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Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.

Safe and effective vaccines have been developed that protect against high-risk strains of HPV, but uptake is relatively low. We previously identified factors such as sexual attitudes and HPV knowledge that impact the intent of Christian parents to vaccinate their children against HPV. We hypothesized that culturally specific interventions in the form of short videos would be effective at improving HPV vaccine intentions and attitudes. We made three short educational videos, one with a Christian focus, one informational about HPV, and one control. Videos were distributed electronically with accompanying surveys, and responses were measured before and after watching a randomly selected video. The religious-focused and educational interventions significantly (p < 0.0001, p = 0.0015) improved intentions towards HPV vaccination. The religiously-focused video also significantly diminished the belief that the HPV vaccine is unnecessary because of a family's values (p = 0.014). Parents significantly credited both interventions with improving their intent to vaccinate their children against HPV (p < 0.001 for both). These results suggest that culturally focused educational interventions are effective at influencing vaccine intentions and attitudes, even when those are based on religious or cultural feelings. Highly specific interventions are likely to be necessary for optimal improvement in vaccine hesitancy., (© 2024. The Author(s).)

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells., Competing Interests: Declaration of interests The authors hold intellectual property through the Massachusetts General Brigham (D.B.M., G.S.O., and J.R.; filing 29618-0390P01) and the University of Oxford (G.S.O., C.S.H., and Y.-L.C.; filings 2116709.3, 2217923.8, 2217924.6). G.S.O. collaborates with UCB and Janssen, and D.B.M. consults for Pfizer., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic changed our world as we know it and continues to be a global problem three years since the pandemic began. Several vaccines were produced, but there was a considerable amount of societal turmoil surrounding them that has affected the way people view not only COVID-19 vaccines but all vaccines. We used a survey to compare how attitudes towards vaccination have changed in college students during the pandemic. An initial survey was administered in 2021, then a follow-up in 2022. Out of 316 respondents who answered the first survey, 192 completed the follow-up. The survey was designed to measure trends in changes to vaccine attitudes since the COVID-19 pandemic began. By comparing the first survey in 2021 and the follow-up, we found that roughly 55% of respondents' vaccine attitudes did not change, roughly 44% of respondents' attitudes towards vaccines became more positive, and only about 1% of the respondents' vaccine attitudes became more negative. Improved view of vaccines was associated with political views and increased trust in medicine and the healthcare system. Worsened opinions of vaccines were associated with a belief that the COVID-19 vaccine affected fertility.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4 + (HLA-DRB1 ∗ 15:01/S191) and CD8 + (HLA-A ∗ 02/S691) T cell epitopes. Antigen-specific CD4 + and CD8 + T cell responses were asynchronous, with the peak CD4 + T cell responses occurring 1 week post the second vaccination (boost), whereas CD8 + T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8 + T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination., Competing Interests: Declaration of interests V.M. and M.M.D. are inventors on a patent application (PCT/US2021/064378) on the spheromer technology described in this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of vaccines has significantly contributed to the success of disease prevention. However, there has been a sharp decline in immunization rates since COVID-19 spread globally. Seemingly overnight, the world shut down and most non-essential medical procedures were postponed. Since the COVID-19 vaccine became available, and the world started going back to normal these vaccine rates have not recovered. In this paper, we review the published literature to explore how convenience factors, perceived risk of vaccination, media or anti-vaccination ideals/movements, and healthcare professionals affect an individual's compliance to be vaccinated to better understand the factors that contribute to the change in overall vaccination rates.

The genetic and molecular basis of flagellar motility has been investigated for several decades, with innovative research strategies propelling advances at a steady pace. Furthermore, as the phenomenon is examined in diverse bacteria, new taxon-specific regulatory and structural features are being elucidated. Motility is also a straightforward bacterial phenotype that can allow undergraduate researchers to explore the palette of molecular genetic tools available to microbiologists. This study, driven primarily by undergraduate researchers, evaluated hundreds of flagellar motility mutants in the Gram-negative plant-associated bacterium Agrobacterium fabrum. The nearly saturating screen implicates a total of 37 genes in flagellar biosynthesis, including genes of previously unknown function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Calvopina-Chavez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States. Most infections are mild and clear without treatment in 1 to 2 years. Some HPV strains result in persistent infection, which can cause various cancers, including cervical, penile, anal, mouth, and throat cancers. Vaccines have been developed that provide protection against the highest risk HPV strains. Despite HPV vaccines having been proven to be safe and effective, uptake has been low. Religiosity has been negatively correlated with HPV vaccine uptake in some studies. It is hypothesized that religiosity and Christian religious affiliation could impact parents' decision to vaccinate their children against HPV via teachings and beliefs about sexual behaviors. A survey was distributed to participants to determine what factors, including religiosity and views about sex, impacted HPV vaccination. The survey results ( n = 442) were analyzed using confirmatory factor analysis, structural equation modeling, and univariate factor analysis. The association between religious practice and vaccine attitudes were complex, with religious practice slightly positively correlated with pro-vaccine attitudes and vaccine knowledge, but also with the belief that religious adherence to expectations surrounding sexual behavior will protect children from HPV infection, as well as more negative views towards vaccines, in general.

With the goal of improving the reproducibility and annotatability of MHC multimer reagent data, we present the establishment of a new data standard: Minimal Information about MHC Multimers (https://miamm.lji.org/). Multimers are engineered reagents composed of a ligand and a MHC, which can be represented in a standardized format using ontology terminology. We provide an online Web site to host the details of the standard, as well as a validation tool to assist with the adoption of the standard. We hope that this publication will bring increased awareness of Minimal Information about MHC Multimers and drive acceptance, ultimately improving the quality and documentation of multimer data in the scientific literature., (Copyright © 2022 by The American Association of Immunologists, Inc.)

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response., Competing Interests: Disclosures: R.N. Cotton reported personal fees from MPM Capital outside the submitted work. G. Ogg reported grants from UCB outside the submitted work. No other disclosures were reported., (© 2021 Cotton et al.)

Intradermal vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) protects infants from disseminated tuberculosis, and i.v. BCG protects nonhuman primates (NHP) against pulmonary and extrapulmonary tuberculosis. In humans and NHP, protection is thought to be mediated by T cells, which typically recognize bacterial peptide Ags bound to MHC proteins. However, during vertebrate evolution, T cells acquired the capacity to recognize lipid Ags bound to CD1a, CD1b, and CD1c proteins expressed on APCs. It is unknown whether BCG induces T cell immunity to mycobacterial lipids and whether CD1-restricted T cells are resident in the lung. In this study, we developed and validated Macaca mulatta ( Mamu ) CD1b and CD1c tetramers to probe ex vivo phenotypes and functions of T cells specific for glucose monomycolate (GMM), an immunodominant mycobacterial lipid Ag. We discovered that CD1b and CD1c present GMM to T cells in both humans and NHP. We show that GMM-specific T cells are expanded in rhesus macaque blood 4 wk after i.v. BCG, which has been shown to protect NHP with near-sterilizing efficacy upon M. tuberculosis challenge. After vaccination, these T cells are detected at high frequency within bronchoalveolar fluid and express CD69 and CD103, markers associated with resident memory T cells. Thus, our data expand the repertoire of T cells known to be induced by whole cell mycobacterial vaccines, such as BCG, and show that lipid Ag-specific T cells are resident in the lungs, where they may contribute to protective immunity., (Copyright © 2021 The Authors.)

Background: Helminth infections can modulate immunity to Mycobacterium tuberculosis (Mtb). However, the effect of helminths, including Schistosoma mansoni (SM), on Mtb infection outcomes is less clear. Furthermore, HIV is a known risk factor for tuberculosis (TB) disease and has been implicated in SM pathogenesis. Therefore, it is important to evaluate whether HIV modifies the association between SM and Mtb infection., Setting: HIV-infected and HIV-uninfected adults were enrolled in Kisumu County, Kenya, between 2014 and 2017 and categorized into 3 groups based on Mtb infection status: Mtb-uninfected healthy controls, latent TB infection (LTBI), and active TB disease. Participants were subsequently evaluated for infection with SM., Methods: We used targeted minimum loss estimation and super learning to estimate a covariate-adjusted association between SM and Mtb infection outcomes, defined as the probability of being Mtb-uninfected healthy controls, LTBI, or TB. HIV status was evaluated as an effect modifier of this association., Results: SM was not associated with differences in baseline demographic or clinical features of participants in this study, nor with additional parasitic infections. Covariate-adjusted analyses indicated that infection with SM was associated with a 4% higher estimated proportion of active TB cases in HIV-uninfected individuals and a 14% higher estimated proportion of active TB cases in HIV-infected individuals. There were no differences in estimated proportions of LTBI cases., Conclusions: We provide evidence that SM infection is associated with a higher probability of active TB disease, particularly in HIV-infected individuals., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC-IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable lentiviral vector-transduced derivatives of HEK-293T cells. The expression design includes allele-specific peptide ligands tethered to the amino-terminus of the MHC-II β chain via a protease-cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide exchange, enabling high-throughput production of many distinct MHC-IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label-free methods, native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp multimer reagents. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and expression line creation Support Protocol 1: Six-well assay for estimation of production cell line yield Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His-tags Basic Protocol 2: Cultures for production of Class II MHC proteins Basic Protocol 3: Purification of Class II MHC proteins by anti-leucine zipper affinity chromatography Alternate Protocol 1: IMAC purification of His-tagged Class II MHC Support Protocol 3: Protein concentration measurements and adjustments Support Protocol 4: Polishing purification by anion-exchange chromatography Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation Basic Protocol 4: Peptide exchange Basic Protocol 5: Analysis of peptide exchange by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Alternate Protocol 2: Native isoelectric focusing to validate MHC-II peptide loading Basic Protocol 6: Multimerization Basic Protocol 7: Staining cells with Class II MHC tetramers., (© 2021 Wiley Periodicals LLC.)

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A' roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.

A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I β2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (K d ) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (K i ) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe K d , inhibitor concentration, and inhibitor K i under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of K i using competition binding data., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αβ and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential., Competing Interests: The authors have declared that no competing interests exist.

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8 + T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08 -positive ( Mamu-B*08 + ) RMs. Here we evaluated if robust vaccine-elicited CD8 + T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08 + RMs following mucosal SIV challenges. Ten Mamu-B*08 + RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8 + T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection. IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8 + T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8 + T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection., (Copyright © 2019 American Society for Microbiology.)

CD1 proteins are expressed on dendritic cells, where they display lipid antigens to T-cell receptors (TCRs). Here we describe T-cell autoreactivity towards ubiquitous human membrane phospholipids presented by CD1b. These T-cells discriminate between two major types of lipids, sphingolipids and phospholipids, but were broadly cross-reactive towards diverse phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. The crystal structure of a representative TCR bound to CD1b-phosphatidylcholine provides a molecular mechanism for this promiscuous recognition. We observe a lateral escape channel in the TCR, which shunted phospholipid head groups sideways along the CD1b-TCR interface, without contacting the TCR. Instead the TCR recognition site involved the neck region phosphate that is common to all major self-phospholipids but absent in sphingolipids. Whereas prior studies have focused on foreign lipids or rare self-lipids, we define a new molecular mechanism of promiscuous recognition of common self-phospholipids including those that are known targets in human autoimmune disease.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17 + and 50% of Mamu-B*08 + RMs control chronic-phase viremia after SIVmac239 infection. Because CD8 + T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08 + RMs, we investigated whether this might also be true for Mamu-B*17 + RMs. Two groups of Mamu-B*17 + RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08 + RMs, preexisting SIV-specific CD8 + T cells alone do not facilitate long-term virologic suppression in Mamu-B*17 + RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17 + RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection. IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17 + macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8 + T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17 + macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8 + T cells for virologic control in Mamu-B*17 + macaques and implicate anti-Env antibodies in containment of SIV infection., (Copyright © 2018 American Society for Microbiology.)

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8., Competing Interests: JG, JA, MR No competing interests declared, SK is affiliated with the Sirona Genomics, where the HLA genotyping for our study was done. The author has no financial interests to declare, (© 2018, Geng et al.)

Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis , yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis -specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis -specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 M. tuberculosis Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of M. tuberculosis -unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis -specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis -specific T cell responses across different states of M. tuberculosis infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of M. tuberculosis -specific T cell responses associated with M. tuberculosis infection outcomes., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Effector memory T cell (T EM ) responses display potent antiviral properties and have been linked to stringent control of simian immunodeficiency virus (SIV) replication. Since recurrent antigen stimulation drives the differentiation of CD8 + T cells toward the T EM phenotype, in this study we incorporated a persistent herpesviral vector into a heterologous prime/boost/boost vaccine approach to maximize the induction of T EM responses. This new regimen resulted in CD8 + T EM -biased responses in four rhesus macaques, three of which controlled viral replication to <1,000 viral RNA copies/ml of plasma for more than 6 months after infection with SIVmac239. Over the course of this study, we made a series of interesting observations in one of these successful controller animals. Indeed, in vivo elimination of CD8αβ + T cells using a new CD8β-depleting antibody did not abrogate virologic control in this monkey. Only after its CD8α + lymphocytes were depleted did SIV rebound, suggesting that CD8αα + but not CD8αβ + cells were controlling viral replication. By 2 weeks postinfection (PI), the only SIV sequences that could be detected in this animal harbored a small in-frame deletion in nef affecting six amino acids. Deep sequencing of the SIVmac239 challenge stock revealed no evidence of this polymorphism. However, sequencing of the rebound virus following CD8α depletion at week 38.4 PI again revealed only the six-amino acid deletion in nef. While any role for immunological pressure on the selection of this deleted variant remains uncertain, our data provide anecdotal evidence that control of SIV replication can be maintained without an intact CD8αβ + T cell compartment.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Mature T lymphocytes of the CD8 or CD4 classes bear αβ T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring antigen-specific T cell immune responses were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as allophycocyanin or R-phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, β2-microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, β2m, and a chosen peptide. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

CD1c is abundantly expressed on human dendritic cells (DC) and B cells, where it binds and displays lipid Ags to T cells. In this study, we report that CD1c tetramers carrying Mycobacterium tuberculosis phosphomycoketide bind γδ TCRs. An unbiased method of ligand-based TCR selection detects interactions only with Vδ1(+) TCRs, and mutational analyses demonstrate a role of the Vδ1 domain during recognition. These results strengthen evidence for a role of CD1c in the γδ T cell response, providing biophysical evidence for CD1c-γδ TCR interactions and a named foreign Ag. Surprisingly, TCRs also bind CD1c complexes formed with diverse lipids such as lysophosphatidylcholine, sulfatide, or mannosyl-phosophomycoketide, but not lipopeptide ligands. Dissection of TCR interactions with CD1c carrying foreign Ags, permissive ligands, and nonpermissive lipid ligands clarifies the molecular basis of the frequently observed but poorly understood phenomenon of mixed self- and foreign Ag reactivity in the CD1 system., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.

Unlabelled: Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques., Importance: Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques—a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: The purpose of this study was to assess the diagnostic accuracy of the instantaneous wave-free ratio (iFR) to characterize, outside of a pre-specified range of values, stenosis severity, as defined by fractional flow reserve (FFR) ≤0.80, in a prospective, independent, controlled, core laboratory-based environment., Background: Studies with methodological heterogeneity have reported some discrepancies in the classification agreement between iFR and FFR. The ADVISE II (ADenosine Vasodilator Independent Stenosis Evaluation II) study was designed to overcome limitations of previous iFR versus FFR comparisons., Methods: A total of 919 intermediate coronary stenoses were investigated during baseline and hyperemia. From these, 690 pressure recordings (n = 598 patients) met core laboratory physiology criteria and are included in this report., Results: The pre-specified iFR cut-off of 0.89 was optimal for the study and correctly classified 82.5% of the stenoses, with a sensitivity of 73.0% and specificity of 87.8% (C statistic: 0.90 [95% confidence interval (CI): 0.88 to 0.92, p < 0.001]). The proportion of stenoses properly classified by iFR outside of the pre-specified treatment (≤0.85) and deferral (≥0.94) values was 91.6% (95% CI: 88.8% to 93.9%). When combined with FFR use within these cut-offs, the percent of stenoses properly classified by such a pre-specified hybrid iFR-FFR approach was 94.2% (95% CI: 92.2% to 95.8%). The hybrid iFR-FFR approach obviated vasodilators from 65.1% (95% CI: 61.1% to 68.9%) of patients and 69.1% (95% CI: 65.5% to 72.6%) of stenoses., Conclusions: The ADVISE II study supports, on the basis rigorous methodology, the diagnostic value of iFR in establishing the functional significance of coronary stenoses, and highlights its complementariness with FFR when used in a hybrid iFR-FFR approach. (ADenosine Vasodilator Independent Stenosis Evaluation II-ADVISE II; NCT01740895)., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens.

T cells autoreactive to the antigen-presenting molecule CD1a are common in human blood and skin, but the search for natural autoantigens has been confounded by background T cell responses to CD1 proteins and self lipids. After capturing CD1a-lipid complexes, we gently eluted ligands while preserving non-ligand-bound CD1a for testing lipids from tissues. CD1a released hundreds of ligands of two types. Inhibitory ligands were ubiquitous membrane lipids with polar head groups, whereas stimulatory compounds were apolar oils. We identified squalene and wax esters, which naturally accumulate in epidermis and sebum, as autoantigens presented by CD1a. The activation of T cells by skin oils suggested that headless mini-antigens nest within CD1a and displace non-antigenic resident lipids with large head groups. Oily autoantigens naturally coat the surface of the skin; thus, this points to a previously unknown mechanism of barrier immunity.

Human CD1a mediates foreign Ag recognition by a T cell clone, but the nature of possible TCR interactions with CD1a/lipid are unknown. After incubating CD1a with a mycobacterial lipopeptide Ag, dideoxymycobactin (DDM), we identified and measured binding to a recombinant TCR (TRAV3/ TRBV3-1, KD of ≈100 μM). Detection of ternary CD1a/lipid/TCR interactions enabled development of CD1a tetramers and CD1a multimers with carbohydrate backbones (dextramers), which specifically stained T cells using a mechanism that was dependent on the precise stereochemistry of the peptide backbone and was blocked with a soluble TCR. Furthermore, sorting of human T cells from unrelated tuberculosis patients for bright DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies demonstrate that the mechanism of T cell activation by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demonstrate the existence of lipopeptide-specific T cells in humans ex vivo.

Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.

CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c-PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo.

Severe arenaviral infections in humans are characterized by clinical findings common to other viral hemorrhagic fevers (VHFs), including thrombocytopenia, leukopenia, skin and internal organ hemorrhages, high viral replication, splenic necrosis, and death. Host responses, rather than direct damage by the arenaviral replication, account for most of the observed pathology, but it is not known what protective roles platelets may have in each of the manifestations. To address this issue in an animal model, we compared nondepleted (100%), partially depleted (15%), and profoundly (< 2.5%) platelet depleted mice infected with the mouse arenavirus lymphocytic choriomeningitis virus (LCMV). Here, we describe that systemic bleedings and death were seen only in those animals receiving the stronger depletion treatment. Furthermore, we showed that the nonhemorrhagic but partially platelet-depleted mice were unable to control the viral replication because of generalized splenic necrosis, affecting innate and adaptive immune cells.These data suggest that, by their supportive roles in hemostasis, platelets may be preventing the severe pathology observed in human arenaviral infections.

Protection against many intracellular pathogens is provided by CD8 T cells, which are thought to need CD4 T cell help to develop into effective memory CD8 T cells. Because murine CD8 T cells do not transcribe MHC class II (MHC-II) genes, several models have proposed antigen presenting cells (APCs) as intermediaries required for CD4 T cells to deliver their help to CD8 T cells. Here, we demonstrate the presence of MHC-II molecules on activated murine CD8 T cells in vitro as well as in vivo. These MHC-II molecules are acquired via trogocytosis by CD8 T cells from their activating APCs, particularly CD11c positive dendritic cells (DCs). Transferred MHC-II molecules on activated murine CD8 T cells were functionally competent in stimulating specific indicator CD4 T cells. CD8 T cells that were "helped" in vitro and subsequently allowed to rest in vivo showed enhanced recall responses upon challenge compared to "helpless" CD8 T cells; in contrast, no differences were seen upon immediate challenge. These data indicate that direct CD8:CD4 T cell interactions may significantly contribute to help for CD8 T cells. Furthermore, this mechanism may enable CD8 T cells to communicate with different subsets of interacting CD4 T cells that could modulate immune responses.

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10(8) PFU) or low-dose (1 × 10(7) PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.

Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human cluster of differentiation (CD) proteins CD1a, CD1b, CD1c, and CD1d bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen-binding grooves influence lipid capture. Although the CD1b groove is distinguished by its unusually large volume (2,200 Å(3)) and the T' tunnel, the average mass of compounds eluted from CD1b was similar to that of lipids from CD1 proteins with smaller grooves. Elution of small ligands from the large CD1b groove might be explained if two small lipids bind simultaneously in the groove. Crystal structures indicate that all CD1 proteins can capture one antigen with its hydrophilic head group exposed for T-cell recognition, but CD1b structures show scaffold lipids seated below the antigen. We found that ligands selectively associated with CD1b lacked the hydrophilic head group that is generally needed for antigen recognition but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrate a function in augmenting presentation of a small glycolipid antigen to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for presenting either two small or one large lipid, allowing presentation of antigens with an unusually broad range of chain lengths.