Characterization and immunogenicity of meningococcal group C conjugate vaccine prepared using hydrazide-activated tetanus toxoid.

The steps to produce, purify and control an immunogenic Brazilian conjugate vaccine against group C meningococcus (MenCPS-TT) using hydrazide-activated tetanus toxoid were developed. The conjugation methodology reduced the reaction time easily allowing scale-up. One freeze-dried pilot vaccine lot purified by tangential filtration, showed satisfactory quality control results including safety and stability. The pilot vaccine was immunogenic in mice in a dose-dependent fashion generating a 10-20-fold rise in IgG response in mice. The vaccine also induced high bactericidal titers. Vaccine concentrations of 1 and 0.1 microg showed higher avidity indices, suggesting induction of immunologic memory. These results support initiation of Phase I clinical studies with the MenCPS-TT conjugate vaccine.

Differential capacities of outer membrane proteins from Neisseria meningitidis B to prime the murine immune system after vaccination.

Understanding the specificity of antibody response to Neisseria meningitidis serogroup B (Men B) is a key requirement for the development of an effective vaccine. This study was designed to investigate the antigen specificity of murine IgG1 and IgG2b antibodies induced by different primary immunization schedules and the booster dose with the Cuban Men B vaccine. Immunoblotting analyses were performed using outer membrane vesicles (OMV) from the vaccine strain (B:4,7:P1.19,15). IgG subclasses binding to PorA, PorB and RmpM were determined by digital scanning of the immunoreactive bands. Bactericidal antibody response after vaccination was also evaluated. The results indicated that IgG2b anti-PorA was the main antibody response induced by two doses of the vaccine. A primary series of three doses was found important for increasing IgG2b as well as IgG1 to PorB and RmpM. The fourth dose favoured the recognition of RmpM as detected by the increase of specific IgG1 and IgG2b. IgG subclasses anti-PorA did not change significantly if animals received two, three or four doses of the vaccine during the primary immunization or after the booster dose for all vaccine groups. The booster response to PorB and RmpM of groups BC2 and BC3 showed a significant increase in IgG2b levels compared with the primary response. However, the recall and the primary response of group BC4 were similar, suggesting a saturated dose-effect response after four doses of vaccine. The same was seen for bactericidal antibody response when human complement source was used in the assay.

Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil

A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60% of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.

Level of maternal antibody required to protect neonates against early-onset disease caused by group B Streptococcus type Ia: a multicenter, seroepidemiology study.

Because of the difficulty of conducting efficacy trials of vaccines against group B streptococcus (GBS), the licensure of these vaccines may have to rely on studies that measure vaccine-induced antibody levels that correlate with protection. This study estimates the level of maternal antibody required to protect neonates against early-onset disease (EOD) caused by GBS type Ia. Levels of maternal serum IgG GBS Ia antibodies, measured by ELISAs in 45 case patients (neonates with EOD caused by GBS Ia) and in 319 control subjects (neonates colonized by GBS Ia but without EOD) born at > or =34 weeks gestation were compared. The probability of developing EOD declined with increasing maternal levels of IgG GBS Ia antibody (P = .03). Neonates whose mothers had levels of IgG GBS Ia antibody > or =5 microg/mL had an 88% lower risk (95% confidence interval, 7%-98%) of developing type-specific EOD, compared with those whose mothers had levels < 0.5 microg/mL. A vaccine that induces IgG GBS Ia antibody levels > or =5 microg/mL in mothers can be predicted to confer a high degree of type-specific immunity to EOD to their infants.

Induction of group 17 specific antibodies by pneumococcal type 17F and 17A polysaccharide vaccines.

Pneumococcal capsular polysaccharide group 17 contains two distinct serotypes, 17F and 17A. Pneumococcal group 17 is present in the licensed 23 valent polysaccharide vaccines. One such vaccine contains type 17A, while the other vaccine contains type 17F. The purpose of these studies was to determine the extent of cross-protection that could be expected, as both type 17F and 17A cause disease. The antibody responses of one group of adults to a vaccine containing type 17F was compared to that of another group that received a type 17A containing vaccine. By ELISA the 17A vaccine induced more cross-reactive antibodies. Opsonophagocytic antibodies are a good predictor of protection and both vaccines induced antibodies opsonic for both 17F and 17A. We conclude that either 17F or 17A will provide similar protection against group 17 disease.

Quantification of bacterial polysaccharides by the purpald assay: measurement of periodate-generated formaldehyde from glycol in the repeating unit.

We have adapted the purpald assay for measurement of bacterial polysaccharides (PS) containing substituted and/or unsubstituted glycol (SG or UG) in residues such as glycerol, ribitol, arabinitol, furanosyl galactose, and sialic acid. For the purpald assay of UG-containing PS, 50 microL of PS samples was consecutively reacted with 50 microL of 16 mM NaIO4 for 20 min, 50 microL of 136 mM purpald reagent in 2 N NaOH for 20 min, and 50 microL of 64 mM NaIO4 for 20 min in a 96-well tissue culture plate followed by a measurement of absorbance at 550 nm with a plate reader. For SG-containing PS, conversion of SG to UG with 25 micro;L of 0.3 N NaOH, 1 h at room temperature for de-O-acetylation followed by 25 microL of 0.6 M H2SO4, 1 h at 80 degrees C for acid hydrolysis of PS precedes the periodate treatment in the purpald assay. The concentration of the samples can be calculated from the sample absorbance and the reference standard curve constructed from the reference concentrations of the same PS (well-characterized) and their corresponding absorbance values assayed in the same plate. The purpald assay provides a tool in addition to the existing ones for the measurement of glycol-containing PS. Among the usefulness of this method are the determinations of the glycerol content in the phospho-glycerol-containing PS and the SG and UG contents and structural integrity in PS and conjugate vaccines.

Immunogenicity in mice of pneumococcal glycoconjugate vaccines using pneumococcal protein carriers.

Antibody response and protective immunity were evaluated in mice immunized with pneumococcal glycoconjugate vaccines using two pneumococcal protein carriers. Mice injected with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) produced high levels of IgG and IgM antibodies to both the PS and the protein carrier. Higher PS antibody titers to the pneumococcal PS conjugates were measured by ELISA using PS-Ply or PS-tetanus toxoid (TT) conjugate as a coating antigen compared with PS mixed with methylated human serum albumin. Type 9V PS (10 microg) inhibited most of the 9V IgM and IgG antibody binding to the 9V-TT coated plate. In contrast, absorption with 19F PS did not inhibit 9V antibody binding. The avidity index of IgG antibodies in the 9V PS-Ply serum was 55.5 +/- 0.9, compared with 47.8 +/- 1.4 for 9V PS-Aly serum. Thus, high avidity of serum antibodies in conjugate-immunized mice can provide more effective functional activity for protection against pneumococcal infection. Mice immunized with these glycoconjugates exhibited rapid bacterial clearance from blood and provided cross-protection against challenge with heterologous serotypes of virulent pneumococci. These results reveal that conjugates using pneumococcal protein carriers can induce opsonophagocytic activity to destroy homologous and heterologous pneumococci, indicating that such conjugates can confer broader protective immunity than conjugates using non-pneumococcal proteins.

Pneumococcal type 22f polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay.

The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

Outer membrane protein vesicle vaccines for meningococcal disease.

Alternative strategies exist for prevention of group B Neisseria meningitidis (meningococcal) disease through vaccination (see Chapters 5 , 8 , 13 , 14 in this volume). However, the most promising approach to date has been the use of outer-membrane vesicle (OMV) vaccines for induction of bactericidal antibodies against cell-surface outer-membrane proteins (OMPs).

Genetic and immunologic characterization of a novel serotype 4, 15 strain of Neisseria meningitidis.

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.

An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study.

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.

Specificity of human antibodies reactive with pneumococcal C polysaccharide.

Antibodies reactive with C polysaccharide (PS) were found in healthy adults, pneumococcal patients, and vaccinees. These antibodies were not directed to the phosphocholine determinant of the C PS, as they appear to be in mice, since the human antibodies were inhibitable only with C PS. We found another population of phosphocholine-specific antibodies inhibitable only by phosphocholine and related compounds.

Immunization with meningococcal outer-membrane protein vesicles containing lipooligosaccharide protects mice against lethal experimental group B Neisseria meningitidis infection and septic shock.

Detergent-treated group B Neisseria meningitidis outer membrane vesicles (D-OMVs) from wild-type M986 and from nonencapsulated mutant M986-non-capsule variant (NCV) were compared as immunogens. Eight weeks after 3 consecutive immunizations with the immunogens, mice were challenged with a lethal dose of purified endotoxin or heat-killed or living N. meningitidis, plus d-galactosamine (400 mg/kg). D-OMVs from M986 induced bactericidal antibodies to both M986 (B : 2a : P1.5,2 : L3,7) and 6275 (B : 2a : P1.2,5 : L3) and protected the animals against both strains, whereas D-OMVs from M986-NCV did not protect the animals against infection with 6275 even when high serum bactericidal activity was induced. Tumor necrosis factor-alpha detected after bacterial infection was high in both protected and unprotected mice; interleukin (IL)-6 was high in mice that died but low in animals that survived. Exogenous administration of recombinant mouse IL-6 reversed the immunogens' protective effects. Protection against infection in mice does not necessarily correlate with the measured levels of serum bactericidal antibody alone, opsonic antibody alone, or cytokine profile alone. A comprehensive assessment of the preclinical efficacy of group B outer-membrane protein vaccines should include monitoring humoral antibodies, cytokine response, and protective effects against lethal infection.

Inhibition enzyme-linked immunosorbent assay for serotyping of group B streptococcal isolates.

Group B Streptococcus (GBS) is one of the most common organisms causing neonatal sepsis as well as serious infections in adults. Serotyping the organism is important in studying the epidemiology of the disease as well as deciding a course of treatment. There are several methods available for serotyping. Most of them need high-titered sera and are not quantitative. We are reporting a new inhibition enzyme-linked immunosorbent assay (ELISA) for serotyping which is sensitive and specific compared to the conventional methods but does not need high-titered serotype-specific antisera, as the specificity is controlled by the polysaccharide coating on the ELISA plates. The method can also be quantitative, and we have measured polysaccharide elaborated by different serotype V strains. Thus, the inhibition ELISA method will be useful in serotyping for epidemiological studies, assessing virulence, and performing strain selection for vaccine production.

Proposed standardization of Neisseria meningitidis PorA variable-region typing nomenclature.

Neisseria meningitidis isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of N. meningitidis, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.

Estimation of group B streptococcus type III polysaccharide-specific antibody concentrations in human sera is antigen dependent.

The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure

Meningococcal disease caused by Neisseria meningitidis serogroup B serotype 4 in São Paulo, Brazil, 1990 to 1996.

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988. A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

The use of oligonucleotide probes for meningococcal serotype characterization.

In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs). Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs) are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A) and 615U (VR3-B) used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

The immunogenicity of Haemophilus influenzae type b conjugate vaccines in children born to human immunodeficiency virus-infected women. Women and Infants Transmission Study Group.

BACKGROUND: Immunocompromise caused by HIV-1 infection increases the importance of receipt of routine childhood vaccines to prevent infections such as invasive Haemophilus influenzae type B (Hib) disease. The objectives of the study were to evaluate the immunogenicity of Hib conjugate vaccines among HIV-infected children according to clinical and immunologic disease progression as well as viral load. METHODS: The concentration of antibody to polyribosylribitol phosphate (PRP) was measured at approximately 9 and 24 months of age in plasma specimens from children of HIV-infected women enrolled in the Women and Infants Transmission Study. RESULTS: Among 227 children (35 HIV-infected, 192 uninfected) at the 9-month study visit who were known to have received age-appropriate immunization with CRM197 mutant Corynebacterium diphtheriae protein-conjugated Hib vaccine, geometric mean antibody concentrations were lower among HIV-infected children (1.64 microg/ml) than among uninfected children (2.70 microg/ml), although the difference was not statistically significant. Anti-PRP antibody concentrations did not vary significantly among these HIV-infected children with predominantly mild-moderate disease progression according to clinical category, immunologic stage or viral load (P > or = 0.48). The proportion of children with antibody concentrations > or = 1.0 microg/ml did not vary significantly according to HIV infection status (73% uninfected, 74% infected) or, if infected, clinical or immunologic disease progression or viral load. Similar results were obtained among 127 children (17 HIV-infected, 110 uninfected) eligible for analysis at the 24-month study visit. Changes in antibody concentrations over time (between 9 and 24 months of age) did not differ significantly among 10 HIV-infected as compared with 72 uninfected children (P=0.81). CONCLUSIONS: These results suggest that HIV-infected children with predominantly mild-moderate disease progression respond reasonably well in terms of a quantitative antibody response to Hib conjugate vaccines during the first 2 years of life. Research to further characterize the immune response to Hib conjugate vaccines and to further delineate the "durability" of anti-PRP antibody concentrations beyond 2 years of life should be pursued.

Correlation between serological and sequencing analyses of the PorB outer membrane protein in the Neisseria meningitidis serotyping system.

The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.