IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pollard, A. J.
Right arrow Articles by Levin, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pollard, A. J.
Right arrow Articles by Levin, M.

 Previous Article  |  Next Article 

Infection and Immunity, May 1999, p. 2452-2463, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cellular Immune Responses to Neisseria meningitidis in Children

Andrew J. Pollard,1,* Rachel Galassini,1 Eileene M. Rouppe van der Voort,2 Martin Hibberd,1 Robert Booy,1 Paul Langford,1 Simon Nadel,1 Catherine Ison,1 J. Simon Kroll,1 Jan Poolman,2,dagger and Michael Levin1

Departments of Paediatrics and Infectious Diseases & Microbiology, Imperial College School of Medicine, St. Mary's Hospital, London W2 1PG, United Kingdom,1 and Laboratory of Vaccine Development and Immune Mechanisms, National Institute of Public Health and the Environment, Bilthoven, The Netherlands2

Received 22 September 1998/Returned for modification 2 December 1998/Accepted 5 February 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

There is an urgent need for effective vaccines against serogroup B Neisseria meningitidis. Current experimental vaccines based on the outer membrane proteins (OMPs) of this organism provide a measure of protection in older children but have been ineffective in infants. We postulated that the inability of OMP vaccines to protect infants might be due to age-dependent defects in cellular immunity. We measured proliferation and in vitro production of gamma interferon (IFN-gamma ), tumor necrosis factor alpha, and interleukin-10 (IL-10) in response to meningococcal antigens by peripheral blood mononuclear cells (PBMCs) from children convalescing from meningococcal disease and from controls. After meningococcal infection, the balance of cytokine production by PBMCs from the youngest children was skewed towards a TH1 response (low IL-10/IFN-gamma ratio), while older children produced more TH2 cytokine (higher IL-10/IFN-gamma ratio). There was a trend to higher proliferative responses by PBMCs from older children. These responses were not influenced by the presence or subtype of class 1 (PorA) OMP or by the presence of class 2/3 (PorB) or class 4 OMP. Even young infants might be expected to develop adequate cellular immune responses to serogroup B N. meningitidis vaccines if a vaccine preparation can be formulated to mimic the immune stimulus of invasive disease, which may include stimulation of TH2 cytokine production.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Neisseria meningitidis is the leading infectious cause of death in children in the United Kingdom and many other countries (5, 45). More than 2,500 cases occur each year in England and Wales (12), with the peak incidence in the first 2 years of life (42) and an overall mortality of 10% (27, 47). Although improvement in intensive care therapy of these children may reduce mortality and morbidity, there remains an urgent need for an effective vaccine to prevent invasive disease.

Serogroup C N. meningitidis infection is likely to be prevented in the next few years by recently developed protein-polysaccharide conjugate vaccines, which have shown excellent immunogenicity in clinical trials (18, 35, 57). Unfortunately, the polysaccharide capsule of the serogroup B meningococcus is chemically and antigenically related to human brain and fetal antigens and is therefore poorly immunogenic in humans (19). Other bacterial components have therefore been considered as vaccine candidates, including outer membrane proteins (OMPs) such as the major porins (43) and iron-regulated proteins (4) and lipopolysaccharide (LPS) (44). The conformational presentation of vaccine antigens might be important in a vaccine (66). The outer membrane of N. meningitidis constantly releases blebs of outer membrane containing a full complement of OMPs and LPS in their natural conformation. Several recent trials have used vaccines based on these blebs of bacterial membrane, which have been treated to reduce the LPS content and to produce outer membrane vesicles (OMVs). Trials of OMP vaccines in Chile (8) and of OMV vaccines in Cuba (52) and Brazil (15) showed efficacy ranging from 51 to 80%. However, subgroup analysis of the Chilean and Brazilian trials showed that the vaccines conferred no protection in those most at risk, i.e., those under 4 years of age. Another OMV vaccine has been evaluated in Norway in teenagers but had an efficacy of only 57% (7). However, immunogenicity studies with this OMV vaccine in Chilean infants were promising, although only strain-specific bactericidal antibody was generated (55). A hexavalent OMV vaccine based on recombinant meningococci expressing multiple PorA proteins, in an attempt to promote immunity to a majority of circulating strains, has been developed in The Netherlands (14). A phase I immunogenicity study with adults, using only one dose of this vaccine, was disappointing, with only half of the vaccinees demonstrating a fourfold increase in bactericidal titer to the six test strains (each with one of the PorA proteins from the vaccine) (43). Results of an immunogenicity trial in the United Kingdom with infants, with four doses of vaccine, are awaited.

We have shown that there is a poor bactericidal antibody response to infection with N. meningitidis (46a) that parallels the poor responses to vaccines in infants (8, 15, 52). The reasons for these poor immune responses in early childhood and infancy are unknown, but they may be a reflection of immunological immaturity. Our data do not suggest that the differences lie in the immunoglobulin subclass produced, but they may represent age-dependent differences in specificity, affinity, or avidity of antibody, which might in turn reflect differences in cellular immune responses. There is evidence that cellular immune responses are different in young children. Antigen-specific T-cell precursors are at lower levels in neonates than in adults (26). Less mitogen-induced interleukin-2 (IL-2), interferon-gamma (IFN-gamma ), IL-4, IL-6, and IL-10 are produced by neonates and children than by adults (11, 13, 33, 37, 64), and in response to recall antigens, IL-2 and IL-4 production from peripheral blood mononuclear cells (PBMCs) is lower in children (37). IFN-gamma production increases during the first few months of life and reaches adult levels by 2 to 5 years of age (22). CD40 ligand expression is also lower than that on adult cells but increases within the first few months of life (16, 23). Neonatal T cells may respond differently to cytokine stimulation and produce different immune responses as a result. Neonatal CD45RA cells are induced to develop an IL-4-producing TH2 phenotype, whereas adult cells develop a TH1 phenotype, following stimulation with IL-12 (51). Neonatal CD4+ T cells are less able to produce help for immunoglobulin synthesis by B cells (28, 53), and this, in combination with the reduced ability of neonatal T cells to produce both TH1 and TH2 cytokines, may partly explain the susceptibility of neonates to infections such as Toxoplasma and herpes simplex virus and the slower antibody response of infants to herpes simplex virus infection and Haemophilus influenzae type b vaccination (25, 54, 64).

We postulated that the poor efficacy of serogroup B meningococcal vaccines in young children and the poor bactericidal response to infection in this age group (46a) might be related to impaired function of the cellular components of the immune system. Age-dependent differences in T-cell help for antibody production or age-related differences in the cytokine milieu following vaccination might be responsible for this observation. In order to establish whether there are age-related differences in cellular responses to meningococcal antigens, we compared T-cell proliferation and cytokine production by PBMCs from children convalescing from natural infection and controls.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Subjects. Venous blood was obtained 9.1 weeks (median) after the onset of meningococcal sepsis from 49 children who presented to the pediatric intensive care and infectious diseases units at St. Mary's Hospital with a clinical diagnosis of meningococcal disease in 1997. All children had typical clinical features of severe meningococcal disease, and microbiological diagnosis was confirmed for 36 children by isolation of N. meningitidis from blood culture or throat swab, by detection of capsular antigen in blood or cerebrospinal fluid, or by detection of the meningococcal genome by PCR analysis of blood. Children without laboratory confirmation had a classical clinical presentation of meningococcal disease and no alternative etiological agent identified. Typing was available for 25 clinical isolates (Table 1). The pediatric risk-of-mortality (PRISM) score (46) was calculated for all children. Blood was also obtained from 22 siblings of meningococcal disease patients and from 19 otherwise healthy children who were undergoing routine surgery. Fourteen adult volunteers from the Department of Paediatrics provided a blood sample, and blood from five umbilical cords was also obtained. Ethical approval from the St. Mary's Hospital local research ethics committee and informed parental consent for blood sampling were obtained.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Study subjects

Bacterial strains. The N. meningitidis strains derived from strain H44/76 (B:15:P1.7,16) are described in Table 2 and were constructed at the Laboratory of Vaccine Development and Immune Mechanisms, National Institute for Public Health and the Environment, Bilthoven, The Netherlands. An Opc-negative variant was selected from strain HI5, a spontaneous PorA-negative mutant from strain H44/76, by colony blotting (58). Class 4 OMP-deficient strains were constructed as described by Rouppe van der Voort et al. (48). Strains were killed by being heated for 1 h at 56°C in a water bath. Whole-cell enzyme-linked immunosorbent assays (ELISAs) (2, 3) were performed with a panel of epitope-specific monoclonal antibodies to verify the pattern of OMP expression for each of the vesicles or bacterial strains (Table 3).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   N. meningitidis strains used for OMV production


                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Antigens used for PBMC stimulation

Antibodies. The PorA-, PorB-, and class 4-specific mouse monoclonal antibodies for whole-cell and vesicle typing (from the Laboratory of Vaccine Development and Immune Mechanisms, National Institute for Public Health and the Environment, Bilthoven, The Netherlands) were as follows: MN16C13F4 (anti-P1.2), MN22A9.19 (anti-P1.5), MN14C11.6 (anti-P1.7), MN20A7.10 (anti-P1.12), MN24H10.75 (anti-P1.13), MN3C5C (anti-P1.15), and MN5C11G (anti-P1.16). Class 4 OMP was detected with MN2D6D, and PorB (serotype 15) was detected with MN15A14H6. B306 anti-Opc was kindly provided by M. Achtman, Berlin, Germany.

Preparation of OMVs. OMVs from N. meningitidis strains were prepared by using a modification of the method described by Claassen et al. (14). N. meningitidis was incubated overnight at 37°C on gonococcal medium base (Difco, Detroit, Mich.) supplemented with IsoVitaleX in a humidified atmosphere containing 5% CO2. The bacteria were inoculated into 200 ml of Mueller-Hinton Broth (Oxoid, Basingstoke, United Kingdom) and incubated at 37°C with shaking at 170 rpm for 3 to 4 h to an optical density at 600 nm of 1.0. The suspension of bacteria was centrifuged at 2,900 × g for 30 min at 14°C, and the pellet was resuspended in 7.5 times the pellet weight of Tris-EDTA buffer (0.1 M Tris-HCl, 10 mM EDTA, pH 8.6). To extract the vesicles, a 1/20 dilution of 10% deoxycholate (DOC) in Tris-EDTA buffer was added with stirring for 30 min, and the suspension was centrifuged at 13,000 × g for 70 min at 10°C in order to pellet the cell debris. The supernatants containing vesicles were concentrated by ultracentrifugation for 65 min at 100,000 × g at 10°C. The ultracentrifugation pellet of vesicles was washed with 0.05 M Tris-HCl-2 mM EDTA-2.5% DOC buffer at pH 9.0 and then resuspended and homogenized with a 30-ml hand-held tissue homogenizer (Fisher Scientific, Loughborough, United Kingdom) in sucrose buffer (0.02 M Tris-HCl, 2 mM EDTA, 1% DOC, 20% sucrose, pH 8.6). The homogenate was ultracentrifuged at 100,000 × g for 65 min at 10°C, and finally the OMV pellet was resuspended in 3% sucrose solution (21). OMV ELISAs (2, 3) and Western blotting were performed by using a panel of epitope-specific monoclonal antibodies to verify the pattern of OMP expression for each of the vesicles (Table 3). OMVs were examined by electron microscopy for successful production.

Antigens. OMVs were stored until use at 4°C in 3% sucrose solution for the 12 months of the study. The protein concentration of the OMVs was estimated by the method of Bradford (9), and endotoxin activity was estimated by the Limulus amebocyte lysate assay (Coatest, Charleston, S.C.). Heat-killed whole meningococci prepared at the start of the study were stored at -70°C at a concentration of 109 CFU/ml in RPMI 1640 (Gibco, Paisley, United Kingdom).

Proliferative responses. PBMCs were separated from fresh whole blood by Histopaque-1077 (Sigma, Poole, United Kingdom) density gradient centrifugation and cultured in 96-well round-bottom tissue culture plates (Nunc, Life Technologies, Paisley, United Kingdom) at 105 cells per well in RPMI 1640 (Gibco) supplemented with 10% human AB serum (Sigma), 2 mM glutamine, 100 Units of penicillin per ml, and 100 µg of streptomycin per ml in a total volume of 200 µl/well with or without antigen. OMVs from N. meningitidis strains at a final concentration of 0.5 µg protein/ml, heat-killed whole meningococci at a final concentration of 5 × 107 CFU/ml, or phytohemagglutinin (PHA) (Sigma) at 10 µg/ml was added to the wells in triplicate. The PBMCs were incubated for 7 days at 37°C in a humidified atmosphere containing 5% CO2 and pulsed with 1 µCi of [3H]thymidine (Amersham, Bucks, United Kingdom) for the last 18 h of culture. All 96 wells were harvested simultaneously with a 96-well sample harvester (Filtermate 196; Packard, Groningen, The Netherlands) onto glass fiber paper (Packard). The fiber papers were dried for 4 min in a microwave oven, and the incorporated radioactivity was measured in a beta counter (Packard Matrix 96) for 3 min. The stimulation index was calculated as the ratio of counts per minute obtained in the presence of antigen to counts per minute in the absence of antigen.

Cytokine production during antigen-induced PBMC proliferation. The pattern of cytokine production by PBMCs after stimulation for 7 days with meningococcal antigens was assessed by ELISA. Supernatants from four to eight culture wells were harvested and pooled for cytokine assay in duplicate after 7 days of stimulation with or without antigen. Concentrations of IL-4, IL-10, IL-12, IFN-gamma , and tumor necrosis factor alpha (TNF-alpha ) (Pharmingen, San Diego, Calif.) and IL-13 (R&D Systems, Abingdon, United Kingdom) were measured by using sandwich ELISA antibody pairs according to the manufacturers' recommendations in 96-well high-binding ELISA plates (Greiner, Stonehouse, United Kingdom). ELISAs were developed by using o-phenylenediamine dihydrochloride (Sigma) at 10 mg/50 ml of citrate buffer with 10 µl of hydrogen peroxide (pH 5) as a substrate, and the absorbance was measured at 490 nm in a Thermomax microplate reader (Molecular Devices, Menlo Park, Calif.). Cytokine concentrations were calculated by using ELISA software (Softmax; Molecular Devices) from a standard curve run in duplicate on every plate. Cytokine concentrations are reported as the concentration with background (supernatants from unstimulated cells) subtracted.

Statistical methods. The differences in PBMC proliferation and cytokine production between the case and the control groups and the effect of age, infecting serogroup, and severity of disease were analyzed. Because the variances were different between groups or the data were not normally distributed, median values were compared by using the Kruskal-Wallis nonparametric test. Epi Info (Centers for Disease Control and Prevention, Atlanta, Ga.) was used for statistical analyses, including linear regression.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Proliferative responses. The background proliferation of PBMCs was 287 ± 95 cpm (mean and 95% confidence interval) after 7 days of in vitro culture in medium alone. In vitro PBMC proliferation to whole meningococci (B:15:P1.7,16) was greater in 21 children convalescing from serogroup B infection (mean stimulation index, 118; 95% confidence interval, ±58) and in 16 children following serogroup C disease (69 ± 37) than in the 19 control children (30 ± 16) (P = 0.003 and P = 0.07, respectively). Serogroup B cases produced greater proliferation than serogroup C cases, but this did not reach significance (P = 0.2). Proliferation to all six different OMVs was also greater in the PBMCs of 49 children convalescing from meningococcal disease (e.g., for P1.7,16 OMVs, 62 ± 27) than in the unrelated control children (25 ± 8) (P = 0.03) (Fig. 1). PBMCs from umbilical cord blood showed the lowest proliferation to meningococcal antigens (29 ± 15 for whole cells and 9 ± 4 for P1.7,16 OMVs). Cells from both adults and siblings of patients also showed greater lymphoproliferative responses to meningococcal antigens than PBMCs from unrelated control children or cord blood, but their responses were not significantly different from those of the children with meningococcal disease.


View larger version (45K):
[in this window]
[in a new window]
  		FIG. 1.   Proliferative responses of PBMCs from 15 adult volunteers, 49 children convalescing from meningococcal disease, 22 siblings of meningococcal disease patients, 19 healthy children, and 5 umbilical cords in response to whole meningococci (bars A) and six different meningococcal OMVs (bars B to G). Results are expressed as the stimulation index (mean and standard error; stimulation index = counts per minute in stimulated cells divided by counts per minute in unstimulated cells for triplicate cultures).

There was no significant difference in proliferation of PBMCs in response to whole cells or OMVs containing four different PorA proteins or a PorA-deficient vesicle (HI5) among either cases or controls. Vesicles deficient in class 3 and class 4 OMPs (P1.7,16 3- 4- vesicles) showed the same responses as vesicles with all OMPs (P1.7,16 vesicles). Variation in the LPS content of the vesicles did not systematically affect proliferative responses (data not shown). We also examined proliferative responses to purified Escherichia coli LPS in order to identify nonspecific proliferation. LPS did not induce proliferation of PBMCs from most children studied. In those where proliferation above background was observed, the stimulation index was 10- to 100-fold lower than that after stimulation with whole meningococci (data not shown).

There was no age-related difference in proliferation to heat-killed meningococci or OMVs for PBMCs from infected children or control children (Fig. 2), although there was a trend towards higher responses in children over 10 years of age. There was no correlation between clinical severity of disease (PRISM) and proliferative responses (data not shown).


View larger version (34K):
[in this window]
[in a new window]
  		FIG. 2.   Proliferative responses of PBMCs from children after natural infection with N. meningitidis in response to whole meningococci (H44/76) or six different meningococcal OMVs in relation to age. Results are expressed as the stimulation index (mean and standard error; stimulation index = counts per minute in stimulated cells divided by counts per minute in unstimulated cells for triplicate cultures).

Cytokine production. Supernatant was available for cytokine analysis for 14 adults, 35 children convalescing from meningococcal disease, 15 siblings of these patients, and 19 healthy children. Intra-assay variation was low, with the coefficient of variation usually less than 10%. Data with coefficients of variation of greater than 20% were rejected. Time course experiments using PBMCs from adult donors showed that cytokine production was at steady state on day 7 of the assay for IL-10 and TNF-beta . IFN-gamma responses peaked on day 7 to 8 as a result of antigen-specific interactions, and IL-10 began to fall as IFN-gamma rose. TNF-alpha peaked on day 1 and fell thereafter to a steady state by day 7. Highest responses were seen with the mitogen PHA for all cytokines. PHA responses peaked earlier than protein antigen-specific responses.

(i) Differences between patients and controls. Production of TNF-alpha , IL-10, and IFN-gamma by PBMCs in response to heat-killed whole meningococci was significantly greater in adults than in meningococcal disease patients or child controls (Fig. 3). All groups tended to produce more IL-10 and IFN-gamma and less TNF-alpha after 7 days of stimulation with whole cells than after stimulation with the OMVs. Adults (2,140 ± 1,087 pg/ml for P1.7,16 OMV) and patients (1,736 ± 480 pg/ml) produced significantly more TNF-alpha in response to OMVs than controls (682 ± 405) (P = 0.001 and P = 0.0008, respectively). Moreover, adults produced more IL-10 (1,420 ± 605 pg/ml) and IFN-gamma (696 ± 257 pg/ml) in response to OMVs than patients (IL-10, 716 ± 197 [P = 0.007]; IFN-gamma , 382 ± 143 [P = 0.01 {for P1.7,16 OMV}]) or controls (IL-10, 698 ± 180 [P = 0.02]; IFN-gamma , 410 ± 325 [P = 0.01]), but patients produced levels of these cytokines similar to those for control children. Cord blood mononuclear cells produced no IFN-gamma in response to OMVs and produced low levels in response to whole cells. IL-10 production was also very low for two of three cords studied, but TNF-alpha production by cord cells was high. The production of IL-10 by PBMCs from siblings of patients was higher than that in patients or controls. The three different OMVs used stimulated production of similar levels of TNF-alpha , IL-10, and IFN-gamma in each group of subjects. There was a trend to a higher IL-10/IFN-gamma ratio for OMVs than for whole cells in each group of subjects, but this did not reach significance (P > 0.2).




View larger version (86K):
[in this window]
[in a new window]
  		FIG. 3.   TNF-alpha (a), IL-10 (b), and IFN-gamma (c) concentrations (means and standard errors) in supernatants after 7 days of stimulation of PBMCs from 14 adult volunteers, 35 children convalescing from meningococcal disease, 15 siblings of meningococcal disease patients, and 19 healthy children with whole meningococci or three different meningococcal OMVs. Data for IFN-gamma levels from cord blood mononuclear cells are shown in panel c.

(ii) Cytokine production in relation to age. In children after meningococcal infection, there was higher IL-10 production by PBMCs in response to whole cells and OMVs with increasing age, although this did not reach significance. There was lower IFN-gamma production with increasing age, which was significant for whole cells (P = 0.02) and P1.7,16 3- 4- OMVs (P = 0.04) in children over 10 years of age. The IL-10/IFN-gamma ratio was calculated to examine the biological interaction between these two cytokines. In response to OMVs, the IL-10/IFN-gamma ratio was significantly higher in children over 4 years of age than in children under 1 year of age (P < 0.05), except for P1.7,16 OMVs (P = 0.07) (Fig. 4a). For whole cells there was also a trend to a higher IL-10/IFN-gamma ratio in older children, but this did not reach significance (P = 0.08). Children after meningococcal infection who produced higher levels of TNF-alpha tended to produce higher levels of IL-10 (correlation coefficient, 0.4), but TNF-alpha production was not significantly related to age (Fig. 5a). However, TNF-alpha production was related to age in the control group, with higher cytokine production in the older children (Fig. 5b). There was also a trend towards a higher IL-10/IFN-gamma ratio in children over 10 years of age compared with children under 4 years of age (Fig. 4b). This trend just reached significance for P1.7,16 OMVs and HI5 OMVs (P = 0.05 and P = 0.04, respectively).


View larger version (32K):
[in this window]
[in a new window]
  		FIG. 4.   IL-10/IFN-gamma ratio in relation to age for patients (a) and controls (b) following stimulation of PBMCs with whole meningococci or three different OMVs. Means and standard errors are shown.


View larger version (37K):
[in this window]
[in a new window]
  		FIG. 5.   TNF-alpha production in relation to age for patients (a) and controls (b) following stimulation of PBMCs with whole meningococci or three different OMVs. Means and standard errors are shown.

(iii) Cytokine production in relation to strain. There was no difference in cytokine production between children infected with serogroup B or serogroup C meningococci. The three different OMVs produced responses similar to each other. Furthermore, the responses to two different strains of whole cells used in the assay, MC58 and H44/76, were the same (data not shown).

(iv) IL-4, IL-12, and IL-13. IL-4, IL-12, and IL-13 were not detectable (limits of detection were 20, 40, and 300 pg/ml, respectively) after stimulation of cell cultures with meningococcal antigens. Stimulation with PHA induced a detectable rise in each cytokine, and there were no differences in levels between children after meningococcal infection, adults, or control children who were studied.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We have found production of IL-10 and IFN-gamma by PBMCs from children after meningococcal infection to be age dependent, with higher IL-10 and lower IFN-gamma production in the oldest children and a trend to higher PBMC proliferative responses in the older children. The balance of cytokine production in the youngest children was skewed towards a TH1 response (low IL-10/IFN-gamma ratio), and the older children produced more TH2 cytokine (high IL-10/IFN-gamma ratio).

Cytokine responses. The balance between TH1 and TH2 cytokines is likely to influence the nature of the immune response to N. meningitidis, since the patterns of cytokines associated with these phenotypes influence B-cell class switching. The main antibodies produced in response to meningococcal infection are of the IgG1 and IgG3 subclasses. IL-10 induces naive human B cells expressing surface IgD to switch to IgG1 and IgG3 production, and IgG1 production by B cells seems also to be enhanced by IL-10 and inhibited by IFN-gamma (10, 31, 32). This would suggest that the T cells from the infants with reduced IL-10 production in this study might be less able to provide help for antibody production and class switching to appropriate complement-fixing antibody. However, there was no difference in the subclass of antibody produced in response to whole meningococci in a parallel study (46a). It may be that the cytokine milieu is still important in determining the nature of the immune response to protective epitopes or the maturation of antibody affinity, and this may explain the poor bactericidal activity that we and others have observed in infants. However, IL-10 may not be the best marker of TH2 activity, as it is not secreted by all TH2 cells and is also produced by other cell populations, including macrophages (1, 6).

PBMCs from adults produced more cytokine in response to meningococcal antigens than PBMCs from children. It has been reported previously that neonates and children produce lower levels of cytokine in response to mitogens and recall antigens compared with adults (17, 22, 34, 37), supporting this finding. This reduced cytokine production in childhood may represent either a less expanded population of antigen-specific responder T cells (26) or a generalized lack of immune stimulation. Indeed, reduced cytokine production in neonates does correlate with lack of a CD45RO+ (memory T-cell) population, which comprises 30 to 40% of adult T cells (34). This may explain the low proliferative responses and IFN-gamma production by umbilical cord mononuclear cells in the present study. However, memory T cells from neonates and young infants produce cytokines as efficiently as memory T cells from adults (11, 16, 17, 51, 64, 65), and similarly, during acute infection with N. meningitidis, plasma levels of TNF-alpha , IL-6, IL-8, and IL-10 are the same in children irrespective of age (29). Our findings showed that there was similar production of cytokines at all ages but that the balance of cytokines produced was different.

TNF-alpha production was strongly age dependent in control children but not in children after meningococcal infection. This suggests that some of the production of this cytokine is antigen specific, as PBMCs from those children with the greatest exposure to antigen (older control children and patients) produced the highest levels. LPS causes TNF-alpha release through binding to CD14 on macrophages, but macrophage activation and cytokine production are enhanced by other cofactors released during the antigen-specific response. The finding that even young children have cytokine responses similar to those in older children, albeit with a different balance, suggests that natural infection induces T-cell responses irrespective of age. There was a correlation between TNF-alpha levels and IL-10, suggesting that release of the latter is involved in the regulation of TNF-alpha , as has been previously suggested (38).

Whole meningococci induced higher levels of IFN-gamma and IL-10 than OMVs in all four groups of subjects. This observation is similar to the findings that vaccination of children with whole-cell pertussis vaccine induces their T cells to secrete IFN-gamma but not IL-5 and that acellular pertussis vaccine induces secretion of both cytokines (50). There was also a trend to a higher IL-10/IFN-gamma ratio for OMVs than for whole cells, suggesting that the presentation of antigens may be important and that a TH2 pattern is induced by OMVs. With both antigens, PBMCs from adults produced more IL-10 and IFN-gamma than those from patients or controls. It is likely that cytokine responses after vaccination will differ in pattern or magnitude depending on the route of administration, type of vaccine (OMV or protein complex), and adjuvant used. Studies of such responses after vaccination may help determine an optimal immunogen that will produce long-lasting memory. In particular, directing immune responses in infants to a TH2 pattern may be important.

Despite the wide variation in the OMP constitutions of the infecting strains, children with serogroup C infections showed no significant difference in in vitro cytokine production in response to meningococcal antigens compared with the serogroup B-infected children. There was a trend for higher responses in the serogroup B-infected children. This is not surprising, since the antigen-specific stimulus involved in these responses is likely to reside in the conserved regions of the antigens shared by both serogroup B and C organisms.

In supernatants from cases, the PorA-deficient OMVs induced a lower IL-10/IFN-gamma ratio than the P1.7,16 replete vesicles, a finding which was reversed for controls. This may be a reflection of the immunomodulatory effects of the meningococcal porin proteins, which have been previously described (59, 60), and may have significance in the induction of appropriate vaccine responses.

Proliferative responses. The proliferative responses of PBMCs from children of different ages convalescing from meningococcal disease were similar in both infants and older children, although there was a trend to higher responses in the older children. Adults produced greater proliferative responses than children did. This may reflect greater numbers, or a wider range, of antigen-specific T cells in adults, generated through continued exposure to neisserial antigens from nasopharyngeal carriage and to cross-reactive antigens from other sources, such as enteric flora (24). The higher responses in siblings of infected children possibly reflect their exposure through carriage of the infecting strain (20). Certainly, adults have greater numbers of antigen-specific, memory phenotype cells than neonates (26), as discussed in the introduction.

The finding that even young children have proliferative responses similar to those in older children suggests that natural infection induces T-cell responses irrespective of age. Older children probably have T-cell memory for some of the antigenic specificities and as a consequence, like adults, produce larger proliferative responses.

In adults, T-cell proliferative responses occur following vaccination with OMV vaccines. Three adults immunized with the Dutch hexavalent PorA meningococcal OMV vaccine showed transiently increased in vitro PBMC proliferative responses and a rise in bactericidal antibody titers (49). Ten adult volunteers with low levels of meningococcal antibody given three doses of the Norwegian serogroup B meningococcal OMV vaccine developed strong primary and booster T-cell responses to both OMV and purified PorA OMP and a lesser response to PorB protein, although immune responses were higher after the second dose than after the third dose (41). The data presented here suggest that similar T-cell responses might be expected in children of all ages with appropriate presentation of meningococcal antigens in a vaccine. The poor booster responses to the Dutch and Norwegian vaccines suggest that the OMV formulation in each case is not ideal.

Despite the wide variation in OMP constitution of the infecting strains (Table 1) and significant age differences, children with serogroup C infections showed no significant difference in in vitro proliferative responses in response to meningococcal antigens compared with the serogroup B-infected children. There was a trend towards higher responses in the serogroup B-infected children. Although the OMPs of different strains of serogroup B and C meningococci are antigenically distinct (on monoclonal antibody typing), most of the amino acid sequence of each of the major OMPs is conserved between strains (39). T-cell epitopes are represented within these conserved regions of the proteins (36, 62, 63). Human T-cell responses to purified meningococcal OMPs were higher to class 5 OMPs (Opa) and Opc than PorA, with some epitopes more widely recognized by different HLA types and some showing greater HLA restriction (61). However, in the present study, OMVs expressing a range of different class 1 OMPs and OMVs deficient in either class 4 or class 3/4 OMP as the antigenic stimulus to PBMCs did not influence proliferative responses or cytokine production. This is presumably because OMVs present many more T-cell epitopes than single purified proteins and only a limited number of conserved T-cell epitope sequences are required to cover all HLA-DR genotypes (63). Wiertz et al. (62) have also demonstrated that PorA T-cell epitopes are in regions of OMPs which are not only conserved between strains but also highly conserved among neisserial porin proteins, making the likelihood of T-cell help for antibody production even greater. Unlike bactericidal antibody specificities, PBMC responses to meningococci are not strain specific, and epitope-specific T cells are likely to respond to a wide range of porins.

Although acquired immunity to meningococci is thought to be mainly through bactericidal antibody, T cells play a vital role in generating these humoral immune responses. Furthermore, the induction of memory, as well as the immunoglobulin isotype pattern (with effects on bactericidal activity), is dependent on the T-cell responses and cytokine production. This study shows that children of all ages can produce in vitro cellular immune responses following infection. Also, OMVs present a stimulus to lymphoproliferation that is not dependent on the infecting strain or on the OMP constitution of the in vitro OMV challenge. Although there was a trend towards higher immune responses in older children, it was not significant and does not provide an obvious explanation for the poor bactericidal or vaccine responses noted in infants.

It seems likely that vaccination with meningococcal OMPs, in a formulation that mimics infection, may be able to produce T-cell memory and, more importantly, protective responses even in infants, but the cytokine balance during the immune response to N. meningitidis may be critical in the generation of bactericidal antibody and vaccine efficacy to serogroup B organisms.


    ACKNOWLEDGMENTS

We thank the medical and nursing staff of the pediatric intensive care and infectious disease units and the pediatric outpatient clinic at St. Mary's Hospital; Nigel Curtis and Anna Goodsall for technical assistance with cell culture; and Jayne Farrant and Kate Dunn for assistance with antigen preparation. We also thank Betsy Kuipers and Harry van Dijken (Laboratory of Vaccine Development and Immune Mechanisms, Bilthoven, The Netherlands) for technical advice.

A.J.P. is funded by an Action Research Fellowship, R.G. is supported by a grant from the Meningitis Research Foundation, and R.B. is supported by a Wellcome Trust Clinical Epidemiology Fellowship.


    FOOTNOTES

* Corresponding author. Mailing address: Paediatric Infectious Diseases Unit, Imperial College School of Medicine, St. Mary's Hospital, Norfolk Place, London W2 1PG, United Kingdom. Phone: 44 171 886 6377. Fax: 44 171 886 6284. E-mail: AJPollard{at}csi.com.

dagger Present address: SmithKline Beecham Biologicals, Rixensart, Belgium.

Editor:   R. N. Moore


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Abbas, A. K., K. M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787-793[Medline].
2. Abdillahi, H., and J. T. Poolman. 1988. Neisseria meningitidis group B serosubtyping using monoclonal antibodies in whole-cell ELISA. Microb. Pathog. 4:27-32[Medline].
3. Abdillahi, H., and J. T. Poolman. 1987. Whole-cell ELISA for typing Neisseria meningitidis with monoclonal antibodies. FEMS Microbiol. Lett. 48:367-371.
4. Ala'Aldeen, D. A. 1996. Transferrin receptors of Neisseria meningitidis: promising candidates for a broadly cross-protective vaccine. J. Med. Microbiol. 44:237-243[Abstract].
5. Anonymous. 1997. 1995 communicable disease statistics, vol. 22. Office of National Statistics HMSO Books, London, United Kingdom.
6. Barnard, A., B. P. Mahon, J. Watkins, K. Redhead, and K. H. Mills. 1996. Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T-cell subsets as Th1, Th2 or Th0. Immunology 87:372-380[Medline].
7. Bjune, G., E. A. Hoiby, J. K. Gronnesby, O. Arnesen, J. H. Fredriksen, A. Halstensen, E. Holten, A. K. Lindbak, H. Nokleby, E. Rosenqvist, et al. 1991. Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway. Lancet 338:1093-1096[Medline].
8. Boslego, J., J. Garcia, C. Cruz, W. Zollinger, B. Brandt, S. Ruiz, M. Martinez, J. Arthur, P. Underwood, W. Silva, et al. 1995. Efficacy, safety, and immunogenicity of a meningococcal group B (15:P1.3) outer membrane protein vaccine in Iquique, Chile. Vaccine 13:821-829[Medline].
9. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254[Medline].
10. Briere, F., C. Servet Delprat, J. M. Bridon, J. M. Saint-Remy, and J. Banchereau. 1994. Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3. J. Exp. Med. 179:757-762[Abstract/Free Full Text].
11. Burchett, S. K., L. Corey, K. M. Mohan, J. Westall, R. Ashley, and C. B. Wilson. 1992. Diminished interferon-gamma and lymphocyte proliferation in neonatal and postpartum primary herpes simplex virus infection. J. Infect. Dis. 165:813-818[Medline].
12. CDSC (Communicable Disease Surveillance Centre). 1998. Notifications of infectious diseases. Communicable Dis. Rep. 8:17-20.
13. Chheda, S., K. H. Palkowetz, R. Garofalo, D. K. Rassin, and A. S. Goldman. 1996. Decreased interleukin-10 production by neonatal monocytes and T cells: relationship to decreased production and expression of tumor necrosis factor-alpha and its receptors. Pediatr. Res. 40:475-483[Medline].
14. Claassen, I., J. Meylis, P. van der Ley, C. Peeters, H. Brons, J. Robert, D. Borsboom, A. van der Ark, I. van Straaten, P. Roholl, B. Kuipers, and J. Poolman. 1996. Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine. Vaccine 14:1001-1008[Medline].
15. de Moraes, J. C., B. A. Perkins, M. C. Camargo, N. T. Hidalgo, H. A. Barbosa, C. T. Sacchi, I. M. Landgraf, V. L. Gattas, H. D. G. Vasconcelos, I. M. Gral, et al. 1992. Protective efficacy of a serogroup B meningococcal vaccine in Sao Paulo, Brazil. Lancet 340:1074-1078[Medline].
16. Durandy, A., G. De Saint Basile, B. Lisowska-Grospierre, J. F. Gauchat, M. Forveille, R. A. Kroczek, J. Y. Bonnefoy, and A. Fischer. 1995. Undetectable CD40 ligand expression on T cells and low B cell responses to CD40 binding agonists in human newborns. J. Immunol. 154:1560-1568[Abstract].
17. Ehlers, S., and K. A. Smith. 1991. Differentiation of T cell lymphokine gene expression: the in vitro acquisition of T cell memory. J. Exp. Med. 173:25-36[Abstract/Free Full Text].
18. Fairley, C. K., N. Begg, R. Borrow, A. J. Fox, D. M. Jones, and K. Cartwright. 1996. Conjugate meningococcal serogroup A and C vaccine: reactogenicity and immunogenicity in United Kingdom infants. J. Infect. Dis. 174:1360-1363[Medline].
19. Frasch, C. E. 1989. Vaccines for prevention of meningococcal disease. Clin. Microbiol. Rev. 2(Suppl.):S134-S138.
20. Frasch, C. E., and L. F. Mocca. 1982. Strains of Neisseria meningitidis isolated from patients and their close contacts. Infect. Immun. 37:155-159[Abstract/Free Full Text].
21. Fredriksen, J. H., E. Rosenqvist, E. Wedege, K. Bryn, G. Bjune, L. O. Froholm, A. K. Lindbak, B. Mogster, E. Namork, U. Rye, et al. 1991. Production, characterization and control of MenB-vaccine "Folkehelsa": an outer membrane vesicle vaccine against group B meningococcal disease. NIPH Ann. 14:67-79[Medline], 79-80.
22. Frenkel, L., and Y. J. Bryson. 1987. Ontogeny of phytohemagglutinin-induced gamma interferon by leukocytes of healthy infants and children: evidence for decreased production in infants younger than 2 months of age. J. Pediatr. 111:97-100[Medline].
23. Fuleihan, R., D. Ahern, and R. S. Geha. 1994. Decreased expression of the ligand for CD40 in newborn lymphocytes. Eur. J. Immunol. 24:1925-1928[Medline].
24. Glode, M. P., J. B. Robbins, T. Y. Liu, E. C. Gotschlich, I. Orskov, and F. Orskov. 1977. Cross-antigenicity and immunogenicity between capsular polysaccharides of group C Neisseria meningitidis and of Escherichia coli K92. J. Infect. Dis. 135:94-104[Medline].
25. Greenberg, D. P., J. M. Lieberman, S. M. Marcy, V. K. Wong, S. Partridge, S. J. Chang, C. Y. Chiu, and J. I. Ward. 1995. Enhanced antibody responses in infants given different sequences of heterogeneous Haemophilus influenzae type b conjugate vaccines. J. Pediatr. 126:206-211[Medline].
26. Hassan, J., and D. J. Reen. 1996. Reduced primary antigen-specific T-cell precursor frequencies in neonates are associated with deficient interleukin-2 production. Immunology 87:604-608[Medline].
27. Havens, P. L., J. S. Garland, M. M. Brook, B. A. Dewitz, E. S. Stremski, and T. J. Troshynski. 1989. Trends in mortality in children hospitalized with meningococcal infections, 1957 to 1987. Pediatr. Infect. Dis. J. 8:8-11[Medline].
28. Hayward, A. R., and J. Kurnick. 1981. Newborn T cell suppression: early appearance, maintenance in culture, and lack of growth factor suppression. J. Immunol. 126:50-53[Abstract].
29. Hazelzet, J. A., I. M. Risseeuw-Appel, R. F. Kornelisse, W. C. Hop, I. Dekker, K. F. Joosten, R. de Groot, and C. E. Hack. 1996. Age-related differences in outcome and severity of DIC in children with septic shock and purpura. Thromb. Haemost. 76:932-938[Medline].
30. Holten, E. 1979. Serotypes of Neisseria meningitidis isolated from patients in Norway during the first six months of 1978. J. Clin. Microbiol. 9:186-188[Abstract/Free Full Text].
31. Kawano, Y., T. Noma, and J. Yata. 1994. Regulation of human IgG subclass production by cytokines. IFN-gamma and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2. J. Immunol. 153:4948-4958[Abstract].
32. Klouche, M., D. Wilhelm, and H. Kirchner. 1994. Cytokines, immunoglobulins, and IgG subclasses in patients with IgG plasmacytomas. Immun. Infekt. 22:149-151[Medline].
33. Lewis, D. B., K. S. Prickett, A. Larsen, K. Grabstein, M. Weaver, and C. B. Wilson. 1988. Restricted production of interleukin 4 by activated human T cells. Proc. Natl. Acad. Sci. USA 85:9743-9747[Abstract/Free Full Text].
34. Lewis, D. B., C. C. Yu, J. Meyer, B. K. English, S. J. Kahn, and C. B. Wilson. 1991. Cellular and molecular mechanisms for reduced interleukin 4 and interferon-gamma production by neonatal T cells. J. Clin. Investig. 87:194-202.
35. Lieberman, J. M., S. S. Chiu, V. K. Wong, S. Partidge, S. J. Chang, C. Y. Chiu, L. L. Gheesling, G. M. Carlone, and J. I. Ward. 1996. Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial. JAMA 275:1499-1503[Abstract].
36. Lifely, M. R., and Z. Wang. 1988. Immune responses in mice to different noncovalent complexes of meningococcal B polysaccharide and outer membrane proteins. Infect. Immun. 56:3221-3227[Abstract/Free Full Text].
37. Lilic, D., A. J. Cant, M. Abinun, J. E. Calvert, and G. P. Spickett. 1997. Cytokine production differs in children and adults. Pediatr. Res. 42:237-240[Medline].
38. Marchant, A., C. Bruyns, P. Vandenabeele, M. Ducarme, C. Gerard, A. Delvaux, D. De Groote, D. Abramowicz, T. Velu, and M. Goldman. 1994. Interleukin-10 controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia. Eur. J. Immunol. 24:1167-1171[Medline].
39. McGuiness, B., A. K. Barlow, I. N. Clarcke, J. E. Farley, A. Anilionis, J. T. Poolman, and J. E. Heckels. 1990. Comparative sequence analysis of the class I protein gene (porA) from three strains of Neisseria meningitidis: synthetic peptides define the epitopes responsible for serosubtype specificity. J. Exp. Med. 171:1871[Abstract/Free Full Text].
40. McGuinness, B. T., I. N. Clarke, P. R. Lambden, A. K. Barlow, J. T. Poolman, D. M. Jones, and J. E. Heckels. 1991. Point mutation in meningococcal por A gene associated with increased endemic disease. Lancet 337:514-517[Medline].
41. Naess, L. M., F. Oftung, A. Aase, L. M. Wetzler, R. Sandin, and T. E. Michaelsen. 1998. Human T-cell responses after vaccination with the Norwegian group B meningococcal outer membrane vesicle vaccine. Infect. Immun. 66:959-965[Abstract/Free Full Text].
42. Noah, N., and M. Connolly. 1996. Surveillance of bacterial meningitis in Europe 1995. King's European Meningitis Surveillance Unit, London, United Kingdom.
43. Peeters, C. C., H. C. Rumke, L. C. Sundermann, E. M. Rouppe van der Voort, J. Meulenbelt, M. Schuller, A. J. Kuipers, P. van der Ley, and J. T. Poolman. 1996. Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 14:1009-1015[Medline].
44. Petrov, A. B., B. F. Semenov, Y. P. Vartanyan, M. M. Zakirov, V. P. Torchilin, V. S. Trubetskoy, N. V. Koshkina, V. L. L'Vov, I. K. Verner, I. V. Lopyrev, et al. 1992. Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes. Infect. Immun. 60:3897-3903[Abstract/Free Full Text].
45. Platt, M. 1997. Child health statistical review. Arch. Dis. Child. 77:542-548[Free Full Text].
46. Pollack, M. M., U. E. Ruttimann, and P. R. Getson. 1988. Pediatric risk of mortality (PRISM) score. Crit. Care Med. 16:1110-1116[Medline].
46a. Pollard, A. J., R. Galassini, E. M. Rouppe van der Voort, R. Booy, P. Langford, S. Nadel, C. Ison, J. S. Kroll, J. Poolman, and M. Levin. 1999. Humoral immune responses to Neisseria meningitidis in children. Infect. Immun. 67:2441-2451[Abstract/Free Full Text].
47. Ramsay, M., E. Kaczmarski, M. Rush, R. Mallard, P. Farrington, and J. White. 1997. Changing patterns of case ascertainment and trends in meningococcal disease in England and Wales. Communicable Dis. Rep. Rev. 7:R49-R54.
48. Rouppe van der Voort, E. M., P. van der Ley, J. van der Biezen, S. George, O. Tunnela, H. van Dijken, B. Kuipers, and J. Poolman. 1996. Specificity of human bactericidal antibodies against PorA P1.7,16 induced with a hexavalent meningococcal outer membrane vesicle vaccine. Infect. Immun. 64:2745-2751[Abstract].
49. Rouppe van der Voort, E. M., H. van Dijken, B. Kuipers, J. van der Biezen, P. van der Ley, J. Meylis, I. Claassen, and J. Poolman. 1997. Human B- and T-cell responses after immunization with a hexavalent PorA meningococcal outer membrane vesicle vaccine. Infect. Immun. 65:5184-5190[Abstract].
50. Ryan, M., G. Murphy, E. Ryan, L. Nilsson, F. Shackley, L. Gothefors, K. Oymar, E. Miller, J. Storsaeter, and K. H. Mills. 1998. Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children. Immunology 93:1-10[Medline].
51. Shu, U., C. E. Demeure, D. G. Byun, F. Podlaski, A. S. Stern, and G. Delespesse. 1994. Interleukin 12 exerts a differential effect on the maturation of neonatal and adult human CD45R0-CD4 T cells. J. Clin. Investig. 94:1352-1358.
52. Sierra, G. V., H. C. Campa, N. M. Varcacel, I. L. Garcia, P. L. Izquierdo, P. F. Sotolongo, G. V. Casanueva, C. O. Rico, C. R. Rodriguez, and M. H. Terry. 1991. Vaccine against group B Neisseria meningitidis: protection trial and mass vaccination results in Cuba. NIPH Ann. 14:195-207[Medline], 208-210.
53. Splawski, J. B., and P. E. Lipsky. 1991. Cytokine regulation of immunoglobulin secretion by neonatal lymphocytes. J. Clin. Investig. 88:967-977.
54. Sullender, W. M., J. L. Miller, L. L. Yasukawa, J. S. Bradley, S. B. Black, A. S. Yeager, and A. M. Arvin. 1987. Humoral and cell-mediated immunity in neonates with herpes simplex virus infection. J. Infect. Dis. 155:28-37[Medline].
55. Tappero, J., R. Lagos, A. Maldonado, P. Herrera, L. Gheesling, D. Williams, G. Carlone, B. Plikaytis, H. Nokleby, J. Holst, G. Sierra, and B. Perkins. 1996. Serum bactericidal activity elicited by two outer membrane protein serogroup B meningococcal vaccines among infants, pre-school children, and adults in Santiago, Chile. Neisseria '96, 110th International Pathogenic Neisseria Conference, Baltimore, Md.
56. Tommassen, J., P. Vermeij, M. Struyve, R. Benz, and J. T. Poolman. 1990. Isolation of Neisseria meningitidis mutants deficient in class 1 (PorA) and class 3 (PorB) outer membrane proteins. Infect. Immun. 58:1355-1359[Abstract/Free Full Text].
57. Twumasi, P. A., Jr., S. Kumah, A. Leach, T. J. O'Dempsey, S. J. Ceesay, J. Todd, C. V. Broome, G. M. Carlone, L. B. Pais, P. K. Holder, et al. 1995. A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants. J. Infect. Dis. 171:632-638[Medline].
58. Van Der Ley, P., and J. T. Poolman. 1992. Construction of a multivalent meningococcal vaccine strain based on the class 1 outer membrane protein. Infect. Immun. 60:3156-3161[Abstract/Free Full Text].
59. Wetzler, L. M. 1994. Immunopotentiating ability of neisserial major outer membrane proteins. Use as an adjuvant for poorly immunogenic substances and potential use in vaccines. Ann. N. Y. Acad. Sci. 730:367-370[Medline].
60. Wetzler, L. M., Y. Ho, H. Reiser, and L. W. Wetzler. 1996. Neisserial porins induce B lymphocytes to express costimulatory B7-2 molecules and to proliferate. J. Exp. Med. 183:1151-1159[Abstract/Free Full Text].
61. Wiertz, E. J., A. Delvig, E. M. Donders, H. F. Brugghe, L. M. van Unen, H. A. Timmermans, M. Achtman, P. Hoogerhout, and J. T. Poolman. 1996. T-cell responses to outer membrane proteins of Neisseria meningitidis: comparative study of the Opa, Opc, and PorA proteins. Infect. Immun. 64:298-304[Abstract].
62. Wiertz, E. J., J. A. van Gaans-van den Brink, H. Gausepohl, A. Prochnicka-Chalufour, P. Hoogerhout, and J. T. Poolman. 1992. Identification of T cell epitopes occurring in a meningococcal class 1 outer membrane protein using overlapping peptides assembled with simultaneous multiple peptide synthesis. J. Exp. Med. 176:79-88[Abstract/Free Full Text].
63. Wiertz, E. J., J. A. van Gaans-van den Brink, G. M. Schreuder, A. A. Termijtelen, P. Hoogerhout, and J. T. Poolman. 1991. T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements. J. Immunol. 147:2012-2018[Abstract].
64. Wilson, C. B. 1991. The ontogeny of T lymphocyte maturation and function. J. Pediatr. 118:S4-S9[Medline].
65. Wu, C. Y., C. Demeure, M. Kiniwa, M. Gately, and G. Delespesse. 1993. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J. Immunol. 151:1938-1949[Abstract].
66. Zollinger, W. D., R. E. Mandrell, J. M. Griffiss, P. Altieri, and S. Berman. 1979. Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man. J. Clin. Investig. 63:836-848.

Infection and Immunity, May 1999, p. 2452-2463, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar