Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo

Division of Retrovirology, NIBSC, South Mimms, Potters Bar, Hertfordshire, UK.
Journal of Medical Primatology (Impact Factor: 0.82). 07/2007; 36(3):131-42. DOI: 10.1111/j.1600-0684.2007.00224.x
Source: PubMed


A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo.
SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo.
These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure.
These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.


Available from: Richard Stebbings
  • Source
    • "Immunohistochemical analyses were performed as previously described [64]. Unmasking of antigens to allow binding of the antibody was undertaken by the optimal technique for each combination of antibody and antigen. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.
    PLoS ONE 08/2014; 9(8):e104390. DOI:10.1371/journal.pone.0104390 · 3.23 Impact Factor
  • Source
    • "A cocktail of either three probes normal to or three probes complementary with SIV transcripts were used within the hybridisation mix as described previously (Canto-Nogues et al. 2001). After extensive washing, bound probes were detected using an alkaline phosphatise/BCIP/NBT chromagenic reaction (Roche, Lewes, UK) for 2 h at room temperature (Ferguson et al. 2007). Sections were washed, counter stained with neutral red, air-dried and mounted in Loctite Super Glue (Denton 1987). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The neuropathology of simian immunodeficiency (SIV) infection in cynomolgus macaques (Macaca fascicularis) was investigated following infection with either T cell tropic SIVmacJ5, SIVmacC8 or macrophage tropic SIVmac17E-Fr. Formalin fixed, paraffin embedded brain tissue sections were analysed using a combination of in situ techniques. Macaques infected with either wild-type SIVmacJ5 or neurovirulent SIVmac17E-Fr showed evidence of neuronal dephosphorylation, loss of oligodendrocyte and CCR5 staining, lack of microglial MHC II expression, infiltration by CD4⁺ and CD8⁺ T cells and mild astrocytosis. SIVmacJ5-infected animals exhibited activation of microglia whilst those infected with SIVmac17E-Fr demonstrated a loss of microglia staining. These results are suggestive of impaired central nervous system (CNS) physiology. Furthermore, infiltration by T cells into the brain parenchyma indicated disruption of the blood brain barrier (BBB). Animals infected with the Δnef-attenuated SIVmacC8 showed microglial activation and astrogliosis indicative of an inflammatory response, lack of MHC II and CCR5 staining and infiltration by CD8⁺ T cells. These results demonstrate that the SIV infection of cynomolgus macaque can be used as a model to replicate the range of CNS pathologies observed following HIV infection of humans and to investigate the pathogenesis of HIV associated neuropathology.
    Journal of NeuroVirology 03/2012; 18(2):100-12. DOI:10.1007/s13365-012-0084-3 · 2.60 Impact Factor
  • Source
    • "Immunohistochemical analysis was performed as previously described [82]. Briefly formaldehyde fixed, paraffin embedded tissue sections were de-waxed, and re-hydrated before being incubated with 50 μg/ml proteinase K (Roche Products Ltd., Welwyn Garden City, UK) in PBS pH7.4 for 15 minutes at 37°C to unmask target antigens followed by immuno-labelling with anti-CCR5 (3A9, BD Biosciences, Oxford, UK). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vaccination with live attenuated SIV can protect against detectable infection with wild-type virus. We have investigated whether target cell depletion contributes to the protection observed. Following vaccination with live attenuated SIV the frequency of intestinal CD4+CCR5+ T cells, an early target of wild-type SIV infection and destruction, was determined at days 3, 7, 10, 21 and 125 post inoculation. In naive controls, modest frequencies of intestinal CD4+CCR5+ T cells were predominantly found within the LPL TTrM-1 and IEL TTrM-2 subsets. At day 3, LPL and IEL CD4+CCR5+ TEM cells were dramatically increased whilst less differentiated subsets were greatly reduced, consistent with activation-induced maturation. CCR5 expression remained high at day 7, although there was a shift in subset balance from CD4+CCR5+ TEM to less differentiated TTrM-2 cells. This increase in intestinal CD4+CCR5+ T cells preceded the peak of SIV RNA plasma loads measured at day 10. Greater than 65.9% depletion of intestinal CD4+CCR5+ T cells followed at day 10, but overall CD4+ T cell homeostasis was maintained by increased CD4+CCR5- T cells. At days 21 and 125, high numbers of intestinal CD4+CCR5- naive TN cells were detected concurrent with greatly increased CD4+CCR5+ LPL TTrM-2 and IEL TEM cells at day 125, yet SIV RNA plasma loads remained low. This increase in intestinal CD4+CCR5+ T cells, following vaccination with live attenuated SIV, does not correlate with target cell depletion as a mechanism of protection. Instead, increased intestinal CD4+CCR5+ T cells may correlate with or contribute to the protection conferred by vaccination with live attenuated SIV.
    Retrovirology 02/2011; 8(1):8. DOI:10.1186/1742-4690-8-8 · 4.19 Impact Factor
Show more