Design and structural bioinformatic analysis of polypeptide antigens useful for the SRLV serodiagnosis.

Due to their intrinsic genetic, structural and phenotypic variability the Lentiviruses, and specifically small ruminant lentiviruses (SRLV), are considered viral quasispecies with a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra or mutant cloud. Immunoenzymatic tests for SRLVs are available but the dynamic heterogeneity of the virus makes the development of a diagnostic "golden standard" extremely difficult. The ELISA reported in the literature have been obtained using proteins derived from a single strain or they are multi-strain based assay that may increase the sensitivity of the serological diagnosis. Hundreds of SRLV protein sequences derived from different viral strains are deposited in GenBank. The aim of this study is to verify if the database can be exploited with the help of bioinformatics in order to have a more systematic approach in the design of a set of representative protein antigens useful in the SRLV serodiagnosis. Clustering, molecular modelling, molecular dynamics, epitope predictions and aggregative/solubility predictions were the main bioinformatic tools used. This approach led to the design of SRLV antigenic proteins that were expressed by recombinant DNA technology using synthetic genes, analyzed by CD spectroscopy, tested by ELISA and preliminarily compared to currently commercially available detection kits.

Aldehyde modification and alum coadjuvancy enhance anti-TNF-α autovaccination and mitigate arthritis in rat.

Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collagen-induced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.

Aldehyde modification of peptide immunogen enhances protein-reactive antibody response to toxic shock syndrome toxin-1.

Introduction of aldehyde groups into protein conjugates enhanced the immune response to a coupled peptide without the use of strong adjuvants. Synthetic peptides representing the N-terminal (residues 1-16) and internal (residues 53-65) epitopes of toxic shock syndrome toxin-1 (TSST-1) were coupled to carrier protein, and carbonyl tags were introduced by Amadori reaction with glycolaldehyde. Modified and unmodified antigens in alum were used to immunize rabbits and the reactivities of antisera were compared. Aldehyde modification augmented the response detected by ELISA, which included enhanced binding to peptides and to native TSST-1. In western blot, TSST-1 was detected by antiserum elicited to the N-terminal peptide, but not that generated to the peptide representing the internal sequence. The same antiserum also neutralized TSST-1 activity in a lymphocyte proliferation assay. The circular dichroism spectrum of the N-terminal peptide indicated a propensity for helical conformation, similar to the structure at the corresponding sequence of the native protein. These data suggest that aldehyde modification can boost immunogenicity of peptide-based vaccines, generating epitope-specific immune responses against the cognate protein antigens without using potent adjuvants.