Simian AIDS: an historical perspective.

The author has had the unique opportunity to participate, over the last 35 years, in the retrovirus research field that proceeded and followed the discovery of human, simian and feline AIDS. The onset of human AIDS was certainly unanticipated, but in retrospect, the warning signs had been present for at least a decade in captive macaques. I will briefly summarize the key scientific knowledge and 'mindset' leading up to these events and will outline the major contributions and unanswered questions arising from the simian model of AIDS.

The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques.

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.

Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells.

Despite recent insights into mucosal human immunodeficiency virus (HIV) transmission, the route used by primate lentiviruses to traverse the stratified squamous epithelium of mucosal surfaces remains undefined. To determine if dendritic cells (DC) are used by primate lentiviruses to traverse the epithelial barrier of the genital tract, rhesus macaques were intravaginally exposed to cell-free simian immunodeficiency virus SIVmac251. We examined formalin-fixed tissues and HLA-DR(+)-enriched cell suspensions to identify the cells containing SIV RNA in the genital tract and draining lymph nodes within the first 24 h of infection. Using SIV-specific fluorescent in situ hybridization combined with immunofluorescent antibody labeling of lineage-specific cell markers, numerous SIV RNA(+) DC were documented in cell suspensions from the vaginal epithelium 18 h after vaginal inoculation. In addition, we determined the minimum time that the SIV inoculum must remain in contact with the genital mucosa for the virus to move from the vaginal lumen into the mucosa. We now show that SIV enters the vaginal mucosa within 60 min of intravaginal exposure, infecting primarily intraepithelial DC and that SIV-infected cells are located in draining lymph nodes within 18 h of intravaginal SIV exposure. The speed with which primate lentiviruses penetrate mucosal surfaces, infect DC, and disseminate to draining lymph nodes poses a serious challenge to HIV vaccine development.

The kinetics of specific immune responses in rhesus monkeys inoculated with live recombinant BCG expressing SIV Gag, Pol, Env, and Nef proteins.

Development of an effective preventive or therapeutic vaccine against HIV-1 is an important goal in the fight against AIDS. Effective virus clearance and inhibition of spread to target organs depends principally on the cellular immune response. Therefore, a vaccine against HIV-1 should elicit virus-specific cytotoxic lymphocyte (CTL) responses to eliminate the virus during the cell-associated stages of its life cycle. The vaccine should also be capable of inducing immunity at the mucosal surfaces, the primary route of transmission. Recombinant Bacille Calmette-Guérin (BCG) expressing viral proteins offers an excellent candidate vaccine in view of its safety and ability to persist intracellularly, resulting in the induction of long-lasting immunity and stimulation of the cellular immune response. BCG can be administered orally to induce HIV-specific immunity at the mucosal surfaces. The immunogenicity of four recombinant BCG constructs expressing simian immunodeficiency virus (SIV) Gag, Pol, Env, and Nef proteins was tested in rhesus macaques. A single simultaneous inoculation of all four recombinants elicited SIV-specific IgA and IgG antibody, and cellular immune responses, including CTL and helper T cell proliferation. Our results demonstrate that BCG recombinant vectors can induce concomitant humoral and cellular immune responses to the major proteins of SIV.

Immunogenicity of a vaccine preparation representing the variable regions of the HIV type 1 envelope glycoprotein.

Variability of the major antigenic sites of the envelope glycoprotein of HIV-1 constitutes a major problem in the formulation of effective vaccines. We have prepared a synthetic peptide vaccine that represents the major hypervariable epitopes (V1 through V5) of the clade B HIV-1 envelope glycoprotein (gp120). We refer to this preparation as variable epitope immunogen or VEI vaccine. This construct takes into consideration the type and frequency of amino acid substitutions found at each epitope during the evolution of the virus in individual patients and in the target population. Immunization of mice, rabbits, and rhesus macaques with the VEI vaccine resulted in the induction of long-lasting, high-titered HIV-1 antibodies, including antibodies that neutralize primary isolates. We also documented lymphocyte proliferative responses to the VEI vaccine, its individual components, analogs, and subtype-specific peptides representing the major hypervariable regions of HIV-1 gp120. Delayed-type hypersensitivity responses to these antigens were also demonstrated in mice. Our results show that this vaccine is highly immunogenic and safe in animals. Our data suggest that this formulation could become an important component of combination vaccine approaches against HIV-1 and other antigenically variable pathogens.

Hypervariable epitope constructs representing variability in envelope glycoprotein of SIV induce a broad humoral immune response in rabbits and rhesus macaques.

Using synthetic peptides, we developed an approach to account for protein epitope variability. We have prepared, in a single synthesis, a cocktail of peptides we have designated a hypervariable epitope construct (HEC), which collectively represents much of the in vivo variability seen in an epitope. Eight HECs representing the in vivo variability seen throughout the envelope glycoprotein of the simian immunodeficiency virus (SIV) were designed and synthesized. The constructs were collectively conjugated to KLH (HEC-KLH) or recombinant gp130 (HEC-rgp130) and used to immunize rabbits and rhesus macaques, respectively. Using sera collected from rabbits immunized with HEC-KLH, we demonstrated that individual components of the immunogen were recognized as antigen in ELISAs, and that the induced antibodies cross-reacted with several strains of SIV as well as with a strain of HIV-2. Following immunization of macaques with HEC-rgp130 antiviral antibodies were induced. These antibodies were still present 9.5 months after the last boost and were also capable of recognizing several different strains of SIV, including SIVmac239, SIVmac251, and SIVsmH3, as well as a strain of HIV-2 (HIV-2ROD). In addition, the antibodies were also capable of neutralizing SIV viral infectivity in vitro. Peripheral blood lymphocytes (PBLs) from immunized macaques proliferated in response to whole proteins and virus. Finally, sera from monkeys immunized with SIV, rgp130, and HIV-2 as well as sera from HIV-2-positive humans recognized HECs in ELISAs, demonstrating the relevance of these epitopes in vivo. This approach can be used as an effective method for generating a strong, broadly cross-reactive humoral response against HIV and can serve as an important component of combination vaccines against HIV and AIDS.

Partial body weight support with treadmill locomotion to improve gait after incomplete spinal cord injury: a single-subject experimental design.

BACKGROUND AND PURPOSE: Gait training with partial body weight support has been used to improve gait. In this study, changes in gait relative to speed, cadence, stride length, and percentages of stance and swing for both lower extremities (LEs) during comfortable walking, fast walking, and running were studied in a subject with an incomplete C-5 on C-6 spinal cord injury. SUBJECT AND METHODS: A single-subject experimental design was used. Following a 6-week period of baseline measurements taken at various intervals (phase AI), the subject ambulated on a treadmill three times a week for 6 weeks with 32% of his body weight supported (phase B). Phase B was followed by a 3-week period without treatment during which measurements were taken at various intervals (phase AII). Gait variables were measured once a week during comfortable walking, fast walking, and running. Heart rate was monitored during treadmill training. RESULTS: During comfortable walking, fast walking, and running, improvements were seen in gait speed. During running, improvements also were seen in stride length and percentages of stance and swing for the right LE. The largest changes were recorded during running. Smaller changes were recorded during comfortable walking and fast walking. CONCLUSION AND DISCUSSION: These results justify testing the efficacy of this technique with larger groups of subjects with neurological impairments.

The history of simian AIDS.

Retrospective data indicate that two separate outbreaks of simian AIDS and associated lymphoma were caused by Simian Immunodeficiency Virus (SIVmac and SIVstm, respectively) in group-housed macaques at the California Regional Primate Research Center (CRPRC) in the early and mid-1970s. Because these epizootics were not then recognized as infectious in nature, surviving healthy SIV carriers were sent to other primate centers where they transmitted the viruses to resident macaques. The source of SIV at the CRPRC was by contact with co-housed seropositive sooty mangabeys. Spread of SIV via saliva and blood while fighting most likely accounted for these epizootics. Separate outbreaks of a somewhat different version of simian AIDS, caused by the simian Type D retrovirus (SRV-1), and spread from healthy carriers via saliva and blood also occurred in the late 1970s and early 1980s in group-housed macaques at the CRPRC. Initially, these SRV-1 outbreaks were also not recognized as infectious.

Origins of simian immunodeficiency virus infection in macaques at the New England Regional Primate Research Center.

A cohort of rhesus macaques (Macaca mulatta), obtained from the California Regional Primate Research Center (CRPRC) and necropsied in 1970-72 with lesions suggestive of simian immunodeficiency virus (SIV) infection, was identified at the New England Regional Primate Research Center (NERPRC). Polymerase chain reaction (PCR), DNA sequence analysis, and in situ hybridization were used to confirm the presence of SIV nucleic acids. This represents the earliest case of SIV infection at the NERPRC and suggests a common source for present day SIV isolates.

Antibodies to the putative SIV infection-enhancing domain diminish beneficial effects of an SIV gp160 vaccine in rhesus macaques.

OBJECTIVE: To demonstrate that antibodies against amino acids (aa) 603-622 of the SIV gp41 transmembrane glycoprotein enhance infection of SIV in vivo. DESIGN: A synthetic peptide derived from aa 603-622 of SIVmac251 gp41 was synthesized and tested for immunogenicity in rabbits and SIV-infected rhesus macaques. Next, SIV-naive animals were immunized with either a recombinant vaccinia virus expressing the SIV gp160 envelope glycoprotein (VVrgp160) and boosted three times with aa 603-622 (group 1, four animals), wild-type vaccinia virus and boosted with aa 603-622 (group 2, two animals), or VVrgp160 followed by three doses of an irrelevant peptide (group 3, two animals). Animals were challenged with SIVmac251. RESULTS: Peptide aa 603-622 was immunogenic in rabbits. SIV-infected rhesus monkeys immunized with the peptide developed two-three log increases in antibodies to this peptide and antibodies that could enhance SIV infection in vitro. SIV-naive rhesus macaques in group 1 had higher levels of antibody to the peptide by enzyme-linked immunosorbent assay and higher levels of enhancing antibodies at the time of SIV challenge than the animals in groups 2 or 3. Following challenge with SIVmac251 the group 1 animals had detectable p27 antigen longer than animals in group 2 and 3 and died of simian AIDS before the respective animals in the two control groups (P < 0.05 by log-rank test). CONCLUSIONS: aa 603-622 of SIV gp41, like aa 579-613 of HIV gp41, can stimulate production of antibodies that enhance SIV and HIV infection in vitro. Furthermore, immunization with this peptide suppressed beneficial effects of a gp160 vaccine and appeared to enhance SIV infection in vivo.

Hypervariable epitope constructs as a means of accounting for epitope variability.

Epitope variability is one of the greatest obstacles to development of synthetic peptide vaccines. Based on a recently described hypervariable epitope (aa 414-434) on the envelope glycoprotein (gp130) to simian immunodeficiency virus (SIVmac142), we have developed a novel approach to account for epitope variability. We have prepared, in a single synthesis, a cocktail of peptides, designated a hypervariable epitope construct (HEC), which collectively represent all the in vivo variability seen in an epitope. The HEC represents permutations of amino acid substitutions found in the epitope and has been able to induce antibodies with enhanced binding to native SIV and broad immunoreactivity to related epitope analogues.

Passive immunization of macaques against SIV infection.

Passive immunization with plasma from an inactivated-whole SIVmac vaccine protected monkey conferred complete or partial protection to rhesus macaques challenged intravenously 4 or 18 hours later with 10 AID50 of homologous cell-free virus. In contrast, passive immunization with inactivated plasma or purified immunoglobulin (Ig) from SIVmac infected asymptomatic monkeys failed to protect any recipients similarly challenged and may have enhanced infection and accelerated disease. Administered 24 hours post challenge, anti-SIV Ig may also have enhanced the infection.

Reduced virus load in rhesus macaques immunized with recombinant gp160 and challenged with simian immunodeficiency virus.

As a safe alternative to inactivated and live-attenuated whole-virus SIV vaccines, we have evaluated the potential of SIVmac239 gp160 expressed by recombinant vaccinia virus (vSIVgp160) and baculovirus (bSIVgp160) to protectively immunize rhesus macaques against intravenous (i.v.) infection with pathogenic SIVmac isolates. Macaques were immunized with live vSIVgp160 and/or bSIVgp160 protein partially purified from insect cells. The challenge viruses, propagated in rhesus peripheral blood mononuclear cells, consisted of the molecular clone SIVmac239 and another genetically similar, uncloned isolate, SIVmac251. Although antibodies that bind gp130 were induced in all animals following immunization with SIVgp160, neutralizing antibodies were undetectable 1 week prior to virus challenge. These results differ from those for macaques vaccinated with inactivated, whole SIV. All animals became infected after i.v. inoculation with 1-10 AID50 of either challenge virus. For animals challenged with SIVmac251, but not those challenged with SIVmac239, the cell-free infectious virus load in plasma of vSIVgp160-primed, bSIVgp160-boosted macaques was significantly lower than in unimmunized controls at 2 weeks postchallenge. Virus virulence, immunization regimen, and challenge with homologous or heterologous virus are factors critical to the outcome of the study. Immunization with surface glycoprotein may not necessarily provide protective immunity against infection but may reduce virus load. The relationship between reduction in virus load by vaccination and delay in onset of disease remains to be determined.

Synthetic peptide in mineral oil adjuvant elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes in rhesus monkeys.

In view of the importance of cell-associated virus in AIDS virus transmission, an HIV vaccine should be able to induce a virus-specific CTL response. Traditional subunit vaccines have not elicited virus-specific CD8+ MHC class I-restricted CTL. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the use of CTL epitope peptide-helper peptide conjugates for the vaccine elicitation of AIDS virus-specific CTL. We found that both the CTL epitope peptide-helper peptide conjugate and the CTL epitope peptide alone, when delivered in an emulsion with IFA, induced CTL epitope-specific CD8+ MHC class I-restricted CTL. These effector cells recognized processed viral protein and were readily cloned from PBL of the immunized monkeys. Moreover, the cloned effector cells inhibited SIVmac replication in PBL. Immunization with the CTL epitope peptide used in this study also elicited a CD4+ PBL proliferative response, suggesting that the peptide also contained a helper epitope. These studies provide further evidence for the potential usefulness of peptide-based AIDS virus vaccines.

Induction of HIVMN neutralizing antibodies in primates using a prime-boost regimen of hybrid synthetic gp120 envelope peptides.

We have tested synthetic peptides composed of Th (T1) and V3 loop B cell neutralizing determinants [SP10 MN(A)] of HIVMN gp120 and the fusogenic (F) domain of gp41 as immunogens in rhesus monkeys. After two immunizations with either HIV env peptide T1-SP10 MN(A) or F-T1-SP10 MN(A), rhesus monkey serum neutralization titers against the HIVMN isolate ranged from 1:160 to 1:1400, and in cell-cell syncytium inhibition assay ranged from 1:20 to 1:80. However, in contrast to animals immunized with T1-SP10 MN(A), animals immunized twice with F-T1-SP10 MN(A) had no rise in anti-gp120 and neutralizing antibodies with an additional immunization with F-T1-SP10 MN(A) peptide. One of 4 rhesus monkeys (18987) had anti-HIVMN antibodies that cross-neutralized divergent HIV isolates HIVIIIB and HIVRF. Serum from animal 18987 neutralized 5 of 10 HIV isolates tested, and neutralizing activity against HIVIIIB of 18987 serum was absorbed with the conserved gp120 loop V3 sequence IGPGRAF. Anti-HIV neutralizing antibodies were boosted after a 6-mo rest by 500 micrograms of T1-SP10 MN(A) in 4 of 4 animals previously immunized with T1-SP10 MN(A) and in 2 of 2 animals previously immunized with F-T1-SP10 MN(A). However, immunization after 6-mo rest of animal 18987 with 500 micrograms of T1-SP10 MN(A) peptide, although boosting anti-HIVMN neutralizing antibodies, selectively did not boost cross-neutralizing anti-HIVIIIB antibodies. Thus, synthetic peptides containing T and B cell epitopes of HIV gp120 can induce high levels of anti-HIVMN neutralizing antibodies in primates.