Viral load does not correlate with CD8 T cell efficacy in vitro.

<p>A) Plasma viral loads were plotted against the percentage of p27+ cells after eight days in culture in wells where cells were not superinfected in vitro. These results display the growth of autologous virus. B) Viral loads were plotted against the normalized percent suppression on autologous target cells. C) Viral loads were plotted against the percentage of target cells that were p27+ after in vitro superinfection in the no effector control wells. D) The normalized % suppresion was plotted against the percentage of p27+ targets in the no infection control wells. E) Normalized percent suppression was plotted against the percent of p27+ targets in the no effector control wells. F) The percent of p27+ targets in the no infection wells was plotted against the percent of p27+ targets in the no effector control wells.</p

Viral suppression by effector cells from homozygous animals.

<p>A) Each plot represents the results from an individual M3/M3 effector animal on each target animal. Each column in the graph displays a different target animal haplotype. B) Table showing the Tukey’s Multiple Comparison Test of the columns in each of the graphs (*p<0.05; **p<0.01; ***p<0.001). C) Each plot represents the results from an individual M1/M1 effector animal on each target animal. Each column in the graph displays a different target animal haplotype. D) Table showing the Tukey’s Multiple Comparison Test of the columns in each of the graphs (*p<0.05; **p<0.01; ***p<0.001).</p

Viral suppression by effector cells from heterozygous animals.

<p>A) Each plot represents the results from an individual M1/M3 effector animal on each target animal. Each column in the graph displays a different target animal haplotype. B) Table showing the Tukey’s Multiple Comparison Test of the columns in each of the graphs (*p<0.05; **p<0.01; ***p<0.001).</p

Allogeneic transfer cell analysis.

<p>A PKH67+ PBMC from recipient MCM3 were sorted from total PBMC by flow cytometry. We performed microsatellite analysis to demonstrate that the mircosatellite profile of PKH67+ cells in the recipient animal matched the microsatellite profile of donor MCM1.</p

<i>In vitro</i> histocompatibility of MHC-matched MCM.

<p>A Microsatellite analysis was used to identify 2-MHC-haplotype-matched, 1-MHC-haplotype-matched, and 0-MHC-haplotype-matched MCM. MHC haplotypes inferred by microsatellite typing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002384#pone.0002384-Wiseman1" target="_blank">[35]</a> are colored to show matching between individuals. Predicted MHC class I and class II alleles for each haplotype are shown. We identified 8 MCM for use in these experiments. B Results from mixed lymphocyte reactions. Whole PBMC from MCM6 was labelled with fluorescent dye PKH67 and incubated with irradiated stimulator cells as indicated above each plot. Cells that divide after recognizing non-self antigen lose fluorescence as indicated by a decrease along the y-axis. Dot plots are gated on lymphocytes according to forward and side scatter.</p

Autologous adoptive transfer.

<p>A Diagram displaying the transfer protocol. B Selection for CD8ß+ cells. The left panel is unmanipulated PBMC. The right panel is the positive fraction collected following CD8ß enrichment gated on lymphocytes. C Transfused cell persistence after transfer. Percentages are of total CD8ß+ cells in the PBMC of the animal based on the lymphocyte gate. D PKH67+ cells in lymph node of animal MCM6. The right inguinal lymph node was processed three days post-transfer to measure persistence of PKH67+ cells. The percentage of PKH67+ cells is based on total CD8ß+ cells in the lymph node lymphocyte gate.</p

Allogeneic adoptive transfer from MCM1 to MCM3, MCM4, and MCM5.

<p>Gating strategy is described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002384#pone-0002384-g002" target="_blank">Figure 2</a>. A Diagram displaying the transfer protocol. B Donor CD8ß+ cells were transferred from MCM1 into 2-MHC-haplotype-matched MCM3, 0-MHC-haplotype-matched MCM4, and 1-MHC-haplotype-matched MCM5 and monitored for persistence in recipient PBMC. 220 million CD8ß+ were transferred to each animal. Donor cells were a composite of CD8ß+ cells enriched from PBMC, mesenteric lymph node and spleen. C Left inguinal lymph node sample 9 days post-transfer.</p

Allogeneic transfer from MCM1 to MCM2.

<p>A Diagram displaying the transfer protocol. B PKH67+ donor cells from MCM1 in PBMC of recipient 2-MHC-haplotype-matched MCM2. Dot plots are gated on lymphocytes and display CD8ß+ lymphocytes only. C Right Axillary lymph node sample 10 days post-transfer.</p

Lymphocytes traffic to peripheral lymphoid tissues after adoptive transfer.

<p>A Diagram displaying the transfer protocol. B Donor bulk PBMC were transferred from MCM7 into MCM6. MCM6 was euthanized 24 hours post transfer and its tissues were processed. Cells selectively trafficked to different peripheral lymphoid organs post-transfer. C Immunohistochemical staining of a lymph node section showing PKH67+ cells (green), CD20 antibody staining (red) and CD8 antibody staining (blue) collected with a confocal microscope at 200X.</p

Vaccination against Endogenous Retrotransposable Element Consensus Sequences Does Not Protect Rhesus Macaques from SIVsmE660 Infection and Replication

<div><p>The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically <i>in vivo</i> following infection with SIVsmE660 and did not recognize SIV-infected targets <i>in vitro</i>, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective <i>TRIM5</i>α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.</p></div