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The upper respiratory tract is an important site for host defense against invading pathogens, since it is the site at which inhaled antigens first come into contact with the immune system (9). Respiratory viruses such as influenza virus affect primarily the upper and lower respiratory tracts, and viremia does not normally occur. Intranasal immunization can elicit antigen-specific immune responses in both the mucosal and systemic compartments following administration of pathogens and even nonreplicating protein antigens (4, 16, 17). Furthermore, intranasal immunization is an effective means of evoking not only local immunity in the respiratory tract but also immunity at distal mucosal sites (10, 15).

The nasal-associated lymphoid tissues (NALT) in the mouse are composed of a pair of organized lymphoid aggregates (O-NALT) located on the palate at the entrance to the nasopharyngeal duct and the less well organized diffuse lymphoid tissue lining the nasal passages (D-NALT) (9). These nasal tissues appear to be functionally equivalent to the Waldeyer's ring of tonsils and adenoids in the human and are most likely responsible for the local immune responses generated following intranasal immunization in the mouse (14). An indication of the importance of the nasopharyngeal lymphoid tissue in humans is the diminished poliovirus-specific antibody levels in nasal secretions from children following tonsillectomy (11). In humans resistance to infection with a cold-adapted vaccine influenza virus has been correlated with antihemagglutinin (anti-HA) immunoglobulin A (IgA) in nasal washes (3). In the mouse model, following intranasal infection, local antibody-forming cell (AFC) production in the NALT of BALB/c mice parallels detection of influenza virus-specific antibodies in the nasal wash and correlates with virus clearance from the nose (14). Furthermore, nasal IgA has been shown to directly mediate local anti-influenza virus immunity in the mouse model, confirming the importance of IgA in protection against virus infection in the upper respiratory tract (12). Specific AFCs secreting antibody to protein antigens can also be detected in the O-NALT of BALB/c mice after repeated intranasal immunization (18).

Previous work has shown that in BALB/c mice both the O-NALT and the D-NALT are composed of roughly similar ratios of T to B cells (2). The majority of T cells express the T-cell receptor (TCR), with few TCR⁺ T cells (1). Studies to date suggest that the O-NALT is rich in unswitched, naive B cells and naive T cells, suggesting that it is a mucosal inductive site, whereas the D-NALT may function as an effector site. It has also been shown that in the O-NALT CD4⁺ T cells are mainly of the TH0 type and that in the D-NALT the CD4⁺ T cells are predominantly of the TH2 type (6, 18).

It is presently unknown whether either the O-NALT or the D-NALT is able to generate long-lasting humoral immunity to pathogens. Such information would be of great benefit to assess the local effectiveness of delivering potential vaccine candidates by the intranasal route. We have examined the longevity of influenza virus-specific antibody responses in the O-NALT and the D-NALT in the mouse following intranasal influenza virus infection. We show that virus-specific AFCs are generated in both the O-NALT and the D-NALT following exposure to virus. However, the frequency of AFCs was much greater in the D-NALT than in the O-NALT over the course of a primary infection with influenza virus, with a higher number of AFCs continuing to secrete antibody for a longer time period. Moreover, long-term virus-specific antibody was still detectable in the D-NALT 18 months after primary infection, whereas no AFCs were detectable in the O-NALT after approximately 5 months postinfection. These results show that the D-NALT is the major B-cell effector site of the nasal tissues and indicate that local long-term virus-specific antibody generated to influenza virus resides in the AFCs lining the nasal passages.

Experimental procedures. Inbred female C57BL/6 mice were obtained from the Institute of Animal Health, Compton, Berkshire, United Kingdom. All mice were held under specific-pathogen-free conditions and were used at 8 to 12 weeks of age. The HKx31 (H3N2) strain of influenza A virus was grown in the allantoic cavities of 10-day-old embryonated eggs and stored at 70°C until use. Mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (Avertin) and then infected intranasally with 30 µl of phosphate-buffered saline containing 5 × 10⁵ 50% egg infectious doses of virus. Following infection mice were kept in filter-top cages until use. No seroconversion was ever observed in sentinel mice stored in open-top cages placed beside experimental cages. At certain time points postinfection, mice were sacrificed for collection of NALT, cervical lymph nodes, lungs, and bone marrow. In all experiments NALT cells from four or five mice were pooled for each time point, and the entire experimental time course was repeated three times. Cell suspensions were prepared from lymph nodes by gently pressing between frosted slides followed by filtration through gauze. The O-NALT and D-NALT cells were extracted by a previously described method (2). Lymphocytes were extracted from the lungs by mincing the tissue followed by incubation with 4 mg of collagenase A (Boehringer-Mannheim, Indianapolis, Ind.) per ml for 30 min and were recovered from the interface of 75 and 40% Percoll gradients.

Phenotype and cellularity of the NALT after influenza virus infection. Both the O-NALT and the D-NALT cell populations isolated from uninfected C57BL/6 mice are largely (70 to 76%) composed of B lymphoid cells (Table 1). This is in contrast to the case for BALB/c mice, where the percentage of B cells is much lower (45 to 50%) (2). In naive C57BL/6 mice fewer than 2% of the T cells in the D-NALT and O-NALT were TCR⁺, and these percentages did not change significantly after infection (results not shown).

Localized antibody production within the NALT after influenza virus infection. We used the ELISPOT assay to determine the isotypes and longevities of virus-specific antibody responses in the nasal tissues of C57BL/6 mice. In the O-NALT there was a dominant IgA response early after infection, peaking at 8 days postinfection. A total of 214 AFCs/5 × 10⁵ cells were detected, of which around 60% were of the IgA isotype. By 12 days postinfection the response was declining, and only negligible numbers of virus-specific cells were still detectable by 5 months postinfection (Fig. 1B), after which virus-specific AFCs could no longer be detected in the O-NALT. The cervical lymph nodes which drain the NALT area generated IgA and IgG virus-specific AFC responses that were also maintained for approximately 5 months postinfection (Fig. 1A). Similarly, virus-specific AFC responses in the lung, first observed by Jones and Ada (8), also lasted for approximately 5 months postinfection (Fig. 2A).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Virus-specific antibody responses in the D-NALT are maintained for at least 18 months after primary intranasal infection with influenza virus. Mice were sacrificed at certain time points postinfection, and virus-specific AFCs in the cervical lymph nodes (A), O-NALT (B), and D-NALT (C) were enumerated by the ELISPOT assay. Cells were pooled from four or five mice per time point. Each panel is representative of one experimental time course. Each time course was carried out three times independently with similar results. D, day; M, months.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Virus-specific AFC frequencies in the lung (A) and bone marrow (B) after primary intranasal infection with influenza virus. Mice were sacrificed at certain time points postinfection, and virus-specific AFCs in the lung and bone marrow were enumerated by the ELISPOT assay. Cells were pooled from four or five mice per time point. Each panel is representative of one experimental time course. Each time course was carried out three times independently with similar results. D, day; M, months.

Virus is cleared more slowly from the D-NALT than from the lung and O-NALT. Interestingly, we found that unlike the case for the lung, where influenza virus is normally cleared by 10 days postinfection and viral RNA sequences are undetectable by PCR 14 days postinfection with influenza virus (5), the virus is not cleared from the D-NALT until approximately 14 to 17 days after infection (Fig. 3). This may be a reflection of the late influx of CD8⁺ T cells into the D-NALT between days 9 and 15 (Table 1). In contrast, influenza virus is cleared from the O-NALT at around 12 days postinfection. Another study (14), which measured influenza virus in the nasal washes, showed virus completely cleared by day 11 postinfection. By analyzing the nasal tissues themselves, however, we were able to determine a more accurate measure of the virus titer.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Influenza virus is cleared later in the D-NALT than in the O-NALT or the lung. Influenza virus titers were determined by inoculation into the allantoic fluid cavities of 10-day-old embryonated hen eggs. HA activity was assayed 72 h later. Assays were performed in triplicate with three mice per time point, and results are the means and standard deviations from three independent experiments. EID50, 50% egg infective dose; D, day.