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. 2012 Mar 13:12:7.
doi: 10.1186/1472-6750-12-7.

M cell targeting by a Claudin 4 targeting peptide can enhance mucosal IgA responses

David D Lo  1 , Jun Ling, A Holly Eckelhoefer
Affiliations

M cell targeting by a Claudin 4 targeting peptide can enhance mucosal IgA responses

David D Lo et al. BMC Biotechnol. .

Abstract

Background: Mucosal immune surveillance is thought to be largely achieved through uptake by specialized epithelial M cells. We recently identified Claudin 4 as an M cell target receptor and developed a Claudin 4 targeting peptide (CPE) that can mediate uptake of nanoparticles through Nasal Associated Lymphoid Tissue (NALT) M cells.

Methods: Recombinant influenza hemagglutinin (HA) and a version with the CPE peptide at the C-terminal end was used to immunize mice by the intranasal route along with a single dose of cholera toxin as an adjuvant. Serum and mucosal IgG and IgA responses were tested for reactivity to HA.

Results: We found that the recombinant HA was immunogenic on intranasal administration, and inclusion of the CPE targeting peptide induced higher mucosal IgA responses. This mucosal administration also induced systemic serum IgG responses with Th2 skewing, but targeting did not enhance IgG responses, suggesting that the IgG response to mucosal immunization is independent of the effects of CPE M cell targeting.

Conclusions: M cell targeting mediated by a Claudin 4-specific targeting peptide can enhance mucosal IgA responses above the response to non-targeted mucosal antigen. Since Claudin 4 has also been found to be regulated in human Peyer's patch M cells, the CPE targeting peptide could be a reasonable platform delivery technology for mucosal vaccination.

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Figures

Figure 1
Figure 1
Immunization with chemical conjugation of CPE to HA antigen. Basic protocol using HA antigen chemically conjugated to the CPE targeting peptide, showing induction of both systemic serum and mucosal antibody responses. A, Immunization time line. B, ELISA results, showing fluorescence units of anti-HA response at specified dilutions.
Figure 2
Figure 2
Recombinant HA trimeric fusion protein. Development of a hybrid recombinant HA fusion protein vaccine incorporating the CPE targeting peptide. The recombinant HA protein forms trimeric complexes detectable in non-denaturing gels. A, Schematic of the recombinant fusion, as detailed in the text. B, Non-denaturing gels with Coomassie stain and Western blot for HA, showing trimeric and monomeric forms of the protein. Note that in non-denaturing gels, the protein molecular weight markers are not as accurate as in denaturing gels, so the molecular weight comparisons are only relative.
Figure 3
Figure 3
Immune response to Recombinant fusion protein. Recombinant protein vaccine and a basic four week intranasal immunization protocol, showing enhanced IgA responses by CPE targeting. Systemic serum IgG responses were also induced, without improvement by CPE targeting. A, Immunization protocol. B, ELISA results, showing increased IgA response to HA-CPE protein.
Figure 4
Figure 4
Persistence of response to recombinant protein vaccine, four dose protocol. After an initial four week course of immunization, mice were rested for ten weeks for later assay. Results showed persistence of both the antibody response and evidence for enhanced IgA response when CPE targeting was used. A, Immunization protocol. B, Response 4 weeks after first dose. C, Response 14 weeks after first dose.
Figure 5
Figure 5
Persistence of response to recombinant protein vaccine, three dose protocol. Using an initial three week course of immunization followed by an eleven week rest period, persistent antibody responses were evident, along with enhanced IgA responses to the CPE targeted vaccine. A, Immunization protocol. B, Response 3 weeks after first dose. C, Response 14 weeks after first dose.
Figure 6
Figure 6
Comparison of intranasal and subcutaneous immunization protocols. Analysis of antibody responses to intranasal versus subcutaneous immunization shows similar Th2 dominance in the IgG isotype response. A, Immunization protocols showing the difference between the multiple dose intranasal administration and single dose subcutaneous alum administration. B, Differences in mucosal response to immunization showing the absence of mucosal responses in subcutaneously immunized mice despite similar systemic IgG responses. C, Titration of isotype specific ELISAs for anti-HA response showing the IgG1 dominance over IgG2a immune responses.
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