Abstract
Adult BALB/c mice were orally inoculated with murine (strain EDIM), simian (strain RRV), or bovine (strain WC3) rotavirus. Six or 16 weeks after inoculation, mice were challenged with EDIM. At the time of challenge and in the days immediately following challenge, production of rotavirus-specific immunoglobulin A (IgA), IgG, and IgM by small intestinal lamina propria lymphocytes (LPL) was determined by fragment culture, and quantities of virus-specific antibodies at the intestinal mucosal surface were determined by intestinal lavage. Mice immunized with EDIM were completely protected against EDIM challenge both 6 and 16 weeks after immunization. Protection was associated with production of high levels of IgA by LPL and detection of virus-specific IgA at the intestinal mucosal surface. In addition, animals immunized and later challenged with EDIM did not develop a boost in antibody responses, suggesting that they were also not reinfected. We also found that in mice immunized with nonmurine rotaviruses, (i) quantities of virus-specific IgA generated following challenge were greater 16 weeks than 6 weeks after immunization, (ii) immunization enhanced the magnitude but did not hasten the onset of production of high quantities of virus-specific IgA by LPL after challenge, and (iii) immunization induced partial protection against challenge; however, protection was not associated with either production of virus-specific antibodies by LPL or detection of virus-specific antibodies at the intestinal mucosal surface.
The importance of rotaviruses as a cause of disease and death in both developed and developing countries has for two decades stimulated interest in disease prevention by vaccine. Development of a successful vaccine may in part depend upon understanding the immunologic mechanism or mechanisms by which the host is protected against infection and disease. For many years, the immunologic correlates of protection against challenge have been a matter of debate (reviewed in reference 21). Recently, using both immunocompetent (5, 17) and immunodeficient (6, 18) mice, investigators found that protection against challenge is mediated by the presence of virus-specific immunoglobulin A (IgA) at the intestinal mucosal surface at the time of challenge. However, these findings are at variance with the fact that levels of virus-specific IgA in the feces or serum of infants have been an unreliable correlate of protection against disease in vaccine trials (1, 29).
In this study, we examined adult, immunocompetent mice orally inoculated with murine or nonmurine rotaviruses and subsequently challenged with murine rotavirus. To determine the relative importance of virus-specific effector and memory B cells in protection against challenge, virus-specific IgA, IgG, and IgM responses were measured both before and immediately after challenge. Virus-specific antibodies produced by small intestinal lamina propria lymphocytes (LPL) were obtained by intestinal fragment culture (13), and lymphocytes present at the intestinal mucosal surface were obtained by intestinal lavage. The use of intestinal fragment cultures allowed for preservation of the native microenvironment of the small intestinal lamina propria and obviated concerns about the use of fluids obtained by intestinal lavage (such as degradation of virus-specific IgA by intestinal proteases, entrapment of secretory IgA in the mucin layer, variable dilution of secretory IgA by osmotic catharsis, and formation of antigen-antibody complexes following challenge).
MATERIALS AND METHODS
Mice.
Adult, 6- to 8-week old, female BALB/c mice and pregnant Swiss Webster mice were obtained from Taconic Breeding Laboratories (Germantown, N.Y.) and housed in separate isolation units.
Cells.
Fetal green monkey kidney cells (MA-104) were grown as previously described (19).
Viruses.
Murine rotavirus strain EDIM (G3[P16]) was obtained from Richard Ward (Children’s Hospital Research Foundation, Cincinnati, Ohio) and inoculated orally into 7-day-old Swiss Webster mice. Small intestines were removed from suckling mice 3 to 4 days after inoculation, and 10% (wt/vol) suspensions were prepared in BHK cell medium (14) (Wistar Institute, Philadelphia, Pa.). Suspensions were homogenized in a PowerGen 125 tissue homogenizer (Fisher Scientific, Pittsburgh, Pa.) and stored at −70°C.
Simian rotavirus strain RRV (G3[P3]), originally obtained from N. Schmidt (Berkeley, Calif.), and bovine rotavirus strain WC3 (G6[P5]) were grown and titered as previously described (19).
Experimental design.
Five groups of 32 adult, female BALB/c mice were inoculated orally with 100 μl each of one of the following: EDIM (6.0 × 104 shedding dose50 [SD50]/mouse [see below]), RRV (either 1.9 × 107 PFU/mouse [high dose] or 1.9 × 196 PFU/mouse [low dose]), WC3 (3.0 × 106 PFU/mouse), or BHK medium by proximal esophageal intubation. Six weeks after inoculation, 16 of the mice from each group were used. Specifically, 12 of the 16 mice were challenged orally with 200 μl of EDIM (1.2 × 105 SD50). The remaining four mice per group were used to determine antibody production by LPL by fragment culture or antibodies secreted onto the intestinal mucosal surface by intestinal lavage. Of the 12 challenged mice, 6 were used for the fragment cultures and 6 were used for intestinal lavages performed 2, 4, and 6 days after challenge. In addition, of the 12 challenged mice, fecal pellets were collected from 4 to 5 mice per group daily for 6 days after challenge. Sixteen weeks after inoculation, a similar series of studies was performed.
In addition, groups of 16 adult (6- to 8-week-old) BALB/c mice were orally inoculated with either 2.7 × 105, 1.3 × 105, or 6.7 × 104 PFU of RRV/mouse in a volume of 100 μl by esophogeal intubation. Six weeks after immunization, eight mice per group were used. Six of these mice were challenged orally with 200 μl of EDIM. Fecal pellets were collected from four mice per group daily for 6 days after challenge. Small intestinal fragment cultures were established at the time of challenge and 4 days after challenge. Similar studies were performed 16 weeks after immunization.
An average of three fecal pellets were collected daily from each mouse for 6 days after challenge, placed in 0.5 ml of Earle’s balanced salt solution, and stored at −20°C. Tubes of Earle’s balanced salt solution plus fecal material weighed an average of 1.44 g with a standard deviation of 0.04. Samples were vortexed to disrupt pellets, and supernatant fluids were tested for the presence of rotaviral antigen by enzyme-linked immunoassay (ELISA) as described below.
Determination of SD50.
Groups of five adult BALB/c mice were inoculated with 100 μl of EDIM derived from intestinal homogenates at dilutions of 1:10, 1:102, 1:103, 1:104, 1:105, and 1:106. Fecal pellets were collected daily for a period of 6 days following inoculation. Quantities of antigen shed were determined by ELISA. Percentages of animals positive for shedding, as calculated by the method of Reed and Muench (22), were 99% (1:10), 93% (1:102), 85% (1:103), 71% (1:104), 44% (1:105), and 2% (1:106). The SD50 was calculated as 1:60,250. Animals were challenged with 200 μl of this preparation.
ELISA to detect rotavirus in feces.
To detect the presence of rotavirus antigen in feces, 96-well, high-binding, flat-bottom plates (Costar, Cambridge, Mass.) were coated with a 1:2,000 dilution of bovine anti-WC3 serum in a solution containing 1.5 mM sodium carbonate and 3.5 mM sodium bicarbonate. Plates were covered and incubated in a humidified atmosphere at 4°C overnight. Plates were washed (MultiReagent Plate Washer; Dynatech, Chantilly, Va.) four times with a wash buffer solution containing 1.73 M NaCl, 0.03 M KH2PO4, 0.13 M Na2HPO4, and 0.25% Tween 20 (Sigma, St. Louis, Mo.) and two times with distilled H2O. Two hundred microliters of a solution containing 0.5% gelatin (Sigma) and 0.05% Tween 20 (blocking buffer) was added to each well, and plates were incubated for 1 h at room temperature. Wells were washed as described above; 50-μl aliquots of undiluted, homogenized fecal supernatant samples were added to experimental wells, and 50-μl aliquots of blocking buffer were added to control wells. After incubation at room temperature for 1 h, wells were washed as described above, 100 μl of rabbit antirotavirus (bovine strain WC3) hyperimmune serum diluted 1:2,000 in blocking buffer was added to each well, and plates were incubated at room temperature for 1 h. Wells were again washed, and 100 μl of alkaline phosphatase-conjugated goat anti-rabbit IgG (Cappel, Durham, N.C.) was added to each well. After incubation at room temperature for 1 h, plates were washed and 100 μl of a solution containing 1 M diethanolamine and p-nitrophenyl phosphate solution (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added to each well. After incubation at room temperature for 1 h, plates were read at a wavelength of 405 nm on an ELISA plate reader (Dynatech MR4000). Samples were considered positive if the optical density (OD) in the experimental well was >0.1 OD units and twofold greater than the OD in the corresponding control well.
Intestinal lavage.
Mice were orally administered four consecutive doses of a 6.8% (wt/vol) solution of Golytely (Braintree Laboratories, Braintree, Mass.) at 15-min intervals. Thirty minutes after the last dose, each mouse received 0.15 ml of pilocarpine HCl (Isopto Carpine, Alcon, Puerto Rico) intraperitoneally. Intestinal secretions from pairs of mice were collected over a period of 20 min in soybean trypsin inhibitor (Sigma). Fluids were diluted in phosphate-buffered saline, vortexed, and centrifuged at 700 × g for 10 min. Phenylmethylsulfonyl fluoride (1 mM; Sigma) was added to supernatants. Samples were centrifuged at 27,000 × g for 20 min. Two percent sodium azide (Sigma) and 100 mM phenylmethylsulfonyl fluoride were both added to supernatants at a final concentration of 1% (vol/vol). Samples were placed on ice for 15 min. Five percent fetal bovine serum (Gibco BRL, Gaithersburg, Md.) and 25% (vol/vol) glycerol (Sigma) were added to supernatants, which were mixed vigorously, aliquoted, and stored at −20°C prior to testing.
Small intestinal fragment cultures.
Cultures of small intestinal lymphoid tissues were prepared as described previously (12). Briefly, small intestines were removed from each animal and placed in Hanks’ balanced salt solution (HBSS; GIBCO Biomedical Research Laboratories, Grand Island, N.Y.). Peyer’s patches were removed, and intestines were cut into several 5-cm fragments. Fat and connective tissues were removed, and each fragment was cut longitudinally to expose the mucosal surface. Fragments were placed in fresh HBSS, allowed to sit on ice for approximately 2 h, and washed twice with HBSS, once with HBSS containing 0.05% EDTA, and twice with HBSS. Eight 1- to 2-mm fragments were isolated and placed in individual wells of a 24-well plate (Becton Dickinson, Lincoln Park, N.J.) with 1 ml of Kennett’s HY medium containing 10% fetal bovine serum (Gibco), 10 mM HEPES, 4 mM l-glutamine, streptomycin (100 μg/ml), gentamicin (50 μg/ml), and amphotericin B (Fungizone); (0.25 μg/ml). All additives were obtained from JRH (Lenexa, Kans.). Plates were incubated for 5 days at 37°C in a chamber containing 95% O2 and 5% CO2. Supernatant fluids were collected and stored at 4°C.
Increased quantities of total IgA were consistently observed between 6- and 16-week samples. This finding is consistent with that of van der Heijden and colleagues (28), who observed that numbers of immunoglobulin-secreting cells in the small intestinal lamina propria were greater in older than in younger mice.
ELISA for detection of rotavirus-specific and total antibody.
Intestinal contents and supernatant fluids from small intestinal lymphoid fragment cultures were tested for the presence of rotavirus-specific and total antibodies as previously described (12). Samples were considered to be positive if the OD in the experimental well was >0.1 OD units and twofold greater than the OD in the corresponding control well. Quantities of virus-specific and total antibodies were determined in comparison with standard curves constructed for each isotype, using purified murine IgA (IgA kappa chain; Sigma), IgG (IgG1 kappa chain; Sigma), or IgM (IgM kappa chain; Sigma). Standard curves were performed with each assay and were subjected to exponential curve fits; only curves with correlation coefficients greater than 0.90 were used.
The lower limit of detection for all isotypes was 1 ng/ml.
Calculations and statistical analysis.
For both fragment culture and intestinal lavage samples, concentrations of virus-specific and total antibodies were calculated based on standard curves for quantities of purified immunoglobulin generated during each assay day. Fragment culture data were adjusted to represent the entire length of the intestine. Sixteen small intestinal fragment cultures were analyzed per group per time point; standard errors were determined based on differences among the percentages of these 16 samples.
Concentrations of rotavirus antigen in feces were also calculated based on standard curves for quantities of purified rotavirus; averages were calculated based on the quantities of antigen shed by all mice in a group on a given day. In addition, Student t tests were performed to determine statistical differences among antigen concentrations; P values less than 0.05 were considered significant.
RESULTS
Mucosal virus-specific IgA responses induced by murine rotaviruses are of greater magnitude and longer duration than those induced by nonmurine rotaviruses.
Six and 16 weeks after oral inoculation of mice with murine strain EDIM, 1.4 and 2.5%, respectively, of all IgA produced by LPL in the small intestine was directed against rotavirus (Table 1). Oral inoculation of mice with a high dose of simian strain RRV (1.9 × 107 PFU/mouse) also induced production of virus-specific IgA by LPL which was detectable at the time of challenge both 6 and 16 weeks after inoculation, but at levels approximately threefold lower than those found after inoculation with EDIM (Table 1). Production of virus-specific IgA by LPL following inoculation with EDIM correlated with detection of virus-specific IgA at the intestinal mucosal surface (Table 2).
TABLE 1.
Ratio of rotavirus-specific IgA to total IgA produced by small intestinal LPL at various intervals after challenge in mice immunized either 6 or 16 weeks previously with murine or nonmurine rotavirusesa
| Immunization | Ratio of virus-specific IgA to total IgA (%) at indicated interval (days) after challenge
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 6 wk postimmunization
|
16 wk postimmunization
|
|||||||
| 0 | 2 | 4 | 6 | 0 | 2 | 4 | 6 | |
| EDIM | 1.4 (±0.07d) (7/490b) | 2.2 (±0.23) (19/880) | 1.5 (±0.09) (12/820) | 2.1 (±0.04) (34/1,590) | 2.5 (±0.16) (83/3,260) | 2.7 (±0.55) (87/3,200) | 2.4 (±0.34) (49/2,080) | 2.1 (±0.07) (20/940) |
| RRV-high | 0.43 (±0.11) (2/460) | 0.23 (±0.07) (2/860) | 0.53 (±0.13) (4/760) | 3.8 (±0.17) (55/1,450) | 0.79 (±0.12) (25/3,180) | 0.06 (±0.02) (3/5,430) | 1.60 (±0.17) (38/2,380) | 14.5 (±1.70) (194/1,340) |
| RRV-low | 0 (0/610)c | 0 (0/960) | 0 (0/1,500) | 0.46 (±0.02) (6/1,300) | 0 (0/2,040) | 0 (0/2,260) | 0 (0/1,320) | 7.9 (±0.80) (75/950) |
| WC3 | 0 (0/430) | 0 (0/820) | 0 (0/790) | 0.21 (±0.06) (3/1,400) | 0 (0/3,380) | 0 (0/4,170) | 0.3 (±0.25) (7/2,390) | 5.3 (±1.27) (60/1,130) |
| Buffer | 0 (0/360) | 0 (0/860) | 0 (0/1,200) | 0.43 (±0.02) (5/1,170) | 0 (0/2,580) | 0 (0/3,710) | 0 (0/1,850) | 0.9 (±0.04) (8/880) |
Groups of 32 animals were inoculated orally with either murine rotavirus strain EDIM, simian strain RRV at two different doses (RRV-high and RRV-low), bovine strain WC3, or buffer; 6 or 16 weeks after immunization, animals were challenged with EDIM. At the time of challenge and 2, 4, and 6 days after challenge, mice were sacrificed and small intestinal fragment cultures were established. Supernatant fluids from these cultures were evaluated for the presence of rotavirus-specific and total IgA.
Quantities (in micrograms per milliliter) of virus-specific IgA to total IgA.
Virus-specific IgA was not detectable at values less than 1 ng/ml. Undetectable levels of virus-specific IgA are shown as 0.
Standard error of the mean based on relative quantities of virus-specific to total IgA from 16 samples.
TABLE 2.
Ratio of rotavirus-specific IgA to total IgA detected at the intestinal mucosal surface at various intervals after challenge in mice immunized either 6 or 16 weeks previously with murine or nonmurine rotavirusesa
| Immunization | Ratio of virus-specific IgA to total IgA (%) at indicated interval (days) after challenge
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 6 wk postimmunization
|
16 wk postimmunization
|
|||||||
| 0 | 2 | 4 | 6 | 0 | 2 | 4 | 6 | |
| EDIM | 1.4 (110/7,760b) | 0.92 (234/25,500) | 2.8 (51/1,800) | 1.5 (219/14,500) | 0.53 (16/3,040) | 0 (0/772)c | 1.09 (71/6,550) | NDd |
| RRV-high | 0.33 (42/12,900) | 1.4 (27/18,900) | 1.33 (47/3,560) | 6.35 (806/12,700) | 0 (0/7,160) | 0 (0/10,300) | 0.52 (29/57,100) | ND |
| RRV-low | 0 (0/15,700) | 0 (0/1,840) | 0 (0/7,170) | 0.87 (122/14,000) | 0 (0/3,720) | 0 (0/21,100) | 0 (0/43,100) | ND |
| WC3 | 0 (0/11,800) | 0 (0/1,750) | 0 (0/11,000) | 1.29 (208/16,200) | 0 (0/4,280) | 0 (0/8,260) | 0 (0/1,700) | ND |
| Buffer | 0 (0/1,790) | 0 (0/1,910) | 0 (0/12,000) | 1.28 (211/16,500) | 0 (0/918) | 0 (0/5,960) | 0 (0/4,060) | ND |
Groups of 32 animals were inoculated orally with either murine rotavirus strain EDIM, simian strain RRV at two different doses (RRV-high and RRV-low), bovine strain WC3, or buffer; 6 or 16 weeks after immunization, animals were challenged with EDIM. At the time of challenge and 2, 4, and 6 days after challenge mice intestinal contents were obtained by lavage as described in Materials and Methods. Intestinal contents were evaluated for the presence of rotavirus-specific and total IgA.
Ratio of virus-specific IgA (in nanograms per milliliter) to total IgA.
Virus-specific IgA was not detectable at values less than 1 ng/ml. Undetectable levels of virus-specific IgA are shown as 0.
ND, not done.
At the time of challenge, virus-specific IgA was neither produced by LPL nor detected at the intestinal mucosal surface 6 or 16 weeks after oral inoculation of mice with a lower dose of RRV (1.9 × 106 PFU/mouse), WC3 (3 × 106 PFU/mouse), or buffer (Tables 1 and 2).
Immunization with nonmurine rotaviruses did not hasten the onset of production of high quantities of virus-specific IgA by LPL following challenge.
In mice inoculated 6 or 16 weeks previously with nonmurine rotaviruses or buffer, a burst of virus-specific IgA production by LPL was first detected 6 days after challenge (Table 1).
Mice challenged 16 weeks after immunization with nonmurine rotaviruses developed greater virus-specific IgA responses than mice challenged 6 weeks after immunization.
Sixteen but not 6 weeks after immunization, quantities of virus-specific IgA produced by LPL 6 days after challenge were greater in animals immunized with nonmurine rotaviruses than in unimmunized animals (Table 1). In addition, following challenge, quantities of virus-specific IgA produced 16 weeks after immunization were greater than those found 6 weeks after immunization (Table 1). Specifically, 6 days after challenge, mice immunized 16 weeks previously with high-dose RRV, low-dose RRV, or WC3 had ratios of virus-specific IgA to total IgA approximately 4-, 17-, and 25-fold greater than in similarly immunized animals challenged 6 weeks after immunization (Table 1).
Both murine rotavirus and nonmurine rotaviruses induced production of virus-specific IgG but not IgM by LPL.
High ratios of virus-specific IgG to total IgG were produced by LPL 16 weeks but not 6 weeks after inoculation of mice with murine or nonmurine rotaviruses (Table 3). At the time of challenge, virus-specific IgG was detected at the intestinal mucosal surface 6 weeks after inoculation with EDIM and 16 weeks after inoculation with EDIM or high-dose RRV (Table 4). Significantly greater percentages of IgG produced by LPL and detected at the intestinal mucosal surface were virus specific compared with those percentages found for virus-specific IgA (Tables 1 to 4).
TABLE 3.
Ratio of rotavirus-specific IgG to total IgG produced by small intestinal LPL at various intervals after challenge in mice immunized either 6 or 16 weeks previously with murine or nonmurine rotavirusesa
| Immunization | Ratio of virus-specific IgG to total IgG (%) at indicated interval (days) after challenge
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 6 wk postimmunization
|
16 wk postimmunization
|
|||||||
| 0 | 2 | 4 | 6 | 0 | 2 | 4 | 6 | |
| EDIM | 0.65 (±0.11d) (1.56/239b) | 0.77 (±0.24) (3.62/470) | 1.3 (±0.67) (1.35/102) | 0.87 (±0.16) (0.7/81) | 24.5 (±2.11) (46/189) | 6.2 (±2.21) (9.4/152) | 8.6 (±1.53) (11/127) | 11.2 (±2.66) (5.4/48.1) |
| RRV-high | 0 (0/14.2) | 0 (0/39)c | 6.9 (±3.40) (1.85/26.9) | 15.5 (±3.73) (4.2/27.2) | 29.9 (±3.31) (58/196) | 24.9 (±3.94) (40.3/162) | 33.7 (±3.64) (43.5/129) | 13.8 (±7.05) (35.4/258) |
| RRV-low | 0 (0/19.9) | 0 (0/151) | 0 (0/29.1) | 1.7 (±0.40) (1.6/94.3) | 17.2 (±4.61) (13/74) | 5.4 (±3.30) (4.3/80.2) | 0.7 (±0.51) (0.6/86.2) | 30.9 (±4.55) (28.2/91.4) |
| WC3 | 0 (0/24.6) | 0 (0/10.1) | 0.53 (±0.19) (0.23/43.3) | 0.84 (±0.48) (0.13/15.4) | 12.2 (±3.84) (22/184) | 16.9 (±2.67) (42.7/253) | 15.7 (±5.24) (26.0/165) | 14 (±4.49) (21.2/152) |
| Buffer | 0 (0/8.7) | 0 (0/18) | 0 (0/54.7) | 0.46 (±0.16) (0.43/93.7) | 0 (0/158) | 0 (0/137) | 0 (0/140) | 0 (0/49.7) |
Groups of 32 animals were inoculated orally with either murine rotavirus strain EDIM, simian strain RRV at two different doses (RRV-high and RRV-low), bovine strain WC3, or buffer; 6 or 16 weeks after immunization, animals were challenged with EDIM. At the time of challenge and 2, 4, and 6 days after challenge, mice were sacrificed and small intestinal fragment cultures were established. Supernatant fluids from these cultures were evaluated for the presence of rotavirus-specific and total IgG.
Quantities of virus-specific IgG (in micrograms per milliliter) to total IgG expressed as percentages.
Virus-specific IgG was not detectable at values less than 1 ng/ml. Undetectable levels of virus-specific IgG are shown as 0.
Standard error of the mean based on relative quantities of virus-specific to total IgG from 16 samples.
TABLE 4.
Ratio of rotavirus-specific IgG to total IgG detected at the intestinal mucosal surface at various intervals after challenge in mice immunized either 6 or 16 weeks previously with murine or nonmurine rotavirusesa
| Immunization | Ratio of virus-specific IgG to total IgG (%) at indicated interval (days) after challenge
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 6 wk postimmunization
|
16 wk postimmunization
|
|||||||
| 0 | 2 | 4 | 6 | 0 | 2 | 4 | 6 | |
| EDIM | 22.4 (1.9/8.5b) | 16.3 (2.6/16) | 13 (1/7.5) | 30.7 (15/48.8) | 6.95 (0.71/10.2) | 0 (0/2.6)c | 5.6 (0.82/14.6) | NDd |
| RRV-high | 0 (0/1.2) | 0 (0/7.8) | 0 (0/9.9) | 79 (16.4/21.3) | 11 (1.12/10.2) | 0 (0/9.2) | 0 (0/12.4) | ND |
| RRV-low | 0 (0/8.3) | NAe | 0 (0/15.2) | 37 (7.4/20) | NA | NA | 0 (0/3.9) | ND |
| WC3 | 0 (0/7.8) | 0 (0/1.8) | 0 (0/15.2) | 58.6 (16/28) | 0 (0/26) | NA | 0 (0/4.1) | ND |
| Buffer | 0 (0/1.1) | 0 (0/7.8) | 0 (0/13.6) | 56.2 (9/16) | 0 (0/19) | 0 (0/8.5) | 0 (0/9.5) | ND |
Groups of 32 animals were inoculated orally with either murine rotavirus strain EDIM, simian strain RRV at two different doses (RRV-high and RRV-low), bovine strain WC3, or buffer; 6 or 16 weeks after immunization, animals were challenged with EDIM. At the time of challenge and 2, 4, and 6 days after challenge, intestinal contents were obtained by lavage as described in Materials and Methods. Intestinal contents were evaluated for the presence of rotavirus-specific and total IgG.
Quantities of virus-specific IgG (in nanograms per milliliter) to total IgG expressed as percentages.
Virus-specific IgG was not detectable at values less than 1 ng/ml. Undetectable levels of virus-specific IgG are shown as 0.
ND, not done.
NA, not applicable (this designation was assigned to data points in which neither virus-specific antibody nor total antibody was detected).
IgM was not detected at significant levels either 6 or 16 weeks after immunization with homologous or heterologous host rotaviruses.
Murine rotavirus induced complete protection against challenge, and this effect was associated with the presence of mucosal, virus-specific IgA at the time of challenge.
Virus-specific IgA was present at the intestinal mucosal surface and produced by LPL at the time of challenge both 6 and 16 weeks after immunization with EDIM (Tables 1 and 2). Production of virus-specific IgA at the time of challenge was associated with complete protection against shedding after challenge (Table 5). Also, as compared with mice immunized with nonmurine rotaviruses, EDIM-immunized animals did not develop an increase in the ratio of virus-specific to total IgA produced by LPL within 6 days of challenge (Table 1).
TABLE 5.
Average quantities of rotavirus antigen shed after challenge in mice previously immunized with murine or nonmurine rotavirusa
| Immunization | Quantity (ng/ml) of rotavirus antigen shed at indicated interval (days) after challenge
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 6 wk postimmunization
|
16 wk postimmunization
|
|||||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | P | 1 | 2 | 3 | 4 | 5 | 6 | P | |
| EDIM | <10b | <10 | <10 | <10 | <10 | <10 | <0.001 | <10 | <10 | <10 | <10 | <10 | <10 | <0.0001 |
| RRV-high | 18 | 44 | <10 | <10 | <10 | <10 | <0.005 | 20 | 28 | 17 | <10 | <10 | <10 | <0.0001 |
| RRV-low | 48 | 178 | 42 | 18 | 13 | 18 | 0.82 | 14 | 39 | 32 | 34 | <10 | <10 | <0.001 |
| WC3 | 23 | 204 | 44 | 16 | 17 | 25 | 0.23 | 39 | 111 | 58 | 66 | 17 | <10 | 0.16 |
| Buffer | 23 | 239 | 51 | 16 | 16 | 49 | <10 | 106 | 157 | 113 | 53 | 19 | ||
Groups of four to five animals were inoculated orally with either murine rotavirus strain EDIM, simian strain RRV at two different doses (RRV-high and RRV-low), bovine strain WC3, or buffer; 6 or 16 weeks after immunization, animals were challenged with EDIM; 1, 2, 3, 4, 5, and 6 days after challenge, fecal pellets were collected and tested for the presence of rotavirus antigen by ELISA.
Values of <10 were assigned a value of 9 for statistical calculations.
Nonmurine rotaviruses induced partial protection against challenge, and this protection occurred in the absence of mucosal, virus-specific IgA at the time of challenge and in the days immediately following challenge.
Significant protection against rotavirus shedding was induced 6 weeks after inoculation with high-dose RRV and 16 weeks after inoculation with high-dose or low-dose RRV (Table 5). Although virus-specific IgA was both produced by LPL and detected at the intestinal mucosal surface 6 and 16 weeks after immunization with high-dose RRV, virus-specific IgA was neither produced by LPL nor detected at the intestinal mucosal surface at the time of challenge (or within 4 days of challenge) in animals immunized 16 weeks previously with low-dose RRV (Tables 1 and 2).
To confirm and extend these findings, we inoculated groups of mice with lesser quantities of RRV (2.7 × 105, 1.3 × 105, or 6.7 × 104 PFU/mouse). Again, mice were protected against challenge 16 but not 6 weeks after immunization. In addition, protection occurred in the absence of virus-specific antibodies either at the time of challenge or in the 4 days following challenge (Table 6).
TABLE 6.
Protection against EDIM challenge and ratios of rotavirus-specific to total antibodies (IgA and IgG) produced by small intestinal LPL at the time of challenge or 4 days after challenge in mice immunized either 6 or 16 weeks previously with simian rotavirus strain RRVa
| Immunization (PFU/mouse) | Ratio of virus-specific IgA to total IgA (%) after challenge
|
Ratio of virus-specific IgG to total IgG (%) after challenge
|
Protection against viral shedding after EDIM challenge (P)d | ||
|---|---|---|---|---|---|
| 0 days | 4 days | 0 days | 4 days | ||
| 6 wk after immunization | |||||
| 2.7 × 105 | 0 (0/1,476b) | 0 (0/7,729) | 0 (0/13) | 0 (0/23) | NSe |
| 1.3 × 105 | 0 (0/1,128) | 0 (0/2,064) | 0 (0/10) | 0 (0/41) | NS |
| 6.7 × 104 | 0 (0/3,529) | 0 (0/2,503) | 0 (0/10) | 0 (0/52) | NS |
| 16 wk after immunization | |||||
| 2.7 × 105 | 0 (0/1,867) | 0 (0/821) | 0 (0/126) | 0 (0/94) | <0.05 |
| 1.3 × 105 | 0 (0/6,658) | 0 (0/6,525) | 0 (0/1,128) | 1.1 (±0.18c) (6/551) | <0.005 |
| 6.7 × 104 | 0 (0/7,298) | 0 (0/7,298) | 0 (0/543) | 0 (0/367) | <0.05 |
Groups of 20 mice were inoculated orally with various doses of simian rotavirus strain RRV. Either 6 or 16 weeks after immunization, mice were orally challenged with murine rotavirus strain EDIM. At the time of challenge and 4 days after challenge, small intestinal fragment cultures were established. Supernatant fluids were evaluated for the presence of rotavirus-specific and total antibodies (IgA and IgG).
Quantities (in micrograms per milliliter) of virus-specific to total antibodies. Virus-specific antibodies were not detectable at values less than 1 ng/ml. Undetectable levels of virus-specific antibodies are shown as 0.
Standard error of the mean based on relative quantities of virus-specific to total IgA from 16 samples.
Based on average quantities of rotaviral antigen shed during the 6 days following challenge with murine strain EDIM. Student t tests were performed, and P values of <0.05 were considered significant.
NS, not significant.
DISCUSSION
Following challenge, mice immunized 16 weeks previously with nonmurine rotaviruses developed greater virus-specific IgA responses than mice immunized 6 weeks previously. Virus-specific, IgA-secreting effector B cells generated following challenge were presumably derived from virus-specific, memory B cells. Because B-cell memory in the absence of antigen is short lived (8), the drive to formation of memory B cells is probably generated by continued presentation of rotavirus antigen by antigen-presenting cells (APCs). Long-lived antigen presentation in the small intestine is most likely mediated by follicular dendritic cells in the germinal centers of Peyer’s patches (2). Although the presence of long-term, antigen-specific memory B cells in the intestine has been previously described (27), to our knowledge we offer the first data suggesting that frequencies of virus-specific memory B cells, helper T cells, or APCs in gut-associated lymphoid tissue may increase significantly well beyond the time when virus replicates. Definition of the mechanisms by which this occurs, including enhanced presentation by APCs or recruitment of new APCs, will be a subject of further study.
Immunization with nonmurine rotaviruses did not significantly hasten the onset of virus-specific IgA responses. Sixteen but not 6 weeks after immunization, we found that quantities of virus-specific IgA produced by LPL 6 days after challenge were greater during a secondary response than during a primary response (Table 1), but the burst of virus-specific IgA produced by LPL occurred 6 days after challenge independent of whether animals had been previously immunized. This phenomenon was consistently observed in animals that lacked effector B-cell responses at the time of challenge. Because both naive and memory B cells require cognate, major histocompatibility complex-restricted T-cell help and because memory B cells respond to lower frequencies of helper T cells, lesser quantities of antigen, and lesser quantities of cytokines than naive B cells (9, 30), one would have predicted that the burst of virus-specific IgA produced by LPL would have occurred significantly earlier in mice immunized previously with rotavirus than in unimmunized mice. We will determine in future studies whether the delay in appearance of virus-specific effector B cells in the lamina propria is due to events associated with activation, differentiation, trafficking, or homing.
There were two patterns of protection against challenge induced by immunization of mice with rotaviruses: animals inoculated with murine strain EDIM did not shed rotavirus after challenge, and animals inoculated with different doses of simian strain RRV shed significantly reduced quantities of rotavirus after challenge. There were clear differences in the relative contribution of rotavirus-specific B-cell responses in protection against shedding following EDIM or RRV immunization. Protection against EDIM challenge following EDIM immunization was associated with high levels of virus-specific IgA both produced by LPL and detected at the intestinal mucosal surface at the time of challenge. These findings are consistent with those of a number of previous investigators (5, 15–18, 24). In addition, unlike the case for mice previously immunized with nonmurine rotaviruses, there was not an increase in the ratio of virus-specific IgA to total IgA within 6 days of challenge. This latter finding is consistent with the hypothesis that mice previously immunized with EDIM not only were protected against shedding but also were not reinfected with EDIM following challenge.
The mechanisms by which mice immunized with nonmurine rotaviruses were protected against challenge remain unclear. Reduced levels of shedding following immunization occurred within 2 days of challenge. It is unlikely that virus-specific B cells were responsible for protection against challenge. First, in mice immunized with low-dose RRV, virus-specific IgA was neither produced by LPL nor detected at the intestinal mucosal surface at the time of challenge. Second, in mice immunized with high- or low-dose RRV, the burst of IgA production by LPL did not occur within 4 days of challenge. It is also unlikely that virus-specific cytotoxic T lymphocytes (CTLs) were responsible for this early reduction of viral shedding. Virus-specific effector CTLs in gut-associated lymphoid tissue are not long lived following inoculation with RRV (20), virus-specific effector CTLs derived from CTL precursors are unlikely to be generated within 3 days of reinfection (10, 20), and mice deficient in the capacity to make virus-specific CTLs (mice lacking the gene expressing β2-microglobulin) are protected against challenge (6, 18). In addition, Franco and colleagues (7) similarly found reduced quantities of rotavirus shedding within 1 to 2 days of challenge in previously immunized antibody-deficient, CD8-depleted mice, further suggesting the lack of a role for CTLs in this early partial protection. One possibility is that reduced shedding is due to antiviral cytokines which are produced either earlier or in greater quantities after reinfection than after primary infection (11, 23). The types, levels, and kinetics of cytokines generated in gut-associated lymphoid tissue during rotavirus infection require further study.
Similar to previous studies (3, 31), we found that virus-specific IgG was produced by LPL after oral inoculation of experimental animals with rotavirus. Greater ratios of virus-specific to total IgG than of virus-specific to total IgA were produced by LPL after oral inoculation with murine and nonmurine rotavirus strains. However, despite the commitment by small intestinal lymphocytes to produce IgG, the role of IgG in protection against rotavirus challenge remains unclear. For example, high quantities of virus-specific IgG were produced by LPL 16 weeks after immunization with WC3 in the absence of protection against challenge. Detection of virus-specific IgG at the intestinal surface of mice immunized with EDIM or high-dose RRV may simply reflect greater quantities of IgG produced by LPL. Alternatively, animals immunized with EDIM or high-dose RRV had greater quantities of virus-specific IgG in serum (data not shown); therefore, detection of IgG at the mucosal surface may represent transudation from serum.
Our findings that virus-specific IgA effector responses were both higher titered and longer lived after immunization with high-dose RRV compared with low-dose RRV or WC3 are consistent with previous studies (5). These differences may be due to relative differences in the capacity of these viruses to replicate in small intestinal epithelial cell (25, 26).
We found that mice immunized orally with a nonmurine rotavirus (RRV) shed reduced quantities of rotavirus (but were not completely protected against shedding) following challenge with a murine rotavirus strain. Partial protection against shedding occurred in the absence of virus-specific IgA production by LPL or detection of virus-specific IgA at the intestinal mucosal surface. Similarly, infants and young children immunized orally with nonhuman rotaviruses are protected against relatively severe disease (but not all disease) following natural challenge with human rotaviruses (1, 4). The contribution of effector arms of the immune response other than B cells (possibly antiviral cytokines) may in part explain the inability to correlate virus-specific antibody responses in either serum or feces with protection against challenge in infant vaccine studies (1, 29).
ACKNOWLEDGMENTS
We thank Kurt Brown, Jeff Brubaker, Andrew Caton, and John Cebra for helpful discussions and comments. We also thank Daniel Sloane, Tashveen Kaur, and Victoria Saakyan for technical assistance.
This work was supported in part by R01 grant AI-26251 from the National Institutes of Health to P.A.O.
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