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.

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In order to develop a successful subunit vaccine against infection with the human immunodeficiency virus (HIV), protective immune effector functions must be identified. Until now, there has been only indirect evidence that HIV-specific cytotoxic T lymphocytes (CTLs) fulfill this role. Using the macaque simian immunodeficiency virus (SIV) model, the protective potential of nef-specific CTLs, stimulated by vaccination, was examined in animals challenged with a high intravenous dose of the pathogenic simian immunodeficiency virus, SIVmac251(32H)(pJ5). An inverse correlation was found between the vaccine-induced nef-specific CTL precursor frequency and virus load measured after challenge. In addition, the early decline in viraemia, observed in both vaccinated and unvaccinated control animals was associated with the development of virus-specific CTL activity and not with the presence of virus-specific neutralizing antibodies. The results imply that vaccines that stimulate strong CTL responses could protect against HIV infection.

Good protection against systemic challenge in the SIVmac model of AIDS has been provided by prior infection with attenuated virus. To determine if such protection extends to intrarectal mucosal challenge two molecular clones, SIVmacC8 and SIVmacJ5, were used in this study. SIVmacC8 has an attenuated phenotype in vivo, due to a 12-bp deletion in the nef/ 3'-LTR, whereas SIVmacJ5 has a full size nef open reading frame and induces AIDS in infected macaques. The J5 molecular clone was shown to infect rhesus macaques following atraumatic intrarectal inoculation. The dynamics were similar to those following intravenous inoculation resulting in early, high, cell-associated viremia and seroconversion. Four macaques previously infected with the attenuated SIVmacC8 resisted superinfection with SIVmacJ5, following intrarectal inoculation. These animals also resisted intrarectal infection with an HIV/SIV chimeric virus (SHIV) composed of SIVmac239 expressing the HXBc2 env, tat, and rev genes, suggesting that immunity to the envelope proteins was unlikely to be involved in the superinfection resistance. Infection with the attenuated SIVmac generated cytotoxic T lymphocytes (CTL) detectable in the peripheral circulation, serum neutralizing antibodies, and SIV-binding antibodies in rectal fluids. SIVmacC8 proviral DNA was found in lymph nodes removed at necropsy but there was no evidence for local sequestration of challenge virus. SIV-specific CTL, were detected in gut-associated lymph nodes and may have a role in limiting superinfection following mucosal exposure.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.

The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, ~pr, tat, rev, env and neJ). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJS) and SIVmae32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmae251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with p J5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nefgene evolved over time in p J5 virusinfected macaques, whereas late in infection in pC8 virus-infected macaques the nefgene remained invariant in sequence. These results support the observation that a nefdeletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for p J5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDSlike symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.

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Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV),؉ 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.