Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm.

OBJECTIVE: To investigate whether vaccination of macaques with attenuated simian immunodeficiency virus (SIV)macC8 could induce long-term protective immunity against rectal exposure to SIVsm and intravenous exposure to the more divergent HIV-2. DESIGN AND METHODS: Eight months after vaccination with live attenuated SIVmacC8, four cynomolgus monkeys were challenged with SIVsm intrarectally and another four vaccinated monkeys were challenged with HIV-2 intravenously. Sixteen months after SIVmacC8 vaccination, another two monkeys were challenged with SIVsm across the rectal mucosa. Two vaccinees shown to be protected against SIVsm were rechallenged 8 months after the first challenge. Ten naive animals were used as controls. Serum antigenaemia, virus isolation, antibody responses, cell-mediated immunity and CD4+ and CD8+ T-cell subpopulations were monitored. PCR-based assays were used to distinguish between virus populations. RESULTS: At the time of challenge, eight out of 10 vaccinees were PCR-positive for SIVmacC8 DNA but no virus could be isolated from peripheral blood mononuclear cells. After SIVsm challenge, three out of six vaccinees were repeatedly SIVsm PCR-negative. In one of the three infected monkeys, the challenge virus was initially suppressed but the monkey ultimately developed AIDS after increased replication of the pathogenic virus. Rechallenged monkeys remained protected. All HIV-2-challenged vaccinees became superinfected. All controls became infected with either SIVsm or HIV-2. At the time of challenge the vaccinees had neutralizing antibodies to SIVmac but no demonstrable cross-neutralizing antibodies to SIVsm or HIV-2. Titres of antigen-binding or neutralizing antibodies did not correlate with protection. Cytotoxic T-cell responses to SIV Gag/Pol and virus-specific T-cell proliferative responses were low. CONCLUSION: The live attenuated SIVmacC8 vaccine was able to induce long-term protection against heterologous intrarectal SIVsm challenge in a proportion of macaques but not against the more divergent HIV-2, which was given intravenously.

Immune responses but no protection against SHIV by gene-gun delivery of HIV-1 DNA followed by recombinant subunit protein boosts.

The efficacy of combining immunization with human immunodeficiency vitus type 1 (HIV-1) DNA and HIV-1 recombinant proteins to obtain protection from chimeric simian/human immunodeficiency virus (SHIV) was determined. Four cynomolgus monkeys received four gene-gun immunizations intraepidermally of plasmid DNA encoding HIV-1lai env (gp160), gag, tat, nef, and rev proteins. Ten micrograms of DNA was used per immunization. The animals were boosted twice intramuscularly with 50 microgram of HIV-1lai Env (MicroGeneSys), Gag, Tat, Nef, and Rev recombinant proteins mixed in Ribi adjuvant. The antibody responses were amplified following the administration of the recombinant subunit boosts. One month after the final subunit immunization, the vaccinated animals together with four control animals were challenged intravenously with 10 monkey infectious doses of SHIV that expresses the env, tat and rev genes of HIV-1 and gag and nef from SIV. However, only low titers of neutralizing antibodies were present at the day of challenge. The consecutive HIV-1 DNA and recombinant protein immunizations induced B- and T-cell responses but not protection against SHIV replication nor reduction of the viral load.

Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein and challenge with a high dose of SHIV-4 virus.

Infection of macaques with chimeric simian-human immunodeficiency viruses (SHIVs) allows evaluation of HIV-1 envelope vaccines. SHIV-4 is based on SIVmac239 but carries the env, tat, and rev genes of HIV-1IIIB. In this study we used Semliki Forest virus (SFV) RNA vectors to express the envelope protein gp160 of HIV-1IIIB in cynomolgus macaques. Monkeys were immunized four times with recombinant suicide SFV. Whereas two of four monkeys showed T cell-proliferative responses, only one monkey had demonstrable levels of antibodies to HIV-1 gp41 and gp120 as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot. The vaccinated monkeys and four control animals were challenged with 10,000 MID100 (100% minimum infectious doses) of cell-free monkey cell-grown SHIV-4 virus. As demonstrated by virus isolation, all macaques became infected after challenge. All vaccinated monkeys showed an HIV-1-specific anamnestic T cell-proliferative response. Three of four vaccines had developed HIV-1-Env-specific antibodies 2 weeks after challenge whereas none of the four controls showed any detectable immune response at this time point. Furthermore, three of four vaccinated monkeys had no demonstrable viral antigenemia and low viral load as opposed to one of the four naive control animals.

Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission.

At present it is not known which form of immunity would be most effective against infection with human immunodeficiency virus (HIV). To evaluate the possible role of cellular immunity, we examined whether four HIV type 2-exposed but seronegative macaques developed cellular immune responses and determined whether these exposed macaques were resistant to mucosal transmission of simian immunodeficiency virus (SIV). Following intrarectal challenge with SIV, 2 monkeys were protected against detectable SIV replication and another showed suppressed viral replication compared to 14 persistently infected controls. The two protected monkeys demonstrated SIV-specific cytotoxic T lymphocytes before as well as after SIV challenge. Here we provide evidence that activation of the cell-mediated arm of the immune system only, without antibody formation, can control SIV replication in macaques. The results imply that vaccines that stimulate a strong and broad cellular immune response could prevent mucosal HIV transmission.

Prevention of simian immunodeficiency virus, SIVsm, or HIV-2 infection in cynomolgus monkeys by pre- and postexposure administration of BEA-005.

OBJECTIVE: To study the possibilities and limitations of postexposure treatment to prevent the establishment of infection after accidental exposure to HIV. DESIGN AND METHODS: The effect of 2,3'-dideoxy-3'-hydroxymethyl cytidine (B1 A-005) was investigated on acute simian immunodeficiency virus (SIV) and HIV-2 infections in macaques in pre- and postexposure treatment experiments. RESULTS: Postexposure treatment with BLA-005 (3 x 10 mg/kg) for as short as 3 days prevented infection with SIVsm after intravenous or rectal inoculation. Infection with HIV-2 could also be blocked by postexposure BFA-005 treatment. CONCLUSION: This study shows that therapeutic intervention can block early systemic and mucosal infections with SIV and HIV-2. Further evaluation is ongoing.

Immunogenicity and protective efficacy of a human immunodeficiency virus type 2 recombinant canarypox (ALVAC) vaccine candidate in cynomolgus monkeys.

The efficacy of a recombinant human immunodeficiency virus (HIV) type 2 canarypox (ALVAC HIV-2) vaccine candidate given alone or in combination with HIV-2 envelope gp125 or HIV-2 V3 synthetic peptides was investigated in 14 cynomolgus monkeys. High antibody titers to HIV-2 gp125 were demonstrated in monkeys given booster immunizations with gp125. Neutralizing antibody titers were low (< or = 20) in all monkeys except 2. Significant lymphocyte proliferative responses to killed HIV-2 virions were observed in monkeys given booster immunizations with gp125. HIV-2-specific cytotoxic T lymphocytes were demonstrated prior to viral challenge in 3 of 12 monkeys. After challenge with homologous cell-free HIV-2 propagated in monkey cells, 4 of 10 monkeys immunized with ALVAC HIV-2 plus HIV-2 gp125 or V3 peptides were protected, as determined by negative virus isolation and polymerase chain reaction for viral DNA. Four monkeys immunized with ALVAC HIV-2 alone were not protected. All 12 control monkeys became infected. There was no correlation between the immunologic parameters studied and protection against infection in the vaccinated monkeys.

Protection against mucosal SIVsm challenge in macaques infected with a chimeric SIV that expresses HIV type 1 envelope.

In a monkey model we used a chimeric SIV expressing the HIV-1 envelope gene (SHIV-4) as a live attenuated vaccine and a virulent SIVsm as a mucosal challenge. Four cynomolgus monkeys were inoculated intravenously with SHIV-4. Virus was repeatedly isolated from blood mononuclear cells of all four animals for 2 to 7 months after the inoculation of SHIV. All monkeys developed neutralizing antibodies to HIV-1 and high antibody titers to HIV-1 envelope glycoproteins. In contrast, no neutralizing antibodies to SIVsm were detected and cross-reacting antibodies to SIV envelope glycoproteins were demonstrable in low titers. Nine to 12 months after the SHIV inoculation the four monkeys and six naive control monkeys were challenged intrarectally with 10 monkey infectious doses of macaque cell-grown SIVsm. After a follow-up period of 1 year, two of four SHIV-infected monkeys were completely protected against SIVsm infection as shown by repeated negative virus isolations and negative polymerase chain reaction for SIV envelope DNA. One naive monkey that received blood from the two protected monkeys showed no signs of infection. The remaining two SHIV-infected monkeys showed an initial infection on challenge with SIVsm, but viral replication was thereafter suppressed. Cytotoxic T lymphocytes to SIV Nef and RT were demonstrable in one of four SHIV-infected monkeys before SIVsm challenge, but this monkey was not protected against SIV infection. All six control animals yielded virus repeatedly after SIVsm challenge and three of them showed declining CD4 cell counts. Thus, infection with SHIV expressing HIV-1 envelope could induce cross-protection against mucosal SIVsm challenge.

Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections.

Sera from human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2)-infected humans were tested with autologous (from the same individual) and heterologous (from other individuals) virus isolates in a neutralization assay. Similarly, sera from experimentally simian immunodeficiency virus (SIVsm from sooty mangabey) or HIV-2SBL6669-infected cynomolgus macaques were tested for neutralizing activity against autologous and heterologous reisolates. In the neutralization assay, the virus dose ranged between 10-75 50% infectious dose (ID50), sera were used in five 2- or 4-fold dilutions, beginning with 1:20, and human peripheral blood mononuclear cells (PBMCs) served as target cells. The readout of the 7-day assay was a HIV-1 or HIV-2 antigen enzyme-linked immunosorbent assay (ELISA). Our results show that SIVsm-inoculated monkeys who develop early immunodeficiency lack serum neutralizing activity or develop a neutralizing antibody response with narrow specificity. Long survival is associated with the ability to neutralize several autologous and heterologous SIVsm reisolates. Infection of macaques with HIV-2SBL6669 did not cause disease within the 5 years observation time and elicited a broadly cross-reactive neutralizing antibody response, including neutralization of other, independently obtained, HIV-2 isolates. In HIV-1-infected humans, neutralizing antibodies can only be detected in up to 50% of cases. Neutralizing activity, whenever present, may show a broad specificity, that is, neutralization may occur across genetic subtypes. Presence of broadly cross-reactive neutralizing antibodies is associated with a lower risk of HIV-1 (subtype B) transmission both from mother to child and sexually from male to female. Unlike HIV-1 infection, serum neutralizing activity is regularly present in HIV-2 infection. In view of the differences between HIV-1 and HIV-2 pathogenesis, we suggest that an effective neutralizing antibody response may contribute to a delay in disease progression and to a decrease in risk of transmission.

SIV infection of monkey spleen cells including follicular dendritic cells in different stages of disease.

Immunoaffinity enriched spleen follicular dendritic cells (FDCs), lymphocytes, and macrophages from SIVsm-inoculated cynomolgus monkeys (Macaca fascicularis) at different stages of disease were compared for latent and productive SIV infection. Analysis of FDCs by in situ hybridization, electron microscopy, and coculture assays indicated that comparatively high levels of virus were associated with the FDC fraction. Polymerase chain reaction (PCR) and RT-PCR results revealed that the levels for SIVpol DNA did not correlate with the level of env mRNA in the various cell subsets, suggesting differences in latency. Limiting dilution assays for spliced env mRNA showed a 10-100-fold higher amount of env mRNA in FDCs than in other spleen cell subsets early during SIV infection. At late stages of disease, the number of productively infected FDCs significantly decreased in parallel with a marked reduction of the FDC network and follicular involution. Our findings indicate that destruction of FDCs probably reflects a cytopathic effect of SIV and/or the activity of specific antiviral cytotoxic T lymphocytes.

Long-term protection against SIV-induced disease in macaques vaccinated with a live attenuated HIV-2 vaccine.

The aim of this study was to test the ability of a live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine to protect cynomolgus monkeys against superinfection with a pathogenic simian immunodeficiency virus (SIVsm). This report is an update on our previously reported observation period of nine months. The new data here show that three of four monkeys vaccinated with live HIV-2 were protected against immunosuppression and SIV-induced disease during more than five years of follow-up. The quality of the immunity was permissive for infection, but monkeys that survived showed restricted viral replication in peripheral blood and lymph nodes. This study shows that it is possible to induce protection against a pathogenic heterologous primate lentivirus and to prevent disease in vaccinated monkeys even if infection is not prevented. These findings provide evidence that protection against AIDS can be achieved by immunization.

Evaluation of HIV-1/HIV-2 immunoblots for detection of HIV-2 antibodies.

OBJECTIVE: To evaluate the sensitivity of commercially available HIV-2 immunoblots and to identify the HIV-2 glycoproteins on Western blots. METHODS: HIV-2 Western blot (WB) strips commercially available from Diagnostic Biotechnology, Diagnostic Pasteur and Cambridge Biotech and in-house HIV-2 WB strips were investigated by monoclonal HIV-2 gp36 and gp125 antibodies for identification of the glycoproteins. The WB strips and commercially available HIV-1/HIV-2 line immunoassays (LIAs) from Diagnostic Pasteur (PEPTI-LAV 1-2), Diagnostic Biotechnology (version 2.2) and Innogenetics (INNO-LIA HIV-1/HIV-2 ab) were analyzed by seroconversion panels from HIV-2 infected cynomolgus monkeys (Macaca fascicularis) to investigate their sensitivity for detection of HIV-2 antibodies. The LIAs were also investigated by use of 100 HIV-2 antibody positive human sera from Guinea Bissau. The in-house WB strips contained HIV-2/SBL-6669 antigen treated with various concentrations of sodium dodecyl sulphate (SDS, 0-2%) at 37 degrees C or 100 degrees C for various times to obtain gp36 in oligomeric and/or monomeric form. RESULTS: By use of monoclonal antibodies, WB strips from Diagnostic Biotechnology and Diagnostic Pasteur were shown to contain gp125 as well as monomeric and oligomeric forms of gp36, whereas Cambridge WB strips contained mainly oligomeric gp36 and no detectable gp125. The sensitivity of the WB strips for detection of HIV-2 seroconversion was similar if WB seropositivity was defined as reactivity with p24 and one envelope protein. When the WHO WB criteria were applied requiring reactivity with at least two envelope proteins for positivity, the sensitivity of the WB strips from Diagnostic Biotechnology and Diagnostic Pasteur was retained, whereas the sensitivity of Cambridge Biotech WB strips was reduced. Among 100 HIV-2 antibody positive human sera all were reactive on PEPTI-LAV 1-2 and INNO-LIA HIV-1/HIV-2 ab, but two of the hundred sera failed to react with the HIV-2 synthetic peptide band on Diagnostic Biotechnology version 2.2 WB strips. On in-house WB strips the relation between monomeric and oligomeric gp36 was changed by altering the SDS concentration and the temperature. Thus the monomeric form increased with the SDS concentration and the temperature. The sensitivity for detection of antibodies during seroconversion did not differ between the monomeric and oligomeric forms of gp36. CONCLUSIONS: The sensitivity for detection of HIV-2 antibodies during seroconversion was independent of the oligomeric or monomeric structure of the transmembrane glycoprotein. One of the three commercial WB kits tested had a lower sensitivity for detection of HIV-2 seroconversion compared with the other two kits when the WHO criteria for WB positivity were used.

Thymic immunopathology and progression of SIVsm infection in cynomolgus monkeys.

Thymuses from 22 cynomolgus monkeys infected with simian immunodeficiency virus (SIVsm) developed characteristic cortical and medullary changes including formation of B-cell follicles (8/21) and accumulation of virus immune complexes. Advanced thymic histopathology was correlated with more pronounced immunodeficiency. SIVsm provirus was detected by polymerase chain reaction (PCR) in most (16/18) thymuses and spliced viral env mRNA in 3 (3/7) thymuses with advanced histopathologic changes indicative of thymic SIVsm replication. By combined in situ hybridization (ISH) and immunohistochemistry, viral RNA was localized mainly to the follicular dendritic network, macrophages, multinucleated giant cells, and lymphocytes of the medullary regions. Latent infection by an Epstein-Barr-related herpesvirus (HVMF1) was also found by PCR and by ISH in medullary regions of three (3 of 8) thymuses with B-cell follicles, suggestive of an inductive role for B-cell proliferation in these thymuses. In a control group of HIV-2-infected nonimmunosuppressed monkeys, no comparable thymic changes were observed. Our results indicate that SIV, and probably by analogy HIV, can have direct and diverse pathogenic effects on the thymus that are important in the development of simian (human) AIDS.

B-cell lymphomagenesis in SIV-immunosuppressed cynomolgus monkeys.

B-cell lymphomas developed frequently (approx. 40%) in SIVsm (SMM3) immunosuppressed monkeys and were mostly extranodal, aggressive and all associated with an EBV-related simian herpes virus operationally designated herpes virus Macaca fascicularis (HVMF-I). Lymphoma tissues from 21 monkeys were studied by PCR and DNA PAGE for mono/oligoclonality of the VDJ-rearranged IgH genes. Most lymphomas (n = 15) showed a monoclonal and approximately 1/3 (n = 6) an oligoclonal VDJ rearrangement pattern. The time after infection to tumor presentation was significantly shorter for oligoclonal than for monoclonal lymphomas, suggesting that oligoclonal selection frequently precedes the outgrowth of a single malignant clone. Comparison of the VDJ rearrangements in an established lymphoma cell line and the original, oligoclonal lymphoma tissue indicated in vitro selection of one HVMF-infected clone. Longitudinal studies of sequential lymph-node biopsies showed that the malignant lymphoma clone in 3 out of 8 lymphomas could be identified as a predominant clone in lymph nodes 2-12 months after SIV infection and 6-10 months before clinical presentation of the lymphomas. VDJ-rearranged DNA corresponding to that of the lymphomas was also detected in most sera at the time of lymphoma manifestation but not in corresponding PBL preparations. Clearly, the SIVsm AIDS model in cynomolgus monkeys represents a powerful tool for biological and clinical studies of herpes-virus-associated lymphomagenesis in immunosuppressed states.

Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus.

A collection of simian immunodeficiency virus (SIV) neutralizing recombinant Fab fragments was generated using the combinatorial antibody library approach. Functional antibody fragments efficiently expressed in Escherichia coli were identified only in the form of chimeric macaque heavy chain gamma 1 and human light chain kappa. The gamma 1 and kappa chains were derived from a clinically healthy long-term surviving SIVsm-infected cynomolgus macaque and from an asymptomatic HIV-2 seropositive individual, respectively. The combinatorial library was constructed on the surface of filamentous phage using the pComb3 phagemid vector and screened against purified SIVsm surface glycoprotein (gp148). Twelve chimeric clones reacting with the antigen were isolated. Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 micrograms/ml. All neutralizing Fab fragments were clonally unrelated as demonstrated by nucleic acid sequencing. These potent neutralizing reagents will be used for prophylactic and therapeutic immune intervention of lentivirus infection in macaques and to map neutralizing determinants of SIV.

Heterologous HIV-2 challenge of rhesus monkeys immunized with recombinant vaccinia viruses and purified recombinant HIV-2 proteins.

In an attempt to analyse the role of anti-envelope immunity in the protection of rhesus monkeys against an HIV-2 intravenous challenge, rhesus macaques were immunized twice with recombinant HIV-2 ROD vaccinia viruses (10(8) p.f.u. each) at days 0 and 30, followed by booster injections of purified HIV-2 proteins at months 8, 9, 15 and 27. One group of five macaques was immunized with the Gag, Pol, Vif and Nef antigens, whereas a second group received the same antigens with the addition of HIV-2 Env protein. Eight months after the last boost, the animals were challenged by intravenous injection of 100 AID50 of a monkey PBMC-grown stock of HIV-2 SBL. None of the animals was protected in spite of high humoral immune responses on day of challenge as determined by ELISA and Western Blot assays.

Broadly reactive HIV-2 and SIVmac specific antibody-dependent cellular cytotoxicity in immunized and infected cynomolgus monkeys.

Antibody-dependent cellular cytotoxicity (ADCC) was analysed in groups of cynomolgus monkeys that had been immunized with either HIV-2 (strains SBL6669 or SBL-K135) or SIVmac. HIV-2- and SIVmac-infected monkeys were also studied for ADCC. Sequential serum samples were collected from the animals, which were followed for 1 to 3 years. Sera from the HIV-2-immunized monkeys had ADCC against both homologous and heterologous HIV-2 strains as well as cross-reactivity against SIVmac-infected target cells. This broadly reactive ADCC response could be detected within the first weeks after immunization. Homologous ADCC was also seen in seven of eight SIVmac-immunized monkeys which were all protected from later challenge with SIVmac or SVsm. ADCC titres sometimes decreased after a few months if the immunized monkeys were not boosted whereas most of the HIV-2-and SIVmac-infected monkeys developed a rapid and persistent ADCC response. The presence of ADCC, like the presence of neutralization in previous studies, did not predict whether the immunized monkeys would be protected upon challenge with live virus.

Long-standing protection of macaques against cell-free HIV-2 with a HIV-2 iscom vaccine.

We investigated the capacity of two immunostimulating-complex (iscom) formulations including inactivated native HIV-2 viral proteins and selected peptides to induce protective immunity against HIV-2 in a nonhuman primate. Four cynomolgus monkeys were first immunized with five i.m. injections of purified detergent-disrupted HIV-2 virions (total dose, 0.7 mg) in iscoms over a period of 16 months. At months 18 and 20, all four macaques were given booster immunizations with iscom-coupled V3-derived synthetic peptides representing a dominating neutralizing region of HIV-2 gp125. Two weeks after the final dose of vaccine, the four vaccinated animals, together with four controls, were challenged i.v. with 10 monkey infectious doses (MID50) of monkey-cell-grown homologous cell-free virus, HIV-2SBL-6669/H5. After the challenge, the four control animals became readily infected; however, three of four vaccinated animals were protected as shown by repeated negative virus isolations and negative polymerase chain reaction for viral DNA and by failure to transmit HIV-2 infection with whole blood and lymph node cells into naive cynomolgus macaques. One of three protected animals showed an anamnestic antibody response to a dominating antigenic site, indicating possible limited virus replication. The vaccine-protected monkeys were subsequently resistant to rechallenge infection at 12, 15, and 18 months after the first challenge, suggesting that a reasonable duration of protective immunity had been induced by the vaccine.