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Previous Article | Next Article 
Infection and Immunity, May 2004, p. 2528-2537, Vol. 72, No. 5
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.5.2528-2537.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Simon Y. C. Wong,1* Bin Liang,1 Lisa Hyland,1 Sam Hou,1 E. Arne Høiby,2 and Svein Rune Andersen1,
The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire RG20 7NN, United Kingdom,1 Division of Infectious Disease Control, National Institute of Public Health, Oslo, Norway2
Received 24 December 2003/ Accepted 13 January 2004
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ABSTRACT |
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INTRODUCTION |
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A universal vaccine for meningococcal diseases caused by serogroup B N. meningitidis is currently unavailable due to the poor immunogenicity of its polysaccharide capsule and the antigenic variability of noncapsular surface components of meningococci (41). Serogroup B vaccines based on detergent extracts of meningococcal outer membrane vesicles (DOMVs) have been used in several countries, but the efficacy of intramuscularly administered DOMV vaccines was variable, and DOMV-induced bactericidal antibodies were strain specific (10, 11, 42, 46). Since the nasopharynx is the only natural habitat of meningococci, intranasal (i.n.) immunization with meningococcal antigens has been suggested to be an effective way of inducing both mucosal and systemic immunity. Recent studies of i.n. administered OMVs in mice and humans have provided support for this strategy. Some studies have shown that i.n. immunizations with DOMV vaccines induce long-lasting elevated levels of serum bactericidal antibodies (SBA) in humans (27; M. Fischer, M., J. Holst, I. S. Aaberge, I. L. Haugen, J. L. Burns, B. A. Perkins, and B. Haneberg, 12th Int. Pathogenic Neisseria Conf., abstr. 113, 2000), albeit the proportion of vaccinees with a 
4-fold increase in bactericidal titers was only between 18 and 40% (Fischer et al., 12th Int. Pathogenic Neisseria Conf.). The safety and immunogenicity of native OMVs (NOMVs) administered i.n. in humans have also been demonstrated (19, 34). NOMVs are outer membrane material shed from meningococci during growth that contain relatively large quantities of lipopolysaccharide (LPS; 25 to 50% by weight relative to protein) compared to DOMVs (5 to 8% LPS). Despite the high level of LPS in NOMVs, these preparations have been well tolerated by humans immunized i.n. (19, 34). However, studies in humans have also shown that DOMVs induced significantly lower SBA levels when administered i.n. than via the intramuscular route (27). Therefore, the effectiveness of mucosal OMV-based vaccines needs to be improved. Determining where and how local and systemic immune responses develop after i.n. immunizations would aid in the assessment and design of mucosal meningococcal vaccines.
The nasal associated lymphoid tissues (NALT) play an important role in local immune responses in the upper respiratory tract. In mice, and other rodents, the NALT is divided into the organized and diffuse NALTs (O-NALT and D-NALT, respectively) (6, 7, 35). O-NALT, which has been described as the equivalent of Waldeyer's ring in humans, is the only well-organized mucosal associated lymphoid tissue in the upper respiratory tract. It consists of paired lymphoid cell aggregates located between the columnar epithelium and the palate. O-NALT is an inductive site with similarities to the Peyer's patches, although these two tissues differentially express certain addressins on their high endothelial venules (15). In contrast, D-NALT is composed of lymphoid tissue lining the nasal passages. D-NALT has been described as an effector site equivalent to the lamina propria of the gut. Responses in tissues other than the NALT, including lymph nodes, spleen and bone marrow, are also of importance upon i.n. immunization as they may contribute to the systemic response. Analysis of immune responses after i.n. immunizations or infections in the upper respiratory tract generally show that the magnitude, kinetics, localization, and longevity of the responses vary with the antigens or microorganisms, immunization protocols, and mouse strains used (28, 34, 53, 54). In the present study we examine the characteristics of humoral responses in various sites, including the NALT, lymph nodes, spleen, and bone marrow after immunization with DOMVs and NOMVs by various routes. We describe for the first time the salient features of the NALT humoral responses toward meningococcal OMVs after i.n. immunization, and we discuss factors that can influence the local and systemic antibody responses induced by OMVs.
(This study was presented in part at the 12th International Pathogenic Neisseria Conference in Galveston, Tex., in September 2000.)
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MATERIALS AND METHODS |
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Preparation of NOMVs and DOMVs. NOMVs were prepared from liquid cultures of Mu-4 as described above. Bacterial cells were collected by centrifugation, and the supernatant was concentrated 10-fold by ultrafiltration (filtration units with a 500-kDa cutoff; A/G Technology Corp., Needham, Mass.). Cell debris was removed from the concentrate by centrifugation (10,000 x g for 30 min). NOMVs were isolated by ultracentrifugation (twice at 100,000 x g for 4 h) in order to remove medium components. The NOMV pellet was resuspended in 0.2-µm (pore-size)-filtered deionized water, divided into aliquots, and stored at –80°C. All preparations were tested for sterility prior to use. DOMVs were prepared by detergent extraction as previously described (22).
Mice. Female C57BL/6 and BALB/c (6 to 8 weeks old) mice were obtained from Charles Rivers Laboratories. Mice were maintained under specific-pathogen-free conditions in the animal care facilities of the Institute of Animal Health, Compton, Berkshire, United Kingdom. Experiments on animals were carried out according to the guidelines of the United Kingdom Home Office and the regulations of the Animals (Scientific Procedures) Act of 1986.
Immunizations and collection of samples. Mice were immunized i.n. and intravenously (i.v.) with NOMVs or DOMVs as indicated in the text. Mice immunized i.n. were first anesthetized with a ketamine-xylazine mixture (100 µl) by the intraperitoneal (i.p.) route. Blood and tissue samples were withdrawn on days 11 or 12 and days 30 or 32 unless otherwise indicated in the text.
Preparation of lymphocytes. Cells from the O- and D-NALTs were extracted as previously described by Asanuma et al. (6, 7). Cell suspensions were prepared from the O-NALT, mediastinal lymph nodes (MLN), and spleen by gently pressing the tissues between frosted glass slides. Bone marrow cells were prepared by flushing marrow of bare bones from hind limbs with Iscove modified Dulbecco medium (IMDM) with a 1-ml syringe with a 25-gauge needle. The cells were dispersed in IMDM or IMDM supplemented with 5% (vol/vol) fetal bovine serum (FBS), 100 U of penicillin/ml, and 0.1 mg of streptomycin/ml (cIMDM). Cell suspensions were then filtered through 35-µm-pore-size nylon gauze (BDH, Leicester, United Kingdom) and washed twice with cIMDM (400 x g for 10 min at room temperature). Red blood cells were lysed by incubating cell suspensions for 5 min at room temperature in the presence of red blood cell lysis buffer (Sigma). Cells were pelleted by centrifugation into a 1-ml cushion of FBS, washed twice in cIMDM as described above, and kept on ice until plated out for enzyme-linked immunospot (ELISPOT) assay analysis as described below.
Lung fragment cultures. Antigen-specific antibodies in lung tissues were detected by a modification of a method used to assay antibody secreted from small intestine segments (14). Briefly, after removal of the lungs under sterile conditions, 1-mm duplicate slices of equivalent size were cut from the middle of one lobe and placed in individual wells of a 24-well plate with 1 ml of cIMDM containing 1% Amphoteracin (Gibco, Paisley, United Kingdom). Samples were incubated for 5 days at 37°C in an atmosphere of 95% O2 and 5% CO2. Supernatants were then removed from each well and assayed for the presence of specific antibodies by enzyme-linked immunosorbent assay (ELISA).
ELISA. Microtiter plates (i.e., flat-bottom 96-well Maxisorp plates; Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 µl of NOMVs per well (4 µg of protein/ml) dissolved in 50 mM sodium carbonate-bicarbonate buffer (pH 9.6). Unoccupied protein-binding sites were blocked with 150 µl/well of 0.5% (wt/vol) bovine serum albumin in phosphate-buffered saline (PBS) for 1 h. Primary antibodies were incubated overnight at 4°C and alkaline phosphatase-conjugated goat anti-mouse immunoglobulin isotype-specific or subclass-specific antibodies (used at 1:1000; Southern Biotechnology Associates, Inc., Birmingham, Ala.) were incubated for 2 h at room temperature. Between each incubation step, the plates were washed five times with PBS containing 0.02% Tween 20. The substrate, p-nitrophenylphosphate (Sigma, St. Louis, Mo.), dissolved at 1 mg/ml in a 0.1 M Tris-HCl buffer (pH 9.8) containing 10% (vol/vol) diethanolamine, 0.1 M NaCl and 5 mM MgCl2] was added at 100 µl per well. The enzymatic reaction was terminated after 30 min by the addition of 50 µl/well of 1 M NaOH. The optical density (OD) was measured at 405 nm (Spectra MAX 340 ELISA plate reader; Molecular Devices Corp., Sunnyvale, Calif.). The results are expressed as log2 reciprocal endpoint titers. A cutoff for positive OD values was calculated as the average (arithmetic mean) plus three standard deviations of all dilutions from nonimmunized control mice (n = 3). The endpoint titer was defined as the reciprocal value of the first dilution below the cut off.
ELISPOT assay. Antibody-forming cells (AFCs) were detected by ELISPOT assay as previously described (16, 43). Microtiter plates (96-well nitrocellulose-based filtration plates; Millipore, Watford, United Kingdom) were coated with NOMVs (100 µl per well of 5 µg of protein/ml of 0.2-µm [pore-size]-filtered PBS) and were incubated at 4°C overnight in a humidified chamber. Plates were washed three times with 200 µl of PBS before unoccupied protein binding sites were blocked with 2% (vol/vol) FBS in PBS for 1 h at room temperature. Plates were then washed as previous, and then 100-µl volumes of lymphocyte suspensions (104 to 105 cells/ml) were added to the plates, and these were incubated overnight at 37°C in a humid 5% CO2 atmosphere. Plates were washed successively once with 200 µl of PBS, once with 200 µl of PBS plus 0.1% Tween 20, and three times with 200 µl of PBS. Goat anti-mouse immunoglobulin isotype-specific or subclass-specific antibodies conjugated with alkaline phosphatase (diluted 1:500) in 5% (wt/vol) bovine serum albumin-PBS were used to detect plaques. The secondary antibodies were incubated for 3 h at room temperature. Plates were then washed as described above before incubating them with the substrate BCIP (5-bromo-4-chloro-3-indolylphosphate; Sigma) dissolved at 1 mg/ml in 0.1 M Tris-HCl (pH 9.5), containing 10% (vol/vol) diethanolamine, 0.1 M NaCl, and 5 mM MgCl2. The enzymatic reaction was stopped, after 30 min incubation at room temperature, by washing the plates four times with PBS. Plaques, which are indicative of AFCs, were enumerated by using a direct light microscope in conjunction with an overhead light source.
Serum bactericidal assays. Assays were performed as previously described (42). Briefly, a bacterial inoculum of 70 to 120 CFU was incubated in the presence of 25% human complement in microtiter plates. Twofold dilution series (starting at 1:8) for nonimmunized or immunized sera were tested for SBA against wild-type strain 44/76. The results are presented as the SBA titer, which is defined as reciprocal of the highest dilution in serum causing more than 50% killing of the target strain.
Statistical methods. Statistical significance between groups was determined by the Student t test if the data passed the normality test. The Mann-Whitney test was used for data that failed the normality test.
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RESULTS |
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The number of immunoglobulin A (IgA)-producing AFCs in the D-NALT was 
10-fold higher than the number of AFCs producing the two other isotypes, IgG2a and IgG2b (Fig. 1, middle and bottom panels). In contrast, the response in the MLN, spleen, and bone marrow showed a significantly higher proportion of AFCs producing the various IgG subclasses and IgM. In these three tissues, at both the early and late time point postimmunization, the IgG2b-producing AFCs dominated over IgG2a-specific AFCs in majority of the mice, which in turn dominated over IgG1- and IgG3-specific AFCs.
NOMVs induced a stronger antibody response and a broader range of immunoglobulin isotypes than DOMVs in serum and lung tissue. Serum pools and individual lung fragment culture supernatants (LFCs) from mice in groups 1 and 2 were analyzed by ELISA with NOMVs as the capturing antigen. Serum pools from mice immunized i.n. with NOMVs had higher levels of NOMV-specific antibodies than those from mice immunized i.n. with DOMVs. Serum pools from the latter group contained only significant titers of IgG2b. In contrast, serum pools from NOMV-immunized mice contained relatively high titers of IgG2b in addition to intermediate titers of IgG1 and IgG2a. In these serum pools the IgG1 titers dominated over IgG2a (Fig. 2). Sera from nonimmunized mice did not contain detectable levels of antibodies to NOMVs (data not shown).
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Serum samples from the i.n. immunized group collected on day 28, 7 days after the last dose, showed that the subclasses IgG2a and IgG2b dominated over IgG1 and IgG3 (8-16-fold higher endpoint titer). A relatively lower endpoint titer for IgA and an insignificant titer for IgM were also detected. In contrast, sera from the i.v. immunized group contained equal levels of IgG1, IgG2a, and IgG2b; slightly lower levels of IgG3; and undetectable levels of IgA and IgM (Fig. 5).
Immunization with meningococcal OMVs induced SBA. Protection against meningococcal diseases correlates with the presence of SBA. Figure 6 shows the bactericidal activities of sera from mice immunized with 0 to 3 doses of NOMVs via the i.n. or i.v. route. We found that i.v. immunization with a single dose of NOMVs generated SBA titers of 1:1,024, and this was further increased to more than 1:8,192 with a second dose given 3 weeks later. The SBA titers for the i.p. immunized mice were almost identical to those obtained for the i.v. immunized mice (data not shown). In contrast, i.n. immunized mice required at least three weekly doses of NOMVs to generate SBA titers of 1:1,024. Mice immunized i.n. with one or two doses did not produce SBA titers more than the control mice whose SBA titers were 1:8 or less. Although sufficient amounts of sera for both ELISA and serum bactericidal assay were not obtained from every immunized group of mice used in the present study, it was clear that NOMV-specific antibodies generated via all three routes were highly bactericidal. The highest levels were found in the group immunized i.v.
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DISCUSSION |
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IgA responses play an important role in mucosal defense against pathogens via a number of potential mechanisms (36). The role of IgA in meningococcal infection and disease is unclear. At the nasopharynx, IgA may mediate antibody-dependent cellular cytotoxicity (38) against meningococci in addition to its general role in providing an immune barrier against microbes at mucosal surfaces (36). Antimeningococcal specific IgA, a non-complement-fixing isotype, also has the potential to block complement-mediated lysis of meningococci by IgG or IgM in serum by competing for the same antigens (26). The present study shows that significant levels of IgA-producing AFCs were induced in various tissues with NOMVs or DOMVs after i.n., but not i.v., immunization. The insignificant or very low levels of IgA detected in serum upon i.n. or i.v. immunization are possibly due to the fact that rodents have very efficient polymeric immunoglobulin receptors that rapidly transport IgA from the circulation to mucosal surfaces (44). The IgA/IgG ratio observed in the D-NALT was much higher than in the O-NALT and other lymphoid tissues studied. Mucosal B cells seem to have a preference for immunoglobulin class switch recombination from µ to 
, probably through stimulation with transforming growth factor ß, interleukin-10, and CD40, as indicated by a number of in vitro studies (13, 18, 33, 40, 50). The mechanism for preferential class switching to IgA is not fully understood, but the O-NALT was recently proposed to possess a unique machinery that provides an enrichment of high-affinity IgA- but not IgG-specific cells in the memory compartment (45). The same study also showed that the frequency of IgG2b-expressing cells in the O-NALT which peaked at day 7 and persisted up to day 11, was about three to four times the number of IgA-producing cells (45). In contrast, our study showed that either only IgA-producing AFCs or equal numbers of IgA- and IgG2b-producing AFCs, were found in the O-NALT on day 11 or 12, and no specific AFCs were detected on day 30 or 33. Our results are consistent with a recent report on the isotype profile and frequency of IgA- and IgG2b-producing cells in the NALT after influenza virus infection (37).
The fate of NOMV-specific IgA-producing AFCs was not determined in the present study, but it has been shown that IgA-producing cells activated in the O-NALT were subsequently found in the posterior cervical lymph nodes and in other nonmucosal lymphoid tissues (45). NOMV-specific IgA-producing AFCs were found in significant numbers in the spleen and bone marrow after i.n, but not i.v., immunization. Intriguingly, these cells increased in numbers in the bone marrow but not in the spleen and persisted in both tissues for at least 75 days. These AFCs may contribute to the long-term mucosal and systemic antibody response against meningococcal antigens, since virus-specific long-lived plasma cells found in spleen and bone marrow have been shown to contribute significantly to the long-term systemic antibody response after influenza virus infection in mice (32, 48). Investigation into the source(s) and fate of NOMV-specific IgA AFCs in the NALT would provide further insights into the generation and maintenance of local antibody responses after i.n. immunization with NOMVs.
Both NOMVs and DOMVs elicited very high, and almost equal, frequencies of AFCs in the D-NALT after i.n. immunization, but striking differences in AFC frequencies between these OMVs were observed among the other lymphoid tissues investigated. NOMVs induced a stronger antibody response than DOMVs in the MLN, spleen, bone marrow, lung, and serum. Also, NOMVs induced a broader range of the IgG subclasses, suggesting that they were more potent inducers of B-cell class-switching events than their DOMV counterparts. It is noteworthy that these differences were only observed when the i.n. administered volume was no less than 50 µl (unpublished results). A previous study on the effect of i.n. administered volume on the distribution of microspheres has indicated that the use of 50 µl but not 10 µl given i.n. resulted in the deposition of microspheres at sites other than the nasal cavity (20). Overflow of OMV antigens in the nasal cavity is a possible means by which cells from other immunological sites, such as the lung, were exposed and responded to i.n. administered NOMVs or DOMVs. Our results suggest that the generation of a strong systemic NOMV-specific antibody response by NOMVs via the i.n. route requires the involvement of lymphoid tissues other than the NALT. Similarly, Katial et al. have found that humans immunized both i.n. and oropharyngeally developed higher levels of both IgG and IgA in the nasal fluid, even though the SBA titers from these vaccinees were not different from those who were only immunized i.n. (35). The use of large volumes to enhance systemic antibody responses elicited by i.n. administered OMV vaccines must proceed with caution since excessive inflammation in the lower respiratory tract has been reported for other antigens and adjuvants (29, 47).
The main difference between NOMVs and DOMVs is that the latter contain 5-fold less LPS than the former due to the detergent extraction process used (49). The less-pronounced difference in the D-NALT responses between mice immunized with NOMVs and DOMVs could be explained by the fact that cells associated with the mucosal surface are less susceptible to the biological activity of LPS. Human cervicovaginal and intestinal epithelial cells have recently been found to be hyporesponsive to LPS due to the absence or low-level expression of MD-2 and Toll-like receptor 4 (TLR-4), two pivotal components of LPS-mediated cell signaling (1, 21). Although the levels of MD-2 and TLR-4 on cells in the respiratory tract have not been determined, LPS hyporesponsiveness could be one of the main reasons for the lack of overt or harmful side effects in human volunteers after i.n. immunization with NOMVs (34). Both NOMVs and DOMVs have components with immunostimulatory effects on local and systemic immune responses since strong antibody responses were induced by OMVs in the absence of any added adjuvant (17). Neisserial porins, which are major components in both preparations, have been found to stimulate B cells and upregulate the surface expression of costimulatory B7-2 (CD86) molecules through a TLR-2- and MyD88-dependent signaling mechanism (39). Interestingly, in contrast to TLR-4, TLR-2 seems to be expressed by cells from various mucosal sites (1, 8, 21). Thus, activation of TLR-2-mediated signaling pathways by non-LPS meningococcal components may be required for the induction of a vigorous antibody response in the NALT. In contrast to its apparent lack of endotoxic effect on the NALT, LPS can act as an adjuvant upon parenteral administration and induce cytokines that may influence the immunoglobulin isotypes and subclasses produced (2). It is not known whether non-LPS meningococcal components have similar influence on antibody production.
NOMV-specific antibodies generated by i.n. immunization with OMVs are bactericidal, with high titers of ca. 1:1,024. This represents a 128-fold increase over nonimmune mouse serum controls. However, it is difficult to compare SBA titers from OMV-immunized mice and humans because SBA titers of 1:4 or greater have been considered to be protective in humans (24, 25, 30). Human vaccine trials have also shown that both NOMVs and the DOMV vaccines induced only modest increases in systemic antibody responses and two- to fourfold increases in SBA titers upon i.n. immunization (19, 27, 34). Nevertheless, the mouse immune responses to i.n. administered OMVs suggest similarity to a phenomenon observed in OMV-immunized humans. In mice primed i.n. with four weekly doses of the Norwegian DOMV vaccine, SBA titers did not increase after a new round of i.n. immunizations (9). We have previously made similar observations when mice were immunized with NOMVs (unpublished results). Human adults with moderately high basal levels of SBA titers of 1:8, due most likely to asymptomatic carriage of meningococci in the nasopharynx, do not respond to i.n. OMV vaccines by increasing SBA titers significantly. Interestingly, a significant increase in SBA titer was found in mice primed i.n. and boosted subcutaneously with one dose of DOMV vaccine (9). Whether humans would benefit from this immunization schedule for OMV vaccines remains to be determined.
The present study has identified the D-NALT as the major site of antimeningococcal AFC production after i.n. immunization with OMVs and showed that the kinetics, magnitude, and immunoglobulin isotype of the systemic antibody response were dependent on the form of OMVs being used and the immunization route. With the exception of the NALT where IgA dominated the isotype of OMV-induced antibodies, NOMVs were better than DOMVs at inducing higher antibody levels and class switching to complement-fixing isotypes. The i.n. route required several doses of NOMVs to generate antimeningococcal systemic antibody responses equivalent to one dose of NOMVs via the i.v. or i.p. route. Although it was more difficult to initiate local and systemic anti-NOMV antibody responses via the i.n. route, the established antibody responses were relatively long-lived, and presumably these antibodies are protective as indicated by their SBA titers and immunoglobulin isotypes. In addition to OMV formulation and immunization route, it is important to determine other factors that influence the initiation of antibody responses against meningococcal antigens in the NALT. It will also be important to identify the immunological tissues other than the NALT that are required to be activated in order to achieve a strong systemic immune response in humans. A better understanding of the underlying mechanisms would thus be critically important not only in the development of improved OMV-based vaccines but also in the design of novel mucosal vaccines for meningococcal diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: Department of Pathology and Microbiology, University of Bristol, Bristol, United Kingdom.
Present address: Vaccine Department, Norwegian Medical Agency, Oslo, Norway.
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) 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.