Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination

Abstract

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Fig 1. The chemical structure of the CCR4 antagonist.

The CCR4 antagonist AF-399/42018025 is a small chemical molecule with a molecular weight of 565.93. It contains containing six 5 or 6 membered aromatic rings and 3 nitrogen, sulfur, and oxygen atoms. The chemical name of the molecule is 4-(1-benzofuran-2-ylcarbonyl)-1-{5-[4-chlorobenzyl)sulfanyl]-1,3,4-thiadiazol-2-yl}-3-hydroxy-5-(2-thienyl)-1,5-dihydro-2H-pyrrol-2-one [5].

Fig 2. The protective efficacy of different adjuvants on the amount of colonizing H. suis bacteria, 3 weeks after challenge, in study 1.

Subcutaneous immunization (SC) was done by mixing H. suis sonicate with Freund’s complete (FC), Freund’s incomplete (FIC) or Curdlan (Curd) and injecting this mixture at the lower back of the mice. Intranasal immunization (IN) was done by mixing H. suis sonicate with Cholera toxin (CT), CpG-DNA (CpG) or Curdlan (Curd) and applying this mixture on the external nares of the mice. Animals in the control groups were sham-immunized with Hank’s Balanced Salt Solution (HBSS). The bacterial load is illustrated as log(10) of H. suis copies/mg stomach tissue. Individual mice are illustrated as dots. Significant differences between immunized and non-immunized challenged animals are noted by * (p<0.05). (con: uninfected, inf: infected).

Fig 3. The protective efficacy of the different immunization protocols on the amount of colonizing H. suis bacteria, 3 weeks after challenge, in study 2.

Subcutaneous immunization (SC) was done by mixing H. suis sonicate with Freund’s complete (FC) or the CCR4 antagonist (CCR4) and injecting this mixture at the lower back of the mice. Intranasal immunization (IN) was done by mixing H. suis sonicate with Cholera toxin (CT) or the CCR4 antagonist (CCR4) and applying this mixture on the external nares of the mice. Sublingual immunizations (SL) were done by mixing H. suis sonicate with Cholera toxin (CT) or the CCR4-antagonist (CCR4). Animals in the control groups were sham-immunized with Hank’s Balanced Salt Solution (HBSS). The amount of bacteria colonizing the stomach of the mice are illustrated as log(10) of H. suis copies/mg stomach. The individual animals are presented as dots. Significant differences between the immunized and challenged groups and the positive control group are noted by ** (p<0.01) and *** (p<0.001). (neg. con.: sham-immunized/not challenged, pos. con.: sham-immunized/challenged).

Fig 4. H&E and PAS staining of a normal fundus.

H&E (A) and PAS staining (B) of a normal fundus of an animal from the negative control group (original magnification: 100x).

Fig 5. Histopathological analysis of fundus by H&E staining.

Normal fundus from an uninfected BALB/c mouse (A). Metaplastic fundus of a Freund’s complete/lysate immunized and H. suis-infected BALB/c mouse, showing severe parietal cell loss, with replacement of the intestinal epithelium by a common glandular epithelium (B). Original magnification: 200x.

Fig 6. H&E and PAS staining of the fundus showing pronounced inflammation and severe pseudo-pyloric metaplasia.

H&E (A) and PAS staining (B) of the fundus of an animal in the FC subcutaneously immunized and challenged group, showing pronounced inflammation and severe pseudo-pyloric metaplasia (original magnification: 100x).

Fig 7. H&E and PAS staining of the fundus, showing mild inflammation and pseudo-pyloric metaplasia.

H&E (A) and PAS staining (B) of the fundus of an animal from the CCR4 antagonist subcutaneously immunized and challenged group, showing mild inflammation and pseudo-pyloric metaplasia (original magnification: 100x).

Fig 8. Relative gene expression of cytokines and chemokines in the stomach of challenged animals after immunization or after sham inoculation in study 1.

The first bar represents the pooled data of the animals that were sham-immunized with HBSS (intranasally and subcutaneously) and that were not challenged with H. suis (Neg. con). The second bar represents the pooled data of the animals that were sham-immunized with HBSS (intranasally and subcutaneously) and that were challenged with H. suis (Pos. con). Bars 3, 4 and 5 represent the groups of animals that were immunized subcutaneously with Freund’s complete (FC/lysate/SC), Freund’s incomplete (FIC/lysate/SC) or Curdlan (Curd/lysate/SC) and challenged with H. suis. Bars 6, 7 and 8 represent the animals that were immunized intranasally with Cholera Toxin (CT/lysate/IN), CpG-DNA (CpG/lysate/IN) or Curdlan (Curd/lysate/IN) and challenged with H. suis. An * (p<0.05) indicates a significant modulation of mRNA expression levels compared to the sham-immunized/challenged groups. Cytokine expression between immunized and challenged groups was also compared with each other. When no differences could be found between the different immunized groups, the same letter designation was attributed.

Fig 9. Relative gene expression in the stomach in challenged animals after immunization or after sham inoculation in study 2.

The first bar represents the pooled data of negative control animals that were sham-immunized with HBSS and remained unchallenged (Neg. con). The second bar represents positive control group where mice were sham-immunized with HBSS (intranasally and subcutaneously) and were subsequently infected with H. suis (Pos. con). Bars 3 and 4 represent Cholera Toxin groups immunized intranasally (CT/lysate/IN) or sublingual routes (CT/lysate/SL) and challenged with H. suis. Bar 5 represents Freund’s complete adjuvant group (FC/lysate/SC). Bars 6 to 8 denote CCR4 antagonists group immunized by intranasal (IN), sublingual (SL) or subcutaneous (SC) routes respectively followed by challenge with H. suis. The letter ‘a’ indicates a significant (p<0.05) difference of mRNA expression levels compared to positive control groups. The letter ‘b’ indicates significant (p<0.05) changes of expression levels compared to the negative control groups.

Fig 10. Correlation between cytokine and chemokine expression and H. suis colonization in study 1.

Shown are the correlation analyses between IL-17, IL-10, MIP-2, LIX, IFN-γ and IL-4 mRNA expression levels on the one hand and the number of H. suis bacteria colonizing stomach of mice in the immunized and challenged groups on the other hand. Correlation was measured by Spearman’s Rho (ρ). Statistical significance between the immunized and challenged groups and the positive control group is noted by the P-value.