JOURNAL OF VIROLOGY, Oct. 2002, p. 10044–10049 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.19.10044–10049.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 19
Immune Responses to the Major Capsid Protein during Parvovirus Infection of Rats Lisa J. Ball-Goodrich,1* Frank X. Paturzo,1 Elizabeth A. Johnson,1 Krista Steger,2 and Robert O. Jacoby1 Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8016,1 and Forge 4ward, Inc., Somerville, New Jersey 088762 Received 16 April 2002/Accepted 27 June 2002
Rat virus (RV) is a common parvovirus of laboratory rodents which can disrupt rat-based research. Prenatal or perinatal infection can be pathogenic or lead to persistent infection, whereas infection of adult rats is typically self-limiting. Effects on the host immune system have been documented during RV infection, but little is known about immune responses necessary for viral clearance. Our studies were conducted to identify humoral and cellular responses to the predominant capsid protein, VP2, during experimental infection of adult rats. We observed VP2-specific proliferation, gamma interferon production, and an immunoglobulin G2a humoral response that is maintained for at least 35 days following RV infection. These results strongly suggest the induction of virus-specific Th1-mediated immunity. Parvoviruses are nonenveloped viruses with a singlestranded genome of approximately 5 kb. Viral replication is dependent on many cellular functions, particularly those expressed during S phase of the cell cycle (7). Rodent parvoviruses encode two classes of proteins: nonstructural (NS) proteins and structural or capsid proteins (VP). NS proteins are involved in viral replication, transcription, and cytotoxicity. VP1 comprises 15% of the icosahedral virion, and its coding sequence contains all of VP2 plus 140 additional N-terminal amino acids. VP2 is the predominant capsid protein, accounting for approximately 85% of the virion. VP3, a cleavage product of VP2, is present in small, varying amounts in DNAcontaining virions. A recent national survey found that parvovirus infections are highly prevalent among laboratory rats and mice (17). Infection with some of these agents can be lethal in fetal or perinatal animals, probably due to high numbers of mitotically active cells that serve as targets for cytolytic viral replication. Larger impacts on biomedical research result from clinically silent infections in infant or adult rodents in which biological processes and immune responses are altered. Specifically, infections with murine parvoviruses such as mouse minute virus and mouse parvovirus are known to inhibit several T-cell effector functions in vitro (2, 8); suppress antigen-induced proliferation of specific cloned T cells (18); augment the rate of tumor, allogeneic, and syngeneic graft rejection; and modulate other T-cell effector functions (19, 21). In addition, infection with rat virus (RV), the prototypic parvovirus of rats, can decrease lymphocyte viability and suppress proliferative responses to alloantigens (4), diminish responses of mixed lymphocyte culture and cytolytic T cells from peripheral and mesenteric lymph nodes (20), alter humoral responses to ovalbumin (5),
* Corresponding author. Mailing address: Section of Comparative Medicine, Yale University School of Medicine, P.O. Box 208016, New Haven, CT 06520-8016. Phone: (203) 737-4196. Fax: (203) 785-7499. E-mail:
[email protected].
and provoke autoimmune diabetes in the diabetes-resistant strain of Biobreeding/Worcester (BB/WOR) rats (3, 5, 13). Despite the impact of these agents on host immunity, little is known about immune responses to rodent parvoviruses. It is known that rats develop antiviral antibodies and perivascular mononuclear cell infiltrates in infected tissues (10, 14). Virusspecific antibodies have been shown to prevent establishment of RV infection but not to prevent or clear previously established infections. Administration of immune sera from RVinfected convalescent rats before or at the time of virus inoculation protected juvenile athymic rats from infection (9). Additionally, transfer of colostral or serum antibodies before or at the time of infection protected infant rats from developing RV infection. In contrast, administration of immune sera to infant rats 1 day after virus inoculation failed to prevent RV infection (11). Furthermore, rats inoculated at 6 days of age develop high anti-RV antibody titers, and yet virus is able to persist (15). Therefore, antiviral antibodies are not sufficient to clear established RV infection. Evidence from previous in vivo studies in our laboratory indicates the importance of T cells in viral clearance. RV infection of adult athymic rats (rnu/rnu) results in the development of a persistent viral infection not observed in immunocompetent adults (9). Additional studies suggest that innate resistance plays a role in survival of acute infection. RV infection of either fetal or neonatal rats, in which innate immunity is immature, induces high mortality, not observed upon infection of either 6-day-old or adult rats (12). Acquired immunity, which is not functionally mature in rats until at least 4 weeks after birth (23), appears to be critical for viral clearance in animals that survive acute infection. This is substantiated in vivo by development of persistent infection in rats inoculated at 6 days of age (15, 16) in contrast to clearance of infection by adult rats (9). We have begun to examine cellular and humoral immune responses during RV infection of adult rats to help clarify the role of host immunity during viral clearance. Specifically, we used proliferation assays, including analysis of cytokine pro-
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FIG. 1. SC proliferation in response to ConA and multiple concentrations of RV VP2. At PID 35, SC from six infected and three uninfected rats were individually purified. Triplicate cultures of 6 ⫻ 105 SC from each rat were incubated in RPMI medium with 10% fetal calf serum (negative control), medium containing ConA (2.5 g/ml; positive control), or increasing concentrations of bacterium-expressed RV VP2 (0.2, 2, 10, and 20 g/ml). In this and the other figures, the SI was calculated for individual rats by dividing the mean counts incorporated in triplicate ConA- or VP2-treated cultures by the mean counts incorporated in the triplicate negative-control cultures. Subsequently, the mean SI for each group (infected and uninfected), SD, and SE were calculated by using Microsoft Excel. The figure shows the mean SIs plus SEs.
duction, to document cellular responses to the major capsid protein, VP2, during acute and clearance phases of RV infection. In addition, isotype profiles of VP2-specific humoral responses were determined. Our data show that a VP2-specific Th1-type response, as indicated by proliferation, gamma interferon (IFN-␥) secretion, and immunoglobulin G2a (IgG2a) antibody production, was generated during clearance of RV infection. Optimization of proliferation assays. Eight-week-old specific-pathogen-free inbred Lewis rats (LEW/SsNHsd; Rattus norvegicus) were obtained from Harlan Sprague-Dawley (Indianapolis, Ind.), housed and husbanded in microisolette cages, and fed autoclaved chow and sterile water ad libitum under standards that met or exceeded those recommended in the Guide for the Care and Use of Laboratory Animals (21a). All experiments were approved by the Institutional Animal Care and Use Committee. Rats were inoculated oronasally by placing 4 ⫻ 104 50% tissue culture infective doses of a suspension of the UMass strain of RV (40 l) on the external nares. RV-UMass had been plaque purified, and stocks were prepared as previously described (13). Initial proliferation assays, using rat splenocytes (SC), were performed to optimize parameters used to test responses to mitogenic stimulation. Single-cell suspensions were generated by homogenization of spleens in a glass tissue grinder prior to purification over a Ficoll-Hypaque gradient (20). Cells were collected, washed in medium, incubated in hypotonic lysis buffer to remove remaining red blood cells, and counted. Triplicate wells were incubated in RPMI medium with 10% fetal calf serum (negative control) or medium containing concanavalin A (ConA) (2.5 g/ml; positive control). At 2, 3, 4, and 5 days after plating, 1 Ci of [3H]thymidine (87 Ci/mmol) was added per well. Approximately 20 h after addition of label,
cells were harvested onto glass fiber filters, washed, and counted in a liquid scintillation counter to determine the number of incorporated counts. A stimulation index (SI) was calculated for each individual rat and for each group (infected or uninfected). After evaluation of various cell concentrations and times of incubation with mitogen, optimal proliferation was seen after incubation of 6 ⫻ 105 rat SC per well for 4 days with ConA prior to addition of [3H]thymidine (data not shown). To examine virus-specific proliferative responses, RV VP2 was expressed with the pET expression system (Novagen, Madison, Wis.) and purified, after solubilization in 8 M urea, by metal chelation chromatography (15). Because urea was cytotoxic to rat SC, purified VP2 protein was dialyzed to a final urea concentration of 0.5 M in phosphate-buffered saline for use in all subsequent assays. Peak VP2-specific proliferation for SC from infected rats occurred after 6 days of incubation with dialyzed VP2 (data not shown). To optimize the VP2 concentration used for proliferation assays, SC from infected and uninfected rats were incubated for 6 days with four VP2 concentrations (Fig. 1). Triplicate cultures were incubated in medium alone (negative control), medium containing ConA (2.5 g/ml; positive control), or increasing concentrations of dialyzed RV VP2 (0.2, 2, 10, and 20 g/ml). SC from uninfected rats had a mean SI of 1.7 or less at all VP2 concentrations tested. Significant proliferation (SI of 25 to 30) from SC of an infected rat was seen in response to VP2 concentrations of 2 g/ml or greater. The proliferative response decreased to an SI of less than 10 when the concentration of VP2 was 0.2 g/ml. In all subsequent experiments, VP2 was used at a concentration of 2.5 g/ml. Induction of antigen-specific proliferation during RV infec-
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tion. Twenty-five 8-week-old Lewis rats were given 4 ⫻ 104 50% tissue culture infective doses of RV-UMass oronasally, and an additional 15 rats were held as uninfected controls. Proliferative responses were examined at 7-day intervals (postinfection days [PID] 7, 14, 21, 28, and 35) for five infected and three control rats. SC from individual rats were purified; counted; plated in either triplicate or quadruplicate; and incubated in culture medium alone (negative control), with ConA (positive control), with bacterially expressed -galactosidase (-Gal) protein (negative antigen control), or with dialyzed VP2 (2.5 g/ml). Negative controls and ConA-treated cultures were labeled following 4 days of incubation, while negative controls and -Gal-treated and VP2-treated cultures were labeled following 6 days of incubation. Mean SIs including standard errors (SEs) are shown in Fig. 2A for SC from uninfected, control rats and in Fig. 2B for SC from RV-infected rats. All cultures proliferated in response to ConA, and levels of proliferation were not statistically significant between infected and uninfected groups (Fig. 2, black bars). This indicates that in vivo infection with RV did not hamper the proliferative responses of SC to ConA. No cultures proliferated after incubation with -Gal at any time point (Fig. 2, white bars). Therefore, RV infection did not stimulate a nonspecific proliferative response to extraneous antigens, nor was there cross-reactive proliferation due to possible residual bacterial proteins. VP2specific proliferation was first evident in SC from infected rats at PID 14, increased at PID 21, and was maintained through PID 35 (Fig. 2B, shaded bars). By Student t test analysis (two tailed; unequal variance), proliferation after incubation with VP2 was statistically significant for SC from infected rats compared to uninfected controls for PID 14 (P ⬍ 0.003), PID 21 (P ⬍ 0.003), PID 28 (P ⬍ 0.02), and PID 35 (P ⬍ 0.001). The significance value on PID 28 was lower due to larger variations in proliferative responses of infected rats (SI ⫽ 28.4 ⫾ 7.4). Proliferative responses observed in a repeat experiment mirrored those presented in Fig. 2. Viral infection of cultured SC. During in vivo infection, RV replication occurs in the spleen (9). It is possible, therefore, that viral replication in SC could alter proliferation results through cytolysis of proliferating SC or incorporation of [3H]thymidine in the viral genome. To assess this possibility, we measured the percentage of virus-infected SC at the time of purification and determined whether levels of infection changed during in vitro proliferation assays. Purified SC, before and after incubation, were deposited on a spot slide, fixed, and hybridized in situ with a random-primed RV DNA probe (1). Random fields were photographed, and a minimum of 300 cells were analyzed per rat per condition. Levels of infection in SC harvested at PID 7 were determined at necropsy and after 4 days of incubation in medium with or without ConA, and mean percent infected cells plus standard deviation (SD) were calculated for each group (Fig. 3). No SC from uninfected rats were positive. In SC from infected rats, there was low-level infection at necropsy (0.8%), which increased to 2.8% after 4 days of incubation with medium alone and to 4.3% after 4 days of incubation with ConA. Nonspecific isotope incorporation after 4 days of incubation in medium alone was comparable for SC from infected and uninfected rats (data not shown), confirming that virus replication did not result in increased incorporation of [3H]thymidine. Mitogenic stimulation causes cell
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proliferation, thus providing a substrate for increased virus infection, which could result in cytolysis. This, in turn, could result in diminished proliferative responses. However, the SI after incubation with ConA was not significantly different between SC from infected rats and those from uninfected rats. To evaluate the effects of viral infection on antigen-specific proliferation, the percentage of RV-infected cells was determined for SC harvested at PID 14, the time at which virusspecific proliferation was first evident. The percentage of RVinfected cells was determined at necropsy, after 4 days with or without ConA, and after 6 days in culture with RV VP2 (Fig. 3). At PID 14, the level of infection at time of necropsy was lower (0.13%) than on PID 7 and two of five rats had no infected cells. Only one rat had a low level of infected SC (0.4%) after 4 days in culture with medium alone, but all cultures had low-level infection after incubation with ConA. After 6 days in culture with VP2, four of five rats were RV DNA positive with a mean percentage of 4.11% infected cells. However, SIs after VP2 stimulation were comparable whether SC were RV DNA positive or not, indicating that proliferative responses were not affected by RV infection. At PID 21, 28, and 35, SC from all rats were negative at necropsy, and SC from one PID 21 rat were positive after incubation with VP2 (data not shown). This concurs with previous data indicating the decline of virus-positive cells as early as PID 10 in adult rats and complete clearance of virus-positive cells by PID 35. Overall, these data show that low-level RV infection of SC does not affect either [3H]thymidine incorporation or proliferation of SC in response to VP2. Cytokine analysis of proliferating SC. We examined cytokine production by SC stimulated with VP2 in order to characterize the type (Th1 versus Th2) of immune response stimulated by RV infection. Prior to the addition of [3H]thymidine on day 7, cultures were frozen at ⫺80°C for cytokine analysis. Plates were thawed, cells were pelleted, and culture supernatants were removed and analyzed with Pharmingen (San Diego, Calif.) OptEIA rat IFN-␥ and interleukin 4 (IL-4) enzyme-linked immunosorbent assay (ELISA) kits. No IL-4 production was detected at any time point. No IFN-␥ was detected in SC cultures incubated with -Gal (data not shown) or SC cultures from uninfected rats incubated with VP2 (Fig. 4). High levels of IFN-␥ were evident, however, in VP2-stimulated SC cultures from RV-infected rats harvested on PID 14, 21, 28, and 35 (Fig. 4). IFN-␥ production peaked at PID 14, a time point when proliferation is first evident at a low level. IFN-␥ levels were reduced but significant at PID 21, 28, and 35. Replicate studies duplicated this pattern of cytokine production. Reduction in cytokine production at later time points was not indicative of decreased proliferation, as SIs remained high at these time points. These data suggest that a limited number of VP2-reactive T cells produce large amounts of IFN-␥ early in infection, coincident with a rapid reduction in the number of RV-positive cells in tissues (R. Jacoby, F. Paturzo, E. Johnson, and L. Ball-Goodrich, unpublished data). Production of high levels of IFN-␥ and no detectable IL-4 is consistent with induction of a Th1-mediated immune response. Seroconversion to RV VP2. All infected rats were seropositive by PID 7 by an immunofluorescence assay (22) at a 1:10 dilution, whereas sera from control rats were seronegative at all time points. Titer and isotype of anti-RV VP2 antibodies
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FIG. 2. Proliferation of SC from uninfected or infected rats. Mean SIs plus SEs at 7-day intervals postinfection are shown for SC from three uninfected rats (A) or five infected rats (B) incubated with ConA, -Gal, or VP2.
were determined for PID 14 through 35 by ELISA with bacterium-expressed RV VP2 as described previously (15). For the initial screen, rat serum was diluted 1:50 and goat anti-rat IgG (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) and mouse anti-rat IgM (Serotec, Raleigh, N.C.) were used at
1:10,000 and 1:2,000 dilutions, respectively. For isotype determination, mouse anti-rat IgG1 (Serotec) and mouse anti-rat IgG2a (Serotec) isotype-specific monoclonal antibodies were used at the recommended dilution (1:2,000). At PID 7, IgM antibodies against VP2 were detected in three of five infected
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FIG. 3. RV infection of SC determined by in situ hybridization. Percent infected SC were determined for individual rats. Mean percent infected cells plus SD were calculated for all groups (PID 7 includes time of necropsy, 4 days in medium, and 4 days in medium with ConA; PID 14 includes the above plus 6 days in medium with VP2).
rats. Two of these IgM-positive rats also had low levels of anti-VP2 IgG antibodies (data not shown). By PID 14, levels of anti-VP2 IgM antibodies peaked in infected rats (Table 1). High levels of anti-VP2 IgG2a antibodies were detected in infected rats at all time points after PID 7. IgG1 antibodies peaked at PID 14 and decreased at subsequent time points. As
expected, initial antibody responses were of the IgM isotype and were subsequently replaced with IgG production. Predominance of the IgG2a isotype further confirms the induction of a Th1-mediated response. Summary. Our earlier studies with athymic and euthymic rats indicated that acquired immunity is essential for RV clear-
FIG. 4. Cytokine production during proliferation of rat SC. Cytokine levels for SC from individual rats (five infected and three uninfected) were determined with a standard curve, and mean cytokine levels (plus SEs) were calculated for each group (medium alone or VP2) per time point.
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TABLE 1. Anti-VP2 antibody titers during infection PID
14 21 28 35
Titer of antibody
a
IgM
IgG
IgG1
IgG2a
6,250 ⫾ 2,236 2,250 ⫾ 2,236 300 ⫾ 542 0
6,250 ⫾ 0 6,250 ⫾ 0 6,250 ⫾ 0 6,250 ⫾ 0
1,050 ⫾ 447 850 ⫾ 548 500 ⫾ 500 500 ⫾ 500
6,250 ⫾ 0 6,250 ⫾ 0 5,250 ⫾ 2,236 6,250 ⫾ 0
a Results are shown as the mean inverses of the last dilution that was ELISA positive ⫾ the SD. Secondary antibodies were horseradish peroxidase linked; the substrate was tetramethylbenzidine peroxidase (Kirkegaard & Perry Laboratories).
ance. This work documents the induction of a Th1-type immune response to VP2 in adult rats following infection with RV. Capsid-specific IgM antibodies were noted on PID 7 followed by high-level IgG responses, of predominantly IgG2a isotype, on PID 14 through PID 35. Stimulation of specific SC proliferation and IFN-␥ production after incubation with RV VP2 was detected as early as PID 14, and it was maintained throughout the time course. Recent experiments with adult rats showed that numbers of infected cells and viral titers in tissues peaked at PID 8, showed a marked decrease between PID 10 and 14, and were cleared by PID 35 (Jacoby et al., unpublished). Our analysis of viral infection of purified SC further confirmed this time course. Thus, kinetics for RV clearance correlate with the induction of virus-specific immune responses in the host: a high IgG response by PID 14 as well as induction of a VP2-specific proliferative response that includes high production of IFN-␥. Similar results, including a Th1-type proliferative response to VP1 and VP2 with production of IFN-␥, were obtained in studies of immune responses during human parvovirus B19 infection (6, 24). Future studies of host immunity during RV infection will focus on delineating the role of T-cell subpopulations (CD4⫹ and CD8⫹) in establishing Th1 immunity and viral clearance. The results from our studies have direct implications for researchers using in vivo or ex vivo rat models of research. Although RV infection of adult rats is clinically silent, we observed induction of a strong Th1-mediated immune response during infection. While the established immune response is virus specific, the Th1 environment, represented by high IFN-␥ levels, may affect immune responses to other antigens. For example, researchers using the rat model for diabetes (DR-BB/WOR) found that RV infection selectively activated Th1-like CD4⫹ T cells and down-regulated Th2-like CD4⫹ T cells (5). In addition, it affected the primary humoral response to ovalbumin by inducing a strong bystander Th1biased activation (5). Therefore, researchers using rat models for infectious disease or immune studies must be aware of possible impacts of RV infection. We thank Kari Nizzardo for expert technical assistance. This work was supported by a National Institutes of Health grant to R.O.J. (RO1-RR11740).
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