Section of Infectious Diseases, The Childrens Hospital of Philadelphia, Abramson Research Building, Room 1205A, 3516 Civic Center Blvd, Philadelphia, PA 19104, USA1
The University of Pennsylvania School of Medicine2 and The Wistar Institute of Anatomy and Biology3, Philadelphia, PA 19104, USA
Author for correspondence: Charlotte Moser. Fax +1 215 590 2025. e-mail moser{at}email.chop.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
We developed a murine model for rotavirus infection that distinguishes the relative protective capacity of virus-specific IgA present at the intestinal mucosal surface at the time of challenge from virus-specific IgA derived from memory B cells after challenge (Moser et al., 1998 ). We found that mice immunized with murine rotavirus strain EDIM or a high dose (1·9x107 p.f.u.) of simian strain RRV produced high levels of virus-specific IgA up to 16 weeks after immunization and were completely protected against shedding following EDIM challenge. In contrast, mice immunized with a lower dose of RRV (1·9x106 p.f.u.) did not produce virus-specific IgA at the intestinal mucosal surface, but were still partially protected after EDIM challenge. Protection against shedding in mice immunized with a lower dose of RRV was mediated by virus-specific IgA. Virus-specific IgA was most likely produced by effector cells generated from virus-specific memory B cells and not from effector cells that continued to produce IgA after immunization. The magnitude of virus-specific IgA produced after challenge by mice immunized 16 weeks previously with a low dose of RRV was greater than that produced by unimmunized mice or by mice immunized 6 weeks previously.
These studies were performed in order to understand better the delay in development of virus-specific IgA memory B cell responses in GALT following infection of mice with RRV. We used a method, similar to that of Slifka & Ahmed (1996) , that employed in vitro stimulation and antibody-secreting cell (ASC) frequency analyses to determine the site and presence of rotavirus-specific memory B cells in GALT after primary immunization. At various intervals after immunization, mice were sacrificed and single-cell suspensions were prepared from various GALT and non-enteric lymphoid tissues. Cells were either tested immediately for the presence of rotavirus-specific IgA-secreting cells (primary effector B cells) or incubated in vitro with or without rotavirus. After 5 days in culture, viable cells were tested by ELISpot assay to determine the number of rotavirus-specific IgA-ASC derived from memory B cells (secondary effector B cells).
Adult, 5- to 6-week-old, female BALB/c mice, obtained from Taconic Breeding Laboratories (Germantown, NY, USA), were inoculated orally by proximal oesophageal intubation with 1·2x106 p.f.u. of rhesus rotavirus strain RRV in a volume of 200 µl. RRV was originally obtained from N. Schmidt (Berkeley, CA, USA) and was grown and titrated in foetal green monkey kidney cells (MA-104) as described previously (Offit et al., 1983 ). At various intervals after inoculation, single-cell suspensions were prepared from tissues including Peyers patches (PP), mesenteric lymph nodes (MLN), spleen, lamina propria (LP) and bone marrow (BM) as described below. PP, MLN and spleens were removed and disrupted using two L-shaped 21-guage needles. LP cells were isolated enzymatically using a method described previously (Offit et al., 1991
). In order to obtain BM, femurs were removed from mice and flushed with RPMI CM [RPMI 1640 (Mediatech) containing 10% FBS (BioWhittaker), 1% HEPES (Gibco BRL), 1% glutamine (BioWhittaker), 20 U/ml penicillin, 20 µg/ml streptomycin, 50 µg/ml gentamicin (all antibiotics from Gibco BRL) and 0·003% 2-mercaptoethanol (Sigma)] using a 26-gauge needle. Clumps were drawn into a 1 ml syringe to separate cells. In the case of all tissues, cell suspensions were washed with RPMI CM and counted by trypan blue exclusion.
Initial studies were performed to determine the presence of primary rotavirus-specific effector B cells by subjecting single-cell suspensions to assay by ELISpot, performed as described previously (Khoury et al., 1994 ). Samples were considered to be positive if there were at least five spots per 106 cells in a well and at least 2-fold more spots in wells coated with purified RRV than in wells coated with BSA. Rotavirus-specific IgA effector B cells were not detected in the PP, MLN, spleen, LP or BM of unimmunized mice or mice immunized 56 weeks or 16 weeks after immunization. However, at 3 weeks after immunization, 55 rotavirus-specific IgA-ASC were detected in the MLN and 180 rotavirus-specific IgA-ASC were detected in the LP.
In order to determine whether secondary rotavirus-specific effector B cells were present, either 48 or 1418 weeks after immunization, groups of three to ten immunized or unimmunized mice were sacrificed and single-cell suspensions were prepared as described above. Cells (1x106) were placed into individual wells of 96-well round-bottomed plates with 100 µl of either medium alone or medium containing 80 ng caesium chloride-purified RRV. Quantities of purified rotavirus were determined by spectrophotometric analysis. Plates were incubated at 37 °C in 5% CO2 for 3 or 5 days prior to assay by ELISpot. In the ELISpot assay, 5x104 viable cells from each lymphoid culture were tested in duplicate wells coated with either purified rotavirus, goat anti-mouse IgA (to determine the total number of IgA-secreting cells as a measure of cell viability) or 1% BSA. By normalizing viable cells recovered following stimulation in vitro, equal numbers of cells potentially capable of secreting antibodies could be assayed by ELISpot and the results compared. Samples were considered to be positive if there were (i) at least five spots per 106 cells in a well, (ii) at least 2-fold more spots in wells coated with purified RRV than in wells coated with BSA and (iii) at least 2-fold more spots in wells containing virus-stimulated compared with unstimulated cells. Data were adjusted by subtracting the mean number of spots in BSA-coated wells from that found in virus-coated wells. These numbers were adjusted further by subtracting numbers calculated for unstimulated cells from the same tissue. Each experiment was performed three to five times and data were log10-transformed and subjected to statistical analysis using two-sample t-tests. Whereas quantities of rotavirus-specific IgA-ASC derived from secondary B cells decreased in PP and spleen between 48 and 1418 weeks after immunization, cell numbers increased in the LP (Table 1). Mechanisms that might account for the development of increased frequencies of virus-specific memory B cells in the LP between 6 and 16 weeks after immunization include the following. Firstly, a decrease in virus-specific memory B cells in the PP and spleen was associated with an increase in virus-specific memory B cells in the LP. These changes may represent a migration of memory B cells from PP and spleen to the LP. Secondly, virus-specific T cells, capable of stimulating virus-specific memory B cells, might migrate slowly to and accumulate in the LP. Thirdly, virus-specific memory B or T cells, already residing in the LP, might increase in frequency over time. Finally, virus-containing antigen-presenting cells, capable of generating or stimulating virus-specific memory B or T cells or both, might migrate to the LP over time. Because the numbers of B cells, T cells and antigen-presenting cells were preserved from each tissue, and not normalized to one specific cell type, none of these mechanisms can be ruled out. Future experiments will help to sort out the likelihood of each of these proposed mechanisms.
|
In order to determine whether virus-specific ASC were generated in vitro, cells were harvested 3 and 5 days after stimulation in vitro. Rotavirus-specific IgA effector B cells derived from virus-specific memory B cells were not detected 3 or 5 days after stimulation of cells from unimmunized mice or 3 days after stimulation of cells from immunized mice. However, in mice immunized with RRV 8 weeks previously, stimulation of PP cells in vitro for 5 days generated 190 rotavirus-specific IgA-ASC and stimulation of splenic cells in vitro generated 80 IgA-ASC. No rotavirus-specific IgG-ASC were detected. Several observations support our detection of in vitro-activated, secondary, rotavirus-specific B cells. Firstly, rotavirus-specific effector B cells were detected 5 but not 3 days after culture with RRV in vitro. Secondly, virus-specific B cells were detected in immunized but not unimmunized mice. Thirdly, primary virus-specific effector B cells were not detected in GALT 16 weeks after immunization when cells were tested immediately at the time of harvest. These findings are also supported by observations from published studies, that memory cells are activated and differentiate to effector cells between 3 and 5 days after stimulation (Arpin et al., 1995 , 1997
).
Virus-specific memory B cells were detected about 4 months after a single oral inoculation of mice with RRV. However, RRV is not detected as infectious virus in GALT or non-enteric tissues beyond 5 days after inoculation (Offit et al., 1991 ). As has been shown for a number of antigens (Gray & Skarvall, 1988
; reviewed in Sprent, 1994
), maintenance of long-term memory in PP and spleen might be mediated by the presence of rotavirus antigens in follicular dendritic cells. Since follicular dendritic cells are not found in the LP, memory B cells in the LP would more likely be derived from memory cells migrating from other sites, such as the PP or spleen.
We found previously that, after a single inoculation of mice with RRV, protection against challenge correlated with development of virus-specific IgA in LP fragment cultures after challenge; virus-specific IgA responses in the LP following challenge were greater 16 weeks compared with 6 weeks after immunization (Moser et al., 1998 ). In these studies, we showed that virus-specific memory B cells were present in the PP and spleen early after immunization, but decreased in frequency several months after immunization. In contrast, virus-specific memory B cells were detected in the LP late, but not early, after immunization. These findings support the hypothesis that protection against shedding is dependent upon the presence of memory B cells in the small intestinal LP. Activation of memory B cells present in the PP or spleen alone is not adequate to ensure protection against mucosal challenge. Strategies of immunization that hasten the onset and enhance the longevity of local virus-specific memory B cells are likely to be important in protection against mucosal pathogens.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Arpin, C., Banchereau, J. & Liu, Y. J. (1997). Memory B cells are biased towards terminal differentiation: a strategy that may prevent repertoire freezing. Journal of Experimental Medicine 186, 931-940.
Benedetti, R., Lev, P., Massouh, E. & Flo, J. (1998). Long-term antibodies after an oral immunization with cholera toxin are synthesized in the bone marrow and may play a role in the regulation of memory B-cell maintenance at systemic and mucosal sites. Research in Immunology 149, 107-118.[Medline]
Coulson, B. S., Grimwood, K., Hudson, I. L., Barnes, G. L. & Bishop, R. F. (1992). Role of coproantibody in clinical protection of children during reinfection with rotavirus. Journal of Clinical Microbiology 30, 1678-1684.[Abstract]
Feng, N., Burns, J. W., Bracy, L. & Greenberg, H. B. (1994). Comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus. Journal of Virology 68, 7766-7773.[Abstract]
Franco, M. A. & Greenberg, H. B. (1995). Role of B cells and cytotoxic T lymphocytes in clearance of and immunity to rotavirus infection in mice. Journal of Virology 69, 7800-7806.[Abstract]
Gray, D. & Skarvall, H. (1988). B-cell memory is short-lived in the absence of antigen. Nature 336, 70-73.[Medline]
Khoury, C. A., Brown, K. A., Kim, J. E. & Offit, P. A. (1994). Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture. Clinical and Diagnostic Laboratory Immunology 1, 722-728.[Abstract]
McNeal, M. M. & Ward, R. L. (1995). Long-term production of rotavirus antibody and protection against reinfection following a single infection of neonatal mice with murine rotavirus. Virology 211, 474-480.[Medline]
McNeal, M. M., Barone, K. S., Rae, M. N. & Ward, R. L. (1995). Effector functions of antibody and CD8+ cells in resolution of rotavirus infection and protection against reinfection in mice. Virology 214, 387-397.[Medline]
Matson, D. O., ORyan, M. L., Herrera, I., Pickering, L. K. & Estes, M. K. (1993). Fecal antibody responses to symptomatic and asymptomatic rotavirus infections. Journal of Infectious Diseases 167, 577-583.[Medline]
Moser, C. A., Cookinham, S., Coffin, S. E., Clark, H. F. & Offit, P. A. (1998). Relative importance of rotavirus-specific effector and memory B cells in protection against challenge. Journal of Virology 72, 1108-1114.
Offit, P. A. (1996). Host factors associated with protection against rotavirus disease: the skies are clearing. Journal of Infectious Diseases 174, S59-S64.[Medline]
Offit, P. A., Clark, H. F., Stroop, W. G., Twist, E. M. & Plotkin, S. A. (1983). The cultivation of human rotavirus, strain Wa, to high titer in cell culture and characterization of the viral structural polypeptides. Journal of Virological Methods 7, 29-40.[Medline]
Offit, P. A., Cunningham, S. L. & Dudzik, K. I. (1991). Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection. Journal of Virology 65, 1318-1324.[Medline]
Ridderstad, A. & Tarlinton, D. M. (1997). B cell memory in xid mice is long-lived despite reduced memory B cell frequency. Scandinavian Journal of Immunology 45, 655-659.[Medline]
Sheridan, J. F., Eydelloth, R. S., Voderfecht, S. L. & Aurelian, L. (1983). Virus-specific immunity in neonatal and adult mouse rotavirus infection. Infection and Immunity 39, 917-927.[Medline]
Slifka, M. K. & Ahmed, R. (1996). Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay. Journal of Immunological Methods 199, 37-46.[Medline]
Sprent, J. (1994). T and B memory cells. Cell 76, 315-322.[Medline]
Starkey, W. G., Collins, J., Wallis, T. S., Clarke, G. J., Spencer, A. J., Haddon, S. J., Osborne, M. P., Candy, D. C. A. & Stephen, J. (1986). Kinetics, tissue specificity and pathological changes in murine rotavirus infection of mice. Journal of General Virology 67, 2625-2634.[Abstract]
Ward, R. L., McNeal, M. M. & Sheridan, J. F. (1992). Evidence that active protection following oral immunization of mice with live rotavirus is not dependent on neutralizing antibody. Virology 188, 57-66.[Medline]
Williams, M. B., Rosé, J. R., Rott, L. S., Franco, M. A., Greenberg, H. B. & Butcher, E. C. (1998). The memory B cell subset responsible for the secretory IgA response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, 4
7. Journal of Immunology 161, 4227-4235.
Received 13 March 2001;
accepted 8 June 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |