Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Virology 331 (2005) 387 – 395 www.elsevier.com/locate/yviro

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions Zhiyi Sha1, Sang-Moo Kang, Richard W. Compans* Department of Microbiology and Immunology, Emory University School of Medicine, 3001 Rollins Research Center, Atlanta, GA 30322, USA Received 22 June 2004; returned to author for revision 13 August 2004; accepted 8 October 2004 Available online 11 November 2004

Abstract Immunoglobulin (Ig) class switching can occur in the absence of ah+ or gy+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice. D 2004 Elsevier Inc. All rights reserved. Keywords: CD4+ T cells; Mucosal immunization; Mice

Introduction CD4+ T cells are believed to play a critical role in the regulation of immunoglobulin (Ig) class switching during an immune response (Bachmann and Zinkernagel, 1996; Oxenius et al., 1998; Parker, 1993). CD4+ T cells can help B cells through two distinct pathways: (1) cognate help for B cells by promoting B-cell activation and Ig isotype switching by direct T–B cell interaction; (2) noncognate or bystander help mediated by the cytokines secreted by the activated CD4+ T cells. It is well established that three cytokines, interleukin 4 (IL-4), gamma-interferon (IFN-g), and transforming growth factor beta (TGF-h), play prominent roles in regulating Ig class switching (Snapper and Mond, 1993). In vitro, IL-4 was found to stimulate the * Corresponding author. Fax: +1 404 727 8250. E-mail address: [email protected] (R.W. Compans). 1 Current address: Department of Neurology, UCLA, Los Angeles, CA 90095. 0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2004.10.014

production of IgG1 and IgE selectively (Coffman and Carty, 1986; Isakson et al., 1982); IFN-g can initiate Ig class switching to IgG2a and IgG3 (McIntyre et al., 1993; Rose et al., 1998; Snapper and Paul, 1987); and TGF-h can specifically enhance the production of IgG2b and IgA (McIntyre et al., 1993; Sonoda et al., 1989). These findings have also been confirmed by in vivo studies (Finkelman et al., 1988, 1990; Schurmans et al., 1990). The idea that Ig class switching is a CD4+ T celldependent process has been challenged recently by the finding that this process can occur in the absence of ah+ T cells (Szomolanyi-Tsuda and Welsh, 1998; SzomolanyiTsuda et al., 1998). Polyomavirus (PyV) infection of TCRh / or TCRhxy / mice, which lack functional ah+ T cells or ah+ and gy+ T cells, respectively, induced IgM and IgG antiviral antibodies; in contrast, noninfectious polyoma virus-like particles or purified viral proteins did not elicit Ig class switching (Szomolanyi-Tsuda and Welsh, 1996, 1998). Studies with vesicular stomatitis virus (VSV) in ah+ T cell-deficient mice have also shown that IgG

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responses could be induced by live VSV infection or recombinant vaccinia virus expressing the VSV glycoprotein, but not by immunization with formalin-inactivated virus (Bachmann et al., 1993, 1994, 1995; Maloy et al., 1998). Our previous study has shown that systemic immunization with inactivated influenza virus can also induce IgG responses in mice in the absence of CD4+ T cells (Sha and Compans, 2000). However, little is known about the ability of inactivated viruses to induce immune responses in serum and mucosal sites after mucosal immunization of immunodeficient mice in the absence of CD4+ T helper cells or ah+ T cells. In this study, we used formalin-inactivated influenza A/PR8/34 virus as an immunogen and investigated the induction of systemic and local mucosal immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice after intranasal immunization. In addition to evaluating IgG and IgA responses in serum and IgA responses in saliva and vaginal secretions in mice lacking CD4+T cells, we determined neutralizing activity in vitro and protection against challenge with a lethal dose of influenza virus.

Results Inactivated influenza virus induces CD4+ T cell-independent IgG and IgA responses after intranasal immunization We compared systemic antibody responses (IgM, IgG, and IgA) after mucosal immunization with inactivated PR8 virus in groups of CD4+ T cell-deficient mice and C57BL/6 immunocompetent mice (Fig. 1). Both groups were immunized with three doses of inactivated PR8 virus intranasally at days 0, 15, and 25. The magnitude of virusspecific IgG and IgA antibodies in sera after each immunization was determined by an ELISA assay. IgM responses, which are considered to be T cell-independent (Bachmann et al., 1993, 1994, 1995), were induced in both CD4+ T cell-deficient mice and C57BL/6. However, levels of IgM did not show significant increases after boost immunizations (ranging from 4.7 F 1.15 to 3.5 F 0.5 Ag/ml for CD4 / vs. 5.5 F 0.2 to 7.2 F 2.4 Ag/ml for wild type) and differences in IgM levels were not statistically significant between CD4+ T cell-deficient and

Fig. 1. Magnitude and isotype profiles of serum antibody responses to intranasal immunization with inactivated influenza virus in CD4+ T cell-deficient or immunocompetent mice. CD4+ T cell-deficient mice (16–20 weeks old) or C57BL/6-immunocompetent mice (10 weeks old) were immunized intranasally at day 0 and boosted at days 15 and 25 with 50 or 10 Ag/mouse of inactivated influenza virus, respectively. CD4/Con, unimmunized CD4+ T cell-deficient mice. CD4/50, CD4+ T cell-deficient mice immunized with 50 Ag of inactivated influenza virus. BL6/10, C57BL/6-immunocompetent mice immunized with 10 Ag of inactivated influenza virus, known to be sufficient for induction of isotype switching of antibodies in wild-type mice (Sha and Compans, 2000). Prime, samples were collected 15 days after the first immunization. Boost, samples were collected 10 days after the first boost. 2nd boost, samples were collected 10 days after the second boost. Statistical analysis was performed by the Microsoft Excel program and standard error bars are indicated for five mice in a group.

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wild-type mice (P N 0.05). IgG and IgA responses were also induced in both groups, indicating that antibody class switching from IgM to IgG and IgA occurred in CD4+ T cell-deficient mice after intranasal immunization with the inactivated virus. All four subtypes of IgG were observed in both groups. In contrast to IgM, the levels of IgG1, IgG2a, IgG2b, and IgA responses were significantly higher in C57BL/6 mice than in CD4+ T cell-deficient mice (87 F 8.3 vs. 34.7 F 21.5 Ag/ml for IgG1, 58 F 14.8 vs. 21.0 F 4.3 Ag/ml for IgG2a, 53 F 8.1 vs. 9.4 F 1.3 Ag/ml for IgG2b, 6.7F2.4 vs. 1.6 F 1.2 Ag/ml for IgA; all P b 0.05), suggesting that although CD4+ T cells are not essential for this process, they can enhance those responses significantly. In contrast, IgG3 levels were found to be comparable in both groups (Fig. 1). These data indicate that intranasal immunization with inactivated PR8 virus can induce Ig class switching to IgG and IgA in mice, and that these responses are independent of the presence of CD4+ T cells. In wild-type mice, intranasal immunization with 10 Ag of inactivated influenza virus antigen was sufficient to induce isotype switching of antibodies (Fig. 1). However,

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the IgG and IgA responses after intranasal immunization in CD4-deficient mice were found to be dependent upon the quantity of the immunogen administered, as determined by the magnitude of the IgG and IgA responses in sera (Figs. 1 and 2). Although similar levels of IgA were observed in groups of mice that received 10 and 50 Ag of inactivated PR8 virus, only the 50-Ag group of CD4deficient mice developed significant levels of all four subtypes of IgG antibody. The levels of IgG and IgA responses were also dependent upon the number of doses administered in CD4-deficient mice. The first boost immunization with 50 Ag of inactivated influenza virus induced low levels of IgG and IgA in CD4-deficient mice. However, the second boost increased the IgG and IgA responses significantly in the 50-Ag group, with antibody levels averaging 3-fold higher than those observed after the first boost. These findings are similar to the results of other studies showing that three immunizations are necessary to induce IgA responses in mucosal secretions (Di Tommaso et al., 1996). These data indicate that, in inducing IgG and IgA responses by intranasal immunization, CD4+ T cell-deficient mice are more dependent on

Fig. 2. Dose dependence of serum antibody responses to intranasal immunization with inactivated influenza virus in CD4+ T cell-deficient mice. CD4+ T celldeficient mice (16–20 weeks old) were immunized intranasally at day 0 and boosted at days 15 and 25 with the same dose using 1, 10, or 50 Ag/mouse of inactivated influenza virus. Prime, samples were collected 15 days after the first immunization. Boost, samples were collected 10 days after the first boost. 2nd boost, samples were collected 10 days after the second boost. Standard error bars are indicated for five mice in a group.

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the quantity of the immunogen and the number of administrations than wild-type mice. Different antigens often lead to the development of preferential immune responses, that is, Th1 vs. Th2 responses (Mosmann and Coffman, 1989). Ratios of IgG1/ IgG2a have been used to indicate the dominance of a Th1 or Th2 type response; Th1 responses are dominated by the production of IgG2a, while IgG1 is associated with a Th2dominant responses (Mosmann and Coffman, 1989). We found that IgG1/IgG2a ratios were from 1 to 1.75 after three immunizations in both immunocompetent C57BL/6 mice and CD4+ T cell-deficient mice, indicating that balanced Th1 vs. Th2 immune responses were induced by the inactivated PR8 virus in both groups. Lack of immunoglobulin class switching in ab T cell-deficient mice The ability of CD4+ T cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8+ T cells by treatment with an anti-CD8 mAb (Sha and Compans, 2000). To determine if isotype class switching occurred in the absence of ah T cells, we immunized ah+ T cell-deficient mice (TCRh / ) intranasally with 50 Ag of PR8 virus three times. Compared to responses induced in CD4-deficient mice, only borderline levels of serum IgG and IgA responses could be detected in TCRh / mice (IgG1, IgG2a b 2 Ag/ml in TCRh / mice vs. 21–34 Ag/ml in CD4 / mice; IgG2b, IgG3, IgA b 0.5 Ag/ml in TCRh / mice vs. 1.6–9.4 Ag/ml in CD4 / mice). This result is consistent with a previous study on systemic immunization (Sha and Compans, 2000). Intranasal immunization with inactivated PR8 virus induces neutralizing antibodies in sera of CD4+ T cell-deficient mice To test whether the sera of mice have neutralizing activity after intranasal immunization with inactivated PR8 virus, a standard plaque reduction assay was performed with MDCK cells. Approximately 100 plaque forming units of influenza PR8 virus was incubated with serial dilutions of sera from immunized mice and then applied to confluent MDCK cell monolayers. The numbers of plaques were counted after 4 days and compared with those observed using sera of unimmunized mice (Fig. 3). The groups immunized with 50 Ag had higher neutralizing titers than the groups immunized with 10 Ag, and the sera after the second boost were found to have higher titers than those after the first boost. The neutralizing titer of the 50-Ag group after the second boost was about 1:500, whereas that from the 10-Ag group after the first boost was less than 1:20. These data indicate that intranasal immunization with inactivated PR8 virus induces virus-neutralizing activity in sera of CD4+ T cell-deficient mice.

Fig. 3. Virus-neutralizing antibody activities in sera of CD4+ T celldeficient mice. CD4+ T cell-deficient mice were intranasally immunized with formalin-inactivated influenza virus (10 or 50 Ag/mouse per dose) at day 0 and boosted at days 15 and 25 with the same dose. Different dilutions of sera from immunized mice (pooled sera from five mice in a group) were incubated with approximately 100 plaque forming unit of PR8 virus for 1 h at room temperature. The mixtures were then applied to monolayers of confluent MDCK cells and a standard plaque reduction assay was performed. Representative data are shown from two independent experiments. CD4/Con, sera from unimmunized CD4+ T cell-deficient mice. CD4/10/1, CD4+ T cell-deficient mice after first boost with 10 Ag/mouse per dose. CD4/10/2, CD4+ T cell-deficient mice after second boost with 10 Ag/mouse per dose. CD4/50/1, CD4+ T celldeficient mice after first boost with 50 Ag/mouse per dose. CD4/50/2, CD4+ T cell-deficient mice after second boost with 50 Ag/mouse per dose. BL6/10/1, C57/BL/6-immunocompetent mice after intranasal priming with 10 Ag/mouse.

Mucosal immunization with inactivated PR8 virus elicits IgA responses in mucosal secretions Groups of CD4+ T cell-deficient mice and C57BL/6 immunocompetent mice were immunized with three doses of inactivated PR8 virus intranasally at days 0, 15, and 25, and the magnitude of virus-specific IgA antibodies after each immunization in mucosal secretions was monitored by measuring PR8-specific IgA concentrations by ELISA (Fig. 4). IgA responses were found to be induced in saliva (Fig. 4A) and vaginal secretions (Fig. 4B) both in C57BL/ 6 immunocompetent mice and CD4+ T cell-deficient mice. The IgA levels in saliva and vaginal secretions in the CD4+ T cell-deficient mice were more than 10-fold higher after three immunizations than those observed after two immunizations. The levels of IgA responses were also found to be dose dependent in the CD4+ T cell-deficient mice in saliva (Fig. 5A) and vaginal secretions (Fig. 5B). Significant increases in virus-specific IgA responses were found in the 50-Ag group in both saliva and vaginal secretions, as well as in saliva in the 10-Ag group after three immunizations (Fig. 5B). These results demonstrate that IgA responses in mucosal secretions are induced by

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intranasal immunization with inactivated influenza virus in CD4+ T cell-deficient mice. Neutralizing activities are induced in mucosal secretions in CD4+ T cell-deficient mice

Fig. 4. Magnitude of IgA responses in mucosal secretions to intranasal immunization with inactivated influenza virus in CD4+ T cell-deficient or immunocompetent mice. CD4+ T cell-deficient mice (16–20 weeks old) or C57BL/6 mice (10 weeks old) were immunized intranasally (five mice per group) at day 0 and boosted at days 15 and 25 with 50 or 10 Ag/mouse of inactivated influenza virus, respectively. CD4/Con, unimmunized CD4+ T cell-deficient mice. CD4/50, CD4+ T cell-deficient mice immunized with 50 Ag of inactivated PR8 virus per dose. BL6/10, C57BL/6-immunocompetent mice immunized with 10 Ag inactivated PR8 virus per dose. (A) Saliva. (B) Vaginal secretions. Standard error bars are indicated for five mice in a group.

Fig. 5. Dose dependence of IgA responses in mucosal secretions to intranasal immunization with inactivated influenza virus in CD4+ T celldeficient mice. CD4+ T cell-deficient mice (16–20 weeks old) or C57BL/6 mice (10 weeks old) were immunized intranasally and samples collected as described in Fig. 2. (A) Saliva. (B) Vaginal secretions. Standard error bars are indicated for five mice in a group.

Evidence has been obtained that IgA responses at mucosal surfaces are important for prevention of viral infections that are initiated at mucosal surfaces (Renegar and Small, 1991a,b; Tamura et al., 1990). To investigate whether the immune responses induced in mucosal secretions have virus-neutralizing activity, plaque reduction assays were performed on MDCK cells. Neutralizing activity was detected in both saliva (Fig. 6A) and vaginal secretions (Fig. 6B) after the second boost of the animals. These neutralizing titers were lower than those observed in sera (Fig. 3), which is consistent with observations of other groups (Di Tommaso et al., 1996; Russell et al., 1996). These data show that mucosal immunization of CD4+ T cell-deficient mice with inactivated PR8 virus results in the induction of neutralizing antibodies in mucosal secretions.

Fig. 6. Virus-neutralizing activity in mucosal secretions in CD4+ T celldeficient mice. CD4+ T cell-deficient mice were intranasally immunized with formalin-inactivated influenza virus (50 Ag/mouse per dose, five mice per group) at day 0 and boosted at days 15 and 25 with the same dose. Different dilutions of mucosal secretions from immunized mice (pooled samples from five mice in a group) were incubated with approximately 100 plaque forming unit of PR8 virus for 1 h at room temperature. The mixture was then applied to monolayers of confluent MDCK cells, and a standard plaque reduction assay was performed. Representative data were shown from two independent experiments. CD4/Con, sera from unimmunized CD4+ T cell-deficient mice. CD4/50/2, CD4+ T cell-deficient mice after second boost with 50 Ag/mouse per dose. BL6/10/2, C57/BL/6-immunocompetent mice after second boost with 10 Ag/mouse per dose. (A) Saliva. (B). Vaginal fluid.

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Fig. 7. Protection of CD4+ T cell-deficient mice against live influenza PR8 virus challenge after intranasal immunization with inactivated PR8 virus. CD4+ T cell-deficient mice (C57BL/6 background) were immunized intranasally three times with inactivated influenza virus and challenged with 10 LD50 doses of live influenza virus under anesthesia 4 weeks after the second boost (four mice per group). Mice were observed daily for 16 days, and all deaths were recorded. *, control, unimmunized CD4+ T cell-deficient mice. 5, CD4/10, CD4+ T cell-deficient mice immunized with 10 Ag/mouse per dose of inactivated influenza virus. x, CD4/50, CD4+ T cell-deficient mice immunized with 50 Ag/mouse per dose of inactivated influenza virus.

CD4+ T cell-deficient mice are protected from lethal dose virus challenge after mucosal immunization with inactivated virus To evaluate whether the immune responses elicited by inactivated PR8 virus after intranasal immunization in CD4+ T cell-deficient mice can protect against live virus challenge, groups of immunized mice were challenged intranasal with 10 LD50 of live PR8 virus 4 weeks after the second boost. Results showed that all mice immunized with 10 or 50 Ag/ dose were protected from lethal challenge (Fig. 7). Not only did these mice survive the challenge, but also minimal signs of virus infection (decrease of activity, ruffling of fur or decrease of body weight) were observed in these mice (data not shown). As expected, most of the unimmunized CD4+ T cell-deficient mice died around day 8 after challenge. Also, ah+ T cell-deficient mice were not protected from the same challenge after three immunizations with 50 Ag of PR8 virus per dose (survival 0/4). These data indicate that CD4+ T cell-deficient mice are protected from lethal dose influenza virus challenge after intranasal immunization with inactivated influenza virus.

Discussion The present study shows that intranasal immunization of CD4+ T cell-deficient mice with formalin-inactivated PR8 virus induces virus-specific IgG and IgA antibodies in serum and IgA responses in mucosal secretions. The sera and mucosal secretions of immunized mice were both found to have virus-neutralizing activities in vitro, and the immunized CD4+ T cell-deficient mice were also protected from challenge with a lethal dose of live virus. To our knowledge, this is the first report demonstrating that mucosal immuni-

zation with an inactivated virus can elicit antibody class switching from IgM to IgG and IgA and induce protective immune responses in the absence of CD4+ T cells. It is important to induce mucosal immunity since the majority of infectious agents enter their host through a mucosal surface. However, very little is known concerning immune responses induced by mucosal delivery of noninfectious virus particles in a CD4+ T cell-deficient condition. Here, we found several differences between immune responses induced by intranasal immunization compared to those by systemic immunization in CD4+ T cell-deficient mice. The most prominent difference is the induction of serum and mucosal IgA antibody by intranasal immunization, which could not be detected following systemic immunization (Sha and Compans, 2000). Second, intranasal immunization required relatively a high dose (50 Ag per mouse) of inactivated influenza virus for induction of IgG antibodies in the absence of CD4+ T cells and the responses were lower than those observed in wild-type mice. Since CD4+ T cell-deficient mice are known to have normal B cell functions in response to lipopolysaccharide indicating no intrinsic defect of B cells, and they exhibit comparable levels of total IgG or IgA expressing B cells as compared with normal mice (Hornquist et al., 1995; Rahemtulla et al., 1991), it is likely that there might be a defect in antigen-specific B cell activation or antigenspecific Ig production from mice lacking conventional CD4 T cells particularly after intranasal (this study) or oral immunization (Hornquist et al., 1995). In contrast, systemic immunization with low doses of inactivated influenza virus in CD4+ T cell-deficient mice induced comparable levels of IgG antibodies to those induced in normal mice (Sha and Compans, 2000). Third, two boosting immunizations were needed for induction of neutralizing antibodies by intranasal immunization in the absence of CD4+ T cells, whereas one boost was sufficient to induce neutralizing antibodies in systemic immunization of CD4+ T cell-deficient mice. Therefore, there are qualitative and quantitative differences in immune responses between mucosal and systemic immunization of CD4+ T cell-deficient mice. Inactivated influenza virus seems to be uniquely effective as an immunogen. In previous studies of CD4+ T celldeficient mice using cholera toxin (CT) as a mucosal immunogen, low levels of antigen-specific mucosal IgA antibody were observed following oral immunization, but the immunized mice remained unprotected against CTinduced diarrhea (Hornquist et al., 1995). Our study demonstrates that intranasal immunization of CD4+ T celldeficient mice with inactivated influenza virus could induce neutralizing antibodies in secretions and provide protection against lethal viral challenge. Taken together, these results suggest that inactivated influenza virus may be a more effective immunogen than CT at inducing mucosal immunity in the absence of CD4+ T cells, although different routes of immunization were used.

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Ig class switching is generally believed to be dependent on the cognate interaction between CD4+ T helper cells and B cells and the cytokines released by the activated CD4+ T cells (Finkelman et al., 1990; Oxenius et al., 1998; Parker, 1993; Snapper and Mond, 1993). However, some studies have shown the T cell-independent induction of IgG antibodies in animals infected with certain live viruses (Maloy et al., 1998; Rahemtulla et al., 1994; SzomolanyiTsuda and Welsh, 1998; Szomolanyi-Tsuda et al., 1998; Viney et al., 1994). Also, it was demonstrated that Rag 1 and Rag 2 knockout mice, which lack both T and B cells, chronically shed 100-fold higher levels of rotavirus antigen than mice lacking only CD4(+) T cell help (VanCott et al., 2001), suggesting that the production of T cell-independent antibody to rotavirus could substantially reduce rotavirus shedding. Polyomavirus infection of ah+ T cell-deficient mice or ah+ and gy+ T cell-deficient mice elicited a protective, T cell-independent IgG response in serum, indicating that neither ah+ nor gy+ T cells are required in this process; however, no IgG responses were elicited by immunization of these mice with polyoma virus-like particles or purified viral protein VP1 (Szomolanyi-Tsuda et al., 1998). In studies with VSV, ah+ T cell-deficient mice produced neutralizing IgG antibodies when infected with live VSV or with a recombinant vaccinia virus expressing the VSV glycoprotein, but not when immunized with inactivated VSV (Maloy et al., 1998). In the present study, ah+ T cell-deficient mice were not observed to produce significant amounts of IgG antibodies upon mucosal immunization with formalin-inactivated PR8 virus. Therefore, signals generated by live virus infection may be essential for induction of IgG antibodies in the absence of ah+ T cells. It has been suggested that CD4 and CD8 doublenegative ah-T cells (DN T cells) that are present in the CD4+ gene-targeted mice play a role in inducing Ig class switching and antigen-specific IgG responses to some antigens (Hornquist et al., 1995; Locksley et al., 1993; Rahemtulla et al., 1994; Sha and Compans, 2000). CD4+ gene-targeted mice were found to have numerous B cell germinal centers in Peyer’s patches and gut-associated lymphoid tissues where DN T cells were co-localized (Hornquist et al., 1995). We also found that such DN T cells constitute about 30% of the total T cell population in 6-month-old CD4+ T-cell deficient mice and about 15% in younger (6-week-old) mice (Sha and Compans, 2000). In contrast, in normal mice, DN T cells represent a very minor population of less than 2% (Sha and Compans, 2000) and seem to differ from DN T cells in CD4+ genetargeted mice (Locksley et al., 1993; Rahemtulla et al., 1994). In support of this idea, when C57BL/6 mice were depleted of CD4+T cells with an anti-CD4 antibody, their ability to generate IgG antibodies after immunization with inactivated influenza virus was abrogated (unpublished observations), indicating that a compensatory mechanism (DN T cells) develops in the congenitally CD4+ T cell-

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deficient mice that is not present in the acutely CD4+ T cell-depleted mice. In summary, the present results indicate that mucosal immunization with a nonreplicating viral antigen can induce neutralizing antibodies and provide protective immune responses even in the absence of normal CD4+ T cells. This finding may have important clinical implications, especially for immunization of immunocompromised patients against infections initiated at mucosal surfaces.

Materials and methods Animals C57BL/6J mice, C57BL/6-Cd4tm1Mak that had a targeted disruption in their CD4 gene, thus, did not express CD4 on cell surfaces, and lacked functional CD4+T cells (Rahemtulla et al., 1991) and C57BL/6J-Tcrbtm1Mom that had a targeted disruption in their TCRh gene and lacked functional ah T cells (Mombaerts et al., 1992), were obtained from Jackson Laboratory (Bar Harbor, ME). Some of the mice were bred in the Department of Animal Resources in Emory University from purchased breeding pairs. Since it was demonstrated that older CD4+ T cell knockout mice produced higher levels of antibody responses (Sha and Compans, 2000), 16- to 20-week-old CD4+ T cell-deficient mice and 10-week-old C57BL/6-immunocompetent mice were used in this study. Virus Embryonated hen’s eggs (9–11 days old) were used to grow influenza virus A/PR8/34 in the allantoic cavity. Virus was purified from allantoic fluid by sucrose gradient (30– 60%) centrifugation at 100 000  g for 3 h (Novak et al., 1993). Inactivation of the purified virus was performed by mixing the virus with formalin at a final concentration at 1:4000 (v/v). The mixture was incubated at 37 8C for 72 h and then dialyzed against PBS with three changes (Novak et al., 1993; Sha and Compans, 2000). The virus stock was stored in aliquots at 80 8C before use. Inactivation of the virus was confirmed by both plaque assay on confluent monolayer MDCK cells and inoculation of the virus into 9to 11-day-old embryonated hen’s eggs. Immunization and sampling Mice (five mice per group) were immunized with 1, 10, or 50 Ag of inactivated influenza virus in 100 Al PBS intranasally (in) at days 0, 15, and 25. Blood samples were collected 15 days after priming and 10 days after each boost. Anesthetized mice were bled from retro-orbital veins to obtain blood samples. Samples were centrifuged at 14 000 rpm for 15 min twice and sera were stored at 20 8C. Saliva was collected by aspiration from the cheek pouch after

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placing four to five drops of 2% pilocarpine under the tongue to stimulate flow in mice, and phenylmethylsulfonyl fluoride (PMSF) in isopropanol (100 mM) was added to the saliva at a final concentration of 1 mM as a protease inhibitor (Russell et al., 1996). Vaginal fluids were collected by flushing the mouse vaginal cavity with 100 Al PBS back and forth 20 times with a blunt-tipped feeding needle; approximately 70 Al of fluid was recovered from each mouse.

mouse Ig(H + L) antibody, then HRP-conjugated goat antibodies against IgA or each IgG subclass were added and colors were developed with ABTS substrate. Concentrations of IgG1, IgG2a, IgG2b, IgG3, and IgA were determined by comparing the reading of the experimental samples with the standard curves, and are represented as the arithmetic mean F1 standard error. The statistical analysis of differences between groups was carried out by the Excel program. Challenge studies

Virus neutralization assay A plaque reduction assay was performed to demonstrate the PR8 virus-specific neutralizing titer of the sera and mucosal secretions as previously described (Sha and Compans, 2000). Briefly, approximately 100 plaque-forming units (PFU) of influenza A/PR8 virus in 200 Al 1 DMEM medium was mixed with sera at 5-, 20-, 100-, or 500-fold dilutions (or mixed with saliva or vaginal secretions at 10-, 20-, 40-, or 80-fold dilutions) and incubated at room temperature for 1 h. Aliquots of 200 Al were added onto confluent MDCK cell monolayers in 6well plates, the plates were incubated at 37 8C for 1 h and shaken gently every 15 min. The inocula were removed, cells were washed once with 1 DMEM medium, then 1.95% white agar in 1 DMEM medium containing 1 Ag trypsin was overlaid on the cells. The plates were incubated at 37 8C for 4 days, stained with neutral red, and plaques in each well were counted. The neutralizing antibody titer was considered as the highest dilution of the serum or mucosal secretions that was found to reduce the number of plaques by at least 50%.

The lethal dose 50 (LD50) in CD4+ T cell-deficient mice for PR8 virus was previously found to be comparable with the LD50 in normal C57BL/6 mice (Sha and Compans, 2000). Challenge experiments were performed as described by Nguyen et al. (1999). Briefly, a mouse adapted antigenically identical strain of A/PR8/34 (provided by Dr. Jiri Mestecky, University of Alabama, Birmingham) was used for intranasal inoculation. 10 LD50 (500 PFU) of virus was administered by instillation into the nostrils of the anesthetized mice in a volume of 50 Al. Mice were observed daily for 16 days, and all deaths were recorded.

Acknowledgments This study was supported by research grants AI 28147 and AI 054337 from the National Institutes of Health, NIAID. We thank Drs. Aron Lukacher, Jacqueline M. Katz, Harriet Robinson, and Periasamy Selvaraj for helpful discussions and suggestions. We also thank Tanya Cassingham for assistance in preparing the manuscript.

Antibody responses References Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of influenza virus-specific antibodies as described (Pertmer et al., 1996). Briefly, flat-bottom 96well plates (Dynatech, Alexandria, VA) were coated with purified PR8 virus in borate-buffered saline (BBS) at a concentration of 2 Ag/ml. After blocking the plates with PBS with 1% BSA, different dilutions of serum, saliva, or vaginal secretions were added to the plates and incubated overnight. The plates were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG or IgA (Southern Biotechnology Associates, Birmingham, AL). After washing, the substrate 2,2’-azino-bis-[3-ethylbenzthiazoline sulfonic acid] (ABTS) (Sigma, St. Louis, MO) in phosphate citrate buffer (3 mg/10 ml) (pH 4.2) containing 0.03% H2O2 was added. After 10- to 20-min incubation at room temperature in the dark, absorbance was measured by an ELISA reader at 405 nm. For determination of the relative levels of PR8-specific IgA and IgG subclass responses, a quantitative assay was performed. Standard curves were obtained as follows: purified mouse IgG1, IgG2a, IgG2b, IgG3, or IgA was added to plates precoated with goat anti-

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