Introduction: steps and challenges in eptiope-based vaccine Production

In the natural pathogen, there is a plethora of antigens, each comprising tens or hundreds of potentail epitopes. Not all antigens or epitopes are equally useful in generating protective immunity. The complexity of antibodies in an individual’s polyclonal serum also correspodns to a relatively limited collection of eptiopes that may not necessarily include those that are the most important for protection. The most dominant epitopes aslo do not necessarily correspond to the most effective neturalizing ones. In fact, natural selection pressure tends to drive occulsion of the most neutralizing epitopes of the pathogen. The pathogen gains a selective advantage when it is able to obscure its weak points and distract the host’s immune system by devising deductive epitopes with little protective value. For eexample, the five variable loops of HIV-1 gp120 are highly immunogenic but, because of their ever chaning nature, allow the swarm of HIV to evade immune surveillance by constantly mutating out of the binding capacity of the antibodies produced after first encounters. Eptiope-based vaccines can contribute to overcoming this problem in that epitopes can be selected for their ability to elicity potent neutralization rather than their natural surface accessibility. Such epitopes are most likely to correspond to conserved aspects of the pathogen that cannot tolerate modificaitons and through natural selection have evolved to be less immunogenic. The production of vaccines can be technically complicated and biohazardous when manufacturers are required to culture large volumes of pathogens. Moreover, each pathogen may have its own idiosyncrasies that translate into the development of custom tailored protocols for production, requiring specific conditions and ragents. A first step in developing an epitopebased vaccine is to identify the epitope itself. The epitope should correspond to the ultimate immuen response desired, i.e., the broad cross neutralization of the genetic diversity of pathogens for which proteiction is required. One such an epitope has been definted, the second step would be to reconstitute the epitope into a functional immunogen. There are a number of considerations that must be taken into account. First, B-cell epitopes (as opposed to T-cell epitopes) are very often discontinuous and highly conformational. In fact, even in the event that a significant porition of an epitope is a short linear peptide, this does not promise that such a peptide represetns the entire epitope or that it does not require a distinct conformation. B cell epitopes typically comprise some 15-20 residues derived form 2-3 discontinous segments of the antigen brought together through folding to produce a contiguous surface that is recognized by the antibody. The task of reconstituting an effective epitope-based vaccine must take these facts into consideration and attempt to position the critical contact residues in a proper spatial oritentation. This is by no means an easy task. (Gershoni, “Eptiope Mapping: The first step in developing epitope-based vaccines” Biodrugs, 2017, 21 (3)). 

Mutagensis

One of the simplest and most popular approaches towards eptiope mapping is site directed mutagensis. The concept is straightforward in that one can replace any amino acid at a given position in the antigen with another, and then test the bidning capacity of the modified protein. Loss of binding is taken to indicate that the modified resiue could be assocaited with the eptiope in question.

Alanine Scanning Mutagensis (ASm or “alanine walking): is the most systematic approach for mutagenesis in that one introduces an alanine for every resiude in a given sequence, one at a time (positions containing alanine in the original sequence are usually replaced with glycine). ASM provides important informaiton on the protien-binding interface, but the mathod is laborius. Many mutants must be produced, purified and evaluated. (Gershoni, “Eptiope Mapping: The first step in developing epitope-based vaccines” Biodrugs, 2017, 21 (3)).

Alanine scanning is a site directed mutagenesis method f replacing amino acid resiudes in a polypeptide with alanine to scan the polypeptide for residues involved in an interaction of itnerest. Alanine scanning has been particularly successful in systematically mapping functional binding epitopes.  (Sidhu, US 2007/0117126). 

Computational Docking

There is an abudnance of structures for isolated antibodies and Fabs, and the produciton of new structures either experimentally or by computer modeling has become reasonably efficient. Thus, in the event that a structure can be obtained for the antigen in question, generating a model of the specific corresponding antibody should not pose a problem. Once the two structures exist separately, a battery of computer algorithms exist that can be used to attempt to dock in silico one structure onto the other. The ultimate goal of all docking algorithms is to determine the structure of a complex from the separately detemriend coordinates of its compoents. In 1982 Kuntz came out with the first widely used docking program, named DOCK. Since then the docking field has flourished with many new alorithms. (Gershoni, “Eptiope Mapping: The first step in developing epitope-based vaccines” Biodrugs, 2017, 21 (3)).

Replacement of Amino Acids: 

In a replacemnet net mapping method an an original amino acid resiude is replaced by any other amino acid residue. Preferably, a plurality of molecules is generated, wherein different amino acids are replaced, either by alanine or by any other amino acid residue. The TDK-alsacan method involves substitution of an original amino acid residue by alanine. 

Peptdies displaced in context of HLA molecules:

Weidanz (US 2006/0034850) discloses peptides displayed in the context of HLA molecules. An effective amount of the immunogen is then adminsitered to a host for eliciting an immune response, serum collected and assayed to determine if desired antibodies that recognize a 3-D presentaiton of the peptide in the binding groove of the MHC molecule are being produced. The desired antibodies can differentaiate the peptide/MHC complex form the MHC molecule alone, the peptide alone and the complex of MHC and irrelvant peptide. Finally, the antibodies are isolated.