Synthesis and immunogenicity of a Helicobacter pylori lipopolysaccharide-based conjugate.

Helicobacter pylori infection in humans is responsible for the onset of severe gastric disorders and a vaccine would be an improvement over current antibiotic-based treatments. Lipopolysaccharide (LPS; O-chain PS→core→lipid A) is a main H. pylori cell wall component, whose O-chain PS exhibits molecular mimicry and therefore any LPS-based vaccine cannot contain O-chain epitopes. Here, the conjugation of de-lipidated H. pylori O:2 LPS to BSA and its immunogenicity in mice is described. IgG antibodies were observed to recognize the LPSs of representative H. pylori serotypes O:1, O:2 and O:5, and more significantly, the core region of H. pylori. This study showed that a monovalent H. pylori LPS conjugate can elicit antibodies that recognize other serotype-specific H. pylori LPSs and specifically the structurally conserved LPS inner-regions.

Assessment of the duration of protection in Campylobacter jejuni experimental infection in humans.

A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 10(5), 10(7), or 10(9) CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 10(9)-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 10(9)-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.

Immune response to and histopathology of Campylobacter jejuni infection in ferrets (Mustela putorius furo).

Campylobacter jejuni is 1 of the most common enteric bacterial pathogens worldwide. The mechanisms of pathogenesis remain obscure, in part because of limitations of small animal models. Young ferrets develop diarrhea when fed C. jejuni, but their pathology and the immune response after infection have not been examined in detail. In the present study, we examined the pathogenesis of C. jejuni CG8421 and associated immune responses in ferrets. After oral infection with C. jejuni CG8421, 86.7% of the animals developed diarrhea and inflammatory responses that were similar to those seen in human infection. Pronounced histopathologic changes in the colonic mucosa of infected animals were observed during the acute phase (days 1 through 3) of infection. Electron micrographs of colonic epithelium revealed disruption of the villi and internalized bacteria that were not within membrane vacuoles. During the acute phase, C. jejuni was isolated from the livers of 7 of 9 (78%) animals, and bacteria were visualized immunohistochemically in the livers from 5 of the 7 animals (71%) from which C. jejuni was isolated. A vigorous systemic and mucosal immune response to Campylobacter antigens was elicited after infection of ferrets. The data presented contribute to the current knowledge of the pathogenicity of and immunologic response to C. jejuni CG8421 in ferrets and better understanding of this model.

Capsule polysaccharide conjugate vaccine against diarrheal disease caused by Campylobacter jejuni.

The capsule polysaccharide (CPS) of Campylobacter jejuni is one of the few identified virulence determinants of this important human pathogen. Since CPS conjugate vaccines have been so effective against other mucosal pathogens, we evaluated this approach using CPSs from two strains of C. jejuni, 81-176 (HS23 and HS36 serotype complex) and CG8486 (HS4 serotype complex). The CPSs of 81-176 and CG8486 were independently linked to the carrier protein CRM(197) by reductive amination between an aldehyde(s), strategically created at the nonreducing end of each CPS, and accessible amines of CRM(197). In both cases, the CPS:CRM(197) ratio used was 2:1 by weight. Mass spectrometry and gel electrophoresis showed that on average, each glycoconjugate preparation contained, at least in part, two to five CPSs attached to one CRM(197). When administered subcutaneously to mice, these vaccines elicited robust immune responses and significantly reduced the disease following intranasal challenge with the homologous strains of C. jejuni. The CPS(81-176)-CRM(197) vaccine also provided 100% protection against diarrhea in the New World monkey Aotus nancymaae following orogastric challenge with C. jejuni 81-176.

Immunogenicity and protective efficacy of recombinant Campylobacter jejuni flagellum-secreted proteins in mice.

Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. Mice were immunized intranasally with each protein with or without LTR192G as the adjuvant and challenged intranasally with C. jejuni 81-176 or CG8486. All three proteins were immunogenic, although FspA1 induced the highest levels of serum immunoglobulin G (IgG) and fecal IgA. Although immunogenic, FlaC provided only 18% protection against disease from C. jejuni 81-176. Immunization with FspA1 resulted in 57.8% protection without adjuvant or 63.8% protection with adjuvant against homologous challenge with 81-176. Alternatively, immunization with FspA2 provided 38.4% (without adjuvant) or 47.2% (with adjuvant) protection against disease from homologous challenge with CG8486. In contrast to FspA2, FspA1 provided some heterologous protection against C. jejuni CG8486 when delivered with (31.2%) or without (44.8%) LTR192G. These results suggest that FspA1 may be a good subunit vaccine candidate against C. jejuni disease.