Cryptic epitope for antibodies should not be forgotten in vaccine design

Introduction

Vaccines have been licensed to prevent infection with approximately 25 different organisms [1]; of these, one-third are subunit vaccines containing critical antigens capable of inducing protective antibodies against single strains. The challenge is that many organisms have multiple strains. For this reason, vaccines have only been developed against two diversified organisms – Streptococcus pneumoniae and Human Papilloma Virus – using a polyvalent vaccine approach.

Plasmodium spp. parasites and Streptococcus pyogenes are two diversified organisms for which highly effective vaccines are not available. They evade immunity by switching antigen expression and/or existing in multiple allelic forms. A virulence factor for P. falciparum is PfEMP1, expressed on the erythrocyte surface and encoded by var genes. It is a target of protective antibodies but approximately 60 variant copies of the gene exist in each parasite [2]. Additionally, most P. falciparum merozoite surface proteins exist in multiple allelic forms rendering antibodies that prevent red cell invasion of one strain ineffectual against other strains [3].

For S. pyogenes, immunity is largely dependent on antibodies to amino-terminal epitopes readily accessible to the immune system on the surface-expressed M protein. Each organism expresses a single strain of M protein but there are over 220 different M-types. While antibodies to the surface of any malaria parasite or streptococcal germ may be lethal to that particular strain, the organisms survive in nature precisely because their hosts generate antibodies to these accessible polymorphic epitopes at the expense of generating antibodies to sensitive, conserved epitopes, which may be partially obscured from ‘immune view’. Some organisms ‘protect’ their sensitive and conserved epitopes by surrounding them with polymorphic residues to attract immune attention, such as the binding pocket of HIV gp120 [4].

Whole organism and hypervalent subunit vaccines circumvent immune decoys for an organism by presenting every possible immune target or every variant of a given immunodominant target antigen, respectively (e.g. [5–7]. While these approaches are promising, these vaccines are logistically very difficult to develop.

For some organisms, polymorphisms are so extensive that a hypervalent vaccine strategy is impossible. An alternative approach is to use highly conserved cryptic epitopes. The important concept is that although these epitopes are poorly immunogenic in the organism, they may be very antigenic. The reason for this difference is that the induction of antibodies (‘immunogenicity’) requires competition for access and growth factors [8]; however, pre-formed antibodies to cryptic epitopes are not competing with other antibodies and have time to find and engage the epitope (‘antigenicity’). The challenge is how to identify such cryptic targets for vaccine design.

Identifying cryptic epitopes

Cryptic epitopes cannot be identified by screening an epitope array with serum from exposed individuals as antibodies will recognize immunodominant polymorphic residues. One approach is to create a comprehensive library of putative epitopes using overlapping synthetic peptides that span the conserved regions of a protein of interest, vaccinate mice with these individually or pooled, then test the antibodies in functional assays in vitro or challenge vaccinated laboratory animals with an infection. This strategy was used in S. pyogenes where a conserved epitope was defined near the carboxylterminus of the M protein [9]. This epitope is partially buried in the capsule of the bacterium and is poorly immunogenic following infection [10]. However, antibodies generated to the isolated epitope are able to kill multiple strains in vitro and protect laboratory animals from multiple strains [11].

Using a similar strategy, a 21-amino acid epitope from the circumsporozoite protein of P. falciparum was defined that induced antibodies capable of blocking invasion of sporozoites into liver cells. This epitope was not immunogenic in the native protein [12].

Cryptic epitopes also include structural antigenic elements exposed specifically in disease states. An epitope (YYR) was identified in the PrP protein that when mis-folded (PrP^SC) causes Transmissible Spongiform Encephalopathies [13]. The epitope discovery was based on the hypothesis that a mis-folded protein would expose hidden residues. To identify these residues, the protein was denatured and tyrosine residues in PrP were exposed. Immunization with the cryptic YYR peptide induced antibodies that immunoprecipitated PrP^SC.

Another strategy is subtractive immunization. Here, a mouse is tolerized to an immunodominant epitope and then immunized with the entire protein or cell. Cryptic epitopes are now recognized because the immune cells reactive to the immunodominant epitopes were removed (reviewed in [14]). This approach was also used to produce diagnostic monoclonal antibodies that recognize cryptic epitopes on pathogenic E. coli [15].

Single chain antibodies, which have flexibility to recognize more concealed epitopes of an antigen, provide a further strategy. Camelid ‘nanobodies’ were used to define epitopes on a malaria-target antigen [16] and IgNARs (‘immunoglobulin new antigen receptors’ – from cartilaginous fishes) were used to probe a hydrophobic cleft on another malaria protein which may lead to a novel vaccine candidate or small molecule inhibitor [17].

Structural and computational biology approaches have been used to generate epitope scaffolds in which the epitope of interest is fused and presented to the immune system in a different environment. This was used to generate structure-specific antibodies against the HIV-1 gp41 epitope [18]. A structural approach was used to design a soluble mini-hemagglutinin stem protein engineered to mimic the structure of the full-length hemagglutinin. This antigen elicited broadly neutralizing antibodies and complete protection from heterologous virus [19].

Cryptic epitopes can thus be exploited to induce antibodies against conserved epitopes shielded from immune selection. Once a candidate is identified, recent advances and established processes in antigen engineering can enable the design of highly immunogenic effective vaccines.

Financial and competing interests disclosure

This work was supported in part by the National Health and Medical Research Council (Australia), the Canadian Institutes of Health Research and Alberta Innovates: Health Solutions. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.