WO1999019501A1 - Non-integrating dna vector of retroviral origin having high-protein expression, and secreted immunogenic antigens - Google Patents

Abstract

A DNA sequence corresponding to a non-integrating retrovirus having high protein expression is taught. The DNA sequence corresponds to a retroviral genome that is reverse transcriptase and/or integrase and where the 3'LTR has been replaced by a heterologous polyadenylation sequence. Preferably, a strong heterologous promoter is inserted in the 5'LTR upstream of the viral enhancer and promoter. In preferred embodiments the retroviral genome is a primate lentivirus or HTLV-1 or 2 genome. Changes are also made to enhance expression of the processed envelope protein. With an HIV genome a stop codon is inserted in the gp160 gene after the cleavage site for gp120. The gp41 reading frame can be inserted in the nef reading frame with a nucleotide encoding a secretory sequence at the N-terminal. The DNA sequence can be used to produce an immune reaction or large amounts of viral protein.

Description

NON-INTEGRATING DNA VECTOR OF RETROVIRAL ORIGIN HAVING HIGH-PROTEIN EXPRESSION AND SECRETED IMMUNOGENIC ANTIGENS

FIELD OF THE INVENTION

The present application is directed to a non-integrating virus having high protein expression and to soluble immunogenic antigens. Preferably, the virus is a provirus corresponding to a retrovirus, more preferably corresponding to a 5 primate immunodeficiency virus such as human immunodeficiency virus (HIV).

BACKGROUND

Human immunodeficiency virus type 1 (HIV-1) and type (HIV-2) are the etiologic agents of acquired immunodeficiency syndrome (AIDS), from the 10 profound depletion of CD4-positive lymphocytes in infected individuals (Barre- Sinoussi, F., Science 1983; Gallo, R.C., et al., Science 1984; Fauci, A.S., et al., Ann Intern Med. 1984).

There have been a number of approaches to dealing with the virus. One 15 approach has focused on the use of drugs such as reverse transcriptase inhibitors and protease inhibitors. Although considerable progress has been made with the use of cocktails containing combinations of different drugs, recent studies have shown failure over time in approximately half those using the drugs. There are additional problems concerning the high cost of the 20 cocktail of drugs, ease of use, and the inability to use them in most of the world because of that cost.

.An alternative approach is to generate an immune response to the virus and use the immune system to fight the virus. A number of strategies have 25 been proposed to generate such immune responses to viruses, particularly retroviruses such as HIV. These strategies include the use of subunit vaccines such as a vaccine to the envelope glycoprotein. However, such approaches have not yet proven clinically successful. Other approaches have been based on approximating the actual virus. For example, using an attenuated virus such as one lacking a critical determinant. For example, Kestler et al., Cell, 65:651:662 (1991) has proposed the use of nef- HIV viruses. However, due to the severe pathogenicity of the virus, numerous health concerns have been voiced with respect to this approach. Moreover, animal tests have shown that over time many of the animals have developed the disease. Haynes et al., U.S. Patent No. 5,439,809 proposed the use of an attenuated HIV virus expressed by HIV-1 expression vectors devoid of long terminal repeat (LTR) elements. It was also proposed that HIV genes could be eliminated with deletions in integrase and υif exemplified. However, such an approach will not result in large scale expression of particles because many important elements in the HIV LTR have been lost.

Other approaches have been directed to using various vectors expressing single and multiple HIV genes. Some of these approaches are based at least in part on generating an immune response to the vector used as well as the HIV proteins.

HIV-1 infects T lymphocytes, monocytes/macrophage, dendritic cells and, in the central nervous system, microglia (Gartner et αl, 1986; Koenig et αl., 1986; Pope et αl., 1994; Weissman et αl, 1995). All of these cells express the CD4 glycoprotein, which serves as the receptor for HIV- 1 and HIV-2 (Dalgleish et αl, 1984; Klatzman et αl, 1984; Maddon et αl, 1986). Efficient entry of HIV- 1 or HIV-2 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gpl20, to the CD4-amino-terminal domain (McDougal et αl., 1986; Helseth et αl., 1990). The exterior envelope glycoprotein, gpl20, and the transmembrane envelope glycoprotein, gp41, are derived from a gpl60 precursor (Earl, P.L., et al., J Virol 1984). The gpl60 glycoprotein results from the addition of N-linked, high mannose sugar chains to the approximately 845-870 amino acid primary translation product of the env gene in the rough endoplasmic reticulum (ER) [Ibid.]. ter virus binding, the HIV envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al, 1987; Stein et al, 1987; Helseth et al, 1990). Membrane fusion directed by HIV envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al, 1986; Sodroski et al, 1986).

Coreceptors such as the chemokine receptor CCR5, in addition to CD4, are necessary for effective HIV envelope glycoprotein-mediated membrane fusion. Some human and animal cells have been shown to be resistant to HIV- 1 infection and syncytium formation even when human CD4 was expressed on the cell surface (Maddon et al, 1986; Ashorn et al, 1990; Chesebro et al, 1990; McKnight et al, 1994) apparently because they lack such a coreceptor. HIV- 1 variants exhibiting distinct differences in the ability to fuse with and to enter particular subsets of CD4-positive cells have been identified (Broder and Berger, 1995). The third variable (V3) region of the gpl20 envelope glycoprotein appears responsible for determining tropsim-related phenotypes and interactions with particular co-receptors. The ability of HIV to mutate and use different cofactors is one of the ways that the virus escapes ready treatment. This is because numerous mutants are still pathogenic. Thus, having a ready and simple means to target a range of HIV strains would be desirable.

It would be desirable to have a simple method for generating immune responses to envelope glycoproteins from a range of different strains of HIV. For example, changes in the viral envelope glycoproteins, in particular in the third variable (V3) region of the gpl20 exterior envelope glycoprotein, can determine tropism-related phenotypes (Cheng-Mayer et al, 1990; O'Brien et al, 1990; Hwang et al, Westervelt et al, 1992; Chesebro et al, 1992; Willey et al, 1994). Amino acid changes in the V3 region (Helseth et al, 1990; Freed et al, 1991; Ivanoff et al, 1991; Bergeron et al, 1992; Grimaila et al, 1992; Page et al, 1992; Travis et al, 1992) and the binding of antibodies to this domain

(Putney et al, 1986; Goudsmit et al, 1988; Linsley et al, 1988; Rusche et al, 1988; Skinner et al, Javeherian et al, 1989) have been shown to disrupt a virus entry process other than CD4 binding. Most primary HIV- 1 viruses that initiate human infection and that persist throughout the course of infection replicate to low levels in PBMC and do not replicate in immortalized T cell lines (Asjo et al, 1986; Schuitemaker et al, 1991; Schuitemaker et al., 1992; Connor et al, 1993, 1994a,b). These viruses are referred to herein as macrophage-tropic primary isolates (sometimes referred to as "M"). In some HIV- 1 -infected individuals, viruses that replicate to higher levels in PBMC and that can infect and induce the formation of syncytia in immortalized CD4-positive cell lines emerge late in the course of infection (Asjo et al, 1986; Schuitemaker et al, 1992; Connor et al, 1993, 1994a,b). These viruses will be referred to herein as T cell line-tropic primary viruses (sometimes referred to as "T") The T cell line-tropic primary viruses, by virtue of their ability to replicate on some immortalized cell lines, serve as precursors to the laboratory- adapted isolates, which have been extensively passaged on such cell lines. Laboratory adaptation, however, results in a loss of the ability of

HIV-1 to replicate in primary monocyte/macrophage cultures (Schuitemaker et al, 1991; Chesebro et al, 1991; Westervelt et al, 1992; Valentin et al, 1994). Thus, while all HIV- 1 isolates replicate on primary T lymphocytes, three groups of virus variants can be defined based on the ability to replicate in primary monocyte/macrophages or in immortalized T cell lines: (1) macrophage-tropic primary viruses that cannot infect T cell lines; (2) laboratory-adapted viruses that cannot infect primary monocytes/macrophages; and (3) T cell line-tropic primary viruses that exhibit dual-tropism for these cell types.

The replication cycle of a primate lentivirus such as of HIV- 1 can be artificially divided into 2 halves. The first half begins after the virus enters a host, followed by converting two single stranded RNA genome into a double stranded DNA (provirus), and ends with integration of the provirus into chromosomal DNA of the host cell. The second half begins with the transcription of the integrated provirus, followed by production of viral proteins and release of viral particles from the host cell, and ends with new viruses binding to and entering in new host cells. While both halves are absolutely required for virus to replicate, it is during the second half of the cycle that all the viral antigens are produced and processed by the host immune system. It would be desirable to mimic the second half of the cycle of replication in vivo as closely as possible of an infectious virus, while completely abolishing the first half of the virus replication for safety.

SUMMARY OF THE INVENTION

We have now discovered a DNA vector that essentially or more preferably completely abolishes the first half of the viral replication cycle of a retrovirus, while mimicking the second half, thereby permitting production of immunogenic antigens such as soluble antigens, e.g. envelope proteins such as HIV gpl20, and capsid proteins such as p55 and p24, etc.

The DNA vector is a provirus corresponding to a retrovirus. Preferably, the retrovirus is a retrovirus containing regulatory proteins as well as structural and enzymatic proteins. More preferably the retrovirus is a lentivirus or a human T-cell leukemia virus such as HTLV-1. This recombinant provirus, preferably contains two independent sets of deletion mutations to abolish the infectivity of the virus. One mutation results from the deletion of reverse transcriptase and the integrase of the virus, both of which play essential roles only in the first half, not the second, of the virus replication cycle. The other mutation results from the deletion of 3' LTR, which has multiple functions in both halves of the replication cycle. A polyadenylation (poly A) signal is provided from a heterologous ("foreign") DNA sequences that provides only the poly A signal substituted for the 3' LTR, thus abolishing the LTR's function in the first half of the replication, such as being as the templates for the reactions of viral reverse transcriptase (RT) and integrase (int) , and for the formation of new LTRs which in turn serves as the transcriptional promoter for all the viral genes. Either one of these two sets of mutations alone is sufficient to inactivate virus replication genetically. Nevertheless, the use of double mutations, which delete more than 1/3 of the viral genome, further assures safety by making the theoretical probability of producing a replicative retrovirus such as HIV- 1 through in vivo recombination neglegable. Thus the invention is a DNA sequence corresponding to a primate lentivirus genome wherein the 3' LTR has been replaced by a heterologous poly A sequence, and wherein the genome is

More preferably at least one of two other mutations are introduced into this recombinant provirus genome wherein the 3' LTR has been replaced by a heterologous poly A sequence, and wherein the genome is int- and/ or RT" enhance its usefulness. The first one is designed to increase the amount of envelope protein, preferably HIV-1 or HIV-2 native gpl20 and gp41, as both contain important protective immuno epitopes. A translation stop codon is introduced at the end of the gpl20 coding region, thus increasing the efficiency of native gρl20 production without the requirement of the very inefficient proteolytic processing of gρl60 into gρl20 and gp41. The gp41, on the other hand, is also produced independently in the same provirus by substituting the gp41 coding portion, for the nef gene.

The second mutation increases the production of viral antigens in human or animal fibroblast cells. A functional efficient promoter for the target cell, e.g., the human cytomegalovirus promoter (hCMV) or the SV40 promoter is incorporated into the 5' LTR in such a way that the initial round of viral transcription is directed by its promoter, for example the hCMV promoter. This can be done by inserting the heterologous promoter upstream of the viral promoter and the viral enhancer. The promoter is selected so that it has a much higher activity in the target cell, e.g. fibroblast, then the viral promoter.

This recombinant retrovirus, e.g. HIV-1, can be used to transform an animal cell line preferably a mammalian cell line, to produce secreted viral antigens, (SVA). The SVAs produced can be used individually or as a group as an immunogen.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of the HIV- 1 and HIV-2 genome. Figure 2 is a schematic of an SIV genome.

Figure 3 is a schematic of an HTLV- 1 genome.

Figure 4 is a schematic of a preferred provirus of the present invention which is an HIV-1 genome that is RT", int-, nef-, has a heterologous polyadenylation sequence, a heterologous promoter substituted for the NRE site in the 5' LTR, a termination codon at the end of gpl20, and a gp41 with a signal sequence substituted for the nef gene.

Figure 5 shows the creation of pNTΔRT/Int.

Figure 6 shows the creation of pNT/ΔRT/Int- 120/41.

Figure 7 shows HIV-1 antigen specific cytotoxic T lymphocyte (CTL) activity in an animal immunized with a DNA vector of the present invention.

Figure 8 shows HIV- 1 antigen specific cytotoxic T lymphocyte (CTL) activity in an animal immunized with a DNA vector of the present invention.

Figure 9 shows HIV- 1 antigen specific cytotoxic T lymphocyte (CTL) activity in an animal immunized with a DNA vector of the present invention.

Figure 10 shows HIV-1 antigen specific cytotoxic T lymphocyte (CTL) activity in an animal immunized with a DNA vector of the present invention.

Figure 11 shows a test from the DNA immunized animals showing the presence of gpl20 specific T helper cells responsible for stimulating cell- mediated immunity.

Figure 12 is a graph that shows the immunized animals developed detectable anti-HIV- 1 antibodies. Figure 13 shows p24 production in 3 different cell lines transformed by pNT, pNT_Δ RT/Int. and pNtΔ RT/Int. 120.

Figure 14 shows qpl20 product in 3 different cell lines transformed by pNT, pNT, pNTΔ RT/Int. 120.

DETAILED DESCRIPTION OF THE INVENTION

The DNA vector contains a DNA sequence corresponding to an RT- and/or int- retrovirus, wherein the 3' LTR has been replaced by a heterologous poly A sequence. Preferably, the retrovirus is a retrovirus containing regulatory proteins as well as structural and enzymatic proteins. More preferably, the retrovirus is a lentivirus such as a primate lentivirus or a human T-cell leukemia virus.

The primate lentiviruses include HIV- 1, HIV-2 and SIV (see Figures 1 and

2). While the exact sequence of these viruses differ they share a common functional and organizational similarity. In addition to proteins such as gag, pol and envelope, the viruses contain regulatory proteins such as tat, rev, vpr, vpu, nef and vif. HTLV-1 and HTLV-2 [Hidaka et al., The EMBO Journal, 7:519- 523 (1988)] contain the regulatory proteins tax and rex as well as structural and regulatory proteins such as gag, pol and env. See Figure 3.

For example, in one embodiment the DNA sequence corresponds to a primate lentivirus genome wherein the 3- LTR has been replaced by a heterologous poly A sequence and is RT- and/or int-, more preferably it is RT- and int-

More specifically, at least one of two mutations is made in the pol region to inactivate the int gene and/ or the RT gene. Preferably both genes are inactivated. This can be done by methods well known in the art such as deletions, additions of nucleotides or substitution of nucleotides. Preferably a region of at least 20 nucleotides from the gene is deleted, added or substituted. More preferably, the entire gene is deleted. If one is leaving a portion of the genes it is preferred that multiple deletions, substitutions and/or additions are made in the gene to avoid the possibility of recombination. Both RT and int play critical functions in the ability of the virus to integrate into a host cells chromosomal DNA. Thus a deletion of either of these genes will disrupt the ability of the virus to effectively enter a host and be converted into a provirus and integrate into the DNA. Making both deletions further reduces the chance of any possible recombination.

A second change from a wild type retrovirus is to delete the 3' LTR. The

3' LTR has a variety of functions in both halves of the virus' replication cycle. For example, in the first half the LTR serves as the template for the reactions of viral RT and int and also for the formation of new LTRs, which in turn serve as transcriptional promoters for the viral genes. In the second half it serves to provide the poly A signal to the viral transcripts. By substituting a heterologous poly A at the 3' end one can still obtain efficient expression of the viral genes and formation of virus particles. Numerous poly A sequences are known and can readily be used based upon this teaching. One would use any eukaryotic or viral poly A with the exception of retroviral poly A. The retroviral poly A is preferably not used because of the theoretical possibility of recombination. For example, human growth hormone poly A, SV40 poly A, HSV tk poly A. For example, one could use one derived from the tk gene of herpes simplex virus (HSV). As a result of this change the virus is unable to successfully replicate upon entering the cell or integrate into the host cell's chromosomes.

.Although either one of these two mutations alone would be sufficient to inactivate the virus' ability to replicate genetically, the use of both sets of mutations provides added safety as it makes the theoretical possibility of producing a replicative virus through in vivo recombination negligible. This results in the ability to use the DNA provirus sequence in humans to generate an immune reaction. Still more preferably, a high efficiency promoter is inserted into the 5' LTR in such a manner to preserve any regulatory sequences such as the TAR element and the enhancer element and the viral promoter. This is accomplished by inserting the heterologous promoter upstream of the viral enhancer and promoter. For example in HIV, one could substitute the promoter for the NRE sequence. Promoters are well known in the art and can be readily be selected depending on the particular target cell. For example, any strong cellular or viral promoter that will work in the target cell can be used including SV40 promoter, CMV promoter and RSV promoter. The human cytomegalovirus (hCMV) promoter, for example, is very effective in fibroblast cells such as CHO or Vero cells. One can delete the NRE sequences of the HIV 5' LTR and insert repeated NF-1 binding sites derived from a CMV promoter, or more preferably substitute the entire promoter thereby preserving the lentiviral transcriptional promoter. Yet, this significantly increases the efficiency of viral gene transcription in a cell such as a fibroblast cell. This is very effective whether you wish to produce large amounts of protein in a host for an immune reactions or to generate large amounts of the protein in a producer cell. Moreover, this retains the TAR element thus permitting trαnsactivation.

For example, it is known that the HIV- 1 LTR is a very weak transcriptional promoter in fibroblast cells such as human muscle cells. However, in the presence of a viral activator such as tat, the viral promoter becomes one of the most effective promoters, and it is self regulated by viral replication as a result of trαnsactivation. In this embodiment the initial round of viral transcription will be directed by the heterologous promoter which has a much higher initial activity in a producer cells such as a fibroblast than the HIV- 1 promoter. However once tat is made, which is as one of the early viral products, inside a transformed cell, the expression of viral antigens directed by the LTR will be irαnsactivated, thereby increasing the production of viral antigens. This produces large amounts of end product. HTLV- 1 and 2 also show trαnsactivation resulting from the interaction between tax and the TAR element in the LTR. The tat/TAR trαnsactivation typically requires at least part of the viral promoter sequence. Consequently, the heterologous promoter should not be substituted for the viral promoter. We have found that because the viral promoter is weak, having it intervening between the heterologous promoter does not destroy the ability of the heterologous promoter to cause expression of the viral gene.

In one embodiment where the envelope glycoprotein is processed into smaller units, a stop codon can be introduced in one segment to increase the efficiency of envelope production by eliminating cleavage of the precursor. For example, with HIV preferably a translation stop codon has been introduced at the end of the gρl20 coding region. This increases the efficiency of gpl20 production without the requirement of the inefficient proteolytic processing of gp 160 into gp 120 and gp41. Still more preferably the nef open reading frame is replaced with a new open reading frame coding for a recombinant gp41 which contains a signal peptide, preferably the gρl20 signal peptide fused to the N- terminus of gp41 referred to as the ssgp41 reading frame. Signal peptides are well known in the art and can readily be constructed by standard means. One could use the nef start codon as long as the open reading coding for the signal peptide gp41 protein is inserted in-frame. aAlternatively, one can delete the entire nef gene including the start codon and insert a ssgp41 gene containing its own start codon. See Figure 4.

For example, the DNA sequence corresponding to the primate lentivirus provirus should contain a TAR element, RRE and CAR elements, Tat, rev, and at least one gene encoding envelope glycoprotein, preferably the gpl20 and still more preferably both the gρl20 and gp41 envelope proteins as well as matrix proteins. Still more preferably with the exception of nef, RT and int, the provirus will contain all the viral proteins. This will permit production of large amounts of viral antigens, while making safety concerns negligible.

In preferred embodiments one can insert a gene encoding envelope glycoproteins from any strains in the DNA provirus backbone. It is the envelope protein that varies most from strain to strain. Moreover, one could also create pseudotypes wherein the envelope protein of different primate lentiviruses are substituted, for example, using an HIV-2 envelope reading frame in an HIV-1 backbone. In this way one can induce immune responses to a range of viruses. For example, one can administer cocktails containing the basic virus, yet with different envelope proteins. Alternatively, one can administer the different envelope encoding DNA sequences at different intervals.

Typically, when one is trying to generate an immune response, one provides subsequent administrations after the initial administration, which are typically referred to as boosts. In one embodiment, one can insert DNA proviruses in producer lines to express large amounts of particles and/or secreted viral antigens (SVA)such as gpl20, p24, p55, etc. These antigens can be harvested from producer cells by standard techniques. Thereafter, in one embodiment the antigens can be administered as a boost.

Alternatively, one could administer the viral antigen itself as an initial agent to stimulate a immune reaction. Typically the viral antigen would be administered along with an adjuvant to stabilize it.

In preferred embodiments one would also wish to add other immuno- stimulatory agents, for example, cytokines such as an interleukin or a co- stimulatory molecule such as B7- 1 and/ or B7-2. The immuno stimulatory agents are well known in the art and can readily be selected.

In view of the functional and organizational similarity of the primate lentiviruses the following exemplification will specifically discuss HIV-1.

However, the skilled artisan can readily make the appropriate modifications to use HIV-2 or SIV. A 3' LTR can be replaced by a fragment of DNA derived from the tk gene of HSV that will serve as the poly A signal for viral transcription (See Figures 4 and 5) . This mutation will not effect expression of viral genes or the formation of viral particles but it will result in the absence U-3 and R sequences at the 3' end of the HIV-1 LTR. A deletion is made in the int or the RT gene, more preferably both the int and the RT genes. This creates a second means of rendering the virus incapable of replication, while not interrupting the expression of HIV- 1 structural and regulatory genes.

A termination codon is preferably introduced at the end of the gpl20 coding region to increase the efficiency of native gpl20 production. The nef reading frame is replaced with a recombinant gp41 containing the region encoding the signal peptide of the gpl20 protein fused to the N-terminus. See Figure 6.

More preferably, the NRE sequence of the 5' LTR is replaced with a promoter that will sufficiently express the gene in the target cell. For example, repeated NF- 1 binding sites from a CMV promoter, thereby preserving the HIV- 1 transcriptional promoter but significantly increasing the efficiency of HIV- 1 gene transcription.

Thus, production of gpl20 and gρ41 is enhanced by eliminating the need for the inefficient processing of the gplδO precursor. Transcription of the viral genes is enhanced by using a heterologous promoter that preserves native viral transcriptors for post- transcription activation and regulation. This DNA provirus can be directly used in a host to generate an immune reaction. For example, as a DNA vaccine in a prime immunization.

It can also be used to produce large amounts of a desired viral antigen or groups of viral antigens. For example, whole particles or various antigens.

The supernatant of such producer cells lines not only contain viral particles but secreted HIV-1 proteins (SVAs) including p24, p55, gpl20, gp41 and tat, all which are collectively referred to herein SVAs. Either individually or collectively an SVA can be used as the prime immunogen or as a boost immunogen preferably the M open reading frame would be derived from a M- tropic strain. Preferred isolates include U455, 89.6 ZAM18, ELI, CM235, NBZ163, etc.

Normally, the gplδO glycoprotein serves as the precursor for gpl20. Following oligomerization, the gplδO glycoprotein is transported to the golgi apparatus where cleavage by a cellular protease generates both the gpl20 and gp41 glycoproteins, which remain associated through non-covalent interactions (Earl, P.L., etα al., J Virol 1991, Kowalski, M., et al., Science 1987). In mammalian host cells, addition of complex sugars to specific, probably surface- exposed, carbohydrate side chains of the envelope glycoproteins occurs in the Golgi apparatus (Leonard, C.K., et al, J Biol Chem 1990). However, the cleavage process is inefficient. By designing a gpl20 "gene" and a ssgp41 "gene" the efficiency of producing the mature proteins should be enhanced.

The mature envelope glycoprotein complex is incorporated into virions, where it mediates virus entry into the host cell. The gpl20 exterior envelope glycoprotein binds the CD4 glycoprotein, which serves as a receptor for the virus (Klatzmann, D., et al., Nature 1984, Dalgleish, A.G., et al, Nature 1984). The binding to CD4 is followed by interaction of the gpl20-CD4 complex with one of the chemokine receptors, which are seven-transmembrane G protein- coupled receptors (Feng, Y., et al., Science 1996; Choe, H., et al., Cell 1996; Doranze, et al., Cell 1996; Dragic, et al., Nature 1996; Alkhatib, G., et al., Science 1996). Chemokine receptor interaction is believed to bring the viral envelope glycoprotein complex nearer to the target cell membrane and to trigger additional conformational changes in the envelope glycoproteins (Wu, L., et al., Nature 1996; Trkola, A., et al., Nature 1996). These changes are believed to result in the interaction of the gp41 glycoprotein with the target cell membrane, culminating in fusion of this membrane with the viral membrane (Kowalski, M., Science 1987; Freed, E.O., Proc Natl Acad Sci 1990; Cao, J., J Virol 1993).

Accordingly, the DNA sequence described herein can be used to produce large amounts of viral antigens. One preferred type of viral antigen is a particle that immunogenically resembles the actual virus, but is safe because the particle does not genetically replicate.

.Another preferred type of viral antigen is a SVA. Particularly preferred SVAs include the gpl20, p24, p55 and ssgp41 proteins. These viral proteins are preferably administered with an adjuvant, e.g. Ribi.

The viral antigens can be used to generate immune reactions in an animal. They can also be used to readily obtain antibodies where the antibodies can be used for a variety of purposes. For example, antibodies to envelope glycoproteins can be used to inhibit cellular entry. Antibodies can be used to monitor specific levels of antigens for diagnostic and/ or prognostic purposes. Such assays can readily be prepared by those of ordinary skill in the .art as discussed later.

Stabilized forms of the antigens can readily be made, for example, by conjugates such as a poly(alkylene oxide) conjugate. The conjugate is preferably formed by covalently bonding the hydroxyl terminals of the poly(alkylene oxide) and for example a free amino group in the gpl20 protein. Other art recognized methods of conjugating these materials include amide or ester linkages. Covalent linkage as well as non-covalent conjugation such as lipophilic or hydrophilic interactions can be used.

The conjugate can be comprised of non-antigenic polymeric substances such as dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyaci l amides or other similar substantially non-immunogenic polymers. Polyethylene gl col(PEG) is preferred. Other poly(alkylenes oxides) include monomethoxy-polyethylene glycol polypropylene glycol, block copolymers of polyethylene glycol, and polypropylene glycol and the like. The polymers can also be distally capped with Cl-4 alkyls instead of monomethoxy groups. The poly(alkylene oxides) used must be soluble in liquid at room temperature. Thus, they preferably have a molecular weight from about 200 to about 20,000 daltons, more preferably about 2,000 to about 10,000 and still more preferably about 5,000.

One can administer the stabilized conjugates, antibodies generated to the viral proteins, the viral particles, the SVAs and the DNA proviruses to individuals by a variety of means. Routes of administration include oral, parenteral, rectal, intravaginal, topical, nasal, ophthalmic, direct injection, etc. For example, these antibodies can be included in vaginal foams or gels that are used as preventives to avoid infection and applied before people have sexual contact.

The viral antigens when used for administration are prepared under aseptic conditions with a pharmaceutically acceptable carrier or diluent.

Doses of the pharmaceutical compositions will vary depending upon the subject and upon the particular route of administration used. Dosages can range from 0J to 100,000μg/kg a day, more preferably 1 to 10,000μg/kg.

Changes in the viral envelope glycoproteins, in particular in the third variable (V3) region of the gpl20 exterior envelope glycoprotein, determine tropism-related phenotypes (Cheng-Mayer et al, 1990; O'Brien et al, 1990; Hwang et al, Westervelt et al, 1992; Chesebro et al, 1992; Willey et al, 1994). Accordingly, one can generate an immune reaction for a particular receptor by substituting different gpl20 gene in the basic viral backbone. For example, one can prepare a standard restriction site for gpl20 in the DNA provirus such as KpnI/BamHI. Various different isolates can be used such as U455, 89.6, Zaml8, ELI, CM235, etc. The particular strains chose will depend upon where the provirus is being used as certain strains are more prevalent in certain countries than others.

Thus, in one embodiment the provirus is used to generate antibodies to specific strains. One can also screen antibodies to choose those with a preferred characteristic, e.g. blocking binding of the virus. One preferred approach is the use of antibodies to the binding site for the chemokine receptors. The antibodies raised to these particles can then be used to block binding. For example, one can use single chain antibodies to target these binding sites.

As used herein the inhibition of HIV infection means that as compared to a control situation infection is reduced, inhibited or prevented. Infection is preferably at least 20% less, more preferably at least 40% less, even more preferably at least 50% less, still more preferably at least 75% less, even more preferably at least 80% less, and yet more preferably at least 90% less than the control.

One preferred use of these molecules is to minimize the risk of HIV transmission. .Another use would be in systemic administration to block HIV- 1 replication in the blood and tissues. The compound could also be administered in combination with other HIV treatments.

An exemplary pharmaceutical composition is a therapeutically effective amount of a DNA provirus, viral particle, SVA, antibody etc. that affects HIV infection optionally included in a pharmaceutically-acceptable and compatible carrier. The term "pharmaceutically-acceptable and compatible carrier" as used herein, and described more fully below, includes (i) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/ or (ii) a system, such as a antibody-DNA vector, capable of delivering the molecule to a target cell. In the present invention, the term "carrier" thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate application. The term "therapeutically-ef ective amount" is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particular condition being treated. Various concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.

The term "compatible", as used herein, means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and/or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.

Dose of the pharmaceutical compositions of the invention will vary depending on the subject and upon particular route of administration used. Dosages can range from OJ to 100,000 μg/kg per day, more preferably 1 to 10,000 μg/kg. By way of an example only, an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use. This dose can be delivered at periodic intervals based upon the composition. For example on at least two separate occasions, preferably spaced apart by about 4 weeks. Other compounds might be administered daily.

Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well-characterized protocols. For example, certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose. See Product Information, Physician's Desk Reference, Merck Sharp & Dohme (1990), at 1442-43. (e.g., Hepatitis B Vaccine-type protocol);

(ii) Recommended administration for children is first dose at elected date (at age 6 weeks old or older); a second dose at 4-8 weeks after first dose; a third dose at 4-8 weeks after second dose; a fourth dose at 6-12 months after third dose; a fifth dose at age 4-6 years old; and additional boosters every 10 years after last dose. See Product Information, Physician's Desk Reference, Merck Sharp δε Dohme (1990), at 879 (e.g., Diphtheria, Tetanus and Pertussis-type vaccine protocols). Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.

The small molecules and polypeptides of the invention may also be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulphonic. .Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Thus, the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/ or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.

The compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention. Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchyrnal injection directly into targeted areas of an organ. Compositions suitable for parenteral administration are preferred. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and/ or polypeptide of the invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.

Preferred compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile mjectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectibles.

.Antibodies The term "antibodies" is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention. The term "selectively reactive" refers to those antibodies that react with one or more antigenic determinants of the virus and do not react with other polypeptides. Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibodies can be used for diagnostic applications or for research purposes.

For example, antibodies may be raised against ammo-terminal (N- terminal) or carboxyl-terminal (C-terminal) peptides of a polypeptide. Most preferably one selects an exposed cell-surface epitope of one of these receptors.

One approach is to use the DNA to generate viral antigens and use, e.g. SVAs that contain an antigenic determinant for use as an immunogen. The immunogen can be attached to a carrier to enhance the immunogenic response. Preferably one uses a cell line expressing high levels of SVA and selects those cells with the highest levels of expression to use the whole cell as an antigen.

For example, a provirus of the invention may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York: 1989) such that 5-20% of the total protein that can be recovered from the host is the desired antigen. For example, recovered SVAs can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization. Alternatively, a protein of interest can be purified by other methods such as, for example, ion exchange hydrophobic, size exclusion, or affinity chromatography.

A particularly preferred method of purification is based upon using hydrophobic interaction chromatography (HIC) . This is a purification technique in which passage of molecules through a column is retarded by hydrophobic interactions between the column support material or a substance bound by the support material. Thus the material to be purified is added to an HIC column, e.g. an ether-HIC or phenyl HIC column, eluted with solutions having sufficient ionic strength (e.g. ammonium sulfate, ammonium acetate, etc.) to cause the material you want purified to be retained in the column and thus separated from the material it was in. Thereafter, the ionic strength of the eluant is lowered to remove the material from the column. We have found that one can use a pH of about 6 to about 8.5, an ionic strength of from about 0.05 to 2.0 M (expressed as NaCl), preferably about OJ M and elution using for example, a gradient from 40% ammonium sulfate. For example, we found that 0.4 M NH₄ Acetate was the optimal condition at which both HIV- 1 p24 and gpl20 are purified. It was discovered that by using this technique multiple SVAs and/ or particles can be purified and obtained in a conformational approximating the wild-type conformation.

Once the protein immunogen is prepared, mice can be immunized twice intraperitoneally with approximately 50 microgra s of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide. For immunohistology, active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin- peroxidase and a chromogenic peroxidase substrate. Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, California. Mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.

For preparation of monoclonal antibodies directed toward polypeptides encoded by a eukaryotic nucleotide sequence of the invention, any technique that provides for the production of antibody molecules by continuous cell lines may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (Nature, 256: 495-497, 1973), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies, and the like, are within the scope of the present invention. See, generally Larrick et al., U.S. Patent 5,001,065 and references cited therein. Further, single-chain antibody (SCA) methods are also available to produce antibodies against polypeptides encoded by a eukaryotic nucleotide sequence of the invention (Ladner et al. U.S. patents 4,704,694 and 4,976,778).

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. The present invention provides for antibody molecules as well as fragments of such antibody molecules.

Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, "Specific killing of lymphocytes that cause experimental Autoimmune Myasthenia Gravis by toxin-acetylcholine receptor conjugates." Jour. Immun. 133: 1335-2549; Jansen, F.K., H.E. Blythman, D. Carriere, P. Casella, O. Gros, P. Gros, J.C. Laurent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H. Vidal, and G.A. Voisin. 1982. "Immunotoxins: Hybrid molecules combining high specificity and potent cytotoxicity". Immunological Reviews 62: 185-216; and Vitetta et al., supra).

Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemoto et al. U.S. Patent 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (l-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio) -toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-ρyridyldithio)- propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo- NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.

Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo- NHS, in particular, can enhance the stability of carbodiimide couplings. Carbodiimide couplings (such as EDC) when used in conjunction with sulfo- NHS, forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone. .Antibodies of the present invention can be detected by appropriate assays, e.g., conventional types of immunoassays. For example, a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase. Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. .After this first incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound materials and interfering substances such as non-specific proteins which may also be present in the sample. The solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as biotin or avidin. Labels for antibodies are well-known in the art and include radionuclides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and the like. The labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti- urea transporter antibody present in the sample. These and other immunoassays can be easily performed by those of ordinary skill in the art.

The following Examples serve to illustrate the present invention, and are not intended to limit the invention in any manner.

EXAMPLES Materials and Methods

The plasmid pCNTΔRT/Int- 120/41 was constructed to introduce 4 genetic modifications into an infectious HIV-1 provirus, thus rendering it not only non-infectious, but also more immunogenic. The construction is divided into the following 4 steps.

Construction of pNT (Figure 5, top half) Plasmid pHXB contains a copy of infectious HIV- 1 provirus that belongs to the B clade of the HIV- 1 family. The digestion of pHXB with restriction enzymes Xhol (nucleotide 8896, HIVHXB) and Xbal (within the cellular flanking sequence at the 3' end of the provirus) resulted in 2 DNA fragments, a 800 base pairs (bp) representing nef open reading frame (orf) and the 3' LTR (long terminal repeat), a 12 kb (kilo bases) fragment containing the rest of the provirus and pBR322 plasmid vector. Plasmid pREP9 (Clontech, California) is a commercially available expression vector that contains a neomycin resistant gene (neo) regulated by the TKp (tyrosine kinase gene of Herpes Simplex Virus 2 promoter) and TK pA (tyrosine kinase gene polyadenylation signal) . A DNA fragment containing only the neo gene and TkpA was produced by PCR (polymerase chain reaction) with primers that introduce a Xhol site immediately upstream of the ATG codon of neo and a Xbal site about 20 bases downstream of the polyadenylation addition site. After digestion with Xhol and Xbal enzymes, the neo-TkpA fragment was ligated with the 12 kb fragment derived from pHXB, thus resulting in plasmid pNT. The expression of neo gene in pNT is regulated by the 5' LTR like all the other viral genes in the provirus. The TkpA now provides the polyadenylation signal for the expression of all viral genes, thus replacing the polyadenylation signal previously provided by 3' LTR.

Construction of pNTΔRT/Int (Figure 5, bottom half)

A 2341 bp DNA fragment coding for RT and Int was deleted from pNT by PCT. Briefly, a PCR fragment was synthesized using primers derived from nucleotide sequence of HIVHBX 4192-5052 with an inserted Xbal site and from the sequences around the Sail site (nucleotide 5785, HIVHXB). A second PCR fragment represents the HIV- 1 sequences from nucleotide 2009 (Apal site) to 2601 with an inserted Xbal site. Plasmid pNT was digested with Apal and Sail and an approximately 7 kb fragment was isolated. After ligation of the 3 DNA fragments via Apal, Xbal, and Sail sites, plasmid pNTΔRT/Int was produced, thus deleting the DNA sequence between 2601 and 4192 that code for RT and Int.

Construction of pNTΔRT/Int- 120/41 (Figure 6) A translation stop codon was introduced into pNTΔRT/Int by site specific mutagenesis at the C-terminus of HIV- 1 g; 120 (nucleotide 7756, HIVHXB), thus creating a new orf for gpl20 alone in plasmid pNTΔRT/Int-120. Meanwhile, a second mutation was introduced into pNTΔRT/Int-120 by site-specific mutagenesis in which a new Xmal site was inserted at the first ATG in the remaining nef orf (nucleotide 8799, HIVHXB). In order to create a new orf for gp41, a Fspl site was introduced into pNTΔRT/Int by site-specific mutagenesis at the N-terminus of gp4 (nucleotide 7759, HIVHXB). A second Fspl site was added to the end of the signal peptide sequences of gpl20 (nucleotide 6311, HIVHXB), thus resulting in plasmid pNTΔRT/Int-FspI. The deletion of the Fspl fragment in pNTΔRT/Int-FspI, which includes the nucleotide between 6311 and 7759, resulted in a new orf that codes for a fusion protein of the signal peptide with its cleavage site and the entire gp41. The plasmid was named pNTΔRT/Int-ss41. The DNA fragment containing the new gp41 fusion orf was isolated by PCR with primers corresponding to nucleotide 6224-6244 and nucleotide 8774-8794 (HIVHXB). A Xmal site was inserted at each of the PCR fragment so that the ss41 fragment can be further prepared with Xmal digestion. Next, the Xmal/Xmal PCR fragment was inserted into the Xmal site in plasmid pNTΔRT/Int-120 to finally create pNTΔRT/ Int- 120/41. As shown in Figure 2, HIV- 1 env gene is now separated into two independent orfs that codes for gp 120 and gp41, respectively. While the translation of gpl20 orf is still directed as the native env gene, it stops at the end of gpl20 without producing gplδO precursor and its subsequent inefficient protease cleavage by cellular protease. On the other hand, a new gp41 orf is translated as if it is the original nef orf, thus making gp41, without producing gpl60 precursor. As the exons coding for tat and rev remain unchanged in this plasmid, the expression and function of TAT and REV are expected to remain intact.

Construction of pCNTΔRT /Int- 120/41 (Figure 6) The DNA fragment containing the 5' LTR in plasmid pNTΔRT/Int- 120/41 was first subcloned into a CAT expression vector using Clal and Hindlll digestion. The Clal site is at upstream sequences of the beginning of the LTR (nucleotide 1, HIVHXB) and the Hindlll site is located after the U3/R junction, at nucleotide 531 (HIVHXB) that corresponds to +80 of the transcription start site (+1 at nucleotide 456, HIVHXB). Consequently, plasmid pTZ5' LTR-CAT contains a functional HIV-1 promoter that directs the expression of CAT gene. The negative regulatory elements (NRE) in the LTR was removed by deleting the EcoRV and Aval fragment, which corresponds to the nucleotides between -420 and -154 from the transcription start site. The larger plasmid fragment carrying the rest of the LTR and the CAT gene was further treated to create a blund end at the Aval site (fill in reaction) for the next step of construction. The DNA fragment containing the functional human Cytomegalovirus (hCMV) promoter was isolated from plasmid pCMVβ (Clontech, California) by Clal and Sad digestion. Both ends were subsequently filled in to create blund ends for cloning into the LTR vector. This Clal/ Sad fragment contains a functional hCMV promoter that spans from -980 to +28. After confirming the orientation of the hCMV promoter is at is the same as the 5' LTR, a hybrid CMV/LTR fragment was isolated by Clal and Hindlll digestion with the latter site being filled in to create a blunt end. Meanwhile, plasmid pNTΔRT/Int- 120/41 was modified to convert one of the multiple Hindlll sites (nucleotide 456, HIXHXB) to a unique NotI site by site-specific mutagenesis. The plasmid pNTΔRT/Int- 120/41 NotI was first digested with NotI, followed by a fill in reaction, and with Clal to create a vector. After ligating the hybrid hCMV/LTR fragment with the vector, plasmid pCNTΔRT/Int- 120/41 was constructed.

This vector can then be used to infect a target cell such as a fibroblast by standard techniques. ter the viral antigens are produced, they can be purified by separating the culture supernatant (CS) by low speed centrifugation (1000 rpm), mixed with 0.4 M NH₄ Acetate at pH 7.0. The CSI salt solution is passed through a 0.25 um filter, 10 ml CSI salt are loaded onto a BS4mini column that is previously washed with the salt solution. The HIC column is then washed with at least 50 ml of the salt solution without the CS. The binding material is eluted with 1 ml PBS at pH 7. This results in high recovery of particles, p24 and gp 120 in a conformationally correct form.

Immunization of Mice with pNTΔ RT/Int 500 μg of PNTΔ RT/Int was injected intramuscularly into each of four Balb/c mouse (8-12 weeks old). The naked DNA immunization was repeated at 4, 8 and 12 weeks after the first immunization. Two weeks after the 4^th immunization, the animals were killed and the spleen tissues were collected for CTL assays and γ-INF release assay and the serum was used for antibody ELISA.

CTL assay and antigen specific γ-INF release assay were conducted to detect any cell-mediated immunity stimulate by DNA immunization of PNTΔ

RT/Int. A synthetic 20 amino acid peptide derived from HIV-1 gag p24 antigen (a.a. 190 to a.a. 210) was used to stimulate the effector cells from the spleen and to prepare peptide-pulsed and ⁵¹Cr-labeled P815 target cells.

Figures 7-10 represent 4 different animals respectively, showing significant HIV- 1 antigen specific CTL activities in these immunized mice. A synthetic 15 amino acid peptide derived from HIV- 1 gpl20 (a.a.310 to a.a. 324) was used to stimulate spleen cells from the immunized animals. Antigen specific y-INF release was measured using a commercially available ELISA kit according to protocols supplied by the manufacturer (Endogen, Woburn, MA). Figure 11 represents a typical test result from these DNA immunized animals, showing the existence of gpl20 specific T helper cells responsible for stimulating cell-mediated immunity. This further shows that the vector caused an immune reaction.

.Antibody response against HIV- 1 antigens in these DNA immunized mice was measured by using an ELISA plate coated with purified HIV- 1 SVA. As shown in Figure 12, all the immunized animals developed detectable level of anti-HIV-1 antibodies .after 4 immunizations with plasmid DNA of PNTΔ RT/Int, thus indicating that the genetically inactivated HIV- 1 provirus is capable of serving as a vaccine that can stimulate both humoral and cell-mediated immunity against HIV- 1. SVA production from Vero cells transformed by different genetically inactivated HIV-1 provirus.

As discussed above, a number of mutations were introduced into the genetically inactivated HIV-1 provirus in order to produce SVA more efficiently in cultured mammalian cells. Figures 13 and 14 shows the comparison of the amount of p24 and gpl20, respectively, in 3 different cell lines transformed by pNT, pNTΔ RT/Int, and PNTΔ RT/Int- 120. They demonstrate the effects of these designed mutations on the production of the 2 key antigens. The antigen- capture ELISA was performed using the kits according to the instructions supplied by the manufacturers (p24: Coulter, Miami, FL. gpl20: ABi, Columbia MD). The deletion of RT and Integrase has a significant negative effect on the production of both gpl20 and p24, showing an approximately 30 fold reduction in p24 production and a 8 fold decrease in gpl20 release. The PNTΔ RT/IntJ20 transformed cells, however, showed a 47 fold increase of gpl20 production compared with its parental plasmid PNTΔ RT/Int and a 5 fold increase over the original pNT transformed cells. This shows that using a vector containing a gene encoding gpl20 truncated protein instead of the gpl20 protein is more effective for envelope production as taught.

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All references described in the above specification are incorporated herein by reference.

This invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modification and improvements within the spirit and scope of the invention.

Claims

We claim:

1. A DNA vector corresponding to a reverse transcriptase (RT)- and/or intergrase (int)- provirus of a retrovirus, wherein the provirus's 3' LTR has been replaced by a heterologous polyadenylation sequence.

2. The DNA vector of claim 1, wherein the retrovirus encodes regulatory proteins as well as capsid and envelope proteins.

3. The DNA vector of claim 2, wherein the retrovirus is a lentivirus.

4. The DNA vector of claim 3, wherein the lentivirus is a primate lentivirus.

5. The DNA vector of claim 4, wherein the primate lentivirus is HIV-1 or HIV-2.

6. The DNA vector of claim 2, wherein the retrovirus is HTLV- 1 or HTLV-2.

7. The DNA vector of claims 2, 3, 4, 5 or 6, wherein a heterologous promoter is inserted in the provirus's 5' LTR in such a manner that it is operably linked to result in gene expression of the retroviral protein while not interfering with positive regulatory sequences.

8. The DNA vector of claims 2, 3, 4, 5, or 6 wherein the wild type retroviral envelope protein is cleaved and a termination codon is inserted at the cleavage site.

9. The DNA vector of claim 4 wherein a heterologous promoter is inserted in the provirus's 5' LTR upstream of the native enhancer and promoter so that the heterologous promoter is operably linked to result in expression by the retroviral gene without interfering with endogenous retroviral positive regulatory sequences.

10. The DNA vector of claim 8, wherein the retrovirus is a primate leatro-virus, and wherein a heterologous promoter is optionally inserted in the 5' LTR upstream of the native proviral enhancer and promoter so that the heterologous promoter is operably linked to result in expression by retroviral genes without interferring with endogenous retroviral positive regulatory sequences.

11. The DNA vector of claim 10, wherein the retrovirus 's nef gene is replaced by a open reading frame encoding a transmembrane envelope protein portion of the cleaved envelope protein with a secretory sequence at its

N-terminal, referred to as ss env.

12. The DNA vector of claim 11, wherein the retrovirus is HIV-1 and the ss env is ssgp41.

13. The DNA vector of claim 10, wherein the heterologous promoter is substituted for the NRE sequence.

14. A method of producing retroviral particles and soluble viral antigen comprising transfecting a cell with the vector of any of claims 1-13.

15. A method of generating an immune reaction comprising injecting an affective amount of the DNA vector of any of claims 1- 13 in a host animal.