Corrected and Republished from: "A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against Lethal Streptococcus pneumoniae Challenge".

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against Lethal Streptococcus pneumoniae Challenge.

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

An ultra scale-down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants.

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.

Identification of an interleukin (IL)-25-dependent cell population that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion.

Type 2 immunity, which involves coordinated regulation of innate and adaptive immune responses, can protect against helminth parasite infection, but may lead to allergy and asthma after inappropriate activation. We demonstrate that il25(-/-) mice display inefficient Nippostrongylus brasiliensis expulsion and delayed cytokine production by T helper 2 cells. We further establish a key role for interleukin (IL)-25 in regulating a novel population of IL-4-, IL-5-, IL-13-producing non-B/non-T (NBNT), c-kit+, FcepsilonR1- cells during helminth infection. A deficit in this population in il25(-/-) mice correlates with inefficient N. brasiliensis expulsion. In contrast, administration of recombinant IL-25 in vivo induces the appearance of NBNT, c-kit+, FcepsilonR1- cells and leads to rapid worm expulsion that is T and B cell independent, but type 2 cytokine dependent. We demonstrate that these IL-25-regulated cells appear rapidly in the draining lymph nodes, implicating them as a source of type 2 cytokines during initiation of worm expulsion.

Immunogenicity and mechanisms of action of PnuBioVax, a multi-antigen serotype-independent prophylactic vaccine against infection with Streptococcus pneumoniae.

Streptococcus pneumoniae has multiple protein antigens on the surface in addition to the serotype specific polysaccharide capsule antigen. Whilst the capsule antigen is the target of the polysaccharide vaccines, bacterial proteins can also act as targets for the immune system. PnuBioVax (PBV) is being developed as a multi-antigen, serotype-independent prophylactic vaccine against S. pneumoniae disease. In this study we have sought to elucidate the immune response to PBV in immunised rabbits. Sera from PBV immunised rabbits contained high levels of IgG antibodies to the PBV vaccine, and pneumococcal antigens PspA, Ply, PsaA and PiuA which are components of PBV, when compared with control sera. The PBV sera supported killing of the vaccine strain TIGR4 in an opsonophagocytic killing assay and heterologous strains 6B, 19F and 15B. In addition, incubation in PBV sera led to agglutination of several strains of pneumococci, inhibition of Ply-mediated lysis of erythrocytes and reduced bacterial invasion of lung epithelial cells in vitro. These data suggest that PBV vaccination generates sera that has multiple mechanisms of action that may provide effective protection against pneumococcal infection and give broader strain coverage than the current polysaccharide based vaccines.

Safety and immunogenicity of a novel multiple antigen pneumococcal vaccine in adults: A Phase 1 randomised clinical trial.

BACKGROUND: Pneumococcal vaccines, combining multiple protein antigens, provide an alternative approach to currently marketed vaccines and may provide broader protection against pneumococcal disease. This trial evaluated the safety and immunogenicity of a novel vaccine candidate PnuBioVax in healthy young adults. METHODS: In a Phase 1 double-blind study, 36 subjects (18-40 years) were randomised to receive 3 doses of PnuBioVax, 28 days apart, at one of three dose levels (50, 200, 500 µg) or placebo. Safety assessments included rates of emergent adverse events (AEs), injection site and systemic reactions. Immunogenicity endpoints included antibody titre against PnuBioVax and selected pneumococcal antigens. RESULTS: In the placebo (n=9) and PnuBioVax (n=27) vaccinated subjects, there were 15 and 72, reported TEAEs, respectively. The majority of TEAEs were classified as common vaccine related AEs. There were no serious AEs. Common vaccine-related AEs occurred in 13 PnuBioVax (48%) and 2 placebo (22%) subjects and were all headaches (mild and moderate). Injection site reactions, mostly pain and tenderness (graded mild or moderate) were reported, in particular in the 200 µg and 500 µg PnuBioVax groups. There were no clinically significant changes in vital signs, ECG or blood chemistries. Subjects receiving the higher dose (200 and 500 µg) demonstrated a greater fold increase in IgG titre compared with the starting dose (50 µg) or the placebo group. The fold-increase was statistically significantly higher for 200 and 500µg PnuBioVax vs 50µg PnuBioVax and placebo at each timepoint post-immunisation. Most subjects receiving 200 and 500 µg PnuBioVax demonstrated a ≥2-fold increase in antibody against pneumolysin (Ply), Pneumococcal surface antigen (PsaA), PiaA (Pneumococcal iron acquisition), PspA (Pneumococcal surface protein A) and pilus proteins (RrgB and RrgA). CONCLUSIONS: All dose levels were considered safe and well tolerated. There was a statistically significant increase in anti-PnuBioVax IgG titres at the 200 and 500 µg dose levels compared to 50 µg and placebo. TRIAL REGISTRATION NUMBER: NCT02572635https://www.clinicaltrials.gov.