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227a Filamentous Phage for Producing Antibody Responses Focused against B2.1, an HIV-1 Vaccine Lead Peptide
N. E. van Houten*1, M. B. Zwick1,2, A. Menendez1, P. W. H. I. Parren2, D. R. Burton2, and J. K. Scott1
1Simon Fraser Univ., Burnaby, BC, Canada and 2The Scripps Res. Inst., La Jolla, CA
Background: The B2.1 peptide was isolated from a phage-displayed peptide library using IgG1 b12, a human antibody that neutralizes a broad spectrum of HIV-1 isolates. Our objective is to use the B2.1 peptide in a vaccine that will elicit b12-like, HIV-1 neutralizing antibodies. Two immunization studies have been performed in mice with the goal of optimizing the response against the B2.1 peptide while minimizing the response against the carrier used for immunization.
Methods: In the first study, a comparison was made of the immunogenicity of synthetic-B2.1 peptide chemically coupled to filamentous phage, synthetic B2.1 coupled to ovalbumin (Ova), and phage-bearing recombinant B2.1 peptide fused to the phage coat protein pVIII. Mice were immunized intraperitoneally without adjuvant every 2 weeks. Sera were tested by ELISA against the peptide, the carrier protein and gp120. The ratios of peptide response to anticarrier response were compared. Group data were averaged and results were analyzed by ANOVA and Tukey test. The second study also used synthetic B2.1 peptide coupled to phage. Dose, injection route, and use of adjuvant were optimized in this study. Results were analyzed in the same fashion.
Results: Although the Ova conjugate produced stronger antipeptide titers, synthetic B2.1 coupled to phage elicited a larger ratio of antipeptide-to-anticarrier responses. Both synthetic conjugates elicited stronger antipeptide titers than the recombinant phage. The second study showed that injection route played a significant role in producing antipeptide titers that were even stronger than the response against the phage carrier. Adjuvant decreased the number of boosts required to reach maximal antipeptide titer, but did not increase the plateau titer for anti-B2.1 peptide antibodies. No significant cross-reactivity against gp120 was observed in either study.
Conclusions: Synthetic B2.1 peptide produces high titer antipeptide antibodies. Filamentous phage as carrier produce a weaker, but more focused antipeptide response compared to Ova. This is probably due to weaker T-cell epitopes associated with filamentous phage. Modulation of factors such as dose, adjuvant, and immunization route can improve the response against B2.1 peptide. Concommitant studies on the antigenicity of synthetic versus recombinant B2.1 peptide have demonstrated that the recombinant peptide has a significantly higher affinity for the b12 antibody than the synthetic one. Thus, recombinant peptide should be used in immunization studies. Further studies are planned specifically with recombinant B2.1 phage bearing engineered T-cell epitopes that will produce stronger antibody responses, and the deletion of B-cell epitopes endogenous to the phage, to focus the B-cell response against displayed peptides or proteins. This will allow the phage to be used in a prime-boost immunization strategy, in which gp120 (bearing the native epitope for the b12 antibody) conjugated to phage will be used in priming immunizations, and phage baring the B2.1 peptide will be used to specifically boost the production of b12-like antibodies that were elicited in the gp120 priming. To assure that b12-like antibodies can be produced, transgenic mice (Xenomice, from Abgenix), which bear the human germline V, D, and J gene segments that encode the b12 antibody, will be used in these immunizations.
