Novel HIV‐1 clade B candidate vaccines designed for HLA‐B*5101+ patients protected mice against chimaeric ecotropic HIV‐1 challenge

Novel candidate HIV‐1 vaccines have been constructed, which are tailor‐designed for HLA‐B^*5101⁺ patients infected with HIV‐1 clade B. These vaccines employ novel immunogen HIVB‐B^*5101 derived from consensus HIV‐1 clade B Gag p17 and p24 regions coupled to two Pol‐derived B^*5101‐restricted epitopes, which are together with a third B^*5101 epitope in Gag dominant in HIV‐1‐infected long‐term non‐progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB‐B^*5101 immunogen in cultured cells. Heterologous DNA prime‐recombinant MVA boost regimen induced efficiently HIV‐1‐specific CD8⁺ T‐cell responses in BALB/c mice. These vaccine‐elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV‐1/NL4‐3 and ecotropic HIV‐1/NDK chimaeric viruses with HIV‐1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti‐HIV‐1 gp120 antibodies. These results support further development of HIVB‐B^*5101 vaccines in combined heterologous‐modality regimens. The use of allele‐specific vaccines in humans is discussed in the context of other developments in the HIV‐1 field.     

HIV gag‐specific immune response mediated by double negative (CD3+CD4−CD8−) T cells in HIV‐exposed seronegative individuals

Double negative (DN) T cells are CD3⁺, CD4^?, CD8^? cells with either T‐cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV‐exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV‐serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV‐specific immune responses mediated by DN T‐cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV‐Gag peptides. Thirty‐five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag‐specific DN T‐cell responses were found in 3/13 (23%) HIV‐exposed seronegative individuals (Group A), involving both DN/αβ⁺ and DN/γδ⁺ T‐cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag‐specific responses but were mediated exclusively by DN/γδ⁺ T‐cells, mainly through IFNγ production. DN T‐cells in Group A individuals can display differential HIV‐specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A. J. Med. Virol. 85:200–209, 2013. © 2012 Wiley Periodicals, Inc.     

HIV‐specific T‐cell responses detected in the genital tract of chronically HIV‐infected women are largely monofunctional

HIV‐specific T cells that produce interferon‐γ (IFN‐γ) are present in the genital tract of HIV‐infected women although these do not provide protection against genital HIV shedding. Because polyfunctional HIV‐specific T cells have been implicated in better HIV control than those with a single function, this study aimed to investigate whether polyfunctional T cells were present at the female genital mucosa. Cervical cytobrush‐derived T cells were obtained from chronically HIV‐infected women and compared with blood. CD3⁺ T cells from both compartments were expanded with Dynal anti‐CD3/CD28 expander beads for 14 days and flow cytometry was used to evaluate four T‐cell functions (CD107a, IFN‐γ, tumour necrosis factor‐α and macrophage inflammatory protein‐1β) from 16 women. The majority of Gag‐specific T‐cell responses in the female genital tract were monofunctional, although low frequencies of HIV Gag‐specific polyfunctional CD8⁺ T cells were detected at the cervix in 81·3% (13/16) of women. The ability of CD8⁺ T cells at both the cervix and in blood to express CD107a and to exhibit polyfunctional responses (two or more functions) following Gag stimulation was inversely associated with plasma viral load and positively associated with blood CD4 counts, suggesting that clinical status impacted on the functionality of HIV‐specific T cells at the mucosa, in a similar way to blood. HIV Gag‐specific cervical T cells were largely monofunctional. Polyfunctional T cells were detected at the cervix in women with high blood CD4 count and low plasma viral load but these did not protect from HIV genital shedding.     

Dimorphic HLA‐B signal peptides differentially influence HLA‐E‐ and natural killer cell‐mediated cytolysis of HIV‐1‐infected target cells

As a mechanism of self‐protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA‐E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK‐mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2‐methionine (P2‐M) having higher HLA‐E binding affinity than P2‐threonine (P2‐T). All HLA‐A and HLA‐C molecules carry P2‐M, whereas HLA‐B products have either P2‐M or P2‐T. Epidemiological evidence suggests that P2‐M is unfavourable in the context of HIV‐1 infection, being associated with accelerated acquisition of HIV‐1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK‐mediated killing of CD4⁺ T cells and monocyte‐derived macrophages infected with two laboratory‐adapted HIV‐1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA‐B P2 genotypes (M/M, M/T and T/T), NK‐mediated cytolysis was elevated consistently for P2‐T in a dose‐dependent manner for all cell and virus combinations tested (P = 0·008–0·03). Treatment of target cells with an anti‐HLA‐E monoclonal antibody restored NK‐mediated cytolysis of cells expressing P2‐M. Observations on cell lysis were also substantiated by measurements of HIV‐1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti‐HIV‐1 function mediated by NK cells is compromised by P2‐M, corroborating the association of HLA‐B genotype encoding P2‐M with accelerated HIV‐1 acquisition.     

KIR‐HLA‐A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina

Activating and inhibitory killer immunoglobulin‐like receptors (KIR) and their ligands HLA‐Bw4 (loci A and B) were studied by way of establishing whether they can contribute to protection against HIV‐1 infection in highly exposed and persistently seronegative (HESN) patients. Twenty‐three HIV‐1 serodiscordant heterosexual couples, 100 HIV‐1⁺ patients and 200 healthy individuals were included in this retrospective case–control study. HLA typing was performed by means of PCR followed by sequence‐specific oligonucleotide probe reverse hybridization. KIR3DL1 and KIR3DS1 were studied by PCR sequence‐specific primers. The frequency of KIR3DS1(3DS1/3DL1)‐Bw4 combination was significantly higher in HESN patients versus the discordant couples (P = 0·0003) and HIV‐1⁺ patients (P = 0·0001). Conversely, the KIR3DL1/KIR3DL1 homozygosity was significantly decreased in HESN patients versus the discordant couples (P = 0·00003), and HIV‐1⁺ patients (P = 0·00066). The frequency of HLA‐A*32 and HLA‐B*44 was higher in HESN versus their discordant couples (P = 0·009; P = 0·049), and HIV‐1⁺ patients (P = 0·00002; P = 0·0001). This had greater significance in combination with KIR3DS1 (3DS1/3DL1). KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV‐1 infection in HESN patients when bound to a specific HLA allele, in this case HLA‐A*32 and HLA‐B*44, both Bw4 alleles. The differences probably arise both in the HLA alleles and in the subtypes of KIR receptors depending on the ethnic group studied.     

NYVAC immunization induces polyfunctional HIV‐specific T‐cell responses in chronically‐infected,ART‐treated HIV patients

We report the results of the Theravac‐01 phase I trial, which was conducted to evaluate the safety and immunogenicity of a poxvirus‐based vector, NYVAC, expressing Gag, Pol, Nef, and Env from an HIV clade B isolate. NYVAC‐B vaccine was injected intra‐muscularly into ten HIV‐infected patients successfully treated with antiretroviral therapy, twice on day 0 and again at week 4. Safety and immunogenicity were monitored for 48 weeks. HIV‐specific T‐cell responses following immunization were quantitatively analyzed using an IFN‐γ ELISPOT assay and qualitatively characterized for their functional profile (including multiple cytokines secretion plus cytotoxic and proliferation capacity) by polychromatic flow cytometry. Our results indicate that the NYVAC‐B vaccine is safe and highly immunogenic, as indicated by increased HIV‐specific T‐cell responses in virtually all vaccinees. Interestingly, both an expansion of preexisting T‐cell responses, and the appearance of newly detected HIV‐specific CD4⁺ and CD8⁺ T‐cell responses were observed. Furthermore, immunization mostly induced an increase in Gag‐specific T‐cell responses. In conclusion, NYVAC‐B immunization induces broad, vigorous, and polyfunctional HIV‐specific T‐cell responses, suggesting that poxvirus‐based vaccine regimens may be instrumental in the therapeutic HIV vaccine field.     

Comprehensive analysis of HIV Gag‐specific IFN‐γ response in HIV‐1‐ and HIV‐2‐infected asymptomatic patients from a clinical cohort in The Gambia

Majority of HIV‐2‐infected individuals meet the criteria of long‐term non‐progressors. This has been linked to superior qualitative HIV‐2‐specific cellular immune responses that correlate with viral control. However, it is unknown whether this is due to frequent targeting of immunodominant Gag epitopes in HIV‐2 than HIV‐1 infection. We describe a comprehensive comparison of the magnitude, breadth and frequency of Gag responses and the degree of cross‐recognition of frequently targeted, immunodominant Gag peptides in a cross‐sectional study of asymptomatic HIV‐1‐ and HIV‐2‐infected individuals. Fresh PBMC from 20 HIV‐1‐ and 20 HIV‐2‐infected patients with similar CD4⁺ T‐cell counts (p=0.36) were stimulated with pools of HIV‐1 and/or HIV‐2 Gag peptides in an IFN‐γ ELISPOT assay. We found no difference in the cumulative magnitude of IFN‐γ responses (p=0.75) despite significantly lower plasma viral loads in HIV‐2‐infected people (p<0.0001). However, Gag211–290 was targeted with significantly higher magnitude in HIV‐2‐infected subjects (p=0.03) although this did not correlate with viral control. There was no difference in frequently targeted Gag peptides, the breadth, immunodominance or cross‐recognition of Gag peptide pools between the two infections. This suggests that other factors may control viral replication in HIV‐2 infection.     

Functional advantage of educated KIR2DL1+ natural killer cells for anti‐HIV‐1 antibody‐dependent activation

Evidence from the RV144 HIV‐1 vaccine trial implicates anti‐HIV‐1 antibody‐dependent cellular cytotoxicity (ADCC) in vaccine‐conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody‐dependent manner is reliant upon several factors. In general, NK cell‐mediated antibody‐dependent activation is most robust in terminally differentiated CD57⁺ NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin‐like receptors (KIR) and their major histocompatibility complex class I [MHC‐I or human leucocyte antigen (HLA‐I)] ligands. With regard to anti‐HIV‐1 antibody‐dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA‐Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA–C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA‐I‐devoid target cells or antibody‐dependent stimulation with HIV‐1 gp140‐pulsed CEM.NKr‐CCR5 target cells in the presence of an anti‐HIV‐1 antibody source. Among donors carrying the HLA‐C2 ligand for KIR2DL1, higher interferon (IFN)‐γ production was observed within KIR2DL1⁺ NK cells than in KIR2DL1^– NK cells upon both direct and antibody‐dependent stimulation. No differences in KIR2DL1⁺ and KIR2DL1^– NK cell activation were observed in HLA‐C1 homozygous donors. Additionally, higher activation in KIR2DL1⁺ than KIR2DL1^– NK cells from HLA–C2 carrying donors was observed within less differentiated CD57^– NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1⁺ NK cells within differentiated CD57⁺ NK cells. These observations are relevant for understanding the regulation of anti‐HIV‐1 antibody‐dependent NK cell responses.     

CTL recognition of HIV‐1‐infected cells via cross‐recognition of multiple overlapping peptides from a single 11‐mer Pol sequence

It is known that overlapping HIV‐1 peptides of different lengths can be presented by a given HLA class I molecule. However, the role of those peptides in CD8⁺ T cells recognition of HIV‐1‐infected cells remains unclear. Here we investigated the recognition of overlapping 8‐mer to 11‐mer peptides of Pol 155–165 by HLA‐B*54:01‐restricted CD8⁺ T cells. The analysis of ex vivo T cells using ELISPOT and tetramer binding assays showed that there were different patterns of CD8⁺ T‐cell responses to these peptides among chronically HIV‐1‐infected HLA‐B*54:01⁺ individuals, though the response to the 9‐mer peptide was the strongest among them. CD8⁺ T‐cell clones with TCRs specific for the 9‐mer, 10‐mer, and/or 11‐mer peptides effectively killed HIV‐1‐infected cells. Together, these results suggest that the 9‐mer and 10‐mer peptides could be predominantly presented by HLA‐B*54:01, though it remains possible that the 11‐mer peptide was also presented by this HLA allele. The present study demonstrates effective CD8⁺ T‐cell recognition of HIV‐1‐infected cells via presentation of multiple overlapping HIV‐1 peptides and cross‐recognition by the CD8⁺ T cells.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

HLA-B*44 Is Associated with a Lower Viral Set Point and Slow CD4 Decline in a Cohort of Chinese Homosexual Men Acutely Infected with HIV-1

HLA class I alleles have been shown to have differential impacts on the viral load and the outcome of HIV-1 disease progression. In this study, HLA class I types from residents of China with acute HIV-1 infection, diagnosed between 2006 and 2011, were identified and the association between expression of individual HLA alleles and the level of the set point viral load was analyzed. A lower level of set point viral load was found to be associated with the Bw4 homozygote on HLA-B alleles. B*44 and B*57 alleles have also been found to be associated with lower set point viral load. The set point viral load of B*44-positive individuals homozygous for Bw4 was significantly lower than that of B*44-negative individuals homozygous for Bw4 (P = 0.030). The CD4 count declined to <350 in fewer B*44-positive individuals than B*44-negative individuals (X² = 7.295, P = 0.026). B*44-positive individuals had a lower magnitude of p24 pool-specific T cell responses than B*44-negative individuals homozygous for Bw4, though this was not statistically significant. The p24 pool-specific T cell responses were also inversely correlated with lower viral load (r_s = −0.88, P = 0.033). Six peptides within p24 were recognized to induce the specific-T cell response in B*44-positive individuals, and the peptide breadth of response was same as that in B*44-negative individuals homozygous for Bw4, but the median magnitude of specific-T cell responses to the recognized peptides in B*44-positive individuals was lower than that in B*44-negative individuals homozygous for Bw4 (P = 0.049). These findings imply that weak p24-specific CD8⁺ T cell responses might play an important role in the control of HIV viremia in B*44 allele-positive individuals. Such studies might contribute to the development of future therapeutic strategies that take into account the genetic background of the patients.     

Highly avid,oligoclonal, early‐differentiated antigen‐specific CD8+ T cells in chronic HIV‐2 infection

HIV‐1‐specific CD8⁺ T cells are present in most HIV‐1‐infected people and play an important role in controlling viral replication, but the characteristics of an effective HIV‐specific T‐cell response are largely unknown. The majority of HIV‐2‐infected people behave as long‐term non‐progressors while those who progress to AIDS do so in a manner indistinguishable from HIV‐1. A detailed study of HIV‐2 infection may identify protective immune responses. Robust gag p26‐specific T‐cell responses are elicited during HIV‐2 infection and correlate with control of viremia. In this study, we analyzed features of an HLA‐B^*3501‐restricted T‐cell response to HIV‐2 p26 that may contribute to virus control. In contrast to HIV‐1, HIV‐2‐specific T cells are at an early stage of differentiation (CD27⁺CD28⁺), a finding that relates directly to CD4⁺ T‐cell levels and inversely to immune activation. The cells demonstrate IFN‐γ secretion, oligoclonal T‐cell receptor Vβ gene segment usage, exceptional avidity and secretion of pro‐inflammatory cytokines. Despite the potentially strong selection pressure imposed on the virus by these cells, there was no evidence of HIV‐2 sequence evolution. We propose that in chronic HIV‐2 infection, the maintenance of early‐differentiated, highly avid CD8⁺ T cells could account for the non‐progressive course of disease. Such responses may be desirable from an HIV vaccine.     

The differential impact of natural killer (NK) cell education via KIR2DL3 and KIR3DL1 on CCL4 secretion in the context of in‐vitro HIV infection

Carriage of certain inhibitory natural killer (NK) cell receptor (iNKR)/HLA ligand pairs is associated with protection from infection and slow time to AIDS implicating NK cells in HIV control. NK cells acquire functional potential through education, which requires the engagement of iNKRs by their human leucocyte antigen (HLA) ligands. HIV infection down‐regulates cell surface HLA‐A/B, but not HLA‐C/E. We investigated how NK cell populations expressing combinations of the iNKRs NKG2A, KIR2DL3 (2DL3) and KIR3DL1 (3DL1) responded to autologous HIV infected CD4 (iCD4) cells. Purified NK cells from HIV‐uninfected individuals were stimulated with autologous HIV iCD4 or uninfected CD4 T cells. Using flow cytometry we gated on each of the 8 NKG2A^+/–2DL3^+/–3DL1^+/‐ populations and analysed all possible combinations of interferon (IFN)‐γ, CCL4 and CD107a functional subsets responding to iCD4 cells. Infected CD4 cells induced differential frequencies of NKG2A^+/–2DL3^+/–3DL1^+/– populations with total IFN‐γ⁺, CCL4⁺ and CD107a⁺ functional profiles. 2DL3⁺NKG2A⁺ NK cells had a higher frequency of responses to iCD4 than other populations studied. A higher frequency of 2DL3⁺ NK cells responded to iCD4 from individuals that were not HLA‐C1 homozygotes. These results show that 2DL3⁺ NK cells are mediators of HIV‐specific responses. Furthermore, responses of NK cell populations to iCD4 are influenced not only by NK cell education through specific KIR/HLA pairs, but also by differential HIV‐mediated changes in HLA expression.     

Plasmacytoid dendritic cell and functional HIV Gag p55‐specific T cells before treatment interruption can inform set‐point plasma HIV viral load after treatment interruption in chronically suppressed HIV‐1+ patients

The identification of immune correlates of HIV control is important for the design of immunotherapies that could support cure or antiretroviral therapy (ART) intensification-related strategies. ART interruptions may facilitate this task through exposure of an ART partially reconstituted immune system to endogenous virus. We investigated the relationship between set-point plasma HIV viral load (VL) during an ART interruption and innate/adaptive parameters before or after interruption. Dendritic cell (DC), natural killer (NK) cell and HIV Gag p55-specific T-cell functional responses were measured in paired cryopreserved peripheral blood mononuclear cells obtained at the beginning (on ART) and at set-point of an open-ended interruption from 31 ART-suppressed chronically HIV-1⁺ patients. Spearman correlation and linear regression modeling were used. Frequencies of plasmacytoid DC (pDC), and HIV Gag p55-specific CD3⁺ CD4⁻ perforin⁺ IFN-γ⁺ cells at the beginning of interruption associated negatively with set-point plasma VL. Inclusion of both variables with interaction into a model resulted in the best fit (adjusted R² = 0·6874). Frequencies of pDC or HIV Gag p55-specific CD3⁺ CD4⁻ CSFE^lo CD107a⁺ cells at set-point associated negatively with set-point plasma VL. The dual contribution of pDC and anti-HIV T-cell responses to viral control, supported by our models, suggests that these variables may serve as immune correlates of viral control and could be integrated in cure or ART-intensification strategies.     

Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indian population

India is at the epicentre of the global HIV/AIDS epidemic in South-east Asia, predominated by subtype C infections. It is important to characterize HIV-1-specific T cell responses in this particular population with the aim of identifying protective correlates of immunity to control HIV-1 infection. In this study, we performed a comprehensive analysis of the breadth and magnitude of T cell responses directed at HIV-1 subtype C Gag, one of the most conserved HIV-1 proteins. The study population consisted of antiretroviral naive, chronic HIV-1 subtype C-infected individuals at various stages of infection. We used recent advanced techniques such as enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining to quantify the total CD4(+) and CD8(+) T cell response to HIV-1 gag at single peptide level, regardless of HLA haplotype of the infected individual. The p24-Gag was identified as the most frequently recognized subunit protein with the greatest magnitude of CD4(+) and CD8(+) T cell responses. Stronger and broader CD8 T cell responses were recognized, contrasting with the weaker and narrower CD4 T cell responses with regard to Gag protein subunits. The magnitude of the HIV-specific interferon (IFN)-gamma responses was observed to be higher than the corresponding interleukin (IL)-2 response, indicating the persistence of antigenic load in chronically infected Indian population due to the probable dysfunction of HIV-specific, IFN-gamma-secreting CD8 T cells in absence of IL-2 help.     

Naturally processed HLA‐DR3‐restricted HHV‐6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 T‐cell responses

Human herpes virus 6B (HHV‐6B) is a widespread virus that infects most people early in infancy and establishes a chronic life‐long infection with periodic reactivation. CD4 T cells have been implicated in control of HHV‐6B, but antigenic targets and functional characteristics of the CD4 T‐cell response are poorly understood. We identified 25 naturally processed MHC‐II peptides, derived from six different HHV‐6B proteins, and showed that they were recognized by CD4 T‐cell responses in HLA‐matched donors. The peptides were identified by mass spectrometry after elution from HLA‐DR molecules isolated from HHV‐6B‐infected T cells. The peptides showed strong binding to matched HLA alleles and elicited recall T‐cell responses in vitro. T‐cell lines expanded in vitro were used for functional characterization of the response. Responding cells were mainly CD3⁺CD4⁺, produced IFN‐γ, TNF‐α, and low levels of IL‐2, alone or in combination, highlighting the presence of polyfunctional T cells in the overall response. Many of the responding cells mobilized CD107a, stored granzyme B, and mediated specific killing of peptide‐pulsed target cells. These results highlight a potential role for polyfunctional cytotoxic CD4 T cells in the long‐term control of HHV‐6B infection.     

Influenza,but not HIV‐specific CTL epitopes,elicits delayed‐type hypersensitivity (DTH) reactions in HIV‐infected patients

The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA‐A*02:01‐restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed‐type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4⁺ T‐cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope‐specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large‐scale HIV vaccine trials.     

Defining the HLA class I‐associated viral antigen repertoire from HIV‐1‐infected human cells

Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High‐throughput definition of HLA class I‐associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T‐cell responses against pathogens such as HIV‐1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo‐assisted database searching to define the HLA class I‐associated immunopeptidome of HIV‐1‐infected human cells. We here report for the first time the identification of 75 HIV‐1‐derived peptides bound to HLA class I complexes that were purified directly from HIV‐1‐infected human primary CD4⁺ T cells and the C8166 human T‐cell line. Importantly, one‐third of eluted HIV‐1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T‐cell responses have previously been reported but for which the precise HLA class I‐binding sequences have not yet been defined. These results validate and expand the current knowledge of virus‐specific antigenic peptide presentation during HIV‐1 infection and provide novel targets for T‐cell vaccine development.     

Polyclonal expansion of cervical cytobrush‐derived T cells to investigate HIV‐specific responses in the female genital tract

Human immunodeficiency virus (HIV) ‐specific T‐cell responses are detectable in the female genital tract of HIV‐infected women but little is known about their frequency or the factors that influence their detection. We investigated the feasibility of polyclonal in vitro expansion of cervical cytobrush‐derived T cells to investigate HIV‐specific responses in the female genital tract in HIV‐infected women. Cytobrush‐derived cervical cells were isolated from 22 HIV‐infected women and expanded with anti‐CD3 and recombinant interleukin‐2. Cervical T‐cell lines were investigated for Gag‐specific responses by interferon‐γ ELISPOT and compared with those detected in matched blood samples. Cervical T‐cell lines were established from 16/22 (72·7%) participants. Although the absolute number of CD3^± cells recovered after expansion was positively associated with the number of cells isolated ex vivo (P = 0·01; R = 0·62), we observed a significant negative correlation between fold expansion and ex vivo cell number (P = 0·004; R = ?0·68). We show that both the magnitude (P = 0·002; R = 0·7) and specific Gag regions targeted by cervical T‐cell lines (P < 0·0001; R = 0·5) correlated significantly with those detected in blood. With one exception, cervical interferon‐γ T‐cell responses to Gag were detected only in HIV‐infected women with blood Gag‐specific response > 1000 spot‐forming units/10⁶ cells. We conclude that cervical Gag‐specific T‐cell responses in expanded lines are most easily detectable in women who have corresponding high‐magnitude Gag‐specific T‐cell responses in blood.     

The microRNA‐9/B‐lymphocyte‐induced maturation protein‐1/IL‐2 axis is differentially regulated in progressive HIV infection

The fine control of T‐cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B‐lymphocyte‐induced maturation protein‐1 (Blimp‐1/Prdm1) is highly expressed in CD4⁺ T cells from chronically HIV‐infected (CHI) patients compared to cells from long‐term nonprogressors or healthy controls. Stimulation through the T‐cell receptor in the presence ofIL‐2 induces Blimp‐1 protein expression. We show here that Blimp‐1 levels are translationally regulated by microRNA‐9 (miR‐9). Overexpression of miR‐9 induces Blimp‐1 repression, restoring IL‐2 secretion in CD4⁺ T cells via reduction in the binding of Blimp‐1 to the il‐2 promoter. In CHI patients where IL‐2 expression is reduced and there is generalized T‐cell dysfunction, we show differential expression of both miR‐9 and Blimp‐1 in CD4⁺ cells compared with levels in long‐term nonprogressors. These data identify a novel miR‐9/Blimp‐1/IL‐2 axis that is dysregulated in progressive HIV infection.