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CD8(+) T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8(+) T cell responses in HIV infection, however, remains unknown. In this study, CD8(+) T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8(+) T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8(+) T cells. Importantly, bulk CD8(+) T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8(+) T cell responses in HIV infection.

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality., Here, using longitudinal pre- and post-infection samples from the RV217 Early Capture HIV Cohort Study, the authors show that mucosa-associated invariant T (MAIT) cells become activated and expand during the early acute phase of HIV infection, with subsequent reprogramming towards innate-like functionality.

[This corrects the article DOI: 10.1371/journal.ppat.1006510.].

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection., Author summary The development of safe, effective, and scalable vaccines and cure strategies to control the HIV-1 pandemic is a major public health concern. The rational design of these preventive and treatment measures requires a profound knowledge of the interaction between HIV-1 and its host during the first weeks that follow viral infection (i.e., acute infection). Here we performed a systematic and in-depth study of individuals whose infection was detected before peak viremia and before the emergence of the first antibody responses. Plasma samples were collected twice weekly during acute infection and we performed next-generation sequencing of the viral swarms. In most participants, we first detected viral escape from the initial adaptive cellular immune responses at the end of peak viremia downslope. Viral escape proceeded at higher rates than previously measured in HIV-1 infection and usually through the exploration of multiple mutational pathways. The analysis of sequences from infections established by multiple viral lineages revealed dramatic shifts in the frequencies of the viruses that composed the HIV-1 population within each host. These results, using early, deep, and frequent sampling, support rapidly changing viral lineages likely in response to both adaptive cellular immunity and possibly other host responses during acute HIV-1 infection.

Natural killer (NK) cells limit viral replication by direct recognition of infected cells, antibody-dependent cellular cytotoxicity (ADCC), and releasing cytokines. Although growing evidence supports NK cell antiviral immunity in HIV-1 infection, further knowledge of their response is necessary. Here we show that NK cells responding to models of direct cell recognition, ADCC, and cytokine activation have unique transcriptional fingerprints. Compared with healthy volunteers, individuals with chronic HIV-1 infection have higher expression of genes commonly associated with activation, and lower expression of genes associated with direct cell recognition and cytokine stimulation in their NK cells. By contrast, NK cell transcriptional profiles of individuals receiving a modified vaccinia Ankara (MVA) vectored HIV-1 vaccine show upregulation of genes associated with direct cell recognition. These findings demonstrate that targeted transcriptional profiling provides a sensitive assessment of NK cell activity, which helps understand how NK cells respond to viral infections and vaccination., Natural killer (NK) cells are important for eliminating cells under stress or infected by virus, and may have a function in anti-HIV immunity. Here the authors show that different NK-activating stimuli induce distinct transcriptional fingerprints in human NK cells that are analogous to changes caused by HIV vaccination or chronic infection.

In this report, we have tested the cytotoxicity of two organotin (OT) compounds by flow cytometry on a panel of immortalized cancer cell lines of human and murine origin. Although the OT compounds exhibited varying levels of cytotoxicity, diphenylmethyltin chloride was more toxic than 1,4-bis (diphenylchlorostannyl)p-xylene on all cell lines tested. The OT compounds were found to be highly cytotoxic to lymphoma cell lines with lower toxicity toward the HeLa cervical cancer cell line. In order to discern the mechanism by which cell death was induced, additional experiments were conducted to monitor characteristic changes consistent with apoptosis and/or necrosis. Cell lines treated with the experimental compounds indicated that there was no consistent mode of cell death induction. However, both compounds induced apoptosis in the pro-B lymphocyte cell line, NFS-70. The work presented here also demonstrates that the two OT compounds possess selective cytotoxicity against distinct transformed cell lines.

Attrition within the CD4 + T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8 + T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8 + T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38 + CD27 − CD8 + T cells characterized by the low expression of the CD8 receptor (CD8 dim ). Interestingly, while high frequencies of HIV-specific CD8 + T cell responses occur within the CD38 + CD27 − CD8 dim T cell population, the minority populations of CD8 bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8 dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8 dim T cells, and the size of this population inversely correlates with the acute loss of CD4 + T cells. These data indicate, for the first time, that early CD4 + T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8 dim T cell population less efficient in controlling HIV viremia. IMPORTANCE A distinct population of activated CD8 + T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8 + T cell dysfunction during acute infection.

Chronic viral infection and persistent antigenic stimulation is associated with a rise of late-stage differentiated CD8 T cells including a unique subset of CD8 T cells expressing FCg receptor IIIa (CD16). Expansion of CD16+ CD8 T cells is observed in other chronic diseases like HCV, but not HIV-1 infection. CD16 mediates antibody dependent cellular cytotoxicity (ADCC), most notably on NK cells, and is associated with protection from HIV-1 acquisition. Using cryopreserved PBMC from healthy and HIV-1 chronically infected donors from Uganda, we compared phenotypic, transcriptional, and functional changes in CD16+ CD8 T cells. We found that chronic, untreated HIV-1 infection is associated with elevated numbers of CD45RA+CD57+ terminal effector CD16+ CD8 T cells compared to healthy individuals, and display a distinct transcriptional profile in between conventional CD8 T cells and NK cells, characterized by high IKZF2 and KLRF1, and low expression of IL7R. This transcriptional profile translates into a NKp80+ IL-7Rα-surface phenotype and high expression of the Helios transcription factor. Additionally, CD16+ CD8 T cells are capable of mediating ADCC at levels similar to that of NK cells. These CD16+ CD8 T cells are highly activated and correlate positively with expansion of the CD8 T cell compartment and plasma levels of soluble factors of antiviral immunity and inflammation, such as IP-10, TNF, IL-6, and TNFRII. These data indicate that terminally differentiated, effector CD8 T cells acquire innate, NK cell-like characteristics during chronic HIV-1 infection, and suggest that HIV-specific ADCC is part of the repertoire of functions CD8 T cells use to combat the ability of HIV-1 to escape the TCR-specific, anti-viral effects of CD8 T cells.

Natural Killer (NK) cells limit viral replication by direct recognition and killing of infected cells, performing Fc mediated ADCC and releasing cytokines to modulate other effector activity. Despite growing evidence that supports the importance of NK cells in HIV-1 disease progression, understanding how these cells engage in vivo remains unclear. Here, we combined flow cytometry and gene expression analysis of NK cells responding to three in vitro models of NK stimulation to reveal distinct transcriptional profiles. While 76 gene transcripts were commonly upregulated following stimulation by direct cellular recognition, ADCC, and cytokine activation, 276, 36, and 293 genes were uniquely upregulated, respectively. Subsequently, a targeted gene expression panel was used to assess NK cell transcriptional content, directly ex vivo, in HIV-1 chronic infection. Transcriptional profiles in HIV-1 chronically infected individuals showed higher expression of genes associated with global activation and lower expression of genes associated with direct cell recognition and cytokine activation, compared to healthy individuals. We next looked at the transcriptional content of NK cells in response to MVA-CMDR vaccination, a live-attenuated viral vectored HIV-1 vaccine. NK cell transcriptional profiles of vaccinated individuals revealed upregulation of genes associated with direct cell recognition. These findings demonstrate that targeted transcriptional profiling provides a sensitive assessment of NK cell activity directly ex vivo that will improve our understanding of how NK cells respond to viral infections and after vaccination.

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8(+) T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8(+) T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N(121-129)) and IL9 (ILDAHSLYL, N165-173), were defined. VT9- and IL9-specific CD8(+) T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+) T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+) T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+) T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

Methods To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from eight seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays.

Recapitulating immunodominant A2-restricted HIV-specific CTLs that do not suppress virus in vivo is unlikely to be an effective vaccine strategy. To detect novel subdominant determinants in HIV-1 Gag, we primed CD8+ T cells from five seronegative donors with autologous dendritic cells transduced to express Gag. T cells were re-stimulated weekly with monocytes pulsed with 123 15-mer overlapping peptides (OLPs) spanning Gag. Responses were identified with OLP matrix pools followed by interrogation at the single OLP level in responsive pools using IFN-γ ELISPOT assays. One OLP (Gag145-159) was recognized by all donors. Of note, this reactivity predominated in three of five T cell cultures. Fine mapping with progressively truncated peptides revealed three overlapping epitopes: RTLNAWVKV (RV9), RTLNAWVKVV (RV10) and TLNAWVKVV (TV9). TV9 is a known epitope that is rarely recognized in vivo. In contrast, RV9 and RV10 are novel. Although the latter bound with lower affinities to HLA-A2 than TV9, RV9- and RV10-cultures were readily generated. RV9- and RV10-T cells were cytotoxic, capable of secreting many cytokines, and suppressive of HIV replication in vitro. In sum, we report two novel naturally processed and presented epitopes in HIV p24 that are recognized by pre-infection T cell repertoires. Further studies are needed to explain why these reactivities are rare during infections and more importantly, whether this conserved region of the HIV proteome has value as a prophylactic vaccine for A2 individuals.