The Immune Responses To Rotavirus Infection In Children

Objective Group A rotavirus is the single most important pathogen leading to dehydrating diarrhea in young children in worldwide scope. One of the major reasons that hindered rotavirus vaccine development is that immunity of this disease is still not completely understood. In this research the adaptive immunity to rotavirus was roundly evaluated by study on rotavirus specific humoral immune response and Th1/Th2 response in children with naturally acquired rotavirus infection. More further, the innate immunity to rotavirus was assessed by study on toll-like receptor (TLR) that recognizes pathogens. The objective of this study was to understand the immune response to rotavirus infection in children which will provide evidence for effective prevention and therapy of rotavirus diarrhea.Methods 1. Blood and fecal samples were collected from 75 children with acute rotavirus diarrhea, and 45 children without any infectious disease recently were chosen as controls. 2. ELISA assay and RT-PCR assay were applied to detect rotavirus antigen and genotypes respectively. 3. Indirect ELISA assay was developed for the determination of rotavirus-specific antibody titer from plasmas and stools. 4. Subgroups of lymphocytes were measured by flow cytometry, and real-time RT-PCR assay was applied for determination of mRNA expression of cytokines. 5. Five TLRs in peripheral blood mononuclear cell (PBMC) were measured by real-time RT-PCR. 6. A human intestinal epithelial cell line of HT-29 was cultured and infected by rotavirus, coxsackie B3 virus and shigella respectively, and then the changes of mRNA expressions for five TLRs were investigated by real-time PCR.Results The main characteristic of the adaptive immune response in children with acute rotavirus infection were as following: 1. 77.3% of G₃ and 82.7% of P[8]were identified in 75 stool specimens with rotavirus infection. Other minor genotypes of G₁, G₂, P[4], P[9]were also found in these samples. 2. The median titers of rotavirus specific IgM/IgG/IgA in plasmas of diarrhea children were significantly higher than that in the control children, and higher titers of rotavirus specific IgA were detected in stools of patients compared with that in plasmas. Moreover, rotavirus specific IgA titers in stools and plasmas were higher in patients with more severity of diarrhea than the other patients. 3. The percentages of CD19⁺ cells were increased in rotavirus diarrhea children (30.8±7.9 % vs. 23.1±7.7% in the control). But the percentage of CD4⁺ cellsdecreased significantly in patients compared with controls. Expression levels of IFN-γ mRNA were significantly increased in the first 3 days of illness in children infected with rotavirus, and IL-12p40 mRNA were in a high level during the entire period of rotavirus diarrhea.The changes of TLR in the innate immune response to rotavirus infection were as following: 1. Significantly higher expression of genes encoding TLR2, TLR3, TLR4, TLR7 and TLR8 were observed in PBMC of patients within 3 days of illness onset than those in control children. After 3 days of illness onset, only TLR3 and TLR8 mRNA expressions were still significantly increased. Both TLR2 and TLR4 mRNA levels were significantly elevated in PBMC of patients with shigellosis during the entire phase of illness. 2. Differential TLR induced cytokines expressions existed between diarrhea children with rotavirus infection or shigellosis. 3. The HT-29 cells cultured in vitro demonstrated significantly increased expressions of TLR2, TLR3, TLR4, TLR7 and TLR8 after infected with rotavirus , and expression levels of TLR2, TLR7 and TLR8 were significantly elevated in HT-29 cells infected by coxsackie B3 virus. HT-29 cells infected by different viral or bacterial pathogens showed obviously differential TLR responses in time course and dose-dependent effect. TLR2 and IL-8 levels in HT-29 cells were significantly elevated at 3hr post invasion of shigella.Conclusions 1. Humoral immunity and especially mucosal immunity is critically important in host immune response to acute rotavirus infection. Th1 response is predominant during early phase of rotavirus infection. 2. After rotavirus infection, at least five TLRs were induced increased and effector cytokine IFN-γ was elevated, it thereby indicated that TLRs play important roles in early immune response to rotavirus infection. 3. The differential TLR expressions in acute diarrhea children infected by rotavirus or shigella and also in cell cultures infected by viral and bacterial pathogens suggested that different TLRs were initiated in host immune response to different pathogens. TLRs may modulate immune response in children with infectious diarrhea by activation of differential cytokines.