BackgroundLewis and secretor histo–blood group antigens (HBGAs) have been associated with decreased susceptibility to P[8] genotype rotavirus (RV) infections. Efficacy of vaccines containing attenuated P[8] strains is decreased in low-income countries. Host phenotype might impact vaccine efficacy (VE) by altering susceptibility to vaccination or RV diarrhea (RVD). We performed a substudy in a monovalent RV vaccine (RV1) efficacy trial in Bangladesh to determine the impact of Lewis and secretor status on risk of RVD and VE.MethodsIn infants randomized to receive RV1 or no RV1 at 10 and 17 weeks with 1 year of complete active diarrheal surveillance, we performed Lewis and secretor phenotyping and genotyped the infecting strain of each episode of RVD.ResultsA vaccine containing P[8] RV protected secretors and nonsecretors similarly. However, unvaccinated nonsecretors had a reduced risk of RVD (relative risk, 0.53 [95% confidence interval, .36–.79]) mediated by complete protection from P[4] but not P[8] RVs. This effect reduced VE in nonsecretors to 31.7%, compared to 56.2% among secretors, and decreased VE for the overall cohort.ConclusionsHost HBGA status may impact VE estimates by altering susceptibility to RV in unvaccinated children; future trials should therefore account for HBGA status.Clinical Trials RegistrationNCT01375647.

Acetate relaxes rat caudal artery and increases tissue adenosine 3',5'-cyclic monophosphate (cAMP) levels. The present study's purpose was to determine whether related ultrashort chain (volatile) fatty acids, propionate (C3), butyrate (C4), and octanoate (C8) would do so as well. It was found that butyrate and propionate had very similar vasorelaxant profiles to acetate; they relaxed caudal artery strips precontracted with KCl, phenylephrine, arginine vasopressin, and prostaglandin F2 alpha. The vasorelaxation was endothelium independent, was not blocked by indomethacin, and was associated with a rise in tissue cAMP levels but not in guanosine 3',5'-cyclic monophosphate (cGMP) levels. The mean effective concentration for butyrate (0.8 mM) was lower than that for acetate (2.0 mM) or propionate (1.9 mM). Propionate-mediated relaxation was blocked by the stilbene inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Vasorelaxant effects of octanoate were also not dependent on endothelium and were not affected by indomethacin. Maximal vasorelaxant effect of octanoate was greater than that of acetate, propionate, or butyrate; vasorelaxant effect of octanoate was only slightly attenuated by DIDS and was associated with only a minimal effect on tissue cAMP levels. Results suggest that vasodilatory effects of acetate generalize to propionate and butyrate, but that vasorelaxant effects of octanoate may be occurring via a separate mechanism.

Myelin-specific induction of tolerance represents a promising means to modify the course of autoimmune inflammatory demyelinating diseases such as multiple sclerosis (MS). Our laboratory has focused on a novel preclinical strategy for the induction of tolerance to the major encephalitogenic epitopes of myelin that cause experimental autoimmune encephalomyelitis (EAE) in rats and mice. This novel approach is based on the use of cytokine-NAg (neuroantigen) fusion proteins comprised of the native cytokine fused either with or without a linker to a NAg domain. Several single-chain cytokine-NAg fusion proteins were tested including GMCSF-NAg, IFNbeta-NAg, NAgIL16, and IL2-NAg. These cytokine-NAg vaccines were tolerogenic, therapeutic vaccines that had tolerogenic activity when given as pre-treatments before encephalitogenic immunization and also were effective as therapeutic interventions during the effector phase of EAE. The rank order of inhibitory activity was as follows: GMCSF-NAg, IFNbeta-NAg > NAgIL16 > IL2-NAg > MCSF-NAg, IL4-NAg, IL-13-NAg, IL1RA-NAg, and NAg. Several cytokine-NAg fusion proteins exhibited antigen-targeting activity. High affinity binding of the cytokine domain to specific cytokine receptors on particular subsets of APC resulted in the concentrated uptake of the NAg domain by those APC which in turn facilitated the enhanced processing and presentation of the NAg domain on cell surface MHC class II glycoproteins. For most cytokine-NAg vaccines, the covalent linkage of the cytokine domain and NAg domain was required for inhibition of EAE, thereby indicating that antigenic targeting of the NAg domain to APC was also required in vivo for tolerogenic activity. Overall, these studies introduced a new concept of cytokine-NAg fusion proteins as a means to induce tolerance and to inhibit the effector phase of autoimmune disease. The approach has broad application for suppressive vaccination as a therapy for autoimmune diseases such as MS.

Environmental enteropathy (EE) is a poorly understood condition that refers to chronic alterations in intestinal permeability, absorption, and inflammation, which mainly affects young children in resource-limited settings. Recently, EE has been linked to suboptimal oral vaccine responses in children, although immunological mechanisms are poorly defined. The objective of this study was to determine host factors associated with immune responses to an oral cholera vaccine (OCV). We measured antibody and memory T cell immune responses to cholera antigens, micronutrient markers in blood, and EE markers in blood and stool from 40 Bangladeshi children aged 3–14 years who received two doses of OCV given 14 days apart. EE markers included stool myeloperoxidase (MPO) and alpha anti-trypsin (AAT), and plasma endotoxin core antibody (EndoCab), intestinal fatty acid binding protein (i-FABP), and soluble CD14 (sCD14). We used multiple linear regression analysis with LASSO regularization to identify host factors, including EE markers, micronutrient (nutritional) status, age, and HAZ score, predictive for each response of interest. We found stool MPO to be positively associated with IgG antibody responses to the B subunit of cholera toxin (P = 0.03) and IgA responses to LPS (P = 0.02); plasma sCD14 to be positively associated with LPS IgG responses (P = 0.07); plasma i-FABP to be positively associated with LPS IgG responses (P = 0.01) and with memory T cell responses specific to cholera toxin (P = 0.01); stool AAT to be negatively associated with IL-10 (regulatory) T cell responses specific to cholera toxin (P = 0.02), and plasma EndoCab to be negatively associated with cholera toxin-specific memory T cell responses (P = 0.02). In summary, in a cohort of children 3–14 years old, we demonstrated that the majority of biomarkers of environmental enteropathy were positively associated with immune responses after vaccination with an OCV.

BackgroundRotavirus (RV)–specific immunoglobulin A (IgA) responses following oral RV vaccination are impaired in low-income countries, where the utility of RV-IgA as a correlate of protection (CoP) remains unclear. In a monovalent oral RV vaccine (Rotarix) efficacy trial among infants in Dhaka, Bangladesh, we identified factors associated with poor RV-IgA responses and explored the utility of RV-IgA as a CoP.MethodsInfants were randomized to receive Rotarix or no Rotarix at 10 and 17 weeks of life and followed with active diarrheal surveillance. RV-IgA concentration, seroconversion, and seropositivity were determined at 18 weeks of life and analyzed for correlation(s) with rotavirus diarrhea (RVD) and for contribution to Rotarix vaccine effect.ResultsAmong vaccinated infants, overall RV-IgA geometric mean concentration was 21 U/mL; only 27% seroconverted and 32% were seropositive after vaccination. Increased RV-specific maternal antibodies significantly impaired immunogenicity. Seroconversion was associated with reduced risk of RVD through 1 year of life, but RV-IgA seropositivity only explained 7.8% of the vaccine effect demonstrated by the clinical endpoint (RVD).ConclusionsRV-IgA responses were low among infants in Bangladesh and were significantly impaired by maternal antibodies. RV-IgA is a suboptimal CoP in this setting; an improved CoP for RV in low-income countries is needed.Clinical Trials RegistrationNCT01375647.

Single-chain fusion proteins comprised of GM-CSF and neuroantigen (NAg) are potent, NAg-specific inhibitors of experimental autoimmune encephalomyelitis (EAE). An important question was whether GMCSF-NAg tolerogenic vaccines retained inhibitory activity within inflammatory environments or were contingent upon steady-state conditions. A GMCSF-MOG fusion protein reversed established paralytic disease in both passive and active models of EAE in C57BL/6 mice. The fusion protein also reversed EAE in CD4-deficient and B cell-deficient mice. Notably, GMCSF-MOG inhibited EAE when co-injected adjacent to the MOG35-55/CFA emulsion. GMCSF-MOG also retained dominant inhibitory activity when directly emulsified with MOG35-55 in the CFA emulsion in both C57BL/6 or B cell-deficient models of EAE. Likewise, when combined with PLP139-151 in CFA, GMCSF-PLP inhibited EAE in SJL mice. When deliberately emulsified in CFA with the NAg, GMCSF-NAg inhibited EAE even though NAg was present at more than a 30-fold molar excess. In vitro studies revealed that the GMCSF domain of GMCSF-MOG stimulated growth and differentiation of inflammatory dendritic cells (DC) and simultaneously targeted the MOG35-55 domain for enhanced presentation by these DC. These inflammatory DC presented MOG35-55 to MOG-specific T cells by an inhibitory mechanism that was mediated in part by IFN-γ signaling and NO production. In conclusion, GMCSF-NAg was tolerogenic in CFA-primed pro-inflammatory environments by a mechanism associated with targeted antigen presentation by inflammatory DC and an inhibitory IFN-γ/ NO pathway. The inhibitory activity of GMCSF-NAg in CFA-primed lymphatics distinguishes GMCSF-NAg fusion proteins as a unique class of inflammation-dependent tolerogens that are mechanistically distinct from naked peptide or protein-based tolerogens.

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-β and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components; IFN-β, NAg, and Alum for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with “IFN-β+NAg in Alum” ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 (B6) mice in both pre-treatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine antigen PLP178-191 or MOG35-55 in PLP and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-β/Alum-based vaccination exhibited hallmarks of infectious tolerance, because “IFN-β+OVA in Alum”-specific vaccination inhibited EAE elicited by “OVA+MOG in CFA” but not by “MOG in CFA”. “IFN-β+NAg in Alum” vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ Treg populations. IFN-β and MOG35-55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti-TGF-β and that resulted in the induction of an ‘effector’ CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. “In vitro IFN-β+MOG-induced Tregs” inhibited EAE when transferred into actively-challenged recipients. Unlike “IFN-β+NAg in Alum” vaccines, vaccination with “TGF-β+MOG35-55 in Alum” did not elevate Treg percentages in vivo. Overall, this study indicates that “IFN-β+NAg in Alum” vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-β has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs.

Secretor status controls mucosal histo–blood group antigen expression and is associated with susceptibility to rotavirus (RV) diarrhea, with nonsecretors less susceptible to symptomatic infection. The role of breast milk secretor status on oral live-attenuated RV vaccine response in breastfed infants has not been explored. In a monovalent G1P[8] RV vaccine (Rotarix) trial in Bangladesh, RV-specific plasma immunoglobulin A antibody seroconversion rates were higher among infants of maternal nonsecretors (39%) than infants of maternal secretors (23%; P = .001). Maternal status remained a significant predictor when correcting for infant status (P = .002). Maternal secretor status should be considered when interpreting oral RV vaccine responses in low- and middle-income settings. Clinical Trials Registration. NCT01375647.