Barbro Mäkitalo

Majority of CD4+ T cells from peripheral blood of HIV-1–infected individuals contain only one HIV DNA molecule

Neither the number of HIV-1 proviruses within individual infected cells in HIV-1–infected patients nor their genetic relatedness within individual infected cells and between cells and plasma virus are well defined. To address these issues we developed a technique to quantify and genetically characterize HIV-1 DNA from single infected cells in vivo. Analysis of peripheral blood CD4+ T cells from nine patients revealed that the majority of infected cells contain only one copy of HIV-1 DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity between HIV populations in CD4+ T cells and plasma implies ongoing exchange between these compartments both early and late after infection.

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.

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.

A Novel Assay for Assessment of HIV-Specific Cytotoxicity by Multiparameter Flow Cytometry

Assessment of CD8+ T‐cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry.

ELISpot and ELISA analysis of spontaneous, mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macaques

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.

Spontaneous production of RANTES and antigen-specific IFN-γ production in macaques vaccinated with SHIV-4 correlates with protection against SIVsm challenge

The β‐chemokines, RANTES, MIP‐1α and MIP‐1β, have been implicated as being some of the protective factors in the immune response against human immunodeficiency virus (HIV) infection. We have presented data previously indicating that these chemokines also play a role in protective immunity against HIV/SIV infection in macaques. The aim of this study was to investigate the production of β‐chemokines in eight cynomolgus macaques vaccinated with non‐pathogenic SHIV‐4 in relation to protection against pathogenic SIVsm challenge. Four control animals were also included in the study. Two of the vaccinated monkeys were completely protected and one was partially protected against the challenge virus. The monkeys that resisted infectious SIVsm virus challenge showed higher spontaneous β‐chemokine production by peripheral blood mononuclear cells and had higher numbers of antigen‐induced IFN‐γ secreting cells compared to the non‐protected animals. Our observations support our previous findings that the genetic background of the host and/or environmental factors are involved in the chemokine production and that β‐chemokines contribute to protection against HIV/SIV infection.

Cross-protection against mucosal simian immunodeficiency virus (SIVsm) challenge in human immunodeficiency virus type 2-vaccinated cynomolgus monkeys

In this study we compared the efficacy of live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine alone versus boosting with live non-pathogenic HIV-2 following priming with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol). Six monkeys were first inoculated intravenously with live HIV-2(SBL-6669) and 7 to 10 months later were challenged intrarectally with 10 MID(50) of cell-free simian immunodeficiency virus (SIV) strain SIVsm. One monkey was completely protected against SIV infection and all five monkeys that became SIV-infected showed a lower virus replication and an initial lower virus load as compared with a parallel group of six control animals. In another experiment five monkeys were immunized either three times with ALVAC HIV-2 alone or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125. The monkeys were then challenged with HIV-2 given intravenously and finally with pathogenic SIVsm given intrarectally. After challenge with SIVsm, three of five monkeys were completely protected against SIVsm infection whereas the remaining two macaques became SIV-infected but with limited virus replication. In conclusion, vaccination with an ALVAC HIV-2 vaccine followed by exposure to live HIV-2 could induce cross-protection against mucosal infection with SIVsm and seemed to be more efficient than immunization with a live HIV-2 vaccine only.

Cell-mediated immunity to low doses of SIVsm in cynomolgus macaques did not confer protection against mucosal rechallenge

Simian immunodeficiency virus (SIV) infection of macaques is a useful model for studies of the roles of different immune responses against viruses that cause (AIDS). In this study, six cynomolgus macaques were inoculated intrarectally with subinfectious or infectious doses of SIVsm to assess the SIV specific immunity, in particular protective immunity against subsequent challenge with a higher dose of SIVsm. Following the first inoculation with SIVsm, the two monkeys given the highest doses of cell-free SIVsm stock and one monkey given the intermediate dose became infected. In the three remaining animals, one animal inoculated with an intermediate dose and two animals given low doses of SIVsm, no overt infection occurred. Nevertheless, SIV specific cytotoxic T-cells against Gag/Pol and Nef proteins and T-cell proliferative responses against HIV-2 whole viral lysate, native HIV-2 gp125, recombinant SIV gp140 and SIV Env synthetic peptides were detected. After intrarectal rechallenge of the uninfected macaques with a higher dose of SIVsm all the animals became infected. These results demonstrate that cell mediated immunity can occur in the absence of detectable infection in monkeys inoculated with a low dose of SIVsm. Despite the presence of cellular immune responses, the animals were not protected when challenged with a higher dose of virus later.

HIV-1 Specific CD4 and CD8 T-cell Responses Associated With Low Viral Load in Treatment-Naïve HIV-1 Infected Individuals

In preparation for monitoring of vaccine-induced responses, we determined HIV-specific cell-mediated immune responses in 17 treatment naive HIV-1 infected individuals with > 400 CD4+ T cells/ml for at least 5 years including 9 patients with low viral load (VL, 5000 copies/ml). HIV-1 specific IFN-γ-production and cytolytic activity were higher in subjects with low VL. The differences between the two groups were statistically significant for CD4+ Tcell responses to Gag and Nef peptides, tested by a longterm (48 h) ICS assay and of border-line significance for the Gag-specific cytolytic responses measured by a flowcytometry assay and a chromium release assay. We also found a significant inverse correlation between VL and IFN-γ-production by CD8+ T-cells in response to Gag as measured by ICS. The ELISpot IFN-γ response was not significantly different in patients with high and low VL. During a median follow-up period of 2.4 years, 6 of 8 subjects with high VL and 1 of 9 with low VL showed decreasing CD4+ T-cell counts, and ARV treatment was more frequently initiated in the former patient group (5 of 8 versus 1 of 9). The CD4 and CD8 T cell immune responses found to be associated with low VL and stable CD4 counts may be of importance for protection. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005