Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response

Abstract

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced. To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses. In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E. coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs. By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.

FIG. 1

A single oral immunization with the Salmonella-CFA/I construct elicits elevated mucosal and serum IgA Ab responses to CFA/I fimbriae, while an attenuated E. coli elicits poor mucosal and serum IgA Ab responses. BALB/c mice were vaccinated with a single dose of designated live Salmonella vector with (strain H696) or without the cfaABCE insert (strain H647) or with a single oral dose of live E. coli-CFA/I vector (strain H695). A single dose of live E. coli-CFA/I vector failed to elicit a mucosal IgA response. The addition of CT along with the E. coli-CFA/I vector failed to elicit a mucosal IgA response 2 weeks after oral immunization. Consequently, an additional oral dose of E. coli-CFA/I with CT was administered, and an adjuvant effect was observed. Fecal and serum IgA anti-CFA/I Ab titers (log₂) were measured by ELISA. Each value represents the mean of 10 mice ± the standard error of the mean. ∗ and ∗∗, where P < 0.001, represent the statistical differences in serum and mucosal IgA anti-CFA/I fimbria levels, respectively, between mice orally immunized with the Salmonella-CFA/I construct and other indicated vaccine strains.

FIG. 2

Oral immunization with the Salmonella-CFA/I vaccine promotes the induction of Th2 cells in Peyer's patch and splenic CD4⁺ T-cell populations. Freshly isolated splenic (A) and Peyer's patch (B) CD4⁺ T cells harvested 1 week after vaccination were examined for cytokine secretion by the cytokine ELISPOT method. Values are expressed as the average number of SFC/10⁶ CD4⁺ T cells from quadruplicate cultures ± the standard deviation (SD). Values were corrected for spontaneous release by cells obtained for normal, uninfected mice. A representative example from three separate experiments is shown. Splenic and Peyer's patch IL-4 and IL-5 SFC were significantly greater in number when compared to IFN-γ SFC (∗, P < 0.001). No IL-10-producing cells were detected. In addition, Peyer's patch IL-6 SFC were significantly elevated (∗, P < 0.001). Splenic (C) and Peyer's patch (D) CD4⁺ T cells obtained 1 week after oral immunization of mice with strain H696 were cultured for 2 days with or without CFA/I fimbriae. These cells were harvested and evaluated for Th1 and Th2 cytokine production by the cytokine ELISPOT method. A representative example from three separate experiments is shown. Values are expressed as the average number of SFC/10⁶ CD4⁺ T cells from quadruplicate cultures ± the SD. Increases in the numbers of IL-4-, IL-5-, and IL-6-producing T cells and minimal IFN-γ-producing T cells were noted compared to unstimulated cells. The numbers of splenic IL-4 and IL-6 (∗∗, P < 0.011), as well as IL-5 SFC (∗, P < 0.001) significantly exceeded splenic IFN-γ SFC; the number of Peyer's patch IL-4, IL-5, IL-6, and IL-10 (∗, P < 0.001) significantly exceeded the numbers of Peyer's patch IFN-γ SFC.

FIG. 3

(A) Antigen-specific serum IgG1 Ab responses increased 1 week after oral immunization with the Salmonella-CFA/I vaccine. (B) While serum IgG2a CFA/I-specific Ab responses are delayed, they become equivalent to serum IgG1 titers by 4 weeks. Serum IgG, IgG1, and IgG2a titers (log₂) were determined by using the ELISA procedure. No serum IgG titers against CFA/I fimbriae were elicited in mice orally immunized with the isogenic, H647 construct (which lacks the cfaABCE insert). Values represent the average of duplicate samples from each of 10 mice/group. ∗, P < 0.001.

FIG. 4

(A) Oral immunization with the E. coli-CFA/I H695 construct failed to elicit elevated serum IgG antibodies against CFA/I fimbriae. A group of 10 BALB/c mice received a single oral dose of the H695 construct, and serum IgG, IgG1, and IgG2a antibody titers were measured by an ELISA method. At 4 weeks after immunization, minimal IgG antibody titers against CFA/I fimbriae were induced, and these antibodies were predominantly IgG1. (B) Oral immunization with the E. coli-CFA/I H695 construct in conjunction with CT on days 0 and 14 resulted in enhanced IgG anti-CFA/I titers. A group of 10 BALB/c mice received a single oral immunization with H695 plus CT showed no IgG antibodies against CFA/I fimbriae 2 weeks after a single oral dose. However, subsequent to receiving an additional oral dose of H695 plus CT resulted in induced CFA/I fimbria-specific IgG, IgG1, and IgG2a antibodies, albeit not as elevated as with the Salmonella-CFA/I H696 construct.

FIG. 5

Four weeks after oral immunization with the Salmonella-CFA/I vaccine, a predominance of IFN-γ-producing CD4⁺ Th cells was detected by the cytokine ELISPOT method. Splenic (A) and Peyer's patch (B) CD4⁺ T cells were cultured for 2 days in medium only, with 10 μg of CFA/I fimbriae, per ml, or with 10 μg of OVA per ml, after which CD4⁺ T cells were evaluated for Th1 and Th2 cytokine production. Representative data from a total of three experiments are shown. Values are expressed as the average number of SFC/10⁶ CD4⁺ T cells from quadruplicate cultures ± the SD. Significantly more splenic IFN-γ SFC were detected than IL-4, IL-5, and IL-6 SFC (P < 0.001). Likewise, significantly more Peyer's patch IFN-γ SFC were detected than IL-4, IL-5, and IL-10 SFC ∗, P < 0.001) but less than IL-6 SFC (∗∗, P < 0.002). (C) CE analysis of RT-PCR amplified IL-4 and IFN-γ mRNA isolated from splenic CD4⁺ T cells harvested from BALB/c mice 1 or 4 weeks after oral immunization with the Salmonella-CFA/I vaccine. Two groups of mice (five/group) were orally immunized with 5 × 10⁹ CFU of Salmonella-CFA/I vaccine; the mice were sacrificed either 1 or 4 weeks postimmunization. Splenic CD4⁺ T cells were isolated and cultured with irradiated T-cell-depleted splenocytes for 2 days with or without 10 μg of CFA/I antigen per ml. After culture, CD4⁺ T cells were isolated by flow cytometry and evaluated for IL-4 and IFN-γ mRNA expression by RT-PCR. The results are expressed as the percent increase in cytokine-specific messages relative to the β-actin mRNA level (taken to be 100) according to the peak areas obtained by CE analysis. The results are representative of three separate experiments. ∗, P < 0.001, amplified IL-4 mRNA is significantly greater than IFN-γ amplified mRNA; ∗∗, P = 0.003, amplified IFN-γ mRNA is significantly greater than amplified IL-4 mRNA.

FIG. 5

Four weeks after oral immunization with the Salmonella-CFA/I vaccine, a predominance of IFN-γ-producing CD4⁺ Th cells was detected by the cytokine ELISPOT method. Splenic (A) and Peyer's patch (B) CD4⁺ T cells were cultured for 2 days in medium only, with 10 μg of CFA/I fimbriae, per ml, or with 10 μg of OVA per ml, after which CD4⁺ T cells were evaluated for Th1 and Th2 cytokine production. Representative data from a total of three experiments are shown. Values are expressed as the average number of SFC/10⁶ CD4⁺ T cells from quadruplicate cultures ± the SD. Significantly more splenic IFN-γ SFC were detected than IL-4, IL-5, and IL-6 SFC (P < 0.001). Likewise, significantly more Peyer's patch IFN-γ SFC were detected than IL-4, IL-5, and IL-10 SFC ∗, P < 0.001) but less than IL-6 SFC (∗∗, P < 0.002). (C) CE analysis of RT-PCR amplified IL-4 and IFN-γ mRNA isolated from splenic CD4⁺ T cells harvested from BALB/c mice 1 or 4 weeks after oral immunization with the Salmonella-CFA/I vaccine. Two groups of mice (five/group) were orally immunized with 5 × 10⁹ CFU of Salmonella-CFA/I vaccine; the mice were sacrificed either 1 or 4 weeks postimmunization. Splenic CD4⁺ T cells were isolated and cultured with irradiated T-cell-depleted splenocytes for 2 days with or without 10 μg of CFA/I antigen per ml. After culture, CD4⁺ T cells were isolated by flow cytometry and evaluated for IL-4 and IFN-γ mRNA expression by RT-PCR. The results are expressed as the percent increase in cytokine-specific messages relative to the β-actin mRNA level (taken to be 100) according to the peak areas obtained by CE analysis. The results are representative of three separate experiments. ∗, P < 0.001, amplified IL-4 mRNA is significantly greater than IFN-γ amplified mRNA; ∗∗, P = 0.003, amplified IFN-γ mRNA is significantly greater than amplified IL-4 mRNA.

FIG. 6

At 4 weeks after oral immunization with the E. coli-CFA/I construct, a predominance of CD4⁺ Th2 cells was detected by the cytokine ELISPOT method. Splenic (A) and Peyer's patch (B) CD4⁺ T cells were cultured for 2 days in medium only, with CFA/I fimbriae, with CT-B, or with OVA, after which CD4⁺ T cells were evaluated for Th1 and Th2 cytokine production. Depicted are representative data from a total of three experiments. Values are expressed as the average number of SFC/10⁶ CD4⁺ T cells from quadruplicate cultures ± the SD. Significantly more CFA/I-specific splenic IL-4, IL-5, and IL-6 SFC were detected than IFN-γ SFC; significantly more CT-B-specific IL-4 and IL-6 SFC were found than IFN-γ SFC. Splenic CD4⁺ T cells cultured in medium only or with OVA showed minimal cytokine induction. For the Peyer's patches, CFA/I fimbria restimulation induced significantly more IL-5 and IL-6 SFC, while CT-B restimulation significantly induced more IL-4, IL-5, and IL-6 SFC compared to the number of IFN-γ SFC induced. Some IL-6 was induced by OVA, but for the remaining cytokines minimal induction was detected. ∗, P ≤ 0.001; ∗∗, P = 0.006; ∗∗∗, P = 0.018.