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

Analysis of ERE mRNA expression in SIV-infected versus uninfected CD4+ T cells.

<p>[<b>A</b>] Flow cytometry analysis staining for SIV Gag p27 and CD4 of uninfected or SIVsmE660-infected CD4+ T cells used for subsequent qPCR analysis [72 h time point is shown]. [<b>B</b>] qPCR analysis of mRNAs of interest in proportion to the housekeeping gene TBP, confirmed SIV infection [top panel] which declined sharply after 72 hours concomitant with a decline in viability of the cell culture. The mRNA levels of the ERE genes of interest were not significantly increased with the exception of SERV-K Env as detected by narrow specificity primers [bottom panel] which showed significant but low level elevation at 72 h. The mean and range at each time point are shown. [<b>C</b>] Summary of the qPCR panel by mean and range fold change compared to uninfected cells. Following correction for multicomparisons only the 2.9-fold increase in mRNA of SERV-K Env as detected by narrow specificity primers and the 1.4-fold decrease in SRVmac Gag remained significant.</p

Vaccine induced LINE1 ORF2-, SERV-K Gag-, and SERV-K Env-specific T cells do not recognize SIV-infected cells in vitro.

<p>[<b>A</b>] An <i>in vitro-</i>generated CD4+ T cell line specific for SERV-K Env_{667–681/671–685} NK15/FN15 does not respond to SIV-infected macrophages. [<b>B</b>] SERV-K Env_25–33 LM9-specific CD8+ T cells do not respond to SIV-infected CD4+ T cells. [<b>C</b>] LINE1 ORF2_221–229 RL9-specific CD8+ T cells do not respond to SIV-infected CD4+ T cells [<b>D</b>] SERV-K Gag_376–383 IL8-specific CD8+ T cells do not respond to SIV-infected CD4+ T cells regardless of the cytokine readout [IFN-γ, TNF-α, or CD107a]. Results are indicative of targets infected with either SIVmac239 or SIVsmE660, except for panel A, which is indicative of both SIVsmE660 and SIVmac316E [a macrophage tropic variant of SIVmac239]. Dot plots were generated by gating on CD3+ CD4+ T cells [panel A] or CD3+ CD8+ T cells [panels B-D]. Percentages are indicative of cytokine producing cells. Exogenous peptide antigen was included as a positive control in all recognition assays.</p

Effect of TRIMα on SIVsmE660 acquisition and replication.

<p>Kaplan-Meyer curve analysis of the effect TRIM5α had on the rate of acquisition of SIVsmE660 infection after repeated limiting-dose intrarectal challenge. The statistical significance of the rate of infection was determined by log rank test. Note that animal r99080 was the only animal with a susceptible phenotype based on TRIM5α and therefore was excluded from all TRIM5α analysis. [<b>B</b>] Comparison of the number of challenges needed to productively infect animals with SIVsmE660 based on the presence of resistant TRIM5α alleles. The statistical significance of the number of challenges required between the groups was performed by generalized gamma model. [<b>C</b>] The geometric mean of the viral loads of each TRIM5α group is shown. A statistically different value was observed between the groups as measured by the area under of the curve. The statistical difference between the groups in area under of curve was performed by one-way ANOVA. Note that animal r07045 was never infected and is, therefore, excluded from this analysis.</p

ERE vaccine-induced T cells do not expand <i>in vivo</i> following SIVsmE660 infection.

<p>Vaccine induced T cell responses detected in ELISPOT above the threshold of 50 IFN-γ spot forming cells (SFCs) at two weeks prior to SIV infection were tracked for the first six weeks post infection and are shown for animals [<b>A</b>] r07015, [<b>B</b>] r99047, and [<b>C</b>] r99080 from vaccine group one and [<b>D</b>] rh1999, [<b>E</b>] r05040, and [<b>F</b>] r99079 from vaccine group two. Similar results were obtained for the remaining animals in both groups. The results shown indicate the mean plus standard deviation of duplicate wells for the indicated peptide pools with the background level subtracted. Time from last ERE vaccination [Ad5 for group 1, DNA for group 2] is indicated at the top of each graph.</p