Per Putkonen

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 (BEA-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 BEA-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 BEA-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.

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.

Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection

Simian immunodeficiency virus (SIV) infection in cynomolgus macaques leads to severe immunodeficiency with a fatal outcome. In contrast, HIV-2 infects these primates without apparently causing any immunological abnormalities. In this study three cynomolgus monkeys were experimentally infected with HIV-2 strain SBL-K135 and 168 days later challenged with 10-100 animal infectious doses of the closely related SIV strain SM to study protective immunity. At the time of SIV challenge the HIV-2-infected monkeys had neutralizing antibodies against HIV-2, but virus could no longer be recovered from their peripheral blood mononuclear cells (PBMCs) and no clinical symptoms or decrease in CD4+ lymphocytes were observed. Follow-up for 9 months after challenge with SIV showed that the HIV-2-infected monkeys were protected against SIV-induced immunodeficiency (no decrease of CD4+ lymphocytes) and lymphadenopathy. However, they were not resistant to SIV infection since virus could be recovered from their PBMCs and they developed anamnestic antibody responses. Four naive control monkeys which were inoculated with the same dose of SIV became persistently infected and developed a decrease of the absolute numbers of CD4+ cells and showed a marked lymphadenopathy. Two out of four control animals died 58-265 days postinfection with an immunosuppressive disease. Immunohistochemical examination showed abundant viral antigen in lymph-node biopsies from the SIV-infected control monkeys but absence of SIV or HIV-2 antigens in the biopsies from the three HIV-2-preinfected and SIV-superinfected monkeys. The present study demonstrates possibilities for induction of immunity against immunodeficiency induced by a primate lentivirus, a concept with application also to HIV infection and AIDS in man.

Clinical features and predictive markers of disease progression in cynomolgus monkeys experimentally infected with simian immunodeficiency virus.

ObjectiveTo study the pathogenicity of simian immunodeficiency virus (SIVsm) in cynomolgus monkeys in order to establish an animal model for human AIDS. MethodsThirty-three cynomolgus monkeys were monitored for more than 2 years following experimental infection with SIVsm. ResultsAll the macaques became SIV-infected, as demonstrated by virus recovery from peripheral blood lymphocytes and by the appearance of viral antibodies. SIVsm was found to be pathogenic, killing 29 out of the 33 monkeys (88%) within 26 months. Clinically, infected monkeys developed lymphadenopathy, splenomegaly, diarrhoea, weight loss, neurological symptoms and a remarkably high incidence (39%) of malignant lymphomas. All lymphomas were high-grade malignant and of B-cell origin. Disease progression was associated with low CD4+ lymphocyte count, involution of initially hyperplastic follicular B-cell areas in lymph nodes, reappearance of viral antigen in serum, loss of anti-Gag antibodies and development of systemic giant cell disease in 55% of the monkeys. ConclusionsThere are many similarities between SIVsm-induced AIDS in cynomolgus monkeys and human AIDS with regard to clinical, virological, immunological and pathological manifestations.

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.

Protection of macaques against simian immunodeficiency virus infection with inactivated vaccines: comparison of adjuvants, doses and challenge viruses

Nine European laboratories contributed a total of 98 macaques towards a collaborative trial to study the ability of formaldehyde-inactivated or subunit SIV vaccines to protect immunized animals against live virus challenges. Four adjuvants, three dose levels and two immunization schedules were compared. Fifty-two of 61 (85%) immunized animals were protected against infection after challenge with either homologous or heterologous virus strains grown in human cells. Optimum protection required a high dose of antigen and a prolonged immunization schedule. On the day of challenge the titres of antibodies to SIV and to host cell components, as well as the titres of neutralizing antibodies, were significantly higher in the protected animals than in the non-protected. Forty-four vaccinated macaques (of which 36 were protected against previous challenges grown in human cells) and 28 naive animals were then challenged with extracellular or cell-associated SIV grown in simian cells. All naive animals and all vaccinees challenged with extracellular SIV became infected. Four of the eight animals challenged with cell-associated viruses were protected. These results clearly indicate that vaccines which potently protect against SIV grown in human cells, do not protect against SIV grown in simian cells. The cell substrate on which challenge viruses are grown is clearly significant in interpreting the results of vaccine trials. This trial has demonstrated that SIV vaccines using different adjuvants can protect macaques against SIV grown in human cells but not against extracellular SIV grown in simian cells. These results have important relevance to the development of HIV vaccines for humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys.

A non-opportunistic, generalized giant cell disease (GCD) was found in 12 out of 25 (48%) cynomologus monkeys infected with SIVsm. Most organs were affected notably the lymph nodes (LN), spleen, gut, liver, lungs and CNS. The multinucleated GC varied considerably in cell size and in the number and cytoplasmic distribution of the nuclei. Immunohistochemically most GC expressed SIV antigens and markers of mononuclear phagocytes (CD68), CD4 and also occasionally the T-cell markers CD45RO, CD43 and CD2. Monkeys with GCD had more pronounced immunosuppression with lower CD4-cell counts, more often demonstrable SIV antigen in the blood and LN and had been infected for a longer time period, as compared to monkeys without GCD. These findings show that SIV infection in cynomolgus monkeys is frequently associated with extensive formation of multinucleated GC of macrophage origin, which appears to be related to the pathogenesis of the infection and the degree of immunosuppression.

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.

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.

Efficacy of inactivated whole HIV-2 vaccines with various adjuvants in cynomolgus monkeys

Twenty‐one cynomolgus monkeys were immunized with whole inactivated HIV‐2 preparations administered with various adjuvants (incomplete Freunds adjuvant, Alum, Ribi, MDP, or Iscoms) and challenged with 10 or 100 MID50 of a homologous monkey‐cell grown, cell‐free HIV‐2. Seven animals were completely protected against infection, three showed reduced virus replication. The vaccines elicited neutralizing and ADCC antibodies; the titers did not correlate with protection. Immunization with a whole inactivated vaccine can protect primates from intravenous challenge with a monkey‐cell grown cell‐free human immunodeficiency virus type 2.