Human T-Cell Responses after Vaccination with the Norwegian Group B Meningococcal Outer Membrane Vesicle Vaccine

We have analyzed human T-cell responses in parallel with serum immunoglobulin G (IgG) antibody levels after systemic vaccination with the Norwegian group B Neisseria meningitidis outer membrane vesicle (OMV) vaccine. Ten adult volunteers, with no or very low levels of serum IgG antibodies against meningococci, received three doses intramuscularly of the OMV vaccine (at weeks 0, 6, and 46). T-cell proliferation against the OMV vaccine, purified outer membrane proteins (PorA and PorB), and control antigens (Mycobacterium bovis BCG vaccine and tetanus toxoid) was measured by thymidine incorporation of peripheral blood mononuclear cells before and after vaccination. The results showed that vaccination with OMV elicits strong primary and booster T-cell responses specific to OMV as well as the PorA (class 1) protein and significant, but markedly lower, responses against the PorB (class 3) protein. The median responses to OMV and PorA were 26 and 16 times the prevaccination levels, respectively. Most of the vaccinees showed low T-cell responses against OMV and PorA before vaccination, and the maximum T-cell responses to all vaccine antigens were usually obtained after the second vaccine dose. We found a positive correlation between T-cell responses and anti-OMV IgG antibody levels (r = 0.50, P < 0.0001, for OMV and PorA). In addition, we observed a progressive increase in the percentage of CD45R0⁺ (memory) CD4-positive T cells (P = 0.002). In conclusion, we have shown that the Norwegian OMV vaccine against meningococcal B disease induced antigen-specific T-cell responses, kinetically accompanied by serum IgG responses, and that vaccination increased the proportion of memory T-helper cells.

Vaccination with protein antigens will usually result in both a cellular (T-cell) and humoral (B-cell) immune response. For protection against extracellular bacterial infections, like Neisseria meningitidis, bactericidal and opsonic antibodies are of crucial importance, while T cells play a more indirect role by regulating the antibody response in terms of immunoglobulin class switch, affinity maturation, and magnitude of response (1). T cells are also necessary for the induction of immunological memory and can indirectly induce killing of bacteria by activating phagocytes (24).Although polysaccharide-based vaccines against serogroup A and C meningococci are available, this principle cannot be applied to the B serogroup (the most prevalent serogroup in Europe, America, and South Australia) due to low immunogenicity of the B polysaccharide in humans (36). Furthermore, polysaccharides fail to induce T-cell responses and give a poor and short-lived immunity in infants due to their immature immune systems (13, 26). This situation has motivated the development of a Norwegian outer membrane vesicle (OMV) vaccine against group B meningococci based on a B:15:P1.7,16 epidemic strain (44/76) in which the major antigens are outer membrane proteins (11). The porin proteins PorA (class 1) and PorB (class 3) are the most abundant neisserial outer membrane proteins (6). They are present in equal amounts and account for about 70% of the proteins (by weight) in the OMV vaccine (11). This vaccine has previously been shown to induce protection against group B meningococcal disease among teenagers in a large, double-blind, placebo-controlled study (4).To address the question of T-cell-mediated help in vaccine-induced protective B-cell responses against meningococci, we have investigated human T-cell responses during a three-dose regimen with the Norwegian OMV vaccine. Proliferative in vitro T-cell responses against the OMV vaccine and PorA and PorB outer membrane proteins were assayed in peripheral lymphocytes from OMV vaccinees and compared with immunoglobulin G (IgG) antibody responses.     

Idiotypic Vaccination: Immunoprotection Mediated by Anti-idiotypic Antibodies with Antibiotic Activity

Anti-Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin. Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin-sensitive Candida alhicans cells. The course of candidosis was studied in comparison with mice non-immunized and immunized with an isotypc-matched unrelated MoAb subdivided into control groups. Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin-like anti-Id antibodies. MoAb KT4 affinity chromatography purified mouse anti-Id antibodies were capable of killing in vitro the yeast ceils of the Candida albicans strain used for the experimental infection.
This is the first report of antimicrobial protection that exploits the role of anti-idiotypic antibodies presumably acting in vivo as antibiotics (idiotypic vaccination).     

Intranasal Administration of a Meningococcal Outer Membrane Vesicle Vaccine Induces Persistent Local Mucosal Antibodies and Serum Antibodies with Strong Bactericidal Activity in Humans

A nasal vaccine, consisting of outer membrane vesicles (OMVs) from group B Neisseria meningitidis, was given to 12 volunteers in the form of nose drops or nasal spray four times at weekly intervals, with a fifth dose 5 months later. Each nasal dose consisted of 250 μg of protein, equivalent to 10 times the intramuscular dose that was administered twice with a 6-week interval to 11 other volunteers. All individuals given the nasal vaccine developed immunoglobulin A (IgA) antibody responses to OMVs in nasal secretions, and eight developed salivary IgA antibodies which persisted for at least 5 months. Intramuscular immunizations did not lead to antibody responses in the secretions. Modest increases in serum IgG antibodies were obtained in 5 volunteers who had been immunized intranasally, while 10 individuals responded strongly to the intramuscular vaccine. Both the serum and secretory antibody responses reached a maximum after two to three doses of the nasal vaccine, with no significant booster effect of the fifth dose. The pattern of serum antibody specificities against the different OMV components after intranasal immunizations was largely similar to that obtained with the intramuscular vaccine. Five and eight vaccinees in the nasal group developed persistent increases in serum bactericidal titers to the homologous meningococcal vaccine strain expressing low and high levels, respectively, of the outer membrane protein Opc. Our results indicate that meningococcal OMVs possess the structures necessary to initiate systemic as well as local mucosal immune responses when presented as a nasal vaccine. Although the serum antibody levels were less conspicuous than those after intramuscular vaccinations, the demonstration of substantial bactericidal activity indicates that a nonproliferating nasal vaccine might induce antibodies of high functional quality.     

Interlaboratory Standardization of the Measurement of Serum Bactericidal Activity by Using Human Complement against Meningococcal Serogroup B, Strain 44/76-SL, before and after Vaccination with the Norwegian MenBvac Outer Membrane Vesicle Vaccine

There is currently no standardized serum bactericidal antibody (SBA) assay for evaluating immune responses to meningococcal outer membrane vesicle or protein vaccines. Four laboratories, Manchester Health Protection Agency (MC HPA), New Zealand Institute of Environmental Science and Research Limited (NZ ESR), Norwegian Institute of Public Health (NIPH), and Chiron Vaccines (Chiron), measured SBA titers in the same panel of human sera (n = 76) from laboratory staff (n = 21) vaccinated with MenBvac. Blood samples were collected prevaccination, prior to each of the three doses of MenBvac given at 6-week intervals, and 6 weeks following the third dose. Initial results showed a number of discrepancies in results between the four participating laboratories. The greatest effect on titers appeared to be due to differences among laboratories in the maintenance of the meningococcal serogroup B test strain, 44/76-SL. A repeat study was conducted using the same frozen isolate (meningococcal serogroup B test strain 44/76-SL), freshly distributed to all four laboratories. Using SBA titers from the tilt method for all samples, and using MC HPA as the comparator, the results were as follows for NZ ESR, NIPH, and Chiron, respectively, using log₁₀ titers: correlation coefficients (r) were 0.966, 0.967, and 0.936; intercepts were 0.08, 0.15, and 0.17; and slopes were 0.930, 0.851, and 0.891. In both prevaccination and postvaccination samples from 15 subjects assayed by all four laboratories, similar increases in SBA (fourfold or greater) were observed (for 11, 11, 9, and 9 subjects for MC HPA, NZ ESR, NIPH, and Chiron, respectively), and similar percentages of subjects with SBA titers of ≥4 prevaccination and 6 weeks following each dose were found. The SBA assay has been harmonized between the four different laboratories with good agreement on seroconversion rates, n-fold changes in titers, and percentages of subjects with SBA titers of ≥4.     

Avidity of IgG Antibodies Against Meningococcal Serogroup A Polysaccharide and Correlations with Bactericidal Activity in Sera From Meningitis Patients and Controls From Ethiopia

Meningococcal meningitis is a significant global health challenge, especially for sub‐Saharan area: the African meningitis belt. Neisseria meningitidis of serogroup A (MenA) is responsible for the large number of epidemics that have been recorded in these countries. To determine the level of antibodies against meningococcal A polysaccharide (APS) that correlates with protection against MenA disease in the African meningitis belt, it may be important to consider antibody avidity along with quantity. In this study, two ELISA methods using the chaotropic agent ammonium thiocyanate were compared and employed to measure avidity indexes (AI) of IgG antibodies against APS in controls and in acute and convalescent sera from Ethiopian meningococcal patients. High statistical correlations between the AIs determined by the two methods were observed. The geometric mean AI (GMAI) increased with time from acute to convalescent sera indicating affinity maturation. GMAI was significantly higher in convalescent sera from the MenA patients and in sera from the controls than in acute sera from patients with meningococcal disease. A significant correlation between serum bactericidal activity titres (SBA) and concentration of IgG antibodies against APS was observed; however, our results did not indicate that determination of antibody avidities by the thiocyanate elution method gave a better correlation with SBA than anti‐APS IgG concentrations determined by the standard ELISA method.     

IgG Subclass Deficiency in Patients with Down''s Syndrome and Aberrant Hepatitis B Vaccine Response

Seventeen adult patients with Down's syndrome (DS) and 19 adult healthy references were vaccinated with a hepatitis B vaccine in order to study the IgG subclass response. An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies specific for IgG subclasses was employed. In spite of normal levels of total IgG1 and normal or even high levels of IgG3 in the DS patients, a significantly lower IgG1 response to the vaccine was observed in trisomic patients than in the references.     

A Flow Cytometric Opsonophagocytic Assay for Measurement of Functional Antibodies Elicited after Vaccination with the 23-Valent Pneumococcal Polysaccharide Vaccine

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5,6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with ≥97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P ≤ 0.01) was found between the two assays for the seven serotypes tested. The flow cytometric assay is rapid (~4 h) with high throughput (~50 serum samples per day per technician) and provides a reproducible measurement of serotype-specific functional antibodies, making it a highly suitable assay for the evaluation of the immune responses elicited by pneumococcal vaccines.     

Opsonophagocytic Activity Induced by Chimeric Antibodies of the Four Human IgG Subclasses With or Without Help from Complement

The Opsonophagocytic activity of the four human IgG subclasses was studied using chimeric mouse-human antibodies with specificity for the hapten NIP. As target cells we used haptenized sheep red blood cells and N. meningitidis , labelled with different amounts of hapten. We used polymorphonuclcar leucocytes (PMN) as effector cells to measure respiratory burst (RB), and U937 to measure phagocytosis/rosette formation. When the target cells were opsonized with antibody only, and PMN used as effector cells, lgG3 was highly efficient, while IgG1 revealed an intermediate activity and IgG2 and IgG4 were negative. The same pattern among the subclasses was obtained in the presence of complement source, when target cells with low hapten concentration were used. However, at high cpitope concentration on the target cells, in the presence of complement source, IgG2 was highly active. while IgG4 was still negative or only slightly positive. When U937 were used as effector cells and complement was omitted, IgG1, IgG3 and lgG4 all revealed high phagocylic/rosette-forming activity, while IgG2 was negative. When the target cells were opsonized with antibody and complement, the phagocytic/rosette-forming activity was often suppressed. Our results reveal that all four human IgG subclasses possess Opsonophagocytic capacity, but with different requirements concerning complement and Fc Rs. They also enlighten us as to how IgG2 might perform its protective effect against harmful bacteria displaying high density of carbohydrate epitopes on their outside surface.     

Importance of Antibodies to Lipopolysaccharide in Natural and Vaccine-Induced Serum Bactericidal Activity against Neisseria meningitidis Group B

Analysis of the specificity of bactericidal antibodies in normal, convalescent, and postvaccination human sera is important in understanding human immunity to meningococcal infections and can aid in the design of an effective group B vaccine. A collection of human sera, including group C and group B convalescent-phase sera, normal sera with naturally occurring cross-reactive bactericidal activity, and some postvaccination sera, was analyzed to determine the specificity of cross-reactive bactericidal antibodies. Analysis of human sera using a bactericidal antibody depletion assay demonstrated that a significant portion of the bactericidal activity could be removed by purified lipopolysaccharide (LPS). LPS homologous to that expressed on the bactericidal test strain was most effective, but partial depletion by heterologous LPS suggested the presence of antibodies with various degrees of cross-reactivity. Binding of anti-L3,7 LPS bactericidal antibodies was affected by modification of the core structure, suggesting that these functional antibodies recognized epitopes consisting of both core structures and lacto-N-neotetraose (LNnT). When the target strain was grown with 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) to increase LPS sialylation, convalescent-phase serum bactericidal titers were decreased by only 2- to 4-fold, and most remaining bactericidal activity was still depleted by LPS. Highly sialylated LPS was ineffective in depleting bactericidal antibodies. We conclude that natural infections caused by strains expressing L3,7 LPS induce persistent, protective bactericidal antibodies and appear to be directed against nonsialylated bacterial epitopes. Additionally, subsets of these bactericidal antibodies are cross-reactive, binding to several different LPS immunotypes, which is a useful characteristic for an effective group B meningococcal vaccine antigen.     

Proportions of IgG Subclasses in Antibodies Formed during Vaccination with Hepatitis B Surface Antigen

Nineteen healthy adults were given three injections of hepatitis B vaccine (days 0, 30, and 180), and serum samples were obtained on days 0, 21, 51, and 201. A fourfold or greater increase of IgG antibodies within 201 days was detected in 18 individuals (geometrical mean increase 26-fold), but final IgG antibody concentrations (0.3-3 micrograms/ml) were low. Some vaccines had almost reached peak concentration on day 21, in others a great increase was observed between days 21 and 51, and in a few individuals between days 51 and 201. IgG1 was the predominant subclass in the antibodies of most individuals. Ninety-five per cent of all IgG antibodies were IgG1 in early samples of early responders. In late samples of all vaccinees the average proportion of IgG1 antibodies was 78%. The proportion of IgG2 antibodies was slightly higher in late samples (average 12%) than in the early samples of the early responders, (average less than 6%, P less than 0.05). Considerable amounts of IgG3 and IgG4 antibodies were found in one vaccine, but on the average the proportion of IgG3 or IgG4 antibodies was low (less than 6%).     

Subclass Distribution of IgA and IgG Antibodies Against Clq in Patients with Rheumatic Diseases

To obtain insight into the immunoregulatory mechanisms in patients with different rheumatic diseases, the occurrence and the subclass distribution of IgA and IgG antibodies against Clq (anti-ClqAb) was determined. In patients with systemic lupus erythaematosus (SLE) the highest frequency of increased serum levels of IgG anti-ClqAb were found, whereas IgA anti-ClqAb were predominantly present in patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis complicated by vasculitis (RV). In all the IgA anti-ClqAb positive AS and RV patients the antibody reactivity involved the IgAl subclass while the IgA2 subclass was found in 47% of the patients. Further characterization of the IgA anti-Clq binding activity in sera of AS patients revealed that both subclasses of IgA anti-ClqAb were predominantly polymeric; the binding of both IgA subclasses with solid phase CI q was inhibitable by aggregated fluid phase Clq; we found no delectable interference of rheumatoid factor in the test system for the measurement of IgA anti-ClqAb. In patients with SLE the IgG anti-ClqAb reactivity was mainly of the IgG2 and IgG3 subclass, whereas in the same patients the IgG anti-tetanus toxoid response was not restricted to these subclasses.
The predominance of IgG2 and IgG3 subclass of anti-ClqAb in sera of SLE patients, suggests a skewing of the anti-ClqAb response. The observation that the IgA anti-ClqAb of both subclasses is predominantly polymeric in nature and the notion that polymeric IgA is associated with activation of inflammation cascades, suggests that IgA anti-ClqAb may contribute to tissue damage.     

Specificity of Bactericidal Antibody Response to Serogroup B Meningococcal Strains in Brazilian Children after Immunization with an Outer Membrane Vaccine

Pre- and postvaccination serum samples from 77 children aged 2 to 6 years, who received the Cuban BC vaccine (B:4:P1.15), were analyzed for bactericidal antibodies against a local B:4:P1.15 strain (N44/89). Sera from 16 individuals with bactericidal antibodies against the B:4:P1.15 strain were tested against 23 Brazilian isolates. These include B:4 strains of distinct serosubtypes: P1.15, P1.7,1, P1.3, P1.9, P1.nt, and a B:8,19,23:P1.16 strain. A Cuban B:4:P1.15 strain (Cu385/83) was also included in the study. The specificities of bactericidal antibodies were analyzed by using mutant strains lacking a class 1 protein (PorA protein) or a class 5 protein or both. The results indicated that PorA and class 5 proteins are the main targets recognized by the bactericidal antibodies of vaccinees. Nonetheless, a complex pattern of recognition by bactericidal antibodies was found, and vaccinees were grouped according to antibody specificity. Antibodies from some individuals recognized PorA of serosubtype P1.15. However, antibodies from these individuals could not kill all P1.15 strains tested. Antibodies from a second group recognized both PorA and class 5 proteins, and antibodies from a third group recognized an as yet unidentified target antigen. The results demonstrate the importance of determining the fine epitope specificity of bactericidal antibodies to improve the existing vaccines against B meningococci.Group B meningococcal disease remains a significant public health problem in Brazil and in many other countries (17, 22). In contrast to polysaccharides A and C, B polysaccharide is poorly immunogenic in humans (18). Development of vaccines against group B meningococcal disease has focused on the use of lipo-oligosaccharide (LOS)-depleted outer membrane proteins (OMPs) (2, 3). Between 1989 and 1990 an OMP vaccine produced in Cuba was used to immunize 2.4 million children ranging from 3 months to 6 years of age in the city of São Paulo, Brazil. Results of a case control study performed from June 1990 to June 1991 (12 months) showed that vaccine efficacy was age dependent. In children aged 24 to 48 months and aged over 48 months, estimated efficacies were 47 and 74%, respectively. There was no vaccine efficacy in children aged up to 23 months (14). In spite of being statistically significant, levels of protection observed in children 24 months or older were far from ideal and did not have a significant impact on public health as the incidence of the disease was not significantly reduced in São Paulo (14). Also, the duration of the protection induced by the vaccine remains unknown.Several factors may account for the performance of this OMP vaccine in Brazil. The fact that only a portion (∼44%) of the bacterial isolates from infected individuals matched the vaccine type strain (B:4:P1.15) could be a factor that reduced its efficacy (14). An analysis of the presence of bactericidal antibodies in the sera of the vaccinated children found that only 40% had bactericidal antibodies to a B:4:P1:1.15 strain (13). As bactericidal antibodies are believed to be important for the immunity of vaccinated individuals (5), the fact that this vaccine failed to elicit bactericidal antibodies in the majority of children may account for its poor performance. In agreement with this possibility is the fact that a correlation between vaccine efficacy and the increasing prevalence of induced bactericidal antibodies with age was found (13).Among the five main classes of proteins found in the outer membrane vesicles (OMVs) (classes 1 through 5), PorA protein and class 5 proteins have been suggested to be of great importance for the induction of bactericidal antibodies after immunization and disease (11, 19, 26). In a recent study (25), the specificity of bactericidal antibodies of individuals vaccinated with hexavalent meningococcal PorA protein vesicle vaccine was evaluated by using isogenic strains differing only in their PorA protein compositions. This study demonstrated that the epitopes that contributed predominantly to the bactericidal activity were present in loops 1 and 4 of PorA protein, which contain variant region 1 (VR1) and VR2, respectively. In a parallel study, Rosenqvist et al. (19) demonstrated that PorA protein and class 5 proteins are the major targets of bactericidal antibodies of individuals vaccinated twice with an OMV vaccine.The present study was designed to evaluate the specificity of bactericidal antibodies from Brazilian children vaccinated with the Cuban OMP vaccine. For that purpose we determined the bactericidal activities of serum samples from selected individuals against local strains as well as against mutant strains lacking either class 1 or class 5 proteins or both.     

Immunogenicity and Safety Testing of a Group B Intranasal Meningococcal Native Outer Membrane Vesicle Vaccine

The presently licensed meningococcal vaccine is a tetravalent capsular polysaccharide vaccine that induces immunity to serogroups A, C, Y, and W-135 but not to group B, which causes nearly half of the meningitis cases in the United States. The purpose of this study was to evaluate the safety and immunogenicity of an intranasal native outer membrane vesicle (NOMV) vaccine prepared from a capsule negative strain of group B of Neisseria meningitidis. In this study all volunteers received the same dose of vaccine, but we evaluated two different immunization schedules and the oropharyngeal and intranasal routes of vaccine delivery, assessed nasal cytology for cellular infiltration, and measured antibody-secreting cells (enzyme-linked immunospot assay [ELISPOT]) as an early marker for systemic immune response. Additionally, both intranasal and serum vaccine-specific antibodies were measured as well as serum bactericidal activity. Four groups with a total of 42 subjects were immunized on days 0, 28, and 56. Group 3 received an additional dose on day 7. Group 2 subjects were immunized both intranasally and oropharyngeally. Group 4 received a different lot of vaccine. All groups received approximately 1,200 microg of vaccine per subject. Patients were evaluated for side effects. The vaccine was well tolerated without evidence of inflammation on nasal cytology. The group receiving the extra vaccine dose showed the maximum increase in bactericidal activity. Thirty of 42 subjects demonstrated an increase in meningococcus-specific intranasal immunoglobulin A (IgA) titers, while 23 of 42 demonstrated an increase in specific IgG titers. The group receiving vaccine intranasally and oropharyngeally showed the highest rise in intranasal titers for both IgA and IgG. Groups 1, 3, and 4 showed a significant increase in antibody-secreting cells on ELISPOT. Eighteen of 42 volunteers demonstrated a fourfold or greater rise in bactericidal titers, with 81% showing an increase over baseline. We have demonstrated the immunogenicity and safety of a group B lipopolysaccharide-containing, intranasal, NOMV vaccine.     

The Transferrin Binding Protein B of Moraxella catarrhalis Elicits Bactericidal Antibodies and Is a Potential Vaccine Antigen

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.     

IgG1 Subclass Restriction of Oligoclonal Measles Virus-Specific IgG Antibodies in Patients with Subacute Sclerosing Panencephalitis and in a Patient with Multiple Sclerosis

A restriction to the IgG1 subclass was demonstrated for measles virus-specific IgG antibodies isolated from the sera of five patients with subacute sclerosing panencephalitis, from the cerebrospinal fluid of one of the patients, and from brain extract from a sixth patient. A predominance of IgG1 was also observed in measles antibodies isolated from the serum and brain extract of a patient with multiple sclerosis. Evidence is presented that the IgG1 restriction is associated with the occurrence of oligoclonal measles antibodies in these patients. A similar restriction to the IgG1 subclass was not observed in measles antibodies isolated from the serum of a measles-convalescent child or from pooled normal serum and IgG.     

Quadrivalent Meningococcal Vaccination of Adults: Phase III Comparison of an Investigational Conjugate Vaccine,MenACWY-CRM,with the Licensed Vaccine,Menactra

Neisseria meningitidis is a leading cause of bacterial meningitis in the United States, with the highest case fatality rates reported for individuals ≥15 years of age. This study compares the safety and immunogenicity of the Novartis Vaccines investigational quadrivalent meningococcal CRM₁₉₇ conjugate vaccine, MenACWY-CRM, to those of the licensed meningococcal conjugate vaccine, Menactra, when administered to healthy adults. In this phase III multicenter study, 1,359 adults 19 to 55 years of age were randomly assigned to one of four groups (1:1:1:1 ratio) to receive a single dose of one of three lots of MenACWY-CRM or a single dose of Menactra. Serum samples obtained at baseline and 1 month postvaccination were tested for serogroup-specific serum bactericidal activity using human complement (hSBA). The hSBA titers following vaccination with MenACWY-CRM and Menactra were compared in noninferiority and prespecified superiority analyses. Reactogenicity was similar in the MenACWY-CRM and Menactra groups, and neither vaccine was associated with a serious adverse event. When compared with Menactra, MenACWY-CRM met the superiority criteria for the proportions of recipients achieving a seroresponse against serogroups C, W-135, and Y and the proportion of subjects achieving postvaccination titers of ≥1:8 for serogroups C and Y. MenACWY-CRM''s immunogenicity was statistically noninferior (the lower limit of the two-sided 95% confidence interval was more than −10%) to that of Menactra for all four serogroups, with the postvaccination hSBA geometric mean titers being consistently higher for MenACWY-CRM than for Menactra. MenACWY-CRM is well tolerated in adults 19 to 55 years of age, with immune responses to each of the serogroups noninferior and, in some cases, statistically superior to those to Menactra.Neisseria meningitidis is a leading cause of bacterial meningitis in the United States (11). While the incidence of meningococcal disease is highest in infants, the highest case fatality rates are observed among older subjects: 4.6% in children <15 years of age, 22.5% in individuals 15 to 24 years of age, and 16.5% in adults >25 years of age (5). In 2007, approximately 72% of all cases of meningococcal disease in the United States were reported in individuals ≥18 years of age (4a).Two quadrivalent meningococcal vaccines, for prevention of meningococcal disease caused by serogroups A, C, W-135, and Y, are available in the United States: an unconjugated polysaccharide vaccine (MPSV4 [Menomune]; Sanofi Pasteur, Inc., Swiftwater, PA) and a diphtheria toxoid protein conjugated vaccine (Menactra; Sanofi Pasteur, Inc., Swiftwater, PA). In the United States, the quadrivalent meningococcal polysaccharide protein conjugate vaccine is currently recommended for all persons 11 to 18 years of age and for those persons 2 to 55 years of age who are at increased risk of meningococcal disease (4). This vaccine is only available in the United States and Canada. Outside North America, polysaccharide vaccines are the only quadrivalent meningococcal vaccines available.To expand the options for prevention of meningococcal disease, an investigational quadrivalent meningococcal CRM₁₉₇ conjugate vaccine (MenACWY-CRM; Novartis Vaccines, Siena, Italy) was recently developed. Studies have shown that MenACWY-CRM is well tolerated and elicits robust immunogenicity when administered to infants as young as 2 months of age (10, 12), as well as children (2a) and adolescents (9). A large, randomized, controlled, direct comparative phase III study was recently completed, examining the safety and immunogenicity of MenACWY-CRM versus those of Menactra when administered to healthy subjects 11 to 55 years of age. Due to differences in expected immune responses between adolescents and adults, the predefined analyses were powered and analyzed separately for the 11- to 18-year-old and 19- to 55-year-old groups. The larger adolescent age stratum included in the study was powered to demonstrate the consistency of the immune response to three lots of MenACWY-CRM, and the results showed that MenACWY-CRM generated a significantly higher immune response to all four serogroups than Menactra (8). Here, we present the analysis of the safety and immunogenicity of MenACWY-CRM versus those of Menactra among the adult subjects 19 to 55 years of age.     

Investigation on the Effect of Immune Selection on Resistance to Bactericidal Antibodies to Group B Meningococci In Vitro

The induction of resistance by immune selective pressure to bactericidal antibodies from humans immunized with Novartis recombinant meningococcal group B vaccines was assessed. Serum bactericidal antibody titers against selected bacteria were within assay variability through a selection event frequency of 1 in 10⁻⁵. No change in antigen expression was observed by Western blotting.The development of a broadly protective vaccine against group B meningococci (MenB) has been hampered by ineffective MenB capsular polysaccharide vaccines and extensive variability of outer membrane proteins targeted by vesicle-based vaccines (1, 11, 15, 16). We have approached this problem by selecting candidate antigens encoded on the MenB genome that can individually serve as targets for protective antibody responses yet are very distinct from each other (6, 13). The use of diverse, highly conserved antigens could reduce the possibility of immune escape due to vaccine-induced selective pressure (17). However, given the high degree of genetic instability observed for group B strains (5, 7, 8, 12), it is possible that resistance to bactericidal antibodies could be induced by vaccination. To address this question, we have selected bacteria with human serum complement and bactericidal antibodies induced by one of two vaccines containing 25 μg each of three recombinant proteins known to elicit bactericidal responses in mice—NadA as a single polypeptide and factor H-binding protein (fHBP) and GNA2132 fused to carrier proteins as GNA2091-fHBP and GNA2132-GNA1030—either alone (6) or in combination with 50 μg outer membrane vesicles from strain H44/76, with each vaccine in 1.5 mg of aluminum hydroxide per 0.5-ml dose. Sera were collected 1 month after immunization with three doses of vaccine given at 1-month intervals, after a fourth immunization given 4 months after the third dose, or after a fifth immunization given 12 months after the fourth dose. Control sera were obtained before the first immunization and pretested to ensure that they lacked naturally acquired bactericidal antibodies against strains H44/76 and 2996.The bactericidal assay was performed using human complement as described previously (14). Briefly, frozen stock cultures of bacteria were grown overnight on chocolate agar. The next day (day 1), 10 to 20 colonies were selected, pooled, and grown in Mueller-Hinton broth (Becton-Dickinson) for approximately 2 h to mid-log phase. Bacteria were then diluted to a concentration of 2.0 × 10⁴/ml for use in the assay. Test sera were serially diluted twofold in 96-well plates starting from a 1:2 dilution and then incubated for 60 min with bacteria and 25% human serum complement lacking intrinsic bactericidal activity. Aliquots were spread onto chocolate agar plates and grown overnight. All bacterial cultures were grown at 37°C in 5% CO₂. On day 2, colonies were counted and the 50% titer of each test specimen relative to that of the time zero inoculum was determined. Surviving bacterial colonies from the serum dilution treatment that resulted in 90% killing of bacteria were collected from the agar plate and pooled to prepare a new broth culture on day 2 that was then immediately reassayed in the next cycle. This process was repeated for up to five rounds of selection. We chose to use five rounds of 10-fold reduction in colony counts (overall, a 10,000-fold selection) based on studies of phase variation in MenB in which individual genes underwent phase variation with frequencies of 1 in 10⁴ to 1 in 10⁵ (9). A pool of surviving colonies was used in each round instead of single colonies being picked posttreatment to optimize the chance of passaging a selection variant. Also, the use of pooled colonies is a standard practice when performing bactericidal assays on meningococci (3). The criterion used to assess induction of resistance was a fourfold or greater reduction in titer compared to that of control bacteria. This criterion was selected because the sera are titrated in a twofold serial dilution and a difference of two titer steps is the accepted criterion for a measurable difference in results (4), which reflects the inherent variability of the assay.The study included two types of planned comparisons. First, we compared the titers of a set of immune serum against bacteria that had been selected over four rounds of selection in that same serum to the titers obtained against bacteria treated with control serum. The effect of this selection process using strains H44/76 and 2996 is shown in Table ​Table1.1. A fourfold or greater reduction in the mean difference in titer was not observed at any of the four selection steps. The mean difference after the fourth cycle of selection was approximately 1.3-fold. Mean differences were calculated using logarithmically transformed titers (logarithm to base 2). Second, we compared the bactericidal titers of 10 immune sera using strain H44/76 after a fifth selection cycle to the results with the control treatment. The mean difference in titers was approximately −2.5-fold (Table ​(Table2).2). Overall, there was a slight trend toward lower titers when bacteria were selected with a serum containing bactericidal antibody.

TABLE 1.

Differences in bactericidal titer obtained with bacteria passaged from 1 to 4 times^a

Poor Correlation between Pneumococcal IgG and IgM Titers and Opsonophagocytic Activity in Vaccinated Patients with Multiple Myeloma and Waldenstrom's Macroglobulinemia

Patients with multiple myeloma and other B cell disorders respond poorly to pneumococcal vaccination. Vaccine responsiveness is commonly determined by measuring pneumococcal serotype-specific antibodies by enzyme-linked immunosorbent assay (ELISA), by a functional opsonophagocytosis assay (OPA), or by both assays. We compared the two methods in vaccinated elderly patients with multiple myeloma, Waldenstrom''s macroglobulinemia, and monoclonal gammopathy of undetermined significance (MGUS). Postvaccination sera from 45 patients (n = 15 from each patient group) and 15 control subjects were analyzed by multiplexed OPA for pneumococcal serotypes 4, 6B, 14, and 23F, and the results were compared to IgG and IgM antibody titers measured by ELISA. While there were significant correlations between pneumococcal OPA and IgG titers for all serotypes among the control subjects (correlation coefficients [r] between 0.51 and 0.85), no significant correlations were seen for any of the investigated serotypes in the myeloma group (r = −0.18 to 0.21) or in the group with Waldenstrom''s macroglobulinemia (borderline significant correlations for 2 of 4 serotypes). The MGUS group resembled the control group by having good agreement between the two test methods for 3 of 4 serotypes (r = 0.53 to 0.80). Pneumococcal postvaccination IgM titers were very low in the myeloma patients compared to the other groups and did not correlate with the OPA results. To summarize, our data indicate that ELISA measurements may overestimate antipneumococcal immunity in elderly subjects with B cell malignancies and that a functional antibody test should be used specifically for myeloma and Waldenstrom''s macroglobulinemia patients.     

Development and Use of a Serum Bactericidal Assay Using Pooled Human Complement To Assess Responses to a Meningococcal Group A Conjugate Vaccine in African Toddlers

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′ lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC′. hSBA titers determined using three independent lots of pooled hC′ were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC′ is feasible and showed that PsA-TT was highly immunogenic in African toddlers.     

Sehistosoma Mansoni in the Baboon: Modulation of Pathology After Vaccination with Polyclonal Anti-Idiotypic Antibodies

Vaccination of five baboons with an anti-idiotypic vaccine to irradiated Schistosoma mansoni cercariae resulted in nearly 19% protection compared to 39% protection conferred to five baboons vaccinated with an irradiated vaccine. Vaccination with the anti-idiotypic antibodies resulted in a significant reduction of pathology and granuloma size following challenge with live unattenuated cercariae. Results presented in this work are considered highly significant because the anti-idiotypic vaccine markedly influenced schistosomiasis morbidity which is the main consideration in this disease.