IgA is important for clearance and critical for protection from rotavirus infection

Abstract

Based on a lack of severe phenotype in human immunoglobulin A (IgA) deficiency syndromes, the role of IgA in controlling respiratory and gastrointestinal (GI) infections has not been clearly defined. C57BL/6 and BALB/c mice lacking IgA (IgA(-/-)) were developed and used to address this question. When exposed to a common GI virus, rotavirus, IgA(-/-) mice exhibited a substantial and significant delay in clearance of the initial infection compared with wild-type mice. IgA(-/-) mice excreted rotavirus in stool up to 3 weeks after the initial exposure compared with 10 days observed in wild-type mice. Importantly, IgA(-/-) mice failed to develop protective immunity against multiple repeat exposures to the virus. All IgA(-/-) mice excreted virus in the stool upon re-exposure to rotavirus, whereas wild-type mice were completely protected against re-infection. These findings clearly indicate a critical role for IgA in the establishment of immunity against a GI viral pathogen.

Figure 1. Induction of fecal IgA coincides with clearance of rotavirus infection in mice

C57BL/6 (A) and BALB/c mice (B) mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus on day 0. Fecal pellets were collected every day and analyzed for the presence of the VP6 middle capsid protein by ELISA (mean values shown by solid line) or the presence of rotavirus-specific IgA (mean value shown by dotted line). Each symbol represents the OD of either antigen (black circles) or antibody (grey squares) present in the stool of an individual mouse. Horizontal line indicates limit of detection of the assay.

Figure 2. IgA is important for clearance of rotavirus infection

C57BL/6 (A) and BALB/c (B) mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus on day 0. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD of VP6 present in an individual wild type (black circles) or IgA^−/− (grey triangles) mouse. The mean OD values for the groups are indicated for wild type (solid line) and IgA^−/− (dotted line) mice. Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. Black bar, wild type; grey bar, IgA^−/−. Each bar represents the mean for each group+SD (n=10–12 mice/group). *, p<0.05 by Kruskal-Wallis equality of populations rank test compared to wild type mice.

Figure 3. IgA is required for protection from a secondary rotavirus infection

C57BL/6 (A) and BALB/c (B) wild type or IgA^−/− mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus and received a secondary inoculation with 10³ ID₅₀ EC_wt either six weeks or twelve weeks later. Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol represents the OD obtained from an individual wild type mouse (black circles) or IgA^−/− mouse at six weeks (black squares) or 12 weeks (grey triangles). The mean antigen OD values for the groups are indicated for wild type (solid line) and IgA^−/− at either six weeks (large dotted line) or twelve weeks (small dotted line). Horizontal line indicates limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bar, wild type mice, six or twelve week challenge (no antigen shedding at either time point); black bar, IgA^−/− mice, six week challenge; grey bar, IgA^−/− mice, twelve week challenge. Each bar represents the mean for each group+SD (n=5–6 mice/group). *, p<0.05 by Kruskal-Wallis equality of populations rank test compared to wild type mice.

Figure 4. Mice lacking IgA are susceptible to multiple infections with rotavirus

Naïve C57BL/6 IgA^−/− (A) or BALB/c IgA^−/− (B) mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus (mean value, solid line) followed by an identical second inoculation six weeks (mean value, large dotted line) and third inoculation twelve weeks later (mean value, small dotted line). Fecal pellets were collected every other day and analyzed for the presence of the VP6 middle capsid protein by ELISA. Each symbol is the OD, representative of the amount of VP6 present in a sample from an individual IgA^−/− mouse following the first (black circles), second (black squares, six weeks), and third (grey triangles, 12 weeks) inoculations. The mean antigen OD values for the groups are indicated following first (solid line), second (large dotted line), or third (small dotted line) inoculation. Horizontal line indicates the limit of detection of the assay. C, the mean number of time points antigen was detected in fecal samples for each group. White bars, initial infection, black bars, six week inoculation, grey bars, twelve week inoculation. Each bar represents the mean for each group+SD (n=5–6 mice per group). *, p<0.05 by Kruskal-Wallis equality of populations rank test compared mice receiving a challenge at 6 weeks.

Figure 5. IgA is not critical for clearance of rotavirus antigenemia

Mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus on day 0. Serum was collected either 4 or 15 days later and analyzed for the presence of the VP6 middle capsid protein by ELISA. C57BL/6 (black bar), C57BL/6 IgA^−/− (white bar), BALB/c (grey bar), and BALB/c IgA^−/− (hatched bar). Each bar represents the mean OD of VP6 present in 5–6 mice + SD. *, p<0.05 by Kruskal-Wallis equality of populations rank test compared to wild type mice.

Figure 6. Mice lacking IgA do not develop detectable compensatory rotavirus-specific IgG or IgM fecal antibody

Mice were orally inoculated with 10³ ID₅₀ of EC_wt rotavirus on day 0. Fecal pellets were analyzed for the presence of rotavirus specific total antibody (IgA, IgM, and IgG) 15 days following viral exposure by ELISA. Each bar represents the geometric mean titer 4–6 mice + SD. *, p<0.05 by Mann Whitney U. Horizontal line indicates the limit of detection of the assay.