Immunisation with recombinant polymorphic membrane protein D elicits robust protection against sexually transmitted Chlamydia trachomatis infection

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen worldwide, responsible for ~90 million new cases of disease each year, with asymptomatic infections giving rise to sequelae such as pelvic inflammatory disease, ectopic pregnancy and infertility in women. Aggressive ‘seek and treat’ public health measures have not stemmed the rise of Ct infections, leading to proposal of the ‘arrested immunity’ hypothesis, where herd immunity within a population is blunted following earlier treatment with antibiotics. This suggests vaccination as the next key step in potentially eliminating Ct infections. Due to the paucity of robust clinical data, protective immune parameters have largely been derived from studies in mice, where pathogen-specific Th1-type immunity involving CD4+ T cells has been the focus of preclinical vaccine development. Our study represents the first preclinical characterization of a novel, rationally designed second-generation lipid adjuvant (SLA) in combination with the highly conserved recombinant Ct polymorphic membrane protein D (rPmpD) antigen. We demonstrate robust protection against urogenital Ct infection in the C57BL/6 murine model, characterized by significantly enhanced resistance to infection and reduction in mean bacterial load. However, enhanced Th1-type immunity was not found to correlate with relative protection, which rather coincided with the presence of robust rPmpD-specific serum and cervico-vaginal IgG titres. Prior to our study, the only convincing evidence of neutralizing antibodies effecting protection against chlamydial infection in vivo has emerged from studies employing the antigenically variable Ct MOMP as a vaccine immunogen. We propose that anti-rPmpD antibodies may play a significant role in vaccine-induced protection against urogenital Ct challenge, and that the role of antibodies warrants further investigation in future Ct vaccine development

The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen, and the leading cause of infectious blindness worldwide. We have recently shown that immunization with the highly conserved antigenic passenger domain of recombinant Ct polymorphic membrane protein D (rPmpD) is protective in the mouse model of Ct genital tract infection, and previously, that ocular anti-rPmpD antibodies are elicited following vaccination. However, the mechanisms governing the assembly and structure-function relationship of PmpD are unknown. Here, we provide a biophysical analysis of this immunogenic 65 kDa passenger domain fragment of PmpD. Using differential cysteine labeling coupled with LC-MS/MS analysis, we show that widespread intra- and intermolecular disulphide interactions play important roles in the preservation of native monomeric secondary structure and the formation of higher-order oligomers. While it has been proposed that FxxN and GGA(I, L,V) repeat motifs in the Pmp21 ortholog in Chlamydia pneumoniae mediate self-interaction, no such role has previously been identified for cysteine residues in chlamydial Pmps. Further characterisation reveals that oligomeric proteoforms and rPmpD monomers adopt β–sheet folds, consistent with previously described Gram-negative bacterial type V secretion systems (T5SSs). We also highlight adhesin-like properties of rPmpD, showing that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate binding of rPmpD-coated beads to mammalian cells in a dose-dependent fashion. Hence, our study provides further evidence that chlamydial Pmps may function as adhesins, while elucidating yet another important mechanism of self-association of bacterial T5SS virulence factors that may be unique to the Chlamydiaceae

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue

Large-Scale Immunopeptidome Analysis Reveals Recurrent Posttranslational Splicing of Cancer- and Immune-Associated Genes

Posttranslational spliced peptides (PTSPs) are a unique class of peptides that have been found to be presented by HLA class-I molecules in cancer. Thus far, no consensus has been reached on the proportion of PTSPs in the immunopeptidome, with estimates ranging from 2% to as high as 45% and stirring significant debate. Furthermore, the role of the HLA class-II pathway in PTSP presentation has been studied only in diabetes. Here, we exploit our large-scale cancer peptidomics database and our newly devised pipeline for filtering spliced peptide predictions to identify recurring spliced peptides, both for HLA class-I and class-II complexes. Our results indicate that HLA class-I–spliced peptides account for a low percentage of the immunopeptidome (less than 3.1%) yet are larger in number relative to other types of identified aberrant peptides. Therefore, spliced peptides significantly contribute to the repertoire of presented peptides in cancer cells. In addition, we identified HLA class-II–bound spliced peptides, but to a lower extent (less than 0.5%). The identified spliced peptides include cancer- and immune-associated genes, such as the MITF oncogene, DAPK1 tumor suppressor, and HLA-E, which were validated using synthetic peptides. The potential immunogenicity of the DAPK1- and HLA-E–derived PTSPs was also confirmed. In addition, a reanalysis of our published mouse single-cell clone immunopeptidome dataset showed that most of the spliced peptides were found repeatedly in a large number of the single-cell clones. Establishing a novel search-scheme for the discovery and evaluation of recurring PTSPs among cancer patients may assist in identifying potential novel targets for immunotherapy

Hypoxic microenvironment shapes HIV-1 replication and latency

Abstract: Viral replication is defined by the cellular microenvironment and one key factor is local oxygen tension, where hypoxia inducible factors (HIFs) regulate the cellular response to oxygen. Human immunodeficiency virus (HIV) infected cells within secondary lymphoid tissues exist in a low-oxygen or hypoxic environment in vivo. However, the majority of studies on HIV replication and latency are performed under laboratory conditions where HIFs are inactive. We show a role for HIF-2α in restricting HIV transcription via direct binding to the viral promoter. Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin, mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. Our data support a model where the low-oxygen environment of the lymph node may suppress HIV replication and promote latency. We identify a mechanism that may contribute to the limited efficacy of latency reversing agents in reactivating HIV and suggest new strategies to control latent HIV-1

Broad and strong memory CD4(+)and CD8(+)T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide–MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design

Impact of micropolymorphism outside the 1 peptide binding groove in the clinically-relevant allele HLA-C*14 on T cell responses in HIV-1 infection

There is increasing evidence for the importance of HLA-C-restricted CD8+ 28 T-cells in HIV-1 control, but these responses are relatively poorly investigated. Numbers of HLA-C-restricted HIV-1 epitopes identified are much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 non-protective alleles. We identified twenty-five 8-11mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+ /14:03+ 34 cells. Analysis of T-cell responses to these peptides identified novel 6 T-cell epitopes targeted in HIV-1-infected HLA-C*14:02+ /14:03+ subjects. Analyses using HLA-stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell-surface stabilization of HLA-C*14:02 than HLA-C*14:03. T-cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals,with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T-cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ one, the present study indicates that these HLA-C*14 subtypes differentially impact on HIV-1 control by T-cells and NK cells

Guía para la evaluación de riesgo ambiental de organismos genéticamente modificados

144 páginasEl propósito de la Guía es que ésta sea utilizada en el día a día, como una referencia rápida y objetiva para resolver las cuestiones que se plantean, permitiendo seguir caminos seguros y específicos para cada evento de transformación generado por la biotecnología moderna. Se compone de dos secciones: en la primera se explican todos los pasos de la evaluación de riesgo y su relación con los demás elementos de análisis de riesgos. En la segunda sección el lector tiene seis ejemplos concretos de aplicación del tutorial proporcionado por la guía. Aunque la misma sigue las directrices generales del Protocolo de Cartagena, mantiene un nivel de simplicidad y claridad sin precedentes y seguramente será una de las herramientas más útiles en la evaluación del riesgo ambiental de OGMs, tanto para principiantes como para los evaluadores más experimentados

Hypoxic microenvironment shapes HIV-1 replication and latency

Abstract: Viral replication is defined by the cellular microenvironment and one key factor is local oxygen tension, where hypoxia inducible factors (HIFs) regulate the cellular response to oxygen. Human immunodeficiency virus (HIV) infected cells within secondary lymphoid tissues exist in a low-oxygen or hypoxic environment in vivo. However, the majority of studies on HIV replication and latency are performed under laboratory conditions where HIFs are inactive. We show a role for HIF-2α in restricting HIV transcription via direct binding to the viral promoter. Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin, mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. Our data support a model where the low-oxygen environment of the lymph node may suppress HIV replication and promote latency. We identify a mechanism that may contribute to the limited efficacy of latency reversing agents in reactivating HIV and suggest new strategies to control latent HIV-1

HLA variants have different preferences to present proteins with specific molecular functions which are complemented in frequent haplotypes

Human leukocyte antigen (HLA) genes are the most polymorphic loci in the human genome and code for proteins that play a key role in guiding adaptive immune responses by presenting foreign and self peptides (ligands) to T cells. Each person carries up to 6 HLA class I variants (maternal and paternal copies of HLA-A, HLA-B and HLA-C genes) and also multiple HLA class II variants, which cumulatively define the landscape of peptides presented to T cells. Each HLA variant has its own repertoire of presented peptides with a certain sequence motif which is mainly defined by peptide anchor residues (typically the second and the last positions for HLA class I ligands) forming key interactions with the peptide-binding groove of HLA. In this study, we aimed to characterize HLA binding preferences in terms of molecular functions of presented proteins. To focus on the ligand presentation bias introduced specifically by HLA-peptide interaction we performed large-scale in silico predictions of binding of all peptides from human proteome for a wide range of HLA variants and established which functions are characteristic for proteins that are more or less preferentially presented by different HLA variants using statistical calculations and gene ontology (GO) analysis. We demonstrated marked distinctions between HLA variants in molecular functions of preferentially presented proteins (e.g. some HLA variants preferentially present membrane and receptor proteins, while others – ribosomal and DNA-binding proteins) and reduced presentation of extracellular matrix and collagen proteins by the majority of HLA variants. To explain these observations we demonstrated that HLA preferentially presents proteins enriched in amino acids which are required as anchor residues for the particular HLA variant. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Taking into consideration that HLA alleles are inherited in haplotypes, we hypothesized that haplotypes composed of a combination of HLA variants with different presentation preferences should be more advantageous as they allow presenting a larger repertoire of peptides and avoiding holes in immunopeptidome. Indeed, we demonstrated that HLA-A/HLA-B and HLA-A/HLA-C haplotypes which have a high frequency in the human population are comprised of HLA variants that are more distinct in terms of functions of preferentially presented proteins than the control pairs