Expansion of Simian Immunodeficiency Virus (SIV)-specific CD8 T cell lines from SIV-naive Mauritian cynomolgus macaques for adoptive transfer

CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replication in vitro. We further cultured cell lines specific for four SIV-derived epitopes, Nef103-111 RM9, Gag389-394 GW9, Env338-346 RF9, and Nef254-262 LT9. These cell lines were up to 94.4% pure, as determined by major histocompatibility complex (MHC) tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks and trafficked to multiple extralymphoid tissues. However, these cells did not impact the acute-phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naive animals provide a unique model for exploring cellular immunity and the control of SIV infection and facilitate a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir. IMPORTANCE: CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens

Pegivirus avoids immune recognition but does not attenuate acute-phase disease in a macaque model of HIV infection

<div><p>Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis–as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses–suggesting that HPgV’s protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses–an understudied group of viruses with a high prevalence in the global human population–and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.</p></div

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques.

BACKGROUND:Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV. METHODOLOGY/PRINCIPAL FINDINGS:Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form. CONCLUSIONS/SIGNIFICANCE:An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains

SIV pathogenesis in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Peripheral CD4+ T cell counts were obtained by multiplying absolute lymphocyte counts by the percentage of lymphocytes that were CD3+ CD4+ CD20- CD8- (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006692#ppat.1006692.g003" target="_blank">Fig 3</a> for gating strategy details). (<b>B</b>) Gut CD4+ T cells were stained within sections of colonic tissues via IHC with an anti-CD4 antibody and manually quantified. Significant differences between the SIV-only and SPgV+SIV groups were analyzed using a two-tailed unpaired t-test (solid line) with error bars representing SEM. Significant changes in all animals over the course of acute SIV infection were quantified using a two-tailed paired t-test (dashed line). (<b>C</b>) A representative set of colonic tissue from Cy0883 (SIV+SPgV) and Cy0887 (SIV-only) are shown pre and post SIV infection at 400x for comparison. Arrows highlight representative cells with membranous CD4 staining.</p

Immune activation following SPgV vs. SIV infection.

<p>(<b>A-C</b>) Fresh whole blood was used for analysis by flow cytometry at each time point. <i>P</i> values are from a two-tailed paired t-test comparing the average immune activation pre-any-virus-infection to the average of all post-SPgV or post-SIV data points within the first 26 days of infection for each virus for which flow cytometry data was available. (<b>D</b>) Proliferating cells were stained within sections of lymph nodes via IHC with an anti-Ki67 antibody and manually quantified (SPgV: day -8 vs day 24; SIV: day -34 vs day 25). Significant changes over time were quantified using a two-tailed paired t-test. (<b>E</b>) Representative set of lymph node tissue from Cy0885 is shown at 400x.</p

SPgV and SIV viral loads in infected macaques.

<p>Titers for each virus were measured from plasma using highly sensitive virus-specific quantitative RT-PCR assays. (<b>A</b>) SPgV titers in the four macaques infected with SPgV+SIV. (<b>B</b>,<b>C</b>,<b>D</b>) SIV titers in four macaques infected with SPgV+SIV (green) and four macaques infected with SIV-only (black). <i>P</i> values reflect a two-tailed unpaired t-test and error bars represent SEM. The symbols used for each animal in this figure are consistent throughout the manuscript.</p

Peripheral immune activation in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Flow cytometry gating strategy used for defining immune cell subsets. Fresh whole blood was used for staining and flow cytometry at each time point. (<b>B-D</b>) Activation of immune cell subsets. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM. Note: Cy0886 did not exhibit a distinct peak or nadir of CD69+ Ki67+ expression in the CD3+ CD8+ T cell population, and so is not included in these analyses.</p

SPgV co-infection does not alter recognition of MHC class I restricted SIV epitopes by CD8+ T cells.

<p>Lymph nodes were collected from macaques at 125/126 days post SIV infection and cells were stained for analysis with MHC class I tetramers folded with SIV peptides that are immunodominant on the M3/M4 MHC background. (<b>A</b>) Flow cytometry gating strategy used for defining tetramer-positive CD8+ T cells. (<b>B</b>) Percentage of CD8+ T cells that were positive for each tetramer. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM.</p