Structural relationships and sialylation among meningococcal L1, L8, and L3,7 lipooligosaccharide serotypes.

Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a). [see text for structure] The more abundant molecule, designated L1, had a trihexose globosyl alpha chain; the less abundant one, designated L1a, had a beta-lactosyl alpha chain and a parallel alpha-lactosaminyl gamma chain. A P(k) globoside (Galalpha1-->4Galbeta1-->4 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P(1) globoside (Galalpha1-->4Galbeta1-->4GlcNAc-R) mAb bound none of them. alpha-Galactosidase caused loss of both L1 structures and creation of L8 structures; beta-galactosidase caused loss of the L8 determinant. The L1/P(k) glycose was partially sialylated. Some LOS also had unsubstituted basal beta-GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes.

Safety and immunogenicity testing of an intranasal group B meningococcal native outer membrane vesicle vaccine in healthy volunteers.

An intranasal vaccine composed of native outer membrane vesicles (NOMV) not exposed to detergent or denaturing agents was prepared from the group B meningococcal strain 9162 SynX(-)(-:15:P1.3:P5.10,11:L3,7,9) and tested in 32 healthy adult volunteers. Four groups of 8 volunteers were vaccinated intranasally with three doses of vaccine. The vaccine was very well tolerated in all dosing groups, despite the presence of lipo-oligosaccharide in the vaccine at a level of 25% relative to protein. The antibody response as measured by ELISA in serum, saliva and nasal wash fluids was relatively low in all 4 groups, but the induced serum antibodies had strong bactericidal activity. Persistent bactericidal antibodies (> or =4-fold increase) were produced in 75% of the recipients. Some of the bactericidal antibodies were cross reactive against divergent group B strains. Most of the bactericidal antibodies appeared to be specific for PorA and L3,7,9 LOS. The vaccine also produced a local antibody response which was detected in the nasal wash fluids of volunteers. These data suggest that nasal immunization with NOMV is a safe and effective approach to induce systemic and local immunity against the group B meningococcus and deserves further study.

Immunogenicity of intranasally administered meningococcal native outer membrane vesicles in mice.

Colonization of the human nasopharyngeal region by Neisseria meningitidis is believed to lead to natural immunity. Although the presence of bactericidal antibody in serum has been correlated with immunity to meningococcal disease, mucosal immunity at the portal of entry may also play an important role. This study was undertaken to examine in mice the possibility of safely using native outer membrane vesicles (NOMV) not exposed to detergent as an intranasal (i.n.) vaccine. The mucosal and systemic responses of mice to intranasal and intraperitoneal (i.p.) vaccination with NOMV were compared over a range of doses from 0.1 to 20 microgram. Intranasal vaccination of mice with NOMV induced a strong systemic bactericidal antibody response, as well as a strong local immunoglobulin A immune response in the lung as determined by assay of lung lavage fluid by enzyme-linked immunosorbent assay and lung antibody secreting cells by enzyme-linked immunospot assay. However, 8- to 10-fold-higher doses of NOMV were required i.n. compared to i.p. to elicit an equivalent bactericidal antibody response in serum. Some NOMV vaccine was aspirated into the lungs of mice during i.n. immunization and resulted in an acute inflammatory response that peaked at 1 to 2 days postimmunization and was cleared by day 7. These results indicate that i.n. delivery of meningococcal NOMV in mice is highly effective in eliciting the production of both a mucosal immune response and a systemic bactericidal antibody response.

Immunological and molecular characterization of three variant subtype P1.14 strains of Neisseria meningitidis.

Epidemic outbreaks of group B meningococcal disease exhibit a clonal nature consisting of a common serotype-subtype. Subtype-specific monoclonal antibodies (MAbs) directed toward two variable regions (VR1 and VR2) of the class 1 protein of Neisseria meningitidis are used in this classification scheme. A new MAb was developed to classify a nonsubtypeable (NST) strain of N. meningitidis, 7967. This MAb bound to both the NST strain and the prototype subtype P1. 14 strain, S3446, by dot blot analysis. However, a MAb produced to the prototype P1.14 strain did not bind to strain 7967. Sixteen additional strains were further identified as P1.14 with the prototype MAb; of these, 15 strains bound both MAbs. Differences in the characteristics of binding of both antibodies to the three apparently diverse P1.14 strains were studied further by using outer membrane complex proteins, immobilized peptides, and soluble peptides. Deduced amino acid analysis suggested that both MAbs bind to VR2 and that single amino acid changes within VR2 (KM, NM, or KK) might explain the differences in binding characteristics. These results demonstrated that minor variations which exist within subtype variable regions may be clearly identified only by a combination of molecular and immunologic testing. The impact of subtype variation will become more evident as subtype-specific vaccines are developed and tested for efficacy.

Confirmation of suspicious cases of meningococcal meningitis by PCR and enzyme-linked immunosorbent assay.

A significant problem in efficacy trials of meningococcal vaccines has been accurate identification of all cases of meningococcal disease that occur in study populations. The accuracy of case determination would be improved by utilizing methods which confirm or disprove suspicious cases of meningococcal disease that are culture negative. A collection of serum and cerebrospinal fluid (CSF) samples from a meningococcal vaccine field trial performed in Iquique, Chile, were utilized to assess the status of patients for whom cultures, Gram stains, and clinical evaluations for meningococcal disease were available. Nested PCRs (nPCRs) for amplification of Neisseria meningitidis DNA in CSF samples and enzyme-linked immunosorbent assays (ELISAs) for quantification of serum immunoglobulin G antibodies specific for N. meningitidis were used in combination to confirm or eliminate cases classified by physicians as suspicious for meningococcal disease. Samples from 12 of 79 patients suspected of having meningococcal meningitis tested positive by both methods; specimens from 61 of the 79 were negative by both methods; and samples from 6 patients yielded ambiguous results, and these cases remained unconfirmed. Direct sequence analysis of amplified DNA from patients suspected of having meningococcal disease confirmed that 2 of the 12 newly confirmed cases were not attributable to the typical epidemic strain (B:15:P1.[7],3) while the others were due to the epidemic strain. A combination of nPCR and ELISA reduced the number of suspicious cases in this study from 79 to 6, thereby improving the potential for assessment of vaccine efficacy. Molecular identification by nPCR in conjunction with immunological assessment of patient response could be considered diagnostic of disease in future testing of meningococcal vaccines to improve efficacy analyses.

Expression of the L8 lipopolysaccharide determinant increases the sensitivity of Neisseria meningitidis to serum bactericidal activity.

The influence of lipopolysaccharide (LPS) expression on the sensitivity of Neisseria meningitidis to serum bactericidal activity was investigated by using a series of variants that expressed different LPS determinants. For each of two different strains, a series of three LPS variants that expressed L3,7, L8, or both were analyzed. LPS variants were identified and monitored by colony blotting with murine monoclonal antibodies specific for the L8 or the L3,7,9 immunotype determinants. Differences in LPS expression were verified by analysis of proteinase K lysates of whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then by silver staining or immunoblotting. The variants were used as test strains in bactericidal assays with human sera and with murine sera and monoclonal antibodies. Expression of the L8 LPS epitope was correlated with increased sensitivity to serum bactericidal activity. The geometric mean bactericidal titers of 12 to 16 sets of pre- and postvaccination sera were determined for each variant. Mean serum titers increased progressively with increased expression of L8 on the target strain. The bactericidal titers of anti-outer membrane protein monoclonal antibodies also increased with increased L8 expression.

Safety and immunogenicity of multiple conventional immunizations administered during early HIV infection.

Twenty-one asymptomatic adults who had recently received multiple polysaccharide, live viral, and protein-derived vaccines were identified as being infected with human immunodeficiency virus (HIV). The mean subject age was 24 years (range 18-33); 20 of 21 (95%) were male. The mean T4 count was 523/mm3 with a mean T4/T8 ratio of 0.6. Serologic responses to immunization with meningococcus group C, adenovirus types 4 and 7, tetanus, and diphtheria were evaluated for the HIV seropositive subjects and were compared with the responses of similarly vaccinated age-, sex-, and race-matched HIV-seronegative controls. Significantly fewer (p less than 0.03) HIV subjects responded to meningococcus C (bactericidal antibody) and adenovirus 4 (neutralizing antibody) vaccines than did normals; the HIV-infected subjects who did respond produced functional antibody comparable to that of normals. Booster responses of HIV subjects to tetanus and diphtheria were comparable to those of normals. HIV-infected vaccine nonresponders did not differ from HIV-infected responders in total white blood cell, T4, T4/T8, total serum IgG, or delayed-type hypersensitivity skin test reactivity. All HIV subjects had negative cultures for live vaccine viruses (rubella, measles, adenovirus, and poliovirus). Postimmunization, no clinically apparent adverse reactions to vaccination were detected.

Diagnosis of human immunodeficiency virus infection by immunoassay using a molecularly cloned and expressed virus envelope polypeptide. Comparison to Western blot on 2707 consecutive serum samples.

To detect human immunodeficiency virus (HIV) antibodies in a simple enzyme-linked immunoassay (CBre3-EIA), we used an Escherichia coli-expressed polypeptide antigen, representing the carboxy-terminal third of the external membrane glycoprotein gene fused with the amino-terminal half of the transmembrane glycoprotein gene. Over a 3-month period, 2707 consecutive serum samples referred for confirmatory testing for human T-lymphotrophic virus type III (HTLV-III) antibodies were evaluated by both Western blot and CBre3-EIA. On a single determination for each sample, the CBre3-EIA was found to have an estimated sensitivity (99.9%) and specificity (99.1%) similar or superior to the more cumbersome Western blot method. This study shows that all HIV-seropositive subjects have antibodies to the virus envelope protein; no other virus antigens are required for construction of highly sensitive immunoassays.

Human bactericidal antibody response to meningococcal outer membrane protein vaccines.

Several different meningococcal outer membrane protein vaccines have been prepared and used in human safety and immunogenicity studies. The results of these studies have led to some general conclusions regarding the human antibody response to these vaccines. A review of these conclusions, however, indicates that a number of important questions and problems still need to be addressed. Two of these are the determination of the protective level of bactericidal antibody in human serum and the impact of phase variation of surface antigens on vaccine strategy. Bactericidal assays using intrinsic complement and high concentrations of serum suggest that the level of natural immunity to group B meningococci is quite high, but is increased by vaccination with outer membrane protein vaccine. Phase variation in meningococcal surface antigens including capsule, class 1 protein, class 5 protein, and lipopolysaccharide was demonstrated using colony blotting with monoclonal antibodies. Phase variation resulted in differences in susceptibility to the bactericidal activity of human sera.

Immune response of infants and children to disseminated infections with Neisseria meningitidis.

Acute- and convalescent-phase sera from 34 children and 10 young adults were studied to determine if, at what age, and to which antigens of Neisseria meningitidis they respond during disseminated disease. Seven children older than two years of age who were infected with group C or Y strains developed significant increases in both binding and bactericidal antibody. Children infected with group B strains infrequently (eight [31%] of 26) had measurable increases in serum antibody to this capsular polysaccharide; response was meager when it did occur, was unrelated to age, and was considerably poorer than that of young adults, of whom 80% responded. Convalescent-phase sera from all children contained bactericidal antibody. Binding capacity for group B polysaccharide accounted for only 35% of the bactericidal activity in convalescent-phase sera of children infected with group B strains. Bactericidal antibody in the sera of children who did not respond to capsular polysaccharides was often to a lipooligosaccharide antigen.

Immunological relationship between the capsular polysaccharides of Neisseria meningitidis serogroups Z and 29E.

The immunological relationship between serogroups 29E and Z of Neisseria meningitidis was investigated using bacterial agglutination, precipitin-in-gel, primary antigen binding and immune lysis assays. The two capsular polysaccharides were both cross immunogenic and cross reactive. Demonstration of the relationship using group Z antisera depended on which assay was used. It was most readily apparent in assays of immune lysis, less apparent when precipitins were sought in gel and inapparent when bacterial agglutination, the standard assay for determining serogroup, was employed. The cross reacting epitope was expressed 10-fold more within the group 29E polysaccharide than within the group Z polysaccharide. These findings imply that inclusion of either polysaccharide in a polyvalent meningococcal vaccine may obviate the need to include the other polysaccharide.

Safety and immunogenicity of a group 29E meningococcal capsular polysaccharide vaccine in adults.

A serogroup 29E Neisseria meningitidis capsular polysaccharide vaccine at a dose of 50 micrograms was injected subcutaneously into 10 adult human volunteers. One vaccinate experienced a mild systemic reaction; one complained of moderate to severe local pain and tenderness. The vaccine induced significant homologous binding and bactericidal antibody by 2 weeks and significant binding antibody against the heterologous serogroup Z capsular polysaccharide by 4 weeks. Although binding antibody rose during the first 4 weeks and then declined slowly over the subsequent 4 months, bactericidal antibody response declined substantially by 4 and 8 weeks for both polysaccharides. The increase in group 29E bactericidal activity was no longer significant at 4 and 8 weeks; loss of bactericidal activity against group Z was significant by 8 weeks. Bactericidal activity again rose between 8 and 26 weeks, becoming significantly increased over prevaccination levels for group 29E and increased, though not significantly (P = 0.085), over prevaccination levels for group Z. As a result of the failure of induced antibody to increase bactericidal activity, only 40% of vaccinates achieved a greater than 2 log2 increase in lytic activity against group 29E, and none achieved this large an increase in lytic activity against group Z.

Human immune response to various doses of group Y and W135 meningococcal polysaccharide vaccines.

A divalent vaccine containing equal weights of Neisseria meningitidis group Y and group W135 capsular polysaccharides was inoculated subcutaneously into groups of 32 military recruit volunteers at doses of 10, 25, 50, and 100 micrograms in 10-microliter/microgram volumes. At 4 weeks, the two higher doses induced significantly greater binding antibody responses than did the two lower doses. Differences in response were not found between the two higher doses or between the two lower doses. An additional 32 volunteers received a dose of 25 micrograms in a 20-microliter/microgram volume. Binding antibody response to this vaccine did not differ from the response to doses of 10 and 25 microgram in 10-microliter/microgram volumes. In contrast, bactericidal antibody responses did not differ among doses. Bactericidal antibody was induced in 100% of individuals with low (greater than 4 log2) preexisting serum bactericidal activity, regardless of dose. Bactericidal antibody nonresponse was restricted to individuals with high preexisting serum bactericidal titers. The discrepant dose response between binding and bactericidal antibody resulted from the induction of nonlytic antibody by the higher doses. We conclude that there are no advantages to doses in excess of 5 micrograms of these two chemically similar polysaccharides for the target population of young adult military recruits.

Safety and immunogenicity of group Y and group W135 meningococcal capsular polysaccharide vaccines in adults.

Serogroup Y and W135 Neisseria meningitidis capsular polysaccharide vaccines were tested as monovalent and divalent preparations in groups of 10 adult human volunteers at a dose of 50 (monovalent) or 100 micrograms (divalent) injected subcutaneously. Reactogenicity was low for the group Y vaccine and the group Y-W135 combined vaccine; 3 of 10 volunteers developed systemic reactions after group W135 vaccination. All three vaccines induced significant homologous and heterologous binding and bactericidal antibody. Except for group W135 bactericidal antibody, homologous responses exceeded heterologous responses, and divalent and monovalent vaccines induced equivalent homologous responses. Homologous bactericidal antibody responses were maintained for 4 weeks in 85% of group W135 vaccinates and in 100% of group Y vaccinates. Bactericidal antibody was induced in 11 of 11 group Y and 12 of 15 group W135 volunteers without preexisting respective bactericidal activities, regardless of which vaccine they received. For all three vaccines, antibody levels declined only slightly over 6 months. Prevaccination antibody levels positively affected postvaccination binding antibody levels, but not bactericidal levels.

Antibody-dependent mononuclear cell-mediated antimeningococcal activity. Comparison of the effects of convalescent and postimmunization immunoglobulins G, M, and A.

We have compared the abilities of immunoglobulin (Ig)G, IgM, and IgA to induce either mononuclear cell-mediated (complement-independent) or complement-mediated (cell-free) antibacterial activity against group C meningococci. In each of these assays, immunoglobulins purified from the sera of individuals immunized with meningococcal group C polysaccharide were compared with those purified from sera of patients convalescing from disseminated meningococcal disease. Our data support three conclusions. First, although nonbactericidal in cooperation with complement, IgA can induce cell-mediated antibacterial activity as well as IgG. Second, the amount of IgG required to induce cell-mediated antibacterial activity is similar to the amount required for complement-mediated killing. Third, although the amount of either postimmunization or convalescent IgM required to induce complement-mediated killing is 16- to 20-fold less than the amount of respective IgG required, IgM is inferior to IgG in its ability to induce cell-mediated antibacterial activity because in the cell-mediated system (a) postimmunization IgM is ineffective; (b) the amount of convalescent IgM required for minimal activity is eightfold more than the amount of convalescent IgG required; and (c) the maximal antibacterial index induced by convalescent IgM is 50% less than that which can be induced by IgG. These data suggest that IgG and IgA may play a greater role than IgM in mononuclear cell-mediated antibacterial host immune defense.

IgA-dependent, monocyte-mediated, antibacterial activity.

IgA purified from the sera of patients convalescing from disseminated group C meningococcal disease induced human monocyte-mediated anti-meningococcal activity in vitro in the absence of complement. Both IgA- and IgG-dependent activity were directed against the group C meningococcal polysaccharide (Csss) capsule. The amount of IgA that was effective bound less than 1 ng of Csss. Antibacterial activity was dependent upon the length and the temperature of the test incubation and on the concentration of monocytes. The implications of this mechanism for local cell-mediated antibacterial immunity are discussed.

Elaboration of both the group W135 and group Y capsular polysccharides by a single strain of Neisseria meningitidis.

A single strain (8021) of Neisseria meningitidis, isolated from a child with disseminated meningococcal disease, was found to elaborate two serogroup-specific capsular polysaccharides-Y and W135. The original isolate as well as the progeny of ten single colony sub-isolates each agglutinated with both group Y and group W135 serogrouping antisera. The capsular polysccharide of strain 8021 contained the chemical constituents of both the W135 and Y capsular polysaccharides in a ratio of about 2.5:1. The patient responded immunologically to both capsular polysaccharides with haemagglutinating antibodies. Analysis by double diffusion in agar revealed that the capsular polysaccharide of strain 8021 contained individual molecules of group W135 and group Y capsular polysaccharides as well as a mosaic molecule containing both antigenic determinants.

Disease due to serogroup W135 Neisseria meningitidis.

Five cases of disseminated meningococcal disease due to serogroup W135 Neisseria meningitidis are presented. The cases ranged in age from 16 months to 23 years, and spanned a clinical spectrum from mild meningitis without rash or evidence of meningococcal septicemia to severe meningoencephalitis with fulminant meningococcemia, disseminated intravascular coagulation, and death. These cases demonstrate that serogroup W135 N meningitidis is fully pathogenic for man and capable of producing the full spectrum of disseminated meningococcal disease associated with other serogroups. Since this serogroup has recently emerged as a significant cause of disease in Europe, attention should be focused on the correct serogroup designation of strains of N meningitidis isolated from clinical material and reported as "nongroupable" by clinical laboratories, so that additional clinical and epidemiologic information may be obtained.

Immunogenicity of serogroup A and C Neisseria meningitidis polysaccharide vaccines administered together in humans.

Serogroup A and C Neisseria meningitidis polysaccharide vaccines were administered to U.S. Army recruit volunteers, either two weeks apart or at the same time into two different sites on the same arm, to determine what effect, if any, the combined administration would have on the immunogenicity of each individual component. The vaccines were found to be as immunogenic when administered together as when given separately; in fact, the data suggest a possible enhancement of the response, as measured by the radioactive antigen binding test, to both components when the two vaccines are given together.