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A major limitation of many current immunomodulation
approaches is that they are not specific and can cause unwanted side effects. Antigen Express has developed novel technology that can be used for antigen-specific
stimulation or suppression of helper T cells. This more selective modulation of the immune system may make it possible to develop better immune therapies for a range of diseases as well as prophylactic
vaccines.
Summary
Linking the immunoregulatory Ii-Key moiety of the MHC class II-associated invariant chain (or Ii protein) to an MHC class II epitope greatly increases the vaccine potency of that epitope peptide. Ii-Key/MHC class II epitope hybrids have increased potency 200 times in vitro for presentation of epitopes to T cell hybridomas and 8 to 10 times in mouse immunizations when assayed by interferon-gamma ELISPOT using immunopurified CD4+ T helper cells. Antigen-specific T helper stimulation has been shown to play a major role in augmenting the production of both antibodies and cytotoxic T cells as well as in providing immunological memory. Here we describe the application of this technology to influenza H5N1 and hypothesize that Ii-Key/MHC class II epitope peptide vaccines, derived from hemagglutinin (HA), will lead to clinically useful T-helper cell
responses
Using an MHC class II epitope predictive algorithm, we anticipate identifying a selected set of three to five hybrid vaccine peptides cumulatively presented by greater than 90 percent of people. Studies with other Ii-Key/MHC class II epitope hybrids with predicted HLA-DR or I-E antigenic epitopes demonstrate that 50 percent to 70 percent of the epitopes are recognized in humans or mice. While comparison of influenza viruses with different HA subtypes indicate that modified MHC class II epitopes from one strain will not protect against the others, the level of conservation within strains suggests that Ii-Key/HA MHC class II hybrids could offer significant protection resulting from antigenic drift. Upon infection, such T-helper cell responses will enhance the induction and magnitude of neutralizing antibodies and may induce long-term protection. Such a selected set of Ii-Key/H5 HA hybrid peptides may be a useful prophylactic against infection as a stand-alone and/or in improving the specific immune response to more conventional H5N1 vaccines (e.g., inactivated virus, subunit, recombinant protein, and/or DNA
vaccines).
Introduction
T-helper cells, which become activated via MHC class II/antigen presentation (signal 1) and costimulatory molecule interaction (signal 2), are essential to a robust antibody and cytotoxic T cell response and long-term immunological memory (1,2). For example, dendritic cells require
"licensing" by CD4+ T cells before they can activate and expand CD8+ cytotoxic T lymphocytes (3), while influenza virosomes enhance class I restricted CTL induction by CD4+ T-helper cell activation (4). T-helper cells also activate and promote differentiation of antibody-producing plasma
cells.
A portion of the MHC class II-associated invariant chain (Ii protein) is used to enhance the antigen-specific stimulation of T helper cells by facilitating direct charging of MHC class II molecules. Prior to class II epitope loading, the antigenic peptide-binding site of class II molecules is blocked by the Ii protein, which occurs at the time of synthesis in the ER of professional antigen presenting cells (APC). The MHC class II molecules with the site-blocking Ii protein traffic intracellularly to a post-Golgi compartment where cleavage of the Ii protein occurs in a concerted process with binding of processed fragments of foreign antigens (5,6), followed by localization to the cell surface for presentation. The Ii-Key portion of the Ii protein was found to regulate tightness of closure of the antigenic peptide-binding site of MHC class II molecules. This segment was identified because of its six positive side chains, no negative side chains, and four proline residues, which together appeared to constitute a signal for a protease or
"exchangease" regulating cleavage and release of Ii protein and antigenic peptide binding (7,8). Mutations in this segment blocked the staged cleavage and release of Ii proteins (9). Many Ii-Key peptide homologues enhanced presentation of synthesized antigenic peptides to murine T hybridomas (10,11). Coupling the Ii-Key peptide to an antigenic peptide enhanced presentation about 200 times in vitro (12). More importantly, such compounds enhanced Th1 type responses 8- to 10-fold in vivo, as judged by IFN-g ELISPOT assays (13). This observation was pivotal in demonstrating the clinical potential of Ii-Key/epitope hybrids.Molecular modeling studies suggest that the Ii-Key moiety binds to an allosteric site on the surface of MHC class II molecules outside of the antigenic peptide-binding trough. The MHC class II epitope of the hybrid then binds into the antigenic peptide-binding site, pulling the Ii-Key moiety from the allosteric site (see Figure). The increased potency of presentation of the MHC class II epitope, afforded by the Ii-Key moiety, might lead to presentation of some epitopes by HLA-DR alleles that otherwise would be judged to be
"low responder" alleles. Such increased "promiscuity" of presentation of an epitope within a Ii-Key/Flu H5 MHC class II epitope hybrid peptide could contribute to coverage of a greater percentage of subjects than predicted with the SYFPEITHI epitope-prediction
program.
Epitope Algorithm
Prediction
Currently, no MHC class II epitopes have been identified for H5N1 HA. We utilized an algorithm-based prediction approach in a manner to maximize the likelihood of identifying promiscuous HA epitopes. Initially, epitopes were predicted for HLA-DRB1 alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, and DRB1*1501) using the Rammensee SYFPEITHI epitope prediction program
(www.syfpeithi.de) from the A/Duck/Anyang/AVL-1/2001 strain of H5N1 (GenBank accession number AF468837). The 40 top-scoring predicted epitopes were ranked on a cumulative basis according to the score reported from the SYFPEITHI program for the alleles
indicated.
In some cases, an epitope predicted for presentation by a second allele overlaps the reported high-scoring MHC class II allele offset by only one or two amino acids. When the second epitope extends one or two amino acids C-terminal to the reference epitope, then
"+1" or "+2," respectively, is given in the overlap-scoring column, labeled
"fl" to the right of the respective allele. When the second epitope extends one or two amino acids N-terminal to the reference epitope, then
"-1" or "-2," respectively, is listed in the overlap-scoring column. Because Ii-Key hybrid peptides are synthesized with two flanking amino acids of the primary sequence at both N- and C-termini of the epitope, additional epitopes including such flanking amino acids might be present. In general, one observes that many high-scoring epitopes might be presented by more than one HLA-DR allele. Such hybrids are likely to be
"promiscuous" either because one epitope is presented by multiple HLA-DR alleles and/or because two or more closely overlapping epitopes (presented by difference HLA-DR alleles) are present within the 2+9+2 = 13 amino acids of the epitope-containing segment of the Ii-Key hybrid
peptide.
From this set of predicted epitopes, a number of epitopes were excluded based on the presence cysteines or an overabundance of residues containing bulky hydrophobic side chains that may reduce solubility. Ii-Key hybrids of the remaining 24 epitopes were synthesized for immunogenicity studies in BALB/c, DR4 transgenic mice and potential screening using PBMC from subjects with a known immunological response to H5N1. While some have been found to be active in stimulating T helper activity and in potentiating neutralizing antibody response to recombinant HA protein (rHA) in BALB/c and DR4 mice (data not shown), their ability to stimulate PBMC ex vivo from subjects known to have an immunological response to the H5N1 has not yet been determined. Given the urgency of a potential H5N1 pandemic coupled with the relative safety of peptide vaccines, one may well argue that a clinical trial of a reasonably small number of Ii-Key hybrid peptide vaccines in people is
warranted.
Discussion
While there is a total absence of identical MHC class II epitopes in the HA among, for instance, H1 and H5 influenza strains, they appear to be well conserved within several historically important H1 strains. This suggests that MHC-class-II-epitope-based vaccines have significant potential to protect against interseasonal drift. The consistency of homologous MHC class II epitopes in various H1 strains, in the face of widely varying antibody recognizing determinants (ARD) recognized by neutralizing antibodies, reflects the lack of a selecting pressure against mutations in MHC class II epitopes. This finding might also reflect pressure against mutations in such epitopes, in part because they are distributed through proteins in segments required for tertiary structure, (e.g.,
alpha-helices). The pattern of hydrophobic residues in MHC class II epitope positions P1, P4, P6, and P9 is preferred in proteins in general (14) and in a-helices in particular (15,16). Since such patterns relate to folding of structural element of proteins, the requirement for structural integrity selects against mutations. This suggests that MHC class II epitope hybrids based on HA will have activity in generating an immunological response against a variety of strains possessing
H5.
Perhaps the greatest value in Ii-Key/H5 HA MHC class II epitope hybrid peptide vaccines lies in their use as a pre-vaccine coupled with the use of more conventional H5N1 vaccines. Such a vaccination strategy may significantly decrease the overall dose of conventional vaccine, thereby increasing availability. In addition, such hybrids may augment in a qualitative manner the antibody response to poorly immunogenic conventional vaccines such as rHA. Giving enough protein to approximate the molarity of MHC II epitopes available via hybrids would lead to recruitment of many low-affinity B cell clones, recognizing low-affinity ARDs. In contrast, inoculation with MHC class II epitope hybrids may lead to potent T helper clones, which stimulate B cells with surface Ig recognizing very low concentrations of rH5, yielding antibodies of very high affinity. Also, repeated injections might create a clinically functional response in persons who fail to mount a neutralizing antibody response after vaccination with a current vaccine
(17).
At the least, Ii-Key/MHC class II epitope hybrids have the potential to offer some clinically measurable protection during mass vaccinations in the face of a potential H5N1 pandemic. Given that there will likely be a lack of traditional vaccines if/when such a pandemic occurs, the importance of even partial protection should not be underestimated, considering the possible high mortality rate in people contracting bird flu. There might be limitations in the available amounts of, or time to prepare, traditional vaccines. H5N1 influenza clearly has serious pandemic potential (18,19). The value of the hybrids as an alternate vaccine is enhanced by the particular difficulties in preparing an H5N1 flu vaccine by methods used for previous vaccines
(20,21).
Contact Eric von Hofe at Antigen Express:
evonhofe@antigenexpress.com.
References
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