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Noroviruses (NoV) are responsible for the majority of acute non-bacterial gastroenteritis worldwide. Previous research demonstrates that some individuals who are genetically susceptible to Norwalk virus, a specific type of NoV, do not become infected after they are challenged with the virus. This research suggests that there is a protective immune response to Norwalk virus infection. However, the particular components of this immune response are not known. Because Norwalk virus infection takes place in the gastrointestinal tract, it is likely that mucosal antibodies play an important role in protection from Norwalk virus infection. Secretory immunoglobulin A (SIgA) is the main type of antibody important to mucosal immunity. There is a need to detect and quantify levels of total and Norwalk virus-specific SIgA to determine the human SIgA response throughout the course of Norwalk virus infection. I am in the process of developing novel enzyme-linked immunosorbent assays (ELISAs) to quantify the amount of total and Norwalk virus-specific SIgA in saliva samples. Subsequent use of these assays will provide information on the human SIgA response to Norwalk virus infection, thereby providing insight into the pathology of NoV and guiding the development of NoV vaccines.
Noroviruses (NoV) pose a significant public health risk. In many industrial countries, NoV are the leading cause of epidemic acute gastroenteritis (Fankhauser, Monroe et al. 2002). NoV are also responsible for a significant proportion of severe diarrhea in young children living in developing countries (Glass, Noel et al. 2000). Previous human challenge studies demonstrate that individuals can acquire short-term immunity, but not long-term immunity, to NoV. One of our previous human challenge studies suggests that there is an association between levels of NoV-specific salivary immunoglobulin A (IgA) and protection from infection (Figure 1). The results suggest that the genetically-susceptible, protected volunteers had acquired a short-term memory immune response. However, some volunteers who did not become infected showed weak salivary IgA responses, suggesting that salivary IgA responses are not the only mechanism involved in protection from NoV.
Specific subtypes of IgA may be primarily responsible for protection from NoV infection. One particular subtype of IgA is secretory IgA (SIgA). SIgA is a critical antibody in mucosal immunity (Figure 2, Brandtzaeg 2003). Since the gastrointestinal tract is the site of Norwalk virus infection, SIgA may play an important role in protection from NoV infection. There is a need to develop NoV-specific SIgA assays to give insight into the human mucosal immune response to NoV and the mechanisms of NoV infection. Subsequent use of this assay could contribute to the development of a NoV vaccine.
Project Goal
To develop enzyme-linked immunosorbent assays (ELISAs) to detect and quantify total and Norwalk virus-specific secretory IgA (SIgA) antibodies in human saliva.
Assay Design
The Norwalk virus-specific and total SIgA assays are designed to detect the secretory component of SIgA (Figure 3). Both SIgA and secretory IgM (SIgM) contain the secretory component. To ensure specificity of our assay to SIgA, I added an IgM-adsoprtion step to remove all IgM from saliva to the Norwalk-virus specific assay (Figure 3A). The total SIgA assay includes an anti-IgA antibody coating step to ensure specificity to SIgA (Figure 3B).
Before developing the assays, we are identifying the most appropriate reagents and confirming the validity of the assay design. First, we are determining the optimal blocking agent. It is possible that some blocking agents contain secretory component and cannot be used in the assays. To determine the best blocking agent, we conducted control experiments to detect SC in blocking agents (Figure 4A). We also measured which blocking agent is most effective at reducing non-specific binding of the anti-SC detection antibody conjugated to HRP (Figure 4B). To confirm that the NV-specific SIgA assay design is appropriate, we will then test if free secretory component binds to NV antigen (Figure 5). If free secretory component binds to NV antigen, the NV-specific SIgA assay will detect both SIgA and free secretory component and the assay design would need to be modified.
The results pertain to the control experiment to determine the optimal blocking agent (Figure 6). The control experiment to determine if blocking solutions contain secretory component was not successful because the controls did not work (data not shown).
Blotto and FBS are potential blocking agents for the proposed assays.
The results presented in Figure 6 suggest that blotto and FBS most effectively reduce non-specific binding of the anti-SC antibody. However, additional experiments are needed for confirmation.
Identify the optimal blocking agent.
Additional experiments will be performed to determine which blocking agent provides the least background activity with the anti-SC antibody when the same blocking solution coats the well. We will also modify the control experiment to determine if secretory component is present in blocking solutions to ensure that the selected blocking agent does not contain secretory component.
Validate the assay design.
We will perform the control experiment to determine if free secretory component binds to NV antigen (Figure 5).
Optimize the IgM adsorption step of the Norwalk virus-specific SIgA assay.
To confirm that the Norwalk virus-specific SIgA assay removes all IgM from saliva, we will determine the upper limit of IgM adsorption and ensure that it is greater than the upper limit of IgM found in saliva.
Optimize and validate the assay.
We will determine the optimal concentrations of all reagents that provide the most distinct optical density data for each antibody titer. After development, we will calculate the reproducibility of the assay by measuring the between-run and within-run coefficient of variation. We will also examine the effect of heat inactivation and freeze-thawing on the reproducibility of the assays. We will measure the specificity of the anti-SC antibody by comparing the antibody binding to monomeric IgA, polymeric IgA, and secretory IgA.
This research is supported by the Howard Hughes Medical Institute under Grant No.52005873, the United States Department of Agriculture under Grant No. 2005-511110-3271, and the Atlanta Clinical and Translational Science Institute. I am grateful to the Summer Undergraduate Research at Emory program for continued support.
Brandtzaeg, P. (2003). "Role of secretory antibodies in the defence against infections." Int J Med Microbiol 293(1): 3-15.
Fankhauser, R. L., S. S. Monroe, et al. (2002). "Epidemiologic and molecular trends of "Norwalk-like viruses" associated with outbreaks of gastroenteritis in the United States." J Infect Dis186(1): 1-7.
Glass, R. R, J. Noel, et al. (2000). "The epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics." J Infect Dis 181:S254-S261.
Lindesmith, L., C. Moe, et al. (2003). "Human susceptibility and resistance to Norwalk virus infection." Nat Med 9(5): 548-53.
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