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Influenza viruses frequently acquire mutations undergoing antigenic drift necessitating annual evaluation of vaccine strains. Highly conserved epitopes have been identified in the hemagglutinin (HA) head and stem regions, however, current influenza vaccines induce only limited responses to these conserved sites. Here, we describe a novel seasonal recombinant HA nanoparticle influenza vaccine (NIV) formulated with a saponin-based adjuvant, Matrix-M™. NIV induced hemagglutination inhibition (HAI) and microneutralizing (MN) antibodies against a broad range of influenza A(H3N2) subtypes. In a comparison of NIV against standard-dose and high-dose inactivated influenza vaccines (IIV and IIV-HD, respectively) in ferrets NIV elicited HAI and MN responses exceeding those induced by IIV-HD against homologous A(H3N2) by 7 fold, A(H1N1) by 26 fold, and B strain viruses by 2 fold. NIV also induced MN responses against all historic A/H3N2 strains tested, spanning more than a decade of viral evolution from the 2000-2017 influenza seasons whereas IIV and IIV-HD induced HAI and MN responses were largely directed against the homologous A(H3N2), A(H1N1), and B virus strains. NIV induced superior protection compared to IIV and IIV-HD in ferrets challenged with a homologous or 10-year drifted influenza A(H3N2) strain. HAI positive and HAI negative neutralizing monoclonal antibodies derived from mice immunized with NIV were active against homologous and drifted influenza A(H3N2) strains. Taken together these observations suggest that NIV can induce responses to one or more highly conserved HA head and stem epitopes and result in highly neutralizing antibodies against both homologous and drift strains., (Copyright © 2017. Published by Elsevier Ltd.)

Clostridium difficile is the number one cause of nosocomial antibiotic-associated diarrhea in developed countries. Historically, pathogenesis was attributed two homologous glucosylating toxins, toxin-A (TcdA) and toxin-B (TcdB). Over the past decade, however, highly virulent epidemic strains of C. difficile (B1/NAP1/027) have emerged and are linked to an increase in morbidity and mortality. Increased virulence is attributed to multiple factors including: increased production of A- and B-toxins; production of binary toxin (CDT); and the emergence of more toxic TcdB variants (TcdB (027) ). TcdB (027) is more cytotoxicity to cells; causes greater tissue damage and toxicity in animals; and is antigenically distinct from historical TcdB (TcdB (003) ). Broadly protective vaccines and therapeutic antibody strategies, therefore, may target TcdA, TcdB variants and CDT. To facilitate the generation of multivalent toxin-based C. difficile vaccines and therapeutic antibodies, we have generated fusion proteins constructed from the receptor binding domains (RBD) of TcdA, TcdB (003) , TcdB (027) and CDT. Herein, we describe the development of a trivalent toxin (T-toxin) vaccine (CDTb/TcdB (003) /TcdA) and quadravalent toxin (Q-toxin) vaccine (CDTb/TcB (003) /TcdA/TcdB (027) ) fusion proteins that retain the protective toxin neutralizing epitopes. Active immunization of mice or hamsters with T-toxin or Q-toxin fusion protein vaccines elicited the generation of toxin neutralizing antibodies to each of the toxins. Hamsters immunized with the Q-toxin vaccine were broadly protected against spore challenge with historical C. difficile 630 (toxinotype 0/ribotype 003) and epidemic NAP1 (toxinotype III/ribotype 027) strains. Fully human polyclonal antitoxin IgG was produced by immunization of transgenic bovine with these fusion proteins. In passive transfer studies, mice were protected against lethal toxin challenge. Hamsters treated with human antitoxin IgG were completely protected when challenged with historical or epidemic strains of C. difficile. The use of chimeric fusion proteins is an attractive approach to producing multivalent antitoxin vaccines and therapeutic polyclonal antibodies for prevention and treatment of C. difficile infections (CDI)., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Novel influenza viruses continue to pose a potential pandemic threat worldwide. In recent years, plants have been used to produce recombinant proteins, including subunit vaccines. A subunit influenza vaccine, HAC1, based on recombinant hemagglutinin from the 2009 pandemic A/California/04/2009 (H1N1) strain of influenza virus, has been manufactured using a plant virus-based transient expression technology in Nicotiana benthamiana plants and demonstrated to be immunogenic and safe in pre-clinical studies (Shoji et al., 2011)., Methods: A first-in-human, Phase 1, single-center, randomized, placebo-controlled, single-blind, dose escalation study was conducted to investigate safety, reactogenicity and immunogenicity of an HAC1 formulation at three escalating dose levels (15 μg, 45 μg and 90 μg) with and without Alhydrogel(®), in healthy adults 18-50 years of age (inclusive). Eighty participants were randomized into six study vaccine groups, a saline placebo group and an approved monovalent H1N1 vaccine group. Recipients received two doses of vaccine or placebo (except for the monovalent H1N1 vaccine cohort, which received a single dose of vaccine, later followed by a dose of placebo)., Results: The experimental vaccine was safe and well tolerated, and comparable to placebo and the approved monovalent H1N1 vaccine. Pain and tenderness at the injection site were the only local solicited reactions reported following vaccinations. Nearly all adverse events were mild to moderate in severity. The HAC1 vaccine was also immunogenic, with the highest seroconversion rates, based on serum hemagglutination-inhibition and virus microneutralization antibody titers, in the 90 μg non-adjuvanted HAC1 vaccine group after the second vaccine dose (78% and 100%, respectively)., Conclusions: This is the first study demonstrating the safety and immunogenicity of a plant-produced subunit H1N1 influenza vaccine in healthy adults. The results support further clinical investigation of the HAC1 vaccine as well as demonstrate the feasibility of the plant-based technology for vaccine antigen production., (Copyright © 2014. Published by Elsevier Ltd.)

Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region termed "LSA-NRC." We describe the first Phase I/II study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system. We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10microg LSA-NRC/0.5ml AS01 (n=5), high dose (HD) LSA-NRC/AS01: 50microg LSA-NRC/0.5ml AS01 (n=13); LD LSA-NRC/AS02: 10microg LSA-NRC/0.5ml AS02 (n=5) and HD LSA-NRC/AS02: 50microg LSA-NRC/0.5ml AS02 (n=13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity controls underwent experimental malaria sporozoite challenge. The vaccines showed a reassuring safety profile but were moderately reactogenic. There were no serious adverse events. All subjects seroconverted after the first immunization. Following the second immunization, LSA-1-specific CD4+ T cells producing two cytokines (IL-2 and IFN-gamma) were found by intra-cellular staining in all subjects in the LD LSA-NRC/AS01B group and in 3 of 5 subjects in the LD LSA-NRC/AS02 group. In contrast, the HD LSA-NRC/AS01 and HD LSA-NRC/AS02 group subjects had fewer LSA-1-specific CD4+ T cells, and minimal to no IFN-gamma responses. There was no increase in LSA-1-specific CD8+ T cells found in any group. Per protocol, 22 high dose vaccinees, but no low dose vaccinees, underwent P. falciparum homologous malaria challenge (3D7 clone). All vaccinees became parasitemic and there was no delay in their pre-patent period versus controls (p=0.95). LSA-NRC/AS01 and LSA-NRC/AS02 elicited antigen-specific antibody and CD4+ T cell responses, but elicited no protective immunity. Although the optimal antigen dose of LSA-NRC may not have been selected for the challenge portion of the protocol, further vaccine development based upon LSA-1 should not be excluded and should include alternative vaccine platforms able to elicit additional effector mechanisms such as CD8+ T cells., (Copyright 2009. Published by Elsevier Ltd.)

Background: Immunization with RTS,S/AS02 consistently protects some vaccinees against malaria infection in experimental challenges and in field trials. A brief immunization schedule against falciparum malaria would be compatible with the Expanded Programme on Immunization, or in combination with other prevention measures, interrupt epidemic malaria or protect individuals upon sudden travel to an endemic area., Methods: We conducted an open label, Phase 2a trial of two different full dose schedules of RTS,S/AS02 in 40 healthy malaria-naïve adults. Cohort 1 (n=20) was immunized on a 0, 1, and 3 month schedule and Cohort 2 (n=20) on a 0, 7, and 28 day schedule. Three weeks later, 38 vaccinees and 12 unimmunized infectivity controls underwent malaria challenge., Results: Both regimens had a good safety and tolerability profile. Peak GMCs of antibody to the circumsporozoite protein (CSP) were similar in Cohort 1 (78 microg/mL; 95% CI: 45-134) and Cohort 2 (65 microg/mL; 95% CI: 40-104). Vaccine efficacy for Cohort 1 was 45% (95% CI: 18-62%) and for Cohort 2, 39% (95% CI: 11-56%). Protected volunteers had a higher GMC of anti-CSP antibody (114 microg/mL) than did volunteers with a 2-day delay (70 microg/mL) or no delay (30 microg/mL) in the time to onset of parasitemia (Kruskal-Wallis, p=0.019). A trend was seen for higher CSP-specific IFN-gamma responses in PBMC from protected volunteers only in Cohort 1, but not in Cohort 2, for ex vivo and for cultured ELISPOT assays., Conclusion: In malaria-naïve adults, the efficacy of three-dose RTS,S/AS02 regimens on either a 0, 1, and 3 month schedule or an abbreviated 0, 7, and 28 day schedule was not discernibly different from two previously reported trials of two-dose regimens given at 0, 1 month that conferred 47% (95% CI: -19 to 76%) protection and in another trial 42% (95% CI: 5-63%). A strong association of CSP-specific antibody with protection against malaria challenge is observed and confirms similar observations made in other studies. Subsequent trials of adjuvanted RTS,S in African children and infants on a 0, 1, and 2 month schedule have demonstrated a favorable safety and efficacy profile.

We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40microg of FMP2.1 in a fixed volume of 0.5mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-gamma and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.

We report the first trial of candidate malaria vaccine antigen FMP1, a 42kDa fragment from the C-terminus of merozoite surface protein-1 (MSP-1) from the 3D7 strain of Plasmodium falciparum, in an endemic area. Forty adult male and female residents of western Kenya were enrolled to receive 3 doses of either FMP1/AS02A or Imovax rabies vaccine by intra-deltoid injection on a 0, 1, 2 month schedule. Thirty-seven volunteers received all three immunizations and 38 completed the 12-month evaluation period. Slightly more recipients of the FMP1/AS02A vaccine experienced any instance of pain at 24 h post-immunization than in the Imovax group (95% versus 65%), but otherwise the two vaccines were equally safe and well-tolerated. Baseline antibody levels were high in both groups and were boosted in the FMP1/AS02A group. Longitudinal models revealed a highly significant difference between groups for both the average post-baseline antibody responses to MSP-1(42) (F1,335=13.16; P<0.001) and the Day 90 responses to MSP-1(42) (F1,335=16.69; P<0.001). The FMP1/AS02A vaccine is safe and immunogenic in adults and should progress to safety testing in children at greatest risk of malaria.

Background: RTS,S/AS02A, a pre-erythrocytic Plasmodium falciparum vaccine based upon the circumsporozoite protein, is the only vaccine demonstrated in field trials to confer partial protection against a range of malaria disease manifestations. Pre-clinical studies are on-going to identify new RTS,S formulations with improved magnitude and duration of specific immunity., Methods: Rhesus macaques were immunized with saline or one of four "RTS,S/adjuvant" formulations at 0, 4, and 12 weeks: RTS,S/AS01B, RTS,S/AS02A-standard (current formulation), RTS,S/AS05 or RTS,S/AS06. An RTS,S/AS02A-accelerated group was immunized at 0, 1, and 4 weeks. Outcomes were safety, RTS,S-specific antibody, and IFN-gamma and IL-5 ELISpots (weeks 14 and 34)., Findings: All regimens were safe and, except for RTS,S/AS06, generated equivalent high titer antibody levels. For IFN-gamma ELISpots, RTS,S/AS01B had the highest geometric mean (GM) values at weeks 14 and 34, and was the only group with an overall GM mean (weeks 14+34) higher than RTS,S/AS02A-standard (p<0.015). For IFN-gamma to IL-5 ELISpot response ratios, RTS,S/AS01B had the highest values at weeks 14 and 34, and was the only group higher than RTS,S/AS02A-standard at each individual time point and overall (weeks 14+34) (p<0.015)., Interpretation: RTS,S/AS01B is a safe and immunogenically superior formulation for cellular responses, in comparison with the RTS,S/AS02A-standard. Phase 1, 2a, and 2b clinical trials are underway to determine if RTS,S/AS01B demonstrates improved immunogenicity and protective efficacy against experimental challenge and natural mosquito-borne malaria.

Background: Studies are underway to identify more immunogenic formulations of the existing anti-falciparum malaria vaccine RTS,S/AS02A. To supplement in vitro immunogenicity assays, cutaneous delayed-type hypersensitivity (DTH) may be a useful indicator of functional, cell-mediated immunogenicity., Methods: Adult rhesus monkeys were immunized with saline or one of four RTS,S/adjuvant formulations: RTS,S/AS01B, RTS,S/AS02A-standard (current formulation), RTS,S/AS05 or RTS,S/AS06 at 0, 4, and 12 weeks. An additional cohort received RTS,S/AS02A-accelerated, at 0, 1, and 4 weeks. Six months after completing immunizations, five vaccine-relevant antigens (high and low doses) and two controls were administered intradermally. DTH reactivity (induration) was measured at 48 and 72h, and selected sites were biopsied for histological confirmation., Results: In comparison with RTS,S/AS02A-standard, RTS,S/AS01B and RTS,S/AS05 each had larger mean reactions (induration) at 5 of 10 (p<0.01, at each site) and 1 of 10 (p<0.05, at the single site) vaccine relevant test sites, respectively. Histologically, perivascular mononuclear cell infiltrates, a cardinal feature of DTH, were largest in the RTS,S/AS01B monkeys., Interpretation: In DTH testing, with histological confirmation, RTS,S/AS01B was immunogenically superior to RTS,S/AS02A-standard and two other novel RTS,S formulations. The DTH outcomes paralleled conventional in vitro cellular immunogenicity assessments in distinguishing among similar RTS,S formulations, even at 6 months after final vaccination.

We report the first safety and immunogenicity trial of the Plasmodium falciparum malaria blood stage vaccine candidate, FMP1/AS02A consisting of the FMP1 antigen, an Escherichia coli-expressed His-tagged fusion protein from the 42 kDa C-terminal fragment from the 3D7 clone of the merozoite surface protein 1 formulated in the AS02A adjuvant. An open label, prospective, single-center Phase I dose escalation trial of FMP1/AS02A was conducted in 15 adult malaria-naïve human volunteers to assess safety, reactogenicity, and immunogenicity. The vaccine was safe and well-tolerated and no serious adverse events were observed. The vaccine induced high-titer ELISA and IFA responses in all volunteers. Proliferative and ELISPOT responses were induced to vaccine antigen. Biologically active antibodies were induced as measured by GIA. This study establishes the foundation to further evaluate and measure the vaccine's ability to reduce morbidity and mortality in target populations directly affected by P. falciparum malaria.

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.