Protection induced by a Francisella tularensis subunit vaccine delivered by glucan particles.

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of ß-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens.

Schistosoma mansoni SmKI-1 or Its C-Terminal Fragment Induces Partial Protection Against S. mansoni Infection in Mice.

Current schistosomiasis control strategies are mainly based on chemotherapy, but the development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. When it comes to vaccine candidates, several genes encoding Schistosoma mansoni proteins expressed at the mammalian host-parasite interface have been tested. Among the most promising molecules are the proteins present on the tegument and digestive tract of the parasite. In this study, we evaluate the potential of SmKI-1, the first Kunitz-type protease inhibitor functionally characterized in S. mansoni, as a vaccine candidate. Bioinformatic analysis points to the C-terminal fragment as the main region of the molecule responsible for the development of a potential protective immune response induced by SmKI-1. Therefore, for the vaccine formulations, we produced the recombinant (r) SmKI-1 and two different fragments, its Kunitz (KI) domain and its C-terminal tail. First, we demonstrate that mice immunized with recombinant SmKI-1 (rSmKI-1) or its fragments, formulated with Freund's adjuvant, induced the production of IgG-specific antibodies. Further, all vaccine formulations tested here also induced a Th1-type of immune response, as suggested by the production of IFN-γ and TNF-α by protein-stimulated cultured splenocytes. However, the protective effect conferred by vaccination was only observed in groups which received rSmKI-1 or C-terminal domain vaccines. Mice administered with rSmKI-1 demonstrated reduction of 47% in worm burden, 36% in egg number in mouse livers, and 33% in area of liver granulomas. Additionally, mice injected with C-terminal domain showed reduction of 28% in worm burden, 38% in egg number in liver, and 25% in area of liver granulomas. In contrast, KI domain immunization was unable to reduce worm burden and ameliorate liver pathology after challenge infection. Taken together, our data demonstrated that SmKI-1 is a potential candidate for use in a vaccine to control schistosomiasis, and its C-terminal tail seems to be the main region of the molecule responsible for protection conferred by this antigen.

Vaccination with Recombinant Cryptococcus Proteins in Glucan Particles Protects Mice against Cryptococcosis in a Manner Dependent upon Mouse Strain and Cryptococcal Species.

Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus-derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli, purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific.IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An effective vaccine could substantially reduce the burden of cryptococcosis. However, a major gap in cryptococcal vaccine development has been the discovery of protective antigens to use in vaccines. Here, six cryptococcal proteins with potential as vaccine antigens were expressed recombinantly and purified. Mice were then vaccinated with glucan particle preparations containing each antigen. Of the six candidate vaccines, four protected mice from a lethal cryptococcal challenge. However, the degree of protection varied as a function of mouse strain and cryptococcal species. These preclinical studies identify cryptococcal proteins that could serve as candidate vaccine antigens and provide a proof of principle regarding the feasibility of protein antigen-based vaccines to protect against cryptococcosis.

In Silico Prediction Analysis of Idiotope-Driven T-B Cell Collaboration in Multiple Sclerosis.

Memory B cells acting as antigen-presenting cells are believed to be important in multiple sclerosis (MS), but the antigen they present remains unknown. We hypothesized that B cells may activate CD4+ T cells in the central nervous system of MS patients by presenting idiotopes from their own immunoglobulin variable regions on human leukocyte antigen (HLA) class II molecules. Here, we use bioinformatics prediction analysis of B cell immunoglobulin variable regions from 11 MS patients and 6 controls with other inflammatory neurological disorders (OINDs), to assess whether the prerequisites for such idiotope-driven T-B cell collaboration are present. Our findings indicate that idiotopes from the complementarity determining region (CDR) 3 of MS patients on average have high predicted affinities for disease associated HLA-DRB1*15:01 molecules and are predicted to be endosomally processed by cathepsin S and L in positions that allows such HLA binding to occur. Additionally, complementarity determining region 3 sequences from cerebrospinal fluid (CSF) B cells from MS patients contain on average more rare T cell-exposed motifs that could potentially escape tolerance and stimulate CD4+ T cells than CSF B cells from OIND patients. Many of these features were associated with preferential use of the IGHV4 gene family by CSF B cells from MS patients. This is the first study to combine high-throughput sequencing of patient immune repertoires with large-scale prediction analysis and provides key indicators for future in vitro and in vivo analyses.

Molecular evolution of Zika virus as it crossed the Pacific to the Americas.

Zika virus was previously considered to cause only a benign infection in humans. Studies of recent outbreaks of Zika virus in the Pacific, South America, Mexico and the Caribbean have associated the virus with severe neuropathology. Viral evolution may be one factor contributing to an apparent change in Zika disease as it spread from Southeast Asia across the Pacific to the Americas. To address this possibility, we have employed computational tools to compare the phylogeny, geography, immunology and RNA structure of Zika virus isolates from Africa, Asia, the Pacific and the Americas. In doing so, we compare and contrast methods and results for tree search and rooting of Zika virus phylogenies. In some phylogenetic analyses we find support for the hypothesis that there is a deep common ancestor between African and Asian clades (the "Asia/Africa" hypothesis). In other phylogenetic analyses, we find that Asian lineages are descendent from African lineages (the "out of Africa" hypothesis). In addition, we identify and evaluate key mutations in viral envelope protein coding and untranslated terminal RNA regions. We find stepwise mutations that have altered both immunological motif sets and regulatory sequence elements. Both of these sets of changes distinguish viruses found in Africa from those in the emergent Asia-Pacific-Americas lineage. These findings support the working hypothesis that mutations acquired by Zika virus in the Pacific and Americas contribute to changes in pathology. These results can inform experiments required to elucidate the role of viral genetic evolution in changes in neuropathology, including microcephaly and other neurological and skeletomuscular issues in infants, and Guillain-Barré syndrome in adults.

Zika Virus: Medical Countermeasure Development Challenges.

INTRODUCTION: Reports of high rates of primary microcephaly and Guillain-Barré syndrome associated with Zika virus infection in French Polynesia and Brazil have raised concerns that the virus circulating in these regions is a rapidly developing neuropathic, teratogenic, emerging infectious public health threat. There are no licensed medical countermeasures (vaccines, therapies or preventive drugs) available for Zika virus infection and disease. The Pan American Health Organization (PAHO) predicts that Zika virus will continue to spread and eventually reach all countries and territories in the Americas with endemic Aedes mosquitoes. This paper reviews the status of the Zika virus outbreak, including medical countermeasure options, with a focus on how the epidemiology, insect vectors, neuropathology, virology and immunology inform options and strategies available for medical countermeasure development and deployment. METHODS: Multiple information sources were employed to support the review. These included publically available literature, patents, official communications, English and Lusophone lay press. Online surveys were distributed to physicians in the US, Mexico and Argentina and responses analyzed. Computational epitope analysis as well as infectious disease outbreak modeling and forecasting were implemented. Field observations in Brazil were compiled and interviews conducted with public health officials.