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

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

Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus

<div><p>The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection <i>in vivo</i>. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions <i>in vivo</i>. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection.</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

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

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

T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

<div><p>Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination.</p></div

Rh159 interferes with intracellular transport of NKG2DL.

<p><b>A)</b> Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. <b>B)</b> Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. <b>C)</b> Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. <b>D-E)</b> Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (<b>D</b>), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments.</p

Deletion of Rh159 rescues intracellular transport and surface expression of MICA and MICB upon RhCMV infection.

<p><b>A)</b> Comparison of NKG2DL surface expression upon infection with RhCMV or ΔRh159. U373-NKG2DL cells were infected with RhCMV (blue) or ΔRh159 (red) (MOI = 3) for 48 h. Cell surface levels of NKG2DL or TfR were determined by flow cytometry, using specific antibodies and compared to isotype control (dotted). Depicted is NKG2DL or TfR surface expression on infected cells gated for RhCMV IE2⁺ expression. The results shown are representative of three or more independent experiments. <b>B)</b> Biosynthesis and maturation of NKG2DL in uninfected U373-NKG2DL cells or upon infection with RhCMV or ΔRh159. U373-NKG2DL cells were uninfected (NI), infected with RhCMV (WT) or ΔRh159 (MOI = 3) for 24 h, verified by light microscopy as having 100% CPE, then metabolically labeled with [35S]cysteine and [35S]methionine for 30 min prior to chasing the label for the indicated times. The indicated NKG2DLs were immunoprecipitated from cell lysates with specific mAbs. Immunoprecipitates were split and digested with EndoH (+) or mock treated (-) then analyzed by SDS-PAGE and autoradiography.</p

rGal-9 partially activates primary CD4+ T cells and induces proliferation primarily in naïve CD4+ T cells.

<p>(<b>A</b>, <b>B</b>) Effects of rGal-9 stimulation on the cell surface expression of CD69 and CD25 activation markers on CD4+ T cells isolated from six ART-suppressed individuals. Mean ± SEM is displayed. Asterisks represent statistically significant differences as compared to DMSO control (p < 0.05, two-tailed Wilcoxon signed-rank test). (<b>C</b>) Effects of rGal-9 stimulation on the proliferation of CD4+ T cells isolated from three ART-suppressed individuals. Primary CD4+ T cells were stained with CFSE and cultured for 5 days, stained with CD4 and CD45RA monoclonal antibodies, and proliferation was quantified as the percentage of CFSE_low cells on CD4+ CD45RA+ (Naïve, Tn) or CD4+ CD45RA- (Memory, Tm) T cells. Mean ± SEM is displayed. (<b>D</b>) Example of the flow cytometry gating strategy. (<b>E</b>, <b>F</b>) Effects of rGal-9 on proliferation of (<b>E)</b>, memory CD4+ T cells, and (<b>F)</b>, naïve CD4+ T cells.</p