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To prepare for the development of the 2021 Centers for Disease Control and Prevention (CDC) sexually transmitted infections treatment guidelines, the CDC convened a committee of expert consultants in June 2019 to discuss recent abstracts and published literature on the epidemiology, diagnosis, and management of sexually transmitted infections.This paper summarizes the key questions, evidence, and recommendations for the diagnosis and management of uncomplicated Chlamydia trachomatis (CT) infections in adolescents and adults that were reviewed and discussed for consideration in developing the guidelines. The evidence reviewed mostly focused on efficacy of doxycycline and azithromycin for urogenital, rectal, and oropharyngeal CT infection, CT risk factors in women, performance of CT nucleic acid amplification tests on self-collected meatal specimens in men, and performance of newer CT point-of-care tests.

The payoff for understanding Chlamydia trachomatis immunology is the development of a vaccine. Two lines of research have contributed to our current understanding of C. trachomatis immunology. The first is the Grayston model of type-specific immunity and genus-specific pathology, which was elaborated by Caldwell and Morrison as the major outer membrane protein and heat shock protein 60 paradigm. The second is the murine model of Chlamydia muridarum infection, which established the essential role of major histocompatibility complex class II and CD4 T cells in immunity. However, neither approach has yielded a vaccine. I review these 2 lines of research and conclude with 6 problem areas in human C. trachomatis immunology whose resolution may result in a vaccine.

The traditional framework in which to study the biology of human infectious diseases involves characterizing interactions and features of the host, pathogen, and environment. Using the tools of epidemiology, immunology, and genomics allows one to study the biology of infectious disease within this framework. The study of Chlamydia trachomatis biology vividly illustrates the usefulness for the approach. I note key findings from my own studies on C. trachomatis epidemiology, immunology, and genomics to show how important light has been shed on its biology and how this has impacted the Chlamydia field generally. In particular, the epidemiology of C. trachomatis diseases in women shows its impact on reproduction and how public health programs to detect and treat infection has reduced that impact but at the cost of arresting the development of protective immunity and increasing the risk of infection and reinfection. Immunological studies demonstrate the importance of CD4 Th1 cells in protection and that antibiotic treatment interferes with the development of protective immunity when given early in the course of infection. Evaluating the T-cell antigen landscape for C. trachomatis and Chlamydia muridarum demonstrates the role of surface proteins such as the major outer-membrane protein and the polymorphic membrane proteins as major protective CD4 T-cell antigens. Genomic studies reveal that the genome of organism has 3 loci of immunological interest. The antigen loci of the major outer-membrane protein and polymorphic membrane proteins are hotspots for both mutation and recombination, and the plasticity zone contains immune evasion genes that are highly variable from species to species. Interestingly, these 3 loci seem to have entered the Chlamydia phylum at the time of the evolution of the Chlamydiaceae when they became pathogens of vertebrates and encountered the adaptive immune system. In aggregate, these 3 approaches have shed light on human C. trachomatis infections and suggest paths for vaccine development. These approaches are likely to remain useful for the further study of C. trachomatis and for other human pathogens.

Background Chlamydia trachomatis and Chlamydia muridarum are intracellular bacterial pathogens of mucosal epithelial cells. CD4 T cells and major histocompatibility complex (MHC) class II molecules are essential for protective immunity against them. Antigens presented by dendritic cells (DCs) expand naive pathogen-specific T cells (inductive phase), whereas antigens presented by epithelial cells identify infected epithelial cells as targets during the effector phase. We previously showed that DCs infected by C trachomatis or C muridarum present epitopes from a limited spectrum of chlamydial proteins recognized by Chlamydia-specific CD4 T cells from immune mice. Methods We hypothesized that Chlamydia-infected DCs and epithelial cells present overlapping sets of Chlamydia-MHC class II epitopes to link inductive and effector phases to generate protective immunity. We tested that hypothesis by infecting an oviductal epithelial cell line with C muridarum, followed by immunoaffinity isolation and sequencing of MHC class I- and II-bound peptides. Results We identified 26 class I-bound and 4 class II-bound Chlamydia-derived peptides from infected epithelial cells. We were surprised to find that none of the epithelial cell class I- and class II-bound chlamydial peptides overlapped with peptides presented by DCs. Conclusions We suggest the discordance between the DC and epithelial cell immunoproteomes has implications for delayed clearance of Chlamydia and design of a Chlamydia vaccine.

Bacterial Infections of the Vagina Bacterial vaginal infections are in two main classes: bacterial vaginosis, associated with a fishy-smelling discharge and increased pH, and desquamative inflammat...

Gynecological and obstetrical infectious diseases are an important component of women's health. A system approach to gynecological and obstetrical infection helps unify and classify microbial etiology and pathogenesis within a clinical anatomical framework of lower and upper genital tract syndromes. The reproductive system of women includes the vulva, vagina, cervix, uterus, fallopian tubes and ovaries. During pregnancy, additional tissues include the chorioamnion and placenta together with the fetus and amniotic fluid. We review in two parts reproductive system infection syndromes in women using selected research results to illustrate the clinical utility of the system approach in terms of diagnosis, treatment and prevention. We conclude that a reproductive system perspective will lead to improvements in understanding, management and prevention of these diseases.

Lower genital tract infection and bloodborne spread of infection are the two principal modes for infection of the upper genital tract or for infection of the fetus, neonate or infant. Treponema pallidum and human immunodeficiency virus (HIV) are the two most common bloodborne pathogens that infect the fetus, neonate or infant. Most infections of the upper genital tract, however, spread along epithelial surfaces from the vagina or cervix to the upper genital tract or chorioamnion, fetus, neonate or infant. These infections are caused by either pathogens associated with a dysbiotic vaginal microbiome or those that are sexually transmitted. The clinical syndromes that these pathogens produce in the lower genital tract were discussed in part one of this review. We now discuss the syndromes and pathogens that affect the upper genital tract of both non-pregnant and pregnant women as well as fetus, neonate and infant.

Background and hypothesis Recently, the world has experienced a rapidly escalating outbreak of infectious syphilis primarily affecting men who have sex with men (MSM); many are taking highly active antiretroviral therapy (HAART) for HIV-1 infection. The prevailing hypothesis is that HAART availability and effectiveness have led to the perception among both individuals who are HIV-1 infected and those who are uninfected that HIV-1 transmission has become much less likely, and the effects of HIV-1 infection less deadly. This is expected to result in increased sexual risk-taking, especially unprotected anal intercourse, leading to more non-HIV-1 STDs, including gonorrhoea, chlamydia and syphilis. However, syphilis incidence has increased more rapidly than other STDs. We hypothesise that HAART downregulates the innate and acquired immune responses to Treponema pallidum and that this biological explanation plays an important role in the syphilis epidemic. Methods We performed a literature search and developed a mathematical model of HIV-1 and T. pallidum confection in a population with two risk groups with assortative mixing to explore the consequence on syphilis prevalence of HAART-induced changes in behaviour versus HAART-induced biological effects. Conclusions and implications Since rising syphilis incidence appears to have outpaced gonorrhoea and chlamydia, predominantly affecting HIV-1 positive MSM, behavioural factors alone may be insufficient to explain the unique, sharp increase in syphilis incidence. HAART agents have the potential to alter the innate and acquired immune responses in ways that may enhance susceptibility to T. pallidum . This raises the possibility that therapeutic and preventative HAART may inadvertently increase the incidence of syphilis, a situation that would have significant and global public health implications. We propose that additional studies investigating the interplay between HAART and enhanced T. pallidum susceptibility are needed. If our hypothesis is correct, HAART should be combined with enhanced patient management including frequent monitoring for pathogens such as T. pallidum .

The Chlamydial outer membrane complex (COMC) from the elementary body (EB) is a protein rich insoluble outer membrane shell from which cytosolic proteins have been extracted with detergent. In this study we conducted mass spectrometry experiments to detect proteins in the COMC prepared from C. muridarum EB. Proteomic analysis showed that the COMC contained 75 proteins that included 10 outer membrane proteins (OMPs) such as the major outer membrane protein (MOMP) and polymorphic membrane proteins (Pmps) that were previously identified as CD4 T cell vaccine candidates. We tested the vaccine efficacy of COMC in comparison to individual or combination of recombinant OMPs formulated with Th1 polarizing adjuvant DDA/MPL in two murine genital tract models (C. muridarum and C. trachomatis) by measuring organismal shedding, tubal pathology and immune responses including neutralizing antibodies. In the C. muridarum model, the COMC vaccine generated broadly reactive immune responses against multiple outer membrane proteins, high levels of EB binding and neutralizing antibody and exhibited superior protection against genital infection and pathology when compared to the recombinant PmpG vaccine. Denaturing the COMC by boiling significantly reduced protection. In the C. trachomatis model, the COMC vaccine also conferred greater protection compared to individual or multiple recombinant outer membrane proteins. Immunization with multiple COMCs from C. trachomatis serovars D, F and J generated neutralizing antibodies against multiple C. trachomatis serovars. We conclude that broader immunogenicity and generation of neutralizing antibody may explain the superior efficacy of COMC vaccine. The study suggests that conformationally intact proteins will be necessary for a successful recombinant OMP vaccine.

Worldwide Salmonella enterica infections result in substantial morbidity and mortality and are the major cause of infant bacteremia in Sub-Saharan Africa. Diseases caused by Salmonella are 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 Salmonella depends primarily on CD4 T-cell-mediated immune responses; therefore, identifying relevant T-cell antigens is necessary for Salmonella vaccine 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 Chlamydia infection 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 enterica strain 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 Salmonella peptides stimulated IFN-γ production by CD4+ T cells from the spleens of mice with persistent Salmonella infection. We conclude that antigens identified by MHC immunoproteomics will be useful for Salmonella immunobiology studies and are potential Salmonella vaccine candidates. Data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD004451.

For almost 2 decades, results from Chlamydia pathogenesis investigations have been conceptualized using a cytokine polarization narrative. Recent viral immunity studies identifying protective tissue-resident memory T cells (Trm) suggest an alternative paradigm based on localized immune networks. As Chlamydia vaccines enter the preclinical pipeline and, in the case of an attenuated trachoma vaccine, are given to human subjects, it may be useful to ask whether cytokine polarization is the appropriate framework for understanding and evaluating vaccine efficacy. In this review, we revisit C. trachomatis pathogenesis data from mice and humans using a Trm narrative and note a comfortable concordance with the Chlamydia pathogenesis literature.

KEY POINTS The revolution in genomics is transforming medicine.[1][1] Among its important contributions is a demonstration — through a greater understanding of the human genome and the microbiome — that evolutionary biology underpins the principles and practice of medicine. Evolutionary medicine

Surveillance and defense of the enormous mucosal interface with the nonsterile world are critical to protecting the host from a wide range of pathogens. Chlamydia trachomatis is 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 Chlamydia MLCs have prominent B cell populations. We investigated the status of genital tract B cells during C. muridarum infections 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.

A successful vaccine could be a cost-effective addition to current screening practices., We explored potential cost-effectiveness of a chlamydia vaccine for young women in the United States by using a compartmental heterosexual transmission model. We tracked health outcomes (acute infections and sequelae measured in quality-adjusted life-years [QALYs]) and determined incremental cost-effectiveness ratios (ICERs) over a 50-year analytic horizon. We assessed vaccination of 14-year-old girls and catch-up vaccination for 15–24-year-old women in the context of an existing chlamydia screening program and assumed 2 prevaccination prevalences of 3.2% by main analysis and 3.7% by additional analysis. Estimated ICERs of vaccinating 14-year-old girls were $35,300/QALY by main analysis and $16,200/QALY by additional analysis compared with only screening. Catch-up vaccination for 15–24-year-old women resulted in estimated ICERs of $53,200/QALY by main analysis and $26,300/QALY by additional analysis. The ICER was most sensitive to prevaccination prevalence for women, followed by cost of vaccination, duration of vaccine-conferred immunity, and vaccine efficacy. Our results suggest that a successful chlamydia vaccine could be cost-effective.

Pelvic inflammatory disease can produce acute symptoms and result in infertility, ectopic pregnancy, and chronic pelvic pain. This review summarizes current approaches to diagnosis and treatment and the future prospects for better prevention strategies.

Chlamydia antigen analysis enables understanding of disease pathogenesis, facilitates development of diagnostic immunoassays and is essential to the design of a subunit Chlamydia trachomatis vaccine. Using an autobiographical narrative, I review over three decades of antigen analysis research findings coming from my research laboratory and provide an outlook to the broader field of Chlamydia seroepidemiology and vaccinology. Based on the experiences of my scientific career I conclude with thoughts for young scientists newly entering the Chlamydia research field.

We appreciate the thoughtful comments of Vicetti Miguel et al. One of the main purposes of writing a review is to generate a discussion, and optimal times for reviews are moments where long held paradigms begin to look a little shaky. We do not disagree with the commentators' critique of the

Attempts to produce a vaccine to protect against Chlamydia trachomatis -induced trachoma were initiated more than 100 years ago and continued for several decades. Using whole organisms, protective responses were obtained. However, upon exposure to C. trachomatis , disease exacerbation developed in some immunized individuals, precluding the implementation of the vaccine. Evidence of the role of C. trachomatis as a sexually transmitted pathogen started to emerge in the 1960s, and it soon became evident that it can cause acute infections and long-term sequelae in women, men, and newborns. The main focus of this minireview is to summarize recent findings and discuss formulations, including antigens, adjuvants, routes, and delivery systems for immunization, primarily explored in the female mouse model, with the goal of implementing a vaccine against C. trachomatis genital infections.

Chlamydia trachomatis (Ct), an intracellular pathogen, is the most common bacterial sexually transmitted infection. In addition to acute cervicitis and urethritis, Ct can lead to serious sequelae of significant public health burden including pelvic inflammatory disease (PID) and infertility. Ct control efforts have not resulted in desired outcomes such as reduced incidence and reinfection, and this highlights the need for the development of an effective Ct vaccine. To this end, NIAID organized a workshop to consider the current status of Ct vaccine research and address critical questions in Ct vaccine design and clinical testing. Topics included the goal(s) of a vaccine and the feasibility of achieving these goals, animal models of infection including mouse and nonhuman primate (NHP) models, and correlates of protection to guide vaccine design. Decades of research have provided both whole cell-based and subunit vaccine candidates for development. At least one is currently in clinical development and efforts now need to be directed toward further development of the most attractive candidates. Overall, the discussions and presentations from the workshop highlighted optimism about the current status of Ct vaccine research and detailed the remaining gaps and questions needed to move vaccines forward.

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.

Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that include tuberculosis, malaria, HIV/AIDS, and others such as Chlamydia trachomatis. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. Genomics and proteomics now provide tools to allow unbiased empirical identification of candidate T cell antigens. This approach represents an advance on bioinformatic searches for candidate T cell antigens. This chapter discusses an immunoproteomic approach we have used to identify Chlamydia T cell antigens. We further discuss how these T cell antigens can be developed into a human vaccine.

Chlamydia trachomatis is the most common preventable cause of tubal infertility in women. In high-income countries, despite public health control efforts, C. trachomatis case rates continue to rise. Most medium and low-income countries lack any Chlamydia control program; therefore, a vaccine is essential for the control of Chlamydia infections. A rationally designed Chlamydia vaccine requires understanding of the immunological correlates of protective immunity, pathological responses to this mucosal pathogen, identification of optimal vaccine antigens and selection of suitable adjuvant delivery systems that engender protective immunity. Fortunately, Chlamydia vaccinology is facilitated by genomic knowledge and by murine models that reproduce many of the features of human C. trachomatis infection. This article reviews recent progress in these areas with a focus on subunit vaccine development.

Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that include tuberculosis, malaria, HIV/AIDS, and others such as Chlamydia trachomatis. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. Genomics and proteomics now provide tools to allow unbiased empirical identification of candidate T cell antigens. This approach represents an advance on bioinformatic searches for candidate T cell antigens. This chapter discusses an immunoproteomic approach we have used to identify Chlamydia T cell antigens. We further discuss how these T cell antigens can be developed into a human vaccine.

Background. Many countries have witnessed a disturbing increase in cases of Chlamydia trachomatis infection despite enhanced control programs. Since the goal of Chlamydia control is to prevent reproductive complications such as pelvic inflammatory disease and ectopic pregnancy, an understanding of recent trends in these conditions is needed to fully evaluate the effect of control efforts. Methods. We analyzed 2 provincial, comprehensive health services administrative databases (encompassing hospitalizations and all physician-delivered services) for pelvic inflammatory disease and ectopic pregnancy trends from 1992 through 2009 in women of reproductive age in British Columbia, Canada. Trends were compared to provincial Chlamydia surveillance data by time-series analysis, using the cross-correlation function method and Granger causality testing. Results. Chlamydia cases substantially increased from 1992 through 2009. Inpatient, outpatient, and total diagnoses of pelvic inflammatory disease and ectopic pregnancy declined from 1992 through 2003. After 2003, pelvic inflammatory disease rates continued to fall, while ectopic pregnancy rates significantly increased. The male Chlamydia urethritis rate increased from 39.4 to 173.6 cases/100 000 from 1996 to 2009. Conclusions. In the context of increasing Chlamydia infection rates, the reproductive complications of Chlamydia infection in women are declining overall. A recent increase in rates of ectopic pregnancies is cause for concern.

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ+–producing CD4+ T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4+ T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.

Major impediments to developing aChlamydiavaccine 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 immunodominantChlamydiaT-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 againstChlamydiainfection in the genital tract in C57BL/6 mice immunized withChlamydia-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 againstChlamydiainfection; 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, aChlamydiavaccine 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.

We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post-infection. Intrapulmonary cytokine patterns also differed at early time-points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post-infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor-alpha and interleukin-17 (IL-17) and a significantly lower level for interferon-gamma than did C57BL/6 mice. In vitro, bone-marrow-derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL-12 and more IL-23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.

Background Between 5 and 25 April 2009, pandemic (H1N1) 2009 caused a substantial, severe outbreak in Mexico, and subsequently developed into the first global pandemic in 41 years. We determined the reproduction number of pandemic (H1N1) 2009 by analyzing the dynamics of the complete case series in Mexico City during this early period. Methods We analyzed three mutually exclusive datasets from Mexico City Distrito Federal which constituted all suspect cases from 15 March to 25 April: confirmed pandemic (H1N1) 2009 infections, non-pandemic influenza A infections and patients who tested negative for influenza. We estimated the initial reproduction number from 497 suspect cases identified prior to 20 April, using a novel contact network methodology incorporating dates of symptom onset and hospitalization, variation in contact rates, extrinsic sociological factors, and uncertainties in underreporting and disease progression. We tested the robustness of this estimate using both the subset of laboratory-confirmed pandemic (H1N1) 2009 infections and an extended case series through 25 April, adjusted for suspected ascertainment bias. Results The initial reproduction number (95% confidence interval range) for this novel virus is 1·51 (1·32–1·71) based on suspected cases and 1·43 (1·29–1·57) based on confirmed cases before 20 April. The longer time series (through 25 April) yielded a higher estimate of 2·04 (1·84–2·25), which reduced to 1·44 (1·38–1·51) after correction for ascertainment bias. Conclusions The estimated transmission characteristics of pandemic (H1N1) 2009 suggest that pharmaceutical and non-pharmaceutical mitigation measures may appreciably limit its spread prior the development of an effective vaccine.

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

Summary We report two novel human leukocyte antigen G (HLA-G) alleles identified in an East African population during sequence-based typing of HLA-G. The novel alleles were confirmed by sequencing multiple polymerase chain reaction products and molecular cloning and subsequent sequencing of multiple clones. The sequence of HLA-G*0110 ( EU290672 ) is identical to G*01010101/01010102/01010103/01010104/01010105 at exons 2, 3, and 4 except for a single nucleotide difference at codon 31 (ACG → ATG), resulting in a coding change from threonine to methionine. The sequence of HLA-G*0111 ( EU290673 ) is identical to G*010404 at exons 2, 3, and 4 except for a single nucleotide difference at codon 31 (ACG → ATG), resulting in a coding change from threonine to methionine. These new alleles are detected in several other individuals in our study population and the functional relevance of these new alleles must be studied.

Background. There is strong evidence that human papillomavirus (HPV) is necessary for the development of cervical cancer. A prophylactic HPV vaccine with high reported efficacy was approved in North America in 2006. Methods. A mathematical model of HPV transmission dynamics was used to simulate different scenarios of natural disease outcomes and intervention strategies. A sensitivity analysis was performed to compensate for uncertainties surrounding key epidemiological parameters. Results. The expected impact that HPV vaccines have on cervical cancer incidence and HPV prevalence in the province of British Columbia in Canada revealed that, for lifelong vaccine-related protection, an immunization routine targeting younger females (grade 6), combined with a 3-year program for adolescent females (grade 9), is the most effective strategy. If vaccine-related protection continues for10 years, then the targeting of adolescent females would be more beneficial than the targeting of younger females. The incremental benefit if boys, as well as girls, are vaccinated is small. Conclusions. Optimization of the design of immunization strategies for treatment of HPV depends substantially on the duration of vaccine-induced immunity. Given the uncertainty in estimating this duration, it may be prudent to assume a value close to the lower limit reported and adjust the program when more-accurate information for the length of vaccine-induced immunity becomes available. Human papillomavirus (HPV) is a sexually transmitted infection that has been linked to a variety of cancers in women and in men [1, 2]. HPV DNA is found in 99% of cervical cancer cases and is said to be the necessary, but insufficient, cause of cancer of the cervix [1–3]. Although the lifetime risk of contracting an HPV infection of the cervix has been reported to be 80%, most of these infections are transient and do not progress to cancer even when they are caused by an oncogenic HPV genotype [4 – 6]. Despite this, almost 500,000 women worldwide are diagnosed with cervical cancer each year and

Dendritic cells (DCs) appear to orchestrate much of the immunobiology ofChlamydiainfection, but most studies ofChlamydia-DC interaction have been limited by the availability and heterogeneity of primary bone marrow-derived DCs (BMDCs). We therefore evaluated the immunobiology ofChlamydia muridaruminfection in an immortal DC line termed JAWS II derived from BMDCs of a C57BL/6 p53-knockout mouse. JAWS II cells were permissive to the developmental cycle ofChlamydia. Infection-induced cell death was 50 to 80% less in JAWS II cells than in BMDCs.Chlamydiainfected JAWS II cells and yielded infectious progeny 10-fold greater than that with primary BMDCs. JAWS II cells showed an expression pattern of cell activation markers and cytokine secretion followingChlamydiainfection similar to that of primary BMDCs by up-regulating the expression of CD86, CD40, and major histocompatibility complex class II and secreting significant amounts of interleukin-12 (IL-12) but not IL-10. JAWS II cells pulsed withChlamydiastimulated immune CD4+T cells to secrete gamma interferon. Adoptive transfer of ex vivoChlamydia-pulsed JAWS II cells conferred levels of immunity on C57BL/6 mice similar to those conferred by primary BMDCs. Taken together, the data show that JAWS II cells exhibit immunobiological characteristics and functions similar to those of primary BMDCs in terms ofChlamydiaantigen presentation in vitro and antigen delivery in vivo. We conclude that the JAWS II cell line can substitute for primary BMDCs inChlamydiaimmunobiological studies.

Are we losing ground in our efforts to control sexually transmitted Chlamydia trachomatis infection? Before we can answer this question, we must first consider recent trends in Chlamydia from around the world to establish a baseline for understanding the possible explanations underlying these data.

Chlamydia trachomatis is the most important infectious cause of infertility in women with important implications in public health and for which a vaccine is urgently needed. Recent immunoproteomic vaccine studies found that four polymorphic membrane proteins (PmpE, PmpF, PmpG and PmpH) are immunodominant, recognized by various MHC class II haplotypes and protective in mouse models. In the present study, we aimed to evaluate genetic and protein features of Pmps (focusing on the N-terminal 600 amino acids where MHC class II epitopes were mapped) in order to understand antigen variation that may emerge following vaccine induced immune selection. We used several bioinformatics platforms to study: i) Pmps' phylogeny and genetic polymorphism; ii) the location and distribution of protein features (GGA(I, L)/FxxN motifs and cysteine residues) that may impact pathogen-host interactions and protein conformation; and iii) the existence of phase variation mechanisms that may impact Pmps' expression. We used a well-characterized collection of 53 fully-sequenced strains that represent the C. trachomatis serovars associated with the three disease groups: ocular (N=8), epithelial-genital (N=25) and lymphogranuloma venereum (LGV) (N=20). We observed that PmpF and PmpE are highly polymorphic between LGV and epithelial-genital strains, and also within populations of the latter. We also found heterogeneous representation among strains for GGA(I, L)/FxxN motifs and cysteine residues, suggesting possible alterations in adhesion properties, tissue specificity and immunogenicity. PmpG and, to a lesser extent, PmpH revealed low polymorphism and high conservation of protein features among the genital strains (including the LGV group). Uniquely among the four Pmps, pmpG has regulatory sequences suggestive of phase variation. In aggregate, the results suggest that PmpG may be the lead vaccine candidate because of sequence conservation but may need to be paired with another protective antigen (like PmpH) in order to prevent immune selection of phase variants. AN is a recipient of a post-doctoral fellowship (SFRH/BPD/75295/2010) from Fundação para a Ciência e a Tecnologia (FCT).

Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ producing CD4 T-cells. By contrast, mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T-cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAP) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b+CD103− dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b−CD103+ DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T-cells, but only mucosal vaccination induced effector T-cells that rapidly seeded uterine mucosa with resident memory T-cells (TRM). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T-cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T-cell subsets with distinct migratory properties.

In 2004, expert groups in North America recommended annual influenza immunization for healthy infants and toddlers aged 6-23 months with a goal of reducing high hospitalization rates (HR). We assess cost-effectiveness of this program in Canada.Analysis was from third-party payer and societal perspectives in preventing hospitalization and other outcomes among 500,000 vaccinated or non-vaccinated infants/toddlers. Base-case assumptions include: 25% attack rate (AR), 1% case hospitalization rate (HR), 0.002% case fatality, vaccine effectiveness (VE) of 66%, CDN dollar 15 per dose for vaccine and administration, half of mothers requiring 2h from work per dose to immunize and two doses required by 100% (first year) or 33% (subsequent years) of infants/toddlers immunized.After the first year, infant/toddler influenza immunization costs the third-party approximately CDN dollar 9 per day of illness averted, CDN dollar 120 per physician visit averted, CDN dollar 7000 per hospitalization averted, CDN dollar 3 million per death averted and CDN dollar 450,000 per life year gained. Corresponding costs from societal perspective are approximately CDN dollar 3, CDN dollar 45, CDN dollar 2500, CDN dollar 1 million and CDN dollar 170,000. The program becomes cost-saving from the third-party perspective at AR55%, HR4%, or cost per dose (for vaccine and its administration)CDN dollar 6.81. From the societal perspective corresponding break-even occurs at 28%, 2% and CDN dollar 11.90, or when half of all mothers require no more than 1.6h to immunize or VE is74%.In the base case, infant/toddler influenza immunization is not cost-saving but could become more cost-effective in settings of higher attack rate and lower immunization cost. In this context, immunization of children in daycare or other group settings should be preferentially considered. Economic analyses should routinely inform expansions to influenza immunization programs.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated byβ-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.

Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.

Purpose Persistence of sexually transmitted infections (STI) in a population is due to the activities of a small proportion of the population with STI, who transmit infection on average to one or more susceptible sex partners during an infectious period. Within these groups, the average number of transmissions by infectious people to susceptible people in a closed group is measured by the reproductive number; a threshold, above which endemic infection is likely occur and below which, in the rest of the population, it is unlikely to occur. We hypothesized that people with repeated bacterial STI's and their sex partners include the theoretical core group and that they differ from singly infected noncore individuals. Methods Data on infected individuals and nominated sex partners for the years 1990–1992 were extracted from the notifiable disease and health insurance registries in Manitoba, Canada. Individuals with repeated gonorrhea, chlamydia and coinfected infections were compared using logistic regression, and reproductive numbers were calculated using sex partner data. Results Of the three groups, the coinfected were youngest, and they were largely of aboriginal descent and had the lowest incomes. Repeaters were older; they had higher incomes, and there were fewer aboriginal people in this group. Chlamydia repeaters had even higher incomes, and this group comprised the least number of aboriginal people. The reproductive numbers showed the same gradient; (1.09, 1.01, and 2.41, respectively.) Conclusions These data show that these groups do not contribute equally to STI endemicity and indicate that sexual network structure affects epidemic thresholds.

Epidemiological, animal, and in vitro investigations suggest that Chlamydia trachomatis infection engenders acquired immunity, the basis for which is incompletely defined, especially in humans. In a prospective cohort study of women at high risk for C. trachomatis infection, we found that, at baseline and after adjustment for age and other potential confounding variables, production of interferon- gamma by peripheral-blood mononuclear cells (PBMCs) stimulated with chlamydia heat-shock protein 60 strongly correlated with protection against incident C. trachomatis infection. This investigation supports a direct role for C. trachomatis-specific immune responses in altering the risk of infection and suggests immune correlates of protection that are potentially useful in vaccine development.