Microbiology & Immunology - Research Publications

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    A randomized, placebo-controlled phase I trial of DNA prime, recombinant fowlpox virus boost prophylactic vaccine for HIV-1
    Kelleher, AD ; Puls, RL ; Bebbington, M ; Boyle, D ; Ffrench, R ; Kent, SJ ; Kippax, S ; Purcell, DFJ ; Thomson, S ; Wand, H ; Cooper, DA ; Emery, S (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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    Rotavirus activates JNK and p38 signaling pathways in intestinal cells, leading to AP-1-driven transcriptional responses and enhanced virus replication
    Holloway, G ; Coulson, BS (AMER SOC MICROBIOLOGY, 2006-11)
    Rotavirus infection is known to regulate transcriptional changes in many cellular genes. The transcription factors NF-kappaB and AP-1 are activated by rotavirus infection, but the upstream processes leading to these events are largely unidentified. We therefore studied the activation state during rotavirus infection of c-Jun NH2-terminal kinase (JNK) and p38, which are kinases known to activate AP-1. As assessed by Western blotting using phospho-specific antibodies, infection with rhesus rotavirus (RRV) or exposure to UV-psoralen-inactivated RRV (I-RRV) resulted in the activation of JNK in HT-29, Caco-2, and MA104 cells. Activation of p38 during RRV infection was observed in Caco-2 and MA104 cells but not in HT-29 cells, whereas exposure to I-RRV did not lead to p38 activation in these cell lines. Rotavirus strains SA11, CRW-8, Wa, and UK also activated JNK and p38. Consistent with the activation of JNK, a corresponding increase in the phosphorylation of the AP-1 component c-Jun was shown. The interleukin-8 (IL-8) and c-jun promoters contain AP-1 binding sequences, and these genes have been shown previously to be transcriptionally up-regulated during rotavirus infection. Using specific inhibitors of JNK (SP600125) and p38 (SB203580) and real-time PCR, we showed that maximal RRV-induced IL-8 and c-jun transcription required JNK and p38 activity. This highlights the importance of JNK and p38 in RRV-induced, AP-1-driven gene expression. Significantly, inhibition of p38 or JNK in Caco-2 cells reduced RRV growth but not viral structural antigen expression, demonstrating the potential importance of JNK and p38 activation for optimal rotavirus replication.
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    Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8
    Scott, SA ; Holloway, G ; Coulson, BS ; Szyczew, AJ ; Kiefel, MJ ; von Itzstein, M ; Blanchard, H (INT UNION CRYSTALLOGRAPHY, 2005-06)
    Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 A resolution, enabling the determination of the VP8* structure by molecular replacement.
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    Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8*carbohydrate-binding protein of the human rotavirus strain Wa
    Kraschnefski, MJ ; Scott, SA ; Holloway, G ; Coulson, BS ; von Itzstein, M ; Blanchard, H (INT UNION CRYSTALLOGRAPHY, 2005-11)
    Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3(2)21 and monoclinic P2(1)) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*(64-223) structure by molecular replacement.
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    MHC class I allele frequencies in pigtail macaques of diverse origin
    Pratt, BF ; O'Connor, DH ; Lafont, BAP ; Mankowski, JL ; Fernandez, CS ; Triastuti, R ; Brooks, AG ; Kent, SJ ; Smith, MZ (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.
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    Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost
    Fernandez, CS ; Stratov, I ; De Rose, R ; Walsh, K ; Dale, CJ ; Smith, MZ ; Agy, MB ; Hu, SL ; Krebs, K ; Watkins, DI ; O'Connor, DH ; Davenport, MP ; Kent, SJ (AMER SOC MICROBIOLOGY, 2005-05)
    Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.
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    CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells
    Zaunders, JJ ; Dyer, WB ; Munier, ML ; Ip, S ; Liu, J ; Amyes, E ; Rawlinson, W ; De Rose, R ; Kent, SJ ; Sullivan, JS ; Cooper, DA ; Kelleher, AD (AMER SOC MICROBIOLOGY, 2006-10)
    The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.
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    Growth of rotaviruses in primary pancreatic cells
    Coulson, BS ; Witterick, PD ; Tan, Y ; Hewish, MJ ; Mountford, JN ; Harrison, LC ; Honeyman, MC (AMER SOC MICROBIOLOGY, 2002-09)
    Rotavirus infection in children at risk of developing type 1 diabetes has been temporally associated with development of pancreatic islet autoantibodies. In this study, nonobese diabetic mice were shown to be susceptible to rhesus rotavirus infection and pancreatic islets from nonobese diabetic mice, nonobese diabetes-resistant mice, fetal pigs, and macaque monkeys supported various degrees of rotavirus growth. Human rotaviruses replicated in monkey islets only. This islet susceptibility shows that rotavirus infection of the pancreas in vivo might be possible.
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    Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice
    Johnson, BJ ; Costelloe, EO ; Fitzpatrick, DR ; Haanen, JBAG ; Schumacher, TNM ; Brown, LE ; Kelso, A (NATL ACAD SCIENCES, 2003-03-04)
    Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8(+) CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-gamma mRNAs in CD8(+) populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-gamma in activated CD8(+) cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8(+) cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-gamma mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8(+) T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.