Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response

Abstract

We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response.

Keywords: Streptococcus agalactiae; antigen; capsular polysaccharide; group B streptococcus; mimotope; synthetic peptide.

Figure 1

Production of VLPs and chemically conjugated VLPs. (A) Transmission electron micrographs of VLPs purified on a density gradient; (B) Coupling of S9 peptide to VLPs. VLPs of ssCCMV were incubated with cross-linking reagent at a 1:1 ratio of cross linker to capsid subunit. Different concentrations of the peptide were then added. After 1.5 hours, the reaction was stopped and samples loaded onto non-reducing 12% SDS-PAGE gels and visualized by silver stain. Free and peptide-conjugated capsid protein was observed; (C) Measurement of S9-peptide on different immunogens. The protein concentration of the different immunogens was determined by the BCA method. Different concentrations were coated onto ELISA wells. Peptide was detected with the S9 mAb, then alkaline phosphatase-conjugated anti-mouse IgM.

Figure 2

Production of antibody to CCMV, S9 peptide, and GBS by mice immunized with different immunogens. Groups of four mice were immunized with ssCCMV VLPs, ssCCMV VLPs conjugated to S9 peptide, subE (monomeric capsid) conjugated to S9 peptide, CPMV VLPs conjugated to S9 peptide, and KLH conjugated to S9 peptide. Mice were immunized either with Freund’s adjuvant, or in the absence of adjuvant. The results of individual mice are shown. Prebleeds are in open bars, sera obtained following immunization in solid bars. ELISA plates were coated with the indicated antigens. Sera were tested at 1:1000 dilution for CCMV, 1:5000 for S9 peptide, and 1:200 for GBS. Binding was detected with alkaline phosphatase conjugated anti-mouse IgG (H + L). Antibodies of all isotypes are detected because of the L chain specificity of the conjugated antiserum.

Figure 3

IgG subclasses of anti-S9 antibody response. Post-immune sera of individual mice immunized with the indicated antigens were tested by ELISA in wells coated with the S9 peptide. Antibodies of the different IgG subclasses were detected with alkaline phosphatase-conjugated subclass-specific antibodies.

Figure 4

Antibody responses in a second experiment. Groups of six mice were immunized with CCMV, CCMV-S9, CPMV-S9, and KLH-S9 with no adjuvant. In panel A, prebleed (open bar) and post-immune serum (filled bar) were tested for binding to CCMV, S9 peptide, and GBS and detected with alkaline phosphatase–conjugated anti-mouse IgG (H + L). In panel B, the sera are tested for IgG subclass of antibody to the S9 peptide.

Figure 5

Cytokine production by lymphocytes from VLP- or carrier-immunized mice. Mice immunized with CCMV-S9 or KLH-S9 were boosted and, four days later, sacrificed. Lymphocytes from mesenteric lymph nodes and spleens were pooled from mice in each group and splenocytes isolated. The cells were incubated with the indicated antigens and cytokines in the cell supernatants measured on day 3 (IFN-γ) or day 5 (IL-4 and IL-10) using cytokine capture ELISAs.