Charlotte Brookes

Phase I safety and immunogenicity study of a candidate meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

ABSTRACT Natural immunity to meningococcal disease in young children is associated epidemiologically with carriage of commensal Neisseria species, including Neisseria lactamica. We have previously demonstrated that outer membrane vesicles (OMVs) from N. lactamica provide protection against lethal challenge in a mouse model of meningococcal septicemia. We evaluated the safety and immunogenicity of an N. lactamica OMV vaccine in a phase I placebo-controlled, double-blinded clinical trial. Ninety-seven healthy young adult male volunteers were randomized to receive three doses of either an OMV vaccine or an Alhydrogel control. Subsequently, some subjects who had received the OMV vaccine also received a fourth dose of OMV vaccine, 6 months after the third dose. Injection site reactions were more frequent in the OMV-receiving group, but all reactions were mild or moderate in intensity. The OMV vaccine was immunogenic, eliciting rises in titers of immunoglobulin G (IgG) against the vaccine OMVs, together with a significant booster response, as determined by an enzyme-linked immunosorbent assay. Additionally, the vaccine induced modest cross-reactive immunity to six diverse strains of serogroup B Neisseria meningitidis, including IgG against meningococcal OMVs, serum bactericidal antibodies, and opsonophagocytic activity. The percentages of subjects showing ≥4-fold rises in bactericidal antibody titer obtained were similar to those previously reported for the Norwegian meningococcal OMV vaccine against the same heterologous meningococcal strain panel. In conclusion, this N. lactamica OMV vaccine is safe and induces a weak but broad humoral immune response to N. meningitidis.

Identification of new meningococcal serogroup B surface antigens through a systematic analysis of neisserial genomes

The difficulty of inducing an effective immune response against the Neisseria meningitidis serogroup B capsular polysaccharide has lead to the search for vaccines for this serogroup based on outer membrane proteins. The availability of the first meningococcal genome (MC58 strain) allowed the expansion of high-throughput methods to explore the protein profile displayed by N. meningitidis. By combining a pan-genome analysis with an extensive experimental validation to identify new potential vaccine candidates, genes coding for antigens likely to be exposed on the surface of the meningococcus were selected after a multistep comparative analysis of entire Neisseria genomes. Eleven novel putative ORF annotations were reported for serogroup B strain MC58. Furthermore, a total of 20 new predicted potential pan-neisserial vaccine candidates were produced as recombinant proteins and evaluated using immunological assays. Potential vaccine candidate coding genes were PCR-amplified from a panel of representative strains and their variability analyzed using maximum likelihood approaches for detecting positive selection. Finally, five proteins all capable of inducing a functional antibody response vs N. meningitidis strain CU385 were identified as new attractive vaccine candidates: NMB0606 a potential YajC orthologue, NMB0928 the neisserial NlpB (BamC), NMB0873 a LolB orthologue, NMB1163 a protein belonging to a curli-like assembly machinery, and NMB0938 (a neisserial specific antigen) with evidence of positive selection appreciated for NMB0928. The new set of vaccine candidates and the novel proposed functions will open a new wave of research in the search for the elusive neisserial vaccine.

Induction of immune responses by purified outer membrane protein complexes from Neisseria meningitidis

A broad-spectrum vaccine against disease caused by serogroup B of Neisseria meningitidis is still a challenge due to antigenic variability. In the present study outer membrane protein complexes and their components were analysed using non-denaturing 2D electrophoresis and identified using LC/MS-MS and MALDI-TOF. Outer membrane protein complexes were purified from both the wild-type strain H44/76 and their knock-out mutants lacking PorA, PorB, RmpM or FetA. The immune responses elicited by the whole outer membrane vesicles (OMV) and the purified complexes were analysed for bactericidal activity, antibody surface binding, antibody-mediated C3b/iC3b deposition, membrane attack complex (MAC) deposition and induction of opsonophagocytosis, both on the homologous and several heterologous strains. The main antigenic complexes found were homomeric, formed by the 60 kDa chaperonin (MSP63) or PorB, or heteromeric, formed by different combinations of PorA, PorB and/or RmpM. The lack of some of these proteins in the OMVs from the knock-out mutants did not affect significantly the immune responses analysed except MAC, which was significantly reduced in the anti-PorA- and anti-PorB- sera, and bactericidal activity, which was absent in the anti-PorA- serum. The sera against purified native complexes showed variable activities against the homologous strain, with greatest responses observed for anti-chaperonin and anti-PorA/PorB/RmpM sera. When tested against heterologous strains, the only anti-complex serum showing consistent responses was that against the 60 kDa chaperonin. The comparison of the responses elicited by the different sera suggests an important role of conformational epitopes, present only in native complexes, in the induction of more effective responses against N. meningitidis.

Assessment of vaccine potential of the Neisseria-specific protein NMB0938

The availability of complete genome sequence of Neisseria meningitidis serogroup B strain MC58 and reverse vaccinology has allowed the discovery of several novel antigens. Here, we have explored the potential of N. meningitidis lipoprotein NMB0938 as a vaccine candidate, based on investigation of gene sequence conservation and the antibody response elicited after immunization in mice. This antigen was previously identified by a genome-based approach as an outer membrane lipoprotein unique to the Neisseria genus. The nmb0938 gene was present in all 37 Neisseria isolates analyzed in this study. Based on amino acid sequence identity, 16 unique sequences were identified which clustered into three variants with identities ranging from 92 to 99%, with one cluster represented by the Neisseria lactamica strains. Recombinant protein NMB0938 (rNMB0938) was expressed in Escherichia coli and purified after solubilization of the insoluble fraction. Antisera produced in mice against purified rNMB0938 reacted with a range of meningococcal strains in whole-cell ELISA and western blotting. Using flow cytometry, it was also shown that anti-rNMB0938 antibodies bound to the surface of the homologous meningococcal strain and activated complement deposition. Moreover, antibodies against rNMB0938 elicited complement-mediated killing of meningococcal strains from both sequence variants and conferred passive protection against meningococcal bacteremia in infant rats. According to our results, NMB0938 represents a promising candidate to be included in a vaccine to prevent meningococcal disease.

Development of a large scale human complement source for use in bacterial immunoassays.

The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.

Structural characterisation, stability and antibody recognition of chimeric NHBA-GNA1030: an investigational vaccine component against Neisseria meningitidis.

A new generation multi-component vaccine, principally directed against serogroup B Neisseria meningitidis (4CMenB), has recently been developed. One of its components, identified through reverse vaccinology, is the neisserial heparin-binding antigen (NHBA) which is included in the formulation as a novel NHBA-GNA1030 fusion protein (NHBA-FP). We describe here the biophysical characteristics of this vaccine antigen to understand better its structural properties in solution and concurrent immunogenicity prior to formulation. By deliberately stressing the protein to lose its immune responses, we were able to study the proteins structural changes at the molecular level. The unmodified NHBA-FP was found to be mainly monomeric with mass of 67kDa and secondary structure dominated by β-sheets and turns (57% average). The antigen was very stable in storage buffer. It could be forced to unfold in a low-salt buffer resulting in the exposure of one of its two tryptophan residues at 50°C. Long-term stress studies (10-15 days at 37°C) showed modification in the chromatographic and electrophoretic profiles with progressive degradation and aggregation. Since there was little change in secondary structure (as monitored by circular dichroism and tryptophan fluorescence spectroscopy), the loss of functional immunogenicity of the thermal stressed protein could be mainly attributed to the observed fragmentation and aggregation. We therefore conclude that the maintenance of the intact, non-fragmented state of the NHBA-FP is important to preserve its functional immunogenicity. This may thus be utilised as an assay for the control testing of the protein.