A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity - PubMed (original) (raw)

A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity

S Yamamoto et al. Proc Natl Acad Sci U S A. 1997.

Free PMC article

Abstract

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4(+) T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4(+) Th2-type cells.

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Figures

Figure 1

Figure 1

Serum OVA- and CT-B-specific IgA, IgM, and IgG (A) and IgG subclass (B) responses on day 21 following intranasal immunization with OVA combined with mCT S61F or nCT as adjuvants were determined by endpoint ELISA. Groups of C57BL/6 mice were immunized with 100 μg of OVA alone (□) or together with 5 μg of rCT-B (▨), 0.5 μg of nCT (░⃞), or 5 μg of mCT S61F (▪) on days 0, 7, and 14. Serum samples were collected 1 week after the last immunization. Bars represent the mean Ab titer ± 1 SE in each group. Each group consisted of five mice, and the data are representative of three separate experiments.

Figure 2

Figure 2

Numbers of OVA- and CT-B-specific IgG (A) and IgA (B) AFC in CLN, lung tissues and spleen following intranasal immunization with OVA combined with mCT S61F or nCT as mucosal adjuvants were determined by ELISPOT assay. Groups of C57BL/6 mice were immunized with 100 μg of OVA alone (□) or together with 5 μg of rCT-B (▨), 0.5 μg of nCT (░⃞), or 5 μg of mCT S61F (▪) on days 0, 7, and 14. Samples were collected 1 week after the last immunization. Bars represent the mean numbers of AFC ± 1 SE and each group contained five mice. The data are representative of three separate experiments.

Figure 3

Figure 3

OVA- and CT-B-specific IgA Ab responses in mucosal secretions were determined by ELISA (A) and numbers of IgA AFC in mucosal tissues by ELISPOT assay (B) following intranasal immunization with OVA combined with mCT S61F or nCT as mucosal adjuvants. Groups of C57BL/6 mice were immunized with 100 μg of OVA alone (□) or together with 5 μg of rCT-B (▨), 0.5 μg of nCT (░⃞), or 5 μg of mCT S61F (▪) on days 0, 7, and 14. Tissue samples and external secretions were taken 1 week after the last immunization. Bars represent the mean Ab titer or numbers of AFC ± 1 SE in each group. Each group contained five mice and the data are representative of three separate experiments.

Figure 4

Figure 4

OVA- and CT-B-specific CD4+ T cell proliferative responses isolated from lung (A) and spleen (B). Groups of C57BL/6 mice were immunized with 100 μg of OVA alone (□) or together with 10 μg of rCT-B (▨), 0.5 μg of nCT (░⃞), or 5 μg of mCT S61F (▪) on days 0, 7, and 14. Bars represent the mean stimulation index ± 1 SE and each group contained five mice. The data were similar and are representative of four separate experiments.

Figure 5

Figure 5

Cytokine production from OVA-specific CD4+ T cells isolated from lung tissues. Molecules of cytokine-specific mRNA were determined by quantitative RT-PCR. Cytokine protein production was determined by ELISA. The scale of each figure corresponds to mRNA molecules and protein levels produced by nonimmunized CD4+ T cells stimulated with anti-CD3 mAb. ND, not detected; IFN-γ, interferon γ. Bars represent the mean cytokine profile ± 1 SE in each group. The data are representative of four separate experiments.

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