Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease.

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student ('Case'), who died of IMD, and a close contact ('Carrier') who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine against serogroup B meningococci have focused on the use of surface-exposed outer membrane proteins. Here we report the use of virus-like particles derived from the core protein of Hepatitis B Virus, HBc, to incorporate antigen domains derived from Factor H binding protein (FHbp) and the adhesin NadA. The extracellular domain of NadA was inserted into the major immunodominant region of HBc, and the C-terminal domain of FHbp at the C-terminus (CFHbp), creating a single polypeptide chain 3.7-fold larger than native HBc. Remarkably, cryoelectron microscopy revealed that the construct formed assemblies that were able to incorporate both antigens with minimal structural changes to native HBc. Electron density was weak for NadA and absent for CFHbp, partly attributable to domain flexibility. Following immunization of mice, three HBc fusions (CFHbp or NadA alone, NadA + CFHbp) were able to induce production of IgG1, IgG2a and IgG2b antibodies reactive against their respective antigens at dilutions in excess of 1:18,000. However, only HBc fusions containing NadA elicited the production of antibodies with serum bactericidal activity. It is hypothesized that this improved immune response is attributable to the adoption of a more native-like folding of crucial conformational epitopes of NadA within the chimeric VLP. This work demonstrates that HBc can incorporate insertions of large antigen domains but that maintenance of their three-dimensional structure is likely to be critical in obtaining a protective response.

Measurement of surface protein antigens, PorA and PorB, in Bexsero vaccine using quantitative mass spectrometry.

Bexsero is a multivalent vaccine containing outer membrane vesicles (OMV) derived from Neisseria meningitidis group B strain NZ98/254 and three recombinant meningococcal proteins, Neisserial adhesin A, Heparin binding antigen and factor H binding protein. OMV production relies on the growth of large-scale cultures of N. meningitidis under controlled conditions. Changes to environmental factors, such as temperature, pH, nutrient availability and trace elements, can impact the growth rate of the meningococcus. Furthermore outer membrane expression levels vary in response to the environmental milieu, thus any changes in environmental conditions can result in changes in OMV protein content. This makes consistent production of OMVs challenging and the ability to measure the protein content of the final product is desirable to ensure product quality. The aim of this work was to develop a mass spectrometry (MS) method for measuring the porin proteins and to evaluate this approach for assessing the batch consistency of Bexsero vaccine. Using isotope dilution MS, we measured the PorA and PorB content in 75 lots of Bexsero vaccine. PorA ranged from 4.0 to 5.95 µg/dose with an average of 4.8 µg/dose. PorB ranged from 5.4 to 8.7 µg/dose with an average of 6.5 µg/dose. This is the first description of the quantitative characterisation of adjuvanted Bexsero vaccine drug product at the final stage of the production process, once the aluminium adjuvanted vaccine has been packaged into syringes, to assess manufacturing consistency. The significance of our findings to quality control in the future is discussed.

Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines.

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus genotype 45 (HPV45). Pseudoviruses (PsVs) representing HPV45 sublineages A1, A2, A3, B1, and B2 exhibited comparable particle-to-infectivity ratios and morphologies but demonstrated both increased (A2, A3, and B1) and decreased (B2) sensitivities to cross-neutralization by HPV vaccine antibodies compared to that of the A1 sublineage. Mutant PsVs identified HI loop residue 357 as being critical for conferring this differential sensitivity.