The effect of probiotics and zinc supplementation on the immune response to oral rotavirus vaccine: A randomized, factorial design, placebo-controlled study among Indian infants.

BACKGROUND: Strategies are needed to improve oral rotavirus vaccine (RV), which provides suboptimal protection in developing countries. Probiotics and zinc supplementation could improve RV immunogenicity by altering the intestinal microbiota and immune function. METHODS: Infants 5weeks old living in urban Vellore, India were enrolled in a randomized, double-blind, placebo-controlled trial with a 4-arm factorial design to assess the effects of daily zinc (5mg), probiotic (1010Lactobacillus rhamnosus GG) or placebo on the immunogenicity of two doses of RV (Rotarix®, GlaxoSmithKline Biologicals) given at 6 and 10weeks of age. Infants were eligible for participation if healthy, available for the study duration and without prior receipt of RV or oral poliovirus vaccine other than the birth dose. The primary outcome was seroconversion to rotavirus at 14weeks of age based on detection of VP6-specific IgA at ≥20U/ml in previously seronegative infants or a fourfold rise in concentration. RESULTS: The study took place during July 2012 to February 2013. 620 infants were randomized equally between study arms and 551 (88.9%) completed per protocol. Seroconversion was recorded in 54/137 (39.4%), 42/136 (30.9%), 40/143 (28.0%), and 37/135 (27.4%) infants receiving (1) probiotic and zinc, (2) probiotic and placebo, (3) placebo and zinc, (4) two placebos. Seroconversion showed a modest improvement among infants receiving probiotic (difference between groups 1, 2 and 3, 4 was 7.5% (97.5% Confidence Interval (CI): -1.4%, 16.2%), p=0.066) but not zinc (difference between groups 1, 3 and 2, 4 was 4.4% (97.5% CI: -4.4%, 13.2%), p=0.272). 16 serious adverse events were recorded, none related to study interventions. CONCLUSIONS: Zinc or probiotic supplementation did not significantly improve the low immunogenicity of rotavirus vaccine given to infants in a poor urban community in India. A modest effect of combined supplementation deserves further investigation. TRIAL REGISTRATION: The trial was registered in India (CTRI/2012/05/002677).

Influence of the intestinal microbiota on the immunogenicity of oral rotavirus vaccine given to infants in south India.

Oral rotavirus vaccines have consistently proven to be less immunogenic among infants in developing countries. Discrepancies in the intestinal microbiota, including a greater burden of enteropathogens and an altered commensal community composition, may contribute to this trend by inhibiting the replication of vaccine viruses. To test this possibility, we performed a nested case-control study in Vellore, India, in which we compared the intestinal microbiota of infants who responded serologically or not after two doses of Rotarix delivered at 6 and 10 weeks of age as part of a clinical trial (CTRI/2012/05/002677). The prevalence of 40 bacterial, viral, and eukaryotic pathogen targets was assessed in pre-vaccination stool samples from 325 infants using singleplex real-time PCR on a Taqman array card (TAC). In a subset of 170 infants, we assessed bacterial microbiota composition by sequencing the 16S rRNA gene V4 region. Contrary to expectations, responders were more likely than non-responders to harbor ≥1 bacterial enteropathogen at dose 1 (26% [40/156] vs 13% [21/157] of infants with TAC results who completed the study per protocol; χ2, P = .006), although this was not apparent at dose 2 (24% [38/158] vs 23% [36/158]; P = .790). Rotavirus shedding after dose 1 was negatively correlated with the replication of co-administered oral poliovirus vaccine (OPV). We observed no consistent differences in composition or diversity of the 16S bacterial microbiota according to serological response, although rotavirus shedding was associated with slightly more bacterial taxa pre-vaccination. Overall, our findings demonstrate an inhibitory effect of co-administered OPV on the first dose of Rotarix, consistent with previous studies, but in the context of OPV co-administration we did not find a strong association between other components of the intestinal microbiota at the time of vaccination and Rotarix immunogenicity.

Natural History of Cryptosporidiosis in a Birth Cohort in Southern India.

BACKGROUND: Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention. METHODS: Children (N = 497) were recruited at birth in semiurban slums in Vellore, India, and followed for 3 years with twice-weekly home visits. Stool samples were collected every 2 weeks and during diarrheal episodes were tested for Cryptosporidium species by polymerase chain reaction (PCR). Serum samples obtained every 6 months were evaluated for seroconversion, defined as a 4-fold increase in immunoglobulin G directed against Cryptosporidium gp15 and/or Cp23 antigens between consecutive sera. RESULTS: Of 410 children completing follow-up, 397 (97%) acquired cryptosporidiosis by 3 years of age. PCR identified 1053 episodes of cryptosporidiosis, with an overall incidence of 0.86 infections per child-year by stool and serology. The median age for the first infection was 9 (interquartile range, 4-17) months, indicating early exposure. Although infections were mainly asymptomatic (693 [66%]), Cryptosporidium was identified in 9.4% of diarrheal episodes. The proportion of reinfected children was high (81%) and there was clustering of asymptomatic and symptomatic infections (P < .0001 for both). Protection against infection increased with the order of infection but was only 69% after 4 infections. Cryptosporidium hominis (73.3%) was the predominant Cryptosporidium species, and there was no species-specific protection. CONCLUSIONS: There is a high burden of endemic cryptosporidiosis in southern India. Clustering of infection is suggestive of host susceptibility. Multiple reinfections conferred some protection against subsequent infection.

Rotavirus specific Salivary and Fecal IgA in Indian Children and Adults.

OBJECTIVE: To compare serum, salivary and fecal IgA responses in infants and adults following rotavirus vaccination. STUDY DESIGN: Laboratory testing of samples from clinical trials. SETTING: Medical College Hospital. PARTICIPANTS: 13 healthy adult volunteers not given vaccine, 20 healthy adult volunteers given one dose of bovine rotavirus tetravalent vaccine (Shantha Biotechnics), and 88 infants given 3 or 5 doses of Rotarix. OUTCOME MEASURES: Serum, salivary and fecal IgA at one or more time points. METHODS: IgA antibodies were estimated in serum, saliva and fecal samples by enzyme-linked immunosorbent assay, and normalized to total IgA in saliva. RESULTS: In naturally infected adult volunteers, comparing serum and salivary IgA showed significant positive correlation (r=0.759; P=0.003). Of 20 vaccinated adults, complete samples showing change were available for 10; among them there was a significant positive correlation (P<0.05) between pre-vaccination serum and pre-vaccination salivary IgA but not between post-vaccination serum and post-vaccination salivary IgA. Of 88 infants given 3 or 5 doses of vaccine, 13 had more than 4-fold IgA response in serum, saliva and fecal samples, 6 had a 2-4 fold increases in all specimens. There was weak correlation between seroconversion rates measured by serum and salivary antibody responses. Salivary and stool assays were able to detect seroconversion in a few children in whom there was no detectable response in serum. CONCLUSIONS: Evaluation of multiple samples is useful for intensive experimental study designs and may help improve our understanding of the induction and dynamics of immune responses to rotavirus vaccination.

Serum Anti-Cryptosporidial gp15 Antibodies in Mothers and Children Less than 2 Years of Age in India.

Little is known about the type and longevity of the humoral response to cryptosporidial infections in developing countries. We evaluated serum antibody response to Cryptosporidium gp15 in 150 sets of maternal, preweaning and postinfection/end-of-follow-up sera from children followed up to 2 years of age to determine the influence of maternal and preweaning serological status on childhood cryptosporidiosis. Fifty two percent (N = 78) of mothers and 20% (N = 30) of children were seropositive preweaning. However, most positive preweaning samples from children were collected early in life indicating transplacental transfer and subsequent rapid waning of antibodies. Although 62% (N = 94) of children had a parasitologically confirmed cryptosporidial infection (detected by stool polymerase chain reaction) during the follow-up, only 54% (N = 51) of children were seropositive postinfection. Given there were striking differences in seropositivity depending on when the sample was collected, even though Cryptosporidium was detected in the stool of the majority of the children, this study indicates that antibodies wane rapidly. During follow-up, the acquisition or severity of cryptosporidial infections was not influenced by maternal (P = 0.331 and 0.720, respectively) as well as the preweaning serological status of the child (P = 0.076 and 0.196, respectively).

Effect of withholding breastfeeding on the immune response to a live oral rotavirus vaccine in North Indian infants.

Interference from transplacental and breast milk antibodies may impede the performance of oral live vaccines. The effect of breastfeeding on the immunogenicity of Rotarix, a two-dose oral monovalent rotavirus vaccine, was examined in a community-based trial in New Delhi, India. Four hundred mother-infant pairs were randomized into two equal groups. Infants were aged 6-7 weeks at enrollment. Mothers were encouraged to either breastfeed or to withhold breastfeeding during the 30 min prior to and after each vaccine dose was administered. We collected blood specimens from infants at enrollment and 4 weeks after the second vaccine dose. Blood and breast milk specimens were obtained from mothers at baseline and breast milk specimens were collected at the time of the second vaccine dose. Seroconversion was defined as infant serum anti-VP6 IgA antibody level of ≥20 IU/mL 4 weeks after the second vaccine dose and a ≥4-fold rise from baseline. There was no difference in the proportion who seroconverted between the two groups (26% vs 27%; p=0.92). The levels of infant serum IgA, maternal serum and breast milk IgA and IgG anti-rotavirus antibodies predicted the anti-rotavirus IgA level in infants at end-study and explained approximately 10% of the variability of the immune response (r(2)=0.10, p<0.001). In this population, the immune response to Rotarix was not enhanced by withholding breastfeeding around the time of vaccination. Maternal anti-rotavirus antibodies explained little of the variability in the immune response to the vaccine. Factors other than maternal anti-rotavirus antibodies probably explain why infants in low-and middle-income settings respond poorly to live oral rotavirus vaccines.