Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa.

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.

New research tools for urogenital schistosomiasis.

Approximately 200,000,000 people have schistosomiasis (schistosome infection). Among the schistosomes, Schistosoma haematobium is responsible for the most infections, which are present in 110 million people globally, mostly in sub-Saharan Africa. This pathogen causes an astonishing breadth of sequelae: hematuria, anemia, dysuria, stunting, uremia, bladder cancer, urosepsis, and human immunodeficiency virus coinfection. Refined estimates of the impact of schistosomiasis on quality of life suggest that it rivals malaria. Despite S. haematobium's importance, relevant research has lagged. Here, we review advances that will deepen knowledge of S. haematobium. Three sets of breakthroughs will accelerate discoveries in the pathogenesis of urogenital schistosomiasis (UGS): (1) comparative genomics, (2) the development of functional genomic tools, and (3) the use of animal models to explore S. haematobium-host interactions. Comparative genomics for S. haematobium is feasible, given the sequencing of multiple schistosome genomes. Features of the S. haematobium genome that are conserved among platyhelminth species and others that are unique to S. haematobium may provide novel diagnostic and drug targets for UGS. Although there are technical hurdles, the integrated use of these approaches can elucidate host-pathogen interactions during this infection and can inform the development of techniques for investigating schistosomes in their human and snail hosts and the development of therapeutics and vaccines for the control of UGS.

4-1BBL enhances CD8+ T cell responses induced by vectored vaccines in mice but fails to improve immunogenicity in rhesus macaques.

T cells play a central role in the immune response to many of the world's major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8(+) T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8(+) immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.

A new mouse model for female genital schistosomiasis.

BACKGROUND: Over 112 million people worldwide are infected with Schistosoma haematobium, one of the most prevalent schistosome species affecting humans. Female genital schistosomiasis (FGS) occurs when S. haematobium eggs are deposited into the female reproductive tract by adult worms, which can lead to pelvic pain, vaginal bleeding, genital disfigurement and infertility. Recent evidence suggests co-infection with S. haematobium increases the risks of contracting sexually transmitted diseases such as HIV. The associated mechanisms remain unclear due to the lack of a tractable animal model. We sought to create a mouse model conducive to the study of immune modulation and genitourinary changes that occur with FGS. METHODS: To model FGS in mice, we injected S. haematobium eggs into the posterior vaginal walls of 30 female BALB/c mice. A control group of 20 female BALB/c mice were injected with uninfected LVG hamster tissue extract. Histology, flow cytometry and serum cytokine levels were assessed at 2, 4, 6, and 8 weeks post egg injection. Voiding studies were performed at 1 week post egg injection. RESULTS: Vaginal wall injection with S. haematobium eggs resulted in synchronous vaginal granuloma development within 2 weeks post-egg injection that persisted for at least 6 additional weeks. Flow cytometric analysis of vaginal granulomata revealed infiltration by CD4+ T cells with variable expression of the HIV co-receptors CXCR4 and CCR5. Granulomata also contained CD11b+F4/80+ cells (macrophages and eosinophils) as well as CXCR4+MerTK+ macrophages. Strikingly, vaginal wall-injected mice featured significant urinary frequency despite the posterior vagina being anatomically distant from the bladder. This may represent a previously unrecognized overactive bladder response to deposition of schistosome eggs in the vagina. CONCLUSION: We have established a new mouse model that could potentially enable novel studies of genital schistosomiasis in females. Ongoing studies will further explore the mechanisms by which HIV target cells may be drawn into FGS-associated vaginal granulomata.

Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma.

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.

Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates.

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass.

Live recombinant viral vectors based on adenoviruses and poxviruses are among the most promising platforms for development of new vaccines against diseases such as malaria, tuberculosis, and HIV-AIDS. Vaccines based on live viruses must remain infectious to be effective, so therefore need continuous refrigeration to maintain stability and viability, a requirement that can be costly and difficult, especially in developing countries. The sugars sucrose and trehalose are commonly used as stabilizing agents and cryoprotectants for biological products. Here, we have exploited the ability of these sugars to vitrify on desiccation to develop a thermostabilization technique for live viral vaccine vectors. By slowly drying vaccines suspended in solutions of these disaccharide stabilizers onto a filter-like support membrane at ambient temperature, an ultrathin glass is deposited on the fibers of the inert matrix. Immobilization of two recombinant vaccine vectors-E1/E3-deleted human adenovirus type 5 and modified vaccinia virus Ankara-in this glass on the membranes enabled complete recovery of viral titer and immunogenicity after storage at up to 45 degrees C for 6 months and even longer with minimal losses. Furthermore, the membrane carrying the stabilized vaccine can be incorporated into a holder attached to a syringe for almost simultaneous reconstitution and injection at point of use. The technology may potentially be developed for the deployment of viral vector-based biopharmaceuticals in resource-poor settings.