Partial protection against chlamydial reproductive tract infection by a recombinant major outer membrane protein/CpG/cholera toxin intranasal vaccine in the guinea pig Chlamydia cavaie model

Chlamydia trachomatis is a major cause of sexually transmitted diseases worldwide. There currently is no vaccine to protect against chlamydial infection of the female reproductive tract. Vaccine development has predominantly involved using the murine model, however infection of female guinea pigs with Chlamydia caviae more closely resembles chlamydial infection of the human female reproductive tract, and presents a better model to assess potential human chlamydial vaccines. We immunised female guinea pigs intranasally with recombinant major outer membrane protein (r-MOMP) combined with CpG-10109 and cholera toxin adjuvants. Both systemic and mucosal immune responses were elicited in immunised animals. MOMP-specific IgG and IgA were present in the vaginal mucosae, and high levels of MOMP-specific IgG were detected in the serum of immunised animals. Antibodies from the vaginal mucosae were also shown to be capable of neutralising C. caviae in vitro. Following immunisation, animals were challenged intravaginally with a live C. caviae infection of 102 inclusion forming units. We observed a decrease in duration of infection and a significant (p<0.025) reduction in infection load in r-MOMP immunised animals, compared to animals immunised with adjuvant only. Importantly, we also observed a marked reduction in upper reproductive tract (URT) pathology in r-MOMP immunised animals. Intranasal immunisation of female guinea pigs with r-MOMP was able to provide partial protection against C. caviae infection, not only by reducing chlamydial burden but also URT pathology. This data demonstrates the value of using the guinea pig model to evaluate potential chlamydial vaccines for protection against infection and disease pathology caused by C. trachomatis in the female reproductive tract.

A mathematical model of chlamydial infection incorporating movement of chlamydial particles

We present a spatiotemporal mathematical model of chlamydial infection, host immune response and spatial movement of infectious particles. The re- sulting partial differential equations model both the dynamics of the infection and changes in infection profile observed spatially along the length of the host genital tract. This model advances previous chlamydia modelling by incorporating spatial change, which we also demonstrate to be essential when the timescale for movement of infectious particles is equal to, or shorter than, the developmental cycle timescale. Numerical solutions and model analysis are carried out, and we present a hypothesis regarding the potential for treatment and prevention of infection by increasing chlamydial particle motility.

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

Chlamydia pneumoniae infection of Dentritic cells and its role in asthma exacerbations

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Agroinoculation Allows the Screening of Rice for Resistance to Rice Tungro Bacilliform Virus

A system for agroinoculating rice tungro bacilliform virus (RTBV), one of the two viruses of the rice tungro disease complex, has been optimised. A nontumour-inducing strain of Agrobacterium (pGV3850) was used in order to conform with biosafety regulations. Fourteen-day-old seedlings survived the mechanical damage of the technique and were still young enough to support virus replication. The level of the bacterial inoculum was important to obtain maximum infection, with a high inoculum level (0.5 × 1012 cells/ml) resulting in up to 100% infection of a susceptible variety that was comparable with infection by insect transmission. Agroinoculation with RTBV was successful for all three rice cultivarss tested; TN1 (tungro susceptible), Balimau Putih (tungro tolerant), and IR26 (RTSV and vector resistant). Agroinoculation enables resistance to RTBV to be distinguished from resistance to the leafhopper vector of the virus, and should prove useful in screening rice germplasm, breeding materials, and transgenic rice lines.

Classification of packet contents for malware detection

Many existing schemes for malware detection are signature-based. Although they can effectively detect known malwares, they cannot detect variants of known malwares or new ones. Most network servers do not expect executable code in their in-bound network traffic, such as on-line shopping malls, Picasa, Youtube, Blogger, etc. Therefore, such network applications can be protected from malware infection by monitoring their ports to see if incoming packets contain any executable contents. This paper proposes a content-classification scheme that identifies executable content in incoming packets. The proposed scheme analyzes the packet payload in two steps. It first analyzes the packet payload to see if it contains multimedia-type data (such as . If not, then it classifies the payload either as text-type (such as or executable. Although in our experiments the proposed scheme shows a low rate of false negatives and positives (4.69% and 2.53%, respectively), the presence of inaccuracies still requires further inspection to efficiently detect the occurrence of malware. In this paper, we also propose simple statistical and combinatorial analysis to deal with false positives and negatives.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

Drugs and the respiratory system

17.1 Drugs for bronchial asthma and Chronic Obstructive Pulmonary Disease (COPD) 17.1.1 Introduction to asthma 17.1.2 Introduction to COPD 17.1.3 Drug delivery by inhalation 17.1.4 Drugs to treat 17.1.4.1 β2-adrenoceptor agonists 17.1.4.2 Muscarinic receptor antagonists 17.1.4.3 Leukotriene receptor antagonists 17.1.4.4 Theophylline 17.1.4.5 Oxygen for COPD 17.1.5 Drugs to prevent asthma 31.5.1 Glucocorticoids 31.5.2 Cromolyn sodium 17.1.6 Combination to treat and prevent asthma 17.1.7 Drug for allergic asthma – omalizumab 17.1.8 Emergency treatment of asthma 17.2. Expectorants, mucolytics, cough and oxygen 17.2.1 Introduction to expectorants and mucolytics 17.2.2 Expectorants 17.2.3 Mucolytics 17.2.4 Cough 17.2.5 Oxygen 17.3. Drugs for rhinitis and rhinorrea 17.3.1 Introduction 17.3.2 Histamine and H1-receptor antagonists 17.3.3 Sympathomimetic 17.3.4 Muscarinic receptor antagonists 17.3.4 Cromolyn sodium 17.3.5 Glucocorticoids

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

Post-discharge surveillance : can patients reliably diagnose surgical wound infections?

Post-discharge surgical wound infection surveillance is an important part of many infection control programs. It is frequently undertaken by patient self-assessment, prompted either by a telephone or postal questionnaire. To assess the reliability of this method, 290 patients were followed for six weeks postoperatively. Their wounds were photographed and also covertly assessed for signs of infection by two experienced infection control nurses (ICNs). Patients also responded to a postal questionnaire seeking evidence of infection at both week four and week six post-surgery. Correlation between the patient's assessment of their wound and the ICNs diagnosis was poor (r=0.37) with a low positive predictive value (28.7%), although negative predictive value was high (98.2%). Assessment of photos for signs of infection by two experienced clinicians also correlated poorly with the ICNs diagnosis of infection (r=0.54). The patient's recall of prescription of an antibiotic by their general practitioner (GP) for wound infection during the postoperative period correlated best with the ICNs diagnosis (r=0.76). This latter measure, particularly when confirmed by the GP in those patients reporting an infection, appears to provide the most valid and resource efficient marker of post-discharge surgical wound infection.

Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other south pacific islands

BACKGROUND: Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP. METHODOLOGY/PRINCIPAL FINDINGS: Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009-2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III. CONCLUSIONS/SIGNIFICANCE: This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

New approaches to making the microenvironment of the female reproductive tract hostile to HIV

The studies presented in this review explore three distinct areas with potential for inhibiting HIV infection in women. Based on emerging information from the physiology, endocrinology and immunology of the female reproductive tract (FRT), we propose unique 'works in progress' for protecting women from HIV. Various aspects of FRT immunity are suppressed by estradiol during the menstrual cycle, making women more susceptible to HIV infection. By engineering commensal Lactobacillus to secrete the anti-HIV molecule Elafin as estradiol levels increase, women could be protected from HIV infection. Selective estrogen response modifiers enhance barrier integrity and enhance secretion of protective anti-HIV molecules. Finally, understanding the interactions and regulation of FRT endogenous antimicrobials, proteases, antiproteases, etc., all of which are under hormonal control, will open new avenues to therapeutic manipulation of the FRT mucosal microenvironment. By seeking new alternatives to preventing HIV infection in women, we may finally disrupt the HIV pandemic.