A typhoid fever protein capsular matrix vaccine candidate formulated with Advax-CpG adjuvant induces a robust and durable anti-typhoid Vi polysaccharide antibody response in mice, rabbits and nonhuman primates.

Typhax is an investigational typhoid fever vaccine candidate that is comprised of Vi polysaccharide from Salmonella enterica serovar typhi (S. Typhi) non-covalently entrapped in a glutaraldehyde catalyzed, cross-linked α-poly-L-lysine and CRM197 protein matrix. A previous Phase 1 trial of an aluminum phosphate adjuvanted Typhax formulation showed it induced Vi IgG after a single dose but that subsequent doses failed to further boost Vi IgG levels. The current study asked whether Advax-CpG adjuvant might instead be able to overcome polysaccharide-induced immune inhibition and improve Typhax immunogenicity. Advax-CpG adjuvanted Typhax elicited high and sustained Vi IgG responses in mice, rabbits and non-human primates (NHP) with levels being boosted by repeated immunization. High Vi antibody responses were lost in CD4 + T cell depleted mice confirming that despite the lack of conjugation of the polysaccharide to the carrier protein, Typhax nevertheless acts in a T cell dependent manner, explaining its ability to induce long-term B cell memory responses to Vi capable of being boosted. In NHP, Advax-CpG adjuvanted Typhax induced up to 100-fold higher Vi IgG levels than the commercial Typhim Vi polysaccharide vaccine. Typhax induced high and sustained serum bactericidal activity against S. Typhi and stimulated robust Vi IgG responses even in animals previously primed with a pure polysaccharide vaccine. Hence Advax-CpG adjuvanted Typhax vaccine is a highly promising candidate to provide robust and durable protection against typhoid fever.

Preclinical in vitro and in vivo profile of a highly-attenuated, broadly efficacious pneumolysin genetic toxoid.

Pneumolysin is a highly conserved, cholesterol-dependent cytolysin that is an important Streptococcus pneumoniae virulence factor and an attractive target for vaccine development. To attenuate pneumolysin toxicity, a genetic toxoid was constructed with two amino acid changes, G293S and L460D, termed PLY-D, that reduced cytolytic activity > 125,000-fold. In mice, PLY-D elicited high anti-PLY IgG antibody titers that neutralized the cytolytic activity of the wild-type toxin in vitro. To evaluate the protective efficacy of PLY-D, mice were immunized intramuscularly and then challenged intranasally with a lethal dose of 28 clinical isolates of S. pneumoniae originating from different geographical locations, disease states (i.e. bacteremia, pneumonia), or body sites (i.e. sputum, blood). PLY-D immunization conferred significant protection from challenge with 17 of 20 serotypes (85%) and 22 of 28 strains (79%). Further, we demonstrated that immunization with PLY-D provided statistically significant improvement in survival against challenge with serotype 4 and 18C strains compared to mice immunized with a pneumococcal conjugate vaccine Prevnar 13® (PCV13). Co-administration of PLY-D and PCV13 conferred greater protection against challenge with a serotype 6B strain than immunization with either vaccine alone. These data indicate that PLY-D is a broadly protective antigen with the potential to serve as a serotype-independent vaccine against invasive pneumococcal disease either alone or in combination with PCVs.

A phase 1 randomized safety, reactogenicity, and immunogenicity study of Typhax: A novel protein capsular matrix vaccine candidate for the prevention of typhoid fever.

BACKGROUND: Typhoid fever remains a significant cause of morbidity and mortality in developing countries especially in children ≤5 years old. Although the widely available unconjugated Vi polysaccharide vaccines are efficacious, they confer limited, short-term protection and are not approved for young children or infants. Vi conjugate vaccines, however, are now licensed in several typhoid endemic countries for use in children >6 months of age. As an alternative to conjugate vaccines, Matrivax has applied its novel 'virtual conjugation' Protein Capsular Matrix Vaccine (PCMV) technology to manufacture Typhax, which is composed of Vi polysaccharide entrapped in a cross-linked CRM197 matrix. METHODOLOGY: A randomized, double-blinded, dose escalating Phase 1 study was performed to compare the safety and immunogenicity of three dose levels of aluminum phosphate adjuvanted Typhax (0.5, 2.5, or 10 µg of Vi antigen) to the FDA licensed vaccine, Typhim Vi, and placebo. Groups of 15 healthy adult subjects aged 18 to 55 years were randomized and received Typhax, Typhim Vi, or placebo at a ratio of 9:3:3. Typhax and placebo were administered in a two-dose regimen (Days 0 and 28) while Typhim Vi was administered as a single-dose on Day 0 with a placebo administered on Day 28. All doses were administered as a 0.5 mL intramuscular (IM) injection in a blinded fashion. The anti-Vi IgG antibody response was determined preimmunization (Day 0) and on Days 14, 28, 42, and 180 by ELISA. Seroconversion was defined as a titer 4-fold or greater above baseline. PRINCIPAL FINDINGS: All Typhax vaccine regimens were well tolerated and adverse events were low in number and primarily characterized as mild in intensity and similar in incidence across the treatment groups. Reactogenicity, primarily pain and tenderness at the injection site, was observed in both the Typhax and Typhim Vi treatment groups; a modest increase in incidence was observed with increasing Typhax doses. Following one dose of Typhax, seroconversion rates at day 28 were 12.5%, 77.8%, 66.7% at the 0.5, 2.5, and 10 µg dose levels, respectively, compared to 55.6% and 0% in the Typhim Vi and placebo groups, respectively. A second dose of Typhax on Day 28 did not elicit a significant increase in GMT or seroconversion at Day 42 or Day 180 at any dose level. CONCLUSIONS: Collectively, the results from this randomized phase 1 clinical trial indicate that Typhax is safe, well tolerated, and immunogenic. After a single dose, Typhax at the 2.5 and 10 µg dose levels elicited comparable anti-Vi IgG titers and seroconversion rates as a single dose of Typhim Vi (25 µg dose). A second dose of Typhax at Day 28 did not elicit a booster response. TRIAL REGISTRATION: ClinicalTrials.gov NCT03926455.

In vitro characterization and preclinical immunogenicity of Typhax, a typhoid fever protein capsular matrix vaccine candidate.

Typhax is an investigational typhoid fever vaccine candidate that was GMP manufactured applying Protein Capsular Matrix Vaccine (PCMV) technology. It consists of Vi polysaccharide antigen, derived from S. Typhi, non-covalently entrapped in a glutaraldehyde catalyzed cross-linked α-poly-L-lysine and CRM197 protein matrix. Analysis of Typhax determined the average molecular weight of the vaccine particles was approximately 6 x 106 Daltons, corresponding to particles containing 1-2 molecules of Vi polysaccharide and 10-20 molecules of CRM197 protein. The ratio of the concentration of Vi to CRM197 protein in Typhax is 2.4:1. Preclinical immunogenicity studies in mice demonstrated that Typhax was immunogenic and elicited a significant increase in anti-Vi IgG antibody titers following each immunization. The anti-Vi IgG antibody response elicited by Typhax in rabbits increased as the dose increased from 0.1 µg to 2.5 µg. Further, at the 2.5 and 10 µg dose levels, the anti-Vi IgG antibody titers increased after the second and third immunizations. At the 10 µg dose level, 100% of rabbits seroconverted. In the non-human primate (NHP) study, 100% seroconversion was observed at both 2.5 µg and 10 µg dose levels after the first immunization. A murine in vivo immunopotency study demonstrated that Typhax stored at 4°C was stable for at least 30 months. Collectively, the Typhax in vitro profile, preclinical immunogenicity studies, and rabbit toxicology study indicate that Typhax is a viable typhoid fever vaccine candidate for Phase 1 clinical trial evaluation.

Conjugate-like immunogens produced as protein capsular matrix vaccines.

Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell-independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a "carrier protein" antigen. Such "conjugate vaccines" efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.

Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist.

Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.