Detection Of Simian Immunodeficiency Virus Research Articles

Immunohistochemical Evaluation of Five Antibodies for the Detection of Simian Immunodeficiency Virus

Similar to humans with HIV who develop encephalitis, rhesus macaques infected with Simian Immunodeficiency Virus (SIV)are susceptible to develop encephalitis characterized by perivascular accumulation of virus‐laden macrophages and multinucleated giant cells, astrogliosis, microgliosis, and neuronal injury. There are no investigative studies in the literature documenting sensitivity and specificity of available Anti‐SIV antibodies for the detection of viral protein in the tissue samples and therefore, a more formal and comprehensive investigation is warranted to advance our understanding of the disease. In this study, a comparative immunohistochemical analysis was carried out on formalin‐fixed, paraffin embedded sections of lymphoidorgans and brain from SIV infected rhesus macaques (Macaca mulatta), experimentally infected with SIVmac251 or SIVmac239. Immunohistochemistry (IHC) was performed using five different antibodies: Anti‐SIV mac251gag mAb (clone FA2), Anti‐SIV mac251 p28 mAb (HRP, clone 3F7), Anti‐SIV mac251 p28 polyclonal antibody, Anti‐SIV mac251 gp130 mAb(clone 5i45) and Anti‐SIV mac251 p17 gag mAb (clone KK59) to determine their efficacy for the detection of SIV viral protein. Immunohistochemical quantitative data are correlated with the viral load to estimate sensitivity of each antibody.

Detection of Simian Immunodeficiency Virus in Semen, Urethra, and Male Reproductive Organs during Efficient Highly Active Antiretroviral Therapy.

A number of men receiving prolonged suppressive highly active antiretroviral therapy (HAART) still shed human immunodeficiency virus (HIV) in semen. To investigate whether this seminal shedding may be due to poor drug penetration and/or viral production by long-lived cells within male genital tissues, we analyzed semen and reproductive tissues from macaques chronically infected with simian immunodeficiency virus mac251 (SIVmac251) who were treated for 4 months with HAART, which was intensified over the last 7 weeks with an integrase inhibitor. We showed that a subset of treated animals continued shedding SIV in semen despite efficient HAART. This shedding was not associated with low antiretroviral drug concentrations in semen or in testis, epididymis, seminal vesicles, and prostate. HAART had no significant impact on SIV RNA in the urethra, whereas it drastically reduced SIV RNA levels in the prostate and vas deferens and to a lesser extent in the epididymis and seminal vesicle. The only detectable SIV RNA-positive cells within the male genital tract after HAART were urethral macrophages. SIV DNA levels in genital tissues were not decreased by HAART, suggesting the presence throughout the male genital tract of nonproductively infected cells. In conclusion, our results demonstrate that 4 months of HAART induced variable and limited control of viral infection in the male reproductive organs, particularly in the urethra, and suggest that infected long-lived cells in the male genital tract may be involved in persistent seminal shedding during HAART. These results pave the way for further investigations of male genital organ infection in long-term-treated infected individuals. A substantial subset of men receiving prolonged HAART suppressing viral loads in the blood still harbor HIV in semen, and cases of sexual transmission have been reported. To understand the origin of this persistence, we analyzed the semen and male reproductive tissues from SIV-infected macaques treated with HAART. We demonstrated that persistent seminal shedding was not linked to poor drug penetration in semen or semen-producing prostate, seminal vesicle, epididymis, and testis. We revealed that HAART decreased SIV RNA to various extents in all male genital organs, with the exception of the urethra, in which SIV RNA(+) macrophages were observed despite HAART. Importantly, HAART did not impact SIV DNA levels in the male genital organs. These results suggest that infection of male genital organs, and particularly the urethra, could be involved in the release of virus in semen during HAART.

A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex® Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%–100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

Early Detection of Simian Immunodeficiency Virus in the Central Nervous System Following Oral Administration to Rhesus Macaques

The timing of HIV dissemination to the central nervous system (CNS) has the potential to have important implications regarding HIV disease progression and treatment. The earlier HIV enters the CNS the more difficult it might be to remove with antiretroviral therapy. Alternatively, HIV may only enter the CNS later in the course of disease as a result of disruption of the blood-brain-barrier. We utilized the simian immunodeficiency virus (SIV) infection of rhesus macaques to evaluate the oral route of infection and the subsequent spread of SIV to the CNS during the acute infection phase. A high dose oral SIV challenge was utilized to ensure a successful infection and permit the evaluation of CNS spread during the first 1–14 days post-infection. Ultrasensitive nested PCR was used to detect SIV gag DNA in the brains of macaques at 1–2 days post-infection and identified SIV gag DNA in the brain tissues from three of four macaques. This SIV DNA was also present following perfusion of the macaque brains, providing evidence that it was not residing in the circulating blood but in the brain tissue itself. The diversity of the viral envelope V1–V2 region at early times post-infection indicated that the brain viral variants were similar to variants obtained from lymph nodes. This genetic similarity between SIV obtained from lymphoid and brain tissues suggests that the founder population of viral species entered and subsequently spread without any evidence of brain-specific SIV selection. The relatively rapid appearance of SIV within the CNS tissue following oral transmission may also occur during HIV transmission where it may impact disease course as well as representing a challenge for long-term therapies and future viral eradication modalities.

Immunohistochemical detection of simian immunodeficiency virus (SIV) in rectal mucosa of experimentally infected rhesus macaques (Macaca mulatta)

Experimental simian immunodeficiency virus (SIV) infection is the most appropriate animal model for human HIV infection. Eight male rhesus monkeys (Macaca mulatta) were intravenously or intrarectally infected with SIVmac251/MPBMC to comparatively investigate the distribution and spread of the virus within the rectum during the course of the disease. SIV-positive cells were immunohistochemically detected in rectal biopsies obtained at days 3 and 7 and week 2, 4 and 12 postinfection. SIV-expressing cells were detected for the first time at one week after experimental infection and were present in the lamina propria and lymph follicles. Numbers of positive cells per individual animal varied strongly in time, with a more rapid rise in animals with rapid progression of the disease. Differences were not observed between intravenous and intrarectal infection. Our observations support the significance of the intestinal tract as target organ in initial pathogenesis of SIV infection.

Detection of simian immunodeficiency virus (SIV)-specific T-cell-mediated cytotoxicity in the peripheral blood from infected cynomolgus monkeys.

We have previously demonstrated that peptide immunization restimulates the memory CD4 T-cell response, but fails to induce cytotoxic T lymphocyte (CTL) in cynomolgus macaques. To examine the nature of protective immunity to simian immunodeficiency virus (SIV) in this study, freshly isolated peripheral blood mononuclear cells (PBMC) from four infected juvenile cynomolgus macaques and from three uninfected control macaques were assessed for CTL activity monthly for 9 consecutive months, beginning 1 month after detection of infection. Target cells consisted of major histocompatibility (MHC) haploidentical parental PBMC which were stimulated with mitogen and then pulsed with heat-killed SIVcyn. CTL activity was demonstrated in PBMCs from all four infected animals. The effector cells are T cells which mediate cytotoxicity against SIVcyn-pulsed target cells in an MHC-restricted manner. Furthermore, the cytotoxicity is virus specific and predominantly, if not exclusively, mediated by CD8+ T cells; it is also MHC class I restricted. Incubation of target cells with pepstatin A during antigen pulsing prior to the cytotoxic assay inhibited target cell generation, suggesting that viral antigens are processed via an endocytic pathway.

Detection of simian immunodeficiency virus (SIV)-specific CD8+ T cells in macaques protected from SIV challenge by prior SIV subunit vaccination.

Vaccines for lentiviruses would ideally induce in the host complete resistance to infection of host cells. However, such sterilizing immunity may be neither readily achievable nor absolutely necessary to provide protection from exposure to the immunodeficiency viruses. To examine the nature of protective immunity to simian immunodeficiency virus (SIV), we studied three macaques that had been immunized with a recombinant vaccinia virus-based SIV subunit vaccine regimen and exhibited protection from a challenge with cell-free SIV (MNE) as determined by viral cultures, serology, and PCR for viral genomes. Peripheral blood mononuclear cells were obtained from the protected macaques and analyzed for CD8+ cytotoxic T-lymphocyte (CTL) responses to SIV proteins. CTL reactive to SIV proteins not included in the subunit vaccine, and thus to which these animals had not been exposed prior to challenge, were detected postchallenge in the vaccine-protected animals and persisted for up to 1 year. These CTL, as reflected by studies of cytolytic lines and derived T-cell clones, were CD8+, did not recognize allogeneic targets, and recognized the SIV proteins in the context of class I major histocompatibility complex molecules. The frequency of precursor CD8+ CTL reactive to SIV proteins was determined by limiting-dilution analysis and demonstrated that the responses elicited following challenge of protected animals to SIV proteins not present in the vaccine were quantitatively similar to those of animals persistently infected with SIV. The presence of these CD8+ CTL responses to SIV proteins present only in the challenge virus suggests that infection of some host cells occurred postchallenge. These results suggest that the development of a low level of SIV infection following exposure of vaccinated hosts to SIV does not preclude protection from lethal SIV disease by vaccine-induced immunity.

The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo

Polymerase chain reaction based assays, which amplify a region of the gag gene, have been developed for the direct detection of simian immunodeficiency virus (SIV) DNA sequences in the blood of experimentally infected cynomolgus macaques. In macaques infected with a characterised virus pool (11/88 pool SIVmac 32H), an assay employing a single round of amplification was found to be highly sensitive and specific. However, in animals infected with the SIV molecular clones J5 and C8 (Rud et al., J. Gen. Virol. 75, 529-543), it was necessary to use two rounds of amplification and nested primer pairs in order to achieve sensitivity > 90%. In order to differentiate macaques infected with either of the two genetically distinct SIV clones, J5 or C8, a third PCR based assay has been developed, which amplifies a 492 bp region of the nef gene. Sequence differences between the nef genes of the two molecular clones enabled the PCR product amplified from each virus to be distinguished by restriction analysis. These sensitive and specific assays complement virological detection of SIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

Method for detection of simian immunodeficiency virus neutralizing antibodies using a noncommercial antigen capture enzyme-linked immunosorbent assay.

A neutralization test (NT) using a noncommercial antigen capture enzyme-linked immunosorbent assay (ELISA) to detect simian immunodeficiency virus (SIV) growth in vitro was developed. The capture antibody was a mixture of purified macaque anti-SIV immunoglobulin G (IgG) and a monoclonal antibody to SIV p27. Captured antigens were detected by using purified macaque anti-SIV IgG conjugated to horseradish peroxidase. The NT reliably and sensitively detected differences when various amounts of SIV were used with positive and negative control macaque sera. Dilutions of sequential sera from a macaque (Macaca nemestrina) that had been experimentally infected with SIV were tested for neutralizing antibody with 300 50% tissue culture infective doses of SIV. In this macaque, neutralizing activity and anti-SIV IgG levels in serum (detected by ELISA) increased with time after SIV inoculation, and high IgG titers were required in serum before neutralization occurred in vitro. This simple NT, which detects the presence of SIV serum neutralizing antibodies at a low cost, will be useful for investigating the role of neutralizing antibodies in the SIV-infected macaque model for AIDS.

Detection of simian immunodeficiency virus RNA from infected rhesus macaques by the polymerase chain reaction

A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIV mac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 × 10 5 PBMC of two animals.

The use of the polymerase chain reaction for the detection of Simian immunodeficiency virus in experimentally infected macaques

A rapid, non-radioactive assay for the detection of proviral Simian immunode-ficiency virus (SIV) in tissue-culture cells is described. The assay is based on the co-amplification of the SIV env and gag genes by the polymerase chain reaction (PCR). When the gag PCR product is blotted onto a nylon membrane and hybridised to a radioactive oligonucleotide probe, the assay can also be used to detect the SIV gag gene in DNA isolated directly from experimentally infected cynomolgus macaque lymphocytes. This provides a valuable assay for the presence of proviral SIV during animal trials of AIDS vaccines and chemotherapeutics.