Pre-clinical evaluation of a whole-parasite vaccine to control human babesiosis.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4+ T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

A Glimmer of Hope: Recent Updates and Future Challenges in Zika Vaccine Development.

The emergence and rapid spread of Zika virus (ZIKV) on a global scale as well as the establishment of a causal link between Zika infection and congenital syndrome and neurological disorders triggered unprecedented efforts towards the development of a safe and effective Zika vaccine. Multiple vaccine platforms, including purified inactivated virus, nucleic acid vaccines, live-attenuated vaccines, and viral-vectored vaccines, have advanced to human clinical trials. In this review, we discuss the recent advances in the field of Zika vaccine development and the challenges for future clinical efficacy trials. We provide a brief overview on Zika vaccine platforms in the pipeline before summarizing the vaccine candidates in clinical trials, with a focus on recent, promising results from vaccine candidates that completed phase I trials. Despite low levels of transmission during recent years, ZIKV has become endemic in the Americas and the potential of large Zika outbreaks remains real. It is important for vaccine developers to continue developing their Zika vaccines, so that a potential vaccine is ready for deployment and clinical efficacy trials when the next ZIKV outbreak occurs.

Immunomodulatory effect of ginseng stem-leaf saponins and selenium on Harderian gland in immunization of chickens to Newcastle disease vaccine.

Our previous study demonstrated that ginseng stem-leaf saponins (GSLS) in combination with selenium (GSLS-Se) have adjuvant effect on the live vaccine of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) in intraocular-and-intranasal immunization in chickens. The present study was to investigate the potential molecular mechanisms involved in the immunomodulation of GSLS-Se on the Harderian gland (HG). It was found that the window allowing animals susceptible to infections due to low antibody titers became smaller or even completely closed because of increased NDV-specific HI titers when NDV vaccine and GSLS-Se were coadministered for immunization at early life in chickens. In addition, NDV-specific sIgA and the numbers of IgG+, IgA+, IgM+ plasma cells were significantly more in GSLS-Se group than the control in the HGs. Transcriptome analysis of HGs identified 1184 differentially expressed genes (DEGs) between GSLS-Se treated and non-treated groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified 42 significantly enriched GO terms and 13 canonical immune pathways. These findings indicated that GSLS-Se might exert immunomodulatory effects through influencing the antioxidant regulation and modulating the activity of immune related enzymes. Besides, Toll-like receptor (TLR) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway might be involved primarily in the immunomodulation. Therefore, enhanced antibody responses in GSLS-Se group may be attributed to the immunomodulatory effects of GSLS-Se on the immune-related gene profile expressed in the immunocompetent cells of the HGs.

Combined adjuvant effect of ginseng stem-leaf saponins and selenium on immune responses to a live bivalent vaccine of Newcastle disease virus and infectious bronchitis virus in chickens.

Vaccination with a live bivalent vaccine of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) is a routine practice in poultry industry in China. This study was designed to evaluate ginseng stem-leaf saponins (GSLS) in combination with selenium (Se) for their adjuvant effect on the immune response to vaccination against NDV and IBV in chickens. A live bivalent vaccine of NDV and IBV was diluted in saline solution containing GSLS or Se or both and used to immunize chickens via a intraocular-and-intranasal route. Results showed that GSLS promoted significantly higher NDV- and IBV-specific antibody responses with the highest antibody response detected in GSLS-Se group. The increased antibody was capable of neutralizing NDV and IBV. In addition, GSLS-Se enhanced lymphocyte proliferation and production of IFN-γ and IL-4. More importantly GSLS-Se was found to promote early production and prolong the duration of the antibody responses. In order to improve the efficacy of vaccination in chicken flocks, the diluent containing GSLS-Se deserves further studies to evaluate its effect on other chicken vaccines.

Ginseng stem-leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine.

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se-containing physiological saline solution (PSS) provoked a significantly stronger gB-specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.

Assessment of the safety and efficacy of an attenuated live vaccine based on highly virulent genotype 2b porcine epidemic diarrhea virus in nursing piglets.

We have previously reported the generation of the attenuated KNU-141112-S DEL5/ORF3 virus by continuous propagation of highly virulent G2b porcine epidemic diarrhea virus (PEDV) in Vero cells. The present study aimed to assess the safety of S DEL5/ORF3 and to evaluate its effectiveness as a live vaccine for prime-booster vaccinations. Reversion to virulence experiments revealed that the S DEL5/ORF3 strain retains its attenuated phenotype and genetic stability after five successive passages in susceptible piglets. Pregnant sows were primed orally with an S DEL5/ORF3 live vaccine and boosted intramuscularly twice with a commercial killed vaccine at 2-week intervals prior to parturition. This sow vaccination regimen completely protected nursing piglets against virulent G2b challenge, as evidenced by the increase in survival rate from 0% to 100% and the significant reduction in diarrhea intensity, including the amount and duration of PEDV fecal shedding. In addition, despite a 2-3 day period of weight loss in piglets from vaccinated sows after challenge, their daily weight gain was recovered at 7 days post-challenge and became similar to that of unchallenged pigs from unvaccinated sows over the course of the experiment. Furthermore, strong antibody responses to PEDV were verified in the sera and colostrum of immunized sows with the prime-boost treatment and their offspring. Altogether, our data demonstrated that the attenuated S DEL5/ORF3 strain guarantees the safety to host animals with no reversion to virulence and is suitable as an effective primary live vaccine providing durable maternal lactogenic immunity for passive piglet protection.

Antibody Responses to Bovine Alphaherpesvirus 1 (BoHV-1) in Passively Immunized Calves.

To date, in countries where infectious bovine rhinotracheitis (IBR) is widespread, its control is associated with deleted marker vaccines. These products lack one or more genes responsible for the synthesis of glycoproteins or enzymes. In Europe, the most widely used marker vaccine is one in which glycoprotein E (gE-) is deleted, and it is marketed in a killed or modified-live form. Using this type of immunization, it is possible to differentiate vaccinated animals (gE-) from those infected or injected with non-deleted (gE+) products using diagnostic tests specific for gE. The disadvantage of using modified-live gE-products is that they may remain latent in immunized animals and be reactivated or excreted following an immunosuppressive stimulus. For this reason, in the last few years, a new marker vaccine became commercially available containing a double deletion related to genes coding for gE and the synthesis of the thymidine-kinase (tk) enzyme, the latter being associated with the reduction of the neurotropism, latency, and reactivation of the vaccine virus. Intramuscularly and intranasally administered marker products induce a humoral immune response; however, the mother-to-calf antibody kinetics after vaccination with marker vaccines is poorly understood. This review discusses several published articles on this topic.

Development and evaluation of in murine model, of an improved live-vaccine candidate against brucellosis from to Brucella melitensis vjbR deletion mutant.

Brucellosis is an infectious disease that brings enormous economic burdens for developing countries. The Brucella melitensis (B. melitensis) M5-90 vaccine strain (M5-90) has been used on a large scale in China, but may cause abortions if given to pregnant goats or sheep subcutaneously during the late stages of gestation. Moreover, the vaccine M5-90 cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent M5-90 vaccine is required. In this study, a vjbR mutant of M5-90 (M5-90ΔvjbR) was constructed and overcame these drawbacks. M5-90ΔvjbR strain showed reduced survival capability in murine macrophages (RAW 264.7) and BALB/c mice and induced high protective immunity in mice. In addition, M5-90ΔvjbR induced an anti-Brucella-specific immunoglobulin G (IgG) response and stimulated the expression of gamma interferon (INF-γ) and interleukin-4 (IL-4) in vaccinated mice. Furthermore, M5-90ΔvjbR induced IgG response and stimulated the secretion of IFN-γ and IL-4 in immunized sheep. Moreover, the VjbR antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90ΔvjbR is an ideal live attenuated and efficacious live vaccine candidate against B. melitensis 16 M infection.

Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment.

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

Live Attenuated Tularemia Vaccines for Protection Against Respiratory Challenge With Virulent F. tularensis subsp. tularensis.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.

The vesicular stomatitis virus-based Ebola virus vaccine: From concept to clinical trials.

The devastating Ebola virus (EBOV) epidemic in West Africa in 2013-2016 accelerated the progress of several vaccines and antivirals through clinical trials, including the replication-competent vesicular stomatitis virus-based vaccine expressing the EBOV glycoprotein (VSV-EBOV). Extensive preclinical testing in animal models demonstrated the prophylactic and post-exposure efficacy of this vaccine, identified the mechanism of protection, and suggested it was safe for human use. Based on these data, VSV-EBOV was extensively tested in phase 1-3 clinical trials in North America, Europe and Africa. Although some side effects of vaccination were observed, these clinical trials showed that the VSV-EBOV was safe and immunogenic in humans. Moreover, the data supported the use of VSV-EBOV as an emergency vaccine in individuals at risk for Ebola virus disease. In this review, we summarize the results of the extensive preclinical and clinical testing of the VSV-EBOV vaccine.

Attenuation and efficacy of live-attenuated Rift Valley fever virus vaccine candidates in non-human primates.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.

Retinoic acid elicits a coordinated expression of gut homing markers on T lymphocytes of Zambian men receiving oral Vivotif, but not Rotarix, Dukoral or OPVERO vaccines.

All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4ß7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4ß7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/ß signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.

Principles of vaccine potency assays.

Compared with biologics, vaccine potency assays represent a special challenge due to their unique compositions, multivalency, long life cycles and global distribution. Historically, vaccines were released using in vivo potency assays requiring immunization of dozens of animals. Modern vaccines use a variety of newer analytical tools including biochemical, cell-based and immunochemical methods to measure potency. The choice of analytics largely depends on the mechanism of action and ability to ensure lot-to-lot consistency. Live vaccines often require cell-based assays to ensure infectivity, whereas recombinant vaccine potency can be reliably monitored with immunoassays. Several case studies are presented to demonstrate the relationship between mechanism of action and potency assay. A high-level decision tree is presented to assist with assay selection.

The effect of probiotics and zinc supplementation on the immune response to oral rotavirus vaccine: A randomized, factorial design, placebo-controlled study among Indian infants.

BACKGROUND: Strategies are needed to improve oral rotavirus vaccine (RV), which provides suboptimal protection in developing countries. Probiotics and zinc supplementation could improve RV immunogenicity by altering the intestinal microbiota and immune function. METHODS: Infants 5weeks old living in urban Vellore, India were enrolled in a randomized, double-blind, placebo-controlled trial with a 4-arm factorial design to assess the effects of daily zinc (5mg), probiotic (1010Lactobacillus rhamnosus GG) or placebo on the immunogenicity of two doses of RV (Rotarix®, GlaxoSmithKline Biologicals) given at 6 and 10weeks of age. Infants were eligible for participation if healthy, available for the study duration and without prior receipt of RV or oral poliovirus vaccine other than the birth dose. The primary outcome was seroconversion to rotavirus at 14weeks of age based on detection of VP6-specific IgA at ≥20U/ml in previously seronegative infants or a fourfold rise in concentration. RESULTS: The study took place during July 2012 to February 2013. 620 infants were randomized equally between study arms and 551 (88.9%) completed per protocol. Seroconversion was recorded in 54/137 (39.4%), 42/136 (30.9%), 40/143 (28.0%), and 37/135 (27.4%) infants receiving (1) probiotic and zinc, (2) probiotic and placebo, (3) placebo and zinc, (4) two placebos. Seroconversion showed a modest improvement among infants receiving probiotic (difference between groups 1, 2 and 3, 4 was 7.5% (97.5% Confidence Interval (CI): -1.4%, 16.2%), p=0.066) but not zinc (difference between groups 1, 3 and 2, 4 was 4.4% (97.5% CI: -4.4%, 13.2%), p=0.272). 16 serious adverse events were recorded, none related to study interventions. CONCLUSIONS: Zinc or probiotic supplementation did not significantly improve the low immunogenicity of rotavirus vaccine given to infants in a poor urban community in India. A modest effect of combined supplementation deserves further investigation. TRIAL REGISTRATION: The trial was registered in India (CTRI/2012/05/002677).

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log10 infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.

A recombinant live attenuated tetravalent vaccine for the prevention of dengue.

INTRODUCTION: Dengue is an important and still growing public health problem associated with substantial morbidity, as well as significant social and economic impact. The present review describes the main features and development of the first dengue vaccine (CYD-TDV, Dengvaxia®), which has been licensed by several dengue-endemic countries in Asia and Latin America for use in populations above 9 years of age. Areas covered: The review focuses on the large clinical development of CYD-TDV, which includes in particular two pivotal phase III efficacy trials conducted in Asia and Latin America and supported vaccine licensure. Based on these clinical data, the WHO Strategic Advisory Group of Experts (SAGE) on Immunization recommended considering introduction of the vaccine in geographic settings (national or subnational) with high burden of disease. Long-term safety follow-up studies of the efficacy trials are currently ongoing, and post-licensure studies will evaluate the vaccine effectiveness and safety in 'real-life' following vaccine introduction. Expert commentary: During vaccine development, a number of complexities were tackled, innovation pursued, and risk managed. These aspects, as well as the potential impact of CYD-TDV on public health are also discussed.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.