MenB Vaccine Approach by Reverse Vaccinology

The first example in which genomic technology has been used for the identification of potential vaccine candidates is the vaccine against the human pathogen Neisseria meningitidis serogroup B (MenB), the major cause of sepsis and meningitis in children and young adults. In the last forty years, conventional vaccinology approaches failed to provide an effective and universal vaccine against MenB. Although for other meningococcal serogroups (A, C, Y, and W135) conjugate vaccines based on the capsular polysaccharide are available and those based on oligosaccharides are under development, in the case of serogroup B the capsular vaccine cannot be used, as its capsule contains a major component (a(2-8)-linked

N-acetylneuraminic acid), which is also a common carbohydrate present on human tissues. The MenB capsule is therefore poorly immunogenic and may elicit autoantibody.

To overcome this obstacle, the new approach named "reverse vaccinology" was applied to MenB [7]. The complete genome of the virulent strain MC58 was sequenced in collaboration with TIGR using the shotgun strategy. The MenB genome consists of 2 272 352 basepairs with an average G+C content of 53%. Eighty-three percent of the genome codes for 2158 ORFs. Out of these, 1158 have a putative biological role assigned on the basis of their similarity with known proteins, whereas the remaining 1000 do not have a predicted function [8]. Based on the concept that surface-exposed antigens are more susceptible to antibody recognition and therefore are the most suitable candidates for a vaccine, the full genome was screened using bioinformatics tools in order to select ORFs coding for putative surface-exposed or secreted proteins.

Within 18 months after the beginning of sequencing, more than 600 potential vaccine candidates had been predicted by computer analysis and classified as: secreted or outer membrane proteins (13%); lipoproteins (20%); periplasmic proteins (27%); inner membrane proteins (34%); and proteins with interesting homology (6%). All these ORFs were amplified by PCR and cloned in Escherichia coli, in order to express them as N-terminal glutathione-S-transferase (GST) or C-terminal histidine-tag fusion. Three hundred and fifty recombinant proteins were successfully expressed, purified, and used to immunize mice. The antisera obtained were tested on whole-cell bacteria using enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) techniques to evaluate the surface localization of the antigens. In addition, the antisera were tested for bactericidal activity, a property known to correlate with protection in humans. Ninety-one novel surface-exposed antigens were identified, 29 of which were able to induce complement-mediated bactericidal antibody response, a strong indication of proteins capable of inducing protective immunity.

One of the main problems to face in the design of a vaccine against MenB is the sequence variability of the antigens among different strains. For example, the most abundant antigen of MenB, PorA, is extremely variable and able to confer protection only against the homologous strain. In view of that, some of the vaccine candidates selected by the reverse vaccinology approach were analyzed for their sequence variability using a representative panel of strains. Each gene was amplified by PCR and sequenced. The sequences were subjected to multiple alignments to verify the level of homology among the different alleles [7, 9, 10]. The conserved antigens were tested for their ability to induce complement-mediated bacterial killing in a subset of strains representative of the global diversity of the N. meningitidis population, demonstrating that the antigens identified by in silico analysis are good candidates for the development of a vaccine against MenB. These promising vaccine candidates are currently under evaluation and have entered into development.

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