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A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes.

ABSTRACT: Factor H binding protein (FHbp) is part of two vaccines recently licensed for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp is classified in three phylogenic variant groups that have limited antigenic cross-reactivity, and FHbp variants in one of the groups have low thermal stability. In the present study, we replaced two amino acid residues, R130 and D133, in a stable FHbp variant with their counterparts (L and G) from a less stable variant. The single and double mutants decreased thermal stability of the amino- (N-) terminal domain compared with the wild-type protein as measured by scanning calorimetry. We introduced the converse substitutions, L130R and G133D, in a less stable wild-type FHbp variant, which increased the transition midpoint (Tm) for the N-terminal domain by 8 and 12 °C; together the substitutions increased the Tm by 21 °C. We determined the crystal structure of the double mutant FHbp to 1.6 Å resolution, which showed that R130 and D133 mediated multiple electrostatic interactions. Monoclonal antibodies specific for FHbp epitopes in the N-terminal domain had higher binding affinity for the recombinant double mutant by surface plasmon resonance and to the mutant expressed on meningococci by flow cytometry. The double mutant also had decreased binding of human complement Factor H, which in previous studies increased the protective antibody responses. The stabilized mutant FHbp thus has the potential to stabilize protective epitopes and increase the protective antibody responses to recombinant FHbp vaccines or native outer membrane vesicle vaccines with overexpressed FHbp.

SUBMITTER: Konar M

PROVIDER: S-EPMC4672778 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes.

Konar Monica M Pajon Rolando R Beernink Peter T PT

Proceedings of the National Academy of Sciences of the United States of America 20151116 48

Factor H binding protein (FHbp) is part of two vaccines recently licensed for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp is classified in three phylogenic variant groups that have limited antigenic cross-reactivity, and FHbp variants in one of the groups have low thermal stability. In the present study, we replaced two amino acid residues, R130 and D133, in a stable FHbp variant with their counterparts (L and G) from a less stable variant. The single and double ...[more]

PMID: 26627237

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Project description:NhhA (Neisseria hia homologue) is an outer membrane protein from Neisseria meningitidis, the causative agent of meningococcal disease. The protein is surface exposed and its expression in a wide range of meningococcal strains suggests it is a promising vaccine candidate. In addition, immunization of mice with outer membrane vesicles of strains that overexpress NhhA in conjunction with one of TbpA, Omp85 or NspA results in synergistic bactericidal responses. We previously showed that the NhhA sequence is highly conserved between strains, with the majority of the differences localized to four distinct variable regions located in the amino-terminal region of the mature protein. In this study, N. meningitidis strains were constructed that over-express wild-type NhhA. Strains expressing truncated versions of NhhA, with deletions from the amino-terminal region that removed the most variable regions, were also made. These expression strains were also modified so that immunodominant, phase- and antigenically-variable outer membrane proteins were not expressed, truncated lipooligosaccharide (LOS) expression was genetically fixed (no phase variability), and capsular polysaccharide expression abolished. Outer membrane vesicles derived from these strains were used to immunize mice. As previously observed, a synergistic effect involving another antigen, TbpA, was required to demonstrate bactericidal activity. The highest bactericidal response against a heterologous strain was obtained with a truncated variant of NhhA. These results indicate that removal of (a) variable region(s) does not reduce bactericidal responses against NhhA, and that bactericidal targets exist in regions other than the variable N-teminus. This provides the basis for future examination of responses against truncated NhhA in protecting against heterologous NhhA strains, and further evaluation of truncated NhhA as a candidate for inclusion in a vaccine against all serogroups of N. meningitidis.

Project description:The majority of current vaccines depend on a continuous "cold chain" of storage and handling between 2 and 8°C. Vaccines experiencing temperature excursions outside this range can suffer from reduced potency. This thermal sensitivity results in significant losses of vaccine material each year and risks the administration of vaccines with diminished protective ability, issues that are heightened in the developing world. Here, using peptide self-assemblies based on the fibril-forming peptide Q11 and containing the epitopes OVA323-339 from ovalbumin or ESAT651-70 from Mycobacterium tuberculosis, the chemical, conformational, and immunological stability of supramolecular peptide materials were investigated. It was expected that these materials would exhibit advantageous thermal stability owing to their adjuvant-free and fully synthetic construction. Neither chemical nor conformational changes were observed for either peptide when stored at 45°C for 7days. ESAT651-70-Q11 was strongly immunogenic whether it was stored as a dry powder or as aqueous nanofibers, showing undiminished immunogenicity even when stored as long as six months at 45°C. This result was in contrast to ESAT651-70 conjugated to a protein carrier and adjuvanted with alum, which demonstrated marked thermal sensitivity in these conditions. Antibody titers and affinities were undiminished in mice for OVA323-339-Q11 if it was stored as assembled nanofibers, yet some diminishment was observed for material stored as a dry powder. The OVA study was done in a different mouse strain and with a different prime/boost regimen, and so it should not be compared directly with the study for the ESAT epitope. This work indicates that peptide self-assemblies can possess attractive thermal stability properties in the context of vaccine development.Almost all current vaccines must be maintained within a tight and refrigerated temperature range, usually between 2 and 8°C. This presents significant challenges for their distribution, especially in the developing world. Here we report on the surprisingly robust thermal stability of a self-assembled peptide vaccine. In particular a self-assembled peptide vaccine containing a tuberculosis epitope maintained all of its potency in mice when exposed to an extreme thermal treatment of six months at 45°C. In a different mouse model, we investigated another model epitope and found some storage conditions where potency was diminished. Overall this study illustrates that some self-assembled peptide vaccines can have remarkable thermal stability.

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A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes.

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A meningococcal vaccine antigen engineered to increase thermal stability and stabilize protective epitopes.

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