Local and systemic antibody responses in mice immunized intranasally with native and detergent-extracted outer membrane vesicles from Neisseria meningitidis

Abstract

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.

FIG. 1.

Frequencies and isotypes of NOMV-specific AFCs in O-NALT, D-NALT, MLN, spleen, and bone marrow after i.n. immunization with NOMVs or DOMVs. In group 1, C57BL/6 mice (n = 3 per group) were immunized with either NOMVs or DOMVs (15 μg of protein/dose in 50 μl of PBS) on days 0, 7, 14, and 21. In group 2, C57BL/6 mice (n = 3) were immunized on days 0, 7, and 32 with NOMVs (15 μg of protein/dose in 50 μl of PBS). Single cell suspensions from mice in each group were pooled and plated out on ELISPOT plates coated with NOMVs.

FIG. 2.

Isotype and subclass distribution of NOMV-specific antibodies in serum after i.n. immunization with NOMVs or DOMVs. Mice (C57BL/6, n = 3 per group) were immunized as described in Fig. 1. Sera from each group were withdrawn on day 11 or 12 and day 30 or 33, pooled, and assayed for NOMV-specific antibodies by ELISA. The results are expressed as the OD at 405 nm.

FIG. 3.

NOMV-specific antibodies in lung fragment culture supernatants after i.n. immunization with NOMVs or DOMVs. Mice (C57BL/6, n = 3 per group) were immunized as described in Fig. 1. Lung fragments from individual mice (numbered 1 to 3) were collected on day 11 or 12 and day 30 or 33 after initial immunization with NOMVs or DOMVs. Endpoint titers were defined as the first dilution to give an OD value below the cutoff value. The cutoff value was defined as the mean OD of all of the dilutions of a negative control (three nonimmunized mice) plus three standard deviations. n.d., Not determined.

FIG. 4.

Development of AFCs producing NOMV-specific antibodies in spleen and bone marrow after i.n. or i.v. immunization with NOMVs. Mice (BALB/c) were immunized i.n. with 15 μg of NOMVs in 30 μl of PBS under anesthetic on days 0, 7, 14, and 21 (a and b) or i.v. with 5 μg of NOMVs in 100 μl on days 0 and 21 (c and d). Single cell suspensions of spleens (a and c) and bone marrow (b and d) from mice (n = 3 per time point assayed) were collected and analyzed individually by ELISPOT assay with NOMVs as the capturing antigen. The results are expressed as the mean number of AFCs with the standard deviations.

FIG. 5.

Isotype and subclass distribution of anti-NOMV serum antibodies on day 28 after i.n. (a) or i.v. (b) immunization. Mice (BALB/c, n = 3 per group) were immunized as described in Fig. 4. Endpoint titers and cutoff values were defined as described for Fig. 3.

FIG. 6.

Bactericidal activity of sera from NOMV-immunized mice. The effect of the immunization route was examined with mice immunized either i.n. (▪) or i.v. (□) with zero to three doses of NOMVs. Mice were immunized i.n. once a week for a maximum of 3 weeks, and sera were collected 7 or 14 days after the last dose for serum bactericidal assays. Mice immunized i.v. with one dose were bled 3 weeks later, and those immunized with two doses 3 weeks apart were bled 7 days after the second dose for serum bactericidal assays.