Question

Reverse vaccinology identifies antigens that are very specific to the pathogen. These different antigens can then be synthesized and individually tested for their potential in creating immunity and can thus be used as vaccines. Identify DNA technologies and their purposes in the identification and production of these antigens for vaccine use, and describe the process of making individual antigens in large quantities.

Answer 1

Biochemical, serological and microbiological methods havebeen used to dissect pathogens and identify the componentsuseful for vaccine development.

Although successful in manycases, this approach is time-consuming and fails when thepathogens cannot be cultivated invitro, or when the mostabundant antigens are variable in sequence.

Now genomicapproaches allow prediction of all antigens, independent oftheir abundance and immunogenicity during infection, withoutthe need to grow the pathogen invitro, This allows vaccinedevelopment using non-conventional antigens and exploitingnon-conventional arms of the immune system.

Many vaccinesimpossible to develop so far will become a reality.

Since theprocess of vaccine discovery starts in silico using the geneticinformation rather than the pathogen itself, this novel processcan be named reverse vaccinology.

Group B meningococcus represents the first example of the successful application of reverse vaccinology.

The conventional approach to vaccine developmentagainst this pathogen had been struggling for four decadeswithout progress.

On the one hand, the capsular polysaccharide used to develop conventional and conjugatevaccines against all other pathogenic meningococci couldnot be used because the MenB capsule, which is chemi-cally identical to an ? 2–8 linked polysialic acid present inmany of our tissues, is poorly immunogenic and a potentialcause of autoimmunity.

On the other hand, the protein-based approach had identified as protective antigens themost abundant proteins of the outer membrane.

However, these abundant surface-exposed proteins usuallycontain many amphipathic domains, which span the outermembrane several times and assume a ? -barrel conforma-tion.

The protective epitopes in these proteinsare located in the loops that are exposed on the externalsurface and are usually formed by the precise conformationof a few amino acids.

Therefore, in order to induce protec-tive immunity these antigens need to be folded within the outer membrane and any change in one of the few amino acids of the loop will result in a different epitope.

Vaccines based on outer membrane vesicles and containing themajor outer membrane proteins have been developedand shown to be efficacious in clinical trials; however,owing to the high sequence variability of the externalloops in different MenB strains, protection is inducedonly against the immunizing strain.

As a consequence,the conventional approach to vaccine development hasfailed to deliver a universal vaccine.

Using reverse vaccinology, fragments of DNA werescreened by computer analysis while the MenB nucleotide genome sequence was being determined.

Six hundred novel genes were predicted to codefor surface-exposed or exported proteins.

These werecloned and expressed in Escherichia coli  as fusions to theglutatione transferase or to a histidine tag.

Of these fusion proteins, 350 were successfully expressed, purified andused to immunize mice.

The sera obtained were used to confirm the surface exposure of the proteins by ELISA and FACS analysis, and to test for the ability to induce complement-mediated in vitro killing of bacteria, a testthat correlates with vaccine efficacy in humans.

Within18months, while the nucleotide sequence was still beingfinalized, 85 novel surface-exposed proteins were discov-ered and 25 of these were shown to induce bactericidal antibodies.

These numbers are impressive if oneconsiders that during the past four decades no more thana dozen of such proteins had been identified.

The sur-prising finding was not only the high number of the newproteins found but also the quality of the new proteins.

Inaddition to the conventional outer membrane protein swith variable surface-exposed loops, many of the new proteins were lipoproteins or other types of surface-associated proteins without membrane-span-ning domains.

These were often conserved insequence, and carried multiple protective epitopes con-served in most strains.

These novel proteins provide an optimal basis for the development of a novel and effective vaccine against MenB.