Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes

BACKGROUND: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines

Cre Levels Limit Packaging Signal Excision Efficiency in the Cre/loxP Helper-Dependent Adenoviral Vector System

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is ≤1% pre- and ≤0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved

Mimotope sequences of affinity-selected phage.

a<p>Polyserum used for mimotope isolation.</p

Antigen structure influences the composition of the polyclonal response.

<p><i>A.</i> ELISA plate wells were coated with recombinant rat ECD and reacted with serial dilutions of mouse sera. <i>B.</i> Recombinant extracellular domain (ECD) of rat HER-2 was treated (<i>reduced protein</i>) or not (<i>native protein</i>) with β-mercaptoethanol and run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000. <i>C.</i> 10⁶ Tubo cells over-expressing rat HER-2 were reacted with serial dilutions of mouse sera, probed with anti-mouse antibody conjugated with PE and fluorescence intensity was measured by flow cytometry. Each point is reflective of at least 10 000 events. <i>D.</i> Recombinant extracellular domain of rat HER-2 or human HER-2 were run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000 or anti-HER-2 antibody. <i>E.</i> 500 TUBO cells were plated into 96 well plate in DMEM medium supplemented with 5% FBS and incubated 1–2 weeks in the presence of IgGs purified from mouse sera at a final concentration of 25 µg/ml.. The cell proliferation was measured according to manufacturer instruction by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay. The error bars reflect the mean+/−sem for 4 samples. These data represent the results of pooled serum from 5 vaccinated mice per group. Each experiment was replicated at least 3 times and representative results are shown.</p

Occurrence of statistically significant amino acids within mimotope collections.

a<p>Polyserum used for mimotope isolation.</p>b<p>The frequency was determined as: (# specific residues/total # of residues in the collection) ×100.</p

Limited cross-reactivity between phage selected with different polysera.

<p>Selected phage were immobilized onto nitrocellulose membranes and reacted with three different sera overnight at 4°C (HER-2_FL – serum prepared from mice immunized with full-length HER-2; HER-2_ECD – serum prepared from mice immunized with soluble protein; HER-2_TUBO – serum from tumor bearing mice). Bound antibodies were detected with goat anti-mouse IgG HRP conjugated antibody and the signals were developed by ECL. <i>A.</i> phages selected with HER-2_FLserum, <i>B.</i> phages selected with HER-2_ECD-serum, <i>C.</i> phages selected with HER-2_TUBO -serum).</p

Three-dimensional modeling of putative epitopes.

<p>Three-dimensional model of rat HER-2 is presented in two orientations. <i>A.</i> clusters found for HER-2_FLserum, <i>B.</i> clusters found for HER-2_ECD-serum, <i>C.</i> clusters found for HER-2_TUBO –serum. Serum- specific clusters of amino acid pairs are numbered. Amino acid are coloured according to RasMol amino acid colour code: red = Glu and Asp, blue = Arg and Lys, brown = Pro, orange = Ser and Thr, Cyan = Asn and Gln, violet = Trp and Phe, purple = His.</p

Theoretical prediction of HER-2 epitopes.

*<p>potential calculated for every amino acid using DiscoTope software and characterizing immunogenic property of a given amino acid.</p