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Vaccination with live attenuated simian immunodeficiency virus (SIV) in non-human primate species provides a means of characterizing the protective processes of retroviral superinfection and may lead to novel advances of human immunodeficiency virus (HIV)/AIDS vaccine design. The minimally attenuated SIVmacC8 vaccine has been demonstrated to elicit early potent protection against pathogenic rechallenge with genetically diverse viral isolates in cynomolgus macaques (Macaca fascicularis). In this study, we have characterized further the biological breadth of this vaccine protection by assessing the ability of both the nef-disrupted SIVmacC8 and its nef-intact counterpart SIVmacJ5 viruses to prevent superinfection with the macrophage/neurotropic SIVmac239/17E-Fr (SIVmac17E-Fr) isolate. Inoculation with either SIVmacC8 or SIVmacJ5 and subsequent detailed characterization of the viral replication kinetics revealed a wide range of virus-host outcomes. Both nef-disrupted and nef-intact immunizing viruses were able to prevent establishment of SIVmac17E-Fr in peripheral blood and secondary lymphoid tissues. Differences in virus kinetics, indicative of an active process, identified uncontrolled replication in one macaque which although able to prevent SIVmac17E-Fr superinfection led to extensive neuropathological complications. The ability to prevent a biologically heterologous, CD4-independent/CCR5+ viral isolate and the macrophage-tropic SIVmac316 strain from establishing infection supports the hypothesis that direct target cell blocking is unlikely to be a central feature of live lentivirus vaccination. These data provide further evidence to demonstrate that inoculation of a live retroviral vaccine can deliver broad spectrum protection against both macrophage-tropic as well as lymphocytotropic viruses. These data add to our knowledge of live attenuated SIV vaccines but further highlight potential safety concerns of vaccinating with a live retrovirus.

Polymorphism in the TRIM5α/TRIMcyp gene, which interacts with the lentiviral capsid, has been shown to impact on simian immunodeficiency virus (SIV) replication in certain macaque species. Here, in the context of a live-attenuated SIV vaccine study conducted in Mauritian-origin cynomolgus macaques (MCM), we demonstrate upregulation of TRIM5α expression in multiple lymphoid tissues immediately following vaccination. Despite this, the restricted range of TRIM5α genotypes and lack of TRIMcyp variants had no or only limited impact on the replication kinetics in vivo of either the SIVmac viral vaccine or wild-type SIVsmE660 challenge. Additionally, there appeared to be no impact of TRIM5α genotype on the outcome of homologous or heterologous vaccination/challenge studies. The limited spectrum of TRIM5α polymorphism in MCM appears to minimize host bias to provide consistency of replication for SIVmac/SIVsm viruses in vivo, and therefore on vaccination and pathogenesis studies conducted in this species.

Vaccination with live attenuated simian immunodeficiency virus (SIVmacC8) confers potent, reproducible protection against homologous wild-type virus challenge (SIVmacJ5). The ability of SIVmacC8 to confer resistance to superinfection with an uncloned ex vivo derivative of SIVmac251 (SIVmac32H/L28) was investigated. In naïve, Mauritian-derived cynomolgus macaques (Macaca fascicularis), SIVmac32H/L28 replicated to high peak titres (>10(8) SIV RNA copies ml(-1)), persisted at high levels and induced distinctive pathology in lymphoid tissues. In cynomolgus macaques vaccinated with SIVmacC8, no evidence of detectable superinfection was observed in 3/8 vaccinates following challenge 3 or 20 weeks later with SIVmac32H/L28. Analyses after SIVmac32H/L28 challenge revealed a significant reduction in viral RNA (P<0.001) and DNA levels between 20 week vaccinates and challenge controls. Amongst 3 week vaccinates, less potent protection was observed. However, analysis of env from breakthrough virus indicated >99% sequence similarity with the vaccine virus. Highly sensitive PCR assays that distinguish vaccine and challenge virus stocks demonstrated restimulation of replication of the vaccine virus SIVmacC8 in the face of potent protection against a vigorous, homologous challenge virus. Vaccine-induced antiviral neutralizing antibodies and anti-Nef CD8+ cytotoxic T cell responses did not correlate with the outcome of the challenge. Defining the mechanism of vaccine protection will need to account for the effective control of a genetically closely related challenge virus whilst remaining unable to suppress replication of the pre-existing vaccine virus. The role of innate and intrinsic anti-retroviral immunity in the protection conferred by live attenuated SIV vaccines warrants careful study.

To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

Human immunodeficiency virus type 2 (HIV-2) infections cause severe immunodeficiency in humans, although HIV-2 is associated frequently with reduced virulence and pathogenicity compared to HIV-1. Genetic determinants that play a role in HIV pathogenesis are relatively poorly understood but nef has been implicated in inducing a more pathogenic phenotype in vivo. However, relatively little is known about the role of nef in HIV-2 pathogenesis. To address this, the genetic composition of 44 nef alleles from 37 HIV-2-infected individuals in Portugal, encompassing a wide spectrum of disease associations, CD4 counts and virus load, has been assessed. All nef alleles were subtype A, with no evidence of gross deletions, truncations or disruptions in the nef-encoding sequence; all were full-length and intact. HIV-2 long terminal repeat sequences were conserved and also indicated subtype A infections. Detailed analysis of motifs that mediate nef function in HIV-1 and simian immunodeficiency virus, such as CD4 downregulation and putative SH2/SH3 interactions, revealed significant natural variation. In particular, the central P(104)xxPLR motif exhibited wide interpatient variation, ranging from an HIV-1-like tetra-proline structure (PxxP)(3) to a disrupted minimal core motif (P(104)xxQLR). The P(107)-->Q substitution was associated with an asymptomatic phenotype (Fisher's exact test, P=0.026) and low virus loads. These data indicate that discrete differences in the nef gene sequence rather than gross structural changes are more likely to play a role in HIV-2 pathogenesis mediated via specific functional interactions.

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID(50) of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0.05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.

The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.