Erling W. Rud

Multispecific Vaccine-Induced Mucosal Cytotoxic T Lymphocytes Reduce Acute-Phase Viral Replication but Fail in Long-Term Control of Simian Immunodeficiency Virus SIVmac239

ABSTRACT Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01+ macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4+ helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01+ controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.

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.

Rapid development of vaccine protection in macaques by live-attenuated simian immunodeficiency virus.

Convincing data on experimental vaccines against AIDS have been obtained in the simian immunodeficiency virus (SIV) macaque model by preinfection with a virus attenuated by a nef deletion. To investigate the efficacy of a nef deletion mutant of SIVmac32H called pC8 as a live-attenuated vaccine after shorter preinfection periods and to learn more about the nature of the immune protection induced, eight rhesus monkeys were infected intravenously with the pC8 virus. All monkeys became persistently infected, exhibiting low cell-associated viral loads, but strong cellular and, in terms of binding antibodies, strong humoral antiviral responses. Two of eight pC8-infected monkeys developed an immunodeficiency and were not challenged. Sequence analysis of their nef revealed complete replenishment of the deletion. The other six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged with pathogenic spleen-derived SIV. Complete protection was achieved in four vaccinees. Virus was consistently detected in two vaccinees from the 22-week-group challenge, however, they remained clinically healthy over a prolonged period. Protection from challenge virus infection or a delayed disease development seemed to be associated with a sustained SIV-specific T helper cell response after challenge. Thus, a sterilizing immunity against superinfection with pathogenic SIV can be induced even after a relatively short waiting period of 22 weeks. Nevertheless, such a vaccine raises severe safety concerns because of its potential to revert to virulence.

Brain Microbial Populations in HIV/AIDS: α-Proteobacteria Predominate Independent of Host Immune Status

The brain is assumed to be a sterile organ in the absence of disease although the impact of immune disruption is uncertain in terms of brain microbial diversity or quantity. To investigate microbial diversity and quantity in the brain, the profile of infectious agents was examined in pathologically normal and abnormal brains from persons with HIV/AIDS [HIV] (n = 12), other disease controls [ODC] (n = 14) and in cerebral surgical resections for epilepsy [SURG] (n = 6). Deep sequencing of cerebral white matter-derived RNA from the HIV (n = 4) and ODC (n = 4) patients and SURG (n = 2) groups revealed bacterially-encoded 16 s RNA sequences in all brain specimens with α-proteobacteria representing over 70% of bacterial sequences while the other 30% of bacterial classes varied widely. Bacterial rRNA was detected in white matter glial cells by in situ hybridization and peptidoglycan immunoreactivity was also localized principally in glia in human brains. Analyses of amplified bacterial 16 s rRNA sequences disclosed that Proteobacteria was the principal bacterial phylum in all human brain samples with similar bacterial rRNA quantities in HIV and ODC groups despite increased host neuroimmune responses in the HIV group. Exogenous viruses including bacteriophage and human herpes viruses-4, -5 and -6 were detected variably in autopsied brains from both clinical groups. Brains from SIV- and SHIV-infected macaques displayed a profile of bacterial phyla also dominated by Proteobacteria but bacterial sequences were not detected in experimentally FIV-infected cat or RAG1−/− mouse brains. Intracerebral implantation of human brain homogenates into RAG1−/− mice revealed a preponderance of α-proteobacteria 16 s RNA sequences in the brains of recipient mice at 7 weeks post-implantation, which was abrogated by prior heat-treatment of the brain homogenate. Thus, α-proteobacteria represented the major bacterial component of the primate brain’s microbiome regardless of underlying immune status, which could be transferred into naïve hosts leading to microbial persistence in the brain.

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in nef. To investigate the efficacy of SIVmac32H(pC8), a nef deletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form.

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro.

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV‐HXBc2 in human T‐cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P<0.05) to in vitro infection by virulent SIVJ5 and SHIV‐HXBc2 than were lymphocytes from naïve controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P<0.01) by depletion of CD8+ T‐cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the β‐chemokines regulated upon activation, normal T‐cell expressed and secreted, macrophage inflammatory protein‐1α and macrophage inflammatory protein‐1β did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.

Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge

Abstract: The aim of this study was to evaluate the role of CTLs in the protection from challenge with pathogenic SHIV in macaques vaccinated with attenuated virus. More specifically, we have analyzed the CTL response in cynomolgus macaques vaccinated/infected with the attenuated SIVmacC8 or the wild‐type SIVmacJ5 and correlated these responses to the protection from SHIV89.6P challenge. SIVmacC8‐vaccinated monkeys demonstrated a broader CTL response than the SIVmacJ5‐infected animals. Nevertheless, CTL against some proteins in SIVmacC8‐vaccinated monkeys became progressively more difficult to detect through the day of challenge. In regards to protection from superinfection with SHIV89.6P, neither the presence of circulating CTL nor the CTL precursor frequency against any of the tested proteins correlated with the outcome of the challenge when SIVmacJ5‐ and SIVmacC8‐infected animals were analyzed together. By analyzing the SIVmacC8‐vaccinated animals separately, only the protected animal had detectable CTL precursors with moderate frequencies against all three tested proteins at the day of challenge.

Mechanisms of protection induced by attenuated simian immunodeficiency virus. IV. Protection against challenge with virus grown in autologous simian cells

Abstract: Attenuated simian immunodeficiency virus (SIV) induces potent protection against infection with wild‐type virus, but the mechanism of this immunity remains obscure. Allogeneic antibodies, which arise within animals as a result of SIV infection, might protect against challenge with exogenous SIV grown in allogeneic cells. To test this hypothesis, eight macaques were infected with attenuated SIV and subsequently challenged with wild‐type SIV grown in autologous cells or heterologous cells.