ZA200603044B

ZA200603044B - DNA vaccine compositions and methods of use

Info

Publication number: ZA200603044B
Application number: ZA200603044A
Authority: ZA
Inventors: Opendra Narayan; Zhenqian Liu
Original assignee: Univ Kansas Medical Center
Priority date: 2003-09-16
Filing date: 2006-04-13
Publication date: 2007-07-25

Description

DNA VACCINE COMPOSITIONS AND METHODS OF USE

Cross-Reference to Related Application

This Application claims benefit of U.S. Provisional Application No. 60/503,197, filed September 16, 2003.

Background of Invention

Statement of Government Rights

This work was supported in part by NIH grant numbers G1401830 and

G1402500. The government of the United States of America may have rights in this invention.

The present invention relates generally to the field of prophylactic vaccines for generating protection from HIV-1 induced disease and infection. More specifically, the present invention relates to DNA vaccines against the Human Immunodeficiency

Virus (HIV).

By the end of the year 2000, an estimated 36.1 million people worldwide were infected with HIV. In that year alone, HIV/AIDS-associated illnesses claimed the lives of approximately 3 million people worldwide. An estimated 500,000 of those deaths were of children under the age of fifteen. The importance of an HIV vaccine with respect to world health cannot be overstated.

It is recognized that effective vaccines that will inhibit or prevent HIV-1 infection or HIV-1 induced disease in humans will be useful for the treatment of certain high-risk populations, and as a general prophylactic vaccination for the general

STLDO1-1099641-1 population that may risk HIV-1 infection or HIV-1 induced disease. A vaccine that will confer long-term protection against the transmission of HIV-1 would be most useful. Unfortunately, numerous problems stand in the way of developing effective vaccines for the prevention of HIV-1 infection and disease. Certain problems are most likely the result of the unique nature of the HIV-1 virus and its functional properties, and as yet no effective vaccine has been developed (for review, see:

Berzofsky et al., Developing Synthetic Peptide Vaccines for HIV-1, Vaccines 95, pps. 135-142, 1995; Cease and Berzofsky, Toward a Vaccine for AIDS: The Emergence of

Immunobiology-Based Vaccine Design, Annual Review of Immunology, 12:923-989;

Berzofsky, Progress Toward Artificial Vaccines for HIV, Vaccines 92, pps. 40-41, 1992).

HIV is a retrovirus, meaning that its genome consists of RNA rather than

DNA. There are two primary strains of the virus, designated HIV-1 and HIV-2, with

HIV-1 being the strain that is primarily responsible for human infection. The RNA genome of HIV is surrounded by a protein shell. The combination of RNA genome and the protein shell is known as the nucleocapsid, which is in turn surrounded by an envelope of both protein and lipid.

Infection of host cells by HIV begins when the gp120 protein of HIV, a highly glycosylated protein located in the viral envelope, binds to the CD4 receptor molecule of ahost cell. This interaction initiates a series of events that allow fusion between the viral and cell membranes and the subsequent entry of the virus into the cell.

Following entry into the host cell, HIV RNA is transcribed into double- stranded DNA by a viral reverse transcriptase enzyme. Once integrated into the host genome, HIV expresses itself through transcription by the host’s RNA Polymerase II

STLDO1-1099641-1 enzyme. Through both transcriptional control and posttranscriptional transcript processing, HIV is able to exert a high level of control over the extent to which it expresses itself.

Studies of the HIV virus have revealed much information about the molecular

S biology of the virus, including information concerning a number of genes and genetic regions important to the pathogenicity of HIV. Among these important genes and regions are rt, int, vif, and the 3° LTR of HIV.

The rt gene of HIV encodes viral reverse transcriptase. This enzyme utilizes the RNA genome of HIV to produce a corresponding linear double-stranded DNA molecule that can be incorporated into the host genome.

The int gene of HIV encodes an integrase. This is the enzyme that actually catalyzes the insertion of the reverse-transcriptase-produced linear double-stranded viral DNA into the host genome. In order to complete integration of the viral DNA into the host genome, the host cell DNA repair mathinery performs a ligation of the host and viral DNAs.

The vif gene of HIV encodes a protein known as the ‘viral infectivity factor.’

This protein is required for the production of infectious virions. The protein likely overcomes a cellular inhibitor that otherwise inhibits HIV-1, and may also enhance the stability of the viral core and the preintegration complex.

The LTR (Long Terminal Repeat) regions of HIV-1 contain promoter regions necessary to drive expression of the HIV genes. The 5’ LTR of HIV-1 contains the promoter that is primarily responsible for driving HIV-1 gene expression, though if the 5 LTR sequence is disrupted, the 3° LTR may assume this function. The 3° LTR is necessary for integration of the viral DNA into the host genome.

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Among other important HIV-1 genes are gag, pol, nef, and vpu.

The gag gene encodes for, among other things, the p27 capsid protein of HIV.

This protein is important in the assembly of viral nucleocapsids. The p27 protein is also known to interact with the HIV cellular protein CyA, which is necessary for viral infectivity. Disruption of the interaction between p27 and CyA has been shown to inhibit viral replication.

The pol gene contains the 77 and int sequences of HIV-1, thus encoding, among other things, reverse transcriptase and integrase.

The nef gene product (known as Negative Factor, or Nef) has a number of potentially important properties. Nef has the ability to downregulate CD4 and MHC

Class I proteins, both of which are important to the body’s ability to recognize virus- infected cells. Nef has also been shown to activate cellular protein kinases, thereby interfering with the signaling processes of the cell. Perhaps most importantly, deletion of nef from a pathogenic clone of Simian Immunodeficiency Virus (SIV) renders the virus nonpathogenic in adult macaque monkeys. Thus, a functional nef gene is crucial for the ability of SIV to cause disease in vivo. Further, studies have shown that HIV positive individuals with large deletions in the nef gene remained healthy for well over ten years, with no reduction in cellular CD4 counts.

The ypu gene encodes a protein of originally unknown function (known as

Viral Protein, Unknown, or Vpu), but which is now known to downregulate CD4 and

MHC Class-I expression as well as promote viral budding. Vpu is also similar to another viral protein that acts as an jon charmel. The vpu gene is present in HIV-1, but is absent in HIV-2.

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In nearly all viral infections, certain segments of the infected population recover and become immune to future viral infection by the same pathogen.

Examples of typical viral pathogens include measles, poliomyelitis, chicken pox, hepatitis B, and small pox. The high mortality rate of HIV-1 infection, and the extremely rare incidence of recovery and protective immunity against HIV-1 infection, has cast doubt on the ability of primates to generate natural immunity to

HIV-1 infection when pathogenic HIV-1 is the unmodified wild-type viral pathogen.

Thus, there is a great need for a vaccine that will confer upon primate populations protective immunity against HIV-1 virus.

A hallmark of resistance to future viral infection is the generation of ‘neutralizing antibodies’ capable of recognizing the viral pathogen. Another measure is cellular immunity against infected cells. In typical viral infections, generation of neutralizing antibodies and cellular immunity heralds recovery from infection. In

HIV-1 infection, however, neutralizing antibodies and cellular immunity appear very early during the infection and have been associated with only a transient decrease in : viral burden. In spite of the generation of neutralizing antibodies and cellular immunity, viral replication in HIV-1 infection rebounds and AIDS (acquired immune deficiency syndrome) develops. Thus, in HIV-1 infection, neutralizing antibodies and cellular immunity are not accurate measures of protective immunity.

A further problem in developing an effective vaccine for HIV-1 is the antigenic diversity of the wild-type virus. There is a strong possibility that vaccines generated via recombinant HIV-1 coat proteins will confer resistance to specific phenotypes of virus and not broad spectrum immunity. Vaccine development using recombinant HIV-1 gp120 peptide, a HIV-1 viral coat protein, has passed phase-one

STLDO1-1099641-1 clinical trials showing no toxicity. Data has indicated, however, that neutralizing antibodies appeared only transiently. Thus, recombinant HIV-1 gp120-peptide vaccines may act only in the short-term, with reversion to susceptibility of infection occurring in the future.

In general, it is accepted that live-virus vaccines induce better immunity : against pathogenic viruses than isolated viral proteins (see, for example, Putkonen et al., Immunization with Live Attenuated SIV mec Can Protect Macaques Against

Mucosal Infection with SIVsm, Vaccines 96, pps. 200-210, 1996; Dimmock and :

Primrose Introduction to Modern Virology, Fourth Ed., Blackwell Science, 1994). :

The use of live lentivirus vaccines, such as HIV-1 vaccine, is resisted because of great : concern that the virus will persist indefinitely in the inoculated population because of integration of the viral DNA into the host DNA of the inoculated individuals (see, for example, Haaft et al., Evidence of Circulating Pathogenic SIV Following Challenge of Macaques Vaccinated with Live Attenuated SIV, Vaccines 96, pps. 219-224, 1996). Thus, a safe and effective vaccine against HIV-1 will encompass ; modifications to prevent the development of virulent pathogenic infection that could occur by either random mutation or other change to the initially non-pathogenic vaccine virus. One possibility for such a vaccine could come in the form of a DNA vaccine against HIV-1.

DNA vaccines are generally injected into host tissues in the form of plasmid

DNA or RNA molecules via needle or particle bombardment. Once delivered, the

DNA induces expression of antigenic proteins within transfected cells. U.S. Patent

No. 6,194,389 describes methods for transferring DNA to vertebrate cells to produce

STLDO1-1099641-1 physiological immune-response producing protein in an animal subject and is incorporated herein in its entirety by reference.

Testing of vaccine efficacy generally requires the challenge of a subject with live virus or DNA. It is ethically and practically difficult to attempt preliminary studies using human subjects. The use of model systems for preliminary design and testing of candidate vaccines has been hampered by various species-specific features of the virus. The HIV-1 virus itself is currently known only to infect certain rate and endangered species of chimpanzees in addition to humans. The feasibility of obtaining sufficient numbers of such endangered animals for full preliminary study of

HIV-1 virus vaccines is quite low. It is preferable to use validated analogous animal model systems.

One analogous model system for HIV-1 has been the SIV imc (Simian

Immunodeficiency Virus, macaque) system. SIV infects a variety of simians, including macaques, but the differences between SIV and HIV make SIV of limited use as a potential human vaccine. There is, therefore, a need for a vaccine made from : a virus that is closely related to HIV, but still infectious in an animal model for purposes of testing.

Chimeric SIV-HIV virus has been developed by placing the envelope proteins of HIV-1 on a background of SIV... The chimeric virus proved to be infectious in monkeys, but did not result in full-blown AIDS or an accurate model to mimic HIV-1 infection monkeys.

As described below, the present invention teaches specific DNA constructs and methods that are effective in generating an immune response to HIV-1in a vaccinated host. ’

STLDO1-1099641-1

Summary of Invention

The present invention is directed to a DNA vaccine for immunization against

HIV. The invention comprises a DNA molecule that has a sequence encoding a plurality of viral proteins capable of stimulating an immune response against HIV.

The DNA molecule is rendered safe for use as a vaccine by the disruption of genes encoding reverse transcriptase, integrase, and Vif. The DNA molecule is further rendered safe by at least a partial deletion of the 3’ LTR.

The DNA molecule of the present invention further includes an Sv40 polyadenylation sequence. In addition, the DNA molecule of the present invention is preferably regulated by a natural SIV promoter sequence.

The present invention is also directed to a method of immunizing an individual against HIV by administering the present DNA vaccine to said individual.

The present invention is further directed toward a vaccine against multiple

HIV subtypes as well as viruses other than HIV, said vaccine produced by substituting genes from said other viruses for the orthologous HIV and/or SIV genes described herein.

Brief Description of Drawings

FIG. 1 is a schematic diagram of the A4-SHIVy,, DNA construct of the present invention.

FIG. 2 is a circular diagram of the A4-SHIVi,» DNA construct of the present invention.

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Detailed Description

The object of the present invention is to provide DNA vaccine compositions and methods that provide either protective immunity to uninfected subjects or therapeutic immunity to infected subjects.

One aspect of the present invention is directed to DNA molecules that encode viral proteins capable of stimulating an immune response against HIV. In preferred embodiments, the DNA vaccine encodes the gag, pro, px, VPT nef, tat proteins of

HIV or SIV.

Importantly, the DNA molecules of the present invention have been disrupted functionally such that the ability to encode proteins that are important in pathogenicity are removed. More specifically, preferred embodiments functionally disrupt the vif, int and rt genes of the DNA vaccine. Other embodiments functionally disrupt the ¢ gene. It is anticipated that the DNA can be disrupted functionally by inserting or : : deleting at least one nucleotide such that the number of nucleotides in the altered sequences differs with respect to the unaltered sequences. It is also anticipated that the

DNA encoding pathogenicity-related proteins can be disrupted functionally by . substituting one or more nucleotides that encode functional amino acids with one or more distinct nucleotides that encode non-functional amino acids. In a preferred embodiment of the present invention, the functional disruption of the DNA encoding pathogenicity-related proteins occurs via deletion of the 7%, int, and vif genes.

Another important aspect of this invention is that it provides for DNA vaccines that disrupt the 3° LTR sequences that enable undesirable integration of

DNA sequences into the host genome. Function of the 3° LTR can also be abolished by substituting functional nucleotides with distinct non-functional nucleotides. The

STLDO1-1099641-1 deleted 3’ LTR region is preferably replaced with an SV40 polyadenylation sequence.

Those skilled in the art will recognize that polyadenylation sites derived from a variety of sources other than SV40 may also be used as substitutes for the 3° LTR sequences.

A further aspect of the invention is the regulation of the DNA molecule of the present invention by use of the SHIV 2 or SIV 5’LTR promoter (SEQ ID NO:7). This promoter drives the expression of viral proteins capable of stimulating an immune response against HIV present in the DNA vaccine. Those skilled in the art will recognize that alternative embodiments of this invention may substitute other functional promoter sequences that will also drive expression of the desired viral : proteins.

Examples:

Example 1. Construction of the A4-SHIV..2 DNA construct.

FIG. 1 is a schematic diagram of the A4-SHIVi2 DNA construct (SEQID

NO: 1) of the present invention. The construction of the present DNA vaccine A4- :

SHIV}.2 DNA construct (SEQ ID NO:1) is performed as follows. The vector used for the present vaccine is pET-9a. The 2.3 kb EcoR I / Xmn I fragment of pET-9a is replaced with the approximately 7.4 kb modified SHIViq2 provirus genome and the approximately 0.5 kb polyadenylation signal sequence of SV40 to yield an intermediate vector. EcoRI and Not I restriction sites are created immediately upstream of the 5’ LTR and at the end of the nef gene, respectively, in another intermediate vector. The reverse transcriptase (r7), integrase (int), and vif genes are eliminated by deletion of an approximately 2.5 kb DNA fragment between the downstream end of the pro gene and upstream of the vpx gene. The approximately

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3.8 kb nucleotide sequence that encodes the envelope (env), nef, and 3° LTR genes of

SHIV; provirus genome is then replaced with the approximately 3.2 kb BcoRV / Not 1 DNA fragment that encodes the env and nef genes of HIV-1. The approximately 2.5 kb Nar I / BstE Il DNA fragment that encodes the leader sequence, gag, and pro genes of SIVmc239 in SHIVy2 is replaced with an approximately 2.4 kb Nar I/ BstE II fragment that encodes the HIV-1 leader sequence, gag, and pro of HIV-1 to yield Ad-

SHIV 2 DNA construct (SEQ ID NO:1). Thus, the 5° LTR, vpx, and vpr genes of the present vaccine are from SIV ac239, and the gag, pro, tat, rev, vpu, env, and nef are from HIV-1. The sequence of a preferred embodiment of the present DNA vaccine A4-SHIVi.2 DNA is designated SEQ ID NO:1.

The information below is provided to detail structure of the A4-SHIV,; PNA construct (SEQ ID NO:1) more completely. A 4,981 bp fragment of SHIViqo that encodes the entire gag, and pol genes (which therefore includes the 7¢ and int portions of the genome), as well as the first 472 bp of the vif gene, is replaced with a 2,376 bp

DNA fragment of HIV-1 in the A4-SHIV,.2 DNA construct. This 2,376 bp fragment encodes the entire HIV-1 gag gene, and a portion of the HIV-1 pol gene (the entire region encoding protease is included; the nucleotides corresponding to the first 104 amino acids of reverse transcriptase have been removed; the int and vif genes have been completely removed. The 4,981 bp fragment of SHIVy, that was replaced is : designated SEQ ID NO:2. The DNA sequence of the first 472 bp of the vif gene of

SHIV,2, which was also replaced is designated SEQ ID NO:3. The DNA sequence of the 2,376 bp fragment of HIV-1 used to replace the deleted 4,981 bp and 472 bp

SHIV, sequences (SEQ ID NO:2 and SEQ ID NO:3, respectively) is designated

SEQ ID NO:4.

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In addition to the above, a 411 bp DNA fragment is deleted from the 3° LTR of SHIViuz to yield the A4-SHIVi,, DNA construct (SEQ ID NO:1). This deleted 3°

LTR sequence is designated SEQ ID NO:5. In the A4-SHIVy,; DNA construct the deleted 3’LTR sequences are replaced with 481 bp DNA sequence of the SV40 polyadenylation signal sequence that is designated SED ID NO:6.

Example 2. Efficacy of the A2-SHIVi2 DNA Vaccine.

Prior to detailing the functional aspects of the present invention, and the experimental results derived from its use, it is necessary to establish the efficacy of the present invention by way of comparison with that which has come before. Prior to the invention of the present vaccine, and subsequent testing thereof, the utility thereof as a vaccine was unknown.

It is known from previous studies conducted by the inventor of the present invention that a live virus vaccine against HIV is highly efficient in eliciting protection against the virus. To establish that a DNA vaccine could be just as : efficient in providing such protection, an experiment utilizing five macaques was conducted. Three of the animals were injected with a A2-SHIVy,; DNA (previously described as the V7 embodiment in Patent Application 10/279,992, herein incorporated by reference in its entirety) wherein the n and 3° LTR had been deleted to increase the safety of the vaccine. The A2-SHIV 2 DNA also replaces the deleted 3’ LTR sequence with the SV40 polyadenylation sequence. The remaining two animals were immunized with a live virus vaccine. The three animals vaccinated with the DNA vaccine were each given 2 mg of the DNA, inj ected intradermally, followed by an intramuscular injection of 5 mg of DNA six weeks later, and a third, 0.5 mg intramuscular DNA injection twelve weeks later. The macaques were challenged

STLDO1-1099641-1 intravenously with an undiluted stock preparation of SHIV 89.6P twelve weeks after the final immunization. It is important to note that the same dose of the SHIV 89.6P causes disease in 100% of inoculated control animals. The two macaques vaccinated with live virus were challenged ten weeks post-vaccination with the same SHIV virus.

When the animals were subsequently studied, it became clear that the DNA vaccine induces ELISPOT™ (Cellular Technology Limited, Cleveland, Ohio) responses against epitopes in the Env and Gag peptides, as well as neutralizing antibodies to SHIViq2. ELISPOT™ responses are hereby defined as measures of the number of cells expressing an indicated epitope. All three animals vaccinated with the

DNA vaccine became infected with SHIV 89.6P, but each developed only low levels of viral RNA in plasma, with no loss of CD4 T-cells. The animals vaccinated with the DNA vaccine A2-SHIVy,2 DNA developed a massive anamnestic ELISPOT™ response following challenge. The infection in these animals has been controlled for more than 28 weeks. At the 28-week point, the three animals that were immunized with DNA vaccine demonstrated protection that was as efficient as animals immunized with the live vaccine. Thus, the DNA vaccine proved to be just as efficient as the live vaccine in eliciting protection against heterologous SHIV 89.6P. Further, the animals receiving the DNA vaccination did not have to bear the burden of prior infection with a live vaccine virus.

Example 3. In Vivo Efficacy of both the A2-SHIViq, and A4-SHIV,; DNA vaccines

Although the experiment described in Example 2 indicated the efficacy of the

DNA vaccine A2 -SHIVy,» DNA lacking the rz gene and 3° LTR, it was not clear whether the current vaccine, the A4-SHIV 2, would be efficacious as a vaccine. The

STLDO1-1099641-1 uncertainty stems from the fact that the current vaccine A4-SHIViaa contains four deletions (71, int, vif, and the 3’ LTR), each deletion corresponding to a portion of the viral genome important in infectivity of the virus. The deletions were made in order to render the virus non-infectious and safe for use, but it was unknown whether these four deletions, in addition to the fact that a DNA rather than a live virus was being used, would render the vaccine incapable of providing protection against HIV-1.

Surprisingly, the present virus proved to be just as efficient at inducing protection against heterologous SHIV 89.6P as the A2-SHIVy,; DNA vaccine described in the live virus comparison of Example 2.

Three macaques were injected intramuscularly with 5 mg of the A2-SHI Vino

DNA, while three other macaques were injected intramuscularly with 5 mg of the present A4-SHIVyo DNA. The injections were repeated eleven weeks later, and the animals were challenged intravenously with undiluted stock of SHIV 89.6P six weeks : after the second immunization. All six of the animals developed ELISPOT ™ responses to the vaccine three weeks after the first injection, the responses declining approximately three weeks later to undetectable levels. The responses appeared once again only one week after the second injection, and again declined to low levels.

Only minimal responses were detected at the time of challenge. By one week after challenge, each of the animals had developed high titers of viral replication, which were matched by a powerful CMI (cell-mediated immune) response. By two weeks post-challenge, the viral burdens in the animals declined to levels between ten- and twenty-fold less than concentrations observed one week earlier. None of the animals

Jost CD4 T-cells. The ability of the DNAs to induce protection after only two injections underscores the potency of the DNA vaccines, and the results of the

STLDO1-1099641-1 experiment clearly showed that that, despite the additional deletions, the A4-SHIViu2

DNA construct DNA vaccine (SEQ ID NO:1) of the present invention was just as effective as the A2-SHIViqz DNA vaccine, which in turn was just as effective as the live virus vaccine.

Example 4. Utility of the SV40 Polyadenylation Sequence as a substitute for the 3’LTR.

A further experiment was performed to compare the utility of the SV40 polyadenylation sequences as substitutes for the 3’ LTR sequence. This was accomplished by comparing the ability of the V5 embodiment SHIVia.2 DNA vaccine with an intact 3’LTR and the V6 embodiment SHIVy,2 DNA vaccine having the 3'LTR substituted with an SV40 polyadenylation sequence (SEQIDNO: 6) to express vector-encoded viral proteins (both the V5 and V6 embodiments are described in Patent Application 10/279,992, herein incorporated by reference in its : entirety). Performance of the two DNA molecules was compared in transfected primary human fibroblasts, the human embryonic kidney epithelial cell line 293, and : in Jurkat cells, for expression of viral proteins in intra and extracellular compartments.

The DNAs were also compared for duration of expression and for the amount of protein production, as well as for post translational modification and cleavage of precursor proteins. It was determined that the 3’ LTR deleted V6 embodiment

SHIV,; DNA vaccine construct, surprisingly; was more efficient in producing viral proteins than the V5 embodiment SHIV,.2 DNA vaccine construct having both LTRs.

The duration of protein production was also longer in the 3’ LTR deleted vaccine.

Immunoprecipitation analysis revealed that deletion of the 3° LTR resulted in rapid cleavage of the gag precursor, yielding double the amount of p27 being exported to

STLDO1-1099641-1 the extracellular compartment. Taken together, these data indicate that deletion of the 3* LTR not only alleviates concerns about integration of the viral genome into host

DNA, but also results in a more efficient expression of viral proteins.

Example 5. Coadministration of Cytokines with A4-SHIV,o2 DNA vaccine.

Next, a study was undertaken to ascertain whether the immune response induced by the present vaccine could be enhanced by co-administration of cytokines (for example, GM-CSF) DNA. BALB/C mice were immunized intramuscularly with a mixture of 100pg of A4 SHIVi,; DNA and 25g of mouse GM-CSF DNA. The injections were given twice, two weeks apart, and the mice were sacrificed one week : after the second immunization. Splenocytes were tested for response to SIV Gag peptides divided into five groups in the ELISPOT ™ assay. Even though the ; immunization doses were low and tissue samples were harvested early, before CMI : - responses could peak, all four animals that received GM-CSF DNA along with the : vaccine DNA developed ELISPOT ™ responses, varying from 20 to 40 cells/10° splenocytes, whereas only 50% of the animals receiving the vaccine DNA alone developed such a response. The GM-CSF caused an impressive chemotactic effect, as evidenced by the large number of mononuclear cells that were concentrated at the site of injection. This effect attracted many more antigen-presenting dendritic cells to the site of the injection that evident in the animals that received the DNA vaccine only.

Surprisingly, however, the mice that received both vaccine DNA and GM-CSF developed lower CMI titers that those receiving the DNA vaccine alone. That is, the number of viral protein specific ELISPOT ™ positive cells generated by the vaccine alone was significantly higher than those generated by the vaccine plus GM-CSF. It is concluded that coadministration of the A4-SHIViw; DNA vaccine with a cytokine

STLDO01-1099641-1 such as GM-CSF may be desirable in instances where it is either prophylactically or therapeutically desirable to increase the pumber of injected subjects that develop activated splenocytes.

Thus, the present DNA vaccine is useful for providing protection against HIV.

The DNA used in the present invention was derived from SHIViuo, 2 virus that has a highly efficient replication strategy, making it highly pathogenic. The transcriptional machinery of the DNA was maintained by preserving the 5’ LTR that houses the promoter/enhancer sequences of the viral DNA. In addition, the 5’ LTR contains binding sites for transcription factors such as NFKB, NFAT, SP-1, and the like, and the binding site for the RNA of tat, a molecule unique to HIV and the lentivirus that is responsible for the transactivation of viral DNA. The integrase gene and the 3° LTR : were deleted to minimize the ability of the DNA to integrate into host cell DNA.

Thus, the DNA cannot persist indefinitely in tissues. Furthermore, the deletion of the : reverse transcriptase and vif genes crippled the ability of the genome to code for infectious virus. At the same time, the viral proteins encoded by the env, gag, vpu, ~ : tat, and nef genes were highly expressed in cells transfected with the DNA. The present DNA vaccine is highly immunogenic in macaques and elicits protective immunity against heterologous viruses. Importantly, the present vaccine can be used not only prophylactically, but also therapeutically in individuals already infected with

HIV because the DNA may be injected at any time during a period when anti- retroviral drug therapy is in place.

The examples and disclosure provided above describe certain embodiments of the present invention, but are not meant to be limiting. It will be apparent to those of <kill in the art, upon reading this disclosure, that the present invention may be

STLDO1-1099641-1 modified in a number of ways without departing from the spirit or scope of the invention. For example, the env, gag, and nef genes described above could be excised and replaced with the corresponding genes from another subtype of HIV. Thus, the present vaccine could be used for immunization against various subtypes of HIV.

Further, the env, gag, nef, and other genes described above could be replaced with genes from other viruses, such as SARS and Hepatitis C. Thus, the present DNA vaccine, described above, could be used as an “engine” to drive expression of viral genes from other than HIV or SIV, thereby providing a DNA vaccine to a variety of other viruses. The present invention is limited only by the claims that follow. “Comprises/comprising™ when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps or components or groups thereof.

The sequence listings attached hereto are to be considered as an integral part of the present specification. 18 AMENDED SHEET

Claims

r © g02845-0004 ire ) ~ wl ~~. | P . ad Saat po. £3 BE {UGLY SUS1E (211 uepds,. (US 21/10/05 EAS SAC oso . -19- if CLAIMS :

1. A DNA immunogenic composition against HIV comprising an isolated DNA molecule having a sequence encoding a plurality of viral proteins capable of stimulating an immune response against HIV, wherein said DNA molecule is unable to encode functional reverse transcriptase, integrase and Vif proteins, and further wherein a 3' long terminal repeat of said DNA molecule is at least partially disrupted.

2. The DNA immunogenic composition of claim 1, wherein the DNA molecule is unable to encode functional reverse transcriptase, integrase, and Vif proteins due to at least a partial deletion of the genes that encode for the reserve transcriptase, integrase, and Vif proteins.

O 3. The DNA immunogenic composition of claim 1, wherein the DNA molecule further comprises a natural SIV promoter sequence. 4, The DNA immunogenic composition of claim 2, wherein the DNA molecule further comprises a natural SIV promoter sequence.

5. The DNA immunogenic composition of any of claim 1 further comprising a pharmaceutically acceptable carrier.

6. A DNA immunogenic composition derived from a viral genome encoding at least one - protein capable of providing-an immune response against HIV and having a 5' long terminal J repeat and a 3' long terminal repeat, a rt gene, an int gene, and a vif gene, wherein the DNA immunogenic composition has been rendered nonpathogenic by disruption of the rt gene, the int gene, the vif gene, and the 3' long terminal repeat. -

7. The DNA immunogenic composition of claim 6 further comprising a pharmaceutically acceptable carrier.

8. A DNA immunogenic composition comprising the nucleotide sequence of SEQ ID NO:1.

9. The DNA immunogenic composition of claim 8 further comprising a pharmaceutically acceptable carrier.

. ) _° 802845-0004 IPEAIUS PLS UGUY, BOS 1 2211 beads. US 21/10/05 -20-

10. A DNA immunogenic composition comprising: (a) the 5' LTR of SIV; (b) the gag gene of HIV; (c) the pro gene of HIV; (d) the vpx gene of SIV; (e) the vpr gene of SIV; (f) the nef gene of HIV; (g) the tat gene of HIV; (h) the rev gene of HIV, (i) the env gene of HIV; and (j) an SV40 polyadenylation sequence.

11. The DNA immunogenic composition of claim 10 further comprising a . pharmaceutically acceptable carrier.

12. A method of providing an immune response against HIV comprising administering to a recipient a DNA immunogenic composition of claim 1. - 13. The method of claim 12 wherein the isolated DNA molecule is unable to encode © functional reverse transcriptase, integrase, and Vif proteins due to at least a partial deletion of the genes that encode for the reserve transcriptase, integrase, and Vif proteins.

14. A method of providing an immune response against HIV comprising administering to a recipient the DNA immunogenic composition of claim 6.

15. A method of providing an immune response against HIV comprising administering to a recipient a DNA immunogenic composition of claim 10.

16. A method of providing an immune response against HIV comprising administering to 3 a recipient a DNA immunogenic composition of claim 8. la

17. The method of claim 12 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. '

18. The method of claim 13 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

19. The method of claim 14 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

20. The method of claim 15 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

21. The method of claim 16 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. AMENDED SHEET

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92. The method of claim 12, which further comprises administering to said recipient a cytokine.

23. The method of claim 22 wherein the isolated DNA molecule is unable to encode functional reverse transcriptase, integrase, and Vif proteins due to at least a partial deletion of the genes that encode for the reserve transcriptase, integrase, and Vif proteins.

24. The method of claim 14, which further comprises administering to said recipient a cytokine.

25. The method of claim 15, which further comprises administering to said recipient a, © cytokine.

26. The method of claim 16, which further comprises administering to said recipient a cytokine.

27. The method of claim 22 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

28. The method of claim 23 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

29. The method of claim 24 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

30. The method of claim 25 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. :

31. The method of claim 26 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

32. A method for the treatment of an HIV-1 positive individual comprising administering to said individual the DNA immunogenic composition of claim 1 and further administering to said recipient a cytokine.

33. The method of claim 32 wherein the isolated DNA molecule is unable to encode functional reverse transcriptase, integrase, and Vif proteins due to at least a partial deletion of the genes that encode for the reserve transcriptase, integrase, and Vif proteins. — a wy 0 8 8 “wr > = A - . :

_ 802845-0004 IPEAUS CL peTouUSUss dusile 21102005 'Us 21/10/05

34. A method for the treatment of an HIV-1 positive individual comprising administering to said individual a DNA immunogenic composition of claim 6.

35. A method for the treatment of an HIV-1 positive individual comprising administering to said individual a DNA immunogenic composition of claim 10.

36. A method for the treatment of an HIV-1 positive individual comprising administering to said individual a DNA immunogenic composition of claim 8.

37. The method of claim 32 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. } wD 38. The method of claim 33 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

39. The method of claim 34 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

40. The method of claim 35 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

41. The method of claim 36 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. a 42. A DNA immunogenic construct for immunization against HIV comprising an isolated DNA molecule having: (a) a promoter sequence that is operably linked to; (b) a sequence encoding a plurality of viral proteins capable of stimulating an immune response against HIV, wherein said DNA molecule is unable to encode functional reverse transcriptase, integrase and Vif proteins and contains at least a partial disruption of the 3' long terminal repeat of said DNA molecule, and which is operably linked to; and c) a polyadenylation sequence.

43. The DNA immunogenic construct of claim 42 wherein the promoter is derived from the 5' LTR of SIV herein identified as SEQ ID. NO: 7.

44. The DNA immunogenic construct of claim 42 wherein the sequence encoding the plurality of viral proteins capable of stimulating an immune response is selected from a group of coding sequences comprising the gag, pro, tat, rev, vpu, env, VpX, vpr and nef genes of either SIV or HIV. AMENDED SHEET

: ~> LA © 02845-0004 AEE : pC usu 2051 ix Lirsaos US 21/10/05 -23- CR —

45. The DNA immunogenic construct of claim 42 wherein the sequence encoding the plurality of viral proteins capable of stimulating an immune response is selected from a group of coding sequences comprising the gag, pro, tat, rev, vpu, env, and nef genes of HIV and the vpx, and vpr genes of SIV.

46. The DNA immunogenic construct of claim 42 wherein the polyadenylation sequence is the SV40 polyadenylation sequence herein identified as SEQ ID NO: 6.

47. The DNA immunogenic construct of claim 47 further comprising a pharmaceutically acceptable carrier. —- 48. The DNA immunogenic composition of claim 6 which further comprises a Ww polyadenylation sequence substituted into the disrupted 3'long terminal repeat region. 49, The DNA immunogenic composition of claim 48 wherein the rt gene, the int gene, the vif gene, and the 3' Jong terminal repeat are functionally disrupted by at least a partial sequence deletion.

50. The DNA immunogenic composition of claim 48 further comprising a pharmaceutically acceptable carrier.

51. The DNA immunogenic composition of claim 49 further comprising a pharmaceutically acceptable carrier. ~ 52, canceled.

53. canceled.

54. A method of providing an immune response against HIV comprising the administering to a recipient the DNA composition of Claim 42.

55. The method of claim 54 wherein the DNA composition is derived from a viral genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered nonpathogenic by functional disruption of the rt gene, the int gene, the vif gene, and the 3' long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3' long terminal repeat region.

56. canceled. AMENDED SHEET

7 802845-0004 IPEAUS PLT UNL S JU de ed does | US 21/10/05

57. The method of claim 54 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

58. The method of claim 55 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

59. canceled.

60. A method of providing an immune response against HIV according to claim 54 which further comprises administering to said recipient a cytokine.

61. The method of claim 60 wherein the DNA composition is derived from a viral <) genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered nonpathogenic by functional disruption of the rt gene, the int gene, the vif gene, and the 3' long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3'long terminal repeat region.

62. The method of claim 60 wherein the DNA composition is comprised of the nucleotide sequence of SEQ ID NO:1.

63. The method of claim 60 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. D 64. The method of claim 61 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. - 65. The method of claim 62 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

66. A method for the treatment of an HIV-1 positive individual comprising administering to said individual the DNA composition of claim 42.

67. The method of claim 66 wherein the DNA composition is derived from a viral genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered nonpathogenic by functional disruption of the rt gene, the int gene, the vif gene, and the 3' long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3'long terminal repeat region.

68. canceled. AMENDED SHEET

,' 802845-0004 PENS PL | USSU, 3U0s1e we tiuenus US 21/10/09

69. The method of claim 66 wherein the DNA composition further comprises & pharmaceutically acceptable carrier.

70. The method of claim 67 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

71. canceled.

72. The DNA immunogenic construct of claim 42 wherein the 3' long terminal repeat is at least partially deleted.

73. The immunogenic composition of claim 1 further comprising a cytokine compris ing GM-CSF.

74. The immunogenic composition of claim 8 further comprising a cytokine comprising GM-CSF.

75. The method of claim 22 wherein said cytokine comprises GM-CSF.

76. The method of claim 25 wherein said cytokine comprises GM-CSF.

77. The method of claim 26 wherein said cytokine comprises GM-CSF.

78. The method of claim 42 wherein said cytokine comprises GM-CSF. ( 79. The DNA immunogenic composition of claim 1 wherein when said composition is administered to a macacque that is later challenged with infectious SHIV 89.6P, the immune response results in no loss of CD4 T-cells.

80. The method of claim 12 wherein said immune response is such that when said composition is administered to a macacque that is later challenged with infectious SHIV

89.6P, the immune response results in no loss of CD4 T-cells.

81. The DNA immunogenic composition of claim 42 wherein the promoter is derived from the 5' LTR of SIV derived from a SHIV with a highly efficient replication strategy. AMENDED SHEET

47. The DNA vaccine of Claim 42 further comprising a pharmaceutically acceptable carrier.

48. The DNA immunogenic composition of claim 6 which further comprises a polyadenylation sequence substituted into the disrupted 3’long terminal repeat region.

49. The DNA vaccine composition of Claim 48 wherein the the rz gene, the int gene, the vif gene, and the 3’ long terminal repeat are functionally disrupted by at least a partial sequence deletion. © 50. The DNA vaccine of Claim 48 further comprising a pharmaceutically acceptable carrier.

51. The DNA vaccine of Claim 49 further comprising a pharmaceutically acceptable carrier.

52. A DNA vaccine composition comprising the nucleotide sequence of SEQ ID NO:1.

53. The DNA vaccine of Claim 52 further comprising a pharmaceutically acceptable carrier.

54. A method of providing vaccination against HIV comprising the administering to a recipient the DNA composition comprising an isolated DNA molecule having: (a) a promoter sequence that is operably linked to; (b) a sequence encoding a plurality of viral proteins capable of stimulating an immune response against HIV, wherein said DNA molecule is unable to encode functional reverse transcriptase, integrase and Vif proteins and contains at least a partial disruption of the 3° long STLDO1-1099641-1 terminal repeat of said DNA molecule, and which is operably linked to; and c) a polyadenylation sequence.

55. The method of Claim 54 wherein the DNA composition is derived from a viral genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered : nonpathogenic by functional disruption of the 7 gene, the int gene, the vif gene, and the 3’ long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3’long terminal repeat region.

56. The method of Claim 54 wherein the DNA composition is comprised of the nucleotide sequence of SEQ ID NO:1. a

57. The method of claim 54 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. oo

58. The method of claim 55 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

59. The method of claim 56 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

60. A method of providing vaccination against HIV according to claim 54 which further comprises administering to said recipient a cytokine.

61. The method of claim 60 wherein the DNA composition is derived from a viral genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered nonpathogenic by functional disruption of the rt gene, the int gene, the vif gene, and STLDO1-1099641-1 the 3’ long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3’long terminal repeat regjon.

62. The method of claim 60 wherein the DNA composition is comprised of the nucleotide sequence of SEQ ID NO:1.

63. The method of claim 60 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

64. The method of claim 61 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

65. The method of claim 62 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

66. A method for the treatment of an HIV-1 positive individual comprising administering to said individual the DNA composition comprising an isolated DNA molecule having: (a) a promoter sequence that is operably linked to; (b) a sequence encoding a plurality of viral proteins capable of stimulating an immune response against HIV, wherein said DNA molecule is unable to encode functional reverse transcriptase, mtegrase and Vif proteins and contains at least a partial disruption of the 3’ long terminal repeat of said DNA molecule, and which is operably linked to; and c) a polyadenylation sequence.

67. The method of claim 66 wherein the DNA composition is derived from a viral genome encoding at least one protein capable of providing an immune response against HIV, wherein the DNA vaccine composition has been rendered nonpathogenic by functional disruption of the 7? gene, the int gene, the vif gene, and STLDO1-1099641-1 the 3 long terminal repeat, and wherein a polyadenylation sequence is substituted into the 3’long terminal repeat region.

68. * The method of Claim 66 wherein the DNA composition is comprised of the nucleotide sequence of SEQ ID NO:1. s 69. Themetodofclaim 66 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

70. The method of claim 67 wherein the DNA composition further comprises a pharmaceutically acceptable carrier.

71. The method of claim 68 wherein the DNA composition further comprises a pharmaceutically acceptable carrier. :

72. The DNA vaccine construct of claim 42 wherein the 3° long terminal repeat is at least partially deleted. J STLDO1-1099641-1