Development of Typhax, a Salmonella Typhi Vi polysaccharide protein capsular matrix vaccine

Matrivax Research & Development Corporation is researching and developing a novel technology termed Protein Capsular Matrix Vaccine (PCMV) as an alternative to polysaccharide-protein conjugate vaccines. In a PCMV, polysaccharide antigens are entrapped in a cross-linked protein ‘carrier’ matrix. This process is simpler than conjugate vaccines and should yield polysaccharide vaccines that elicit TH-cell ‘memory’, are highly efficacious, and less expensive to manufacture. Typhoid fever, caused by Salmonella enterica serovar Typhi, is a disease that afflicts ~16 million people worldwide, resulting in 600,000 deaths, annually. Although typhoid fever vaccines are commercially available, there are significant limitations. Ty21a is an oral vaccine that requires a multi-dose regimen; whereas Typherix® and TyphimVi® are parenteral and associated with local reactogenicity. The existing typhoid vaccines confer variable, ~70%, protective efficacy, do not protect young children (\u3c2 years old), and are not used for routine immunization. Please click Additional Files below to see the full abstract

Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells

The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40°C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36°C, the optimum growth temperature of M. xanthus. Cells preincubated at 36°C for 1 h before being shifted to 40°C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40°C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40°C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40°C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells

DEVELOPMENT OF PROTEIN CAPSULAR MATRIX VACCINE (PCMV) TECHNOLOGY

Matrivax R&D Corp. is a start-up biotechnology company with R&D operations located in Boston, USA and a vaccine pilot facility in Haikou, China. We are developing a proprietary vaccine process that entraps polysaccharides in a cross-linked protein ‘carrier’ or matrix, termed Protein Capsular Matrix Vaccine (PCMV), as an alternative to conjugate vaccine technology. Despite highly efficacious pneumococcal vaccines such as Prevnar®, S. pneumoniae causes \u3e 1 million deaths worldwide annually. Likewise, typhoid fever afflicts ~16 million people, resulting in 600,000 deaths despite effective vaccines such as Typhim Vi® and Ty21a. The premise is that inexpensive,efficacious polysaccharide vaccines that elicit TH-cell ‘memory’ will actively displace their unconjugated and conjugated vaccine counterparts. Towards this end, Matrivax is actively research and developing pneumococcal, enteric fever, and meningococcal PCMV candidates. In preliminary studies, a pneumococcal capsular polysaccharide (PPS) 14 ‘whole reaction’ PCMV employing ‘unoptimized chemistry’ elicited an anti-PPS14 reciprocal IgG antibody titer of ~7,000. Functional antibodies were elicited as evidenced by anti-sera facilitated opsonization and passively transferred antibodies conferring protection against lethal pneumococcal challenge. Recently Matrivax devised ‘optimized’ PCMV chemical reaction conditions improving polysaccharide incorporation into protein matrices as well as separated ‘whole reaction’ PCMV by size-exclusion chromatography yielding ‘size-fractionated’ PCMV particles. Size fractionated PPS14 PCMVs and Prevnar® were used to immunize mice in a three dose, bi-weekly regimen. Particle sized PCMV containing 0.12 and 0.03 ug PPS14 elicited anti-PPS14 reciprocal antibody GMT of 617,077 and 501,103,respectively, compared to Prevnar® (2 ug PPS14) which elicited a titer of 776,047. Thus, optimized PCMVs containing 1.5% or 6% the amount of PPS14 contained in Prevnar® elicited a comparable anti-PPS14 antibody response. Matrivax next evaluated PCMV’s applicability to Vi antigen. SDS-PAGE data demonstrated that Vi was captured in a DNI matrix and capture ELISA further indicated that Vi antigen was localized at the surface of PCMV particles. Size fractionated Vi-DNI PMCV was compared to Typhim Vi® following a three dose, bi-weekly vaccine regimen in a murine immunogenicity study. Ten (10) ug Typhim Vi elicited an anti-Vi reciprocal antibody GMT of 200 whereas size-fractionated PCMVs containing ~2 ug Vi elicited an anti-Vi antibody GMT of \u3e600. A Vi PCMV Phase 1 clinical trial is scheduled for 2Q11

MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling

Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers

Determining crystal structures through crowdsourcing and coursework

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality

Geolocation Assisted Routing Protocols for Vehicular Networks

International Conference on Connected Vehicles (ICCVE)The class of flooding-based DTN routing protocols that leverage (transitive) encounter probabilities have been shown to perform well in selected simulations and scenarios, however they are especially sensitive to heterogeneous mobility models in which some nodes’ mobility pattern is on a significantly differ- ent timescale than others. In particular, military and disaster response scenarios can exhibit abrupt topology changes. We analytically show that the worst-case inputs to these existing DTN routing algorithms can drastically reduce their performance. In light of such scenarios, we develop new protocols that inherit the benefits of existing schemes, while leveraging geographic assis- tance to enable faster recovery from abrupt topology changes.This work was funded in part by the US Marine Corps and US Nav

Low threshold voltage vertical cavity surface-emitting laser

Includes bibliographical references (page 586).Vertical-cavity surface emitting laser diodes with threshold voltages as low as 1•48 V are demonstrated. The devices have low-resistance epitaxial mirrors, and current passes through the entire mirror stack. The low threshold voltage results from both low threshold current densities and mirrors with low resistivities even at low current densities. The laser fabrication sequence is relatively quick and simple and allows for the rapid characterization of vertical-cavity surface-emittin glaser material

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

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