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

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

Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag, Nef, and Vif in Indian Rhesus Macaques

<div><p>An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated <em>in vivo,</em> but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8⁺ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4⁺ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D – a clinically relevant vaccine vector – can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.</p> </div

Magnitude of SIV-specific T-cell responses in animals vaccinated with single rYF17D/SIV constructs.

<p>We carried out IFN-γ ELISPOT at days 14 (white bars) and 17 (black bars) after the rYF17D vaccination using peptide pools (ten 15mers overlapping by 11 amino acids in each pool) spanning the regions of SIVmac239 Gag, Vif, and Nef encoded in each rYF17D vector. We adjusted these pools for each macaque depending on the region and SIV protein expressed by the rYF17D viruses. Bar graphs indicate the magnitude of IFN-γ-producing cells in PBMC (SFC/10⁶ PBMC) in macaques immunized with rYF17D/Gag constructs (A), rYF17D/Vif constructs (B), and the rYF17D/Nef construct (C). Responses measured at days 14 and 17 following vaccination with the rYF17D/SIV vectors are shown by white and black bars, respectively. Of note, the vaccine candidates rYF17D/Gag(44–84), rYF17D/Vif(1–110), and rYF17D/Nef(45–210) encode three Mamu-A*02-restricted, CD8⁺ T-cell epitopes: Gag_71–79GY9, Vif_97–104WY8, and Nef_159–167YY9, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Loffredo2" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Vogel1" target="_blank">[51]</a>. Since the recipients of these vectors were <i>Mamu-A*02⁺</i> (r04147, r05089, and r05070; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone-0054434-t001" target="_blank">Table 1</a>), we used minimal optimal peptides in their IFN-γ ELISPOT assays to determine the frequency of CD8⁺ T-cells recognizing the Gag_71–79GY9, Vif_97–104WY8, and Nef_159–167YY9 epitopes. We also assessed vector-specific cellular responses by using synthetic peptides corresponding to four Mamu-A*01-restricted CD8⁺ T-cell epitopes in the backbone of YF17D identified in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054434#pone.0054434-Bonaldo3" target="_blank">[37]</a>. The amino acid sequence of these peptides and their corresponding position in the YF17D polyprotein are as follows: LTPVTMAEV (LV9_1285–1293), VSPGNGWMI (VI9_3250–3258), MSPKGISRM (MM9_2179–2187), and TTPFGQQRVF (TF10_2853–2862). We measured responses to these four epitopes in the <i>Mamu-A*01⁺</i> macaques r04170, r04136, r05079, and r04109. An asterisk (*) on top of a bar indicates a positive response detected in CD8-depleted PBMC, which likely represents SIV-specific CD4⁺ T-cells. The number of IFN-γ-producing cells in positive control wells stimulated with Concanavalin A at days 14 and 17 were 13,321 and 6,194 SFC/10⁶ PBMC, respectively.</p

Animal details and vaccination doses.

<p>N.A., not applicable.</p

Breadth of vaccine-induced, SIV-specific T-cell responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>We determined the breadth of T-cell responses in each animal by adding the number of positive IFN-γ ELISPOT assay responses to peptide pools (15mers overlapping by 11 amino acids) spanning the regions of Gag, Nef, and Vif covered in the SIVmac239 inserts. Panels A and B show the median breadth of T-cell responses to Gag, Nef, Vif, and all three proteins combined in the rYF17D/rAd5 (A) and rAd5 (B) groups. C) Comparison of the total number of peptide pools recognized in rYF17D/rAd5- and rAd5-vaccinated animals. Lines represent the median total number of responses in each group. We used the Mann-Whitney test to determine the p value.</p

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

Functional profile of CD8⁺ cellular responses in rYF17D/rAd5 and rAd5 vaccinees.

<p>We carried out multi-parameter ICS at week 3 after the rAd5 immunization to determine the ability of SIV-specific CD8⁺ T-cells to degranulate (CD107a) and secrete IFN-γ, TNF-α, MIP-1β, and IL-2. The antigen stimuli in this assay consisted of peptide mixtures spanning (i) amino acids 1–291 of Gag, (ii) amino acids 281–510 of Gag, (iii) the Vif ORF, and (iv) the Nef ORF. Bar graphs indicate the mean total frequency of CD8⁺ lymphocytes specific to Gag, Nef, and Vif capable of producing each combination of functions tested. Pie graphs for each animal indicate the percentage of their CD8⁺ lymphocytes that are specific to Gag, Nef, and Vif and that were positive for one (yellow), two (green), three (orange), four (red), and five (black) immune parameters. A) Functional profile of CD8⁺ cellular responses in rYF17D/rAd5 vaccinees. B) Functional profile of CD8⁺ cellular responses in rAd5 vaccinees. Error bars represent the standard error of the mean.</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