Role of immune responses against the envelope and the core antigens of simian immunodeficiency virus SIVmne in protection against homologous cloned and uncloned virus challenge in Macaques.

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

Protection of macaques against intrarectal infection by a combination immunization regimen with recombinant simian immunodeficiency virus SIVmne gp160 vaccines.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.

Limited breadth of the protective immunity elicited by simian immunodeficiency virus SIVmne gp160 vaccines in a combination immunization regimen.

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection.

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).

Analysis of cytotoxic T lymphocyte responses to SIV proteins in SIV-infected macaques using antigen-specific stimulation with recombinant vaccinia and fowl poxviruses.

Methods to analyze CD8+ CTL responses to simian immunodeficiency virus (SIV)-encoded proteins are essential to understand lentivirus immunopathogenesis and protective immune responses. Recombinant infectious shuttle vectors are useful for analyzing CTL responses to many viruses, including HIV. Therefore, CTL responses in SIV-infected Macaca fascicularis to SIV env and SIV gag/pol were evaluated using specific antigen stimulation with recombinant vaccinia (rVV) and fowl poxviruses (rFPV) containing SIV genes. Peripheral blood mononuclear cells from SIV-infected animals were stimulated with autologous cells infected with rVV expressing SIV env/gag/pol, and CTLs specific for SIV env and for SIV gag/pol were detected by testing for lytic activity in target cells expressing these genes separately. Lymphocyte subset purifications from the effector population demonstrated that the CTL response was mediated by CD8+ cells, and the use of brefeldin A to selectively block antigen presentation in association with MHC class I products affirmed this cytolytic activity was class I restricted. The use of rVV to analyze responses to SIV genes is potentially problematic in hosts immunized to vaccinia. Fowl poxvirus is an alternative virus that has many of the molecular advantages of vaccinia virus but is genomically distinct. Therefore, the ability of rFPV to expand and detect SIV-specific CTLs was evaluated. Although there was no cytopathic effect following infection with rFPV, macaque cells infected with this vector did express rFPV gene products, and could be used as stimulator and target cells to detect SIV-specific CD8+ CTLs. The results suggest that these recombinant viral vectors can be used to specifically stimulate CD8+, MHC class I-restricted CTLs reactive to SIV proteins, and should facilitate evaluating CTL responses in both SIV-infected animals and animals vaccinated against SIV.

Protection of vaccinia-primed macaques against SIVmne infection by combination immunization with recombinant vaccinia virus and SIVmne gp160.

Two Macaca fascicularis with preexisting immunity to vaccinia virus were immunized twice with recombinant vaccinia virus expressing SIVmne gp160. Their SIV-specific antibody responses were lower than that of vaccinia-naive animals immunized similarly. Upon repeated boosting with gp160, the SIV-specific antibody titers in vaccinia-primed animals reached similar levels as vaccinia-naive animals and with comparable neutralizing titers. Both animals were protected against repeated intravenous challenge with low-dose SIVmne E11S. These results are significant because SIVmne E11S infection in M. fascicularis is pathogenic and leads to AIDS-like diseases.

Recombinant-expressed virus-like particle pseudotypes as an approach to vaccine development.

Coinfection of a cell with two different types of enveloped virus can result in the generation of infectious virus particle pseudotypes having the internal proteins of one virus and the envelope proteins of the other virus. Vaccinia virus recombinants expressing either non-infectious virus-like particles of simian immunodeficiency virus (SIV) or the gD2 glycoprotein of herpes simplex virus were used to coinfect cells to determine if virus-like particle pseudotypes would be formed. Sucrose gradient sedimentation analysis and immunoprecipitation with a monoclonal antibody provided independent evidence of virus-like particle pseudotype formation. Preparations of such particles were immunogenic in mice. Recombinant-expressed virus-like particles thus represent a novel vaccine approach to presenting envelope glycoprotein antigens in a non-infectious state that mimics natural infection.

Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences.

A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus K1L (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3' end of the K1L gene and the adjacent M2L gene deleted and replaced with the beta-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the K1L gene was used to insert the HBs gene into vAbT33. The M2L negative K1L positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The K1L gene selection system allows the isolation of recombinants arising at frequencies as low as 1/100,000. It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3' end of the K1L gene. The K1L gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the K1L gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5' end of the M2L gene transcripts. Although the insertion lengthened the 3' end and lowered the accumulation of K1L transcripts it altered neither the virulence nor the immunogenicity of the recombinant.

Structure and pathogenicity of individual variants within an immunodeficiency disease-inducing isolate of FeLV.

We previously described the molecular cloning of a replication-defective variant of feline leukemia virus (FeLV) that induced fatal immunodeficiency in cats. Eighteen proviruses have now been molecularly cloned from cats inoculated with the original isolate (FeLV-FAIDS) or its in vivo passages. Three were replication-competent and each of these was noncytopathic for the feline T-cell line, 3201. Replication of the prototype, FeLV-61E, in cats was associated with development of T cell tumors in some cats. The remaining 15 proviruses were replication-defective, but each of six of these tested was found to be cytopathic for 3201 cells when rescued with the noncytopathic helper virus, 61E. Three defective/helper virus mixtures were inoculated into cats and all induced fatal immunodeficiency, but with varied efficiency and kinetics. Each of these virus mixtures was attenuated relative to a mixture containing 61E and the intestine-targeted, FeLV-FAIDS-61C prototype defective molecular clone. Furthermore, one replication-competent virus chimera generated using the envelope and LTR of the defective pathogenic variant was incapable of inducing viremia in cats. The observed differences in the biological activity between the defective viruses could be attributed to no more than 10 scattered amino acid changes in envelope and either one or two nucleotide changes in the LTR.

Evaluation of protective efficacy of recombinant subunit vaccines against simian immunodeficiency virus infection of macaques.

Simian immunodeficiency virus (SIV) was used as a model to study the protective efficacy of an immunization regimen currently being evaluated as candidate vaccines against HIV in human subjects. Four Macaca fascicularis were first immunized with recombinant vaccinia virus expressing the envelope glycoprotein gp160 of SIVmne and then boosted with subunit gp160. Both cell-mediated and humoral immune responses against SIV, including neutralizing antibodies, were elicited. The macaques were shown to be protected from a homologous virus infection as determined by serology, lymphocyte cocultivation, polymerase chain reactions and in vivo transmission analyses. Four unimmunized control animals were readily infected. However, viremia in infected control animals could decrease substantially following the initial phase of infection so that persistent infection might not be readily detectable.

Utilization of DNA recombination for the two-step replacement of growth factor sequences in the vaccinia virus genome.

An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated. Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae. This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome. Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis. Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression. Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers.

Recombinant virus vaccine-induced SIV-specific CD8+ cytotoxic T lymphocytes.

Evidence indicates that cytotoxic T lymphocytes (CTLs) may be important in containing the spread of the human immunodeficiency virus (HIV) in the infected host. Although the use of recombinant viruses has been proposed as an approach to elicit protective immunity against HIV, the ability of recombinant viral constructs to elicit CD8+ CTL responses in higher primates has never been demonstrated. A live recombinant virus, vaccinia-simian immunodeficiency virus of macaques (SIVmac), was used to determine whether such a genetically restricted, T lymphocyte-mediated antiviral response could be generated in a primate. Vaccinia-SIVmac vaccination elicited an SIVmac Gag-specific, CD8+ CTL response in rhesus monkeys. These CTLs recognized a peptide fragment that spans residues 171 to 195 of the Gag protein. The rhesus monkey major histocompatibility complex (MHC) class I gene product restricting this CTL response was defined. Both the vaccinated and SIVmac-infected monkeys that shared this MHC class I gene product developed CTLs with the same Gag epitope specificity. These findings support the use of recombinant virus vaccines for the prevention of HIV infections in humans.

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.

The ability of poxviruses to undergo intramolecular recombination within tandemly arranged homologous sequences can be used to generate chimeric genes and proteins. Genes containing regions of nucleotide homology will recombine to yield a single sequence composed of portions of both original genes. A recombinant virus containing two genes with a number of conserved regions will yield a population of recombinant viruses containing a spectrum of hybrid sequences derived by recombination between the original genes. This scheme has been used to generate hybrid human immunodeficiency virus type 1 env genes. Recombinant vaccinia viruses that contain two divergent env genes in tandem array have been constructed. In the absence of selective pressure to maintain both genes, recombination between conserved homologous regions in these genes generated a wide range of progeny, each of which expressed a novel variant polypeptide encoded by the newly created hybrid env gene. Poxvirus-mediated recombination may be applied to map type-specific epitopes, to create novel pharmaceuticals such as hybrid interferons, to study receptor-binding or enzyme substrate specificities, or to mimic the antigenic diversity found in numerous pathogens.

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques.

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.

Structure, origin, and transforming activity of feline leukemia virus-myc recombinant provirus FTT.

A myc-containing recombinant feline leukemia provirus, designated FTT, was molecularly cloned from the cat T-cell lymphoma line F422. Its transforming activity, as well as the nucleotide sequence of the 3' 2.7 kilobases of FTT, including v-myc, was determined. The predicted v-myc protein differs from feline c-myc by three amino acid changes and is truncated by two amino acids at the carboxyl terminus. Comparison with feline leukemia virus (FeLV), feline c-myc, and other FeLV proviruses indicates that recombination junctions involved in the generation of FeLV-onc viruses occur at preferred locations within the virus. They usually follow or occur within the sequence ACCCC at 5' junctions and may result from homologous recombination between sequences of marked purine-pyrimidine strand bias, especially at 3' junctions. Some recombination sites also resemble recombinase recognition sequences utilized in immunoglobulin and T-cell receptor variable-region joining. Transfection of primary rat embryo fibroblasts and subsequent in vivo analysis revealed that morphologic and tumorigenic transformation require cotransfection of FTT with human EJ-ras DNA; neither gene alone is sufficient. FTT v-myc is expressed in these transformed rat cells as a 3.0-kilobase subgenomic RNA; however, in contrast to the depressed level of c-myc expression in v-myc-involved feline tumors, steady-state levels of rat c-myc RNA and protein are apparently unaltered.