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Worldwide Salmonella entericainfections result in substantial morbidity and mortality and are the major cause of infant bacteremia in Sub-Saharan Africa. Diseases caused by Salmonellaare treatable with antibiotics, but successful antibiotic treatment has become difficult due to antimicrobial resistance and collateral effects on the microbiome. An effective vaccine together with public health efforts may be a better strategy to control these infections. Protective immunity against Salmonelladepends primarily on CD4 T-cell-mediated immune responses; therefore, identifying relevant T-cell antigens is necessary for Salmonellavaccine development. We previously used a dendritic-cell-based immunoproteomics approach in our laboratory to identify T-cell antigens. The testing of these antigens as vaccine candidates against Chlamydiainfection in mice yielded positive results. We applied this technology in the present study by infecting murine bone-marrow-derived dendritic cells from C57BL/6 mice with Salmonella entericastrain SL1344, followed by immunoaffinity isolation of MHC class I and II molecules and elution of bound peptides. The sequences of the peptides were identified using tandem mass spectrometry. We identified 87 MHC class-II- and 23 MHC class-I-binding Salmonella-derived peptides. Four of the 12 highest scoring class-II-binding Salmonellapeptides stimulated IFN-γ production by CD4+T cells from the spleens of mice with persistent Salmonellainfection. We conclude that antigens identified by MHC immunoproteomics will be useful for Salmonellaimmunobiology studies and are potential Salmonellavaccine candidates. Data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD004451.

ABSTRACTUrogenital Chlamydiaserovars replicating in reproductive epithelium pose a unique challenge to host immunity and vaccine development. Previous studies have shown that CD4 T cells are necessary and sufficient to clear primary Chlamydia muridarumgenital tract infections in the mouse model, making a protective CD4 T cell response a logical endpoint for vaccine development. Our previous proteomics studies identified 13 candidate Chlamydiaproteins for subunit vaccines. Of those, PmpG-1 is the most promising vaccine candidate. To further that work, we derived a PmpG303-311-specific multifunctional Th1 T cell clone, designated PmpG1.1, from an immune C57BL/6 mouse and used it to investigate the presentation of the PmpG303-311epitope by infected epithelial cells. Epithelial presentation of the PmpG303-311epitope required bacterial replication, occurred 15 to 18 h postinfection, and was unaffected by gamma interferon (IFN-?) pretreatment. Unlike epitopes recognized by other Chlamydia-specific CD4 T cell clones, the PmpG303-311epitope persisted on splenic antigen-presenting cells (APC) of mice that cleared primary genital tract infections. PmpG1.1 was activated by unmanipulated irradiated splenocytes from immune mice without addition of exogenous Chlamydiaantigen, and remarkably, activation of PmpG1.1 by unmanipulated immune splenocytes was stronger 6 months postinfection than it was 3 weeks postinfection. Enhanced presentation of PmpG303-311epitope on splenic APC 6 months postinfection reflects some type of “consolidation” of a protective immune response. Understanding the antigen-presenting cell populations responsible for presenting PmpG303-311early (3 weeks) and late (6 months) postinfection will likely provide important insights into stable protective immunity against Chlamydiainfections of the genital tract.

ABSTRACTMajor impediments to a Chlamydiavaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model against Chlamydiainfection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for a Chlamydiavaccine, we evaluated 10 new ChlamydiaT cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6'-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720–CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4+T cells coexpressing gamma interferon (IFN-?) and tumor necrosis factor alpha (TNF-a), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13 ChlamydiaT cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly defined Chlamydiavaccine.

ABSTRACTMajor impediments to developing a Chlamydiavaccine lie in identifying immunologically relevant T-cell antigens and delivery in a manner to stimulate protective immunity. Using an immunoproteomic approach, we previously identified three immunodominant ChlamydiaT-cell antigens (PmpG-1, PmpE/F-2, and RplF). Because RplF has high homology to a human ortholog, it may not be suitable for human vaccine development. Therefore, in this study, we evaluated protection against Chlamydiainfection in the genital tract in C57BL/6 mice immunized with Chlamydia-specific membrane proteins PmpG-1, PmpE/F-2, and major outer membrane protein (MOMP; as a reference) or a combination of them formulated with one of three adjuvants, CpG oligodeoxynucleotide (CpG-ODN), AbISCO-100 (AbISCO), or DDA/TDB (dimethyldioctadecylammonium bromide/d-(+)-trehalose 6,6′-dibehenate). The results show that immunization with the CpG-ODN formulation failed to provide protection against Chlamydiainfection; the AbISCO formulation conferred moderate protection, and the DDA/TDB formulation showed the highest degree of protective efficacy. The combination of PmpG-1, PmpE/F-2, and MOMP proteins formulated with DDA/TDB exhibited the greatest degree of protection among all vaccine groups studied. Moreover, this vaccine combination also engendered significant protection in BALB/c mice, which have a different major histocompatibility complex (MHC) background. We measured cell-mediated immune cytokine responses in mice immunized with PmpG-1 mixed with each of the three adjuvants. The results demonstrate that mice immunized with the DDA/TDB formulation induced the strongest gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses, characterized by the highest frequency of IFN-γ/tumor necrosis factor alpha (TNF-α) and IFN-γ/IL-17 double-positive CD4+T cells. In conclusion, a Chlamydiavaccine based on the recombinant proteins PmpG-1, PmpE/F-2, and MOMP delivered in a DDA/TDB adjuvant conferred protection against infection that correlated with IFN-γ/TNF-α and IFN-γ/IL-17 double-positive CD4+T cells.

Major impediments to developing a Chlamydia vaccine lie in identifying immunologically relevant T-cell antigens and delivery in a manner to stimulate protective immunity. Using an immunoproteomic approach, we previously identified three immunodominant Chlamydia T-cell antigens (PmpG-1, PmpE/F-2, and RplF). Because RplF has high homology to a human ortholog, it may not be suitable for human vaccine development. Therefore, in this study, we evaluated protection against Chlamydia infection in the genital tract in C57BL/6 mice immunized with Chlamydia-specific membrane proteins PmpG-1, PmpE/F-2, and major outer membrane protein (MOMP; as a reference) or a combination of them formulated with one of three adjuvants, CpG oligodeoxynucleotide (CpG-ODN), AbISCO-100 (AbISCO), or DDA/TDB (dimethyldioctadecylammonium bromide/D-(+)-trehalose 6,6'-dibehenate). The results show that immunization with the CpG-ODN formulation failed to provide protection against Chlamydia infection; the AbISCO formulation conferred moderate protection, and the DDA/TDB formulation showed the highest degree of protective efficacy. The combination of PmpG-1, PmpE/F-2, and MOMP proteins formulated with DDA/TDB exhibited the greatest degree of protection among all vaccine groups studied. Moreover, this vaccine combination also engendered significant protection in BALB/c mice, which have a different major histocompatibility complex (MHC) background. We measured cell-mediated immune cytokine responses in mice immunized with PmpG-1 mixed with each of the three adjuvants. The results demonstrate that mice immunized with the DDA/TDB formulation induced the strongest gamma interferon (IFN-) and interleukin-17 (IL-17) responses, characterized by the highest frequency of IFN-/tumor necrosis factor alpha (TNF-) and IFN-/IL-17 double-positive CD4+T cells. In conclusion, a Chlamydia vaccine based on the recombinant proteins PmpG-1, PmpE/F-2, and MOMP delivered in a DDA/TDB adjuvant conferred protection against infection that correlated with IFN-/TNF- and IFN-/IL-17 double-positive CD4+T cells.

ABSTRACTHuman papillomavirus (HPV) type 16 and 18 neutralizing antibody (NAb) titers were measured in 1,020 prenatal women in British Columbia aged 15 to 39. HPV 16 and 18 NAbs were detected in 183/1,020 (17.9%) and 97/1,020 (9.5%), respectively, and 39 (3.8%) had NAbs to both types. Titers were similar across age strata.

ABSTRACTRecent whole-genome analysis has demonstrated limited genetic variation in Chlamydia pneumoniae, with one strain (AR39) containing a 4,524 nucleotide single-stranded DNA bacteriophage, FCpn1. Using PCR, reverse transcription (RT)-PCR, and Western blotting, we confirmed the presence and functional expression of FCpn1 in C. pneumoniaestrain AR39 and its absence in strain CWL029. Six additional epidemiologically distinct clinical isolates of C. pneumoniaealso did not contain FCpn1. We generated recombinant viral protein 1 (Vp1) from FCpn1 in Escherichia coliand showed that Vp1 antigen is highly immunogenic in mice and that murine antisera readily recognize native Vp1 from C. pneumoniaestrain AR39 elementary bodies (EB). We developed an enzyme-linked immunosorbent assay (ELISA) to measure antibodies to recombinant Vp1 in human sera collected from 32 patients with abdominal aortic aneurysm (AAA) and 40 controls. Among the 72 subjects, 61 had C. pneumoniaeEB antibodies shown by ELISA. Antibodies to Vp1 were found in 39 of the 61 (64%) seropositive individuals and were significantly correlated with AAA (adjusted odds ratio, 13.9; 95% confidence interval, 1.1 to 175). Our studies indicate that phage-containing strains of C. pneumoniaeare uncommonly found by isolation but may commonly infect individuals with vascular disease.

ABSTRACTSurveillance and defense of the enormous mucosal interface with the nonsterile world are critical to protecting the host from a wide range of pathogens. Chlamydia trachomatisis an intracellular bacterial pathogen that replicates almost exclusively in the epithelium of the reproductive tract. The fallopian tubes and vagina are poorly suited to surveillance and defense, with limited immune infrastructure positioned near the epithelium. However, a dynamic process during clearing primary infections leaves behind new lymphoid clusters immediately beneath the epithelium. These memory lymphocyte clusters (MLCs) harboring tissue-resident memory (Trm) T cells are presumed to play an important role in protection from subsequent infections. Histologically, human ChlamydiaMLCs have prominent B cell populations. We investigated the status of genital tract B cells during C. muridaruminfections and the nature of T cells recovered from immune mice using immune B cells as antigen-presenting cells (APCs). These studies revealed a genital tract plasma B cell population and a novel genital tract CD4 T cell subset producing both gamma interferon (IFN-γ) and interleukin-13 (IL-13). A panel of CD4 T cell clones and microarray analysis showed that the molecular fingerprint of CD4γ13 T cells includes a Trm-like transcriptome. Adoptive transfer of a Chlamydia-specific CD4γ13 T cell clone completely prevented oviduct immunopathology without accelerating bacterial clearance. Existence of a CD4γ13 T cell subset provides a plausible explanation for the observation that human peripheral blood mononuclear cell (PBMC) Chlamydia-specific IFN-γ and IL-13 responses predict resistance to reinfection.

BACKGROUND: A relationship between Chlamydia pneumoniae infection and acute coronary syndromes has not been consistently found in published studies. It has been hypothesized that a bacteriophage-infected subset of C pneumoniae may be uniquely equipped to promote atherosclerosis and acute coronary syndromes through the expression of phage genes. METHODS: The authors performed a pilot case-control study of acute coronary events. Case and control subjects were characterized demographically and according to recognized coronary risk factors. These subjects also provided serum for the detection of antibody to the elementary bodies of C pneumoniae and antibody to the Vp1 protein coded by the phage. Bivariate and multivariate comparisons were performed using statistics appropriate for paired analyses. RESULTS: Antibodies to C pneumoniae, Vp1 protein or both were not associated with acute coronary events by bivariate or multivariate analysis. As expected, case subjects were significantly more likely to have hypertension, hypercholesterolemia or diabetes mellitus. CONCLUSION: The present study adds to a growing body of literature that does not support the hypothesized relationship between C pneumoniae (or a phage-infected subset of C pneumoniae) and acute coronary syndromes.