Immunization with Live Neisseria lactamica Protects Mice against Menin
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《感染与免疫杂志》
Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom
Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom
ABSTRACT
Natural immunity against Neisseria meningitidis is thought to develop following nasopharyngeal colonization with this bacterium or other microbes expressing cross-reactive antigens. Neisseria lactamica is a commensal of the upper respiratory tract which is often carried by infants and young children; epidemiological evidence indicates that colonization with this bacterium can elicit serum bactericidal activity (SBA) against Neisseria meningitidis, the most validated correlate of protective immunity. Here we demonstrate experimentally that immunization of mice with live N. lactamica protects animals against lethal meningococcal challenge and that some, but not all, strains of N. lactamica elicit detectable SBA in immunized animals regardless of the serogroup of N. meningitidis. While it is unlikely that immunization with live N. lactamica will be implemented as a vaccine against meningococcal disease, understanding the basis for the induction of cross-protective immunity and SBA should be valuable in the design of subunit vaccines for the prevention of this important human infection.
INTRODUCTION
Neisseria meningitidis is a gram-negative bacterium that is found in the nasopharynx in 10 to 40% of healthy adults (10, 45, 53). Through unknown mechanisms, certain strains of this bacterium can penetrate the mucosal epithelium and gain access to the bloodstream to cause invasive infection, which continues to be a major public health problem. There are approximately 1.2 million cases of disseminated meningococcal infection each year worldwide, leading to an estimated 135,000 deaths (4). Therefore, there is a pressing need to develop new strategies to reduce the incidence, mortality, and morbidity of invasive meningococcal disease. The sporadic nature, rapid onset, nonspecific initial presentation, and relentless progression of the disease in some patients make meningococcal infection difficult to diagnosis and treat effectively. Prophylactic vaccination potentially offers the most effective method to reduce the burden of disease caused by this important human pathogen (13, 26).
Thirteen serogroups of the meningococci have been identified, based on the chemical and antigenic differences of their capsular polysaccharide (51). Five serogroups (A, C, B, W135, and Y) account for the overwhelming majority of human disease, and there are currently licensed vaccines available against four of these five serogroups (i.e., A, C, Y, and W135) which are directed at their capsular antigens (26). Unfortunately, there is still no universal vaccine against serogroup B strains, the most common cause of meningococcal disease in developed countries, although there has been success with outer membrane vesicle (OMV) vaccines to prevent infections with caused by a single strain (24). Strategies based on the serogroup B capsule, a polymer of 1-8-linked sialic acid, are hampered by its poor immunogenicity and relatedness to a modification of a human neural cell adhesion molecule, NCAM-1 (17, 18). Instead, approaches for developing vaccines against serogroup B infections have focused on surface-exposed, noncapsular antigens including lipopolysaccharide and outer membrane proteins, either individually or in complex preparations such as OMVs (37-41). Several OMV vaccines have undergone clinical trials but are limited by their restricted cross-reactivity against a range of meningococcal isolates and inconsistent immunogenicity among children under 4 years old, the most vulnerable group (7, 48).
The genetic and antigenic diversity of N. meningitidis is a further significant obstacle for the development of vaccines (13). For example, responses against OMVs are mainly directed at highly variable surface proteins and are usually effective against only a subset of closely related strains or those expressing the same variants of surface antigens as in the OMVs. A recent approach based on bacterial genome sequences, termed reverse vaccinology, has identified several promising candidates that elicit the bactericidal antibodies in mice (19). To date, these methods have not led to the production of an effective vaccine against serogroup B N. meningitidis. Therefore, alternative strategies are required for comprehensive vaccines that protect individuals against N. meningitidis disease.
Host defense against meningococcal disease is dependent on both humoral and cellular immune responses (8, 26). Most evidence indicates that the primary mechanism of protection against meningococcal disease is antibody- and complement-mediated bacteriolysis and/or opsonophagocytosis. Protective antibodies can be detected in vitro by measuring serum bactericidal activity (SBA) and using an opsonophagocytosis assay (OPA). Bacterial lysis in the presence of bactericidal antibodies follows the insertion of the membrane attack complex of the complement system into the bacterial outer membrane (21), while the OPA measures the uptake and/or intracellular destruction of N. meningitidis by phagocytic cells (31).
Mucosal colonization with a complex microbial ecosystem is often necessary for the development and maturation of natural immunity against infectious agents. The development of natural immunity against meningococcal disease occurs in all populations, with protection thought to be elicited by nasopharyngeal carriage of N. meningitidis or nonpathogenic Neisseria spp. (53) and other bacteria expressing cross-reactive antigens. Neisseria lactamica is a commensal of the human upper respiratory tract that is closely related to N. meningitidis (5, 47), and colonization with N. lactamica is proposed to promote immunity against the meningococcus (20). N. lactamica is often carried by infants and young children, but colonization rates then decline with age (20); this is the converse of age-specific carriage rates for N. meningitidis (10). However, levels of serum bactericidal antibodies against the meningococcus rise steadily during infancy despite low rates of N. meningitidis carriage. In a longitudinal study, individuals colonized with N. lactamica developed SBA against several serogroups of meningococcus, suggesting that strains of N. lactamica can induce protective immune responses against N. meningitidis (20, 44, 50). Furthermore, it has been shown recently that immunization of mice with OMVs derived from N. lactamica induces protective immunity in the absence of detectable SBA (22, 36).
In the present study, we provide further evidence that N. lactamica contributes to protective immunity against meningococcal infection. Using a murine challenge model, we demonstrate that systemic immunization with N. lactamica generates cross-protective immunity against meningococcal infection and detectable SBA against three important serogroups of N. meningitidis.
MATERIALS AND METHODS
Bacterial strains and growth conditions. N. lactamica strain L13 was obtained from the National Collection of Type Cultures, London, United Kingdom, while strain L18 was from the Centre for Disease Control, Ottawa, Canada. N. lactamica Y92-1009 (sequence type 613 complex 3) was originally isolated during a carriage study in Northern Ireland and was from the Meningococcal Reference Unit, Manchester, United Kingdom. N. meningitidis serogroup A (Z2491), B (MC58), and C (FAM18) strains used this study were isolated from patients with meningococcal disease (11, 49, 52). The live attenuated N. meningitidis strain YH102 (MC58 siaD rfaF) has been described previously (32). All bacteria were grown on brain heart infusion (BHI) plates with Levanthal's supplement in 5% CO2 at 37°C overnight.
Immunization with live N. lactamica or N. meningitidis. Groups of 10 to 15 6-week old female BALB/c mice (Harlan) were immunized on days 1, 21, and 28 by intraperitoneal (i.p.) injection of N. lactamica or N. meningitidis. Bacteria were grown overnight on a solid medium and then harvested into phosphate-buffered saline (PBS). The number of CFU was estimated by measuring the absorbance at 260 nm of a lysate of the suspension in 1% sodium dodecyl sulfate (SDS)-0.1% NaOH, and the number of viable bacteria was confirmed by plating to solid media. Animals received 1 x 106 CFU of either N. lactamica or N. meningitidis in BHI medium containing 0.5% (vol/vol) iron dextran (Sigma, Poole, United Kingdom). Control groups consisted of mice given BHI medium with iron dextran alone. On day 43, the animals were challenged with 5 x 106 CFU of the serogroup B N. meningitidis strain MC58. Blood was collected from animals on day 0 by tail vein bleed or on day 35 by cardiac puncture under terminal anesthesia. All animal experiments were carried out under protocols reviewed and approved by the Home Office, United Kingdom. A one-tailed Student's t test was used to detect statistically significant differences in survival.
Whole-cell ELISAs. Whole-cell enzyme-linked immunosorbent assays (ELISAs) were performed to detect antibodies in serum binding to N. meningitidis by previously described protocols with minor modifications (32). Briefly, N. meningitidis and N. lactamica were grown on solid medium, resuspended in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6 [catalog no. 3041; Sigma]), and then heat inactivated at 56°C for 60 min (50). Bacteria were added to the wells of microtiter plates and left overnight at 4°C. After washing with PBS-0.1% Tween 20 (PBS-T), dilutions of control or immune sera were added to the wells, which were incubated for 2 h, and the plate was washed with PBS-T. The binding of antibodies was detected with a horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (1:200; DAKO, Cambridgeshire, United Kingdom). After a final wash with PBS-T, o-phenylenediamine (Sigma, Poole, United Kingdom) was added for color development at room temperature and readings were taken at 492 nm. All assays were performed in duplicate, and readings for replicate samples analyzed on separate occasions were within 10% of each other. Results were considered positive when the optical density (OD) was more than three times above the maximum value from either wells with no antigen added or those with sera taken before immunization or following immunization with medium alone.
Preparation of bacterial lysates and Western blot analysis. Bacteria were grown overnight, collected, washed, and then adjusted to an OD at 600 nm (OD600) of 6 in PBS. The cells were boiled in the presence of loading buffer (1:1 [vol/vol], 50 mM Tris, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol) for 10 min. The samples were resolved by polyacrylamide gel electrophoresis on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Stonehouse, United Kingdom) (12). After blocking in PBS-T containing 5% dry milk at 4°C overnight, membranes were incubated with control or immune sera for 2 h at room temperature. Membranes were briefly washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:200; DAKO, Cambridgeshire, United Kingdom) for another 2 h at room temperature. Following washing, reactivity was detected using the ECL enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
FACS analysis. Strains were grown as described above, collected, fixed in 3% paraformaldehyde for 15 min at room temperature, and then washed three times with PBS. Next, 107 CFU were incubated with dilutions of control or immune sera in a total volume of 100 μl for 30 min at 37°C, washed twice, and then resuspended in PBS-T containing a fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse polyclonal antibody (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc., Bath, United Kingdom) and incubated for 30 min on ice. After washing in PBS-T, fluorescence was measured using a FACSCalibur fluorescence-activated cell sorter (Becton Dickson), recording at least 104 events. Results are presented as the percentage of gated cells; the gate was set at around 5% of cells after incubation in preimmune serum.
Opsonophagocytosis assay. N. meningitidis was grown overnight, harvested, washed with PBS, and then killed by incubation in 3% paraformaldehyde overnight at 4°C. Bacteria were labeled by incubation in 0.25 mg/ml FITC (Fluka BioChemika, Poole, United Kingdom), washed thoroughly in PBS, resuspended in 10% glycerol in PBS, and kept at –80°C until use. HL-60 is a polymorphonuclear neutrophil leukocyte (PMN) cell line derived from a patient with acute promyelocytic leukemia (European Collection of Cell Culture) (9). Cells were differentiated by incubation for 5 days in Hanks' balanced salt solution medium (Gibco, Paisley, United Kingdom) with 0.2% (vol/vol) bovine serum albumin (BSA). For opsonophagocytosis, 20 μl of bacteria (concentration, 108 CFU/ml) was added to the same volume of control or immune serum diluted 1:20 in PBS in the wells of a microtiter plate. The plate was then incubated for 30 min at 37°C with gentle agitation, and then 10 μl of rabbit complement (Mast Diagnostics) was added to each well followed by another incubation for 15 min. Finally, 40 μl of a suspension of differentiated HL-60 cells (concentration, 2.5 x 106/ml) was added to the wells and incubated for 30 min. The reaction was stopped by the addition of 160 μl of ice-cold Hanks' balanced salt solution, and the contents of each well was transferred to an Eppendorf tube containing 400 μl of ice-cold Dulbecco's PBS with 0.02% (wt/vol) EDTA. Samples were kept on ice until analyzed by flow cytometry; at least 104 cells from each sample were evaluated with a horizontal fluorescence gate set to include 5% of the population of cells incubated in the absence of serum. The relative fluorescence index (FI) was calculated (FI = percentage of gated cells multiplied by the geometric mean fluorescence); significant differences were detected with a Student's t test. The FI ratio was calculated by taking the mean of FI of each serum and dividing it by the mean FI of the complement-only controls. Thus, background (complement-only) levels of phagocytosis have a value of 1. All opsonophagocytosis experiments were repeated on at least two occasions.
SBA. Serum bactericidal activity was measured according to internationally standardized methods (8, 25). Twofold dilutions of sera were prepared in sterile 96-well microtiter plates to which were added N. meningitidis strains with rabbit complement (Pel-freeze, Brown Deer, Wis.) and incubated for 1 h. The number of surviving bacteria was determined by plating aliquots of the suspension to solid medium. End point titers were calculated as the reciprocal of the dilution of serum yielding more than 50% bacterial killing; the lower limit of detection for SBA was 4. Each assay included sera from animals before immunization or those immunized with growth medium alone and sera from those vaccinated with a live attenuated N. meningitidis strain. Assays were performed at least in triplicate.
RESULTS
Immunization with live N. lactamica protects against live bacterial challenge. To establish whether live N. lactamica can confer protective immunity, groups of mice (15 animals per group) received 106 CFU of either N. lactamica L13 or L18 by i.p. administration on days 0, 21, and 28. Control groups were given either medium alone or a characterized live attenuated N. meningitidis strain, YH102 (32). On day 43, animals were challenged with live serogroup B N. meningitidis, MC58. As described previously, all animals immunized with the attenuated N. meningitidis strain survived challenge with the homologous strain (32). Vaccination with both live N. lactamica strains, L13 and L18, conferred partial yet significant protection compared with that in animals that received medium alone (Fig. 1; P = 0.022 and 0.034 for L13 and L18, respectively).
Cross-reactive antibodies raised against N. lactamica recognize antigens on the meningococcal cell surface. Next we established whether antibodies elicited by immunization with N. lactamica recognized cross-reactive epitopes on representatives of three different serogroups of N. meningitidis. Initially whole-cell ELISAs were performed to determine whether immune sera recognized antigens on heat-inactivated bacteria. As expected, the highest titers were detected against the homologous N. lactamica strain used for immunization (Table 1). However, high levels of anti-N. meningitidis antibodies were also detected in sera from animals immunized with N. lactamica, with similar antibody titers found against the meningococcal strains regardless of serogroup (Table 1). Titers were comparable to those obtained with sera from animals immunized with a live attenuated serogroup B N. meningitidis strain, YH102. The cross-reactivity of the antibodies was further demonstrated by Western blot analysis of whole-cell lysates. Multiple bands were recognized in lysates from serogroup A, B, and C meningococcal strains by sera raised against N. lactamica (Fig. 2); no bands were detected with preimmune sera or sera from animals that had received medium alone (not shown). Of note, the profiles of N. meningitidis proteins detected by sera raised against N. lactamica L13 and L18 were distinct. This is most obvious when comparing low-molecular-mass antigens (Fig. 2).
As whole-cell ELISAs and Western blot analysis also can detect cytoplasmic antigens, we undertook flow cytometry analysis to determine whether antibodies in immune sera recognize antigens on intact N. meningitidis. Bacteria were incubated with antisera, and immunoglobin binding was detected with an FITC-conjugated secondary antibody. As described previously (32), flow cytometry demonstrated that immunization with the live attenuated meningococcal strain elicited antibodies that recognized surface antigens on N. meningitidis (Fig. 3). Furthermore, antibody binding was clearly detected to all serogroups of N. meningitidis (witnessed by an obvious shift in the population of cells upon incubation with the sera from immunized animals), compared with preimmune sera from the same animals (Fig. 3). Binding to the serogroup C strain, FAM18, was less marked than to the serogroup A and B strains.
Immunologic correlates of protection elicited by live N. lactamica. Next we next investigated the functional activity of sera from animals vaccinated with live N. lactamica. Initially we examined whether heat-inactivated sera possessed opsonophagocytic activity. Unfortunately there was considerable nonopsonic uptake by the PMN cell line HL-60 of the serogroup A and C strains, FAM18 and Z2491, respectively (not shown), making it difficult to assess the effect of serum or complement on the uptake of these isolates. Therefore, all subsequent experiments were performed with the serogroup B strain, MC58. Sera from animals immunized with the live N. lactamica strain L13 promoted the opsonophagocytosis of serogroup B N. meningitidis at levels similar to those produced by immunization with the homologous strain (Fig. 4); the degree of opsonophagocytosis induced by vaccination with L18 was less marked than those induced by with immunization with the other strains.
SBA is a proven immunological correlate for protective immunity of individuals against serogroup C meningococcal infection (21), and SBA is also important for immunity against serogroup B strains (8). Therefore, we determined whether protected animals had detectable SBA against serogroup A, B, and C meningococcal strains. Preimmune sera and sera from animals in the negative control group (immunized with medium only) failed to mediate killing of any strains (titers of <4). As shown in Table 2, immunization with N. lactamica strain L13 elicited significant serum bactericidal antibodies against all three meningococcal strains; levels were similar to those raised by live attenuated N. meningitidis, except for titers against the homologous strain, MC58. In contrast, there was no detectable SBA among animals receiving N. lactamica L18. This lack of SBA in animals vaccinated with N. lactamica L18 was confirmed with five other serogroup B (electrophoretic type [ET] and ST included) strains (C311, ET-5, ST unknown; BZ83, ET-5, ST-32; H44/76, ET-5, ST-32; BZ169, ET-5, ST-32; and BZ232, ET unknown, ST-38), while sera from animals immunized with L13 had detectable SBA against all of these strains except BZ232 (data not shown).
Previous work has shown that OMVs derived from another strain of N. lactamica, Y92-1009, elicit protection in mice against disseminated meningococcal disease without detectable SBA (36). Therefore, we next investigated the basis for the failure of OMVs from this strain to elicit SBA. Groups of animals were immunized with live Y92-1009 via the intraperitoneal route as above. Antibodies in sera from immunized animals recognized surface antigens on serogroup A, B, and C N. meningitidis strains by ELISA (not shown) and FACS (percentages of gated cells of 42.7%, 31.6%, and 53.3% for MC58, FAM18, and Z2491, respectively). Furthermore, similar to L13, vaccination with this strain was able to elicit both SBA (Table 2) and opsonophagocytic activity (not shown).
DISCUSSION
This study provides further experimental data to support the notion that infection with N. lactamica promotes the natural immunity against N. meningitidis which develops progressively throughout childhood. Longitudinal studies have provided strong epidemiological evidence indicating that antigens on the surface of this commensal can elicit bactericidal antibodies against the meningococcus in nonimmune hosts (20); this is demonstrated here experimentally, and the results should aid in defining the antigens that mediate this cross-protective response.
Although closely related to the meningococcus, N. lactamica does not express a polysaccharide capsule (29). This may be a significant advantage for the development of natural immunity, as the capsule is widely considered to be an unsuitable antigen for preventing serogroup B infections and expression of the capsule could impede the immunogenicity of other surface antigens. Furthermore, N. lactamica does not express PorA, a highly immunogenic and, therefore, highly variable protein on the surface of N. meningitidis (42, 43). Vaccine preparations that contain PorA (such as certain OMVs) tend to elicit responses which are largely directed at this protein and can thus be limited by their lack of coverage against strains expressing other PorA variants (39). Immunization with N. lactamica products therefore has several potential immunologic advantages over preparations of N. meningitidis and could be used to replicate and accelerate the development of natural immunity (23). The sporadic carriage with this commensal during early childhood may explain the susceptibility of some individuals to meningococcal disease; population-based vaccination programs could be used to ensure that coverage is comprehensive. However, there is a potential drawback to using N. lactamica products to prevent meningococcal disease. If the vaccine proved substantially more effective at preventing carriage with N. lactamica than N. meningitidis, the vaccine might afford only partial protection against meningococcal carriage and disease while entirely abolishing the beneficial effects of colonization with circulating N. lactamica.
The only validated immunologic correlate of protection against meningococcal disease is detectable SBA (3, 8, 21). Importantly for vaccine development, SBA is a ready marker of immunity for preclinical assessment of vaccines and provides a suitable end point in clinical trials. The importance of opsonophagocytic killing as a defense mechanism against the meningococcus has been demonstrated recently, with titers of opsonophagocytosis of beads coated with capsular antigen shown to be significantly correlated with SBA directed against capsular antigens (34). In the present study, we have shown that systemic immunization with two strains of N. lactamica (L13 and Y92-1009) elicits high-level bactericidal activity in mice against N. meningitidis strains, including the serogroup B strain, MC58. There are two important implications from this result. First, N. lactamica L18 failed to elicit bactericidal antibodies against any of three meningococcal strains even though the bacterium was given to animals by using precisely the same schedule as the other strains. This indicates that the profiles of antigens expressed by strains L13 and Y92-1009 are distinct from those produced by L18; therefore, N. lactamica strains differ in their immunogenic properties, and it is likely that not all strains can confer protection against N. meningitidis. The likely explanation for this is the considerable genetic diversity among N. lactamica as evidenced by both multilocus sequence typing and differences in gene content and antigen expression (2, 6). This is reflected in the meningococcal antigens recognized by immune sera raised against N. lactamica L13 and L18 (seen in Fig. 2). While immune sera against L13 and L18 reacted with multiple antigens by Western blot analysis, the profile of N. meningitidis proteins detected was distinct, and these differences may account for whether the N. lactamica strains elicit SBA or not. Therefore, knowledge of the antigenic profile of strains of N. lactamica is vital when deciding upon the choice of strain for N. lactamica-based vaccines against N. meningitidis. Of the N. lactamica strains we examined, L13 and Y92-1009 elicited functional responses against the three meningococcal strains. Second, we found that N. lactamica Y92-1009 elicited SBA when administered as a live organism, in contrast to when OMVs or killed cells from this bacterium were used to immunize animals. This could be explained by different routes of administration of the vaccine or different mouse genotypes used in this and a previous study (36). However, the most probable reason for this difference in the immune response is that the antigenic profile of the bacterium grown in vivo is distinct from that during growth in vitro when OMVs or killed whole cells were prepared. The coordinated control of gene expression is vital for microbial virulence (16), and the profile of antigens on the surface of pathogens varies at sites in the host and differs considerably during growth in the laboratory (46).
Although this study provides proof in principle that systemic immunization with live N. lactamica can induce SBA, this is not necessarily the optimal approach for preventing meningococcal infection with vaccines based on this commensal. Systemic immunization with a live organism can be reactogenic, and there have been reported cases of endocarditis and meningitis with N. lactamica (14, 30). Furthermore, there is clear evidence of horizontal DNA transfer between commensal and pathogenic species of Neisseria (15, 33), raising the concern that a vaccine strain could acquire virulence traits from pathogenic species. A more feasible approach might be the mucosal administration of N. lactamica; further studies are under way to determine the susceptibility of experimental animals to colonization with this commensal and its ability to generate systemic immune responses. N. lactamica is known to harbor genes related to those necessary for expression of type IV pili (1), which mediate host-specific adhesion of the meningococcus to human cells (35). Therefore colonization with N. lactamica may be inefficient unless the experimental host expresses an appropriate form of CD46 (27), the receptor proposed to be recognized by type IV pili (28).
Of much more potential value for vaccine design is that our findings demonstrate that strains of N. lactamica differ in their ability to produce functional immune responses against N. meningitidis. Understanding the basis for this observation should lead to the identification of those N. lactamica antigens that mediate cross-reactive protective immunity and the development of natural immunity. Additionally, defining the reason why live bacteria, but not OMVs or heat-killed bacteria, can elicit SBA in immunized animals may also help to pinpoint important antigens. These could then be incorporated into subunit vaccines for protecting of individuals against disseminated meningococcal infection.
ACKNOWLEDGMENTS
We are grateful to Rachel Exley for critical review of the manuscript.
This work was supported by the Meningitis Research Foundation.
FOOTNOTES
Corresponding author. Mailing address: Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom. Phone: (44) 207-594-3072. Fax: (44) 207-594-3076. E-mail: c.tang@imperial.ac.uk.
Published ahead of print on 11 September 2006.
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Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom
ABSTRACT
Natural immunity against Neisseria meningitidis is thought to develop following nasopharyngeal colonization with this bacterium or other microbes expressing cross-reactive antigens. Neisseria lactamica is a commensal of the upper respiratory tract which is often carried by infants and young children; epidemiological evidence indicates that colonization with this bacterium can elicit serum bactericidal activity (SBA) against Neisseria meningitidis, the most validated correlate of protective immunity. Here we demonstrate experimentally that immunization of mice with live N. lactamica protects animals against lethal meningococcal challenge and that some, but not all, strains of N. lactamica elicit detectable SBA in immunized animals regardless of the serogroup of N. meningitidis. While it is unlikely that immunization with live N. lactamica will be implemented as a vaccine against meningococcal disease, understanding the basis for the induction of cross-protective immunity and SBA should be valuable in the design of subunit vaccines for the prevention of this important human infection.
INTRODUCTION
Neisseria meningitidis is a gram-negative bacterium that is found in the nasopharynx in 10 to 40% of healthy adults (10, 45, 53). Through unknown mechanisms, certain strains of this bacterium can penetrate the mucosal epithelium and gain access to the bloodstream to cause invasive infection, which continues to be a major public health problem. There are approximately 1.2 million cases of disseminated meningococcal infection each year worldwide, leading to an estimated 135,000 deaths (4). Therefore, there is a pressing need to develop new strategies to reduce the incidence, mortality, and morbidity of invasive meningococcal disease. The sporadic nature, rapid onset, nonspecific initial presentation, and relentless progression of the disease in some patients make meningococcal infection difficult to diagnosis and treat effectively. Prophylactic vaccination potentially offers the most effective method to reduce the burden of disease caused by this important human pathogen (13, 26).
Thirteen serogroups of the meningococci have been identified, based on the chemical and antigenic differences of their capsular polysaccharide (51). Five serogroups (A, C, B, W135, and Y) account for the overwhelming majority of human disease, and there are currently licensed vaccines available against four of these five serogroups (i.e., A, C, Y, and W135) which are directed at their capsular antigens (26). Unfortunately, there is still no universal vaccine against serogroup B strains, the most common cause of meningococcal disease in developed countries, although there has been success with outer membrane vesicle (OMV) vaccines to prevent infections with caused by a single strain (24). Strategies based on the serogroup B capsule, a polymer of 1-8-linked sialic acid, are hampered by its poor immunogenicity and relatedness to a modification of a human neural cell adhesion molecule, NCAM-1 (17, 18). Instead, approaches for developing vaccines against serogroup B infections have focused on surface-exposed, noncapsular antigens including lipopolysaccharide and outer membrane proteins, either individually or in complex preparations such as OMVs (37-41). Several OMV vaccines have undergone clinical trials but are limited by their restricted cross-reactivity against a range of meningococcal isolates and inconsistent immunogenicity among children under 4 years old, the most vulnerable group (7, 48).
The genetic and antigenic diversity of N. meningitidis is a further significant obstacle for the development of vaccines (13). For example, responses against OMVs are mainly directed at highly variable surface proteins and are usually effective against only a subset of closely related strains or those expressing the same variants of surface antigens as in the OMVs. A recent approach based on bacterial genome sequences, termed reverse vaccinology, has identified several promising candidates that elicit the bactericidal antibodies in mice (19). To date, these methods have not led to the production of an effective vaccine against serogroup B N. meningitidis. Therefore, alternative strategies are required for comprehensive vaccines that protect individuals against N. meningitidis disease.
Host defense against meningococcal disease is dependent on both humoral and cellular immune responses (8, 26). Most evidence indicates that the primary mechanism of protection against meningococcal disease is antibody- and complement-mediated bacteriolysis and/or opsonophagocytosis. Protective antibodies can be detected in vitro by measuring serum bactericidal activity (SBA) and using an opsonophagocytosis assay (OPA). Bacterial lysis in the presence of bactericidal antibodies follows the insertion of the membrane attack complex of the complement system into the bacterial outer membrane (21), while the OPA measures the uptake and/or intracellular destruction of N. meningitidis by phagocytic cells (31).
Mucosal colonization with a complex microbial ecosystem is often necessary for the development and maturation of natural immunity against infectious agents. The development of natural immunity against meningococcal disease occurs in all populations, with protection thought to be elicited by nasopharyngeal carriage of N. meningitidis or nonpathogenic Neisseria spp. (53) and other bacteria expressing cross-reactive antigens. Neisseria lactamica is a commensal of the human upper respiratory tract that is closely related to N. meningitidis (5, 47), and colonization with N. lactamica is proposed to promote immunity against the meningococcus (20). N. lactamica is often carried by infants and young children, but colonization rates then decline with age (20); this is the converse of age-specific carriage rates for N. meningitidis (10). However, levels of serum bactericidal antibodies against the meningococcus rise steadily during infancy despite low rates of N. meningitidis carriage. In a longitudinal study, individuals colonized with N. lactamica developed SBA against several serogroups of meningococcus, suggesting that strains of N. lactamica can induce protective immune responses against N. meningitidis (20, 44, 50). Furthermore, it has been shown recently that immunization of mice with OMVs derived from N. lactamica induces protective immunity in the absence of detectable SBA (22, 36).
In the present study, we provide further evidence that N. lactamica contributes to protective immunity against meningococcal infection. Using a murine challenge model, we demonstrate that systemic immunization with N. lactamica generates cross-protective immunity against meningococcal infection and detectable SBA against three important serogroups of N. meningitidis.
MATERIALS AND METHODS
Bacterial strains and growth conditions. N. lactamica strain L13 was obtained from the National Collection of Type Cultures, London, United Kingdom, while strain L18 was from the Centre for Disease Control, Ottawa, Canada. N. lactamica Y92-1009 (sequence type 613 complex 3) was originally isolated during a carriage study in Northern Ireland and was from the Meningococcal Reference Unit, Manchester, United Kingdom. N. meningitidis serogroup A (Z2491), B (MC58), and C (FAM18) strains used this study were isolated from patients with meningococcal disease (11, 49, 52). The live attenuated N. meningitidis strain YH102 (MC58 siaD rfaF) has been described previously (32). All bacteria were grown on brain heart infusion (BHI) plates with Levanthal's supplement in 5% CO2 at 37°C overnight.
Immunization with live N. lactamica or N. meningitidis. Groups of 10 to 15 6-week old female BALB/c mice (Harlan) were immunized on days 1, 21, and 28 by intraperitoneal (i.p.) injection of N. lactamica or N. meningitidis. Bacteria were grown overnight on a solid medium and then harvested into phosphate-buffered saline (PBS). The number of CFU was estimated by measuring the absorbance at 260 nm of a lysate of the suspension in 1% sodium dodecyl sulfate (SDS)-0.1% NaOH, and the number of viable bacteria was confirmed by plating to solid media. Animals received 1 x 106 CFU of either N. lactamica or N. meningitidis in BHI medium containing 0.5% (vol/vol) iron dextran (Sigma, Poole, United Kingdom). Control groups consisted of mice given BHI medium with iron dextran alone. On day 43, the animals were challenged with 5 x 106 CFU of the serogroup B N. meningitidis strain MC58. Blood was collected from animals on day 0 by tail vein bleed or on day 35 by cardiac puncture under terminal anesthesia. All animal experiments were carried out under protocols reviewed and approved by the Home Office, United Kingdom. A one-tailed Student's t test was used to detect statistically significant differences in survival.
Whole-cell ELISAs. Whole-cell enzyme-linked immunosorbent assays (ELISAs) were performed to detect antibodies in serum binding to N. meningitidis by previously described protocols with minor modifications (32). Briefly, N. meningitidis and N. lactamica were grown on solid medium, resuspended in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6 [catalog no. 3041; Sigma]), and then heat inactivated at 56°C for 60 min (50). Bacteria were added to the wells of microtiter plates and left overnight at 4°C. After washing with PBS-0.1% Tween 20 (PBS-T), dilutions of control or immune sera were added to the wells, which were incubated for 2 h, and the plate was washed with PBS-T. The binding of antibodies was detected with a horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (1:200; DAKO, Cambridgeshire, United Kingdom). After a final wash with PBS-T, o-phenylenediamine (Sigma, Poole, United Kingdom) was added for color development at room temperature and readings were taken at 492 nm. All assays were performed in duplicate, and readings for replicate samples analyzed on separate occasions were within 10% of each other. Results were considered positive when the optical density (OD) was more than three times above the maximum value from either wells with no antigen added or those with sera taken before immunization or following immunization with medium alone.
Preparation of bacterial lysates and Western blot analysis. Bacteria were grown overnight, collected, washed, and then adjusted to an OD at 600 nm (OD600) of 6 in PBS. The cells were boiled in the presence of loading buffer (1:1 [vol/vol], 50 mM Tris, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol) for 10 min. The samples were resolved by polyacrylamide gel electrophoresis on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Stonehouse, United Kingdom) (12). After blocking in PBS-T containing 5% dry milk at 4°C overnight, membranes were incubated with control or immune sera for 2 h at room temperature. Membranes were briefly washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:200; DAKO, Cambridgeshire, United Kingdom) for another 2 h at room temperature. Following washing, reactivity was detected using the ECL enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
FACS analysis. Strains were grown as described above, collected, fixed in 3% paraformaldehyde for 15 min at room temperature, and then washed three times with PBS. Next, 107 CFU were incubated with dilutions of control or immune sera in a total volume of 100 μl for 30 min at 37°C, washed twice, and then resuspended in PBS-T containing a fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse polyclonal antibody (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc., Bath, United Kingdom) and incubated for 30 min on ice. After washing in PBS-T, fluorescence was measured using a FACSCalibur fluorescence-activated cell sorter (Becton Dickson), recording at least 104 events. Results are presented as the percentage of gated cells; the gate was set at around 5% of cells after incubation in preimmune serum.
Opsonophagocytosis assay. N. meningitidis was grown overnight, harvested, washed with PBS, and then killed by incubation in 3% paraformaldehyde overnight at 4°C. Bacteria were labeled by incubation in 0.25 mg/ml FITC (Fluka BioChemika, Poole, United Kingdom), washed thoroughly in PBS, resuspended in 10% glycerol in PBS, and kept at –80°C until use. HL-60 is a polymorphonuclear neutrophil leukocyte (PMN) cell line derived from a patient with acute promyelocytic leukemia (European Collection of Cell Culture) (9). Cells were differentiated by incubation for 5 days in Hanks' balanced salt solution medium (Gibco, Paisley, United Kingdom) with 0.2% (vol/vol) bovine serum albumin (BSA). For opsonophagocytosis, 20 μl of bacteria (concentration, 108 CFU/ml) was added to the same volume of control or immune serum diluted 1:20 in PBS in the wells of a microtiter plate. The plate was then incubated for 30 min at 37°C with gentle agitation, and then 10 μl of rabbit complement (Mast Diagnostics) was added to each well followed by another incubation for 15 min. Finally, 40 μl of a suspension of differentiated HL-60 cells (concentration, 2.5 x 106/ml) was added to the wells and incubated for 30 min. The reaction was stopped by the addition of 160 μl of ice-cold Hanks' balanced salt solution, and the contents of each well was transferred to an Eppendorf tube containing 400 μl of ice-cold Dulbecco's PBS with 0.02% (wt/vol) EDTA. Samples were kept on ice until analyzed by flow cytometry; at least 104 cells from each sample were evaluated with a horizontal fluorescence gate set to include 5% of the population of cells incubated in the absence of serum. The relative fluorescence index (FI) was calculated (FI = percentage of gated cells multiplied by the geometric mean fluorescence); significant differences were detected with a Student's t test. The FI ratio was calculated by taking the mean of FI of each serum and dividing it by the mean FI of the complement-only controls. Thus, background (complement-only) levels of phagocytosis have a value of 1. All opsonophagocytosis experiments were repeated on at least two occasions.
SBA. Serum bactericidal activity was measured according to internationally standardized methods (8, 25). Twofold dilutions of sera were prepared in sterile 96-well microtiter plates to which were added N. meningitidis strains with rabbit complement (Pel-freeze, Brown Deer, Wis.) and incubated for 1 h. The number of surviving bacteria was determined by plating aliquots of the suspension to solid medium. End point titers were calculated as the reciprocal of the dilution of serum yielding more than 50% bacterial killing; the lower limit of detection for SBA was 4. Each assay included sera from animals before immunization or those immunized with growth medium alone and sera from those vaccinated with a live attenuated N. meningitidis strain. Assays were performed at least in triplicate.
RESULTS
Immunization with live N. lactamica protects against live bacterial challenge. To establish whether live N. lactamica can confer protective immunity, groups of mice (15 animals per group) received 106 CFU of either N. lactamica L13 or L18 by i.p. administration on days 0, 21, and 28. Control groups were given either medium alone or a characterized live attenuated N. meningitidis strain, YH102 (32). On day 43, animals were challenged with live serogroup B N. meningitidis, MC58. As described previously, all animals immunized with the attenuated N. meningitidis strain survived challenge with the homologous strain (32). Vaccination with both live N. lactamica strains, L13 and L18, conferred partial yet significant protection compared with that in animals that received medium alone (Fig. 1; P = 0.022 and 0.034 for L13 and L18, respectively).
Cross-reactive antibodies raised against N. lactamica recognize antigens on the meningococcal cell surface. Next we established whether antibodies elicited by immunization with N. lactamica recognized cross-reactive epitopes on representatives of three different serogroups of N. meningitidis. Initially whole-cell ELISAs were performed to determine whether immune sera recognized antigens on heat-inactivated bacteria. As expected, the highest titers were detected against the homologous N. lactamica strain used for immunization (Table 1). However, high levels of anti-N. meningitidis antibodies were also detected in sera from animals immunized with N. lactamica, with similar antibody titers found against the meningococcal strains regardless of serogroup (Table 1). Titers were comparable to those obtained with sera from animals immunized with a live attenuated serogroup B N. meningitidis strain, YH102. The cross-reactivity of the antibodies was further demonstrated by Western blot analysis of whole-cell lysates. Multiple bands were recognized in lysates from serogroup A, B, and C meningococcal strains by sera raised against N. lactamica (Fig. 2); no bands were detected with preimmune sera or sera from animals that had received medium alone (not shown). Of note, the profiles of N. meningitidis proteins detected by sera raised against N. lactamica L13 and L18 were distinct. This is most obvious when comparing low-molecular-mass antigens (Fig. 2).
As whole-cell ELISAs and Western blot analysis also can detect cytoplasmic antigens, we undertook flow cytometry analysis to determine whether antibodies in immune sera recognize antigens on intact N. meningitidis. Bacteria were incubated with antisera, and immunoglobin binding was detected with an FITC-conjugated secondary antibody. As described previously (32), flow cytometry demonstrated that immunization with the live attenuated meningococcal strain elicited antibodies that recognized surface antigens on N. meningitidis (Fig. 3). Furthermore, antibody binding was clearly detected to all serogroups of N. meningitidis (witnessed by an obvious shift in the population of cells upon incubation with the sera from immunized animals), compared with preimmune sera from the same animals (Fig. 3). Binding to the serogroup C strain, FAM18, was less marked than to the serogroup A and B strains.
Immunologic correlates of protection elicited by live N. lactamica. Next we next investigated the functional activity of sera from animals vaccinated with live N. lactamica. Initially we examined whether heat-inactivated sera possessed opsonophagocytic activity. Unfortunately there was considerable nonopsonic uptake by the PMN cell line HL-60 of the serogroup A and C strains, FAM18 and Z2491, respectively (not shown), making it difficult to assess the effect of serum or complement on the uptake of these isolates. Therefore, all subsequent experiments were performed with the serogroup B strain, MC58. Sera from animals immunized with the live N. lactamica strain L13 promoted the opsonophagocytosis of serogroup B N. meningitidis at levels similar to those produced by immunization with the homologous strain (Fig. 4); the degree of opsonophagocytosis induced by vaccination with L18 was less marked than those induced by with immunization with the other strains.
SBA is a proven immunological correlate for protective immunity of individuals against serogroup C meningococcal infection (21), and SBA is also important for immunity against serogroup B strains (8). Therefore, we determined whether protected animals had detectable SBA against serogroup A, B, and C meningococcal strains. Preimmune sera and sera from animals in the negative control group (immunized with medium only) failed to mediate killing of any strains (titers of <4). As shown in Table 2, immunization with N. lactamica strain L13 elicited significant serum bactericidal antibodies against all three meningococcal strains; levels were similar to those raised by live attenuated N. meningitidis, except for titers against the homologous strain, MC58. In contrast, there was no detectable SBA among animals receiving N. lactamica L18. This lack of SBA in animals vaccinated with N. lactamica L18 was confirmed with five other serogroup B (electrophoretic type [ET] and ST included) strains (C311, ET-5, ST unknown; BZ83, ET-5, ST-32; H44/76, ET-5, ST-32; BZ169, ET-5, ST-32; and BZ232, ET unknown, ST-38), while sera from animals immunized with L13 had detectable SBA against all of these strains except BZ232 (data not shown).
Previous work has shown that OMVs derived from another strain of N. lactamica, Y92-1009, elicit protection in mice against disseminated meningococcal disease without detectable SBA (36). Therefore, we next investigated the basis for the failure of OMVs from this strain to elicit SBA. Groups of animals were immunized with live Y92-1009 via the intraperitoneal route as above. Antibodies in sera from immunized animals recognized surface antigens on serogroup A, B, and C N. meningitidis strains by ELISA (not shown) and FACS (percentages of gated cells of 42.7%, 31.6%, and 53.3% for MC58, FAM18, and Z2491, respectively). Furthermore, similar to L13, vaccination with this strain was able to elicit both SBA (Table 2) and opsonophagocytic activity (not shown).
DISCUSSION
This study provides further experimental data to support the notion that infection with N. lactamica promotes the natural immunity against N. meningitidis which develops progressively throughout childhood. Longitudinal studies have provided strong epidemiological evidence indicating that antigens on the surface of this commensal can elicit bactericidal antibodies against the meningococcus in nonimmune hosts (20); this is demonstrated here experimentally, and the results should aid in defining the antigens that mediate this cross-protective response.
Although closely related to the meningococcus, N. lactamica does not express a polysaccharide capsule (29). This may be a significant advantage for the development of natural immunity, as the capsule is widely considered to be an unsuitable antigen for preventing serogroup B infections and expression of the capsule could impede the immunogenicity of other surface antigens. Furthermore, N. lactamica does not express PorA, a highly immunogenic and, therefore, highly variable protein on the surface of N. meningitidis (42, 43). Vaccine preparations that contain PorA (such as certain OMVs) tend to elicit responses which are largely directed at this protein and can thus be limited by their lack of coverage against strains expressing other PorA variants (39). Immunization with N. lactamica products therefore has several potential immunologic advantages over preparations of N. meningitidis and could be used to replicate and accelerate the development of natural immunity (23). The sporadic carriage with this commensal during early childhood may explain the susceptibility of some individuals to meningococcal disease; population-based vaccination programs could be used to ensure that coverage is comprehensive. However, there is a potential drawback to using N. lactamica products to prevent meningococcal disease. If the vaccine proved substantially more effective at preventing carriage with N. lactamica than N. meningitidis, the vaccine might afford only partial protection against meningococcal carriage and disease while entirely abolishing the beneficial effects of colonization with circulating N. lactamica.
The only validated immunologic correlate of protection against meningococcal disease is detectable SBA (3, 8, 21). Importantly for vaccine development, SBA is a ready marker of immunity for preclinical assessment of vaccines and provides a suitable end point in clinical trials. The importance of opsonophagocytic killing as a defense mechanism against the meningococcus has been demonstrated recently, with titers of opsonophagocytosis of beads coated with capsular antigen shown to be significantly correlated with SBA directed against capsular antigens (34). In the present study, we have shown that systemic immunization with two strains of N. lactamica (L13 and Y92-1009) elicits high-level bactericidal activity in mice against N. meningitidis strains, including the serogroup B strain, MC58. There are two important implications from this result. First, N. lactamica L18 failed to elicit bactericidal antibodies against any of three meningococcal strains even though the bacterium was given to animals by using precisely the same schedule as the other strains. This indicates that the profiles of antigens expressed by strains L13 and Y92-1009 are distinct from those produced by L18; therefore, N. lactamica strains differ in their immunogenic properties, and it is likely that not all strains can confer protection against N. meningitidis. The likely explanation for this is the considerable genetic diversity among N. lactamica as evidenced by both multilocus sequence typing and differences in gene content and antigen expression (2, 6). This is reflected in the meningococcal antigens recognized by immune sera raised against N. lactamica L13 and L18 (seen in Fig. 2). While immune sera against L13 and L18 reacted with multiple antigens by Western blot analysis, the profile of N. meningitidis proteins detected was distinct, and these differences may account for whether the N. lactamica strains elicit SBA or not. Therefore, knowledge of the antigenic profile of strains of N. lactamica is vital when deciding upon the choice of strain for N. lactamica-based vaccines against N. meningitidis. Of the N. lactamica strains we examined, L13 and Y92-1009 elicited functional responses against the three meningococcal strains. Second, we found that N. lactamica Y92-1009 elicited SBA when administered as a live organism, in contrast to when OMVs or killed cells from this bacterium were used to immunize animals. This could be explained by different routes of administration of the vaccine or different mouse genotypes used in this and a previous study (36). However, the most probable reason for this difference in the immune response is that the antigenic profile of the bacterium grown in vivo is distinct from that during growth in vitro when OMVs or killed whole cells were prepared. The coordinated control of gene expression is vital for microbial virulence (16), and the profile of antigens on the surface of pathogens varies at sites in the host and differs considerably during growth in the laboratory (46).
Although this study provides proof in principle that systemic immunization with live N. lactamica can induce SBA, this is not necessarily the optimal approach for preventing meningococcal infection with vaccines based on this commensal. Systemic immunization with a live organism can be reactogenic, and there have been reported cases of endocarditis and meningitis with N. lactamica (14, 30). Furthermore, there is clear evidence of horizontal DNA transfer between commensal and pathogenic species of Neisseria (15, 33), raising the concern that a vaccine strain could acquire virulence traits from pathogenic species. A more feasible approach might be the mucosal administration of N. lactamica; further studies are under way to determine the susceptibility of experimental animals to colonization with this commensal and its ability to generate systemic immune responses. N. lactamica is known to harbor genes related to those necessary for expression of type IV pili (1), which mediate host-specific adhesion of the meningococcus to human cells (35). Therefore colonization with N. lactamica may be inefficient unless the experimental host expresses an appropriate form of CD46 (27), the receptor proposed to be recognized by type IV pili (28).
Of much more potential value for vaccine design is that our findings demonstrate that strains of N. lactamica differ in their ability to produce functional immune responses against N. meningitidis. Understanding the basis for this observation should lead to the identification of those N. lactamica antigens that mediate cross-reactive protective immunity and the development of natural immunity. Additionally, defining the reason why live bacteria, but not OMVs or heat-killed bacteria, can elicit SBA in immunized animals may also help to pinpoint important antigens. These could then be incorporated into subunit vaccines for protecting of individuals against disseminated meningococcal infection.
ACKNOWLEDGMENTS
We are grateful to Rachel Exley for critical review of the manuscript.
This work was supported by the Meningitis Research Foundation.
FOOTNOTES
Corresponding author. Mailing address: Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, Flowers Building, Imperial College London, London SW7 2AZ, United Kingdom. Phone: (44) 207-594-3072. Fax: (44) 207-594-3076. E-mail: c.tang@imperial.ac.uk.
Published ahead of print on 11 September 2006.
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