Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

BackgroundThe current generation of Human Papillomavirus (HPV) vaccines, Cervarix® and Gardasil®, exhibit a high degree of efficacy in clinical trials against the two high-risk (HR) genotypes represented in the vaccines (HPV16 and HPV18). High levels of neutralizing antibodies are elicited against the vaccine types, consistent with preclinical data showing that neutralizing antibodies can mediate type-specific protection in the absence of other immune effectors. The vaccines also confer protection against some closely related non-vaccine HR HPV types, although the vaccines appear to differ in their degree of cross-protection. The mechanism of vaccine-induced cross-protection is unknown. This study sought to compare the breadth and magnitudes of neutralizing antibodies against non-vaccine types elicited by both vaccines and establish whether such antibodies could be detected in the genital secretions of vaccinated individuals.Methods and FindingsSerum and genital samples were collected from 12–15 year old girls following vaccination with either Cervarix® (n = 96) or Gardasil® (n = 102) HPV vaccine. Serum-neutralizing antibody responses against non-vaccine HPV types were broader and of higher magnitude in the Cervarix®, compared to the Gardasil®, vaccinated individuals. Levels of neutralizing and binding antibodies in genital secretions were closely associated with those found in the serum (r = 0.869), with Cervarix® having a median 2.5 (inter-quartile range, 1.7–3.5) fold higher geometric mean HPV-specific IgG ratio in serum and genital samples than Gardasil® (p = 0.0047). There was a strong positive association between cross-neutralizing antibody seropositivity and available HPV vaccine trial efficacy data against non-vaccine types.ConclusionsThese data demonstrate for the first time that cross-neutralizing antibodies can be detected at the genital site of infection and support the possibility that cross-neutralizing antibodies play a role in the cross-protection against HPV infection and disease that has been reported for the current HPV vaccines.Trial RegistrationClinicalTrials.gov NCT00956553

Polyunsaturated fatty acids (PUFA) have been shown to modulate immune responses and have therapeutic effects in inflammatory disorders. However, the influence of PUFA on dendritic cells (DC), key cells of the innate immune system in shaping adaptive immune responses, has not yet been defined. In this study, we examine the effects of the cis-9, trans-11 isomer of conjugated linoleic acid (c9, t11-CLA), a dietary PUFA found in meat and dairy products, on murine DC activation. Treatment of DC with c9, t11-CLA suppressed LPS-induced IL-12, enhanced IL-10R expression, and enhanced IL-10 production at the transcriptional and protein level. The suppression of IL-12 by c9, t11-CLA was found to be IL-10 dependent. We investigated the involvement of the MAPK, ERK, and the transcription factor, NF-κB, in this IL-10-mediated effect. c9, t11-CLA enhanced ERK activation after LPS stimulation, and inhibition of ERK resulted in abrogation of IL-10 and recovery of IL-12 production. c9, t11-CLA decreased NF-κB:DNA binding after LPS stimulation, which was concomitant with delayed translocation of NF-κBp65 into the nucleus and an increase in IκBα. These effects were reversed by addition of a neutralizing anti-IL-10 Ab. Our findings demonstrate that c9, t11-CLA suppresses IL-12 production by LPS-stimulated DC by ERK mediated IL-10-induction. Furthermore, these IL-10-mediated effects are dependent on inhibition of NF-κB activation. This is the first study to demonstrate that c9, t11-CLA can enhance transcription and production of the anti-inflammatory cytokine IL-10, while inhibiting the Th1-promoting cytokine IL-12, and may explain certain of its immunosuppressive properties.

Highlights► We examined neutralizing antibody in girls immunized with bivalent HPV vaccine. ► Antibody cross-reactivity dependent on response to vaccine types. ► Significant cross-reactivity against HPV31, HPV33, HPV45. ► Cross-reactive levels are low at <1% of vaccine type antibody responses. ► Potential role for cross-reactive antibodies as surrogate of protection.

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