Major histocompatibility complex class I-associated vaccine protection from simian immunodeficiency virus-infected peripheral blood cells.

To evaluate the effectiveness of vaccine protection from infected cells from another individual of the same species, vaccinated rhesus macaques (Macaca mulatta) were challenged with peripheral blood mononuclear cells from another animal diagnosed with acquired immune deficiency syndrome (AIDS). Half of the simian immunodeficiency virus (SIV)-vaccinated animals challenged were protected, whereas unprotected vaccinates progressed as rapidly to AIDS. Protection was unrelated to either total antibody titers to human cells, used in the production of the vaccine, to HLA antibodies or to virus neutralizing activity. However, analysis of the serotype of each animal revealed that all animals protected against cell-associated virus challenge were those which were SIV vaccinated and which shared a particular major histocompatibility complex (MHC) class I allele (Mamu-A26) with the donor of the infected cells. Cytotoxic T lymphocytes (CTL) specific for SIV envelope protein were detected in three of four protected animals vs. one of four unprotected animals, suggesting a possible role of MHC class I-restricted CTL in protection from infected blood cells. These findings have possible implications for the design of vaccines for intracellular pathogens such as human immunodeficiency virus (HIV).

Protection from HIV-1 envelope-bearing chimeric simian immunodeficiency virus (SHIV) in rhesus macaques infected with attenuated SIV: consequences of challenge.

OBJECTIVES: To determine whether prior infection with simian immunodeficiency virus (SIV)BK28 protects macaques from subsequent exposure to an HIV-1 envelope chimeric SIV (SHIV). Also, to determine the consequences of viral challenge on CD4 numbers and virus load on the current SIV infection. DESIGN AND METHODS: A total of 12 mature outbred Macacca mulatta were studied. Four naive controls and four previously infected with attenuated SIVBK28 were challenged with SHIV; four naive controls were not infected with SHIV. Sampling occurred twice monthly, and monthly thereafter. Changes in virus load, CD4 and CD8 populations were monitored. Highly sensitive and specific discriminative polymerase chain reaction (PCR) assays were used to distinguish between virus populations. RESULTS: SHIV readily infected challenged control animals, which developed a peak in virus load and a decline in CD4+ cell numbers. In controls, viral load declined and CD4 cell numbers rose to near normal levels after seroconversion. In contrast, in SIV-infected animals there was only a minor increase in viral load in only two out of four animals, 100-1000-fold lower than in naive animals. Interestingly, a decline in CD4 cells occurred in all four SIV-infected animals after SHIV challenge, which appeared more pronounced than in animals infected by SHIV alone. One SIV-infected animal which had low CD4 cell numbers at the time of SHIV challenge, developed a further decline in CD4 cells with a rising viral load. Discriminative PCR did not reveal SHIV in the challenged SIV animals. Interestingly the increase in viral load was due to SIV and not SHIV. CONCLUSIONS: Broad protection of animals previously infected with live attenuated SIV was demonstrated with protection from subsequent infection with HIV-1 envelope-bearing chimeric SIV. Subsequent exposure in cases with low CD4 cell numbers reveal the possibility of activation of the vaccine strain with the possible risk of inducing disease progression.

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

OBJECTIVE: A comparative study of the replication kinetics of different HIV-1 variants (including SIV(cpz)) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DESIGN: Plasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIV(cpz-ant)-infected chimpanzees. METHODS: A pan-clade quantitative competitive reverse transcriptase-polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIV(cpz) isolates. RESULTS: Important differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIV(cpz-ant) were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIV(cpz)) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. CONCLUSION: Infection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 10(4) RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

A clinically relevant HIV-1 subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection.

OBJECTIVES: To investigate whether immunization with recombinant HIV-1 envelope protein derived from a clinical isolate could protect macaques from infection with an in vivo passaged chimeric simian-human immunodeficiency virus (SHIV). DESIGN AND METHODS: A total of 16 animals were studied from which three groups of four animals were immunized with vaccine formulations of the CC-chemokine receptor-5-binding recombinant gp120 of HIV-1W6.1D. Four weeks after the last immunization, all 16 animals were intravenously challenged with in vivo passaged SHIV derived from the same HIV-1 group B clinical isolate (W6.1D) as the vaccines. RESULTS: Vaccine protection from infection was demonstrated in 10 out of 12 macaques immunized with recombinant gp120. Complete protection from infection was achieved with all of the animals that received the SBAS2-W6.1D formulation, a potent inducer of both T-cell and humoral immune responses. Partial protection was achieved with SBAS1-W6.1D, a formulation based on immunomodulators known to induce T-cell responses in humans. In vaccinated animals that were infected, virus load was reduced and infection was delayed. CONCLUSIONS: In a relatively large number of primates, vaccine efficacy was demonstrated with a clinically relevant HIV-1 vaccine. These results reveal that it is possible to induce sterilizing immunity sufficient to protect from infection with SHIV which was passaged multiple times in vivo. Our findings have implications for current HIV-1 clinical vaccine trials and ongoing efforts to develop safe prophylactic AIDS vaccines.

Immune strategies utilized by lentivirus infected chimpanzees to resist progression to AIDS.

HIV-1 infected chimpanzees are relatively resistant to the development of AIDS despite their close genetic relatedness to humans and their susceptibility to HIV-1 infection. We have systematically studied possible reasons for their relative ability to maintain T helper (Th) cell numbers and immune competence in the presence of chronic HIV-1 infection. Factors which may alone or together cause the loss in T-cell dependent immunity include: (i) the loss of Th cell function; (ii) the loss of Th cells; and (iii) the loss of capacity for Th cell renewal. Differences in the in vivo and in vitro responses of T lymphocytes from chimpanzees and humans were compared for evidence of HIV-1 related T-cell dysfunction. In contrast to HIV infected individuals, HIV-1 infected chimpanzees maintained strong Th cell proliferative and cytokine responses after receiving tetanus toxoid boosts. In addition there was no abnormal Th1 to Th2 shift as is suggested to occur in AIDS patients. There was no evidence of Th cell dysfunction such as increased level of programmed cell death (PCD) or immune activation in HIV-1 infected chimpanzees in contrast to HIV-1 infected asymptomatic humans. Anergy could be induced with HIV-1 gp120 in human but not chimpanzee Th lymphocytes. We then asked if there was a direct loss of chimpanzee CD4+ cells due to HIV-1 infection in vitro. Infection of chimpanzee CD4+ lymphocyte cultures with HIV-1 in the absence of CD8+ cells resulted in marked cytopathic effect with complete lysis and loss of cells within 3 weeks. We concluded that most chronic HIV-1 infected chimpanzees were able to maintain relatively stable CD4+ lymphocyte numbers despite CD4+ lymphocyte destruction due to direct effects of the virus. Furthermore, there was no evidence of indirect Th cell loss, since neither increased levels of anergy nor apoptosis were observed. Lymph node biopsies from HIV-1 infected chimpanzees revealed that MHC class II rich regions of lymph nodes remained intact, in contrast to the involution of these regions in infected humans. This suggested that chimpanzees may maintain the capacity for Th cell renewal by preserving this MHC class II lymphoid environment. The data presented in this paper suggests that chimpanzees may preserve this critical MHC class II-Th cell environment by dramatically suppressing extra-cellular virus load and that this may be in part mediated by soluble lentivirus suppressing factors.

An in vivo model for HIV resistance development.

Treatment of human immunodeficiency virus (HIV-1) with drugs targeted to the reverse transcriptase (RT) rapidly selects for drug-resistant virus. It is essential to develop a suitable animal model that allows the study of the emergence and reversal of drug resistance. A monkey model was previously developed on the basis of a hybrid virus (RT-SHIV) of simian immunodeficiency virus (SIV) with its RT exchanged for HIV-1 RT. In the present study cynomolgus monkeys infected with RT-SHIV were treated with varying doses of the non-nucleoside RT inhibitor nevirapine. The drug was administered for 2-3 weeks, in agreement with clinical experience of resistance development during nevirapine monotherapy. This resulted in the selection of mutants with Y181C and K103N changes in RT, which correspond to the HIV-1 mutations in nevirapine-resistant HIV-1 patients. The mutants coexisted at varying levels with wild-type virus and fluctuations in the proportion of mutants could be closely monitored. Low-dose treatment was not more efficient in induction of mutations than a virus-inhibiting dose. Structured therapy interruptions could be performed. The monkey RT-SHIV infection offers an in vivo model to determine effects of therapies on resistance development.

Vaccine protection and reduced virus load from heterologous macaque-propagated SIV challenge.

The efficacy of vaccine protection afforded by live attenuated vaccines was tested by heterologous SIVtno8980 challenge following successful protection against homologous SIVmac32H challenge. Animals immunized with the attenuated SIVmacBK28 molecular clone were asymptomatic and virus isolation negative by quantitative virus isolation prior to challenge. Two groups of four animals previously immunized 5 years and 4 months (respectively) were challenged with 100 MID50 of SIVtno8980, as was a third group of four naive controls. All control animals that were challenged developed high levels of plasma antigenemia within 2 weeks of challenge and developed rapid Th/m cell loss whereas vaccinated animals did not. Quantitative virus isolation from peripheral blood mononuclear cells revealed that one of four animals in each group became virus isolation positive but that the virus load in the two vaccinated animals was markedly lower than in nonvaccinated controls. Studies are underway to determine the duration and immunological correlates of protection from AIDS.

The rate of progression to AIDS is independent of virus dose in simian immunodeficiency virus-infected macaques.

Of the viral factors that are proposed to influence the rate of progression to AIDS, the role of infectious dose remains unresolved. Intravenous infection of outbred Macaca mulatta with various doses of simian immunodeficiency virus isolate 8980 (SIV(8980)) revealed an endpoint from which an infectious dose 50 (ID(50)) was defined. In the six infected animals, the time to develop AIDS was variable with a spectrum of rapid, intermediate and slow progressors. High and sustained plasma viraemia with marked loss of CD4(+) T-cells was a distinguishing feature between rapid versus intermediate and slow progressors. Animals that received the highest doses did not develop the highest sustained viral loads, nor did they progress more rapidly to disease. Similarly, animals infected with lower doses did not uniformly develop lower viral loads or progress more slowly to AIDS. Furthermore, compiled data from more than 21 animals infected with different doses of the same virus administered by the same route failed to reveal any correlation of infectious dose with survival. Indeed, host factors of these outbred animals, rather than dose of the initial inoculum, were probably an important factor influencing the rate of disease progression in each individual animal. Comparison of animals infected with SIV(B670), from which SIV(8980) was derived, revealed marked differences in disease progression. Clearly, although dose did not influence viral loads nor disease progression, the virulence of the initial inoculum was a major determinant of the rate of progression to AIDS.

A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques.

To determine if a specific pathogenic threshold of plasma viral RNA could be defined irrespective of virus strain, RNA levels in the plasma of more than 50 infected rhesus macaques (Macaca mulatta) were measured. Animals were inoculated intravenously with either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) strains of known pathogenic potential (SIV8980, SIVsmm-3, SIVmac32H/J5, SIVmac32H/1XC, reverse transcriptase-SHIV, SHIV89.6p) or with attenuated strains (SHIVW6.1D, SHIVsf13, SHIVhan-2, SIVmacDeltanef, SHIVsf33). In animals inoculated with nonpathogenic strains, shortly after the primary peak of viremia viral RNA levels declined and remained below 10(4) RNA equivalents/ml of plasma between 6 and 12 weeks postinoculation. Animals infected with documented pathogenic strains maintained viral RNA levels higher than 10(5) RNA equivalents/ml of plasma. In animals infected with strains with low virulence, a decline in plasma RNA levels was observed, but with notable individual variation. Our results demonstrate that the disease-causing potential was predicted and determined by a threshold plasma virus load which remained greater than 10(5) RNA equivalents/ml of plasma 6 to 12 weeks after inoculation. A threshold virus load value which remained below 10(4) RNA equivalents/ml of plasma was indicative of a nonpathogenic course of infection.

Co-receptor usage of BOB/GPR15 in addition to CCR5 has no significant effect on replication of simian immunodeficiency virus in vivo.

Human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency viruses (SIVs) frequently use the orphan receptor BOB/GPR15 in addition to the chemokine receptor CCR5 for efficient entry and replication. However, the role of BOB/GPR15 in replication and pathogenesis of HIV-2 and SIV in vivo is unclear. This study shows that a single amino acid substitution in the V3 loop of the pathogenic SIVmac239 clone, 321P-->S, impaired the ability to use BOB/GPR15 for entry and replication but had little effect on the ability to use CCR5. This envelope variant replicated with an efficiency comparable with the parental SIVmac239 isolate in rhesus macaques. Furthermore, the mutant genotype and phenotype remained stable even after the onset of immunodeficiency. These results suggest that this cofactor plays only a minor role for the pathogenicity of the HIV-2/SIVmac/SIVsm group of primate lentiviruses.

Evidence for viral virulence as a predominant factor limiting human immunodeficiency virus vaccine efficacy.

Current strategies in human immunodeficiency virus type 1 (HIV-1) vaccine development are often based on the production of different vaccine antigens according to particular genetic clades of HIV-1 variants. To determine if virus virulence or genetic distance had a greater impact on HIV-1 vaccine efficacy, we designed a series of heterologous chimeric simian/human immunodeficiency virus (SHIV) challenge experiments in HIV-1 subunit-vaccinated rhesus macaques. Of a total of 22 animals, 10 nonimmunized animals served as controls; the remainder were vaccinated with the CCR5 binding envelope of HIV-1(W6.1D). In the first study, heterologous challenge included two nonpathogenic SHIV chimeras encoding the envelopes of the divergent clade B HIV-1(han2) and HIV-1(sf13) strains. In the second study, all immunized animals were rechallenged with SHIV(89. 6p), a virus closely related to the vaccine strain but highly virulent. Protection from either of the divergent SHIV(sf13) or SHIV(han2) challenges was demonstrated in the majority of the vaccinated animals. In contrast, upon challenge with the more related but virulent SHIV(89.6p), protection was achieved in only one of the previously protected vaccinees. A secondary but beneficial effect of immunization on virus load and CD4(+) T-cell counts was observed despite failure to protect from infection. In addition to revealing different levels of protective immunity, these results suggest the importance of developing vaccine strategies capable of protecting from particularly virulent variants of HIV-1.

Virus load in chimpanzees infected with human immunodeficiency virus type 1: effect of pre-exposure vaccination.

Many reports indicate that a long-term asymptomatic state following human immunodeficiency virus type 1 (HIV-1) infection is associated with a low amount of circulating virus. To evaluate the possible effect of stabilizing a low virus load by non-sterilizing pre-exposure vaccination, a quantitative virus isolation method was developed and evaluated in four chronically infected chimpanzees infected with a variety of HIV-1 related isolates. This assay was then used to monitor a group of chimpanzees (n = 6) challenged with HIV-1 following vaccination with gp120 or gp160. Data indicated that of the three vaccinated animals which became infected after challenge, the animal with the lowest neutralizing titre at the time of challenge acquired a virus load similar to the control animals, whereas the two other chimpanzees had reduced numbers of virus producing cells in their peripheral circulation. One animal became virus isolation negative, developed an indeterminant PCR signal on lymph node DNA and subsequently became negative for HIV-1 DNA as determined by PCR on PBMC (peripheral blood mononuclear cells) and bone marrow DNA. Recently, the second animal has also become PCR negative. To confirm observations from quantitative virus isolations, quantification of HIV-1 DNA in PBMC and virus RNA in serum was performed by PCR on serially diluted samples at two different time points. Comparison of virus load as determined by these three methods confirmed that there was an effect of vaccination in reducing virus load and demonstrated a correlation between decreased numbers of virus producing cells, HIV-1 DNA containing cells and virus RNA molecules in serum.

Protection of rhesus macaques from SIV infection by immunization with different experimental SIV vaccines.

The immunogenicity and efficacy of an inactivated whole SIVmac (32H) preparation adjuvanted with muramyl dipeptide (SIV-MDP) and a gp120-enriched SIVmac (32H) ISCOM preparation (SIV-ISCOM), were compared by immunizing four rhesus macaques (Macaca mulatta) four times with SIV-MDP and four others in the same way with SIV-ISCOM. Two monkeys immunized with whole inactivated measles virus (MV) adjuvanted with MDP (MV-MDP) and two monkeys immunized with MV-ISCOM served as controls. In the SIV-ISCOM-immunized monkeys higher SIV-specific serum antibody titres were found than in the SIV-MDP-immunized monkeys. In contrast to the MV-immunized monkeys all SIV-MDP- and SIV-ISCOM-immunized monkeys were protected against intravenous challenge 2 weeks after the last immunization with 10 median monkey infectious doses (MID50) of a cell-free SIVmac (32H) challenge stock propagated in the human T-cell line C8166. After 43 weeks the protected monkeys were reboosted and 2 weeks later rechallenged with 10 MID50 of the same virus produced in peripheral blood mononuclear cells (PBMC) from a rhesus macaque. None of these animals proved to be protected against this challenge. In a parallel experiment in which the same numbers of monkeys were immunized in the same way, the animals were challenged intravenously with 10 MID50 of PBMC from an SIVmac (32H)-infected rhesus macaque. Two out of four SIV-MDP- and two out of four SIV-ISCOM-immunized monkeys proved to be protected from SIV infection.

Comparison of protection from homologous cell-free vs cell-associated SIV challenge afforded by inactivated whole SIV vaccines.

This study attempted to determine if SIV vaccines could protect against challenge with peripheral blood mononuclear cells (PBMCs) from an SIV infected rhesus monkey. Mature Macaca mulatta were vaccinated four times with formalin inactivated SIVmac32H administered in MDP adjuvant (n = 8) or SIVmac32H ISCOM vaccine (n = 8). Controls included animals vaccinated with measles virus in MDP adjuvant (n = 4) or ISCOM (n = 4) preparations. Of each group, half were challenged intravenously (IV) with ten MID50 of the cell-free SIVmac32H (11-88) SIV stock and half were challenged with ten MID50 of PBMCs from the SIVmac32H infected macaque 1XC. All SIV vaccinated animals challenged with the 11-88 cell free stock of SIVmac32H were protected, whereas only half of the SIV vaccinated monkeys receiving the same infectious dose of the 1XC cell stock were protected.

CCR5 targeted SIV vaccination strategy preventing or inhibiting SIV infection.

Cell-surface CCR5 is a major coreceptor with CD4 glycoprotein, mediating cellular entry of CCR5 strains of HIV-1 or SIV. We targeted the SIV CCR5 coreceptor in a combined CCR5-SIV antigen immunization strategy. Rhesus macaques were immunized i.m. with the 70 kDa heat shock protein (HSP70) covalently linked to the CCR5 peptides, SIV gpl20 and p27. Intravenous challenge with SIV mac 8980 prevented SIV infection or decreased the viral load with the CCR5-SIV combined vaccine. CC chemokines and antibodies which block and downmodulateCCR5 were induced, as well as immune responses to the subunit SIV antigens. This novel vaccination strategy complements cognate immunity to SIV with innate immunity to the CCR5 coreceptor of SIV.

Simian immunodeficiency virus in which nef and U3 sequences do not overlap replicates efficiently in vitro and in vivo in rhesus macaques.

The nef genes of human immunodeficiency virus and simian immunodeficiency virus (SIV) overlap about 80% of the U3 region of the 3' long terminal repeat (LTR) and contain several essential cis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silent point mutations. To restore viral infectivity, intact cis-regulatory elements were inserted just downstream of the mutated nef gene. The resulting SIV genome contains U3 regions that are 384 bp shorter than the 517-bp 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences truncates the proviral genome by 350 bp. Nonetheless, it contains all known coding sequences and cis-acting elements. The TPI mutant virus expressed functional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restrictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nef and the U3 region. We demonstrate that this conserved genomic organization is not obligatory for efficient viral replication and pathogenicity.

Protection from secondary human immunodeficiency virus type 1 infection in chimpanzees suggests the importance of antigenic boosting and a possible role for cytotoxic T cells.

Recent evidence suggests a much higher prevalence of human immunodeficiency virus type 1 (HIV-1) recombinants than previously anticipated. These recombinants arise from secondary HIV infections in individuals already infected with the virus. It remains unclear why some individuals acquire secondary HIV-1 infections and others do not. To address this question, a study was undertaken of a small cohort of chimpanzees with well-defined HIV-1 infection. After exposure to an infectious dose of heterologous primary isolate, 4 of 8 HIV-1 seropositive chimpanzees resisted secondary infection, whereas 2 naive controls became readily infected. Only animals who were immunologically boosted were protected. Protection from heterologous secondary exposure appeared to be related to the repertoire of the cytolytic CD8(+) T cell responses to HIV-1. Data suggested that immunologic boosting by HIV-1 antigens or exposure to subinfectious doses of virus may be important events in sustaining sufficient immunity to prevent secondary infections from occurring.

Enhanced simian immunodeficiency virus-specific immune responses in macaques induced by priming with recombinant Semliki Forest virus and boosting with modified vaccinia virus Ankara.

The immunogenicity of two vector-based vaccines, either given alone or in a prime-boost regimen, was investigated. Cynomolgus macaques were immunised with modified vaccinia virus Ankara (MVA) expressing simian immunodeficiency virus (SIV)macJ5 env, gag-pol, nef, rev, and tat genes (MVA-SIVmac) or primed with a Semliki forest virus (SFV) vaccine expressing the same genes (SFV-SIVmac) and boosted with MVA-SIVmac. Generally, antibody responses, T-cell proliferative responses and cytotoxic T-cell responses remained low or undetectable in vaccinees receiving MVA-SIVmac or SFV-SIVmac alone. In contrast, monkeys who first received SFV-SIVmac twice and then were boosted with MVA-SIVmac showed increased antibody responses as well as high T-cell proliferative responses. Three of these vaccinees had cytotoxic T-lymphocytes directed against three or four of the gene products. No evidence of protection was seen against an intrarectal heterologous SIVsm challenge given 3 months after the last immunisation. The study demonstrates a prime-boost strategy that efficiently induces both humoral and cellular immune responses.