Microbiology & Immunology - Research Publications
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ItemComplexity of the Inoculum Determines the Rate of Reversion of SIV Gag CD8 T Cell Mutant Virus and Outcome of Infection(PUBLIC LIBRARY SCIENCE, 2009-04)Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively "fit" compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing > or =10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIV(mn229) viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIV(mac239), demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until > or =8 days after inoculation and took > or =8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection.
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ItemNatural Host Genetic Resistance to Lentiviral CNS Disease: A Neuroprotective MHC Class I Allele in SIV-Infected Macaques(PUBLIC LIBRARY SCIENCE, 2008-11-03)Human immunodeficiency virus (HIV) infection frequently causes neurologic disease even with anti-retroviral treatment. Although associations between MHC class I alleles and acquired immunodeficiency syndrome (AIDS) have been reported, the role MHC class I alleles play in restricting development of HIV-induced organ-specific diseases, including neurologic disease, has not been characterized. This study examined the relationship between expression of the MHC class I allele Mane-A*10 and development of lentiviral-induced central nervous system (CNS) disease using a well-characterized simian immunodeficiency (SIV)/pigtailed macaque model. The risk of developing CNS disease (SIV encephalitis) was 2.5 times higher for animals that did not express the MHC class I allele Mane-A*10 (P = 0.002; RR = 2.5). Animals expressing the Mane-A*10 allele had significantly lower amounts of activated macrophages, SIV RNA, and neuronal dysfunction in the CNS than Mane-A*10 negative animals (P<0.001). Mane-A*10 positive animals with the highest CNS viral burdens contained SIV gag escape mutants at the Mane-A*10-restricted KP9 epitope in the CNS whereas wild type KP9 sequences dominated in the brain of Mane-A*10 negative animals with comparable CNS viral burdens. These concordant findings demonstrate that particular MHC class I alleles play major neuroprotective roles in lentiviral-induced CNS disease.
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ItemControl of viremia and prevention of AIDS following immunotherapy of SIV-infected macaques with peptide-pulsed blood(PUBLIC LIBRARY SCIENCE, 2008-05)Effective immunotherapies for HIV are needed. Drug therapies are life-long with significant toxicities. Dendritic-cell based immunotherapy approaches are promising but impractical for widespread use. A simple immunotherapy, reinfusing fresh autologous blood cells exposed to overlapping SIV peptides for 1 hour ex vivo, was assessed for the control of SIV(mac251) replication in 36 pigtail macaques. An initial set of four immunizations was administered under antiretroviral cover and a booster set of three immunizations administered 6 months later. Vaccinated animals were randomized to receive Gag peptides alone or peptides spanning all nine SIV proteins. High-level, SIV-specific CD4 and CD8 T-cell immunity was induced following immunization, both during antiretroviral cover and without. Virus levels were durably approximately 10-fold lower for 1 year in immunized animals compared to controls, and a significant delay in AIDS-related mortality resulted. Broader immunity resulted following immunizations with peptides spanning all nine SIV proteins, but the responses to Gag were weaker in comparison to animals only immunized with Gag. No difference in viral outcome occurred in animals immunized with all SIV proteins compared to animals immunized against Gag alone. Peptide-pulsed blood cells are an immunogenic and effective immunotherapy in SIV-infected macaques. Our results suggest Gag alone is an effective antigen for T-cell immunotherapy. Fresh blood cells pulsed with overlapping Gag peptides is proceeding into trials in HIV-infected humans.
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ItemVaccination and timing influence SIV immune escape viral dynamics in vivo(PUBLIC LIBRARY SCIENCE, 2008-01)CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag164-172 KP9 wild-type (WT) and escape mutant (EM) variants in pigtail macaques. Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder (lacking the restricting Mane-A*10 allele) macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.
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ItemNo Preview AvailableA Protective Vaccine Delivery System for In Vivo T Cell Stimulation Using Nanoengineered Polymer Hydrogel Capsules(AMER CHEMICAL SOC, 2009-11)Successful delivery of labile vaccine antigens, such as peptides and proteins, to stimulate CD4 and CD8 T cell immunity could improve vaccine strategies against chronic infections such as HIV and Hepatitis C. Layer-by-layer (LbL)-assembled nanoengineered hydrogel capsules represent a novel and promising technology for the protection and delivery of labile vaccine candidates to antigen-presenting cells (APCs). Here we report on the in vitro and in vivo immunostimulatory capabilities of LbL-assembled disulfide cross-linked poly(methacrylic acid) (PMA(SH)) hydrogel capsules as a delivery strategy for protein and peptide vaccines using robust transgenic mice models and ovalbumin (OVA) as a model vaccine. We demonstrate that OVA protein as well as multiple OVA peptides can be successfully encapsulated within nanoengineered PMA(SH) hydrogel capsules. OVA-containing PMA(SH) capsules are internalized by mouse APCs, resulting in presentation of OVA epitopes and subsequent activation of OVA-specific CD4 and CD8 T cells in vitro. OVA-specific CD4 and CD8 T cells are also activated to proliferate in vivo following intravenous vaccination of mice with OVA protein- and OVA peptide-loaded PMA(SH) hydrogel capsules. Furthermore, we show that OVA encapsulated within the PMA(SH) capsules resulted in at least 6-fold greater proliferation of OVA-specific CD8 T cells and 70-fold greater proliferation of OVA-specific CD4 T cells in vivo compared to the equivalent amount of OVA protein administered alone. These results highlight the potential of nanoengineered hydrogel capsules for vaccine delivery.
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ItemNo Preview AvailableP17-03. Nanoengineered layer-by-layer capsules as a novel delivery system for HIV vaccines(Springer Science and Business Media LLC, 2009-10-22)
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ItemThe testis and epididymis are productively infected by SIV and SHIV in juvenile macaques during the post-acute stage of infection(BMC, 2007-01-31)BACKGROUND: Little is known about the progression and pathogenesis of HIV-1 infection within the male genital tract (MGT), particularly during the early stages of infection. RESULTS: To study HIV pathogenesis in the testis and epididymis, 12 juvenile monkeys (Macacca nemestrina, 4-4.5 years old) were infected with Simian Immunodeficiency Virus mac 251 (SIVmac251) (n = 6) or Simian/Human Immunodeficiency Virus (SHIVmn229) (n = 6). Testes and epididymides were collected and examined by light microscopy and electron microscopy, at weeks 11-13 (SHIV) and 23 (SIV) following infection. Differences were found in the maturation status of the MGT of the monkeys, ranging from prepubertal (lacking post-meiotic germ cells) to post-pubertal (having mature sperm in the epididymal duct). Variable levels of viral RNA were identified in the lymph node, epididymis and testis following infection with both SHIVmn229 and SIVmac251. Viral protein was detected via immunofluorescence histochemistry using specific antibodies to SIV (anti-gp41) and HIV-1 (capsid/p24) protein. SIV and SHIV infected macrophages, potentially dendritic cells and T cells in the testicular interstitial tissue were identified by co-localisation studies using antibodies to CD68, DC-SIGN, alphabetaTCR. Infection of spermatogonia, but not more mature spermatogenic cells, was also observed. Leukocytic infiltrates were observed within the epididymal stroma of the infected animals. CONCLUSION: These data show that the testis and epididymis of juvenile macaques are a target for SIV and SHIV during the post-acute stage of infection and represent a potential model for studying HIV-1 pathogenesis and its effect on spermatogenesis and the MGT in general.
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ItemA randomized, placebo-controlled phase I trial of DNA prime, recombinant fowlpox virus boost prophylactic vaccine for HIV-1(LIPPINCOTT WILLIAMS & WILKINS, 2006-01-09)An HIV-vaccine consisting of a DNA prime, recombinant fowlpox virus (rFPV) boost was evaluated in a double-blind placebo controlled trial. One milligram of pHIS-HIV-B expressing mutated gag, pol, env, vpu, tat and rev was administered at weeks 0 and 4 boosted by 5 x 10(7) pfu rFPV-HIV-B expressing gag/pol at week 8. The vaccine regimen was safe, but there was no difference between vaccine (n = 18) and placebo recipients (n = 6) for Gag or Pol-specific T-cell immune responses at week 9.
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ItemSubtype AE HIV-1 DNA and recombinant Fowlpoxvirus vaccines encoding five shared HIV-1 genes: safety and T cell immunogenicity in macaques(ELSEVIER SCI LTD, 2005-03-14)To induce broad T cell immunity to HIV-1, we evaluated the safety, immunogenicity and dose-response relationship of DNA and recombinant Fowlpoxvirus (rFPV) vaccines encoding five shared HIV subtype AE genes (Gag, Pol, Env, Tat, Rev) in pigtail macaques. The DNA (three doses of either 1 mg or 4.5 mg) and rFPV (a single boost of either 5 x 10(7) or 2 x 10(8) plaque forming units) vaccines were administered intramuscularly without adjuvants. Broadly reactive HIV-specific T cell immunity was stimulated by all doses of the vaccines administered, without significant differences between the high and low doses studied. The vaccines induced both CD4 and CD8 T cell responses to Gag, Pol, Env and Tat/Rev proteins, with CD4 T cell responses being greater in magnitude than CD8 T cell responses. The vaccine-induced T cell responses had significant cross-recognition of heterologous HIV-1 proteins from non-AE HIV-1 subtypes. In conclusion, these subtype AE HIV-1 DNA and rFPV vaccines were safe, induced broad T-cell immunity in macaques, and are suitable for progression into clinical trials.
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ItemMHC class I allele frequencies in pigtail macaques of diverse origin(SPRINGER, 2006-12)Pigtail macaques (Macaca nemestrina) are an increasingly common primate model for the study of human AIDS. Major Histocompatibility complex (MHC) class I-restricted CD8(+) T cell responses are a critical part of the adaptive immune response to HIV-1 in humans and simian immunodeficiency virus (SIV) in macaques; however, MHC class I alleles have not yet been comprehensively characterized in pigtail macaques. The frequencies of ten previously defined alleles (four Mane-A and six Mane-B) were investigated in detail in 109 pigtail macaques using reference strand-mediated conformational analysis (RSCA). The macaques were derived from three separate breeding colonies in the USA, Indonesia and Australia, and allele frequencies were analysed within and between these groups. Mane-A*10, an allele that restricts the immunodominant SIV Gag epitope KP9, was the most common allele, present in 32.1% of the animals overall, with similar frequencies across the three cohorts. Additionally, RSCA identified a new allele (Mane-A*17) common to three Indonesian pigtail macaques responding to the same Gag CD8(+) T cell epitope. This broad characterization of common MHC class I alleles in more than 100 pigtail macaques further develops this animal model for the study of virus-specific CD8(+) T cell responses.