We previously demonstrated that immunization with H. suis whole-cell lysate protected mice against a subsequent experimental H. suis infection and resulted in high serum anti-H. suis IgG titers. An H. suis infection, on the other hand, did not result in protective immunity, whereby significantly lower serum IgG titers were observed compared to H. suis protected animals. In order to identify possible vaccine candidates, 2D-gel electrophoresis of H. suis proteins was performed followed by immunoblotting with pooled sera from H. suis- infected mice or mice immunized with H. suis whole-cell lysate. To our knowledge, this is the first study describing the immunoproteome of H. suis. The UreB protein showed a pronounced reactivity against sera from immunized mice and was not detected with sera from infected mice (Figure 2b and c). This protein was therefore selected for further evaluation of its protective efficacy. We found that immunization with rUreB resulted in a significant reduction of H. suis colonization in the stomach. The urease protein is known to be crucial for the survival of gastric Helicobacter species[2, 18] and vaccination with its subunit B (either natural or recombinant) also induced partial protection against H. pylori, H. felis and H. heilmannii[19–23].
Vaccination with rUreB did not induce complete protection against an experimental H. suis infection. In contrast, a complete clearance was observed in 50% of mice immunized with whole-cell lysate, which is in line with previously observed results. Most probably, in order to obtain a degree of protection which is similar to or better than that induced by whole-cell lysate, additional H. suis antigens will have to be included in subunit vaccines. The H. suis chaperonin GroEL (spots 9 and 10) is another protein that showed strong reactivity with sera from lysate-immunized mice and might therefore also be a candidate for inclusion in a subunit vaccine. Indeed, oral vaccination with H. pylori Hsp60 or E. coli GroEL induced a partial protection against H. pylori challenge[24, 25]. However, vaccination with this protein has also been associated with post-immunization gastritis. Additionally, it has been demonstrated that antibodies against H. pylori Hsp60 may be associated with gastric cancer and - inflammation in humans[26–28]. Other immunoreactive protein spots identified in this study include UreA, UreH, FlaA, trigger factor, hydrogenase expression/formation protein, methyl-accepting chemotaxis protein and elongation factor G. All these proteins have also been identified in immunoproteomic studies of H. pylori and seem to be essential for gastric colonization of this bacterium. Future research is needed to determine the protective efficacy and possible side effects of vaccination with (combinations of) these proteins.
NapA has been recognized as a key modulator in H. pylori-induced gastritis and has been proposed as a protective antigen and promising vaccine candidate against H. pylori infections[31, 32]. In the present study, NapA was not recognized by the pooled sera from lysate-immunized mice and intranasal immunization with rNapA did not result in protection against H. suis challenge, although it induced anti-rNapA IgG. The reason for the different outcome in protection studies with this protein in H. suis and H. pylori remains unclear. Differences in vaccine preparations, adjuvants and experimental infection models used may play a role. Although the H. suis napA gene shows strong homology with its H. pylori equivalent (99% of sequence aligned, of which 83% conserved), the role of NapA in the pathogenesis of H. suis infections has not yet been determined and is not necessarily identical to that of H. pylori.
Different immune mechanisms may be involved in protection induced by the vaccines tested here. Serum antibodies against rUreB or antigens present in H. suis lysate were detected in mice vaccinated with rUreB or lysate, respectively, while they were absent (rUreB) or remarkably lower in non-vaccinated, infected mice. In future studies it may be interesting to also determine IgA antibody titers locally produced in the stomach. The role of local and serum antibodies in protection against a Helicobacter infection is, however, controversial. Although several authors mentioned that they may play a role in protection[33–38], results of other studies indicate that prophylactic immunization against Helicobacter species does not require antibodies[39, 40]. Whether circulating and/or local antibodies play a role in protection against H. suis infections may be determined by using antibody-deficient mice or by passive administration of serum antibodies[34, 35, 39–41].
In mice vaccinated with rUreB or lysate, mRNA expression of IFN-γ, a signature Th1 cytokine, was significantly higher after challenge with H. suis compared to sham-immunized mice, and this was not demonstrated for rNapA-immunized, not protected mice. Moreover, a clear inverse correlation was observed between the bacterial load and IFN-γ mRNA expression levels. In non-vaccinated mice, an H. suis infection does not induce a Th1 response and does not result in clearance of the infection. This indicates that production of IFN-γ, elicited by immunization, could play a role in suppression and clearance of H. suis.
Expression levels of IL-17 after challenge with H. suis were elevated in mice immunized with rUreB, rNapA and lysate, compared to sham-immunized mice and also for this cytokine, an inverse correlation with H. suis colonization was observed. In non-vaccinated mice, an H. suis infection mainly results in a Th17 response and a secondary Th2 response, which is not able to eradicate the infection although the Th17 response inversely correlates with bacterial load. This might indicate that for a strong suppression or clearance of H. suis, a combined Th17 and Th1 response in the stomach is necessary, as was observed in the rUreB- and lysate-vaccinated groups.
We observed that decreased expression levels of IL-10 were correlated with a reduction in gastric H. suis colonization. This is not entirely unexpected, since IL-10 is a suppressive cytokine for Th17 and Th1. Additionally, it has been shown that IL-10-deficient mice are able to eradicate H. pylori infection.
After infection with H. suis, expression of IL-4, a marker of a Th2 response, was higher in the lysate-immunized group than in groups vaccinated with rUreB and rNapA. Taken all results of the present study together, there are indications that in addition to a local Th1 and Th17 response, a Th2 response, probably resulting in local production of antibodies, may help to eradicate H. suis from the stomach. Indeed, only in the lysate-immunized group, mice were able to clear H. suis from the stomach. Further studies are, however, necessary to confirm this hypothesis.
In lysate-immunized mice, H. suis colonization was significantly lower than in the other experimentally infected groups. However, histological examination revealed that the inflammatory response in this group was almost similar to that in sham-immunized, H. suis-infected mice. For H. pylori too, a transient gastritis is often seen after challenge of immunized mice. It remains to be investigated whether gastritis levels of lysate-immunized mice would drop below gastritis levels of sham-immunized animals after a longer period post challenge.
In conclusion, sera from lysate-immunized, protected mice strongly react with H. suis UreB and immunization with this antigen induced a significant reduction in gastric H. suis colonization in challenged mice. Although rUreB is a promising antigen candidate for the use in vaccines against H. suis infections, further studies are necessary to elucidate if inclusion of additional H. suis antigens may improve the protective efficacy of subunit vaccines. Also, results obtained in this mouse model should be confirmed in pigs, which are the natural host of H. suis. Probably, a combination of local Th1 and Th17 responses, complemented by antibody responses play a role in the protective immunity against H. suis infections. The exact mechanism by which protection against an H. suis infection is mediated remains however to be elucidated.