Association of serum antibodies with protection against rotavirus infection and disease in South Indian children ^☆

Keywords

• Antibody;
• Immunity;
• Protection;
• Rotavirus;
• Diarrhea

1. Introduction

Infection with both natural rotavirus and oral rotavirus vaccines stimulates production of IgM, IgA and IgG antibodies. While it is thought that secretory IgA production in the small intestine is directly involved with protection against infection and disease, infection also stimulates humoral responses that may correlate with protection. Both animal models and studies in humans have indicated that serum antibodies have a relationship with protection against rotavirus infections and disease [1]. An apparent relation of serum IgA response with protection against disease has served as a tool in the clinical development of live oral vaccines for rotavirus [2]. Currently, two live oral rotavirus vaccines (Rotarix [RV1] GSK biologicals and Rotateq [RV5] Merck & Co.) have been successfully evaluated in large-scale clinical trials, are licensed in over 100 countries and are in routine use in over 40 countries [3] ;  [4]. However, both vaccine protection and immunogenicity is considerably reduced in middle and lower income countries compared to high income settings [5]; [6] ;  [7].

Past studies have attempted to identify how serum antibodies protect against natural rotavirus infections. However, findings from these studies have been inconsistent and mechanisms or effectors of protection from natural infection remain unclear. Studies have found reduced risk was associated with serum IgA and not IgG [8], with IgG and not IgA [9] while others did not find association with serum antibodies at all [10]. Further, it is unclear whether serum antibodies are directly involved in protection or merely reflect a recent infection. This incomplete understanding of the correlates of protection from rotavirus infection and disease remains a critical limiting factor in understanding differences in rotavirus vaccine effectiveness. Moreover, identification of a suitable correlate of protection would facilitate evaluation of new vaccines or new vaccine strategies (e.g. alternative schedules) by reducing the need for large scale trials with disease endpoints. This is particularly important as many candidate rotavirus vaccines are currently in development and testing of efficacy of these vaccines in a placebo controlled trial may raise ethical and logistical concerns given the availability of licensed vaccines in many countries.

In this study, we aim to assess the association between pre-existing serum antibody levels and subsequent rotavirus infection and disease risk in a birth cohort from an impoverished community in south India, where natural rotavirus infection has been shown to confer less protection than in higher income settings [11] ;  [12].

2. Materials and methods

2.1. Study design

The study was conducted in the urban slums of Vellore, as described elsewhere [13]. Briefly, a cohort of 452 newborns was recruited at birth from March 2002 to August 2003. The children were followed until 36 months of age. Written, informed consent was obtained from a parent of each child before enrollment. Stool samples were collected during all diarrheal episodes. Surveillance stool samples were collected once in two weeks. A blood sample was collected from the mother at time of child birth. For all children in the cohort, a blood sample was collected at the time of birth (cord blood) or within the first week and at least every six months for three years.

2.2. Definitions

An ‘episode of diarrhea’ was defined as three or more watery stools in 24 h or, in breastfed children, an increased number of daily stools considered to be diarrhea by the mother. The day following the return of the child's bowel movements to normal marked the end of a diarrheal episode. Two episodes of diarrhea were separated by an interval of at least 48 h of normal bowel movements. A ‘rotavirus infection’ was defined as the detection of virus in stool or a fourfold increase in anti-rotavirus IgG or a three-fold change in IgA levels in sequential sera. An infection was considered asymptomatic if a child did not have diarrhea for the week prior to or following detection of rotavirus in stool, or if a child seroconverted with no diarrhea between the two serum sample collections. An infection was defined as symptomatic when rotavirus was identified in stool during the week preceding or following the diarrheal episode.

2.3. Laboratory methods

2.3.1. Detection and characterization of rotavirus in stool

All surveillance and diarrheal stool samples were screened for rotavirus antigen by enzyme linked immunosorbent assay (ELISA, Rotavirus IDEIA, UK). All rotavirus positive surveillance stool samples were retested with the use of same ELISA to improve specificity. Surveillance samples that were positive by both ELISA were genotyped by means of reverse transcription polymerase chain reaction (RT-PCR). All diarrheal stool samples were screened by means of ELISA and tested by RT-PCR assay even if the screening ELISA was negative. A stool sample was considered positive when positive either by two ELISA tests or by RT-PCR (Fig. 1).

2.3.2. Testing for anti-rotavirus IgA and IgG in serum

For each child, the serum specimens obtained at birth and at 6-month intervals thereafter were analyzed for antirotavirus IgA and IgG antibodies by means of an antibody-sandwich enzyme immunoassay. The IgA or IgG titer was determined by comparing the optical density values from sample wells with a standard curve based on pooled human serum samples.