Combined vaccine regimen based on parenteral priming with a DNA vaccine and administration of an oral booster consisting of a recombinant Salmonella enterica serovar Typhimurium vaccine strain for immunization against infection with human-derived enterotoxigenic Escherichia coli strains

Abstract

Repeated evidence has demonstrated that combined primer-booster immunization regimens can improve both secreted and humoral immune responses to antigens derived from viral, bacterial, and parasitic pathogens. For the present work, we evaluated the synergic serum immunoglobulin G (IgG) and fecal IgA antibody responses elicited in BALB/c mice who were intramuscularly primed with a DNA vaccine, pRECFA, followed by oral boosting with an attenuated Salmonella enterica serovar Typhimurium vaccine (HG3) strain, with both vaccines encoding the structural subunit (CfaB) of the CFA/I fimbriae produced by human-derived enterotoxigenic Escherichia coli (ETEC) strains. The immunological properties of the vaccine regimen were evaluated according to the order of the administered vaccines, the nature of the oral antigen carrier, the age of the vaccinated animals, the interval between the priming and boosting doses, and the amount of injected DNA. The production of gamma interferon and the IgG2a subclass in serum indicated that mice immunized with the primer-booster regimen developed prevailing type 1 T-cell-dependent immune responses. The synergic effect of the vaccine regimen on the induced antibody responses was also revealed by its ability to block the adhesive properties of CFA/I fimbriae expressed by live bacteria, as shown by the inhibition of Caco-2 cell and human erythrocyte binding. Moreover, DBA2 newborn mice were protected from lethal challenges with a CFA/I+ ETEC strain after the incubation of live bacteria with serum samples harvested from mice who were subjected to the primer-booster regimen. We propose, therefore, that the DNA primer-Salmonella booster regimen represents an alternative for the development of vaccines requiring both mucosal and systemic antibody responses for immunological protection.

FIG. 1.

Schematic representation of CfaB-encoding plasmids and in vitro expression of the encoded peptides. (A) The pRECFA vaccine was obtained after cloning of the PCR-amplified cfaB gene into the pRE4 vector after PvuII-ApaI double digestion of both the fragment and the plasmid (1). The encoded CfaB protein was expressed as an in-frame sandwich fusion with the HSV-1 gD protein under control of the RSV late promoter. (B) The pCFA-1 plasmid was generated after cloning of a PCR-generated DNA fragment encoding the mature portion (without the signal peptide) of the structural subunit of the CFA/I fimbriae into the SmaI-digested pKK223-3 bacterial expression vector (17, 18). The peptide expressed by transformed Salmonella strains accumulated as an intracellular protein that was unable to form native CFA/I fimbriae. (C) Immunological detection of CfaB expression in whole-cell extracts in a Western blot developed with a specific monoclonal antibody (MAb 84). Samples: 1, purified CFA/I fimbriae isolated from an ETEC strain; 2, BHK-21 whole-cell extract; 3, pRE4-transfected BHK-21 cells; 4, pRECFA-transfected BHK-21 cells; 5, S. enterica serovar Typhimurium SL3261 strain; 6, S. enterica serovar Typhimurium HG3 strain cultivated without inducer (IPTG); 7, S. enterica serovar Typhimurium HG3 cultivated for 4 h in the presence of 0.5 M IPTG. Each lane was loaded with approximately 25 μg of total protein.

FIG. 2.

Systemic (serum IgG) and secreted (fecal IgA) CfaB-specific antibody responses elicited in BALB/c mice subjected to different immunization regimens. Serum IgG (A) and fecal IgA (B) anti-CfaB responses were measured in ELISA plates coated with heat-denatured CfaB protein purified from a CFA/I⁺ ETEC strain. Sera were collected from the following immunization groups (at least five animals per group): pRECFA (▴), mice immunized with two i.m. doses of pRECFA (100 μg/dose); HG3 (▪), mice immunized with two p.o. doses of IPTG-induced S. enterica serovar Typhimurium HG3 (10¹⁰ CFU/dose); pRE4 + HG3 (+), mice immunized with two i.m. doses of the empty vector pRE4 (100 μg/dose) followed by two p.o. doses of the HG3 strain; pRECFA + HG3 (⧫), the complete primer-booster immunization regimen, with mice immunized with two i.m. doses of pRECFA followed by two p.o. doses of the HG3 strain; HG3 + pRECFA (*), inverse primer-booster immunization regimen consisting of two p.o. doses of the HG3 strain followed by two i.m. pRECFA doses. Data corresponding to three determinations are presented as means ± standard errors (SE) of the titers (serum IgG) or of absorbance values at 492 nm of the reactions with diluted fecal extracts (fecal IgA). All values were deducted from the background values obtained with nonimmune sera. All bars marked with an asterisk show statistically significantly different values (P < 0.05) compared to the results of the pRECFA (serum IgG)- or HG3 (fecal IgA)-vaccinated mouse group.

FIG. 3.

Factors affecting serum anti-CfaB IgG responses elicited in mice subjected to the DNA priming-Salmonella boosting immunization regimen. (A) Nature of the bacterial carrier given to mice primed with pRECFA. Mice were boosted p.o. with live (HG3) or ethanol-inactivated (HG3 k) S. enterica serovar Typhimurium HG3 or with live (ETEC) or ethanol-inactivated (ETEC k) ETEC 258909-3. As a control, the mean CfaB-specific serum IgG titer of mice immunized with two doses of pRECFA is indicated (pRECFA). (B) Age-dependent CfaB-specific antibody responses elicited in mice who were first immunized with pRECFA at the age of 1 week (young), 6 weeks (adult), or 52 weeks (old). Mice were immunized with two i.m. doses (100 μg/dose) of pRECFA (black bars) or with the complete primer-booster regimen (white bars). (C) Interval between the last priming dose with pRECFA and the first oral booster with the CfaB-expressing HG3 strain. CfaB-specific antibody levels detected for mouse groups subjected to the primer-booster regimens with intervals of 2, 4, 8, 16, and 52 weeks between the priming and boosting doses are indicated by white bars. As controls, anti-CfaB antibody titers detected in sera of mice who were vaccinated only with two doses of pRECFA and then bled at the same periods as those subjected to the complete primer-booster regimen are indicated by black bars. (D) Amount of DNA required to prime CfaB-specific antibody responses in mice receiving oral boosters with the HG3 strain. The amount of DNA inoculated in each dose is indicated (100, 50, 10, or 1 μg). Data corresponding to one representative determination are presented as means ± SE of the titers. All bars marked with an asterisk show statistically significantly different values (P < 0.05) compared to the results obtained with pRECFA-vaccinated mouse groups that were subjected to the same testing conditions.

FIG. 4.

Factors affecting secreted anti-CfaB fecal IgA responses elicited in mice subjected to the DNA priming-Salmonella boosting immunization regimen. (A) Nature of the bacterial carrier given to pRECFA-primed mice. Mice were boosted p.o. with live (▪, pRECFA + HG3) or ethanol-inactivated (+, pRECFA + HG3 k) S. enterica serovar Typhimurium HG3 or with live (⧫, pRECFA + ETEC) or ethanol-inactivated (*, pRECFA + ETEC k) ETEC 258909-3. As a control, CfaB-specific fecal IgA titers for a mouse group that was immunized with two doses of pRECFA are indicated (▴, pRECFA). (B) Age-dependent CfaB-specific antibody responses elicited in mice who were first immunized with pRECFA at an age of 1 week (▴, young), 6 weeks (▪, adult), or 52 weeks (⧫, old) and then boosted with two doses of the HG3 strain given 2 weeks apart (pRECFA + HG3). As controls, age-matched mouse groups (pRECFA) were immunized with two i.m. doses (100 μg/dose) of pRECFA, as indicated by the open symbols. (C) Interval between the last priming dose with pRECFA and the first oral booster with the CfaB-expressing HG3 strain. CfaB-specific fecal IgA levels detected in mouse groups that were subjected to intervals of 2 (▴), 4 (▪), 8 (+), 16 (⧫), and 52 (*) weeks are indicated. (D) Amount of DNA required to prime CfaB-specific fecal IgA responses in mice receiving oral boosters with the HG3 strain. The amount of DNA inoculated in each dose was 100 μg (▴), 50 μg (▪), 10 μg (+), or 1 μg (⧫). Experiments were performed with pools of fecal extract collected from different immunization groups. The results shown are based on a representative experiment and are expressed as A₄₉₂ values for twofold serial dilutions. Statistically significant results (P > 0.05) with regard to mouse groups primed with pRE4 and boosted with the HG3 strain at the lowest tested dilutions were obtained for the following groups: A, pRECFA + HG3; B, pRECFA + HG3 adult and pRECFA + HG3 young; C, all groups except the one with an interval of 52 weeks; D, mouse groups that were immunized with 100 or 50 μg of pRECFA.

FIG. 5.

Activation of Th1-type responses in mice subjected to the primer-booster immunization regimen. (A) Determination of anti-CfaB IgG1 (white bars) and IgG2a (black bars) subclasses in serum samples collected from mice who were immunized only with pRECFA (pRECFA) or the CfaB-expressing HG3 strain (HG3), primed with pRE4 and boosted with the HG3 strain (pRE4 + HG3), or subjected to the complete primer-booster regimen (pRECFA + HG3). (B) Determination of IFN-γ (black bars) and IL-4 (white bars) production in spleen homogenates harvested from mice who were immunized only with pRECFA (pRECFA) or the CfaB-expressing HG3 strain (HG3), primed with pRE4 and boosted with the HG3 strain (pRE4 + HG3), or subjected to the complete primer-booster regimen (pRECFA + HG3). Cell cultures were stimulated with 25 μg of purified heat-denatured CFA/I fimbriae/ml. IgG subclass (two determinations) and cytokine (one determination) detection values are represented by end-point reverse titers ± SE and mean nanograms per milliliter ± SE, respectively. *, statistically different values (P < 0.05) compared to results obtained with pRECFA-vaccinated mouse groups.

FIG. 6.

Inhibition of binding properties of CFA/I fimbriae by serum samples collected from mice subjected to different immunization regimens. The CFA/I⁺ ETEC 258990-3 strain was incubated with different serum samples for 1 h at 37°C, at a final dilution of 1:3, before being added to Caco-2 cell cultures. The inhibition of bacterial binding was evaluated by assays using serum pools from nonimmunized mice (A), serum pools from mice who were immunized with two i.m. pRECFA doses (titer of 4,268) (B), serum pools from mice who were immunized with two p.o. doses of the HG3 strain (reverse titer of 305) (C), or PBS-diluted serum pools from mice subjected to the pRECFA priming-HG3 strain boosting immunization regimen with a two-week interval between the priming and boosting doses (titer of 4,500) (D). Magnification, ×2,000. The experiment was repeated twice with the same observed results.