JOURNAL OF VIROLOGY, Feb. 2008, p. 1350–1359 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.01615-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 3
Induction of Heterosubtypic Immunity to Influenza Virus by Intranasal Immunization䌤 Fu-Shi Quan,1 Richard W. Compans,1 Huan H. Nguyen,2† and Sang-Moo Kang1* Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia,1 and Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama2 Received 24 July 2007/Accepted 8 November 2007
Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4ⴙ or CD8ⴙ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses. erosubtypic immunity has not been fully defined, effector CD8⫹ cytotoxic T lymphocytes (CTL) were considered to contribute to this immunity (12, 13, 18, 23, 37). These CTL recognize epitopes of internal proteins conserved among influenza A viruses, such as the nucleoprotein. However, mice depleted of CD8⫹ T cells in vivo were protected against heterosubtypic lethal challenge (6, 14). In addition, heterosubtypic immunity has been demonstrated for 2-microglobulin-deficient, T-cell-depleted, or gamma interferon (IFN-␥)-deficient mice (6, 20, 21). These previous reports suggest that immune components other than T cells also contribute to heterosubtypic protection. In contrast to the live virus infection model, heterosubtypic protection and immune correlates after immunization with an inactivated virus have not yet been well identified. In general, inactivated influenza virus has been considered incapable of inducing heterosubtypic immunity, since inactivated antigens do not induce strong CTL responses. Nevertheless, some cross-protection against different subtypes was demonstrated, accompanying a certain degree of morbidity, when mice were immunized intranasally with high doses of an inactivated virus or coimmunized intranasally with an adjuvant (31, 32, 34). Although these studies indicate a role of B cells, the mechanisms by which inactivated influenza virus vaccine induces cross-protective immune responses have not been characterized. Understanding the cross-protective immune responses induced by inactivated influenza virus is critical for developing more effective influenza vaccines. In this study, we investigated how immunization with inactivated influenza A/PR8 virus (H1N1) can induce immune responses conferring protection against challenge with the heterologous influenza strain A/WSN (H1N1) or the heterosubtypic strain A/Philippines (H3N2) in comparison with live virus infection. We found that intranasal immunization with inactivated influenza virus in the presence of cholera toxin (CT) induced enhanced cross-reactive binding and neutralizing antibodies in both mucosal and systemic compartments. Importantly, our results suggest that the presence of cross-reactive neutralizing
Influenza A virus hemagglutinin and neuraminidase glycoproteins are the major targets of neutralizing antibodies. Based on the antigenic variation of these two proteins, diverse influenza A viruses with different combinations of hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) subtypes have been identified (7). Influenza virus infection or live intranasal vaccines induce immune responses that provide not only protection against the homologous virus but also cross-protection against lethal infection with some heterologous strains of different subtypes in mice (1, 4, 6, 19, 20, 23, 26). In humans, natural infection or intranasal vaccination with live-attenuated viruses can also confer resistance to heterologous virus infection to a certain degree (3, 8). The currently licensed inactivated influenza virus vaccines for human use are whole virus or disrupted viral antigens containing the viral surface glycoproteins. The induction of strain-specific neutralizing antibodies is the basis of protective immunity provided by the current, parenterally administered influenza vaccine. The vaccine provides protection against viruses that are closely matched antigenically with those in the vaccine. However, the inactivated parenteral vaccine is less protective against antigenic drift variants within a subtype of the influenza virus and does not provide protection against viruses from different subtypes (30, 33, 34), and thus the annual production of new vaccines is required. Several groups have studied the role of T cells in the induction of heterosubtypic immunity by live virus infections (4, 6, 14, 20, 23). Although the precise immune effector(s) responsible for het-
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. Phone: (404) 727-3228. Fax: (404) 727-3659. E-mail:
[email protected]. † Present address: International Vaccine Institute, Seoul National University (SNU) Research Park, San 4-8, Bongcheon-7-dong, Kwanak-gu, Seoul 151-919, Korea. 䌤 Published ahead of print on 21 November 2007. 1350
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TABLE 1. Protective efficacy of intranasal immunization with inactivated A/PR8 virusa Mouse immunization group Naı¨ve mouse control
PR8i (25 g)
PR8i (5 g) ⫹ CT (2 g) PR8i (25 g) ⫹ CT (2 g) PR8 sublethal infection
Virus and dose (LD50) used for challenge
Clinical signsb
No. of survivors/total no. of mice
% Protection
A/PR8 (H1N1) (3 ⫻ 102) A/PR8 (H1N1) (10) A/WSN (H1N1) (3 ⫻ 102) A/WSN (H1N1) (10) A/Philippines (H3N2) (60) A/Philippines (H3N2) (40) A/PR8 (H1N1) (3 ⫻ 102) A/WSN (H1N1) (3 ⫻ 102) A/Philippines (H3N2) (60) A/Philippines (H3N2) (40) A/Philippines (H3N2) (60) A/Philippines (H3N2) (40) A/Philippines (H3N2) (60) A/Philippines (H3N2) (40) A/Philippines (H3N2) (60) A/Philippines (H3N2) (40)
Sick (⫹⫹⫹) Sick (⫹⫹⫹) Sick (⫹⫹⫹) Sick (⫹⫹⫹) Sick (⫹⫹⫹) Sick (⫹⫹⫹) Healthy Healthy Sick (⫹⫹⫹) Sick (⫹⫹⫹) Sick (⫹⫹) Sick (⫹⫹) Sick (⫾) Healthy Sick (⫾) Healthy
0/6 0/6 0/6 0/6 0/6 0/6 6/6 6/6 0/6 0/6 5/6 6/6 6/6 6/6 6/6 6/6
0 0 0 0 0 0 100 100 0 0 83 100 100 100 100 100
a
Mice were immunized intranasally with inactivated A/PR8 virus (H1N1), alone or in combination with CT adjuvant, at weeks 0 and 2. Mice with ⫹⫹⫹ signs showed severe illness. Clinical signs were determined by body weight losses and mouse symptoms of illness. ⫹⫹⫹, losses in body weight of over 30%; ⫹⫹, losses in body weight of between 20% and 30%; ⫾, ⬍10% decreases in body weight. b
antibodies in the lungs and in blood plays a critical role in providing heterosubtypic cross-protective immunity. The underlying potential mechanism and advantages of mucosal delivery of influenza vaccine are discussed. MATERIALS AND METHODS Viruses. Influenza virus A/PR8/34 (H1N1) was grown in 10-day-old embryonated hen’s eggs and purified from allantoic fluid by using a discontinuous sucrose gradient (15%, 30%, and 60%). Inactivation of the purified virus was performed by mixing the virus with formalin at a final concentration of 1:4,000 (vol/vol) as described previously (22, 27). Inactivation of the virus was confirmed by plaque assay on confluent monolayers of Madin-Darby canine kidney (MDCK) cells and by inoculation of the virus into 10-day-old embryonated hen’s eggs. For challenge experiments, mouse-adapted A/PR8/34 (H1N1; PR8), A/WSN (H1N1; WSN), and A/Philippines/2/82/X-79 (H3N2) were prepared as lung homogenates from intranasally infected mice and used for challenge. Immunization and challenge. Female inbred BALB/c mice (Charles River) aged 6 to 8 weeks were used. Isoflurane-anesthetized mice were immunized intranasally with 50 l phosphate-buffered saline (PBS) containing either 5 g or 25 g of inactivated PR8 virus (PR8i) in the presence or absence of CT (2 g) on days 0 and 14. For sublethal infection, mice were infected intranasally with a 50% lethal dose (LD50) in 50 l PBS. As shown in Table 1, isoflurane-anesthetized mice were challenged with different doses of homologous virus (A/PR8; H1N1), heterologous virus (A/WSN; H1N1), or a heterosubtypic strain (A/ Philippines; H3N2) in 50 l of PBS per mouse on week 4 after the booster immunization. Mice were observed daily to monitor changes in body weight and to record death. Sample collection. Blood samples were collected by retro-orbital plexus puncture before immunization and 2 weeks after priming and booster immunizations. For measurements of viral replication and immune response parameters, six mice from each group were sacrificed at day 4 postchallenge. Nose and tracheal tissue samples were harvested from mice prior to challenge infection and on day 4 after challenge infection, suspended in 200 l PBS, and kept at ⫺20°C until used for immunoglobulin A (IgA) and IgG detection. The whole lungs were collected individually prior to challenge and at day 4 postchallenge and were used to prepare lung homogenates. Lung homogenates in 1 ml Dulbecco’s modified Eagle’s medium were centrifuged at 1,000 rpm for 10 min to collect supernatants, which were frozen and kept at ⫺80°C until used for virus titer and cytokine assays. Lymphocytes from spleen samples were collected from sacrificed mice for enzyme-linked immunospot (ELISPOT) cytokine analysis. Bone marrow cells were obtained by flushing the cavities of both femoral and tibial bones with medium and were used to detect influenza virus-specific antibody-secreting plasma cells. Antibody responses. IgG and IgA antibody levels specific to influenza virus in the serum were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (24, 25). Ninety-six-well microtiter plates
(MaxiSorp immunoplate; Nunc Life Technologies, Basel, Switzerland) were coated with 100 l of inactivated virus (4 g/ml) in coating buffer (0.1 M sodium carbonate, pH 9.5) at 4°C overnight. Serially diluted sera (100 l) were added to wells to determine influenza virus-specific binding antibody levels. Color was developed using the substrate o-phenylenediamine (Zymed, San Francisco, CA), and the optical density was read at 450 nm. For antibody titers, dilutions giving values higher than the mean plus two standard deviations of that of the background (naı¨ve mouse control) were considered positive. Lung viral titer and virus neutralization assays. Lung viral titer and neutralization assays were performed using MDCK cells following a previously described procedure (24, 25). Briefly, 190 l of a serially diluted serum sample was mixed with 10 l of diluted virus stock containing approximately 100 infectious particles, incubated at 37°C for 1 h, and then added to the cell monolayers. After incubation for 2 to 3 days, the cells were fixed with 0.25% glutaraldehyde and stained with 1% crystal violet to visualize plaques. The neutralization activity was expressed as the percentage of plaque reduction. Detection of antibody-secreting cells in the bone marrow. To detect influenza virus-specific antibody-secreting cells, PR8i viral antigen was used to coat Multiscreen 96-well filtration ELISPOT plates (Millipore). Freshly isolated cells from bone marrow (1 ⫻ 106 cells) were added to each well and incubated for 48 h at 37°C with 5% CO2. Using horseradish peroxidase-conjugated anti-mouse immunoglobulin antibodies and the ELISPOT substrate diaminobenzidine (Invitrogen, Carlsbad, CA), color was developed following the manufacturer’s instructions, and counting of ELISPOTs was performed as described previously (25). Heterosubtypic protective role of immune sera and lung extracts. Lung and serum samples from immunized mice prior to challenge or at day 4 postchallenge were heated at 56°C for 30 min. Twenty microliters of heat-treated sample was incubated with 40 l of A/Philippines virus (2,000 PFU [40⫻ LD50]) at 30°C for 30 min. The reaction mixtures (40 l) were administered to naı¨ve mice, and body weight changes and survival rates were monitored daily. Naı¨ve mice infected with a similar mixture of A/Philippines and lung or serum samples from naı¨ve mice were used as controls. Depletion of CD4ⴙ or CD8ⴙ T cells in immunized mice. Mice were immunized with 25 g of inactivated A/PR8 (H1N1) virus plus CT at weeks 0 and 2. To deplete CD4⫹ T cells, immunized mice were administered anti-CD4 GK1.5 antibody (800 g for each mouse) intraperitoneally at days ⫺3, ⫺1, and ⫹2 postinfection. For depletion of CD8⫹ T cells, PR8-plus-CT-immunized mice were treated intraperitoneally with anti-CD8 antibody (clone 53.6.72; Bio-Express, West Lebanon, NH) at a dose of 550 g per mouse at days ⫺3, ⫺1, and ⫹2 postinfection. Anti-CD4 or -CD8 antibody-treated mice were challenged with a lethal dose of heterosubtypic virus A/Philippines (H3N2), and morbidity and mortality were monitored daily. Statistics. All parameters were recorded for individuals within all groups. Statistical comparisons of data were carried out using the analysis of variance and Npar one-way Kruskal-Wallis tests of the PC-SAS system. P values of ⬍0.05 were considered significant.
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FIG. 1. Virus titers in lungs at day 4 postchallenge. Mice immunized intranasally with PR8i alone (25 g) or with CT (2 g) adjuvant (PR8i⫹CT) were challenged with a high dose of A/PR8 (300 LD50), A/WSN (300 LD50), or A/Philippines (40 LD50) (groups PR8i⫹PR8, PR8i⫹WSN, PR8i⫹Phil, and PR8i⫹CT⫹Phil). Naı¨ve mice were infected with a lethal dose of A/PR8 (10 LD50), A/WSN (10 LD50), or A/Philippines (10 LD50) (groups Naive⫹PR8, Naive⫹WSN, and Naive⫹Phil).
RESULTS Cross-protective efficacy of immunization with inactivated A/PR8. We investigated whether inactivated A/PR8 immunization can induce cross-protective immunity against heterosubtypic influenza virus challenge and examined the immune correlates that are involved in heterosubtypic immunity. Mice were immunized intranasally with inactivated A/PR8 virus (5 or 25 g per mouse), alone or in combination with CT (2 g), at weeks 0 and 2. To evaluate the protective efficacy against strains within the same subtype, mice were challenged with high lethal doses of either A/PR8 (H1N1) or A/WSN (H1N1) virus and monitored daily for morbidity and mortality (Table 1). Intranasal immunization of mice conferred complete protection against a high lethal dose of PR8 or WSN (6 ⫻ 104 PFU per mouse; equivalent to 3 ⫻ 102 LD50). Next, to determine heterosubtypic immunity under stringent conditions, high doses (3 ⫻ 103 PFU and 2 ⫻ 103 PFU; equivalent to 60 LD50 and 40 LD50, respectively) of A/Philippines (H3N2) were used to challenge the immunized mice. All mice immunized with A/PR8 alone succumbed to death after showing clinical symptoms. In contrast, groups of mice receiving CT as an adjuvant with a low dose of PR8 immunogen (5 g) showed high rates of protection, with minor clinical symptoms, after A/Philippines challenge (83% to 100%, depending on the challenge dose). Mice immunized with a dose of 25 g PR8i with CT adjuvant showed 100% protection, without any clinical signs, after challenge with the same dose of A/Philippines (2 ⫻ 103 PFU). As another parameter for evaluating protective effects, body weight changes were monitored after challenge. Mice immunized with PR8i (H1N1) alone did not show any changes in body weight when challenged with higher or lower lethal doses of PR8 (H1N1) or WSN (H1N1) virus. However, the same group of PR8-immunized mice showed a significant loss in body weight when challenged with A/Philippines (H3N2), indicating that these mice suffered severe illness. In contrast, mice immunized with inactivated A/PR8 plus CT adjuvant showed slight changes (5-g group) or no changes (25-g group) in body weight after a lethal dose challenge with A/Philippines (2 ⫻ 103 PFU); all survived, except for the group that received 5 g inactivated A/PR8 and was challenged with a high dose, which showed 83% protection. Thus, coadministra-
tion of vaccine with CT plays a role in providing cross-protective immunity. Lung viral titers after challenge in mice immunized with inactivated A/PR8. Lung viral titers postchallenge were analyzed to determine the replication of challenge virus at day 4 postchallenge (Fig. 1). The naı¨ve mouse group receiving challenge virus A/PR8 or A/WSN showed high viral titers, reaching around 6 log, while lower viral titers (2 log or lower) were detected in immunized mice. However, mice from both the naı¨ve and PR8i vaccine groups showed ⬎6-log virus titers when challenged with a lethal dose of A/Philippines virus. Interestingly, the group receiving the CT adjuvant showed low virus titers in the lung even with A/Philippines challenge. These results indicate that mice coimmunized with inactivated A/PR8 (H1N1) and CT adjuvant induce protective immune responses that can inhibit the replication of the heterosubtypic A/Philippines virus (H3N2) and that heterosubtypic immunity is an achievable goal with a reasonable dose of inactivated influenza virus vaccine in combination with an effective adjuvant. Higher levels of antibodies are induced in the CT adjuvant group. Next, we investigated the immune correlates responsible for heterosubtypic immunity against A/Philippines to gain insight into the possible mechanisms of heterosubtypic immunity. We initially detected the binding antibodies induced by intranasal immunization with PR8i virus. The CT adjuvant group (A/Philippines-protected group) showed significantly increased levels of serum binding antibodies against the A/PR8 homologous antigen (average serum IgG titer, 3.2 ⫻ 106; n ⫽ 6) as well as the heterologous A/WSN and A/Philippines antigens (average serum IgG titer, 1.2 ⫻ 105; n ⫽ 6), which were comparable to those induced in mice infected with A/PR8 live virus (average serum IgG titer, 1.02 ⫻ 105; n ⫽ 6) after recovery from infection. The PR8-only immunized group induced sixfold lower levels of cross-reactive antibodies (average serum IgG titer, 6.4 ⫻ 103; n ⫽ 6) than did the CT adjuvant group (Table 2). However, similar binding antibody levels were observed between the WSN (H1N1) and Philippines (H3N2) strains, although there were significant differences in protective efficacy (Table 1). These results suggest that levels of binding antibodies in immune sera may not be a major determining factor responsible for conferring heterosubtypic immunity. Intranasal immunization is known to induce immune re-
TABLE 2. Increases in binding antibodies upon heterosubtypic viral challengea Sample
Group
Titer before challenge/titer after challenge (fold increaseb) IgG
IgA
4 ⫻ 10 /1.6 ⫻ 103 (4) Lung PR8i 4 ⫻ 10 /2.4 ⫻ 10 (6) PR8i ⫹ CT 2.6 ⫻ 104/2 ⫻ 105 (8) 6.4 ⫻ 103/1 ⫻ 106 (16) Sera PR8i 6.4 ⫻ 103/2.56 ⫻ 104 (4) 4 ⫻ 102/8 ⫻ 102 (2) PR8i ⫹ CT 1.02 ⫻ 105/8.1 ⫻ 105 (8) 8 ⫻ 103/3.2 ⫻ 104 (4) 2
3
2
a Cross-reactive binding antibody responses against A/Philippines (H3N2) virus were determined for lung and serum samples collected from inactivated PR8-immunized mice prior to and after challenge infections (n ⫽ 6). Inactivated A/Philippines virus was used as an ELISA coating antigen for IgG and IgA titer determinations. b Increase in titer on day 4 after challenge infection with A/Philippines compared to the titer prior to challenge.
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FIG. 2. Cross-reactive mucosal antibodies detected by ELISA. Mucosal antibodies induced by intranasal immunization with PR8i alone (25 g) or PR8i plus CT (PR8i⫹CT) were tested for reactivity to homologous A/PR8, heterologous A/WSN, and heterosubtypic A/Philippines as ELISA coating antigens. IgG and IgA antibody titers are expressed as the highest dilution of serum having a mean optical density at 450 nm greater than the mean plus 2 standard deviations for similarly diluted naı¨ve serum samples. (A) IgG to A/PR8; (B) IgA to A/PR8; (C) IgG to A/WSN; (D) IgA to A/WSN; (E) IgG to A/Philippines; (F) IgA to A/Philippines.
sponses in the respiratory mucosal sites where influenza virus enters and replicates. We determined the binding antibody titers against homologous (A/PR8) and heterologous (A/WSN and A/Philippines) antigens in mucosal samples and lung extracts collected from mice immunized intranasally with PR8i (Fig. 2). Significant levels of IgG and IgA antibody binding to PR8 were observed in both the upper (nose washes) and lower (tracheal and lung extracts) respiratory tracts. The inclusion of CT increased levels of mucosal antibodies in the nose and tracheal washes two- to eightfold (Fig. 2A to D). It is important that coadministration with CT induced significantly enhanced levels of IgG and IgA antibody binding to A/Philippines in lungs, which were increased over 60-fold compared to those in the group without CT, as shown in Fig. 2E and F. Infection of mice with a sublethal dose of live A/PR8 also induced heterosubtypic immunity against A/Philippines (Table 1). It is noteworthy that levels of lung IgG and IgA titers against A/Philippines were equivalent to those in the PR8-infected group (2.6 ⫻ 104 for IgG and 6.4 ⫻ 103 for IgA; n ⫽ 6). Therefore, inclusion of CT in the PR8i virus vaccine was able to induce similar antibody responses cross-reactive to A/Philippines to those in the PR8 sublethal infection group. These results indicate that high levels of cross-reacting mucosal antibodies
may contribute to conferring heterosubtypic immunity against influenza viruses. Neutralizing activities correlate with cross-protection. Neutralizing activity against influenza virus may provide an implicative barometer for protection. As shown in Fig. 3, PR8immunized groups showed the highest serum neutralizing activities against the homologous virus, A/PR8 (neutralizing titers over 800). Moderate neutralizing titers were observed against A/WSN (90 for PR8 alone and 270 for the CT adjuvant group). Although neutralizing titers against the heterosubtypic strain A/Philippines were found to be low, the CT adjuvant (A/Philippines-protected) group showed a titer of 60, which is higher than that of the PR8-only group (below 20). Importantly, the CT adjuvant group showed similar neutralizing activities to those induced by live A/PR8 virus infection with a sublethal dose (Fig. 3C), which also showed heterosubtypic cross-protection against A/Philippines challenge infection in a comparative study (Table 1). Since influenza virus enters the body through the respiratory tract, determining the presence of neutralizing antibodies in secretory mucosal samples is important. We observed high levels of neutralizing activity against the homologous virus, A/PR8, in all samples, and inclusion of the CT adjuvant in-
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FIG. 3. Serum neutralizing activities. Sera from groups of mice immunized with PR8i alone or coimmunized with CT (PR8i⫹CT) were collected 2 weeks after the second immunization. Infected sera were collected from mice infected with a sublethal dose (0.5 LD50) of A/PR8 virus at day 30 (PR8 infection). Diluted serum samples were incubated with 100 PFU of PR8, WSN, or Philippines virus, and a standard plaque reduction assay was performed using MDCK cells.
creased the titers two- to eightfold (Table 3). Significantly higher levels of neutralizing activity against the heterologous strain A/WSN were detected in lung samples in the CT adjuvant group (titers of 200 to 400, depending on the PR8 antigen dose) (Table 3), and they were threefold higher than those in the PR8-only group but lower than those against the homologous strain PR8. Also, the CT adjuvant group showed higher neutralizing titers against the A/WSN strain, ranging from 30 to 60 in the trachea and 70 to 80 in the nose, depending on the antigen dose used (Table 3), which were higher than those of the nonadjuvant group. Importantly, low but significant levels (titer, 25; n ⫽ 6) of neutralizing activity against A/Philippines
(H3N2) were detected in lung samples from the group of mice coimmunized with CT, which provided 100% protection against A/Philippines (Table 3). These cross-neutralizing titers against A/Philippines in lungs of the PR8i-plus-CT-immunized group were also comparable to those observed in the PR8 sublethal infection group (titers, 20 to 25; n ⫽ 6). However, no neutralizing titers against A/Philippines were detected in the PR8-only immunized group. These results suggest that the presence of neutralizing activity in mucosal samples, particularly in the lung, plays a critical role in providing cross-protective immunity against different subtypes of influenza virus. Antibody-secreting bone marrow plasma cells in immunized mice. An important goal of vaccination is to induce memory immune responses. During the differentiation and development of B-cell memory, a fraction of memory B lymphocytes that develop in the secondary lymphoid organs migrate to the bone marrow. Antibody-secreting plasma cells in the bone marrow are long-lived and responsible for maintaining serum antibodies (28, 29). As shown in Fig. 4, significant levels of influenza A/PR8 virus-specific IgG and IgA antibody-secreting cells were detected in bone marrow from immunized mice at 2 months postimmunization. Compared to the PR8-only group, the group of mice immunized with PR8i plus CT showed threefold higher levels of IgG- and IgA-secreting cells, similar to the levels observed in the sublethal infection group (Fig. 4), indicating that CT can enhance the levels of long-lived plasma cells in bone marrow. When we analyzed antibody-secreting cells cross-reactive to A/Philippines, moderately increased levels were found in the CT adjuvant group (PR8i plus CT) and in PR8-infected mice compared to those in the PR8i-immunized group. These results indicate that the CT adjuvant can induce antigen-specific (and cross-reactive) antibody-secreting B cells to levels similar to those in the sublethal infection group. Heat-stable immune components provide protection against A/Philippines. To better understand the immune correlates providing heterosubtypic protective immunity by intranasal immunization with inactivated influenza virus, lung and serum antibodies were tested for protective efficacy against A/Philippines. Naı¨ve mice that received a mixture of a lethal dose of A/Philippines virus (40 LD50) and heat-inactivated sera or lung extracts collected from the CT adjuvant group prior to challenge were not protected and died of illness (data not shown). Although the CT adjuvant group showed 100% protection against A/Philippines (Table 1), low neutralizing titers (Fig. 3 and Table 3) against A/Philippines that were present in the
TABLE 3. Mucosal neutralizing titersa Neutralizing titer Group2
Preimmune PR8i (5 g) PR8i (5 g) ⫹ CT (2 g) PR8i (25 g) PR8i (25 g) ⫹ CT (2 g)
PR8
WSN
Philippines
Lung
Trachea
Nose
Lung
Trachea
Nose
Lung
Trachea
Nose
10 70 600 75 ⬎800
0 70 150 175 500
10 270 400 400 800
10 50 200 150 480
0 26 30 45 60
10 55 70 60 80
0 0 ⬍10 0 25
0 0 0 0 0
0 0 0 0 0
a Virus neutralizing titers resulting in 50% plaque formation compared to controls (wells containing viruses without mucosal samples). Plaque reduction assays were performed using pooled samples. Representative data from three independent experiments are shown. Neutralizing titers were determined using the following target viruses: homologous A/PR8 (H1N1), heterologous A/WSN (H1N1), and heterosubtypic A/Philippines (H3N2) viruses.
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FIG. 4. Antibody-secreting cells in the bone marrow. (A) IgA antibody-secreting cells. (B) IgG antibody-secreting cells. Bone marrow cells were collected from mice immunized with PR8i. For determination of virus-specific antibody-secreting cells, ELISPOT plates were coated with the corresponding inactivated virus. “PR8 infection” indicates a group of mice that recovered from infection with a sublethal dose of PR8 (0.5 LD50). The spots for antibody-producing cells from the bone marrow cells were counted and expressed based on 1.5 ⫻ 106 total bone marrow cells per well.
sera or lung extracts prior to challenge were insufficient to neutralize a high dose of A/Philippines virus and to provide protection in naı¨ve mice. Interestingly, when heat-treated (56°C for 30 min to inactivate complement and cytokines) sera or lung extracts (40-l samples) from the CT adjuvant group (but not the PR8i-immunized group) collected at day 4 postchallenge (with A/Philippines) were mixed with a lethal dose of A/Philippines (40 LD50), naı¨ve mice that received the mixture of A/Philippines and sera or lung extracts were completely protected against A/Philippines (Fig. 5). Diluted lung samples from either the PR8i-plus-CT group or the PR8-infected group were found not to be protective against the dose used for the challenge infection (40 LD50 of A/Philippines). In contrast, mice that received a mixture of A/Philippines virus and twofold-diluted sera (from either the PR8i-plus-CT group or the PR8-infected group) were protected from A/Philippines challenge infection but showed a certain degree of morbidity. These results at day 4 postchallenge are consistent with higher levels of cross-neutralizing titers in sera than in the lungs (Fig. 6). Samples of lungs and sera collected from the PR8-infected group on day 4 after A/Philippines challenge showed similar protective effects to those for the CT adjuvant group (data not shown). To better understand the differences in lung and serum components between pre- and postchallenge infections with A/Philippines for the CT adjuvant group, we compared the levels of binding and neutralizing antibodies (Table 2 and Fig. 6). The titers of IgA antibody specific to A/Philippines were increased 16-fold, while the IgG antibody titers were increased
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FIG. 5. Heterosubtypic protective effects of immune sera or lung extracts. Sera and lung samples collected from PR8i-immunized mice (groups PR8i and PR8i⫹CT) on day 4 postchallenge with A/Philippines were heated (56°C for 30 min) and then incubated with 2,000 PFU A/Philippines virus (40 LD50) at 37°C for 30 min. Naı¨ve mice were infected with a mixture of A/Philippines virus and lung or serum samples and monitored for morbidity (A) and mortality (B).
8-fold, in both lung and serum samples at day 4 postchallenge. In particular, mucosal IgG and IgA antibody titers were 100and 1,000-fold higher, respectively, than those in the group without CT (Table 2). Most importantly, the cross-reactive neutralizing antibody titers to A/Philippines were increased significantly in both sera (40-fold) and lungs (5-fold) from the CT adjuvant group (Fig. 6). Infected mice showed similar levels of A/Philippines virus neutralizing titers in lungs and sera to those in the PR8i-plus-CT group on day 4 (data not shown). These results suggest that heat-stable components, most likely cross-reactive neutralizing antibodies, were rapidly induced upon challenge infection and played an essential role in providing heterosubtypic protection against A/Philippines virus infection. Overall, the inclusion of CT adjuvant induced equivalent levels of cross-protective immune responses to those observed in the group that recovered from live virus infection. Protection in CD4ⴙ or CD8ⴙ T-cell-depleted mice. The result showing that sera and lung extracts collected from the CT adjuvant group prior to challenge infection were not protective may indicate a potential role of cellular components in providing heterosubtypic protection. To determine the potential role of CD4⫹ T cells in conferring cross-protective immunity, immunized mice were treated with anti-CD4 antibody prior to challenge infection, which resulted in the depletion of most CD4⫹ T cells in both lungs and spleens (Fig. 7A and B).
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Similarly, a group of mice treated with anti-CD8 antibody showed no detectable CD8 T cells in either the spleen or lungs (Fig. 8A and B). Following challenge with a lethal dose of the heterosubtypic virus A/Philippines (40 LD50), lung viral titers and survival rates were determined (Fig. 7C and D). Groups of mice that were immunized with PR8i plus CT and then treated with anti-CD4 or -CD8 antibody showed similar efficacies of protection against A/Philippines to those for nontreated mice. Immune mice with depletion of CD4⫹ T cells resulted in similarly reduced lung viral titers to those of the nontreated group. However, CD8 T-cell depletion was found to be somewhat less effective at inhibiting lung viral replication, since titers observed in the anti-CD8-treated group were 100-fold higher than those in the nontreated group (Fig. 8C). These results suggest that neither CD4⫹ nor CD8⫹ T cells are required for heterosubtypic protection against A/Philippines virus, although the effector CD8⫹ T cells in the immune mice (PR8i plus CT) may partially contribute to inhibiting the replication of the heterosubtypic challenge virus.
DISCUSSION
FIG. 6. Rapid increases in cross-reactive neutralizing activity upon challenge infection. PR8i, mice immunized intranasally with PR8i alone; PR8i⫹CT, mice immunized with PR8i plus CT. Neutralizing titers were determined using A/Philippines virus (H3N2) and serum and lung samples collected from immunized mice prior to challenge (immunized) and on day 4 after challenge with A/Philippines (immunized ⫹ Cha).
The underlying mechanism responsible for inducing heterosubtypic immunity is unknown, although previous studies have shown that B cells and mucosal immune responses are important in mice intranasally vaccinated with inactivated influenza virus (32, 34). The current study demonstrates for the first time that intranasal coimmunization with PR8i (H1N1) virus and CT adjuvant induced cross-neutralizing antibody responses against A/Philippines (H3N2) in both lungs and blood and
FIG. 7. Effects of CD4⫹ T-cell depletion in conferring heterosubtypic protection. Mice immunized with PR8i and CT were treated with either PBS or anti-mouse CD4 antibody (GK1.5) prior to challenge infection with A/Philippines virus. (A) Depletion of CD4⫹ T cells in spleen (at day 4 postchallenge). (B) Depletion of CD4⫹ T cells in lung (at day 4 postchallenge). (C) Lung viral titers on day 4 after challenge with A/Philippines (40 LD50). (D) Survival rates after challenge infection with A/Philippines (40 LD50).
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FIG. 8. Effects of CD8⫹ T-cell depletion in conferring heterosubtypic protection. Mice immunized with PR8i plus CT were treated with either PBS or anti-mouse CD8 antibody (clone 53.6.72) prior to challenge infection with A/Philippines virus. (A) Depletion of CD8⫹ T cells in spleen (at day 4 postchallenge). (B) Depletion of CD8⫹ T cells in lung (at day 4 postchallenge). (C) Lung viral titers on day 4 after challenge with A/Philippines (40 LD50). (D) Survival rates after challenge infection with A/Philippines (40 LD50).
provided the immunized mice with heterosubtypic protection. Low levels of cross-reactive neutralizing antibodies present prior to challenge in the CT adjuvant group were significantly increased upon heterosubtypic challenge infection, which indicates that a subset of B cells secreting cross-reactive neutralizing antibodies was rapidly expanded. Importantly, this heterosubtypic protection was also observed under conditions of CD4⫹ or CD8⫹ T-cell depletion prior to challenge. We observed that inactivated A/PR8 (H1N1) immunization conferred complete protection against high doses of A/PR8 and A/WSN (H1N1) viruses without any clinical signs of disease but did not provide protection against the heterosubtypic strain A/Philippines (H3N2). Although it is not possible to compare the results directly from other studies, Takada et al. demonstrated that intranasal immunization with high doses of inactivated H5N1 virus (300 g) provided partial to complete protection against viruses of different subtypes, with some clinical symptoms (32). This immunization dose may be too high to be applicable to humans. In another study, intranasal immunization with a moderate dose of 60 g of inactivated H3N2 virus in the absence of adjuvant showed partial protection against low-dose challenge with H5N1 virus (34). Our studies were performed under stringent conditions with high doses of challenge viruses, and the PR8i-only group showed no protection against heterosubtypic challenge. Therefore, the dose and frequency of immunization with inactivated influenza virus seem to affect the protective efficacy against heterosubtypic
virus. However, the immune components responsible for conferring heterosubtypic immunity have not previously been identified clearly. In live influenza virus infection studies using CD4⫹ or CD8⫹ T-cell-, natural killer cell-, or IFN-␥-deficient mice, immune components other than T cells have been suggested to play a role in providing heterosubtypic immunity (4, 6, 14, 20, 21). Protection by the current inactivated influenza virus vaccine is based mainly on induction of neutralizing antibodies against the homologous virus. However, previous studies using mice mucosally vaccinated with inactivated virus suggested that heterosubtypic immunity can occur in the absence of cross-neutralizing antibodies (32, 34). In our study, coadministration of inactivated A/PR8 virus with CT provided 100% protection against the heterosubtypic strain A/Philippines, whereas inactivated A/PR8 alone provided no protection. Analysis of serum antibodies showed cross-reactive serum neutralizing antibody activity against A/Philippines at a low level in the CT-coimmunized group. However, mice immunized with A/PR8 alone did not show detectable levels of cross-neutralizing activity against A/Philippines. Thus, the presence of cross-neutralizing antibodies seems likely to play an important role in inducing heterosubtypic immunity. Intranasal immunization with inactivated influenza virus was demonstrated to be superior to parenteral immunization in inducing protective heterosubtypic immunity. However, the mechanism for this observation is not well understood. One
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obvious factor is the induction of mucosal immune responses, which are important as a first line of immune defense against influenza virus infection and are not induced effectively by parenteral immunization (32, 34). It is known that polymeric IgA in secretions has broader neutralizing activity than does serum IgG (17). The CT adjuvant group immunized with inactivated A/PR8 that exhibited 100% protection against the heterosubtypic virus A/Philippines showed significantly enhanced levels of both IgG and IgA antibody binding to A/Philippines in the mucosal respiratory tract, particularly in the lungs, which was not observed in the PR8 group. Importantly, we detected cross-reactive neutralizing activity against A/Philippines in lung samples only for the group receiving the CT adjuvant. These cross-reactive neutralizing antibodies (in both lungs and sera) against the heterosubtypic strain, although present at low levels, might play a critical role in inducing protective immunity against heterosubtypic challenge. To better understand the immune components responsible for heterosubtypic immunity, immune sera or lung extracts from the CT adjuvant group prior to challenge were tested for their protective effects in naı¨ve mice after incubation with live A/Philippines virus (40 LD50) in vitro, and we found that low levels of cross-reactive neutralizing titers were not high enough to be protective (data not shown). To investigate other potential components contributing to heterosubtypic protection, we determined the potential role of CD4⫹ or CD8⫹ T cells in heterosubtypic immunity by acute depletion prior to challenge infection. Depletion of CD4⫹ or CD8⫹ T cells did not affect the heterosubtypic protective efficacy observed for the CT adjuvant group. These results are consistent with a previous study showing that acute depletion of CD4⫹ T cells or genetic CD8⫹ T-cell deficiency did not significantly reduce heterosubtypic protection in mice immunized with inactivated virus and adjuvant (34), although we cannot exclude the possible role of effector CD8⫹ T cells in lowering the viral load, as the CD8⫹ T-cell-depleted immune mice showed higher lung viral titers than did nontreated mice. When we analyzed the reactive immune responses against A/Philippines in sera and lung extracts at early times postchallenge, the most striking feature was a rapid increase in neutralizing activity against A/Philippines in sera (40-fold) as well as in lung samples (5-fold) from the CT adjuvant-PR8 group at day 4 postchallenge, which was not observed in the PR8-immunized postchallenge group. This increased neutralizing activity correlated with complete protection when naı¨ve mice were infected with high doses of A/Philippines virus (40 LD50) after incubation with immune sera or lung extracts collected at day 4 postchallenge from the CT adjuvant group. Overall, our results indicate that the presence of neutralizing antibodies cross-reactive to the heterosubtypic strain can provide the host with the ability to induce rapid protective immunity upon heterosubtypic viral infection. The CT holotoxin consists of a toxigenic A subunit (CTA) with ribosyltransferase activity and a pentameric B subunit (CTB), which is responsible for CT binding to GM1 gangliosides present on most nucleated cells (5). The ribosyltransferase activity increases the intracellular cyclic AMP acting on several GTP-binding proteins, which may play an important role in its adjuvant property. CT can induce phenotypic and functional maturation of dendritic cells, promoting up-regulation of major histocompatibility complex class II and stimula-
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tory CD86 molecules (2, 10, 11). CT can also activate epithelial cells, inducing the production of chemokines (macrophage chemoattractant protein 1, macrophage inflammatory protein 1␣/, and RANTES), and can augment priming of CD4⫹ T cells secreting cytokines (IFN-␥, interleukin-4 [IL-4], IL-5, and IL-10) and the antigen presentation by dendritic and B cells (9, 15). CT strongly promotes germinal center formation and enhances the development of immunological memory (16, 35, 36), which might have contributed to the increased levels of bone marrow antibody-secreting cells in the CT adjuvant group shown in this study. It is generally considered that the potency of CTB without the ribosyltransferase activity is significantly weaker than that of the intact CT holotoxin. We also observed that CTB was less effective than CT in inducing cross-protective heterosubtypic immunity with the influenza vaccine (data not shown). Thus, inclusion of an effective adjuvant will be an advantage in broadening protective immunity, as shown in this and other studies (34). This approach may improve the efficacy of inactivated influenza virus vaccines, abolish the safety concerns inherent in the use of live virus or viral vector-based vaccines, and importantly, confer protective immunity to viruses with new pandemic potential. ACKNOWLEDGMENTS This work was supported in part by NIH/NIAID grants AI57015 (S.-M.K.) and AI0680003 (R.W.C.). We thank Yumiko Matsuoka for the influenza A/WSN/33 strain and Robert Mittler for anti-CD4 antibody (GK1.5). REFERENCES 1. Armerding, D., H. Rossiter, I. Ghazzouli, and E. Liehl. 1982. Evaluation of live and inactivated influenza A virus vaccines in a mouse model. J. Infect. Dis. 145:320–330. 2. Bagley, K. C., S. F. Abdelwahab, R. G. Tuskan, and G. K. Lewis. 2004. Calcium signaling through phospholipase C activates dendritic cells to mature and is necessary for the activation and maturation of dendritic cells induced by diverse agonists. Clin. Diagn. Lab. Immunol. 11:77–82. 3. Belshe, R. B. 2004. Current status of live attenuated influenza virus vaccine in the US. Virus Res. 103:177–185. 4. Benton, K. A., J. A. Misplon, C. Y. Lo, R. R. Brutkiewicz, S. A. Prasad, and S. L. Epstein. 2001. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or gamma delta T cells. J. Immunol. 166:7437– 7445. 5. Cuatrecasas, P. 1973. Gangliosides and membrane receptors for cholera toxin. Biochemistry 12:3558–3566. 6. Epstein, S. L., C. Y. Lo, J. A. Misplon, C. M. Lawson, B. A. Hendrickson, E. E. Max, and K. Subbarao. 1997. Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T celldepleted, beta2-microglobulin-deficient, and J chain-deficient mice. J. Immunol. 158:1222–1230. 7. Fouchier, R. A., V. Munster, A. Wallensten, T. M. Bestebroer, S. Herfst, D. Smith, G. F. Rimmelzwaan, B. Olsen, and A. D. Osterhaus. 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79:2814–2822. 8. Frank, A. L., and L. H. Taber. 1983. Variation in frequency of natural reinfection with influenza A viruses. J. Med. Virol. 12:17–23. 9. Freytag, L. C., and J. D. Clements. 2005. Mucosal adjuvants. Vaccine 23: 1804–1813. 10. Gagliardi, M. C., F. Sallusto, M. Marinaro, A. Langenkamp, A. Lanzavecchia, and M. T. De Magistris. 2000. Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming. Eur. J. Immunol. 30:2394– 2403. 11. Guebre-Xabier, M., S. A. Hammond, D. E. Epperson, J. Yu, L. Ellingsworth, and G. M. Glenn. 2003. Immunostimulant patch containing heat-labile enterotoxin from Escherichia coli enhances immune responses to injected influenza virus vaccine through activation of skin dendritic cells. J. Virol. 77:5218–5225. 12. Heinen, P. P., E. A. de Boer-Luijtze, and A. T. Bianchi. 2001. Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection. J. Gen. Virol. 82:2697–2707. 13. Hogan, R. J., L. S. Cauley, K. H. Ely, T. Cookenham, A. D. Roberts, J. W. Brennan, S. Monard, and D. L. Woodland. 2002. Long-term maintenance of
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