 022~8~68 l998-l2-l8 W 097/48370 PCT~US97/10~17 (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids Sl~'~ SHI~(~U1~26) CA 022~8~68 1998-12-18 W097/48370 PCT~US97110517 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys l 5 l0 (2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Arg l 5 l0 15 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3547 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG l80 SU~ S~ uaE26) CA 022~8~68 1998-12-18 W O 97/48370 PCT~US97/10517 CCCATTGACG TCAATGGGAG 'lll'~ll"l"lGG CACCAAAATC AACGGGACTT TCCAAAATGT 600 ATAAGCAGAG ~lC~lllAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACG~l~ll'll 720 AA~l~l'~l"l"l' ATTGGCTATA TGCCAATACA CTGTCCTTCA GAGACTGACA CGGACTCTGT 1080 AAGGCAGCGG CAGAAGAAGA TGCAGGCAGC TGA~ll~ll~ 'l~l"l'~lGATA AGAGTCAGAG 1500 GGT~ll"l"l~"l' GCAGTCACCG TCCTTAGATC TGCTGTGCCT TCTAGTTGCC AGCCATCTGT 1680 SUBSIIlul~ SHEr (~ULE 2C) CA 022~8~68 l998-l2-l8 W O 97/48370 PCTruS97/l0517 CTTACCGGAT ACCTGTCCGC ~ 'l'CCCT TCGGGAAGCG TGGCGCTTTC TCAATGCTCA 2160 TATGTAGGCG GTGCTACAGA ~llCll~AAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG 2400 TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 'l"1~l"l"l'~'ll"l'~ CAAGCAGCAG 2520 ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 'l~"l"l"l"l~'l'AC GTGATCCCGT 2580 AAGCTTATGC A'lll~l'll'CC AGA~'l"l~llC AACAGGCCAG CCATTACGCT CGTCATCAAA 2940 SUBSIllU~ Sh~ (RUIE26) CA 022~8~68 1998-12-18 W O 97/48370 PCTrUS97/10517 (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "olignucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide~

Sl~~ S~ (BUIE26) CA 022~8~68 1998-12-18 W 097/48370 PCTrUS97/10517 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
GGTACAGATA TCGGAAAGCC ACGll~l~lC TCAAAATC 38 t2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: ~desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

SUBSIIlul~Sh~ ULE2~) CA 022~8~68 l998-l2-l8 W 097/48370 PCT~US97/10~17 (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

_ 95 _ SUBSIllul~ SI~ (RUL~26) W 097/48370 PCTrUS97/10517 (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Glu Leu Asp Lys Trp Ala l 5 SUBSIllul~sHE~ (~ULE26)

Claims (19)

WHAT IS CLAIMED IS:

1. A synthetic polynucleotide comprising a DNA
sequence encoding a peptide or protein, the DNA sequence comprising codons optimized for expression in a nonhomologous host.

2. The synthetic polynucleotide of Claim 1 wherein the protein is an HIV protein.

3. The synthetic polynucleotide of Claim l wherein the DNA sequence encodes HIV env protein or a fragment thereof, the DNA sequence comprising codons optimized for expression in a mammalian host.

4. The polynucleotide of Claim 3 which is selected from:
VlJns-tPA-HIV MN gpl20;
VlJns-tPA-HIV IIIB gpl20;
V 1 Jns-tPA-gp 140/mutRRE-A/SRV - 1 3 ' -UTR;
V 1 Jns-tPA-gp 140/mutRRE-B/SRV- 1 3 '-UTR;
V 1 Jns-tPA-gp 140/opt30-A;
VlJns-tPA-gpl40/opt30-B;
VlJns-tPA-gpl40/opt all-A;
VlJns-tPA-gpl40/opt all-B;
VlJns-tPA-gpl40/opt all-A;
VlJns-tPA-gpl40/opt all-B;
V lJns-rev/env:;
VlJns-gpl60;
V lJns-tPA-gp 160;
VlJns-tPA-gpl60/opt Cl/opt41 -A;
VlJns-tPA-gpl60/opt Cl/opt41-B;
VlJns-tPA-gpl60/opt all-A;
VlJns-tPA-gpl60/opt all-B;
VlJns-tPA-gpl60/opt all-A;

VlJns-tPA-gpl60/opt all-B;
VlJns-tPA-gpl43;
V 1 Jns-tPA-gp 143/mutRRE-A;
VlJns-tPA-gp143/mutRRE-B;
VlJns-tPA-gpl43/opt32-A;
VlJns-tPA-gp 143/opt32-B;
VlJns-tPA-gpl43/SRV-1 3'-UTR;
VlJns-tPA-gpl43/opt Cl/opt32A;
VlJns-tPA-gpl43/opt Cl/opt32B;
VlJns-tPA-gpl43/opt all-A;
VlJns-tPA-gpl43/opt all-B;
VlJns-tPA-gpl43/opt all-A;
VlJns-tPA-gpl43/opt all-B;
VlJns-tPA-gpl43/opt32-A/glyB;
VlJns-tPA-gpl43/opt32-B/glyB;
VlJns-tPA-gpl43/opt Cl/opt32-A/glyB;
VlJns-tPA-gpl43/opt Cl/opt32-B/glyB;
V 1 Jns-tPA-gp 143/opt all-A/glyB;
VlJns-tPA-gpl43/opt all-B/glyB:
V lJns-tPA-gp 143/opt all-A/glyB;
VlJns-tPA-gpl43/opt all-B/glyB; and combinations thereof.

5. The polynucleotide of Claim 2 which induces anti-HIV
neutralizing antibody, HIV specific T-cell immune responses, or protective immune responses upon introduction into vertebrate tissue, including human tissue in vivo, wherein said polynucleotide comprises a gene encoding an HIV gag, HIV protease and combinations thereof.

6. A method for inducing immune responses in a vertebrate against HIV epitopes which comprises introducing between 1 ng and 100 mg of the polynucleotide of Claim 2 into the tissue of the vertebrate.

7. A method for using a rev independent HIV gene to induce immune responses in vivo which comprises:
a) synthesizing the rev independent HIV gene;
b) linking the synthesized gene to regulatory sequences such that the gene is expressible by virtue of being operatively linked to control sequences which, when introduced into a living tissue, direct the transcription initiation and subsequent translation of the gene;

8. A method for inducing immune responses against infection or disease caused by virulent strains of HIV which comprises introducing into the tissue of a vertebrate the polynucleotide of Claim 2.

9. A vaccine for inducing immune responses against HIV infection which comprises the polynucleotide of Claim 2 and a pharmaceutically acceptable carrier.

10. A method for inducing anti-HIV immune responses in a primate which comprises introducing the polynucleotide of Claim 2 into the tissue of the primate and concurrently administering interleukin 12, GM-CSF, or combinations thereof parenterally.

11. A method of inducing an antigen presenting cell to stimulate cytotoxic and helper T-cell proliferation an effector functions including lymphokine secretion specific to HIV antigens which comprises exposing cells of a vertebrate in vivo to the polynucleotide of Claim 2.

12. A method of increasing rev independent in vivo expression of DNA encoding HIV env or a fragment thereof, comprising (a) identifying placement of codons for proper open reading frame;

(b) comparing wild type codons for observed frequency of use by human genes;
(c) replacing wild-type codons with codons optimized for high expression of human genes; and (d) testing for improved expression.

13. A vaccine for inducing immune responses against HIV infection which comprises the polynucleotide of Claim 2 wherein the polynucleotide is delivered by a canarypox, vaccinia virus, adenovirus, adeno-associated virus, retrovirus, Listeria, Shigella, specific ligand, BCG, or salmonella.

14. A method of inducing an immune response to HIV
which comprises administration of the polynucleotide of Claim 2 and administration of an attenuated HIV, a killed HIV, an HIV protein, a fragment of an HIV protein, or combinations thereof, wherein the administration of the polynucleotide is prior to or simultaneous with or subsequent to the administration of the attenuated HIV, the killed HIV, the HIV protein, the fragment of the HIV protein or the combinations thereof.

15. A method of inducing an immune response to HIV
which comprises administration of the polynucleotide of Claim 2 with an adjuvant.

16. A method of treating HIV infection which comprises administration of the polynucleotide of Claim 2 to a patient and administration of an anti-HIV compound to the patient, wherein the administration of the polynucleotide is prior to or simultaneous with or subsequent to the administration of the anti-HIV compound.

17. A method of increasing expression of a gene in a nonhomologous host, comprising:

a) comparing codons of a wild type gene to codons preferred by the nonhomologous host;
b) replacing codons of the wild type gene with new codons, the new codons having a DNA sequence preferred by the nonhomologous host:
c) inspecting third nucleotides of the new codons and first nucleotides of adjacent new codon immediately 3'- of the first, and if a 5'-CG-3' pairing has been created by the new codon selection, replacing it;
d) eliminating undesired sequences to yield a synthetic optimized gene; and e) inserting the synthetic gene into the nonhomologous host.

18. A method of expressing a peptide in a host comprising administration of the synthetic polynucleotide of Claim 1 to the host.

19. A method of increasing production of a recombinant protein by a host, comprising:
a) transforming a host cell with the synthetic polynucleotide of Claim 1 to produce a transformed host; and b) cultivating the transformed host under conditions that permit expression of the synthetic polynucleotide and production of the recombinant protein.