RESUMEN
Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Meningitis Meningocócica/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/metabolismo , Uretritis/inmunología , Uretritis/microbiología , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Supervivencia Celular/genética , Factor H de Complemento/inmunología , Bases de Datos Genéticas , Genómica , Humanos , Infecciones Meningocócicas , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidad , Neisseria meningitidis Serogrupo B/inmunología , Neisseria meningitidis Serogrupo B/aislamiento & purificación , Filogenia , Unión Proteica , Alineación de Secuencia , CatelicidinasRESUMEN
BACKGROUND: Meningococcal outer membrane vesicle (OMV) vaccines are prepared with detergents to remove endotoxin, which also remove desirable antigens such as factor H binding protein (FHbp). Native OMV (NOMV) vaccines with genetically attenuated endotoxin do not require detergent treatment and elicit broader serum bactericidal antibody (SBA) responses than OMV or recombinant FHbp (rFHbp) vaccines. METHODS: We measured human complement-mediated SBA responses in mice immunized with NOMV with overexpressed FHbp subfamily B (NOMV-FHbp), NOMV with FHbp genetically inactivated (NOMV-KO), and/or a control rFHbp vaccine against meningococcal and gonococcal strains. RESULTS: Despite having 36-fold less FHbp per dose, the NOMV-FHbp vaccine elicited a ≥3-fold higher serum IgG anti-FHbp geometric mean titer than control vaccines containing rFHbp (P ≤ .003). Against 2 meningococcal outbreak strains with mismatched PorA and heterologous FHbp subfamily B sequence variants, the NOMV-FHbp vaccine produced ≥30-fold higher SBA titers than control vaccines. Mice immunized with NOMV-FHbp and NOMV-KO vaccines also elicited SBA against a gonococcal strain (P < .0001 vs the adjuvant-only control group). In contrast, 2 licensed meningococcal serogroup B vaccines, including one containing detergent-extracted OMV, did not produce gonococcal SBA in humans. CONCLUSIONS: A meningococcal NOMV vaccine elicits SBA against gonococci and with overexpressed FHbp elicits SBA against meningococci.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Vacunas Meningococicas/inmunología , Neisseria gonorrhoeae/inmunología , Neisseria meningitidis/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Femenino , Técnicas de Inactivación de Genes , Humanos , Inmunoglobulina G/sangre , Ratones , Vacunas Atenuadas/inmunologíaRESUMEN
Eculizumab, a humanized anti-complement C5 monoclonal antibody (mAb) for treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome, blocks the terminal complement pathway required for serum bactericidal activity (SBA). Because treated patients are at >1000-fold increased risk of meningococcal disease, vaccination is recommended; whether vaccination can protect by opsonophagocytic activity in the absence of SBA is not known. Meningococci were added to anticoagulated blood from 12 healthy adults vaccinated with meningococcal serogroup B and serogroup A, C, W, Y vaccines. Bacterial survival was measured after 3-hour incubation in the presence of eculizumab or control complement factor D inhibitor ACH-4471, which blocks the complement alternative pathway (AP) and is in phase 2 development for treatment of PNH. In the absence of inhibitors, colony formation units (CFUs) per milliliter in blood from all 12 immunized subjects decreased from â¼4000 at time 0 to sterile cultures at 3 hours. In the presence of eculizumab, there was a >22-fold increase in geometric mean CFUs per milliliter (90 596 and 114 683 CFU/mL for serogroup B and C strains, respectively; P < .0001 compared with time 0). In the presence of ACH-4471, there was a >12-fold decrease (23 and 331 CFU/mL, respectively; P < .0001). The lack of meningococci killing by blood containing eculizumab resulted from inhibition of release of C5a, a C5 split product needed for upregulation of phagocytosis. The results provide an explanation for the large number of cases of meningococcal disease in immunized patients being treated with eculizumab and suggest that vaccination may provide better protection against meningococcal disease in patients treated with an AP-specific inhibitor.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Inmunización , Viabilidad Microbiana/efectos de los fármacos , Neisseria meningitidis/efectos de los fármacos , Proteínas Opsoninas/metabolismo , Fagocitosis/efectos de los fármacos , Adulto , Anciano , Vía Alternativa del Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Adulto JovenRESUMEN
Patients receiving eculizumab have an increased risk for meningococcal disease, but most reported cases are attributable to encapsulated meningococcal strains. We describe a case in which a nongroupable meningococcal strain, which rarely causes disease in healthy persons, caused fatal disease in an eculizumab recipient despite meningococcal vaccination.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Hemoglobinuria Paroxística/tratamiento farmacológico , Meningitis Meningocócica/microbiología , Meningitis Meningocócica/patología , Vacunas Meningococicas/inmunología , Adolescente , Anticuerpos Antibacterianos/sangre , Resultado Fatal , Femenino , Humanos , Inmunoglobulina G/sangre , Meningitis Meningocócica/prevención & control , Neisseria meningitidisRESUMEN
Background: MenB-4C is a recently licensed meningococcal serogroup B vaccine. For vaccine licensure, short-term efficacy was inferred from serum bactericidal antibody (SBA) titers against 3 antigen-specific indicator strains, which are not necessarily representative of US disease-causing strains. Methods: A total of 4923 students were immunized with MenB-4C in response to an outbreak at a university. Serum samples were obtained at 1.5-2 months from 106 students who received the recommended 2 doses and 52 unvaccinated students. Follow-up serum samples were obtained at 7 months from 42 vaccinated and 24 unvaccinated participants. SBA was measured against strains from 4 university outbreaks. Results: At 1.5-2 months, the proportion of immunized students with protective titers ≥1:4 against an isolate from the campus outbreak was 93% (95% confidence interval [CI], 87%-97%) vs 37% (95% CI, 24%-51%) in unvaccinated students. The proportion with protective titers against strains from 3 other university outbreaks was 73% (95% CI, 62%-82%) vs 26% (95% CI, 14%-41%) in unvaccinated; 71% (95% CI, 61%-79%) vs 19% (95% CI, 10%-33%) in unvaccinated; and 53% (95% CI, 42%-64%) vs 9% (95% CI, 3%-22%) in unvaccinated (P < .0001 for each strain). At 7 months, the proportion of immunized students with titers ≥1:4 was 86% (95% CI, 71%-95%) against the isolate from the campus outbreak and 57% (95% CI, 41%-72%), 38% (95% CI, 24%-54%), and 31% (95% CI, 18%-47%), respectively, for the other 3 outbreak strains. Conclusions: MenB-4C elicited short-term protective titers against 4 strains responsible for recent university campus outbreaks. By 7 months the prevalence of protective titers was <40% for 2 of the 4 outbreak strains. A booster dose of MenB-4C may be needed to maintain protective titers.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Formación de Anticuerpos/inmunología , Actividad Bactericida de la Sangre/inmunología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Adolescente , Adulto , Antígenos Bacterianos/inmunología , Brotes de Enfermedades/prevención & control , Femenino , Humanos , Inmunización/métodos , Masculino , Serogrupo , Estudiantes , Universidades , Vacunación/métodos , Adulto JovenRESUMEN
Neisserial surface protein A (NspA) is a highly conserved outer membrane protein previously investigated as a meningococcal vaccine candidate. Despite eliciting serum bactericidal activity in mice, a recombinant NspA vaccine failed to elicit serum bactericidal antibodies in a phase 1 clinical trial in humans. The discordant results may be explained by the recent discovery that NspA is a human-specific ligand of the complement inhibitor factor H (FH). Therefore, in humans but not mice, NspA would be expected to form a complex with FH, which could impair human anti-NspA protective antibody responses. To investigate this question, we immunized human FH transgenic BALB/c mice with three doses of recombinant NspA expressed in Escherichia coli microvesicles, with each dose being separated by 3 weeks. Three of 12 (25%) transgenic mice and 13 of 14 wild-type mice responded with bactericidal titers of ≥1:10 in postimmunization sera (P = 0.0008, Fisher's exact test). In contrast, human FH transgenic and wild-type mice immunized with a control meningococcal native outer membrane vesicle vaccine had similar serum bactericidal antibody responses directed at PorA, which is not known to bind human FH, and a mutant factor H binding protein (FHbp) antigen with a >50-fold lower level of FH binding than wild-type FHbp antigen binding.Thus, human FH can impair anti-NspA serum bactericidal antibody responses, which may explain the poor immunogenicity of the NspA vaccine previously tested in humans. A mutant NspA vaccine engineered to have decreased binding to human FH may increase protective antibody responses in humans.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Escherichia coli/genética , Efecto Fundador , Humanos , Inmunización , Vacunas Meningococicas/inmunología , Ratones Endogámicos BALB C , Ratones Transgénicos , Porinas/inmunología , Proteínas Recombinantes/inmunología , Determinación de Anticuerpos Séricos BactericidasRESUMEN
BACKGROUND: The meningococcal vaccine antigen, factor H (FH)-binding protein (FHbp), binds human complement FH. In human FH transgenic mice, binding decreased protective antibody responses. METHODS: To investigate the effect of primate FH binding, we immunized rhesus macaques with a 4-component serogroup B vaccine (4CMenB). Serum FH in 6 animals bound strongly to FHbp (FHbp-FH(high)) and, in 6 animals, bound weakly to FHbp (FHbp-FH(low)). RESULTS: There were no significant differences between the respective serum bactericidal responses of the 2 groups against meningococcal strains susceptible to antibody to the NadA or PorA vaccine antigens. In contrast, anti-FHbp bactericidal titers were 2-fold lower in FHbp-FH(high) macaques against a strain with an exact FHbp match to the vaccine (P = .08) and were ≥4-fold lower against 4 mutants with other FHbp sequence variants (P ≤ .005, compared with FHbp-FH(low) macaques). Unexpectedly, postimmunization sera from all 12 macaques enhanced FH binding to meningococci. In contrast, serum anti-FHbp antibodies elicited by 4CMenB in mice whose mouse FH did not bind to the vaccine antigen inhibited FH binding. CONCLUSIONS: Binding of FH to FHbp decreases protective anti-FHbp antibody responses of macaques to 4CMenB. Even low levels of FH binding skew the antibody repertoire to FHbp epitopes outside of the FH-binding site, which enhance FH binding.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Factor H de Complemento/metabolismo , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Animales , Animales Recién Nacidos , Actividad Bactericida de la Sangre , Tolerancia Inmunológica , Macaca mulatta , Vacunas Meningococicas/administración & dosificación , Ratones Transgénicos , Unión ProteicaRESUMEN
Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Factor H de Complemento/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Porinas/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Técnicas de Inactivación de Genes , Humanos , Meningitis Meningocócica/inmunología , Meningitis Meningocócica/prevención & control , Vacunas Meningococicas/inmunología , Ratones , Mutación , Neisseria meningitidis Serogrupo B/aislamiento & purificación , Unión Proteica , Ratas , Ratas Wistar , Análisis de Secuencia de ADNRESUMEN
Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats.
Asunto(s)
Convertasas de Complemento C3-C5 de la Vía Alternativa/fisiología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/fisiología , Porinas/fisiología , Análisis de Varianza , Animales , Antígenos Bacterianos/fisiología , Proteínas Bacterianas/fisiología , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Infecciones Meningocócicas/metabolismo , Ratones , Neisseria meningitidis/patogenicidad , Porinas/metabolismo , Conejos , Ratas , Suero/microbiología , VirulenciaRESUMEN
The binding of human complement inhibitors to vaccine antigens in vivo could diminish their immunogenicity. A meningococcal ligand for the complement down-regulator, factor H (fH), is fH-binding protein (fHbp), which is specific for human fH. Vaccines containing recombinant fHbp or native outer membrane vesicles (NOMV) from mutant strains with over-expressed fHbp are in clinical development. In a previous study in transgenic mice, the presence of human fH impaired the immunogenicity of a recombinant fHbp vaccine. In the present study, we prepared two NOMV vaccines from mutant group B strains with over-expressed wild-type fHbp or an R41S mutant fHbp with no detectable fH binding. In wild-type mice in which mouse fH did not bind to fHbp in either vaccine, the NOMV vaccine with wild-type fHbp elicited 2-fold higher serum IgG anti-fHbp titers (Pâ=â0.001) and 4-fold higher complement-mediated bactericidal titers against a PorA-heterologous strain than the NOMV with the mutant fHbp (Pâ=â0.003). By adsorption, the bactericidal antibodies were shown to be directed at fHbp. In transgenic mice in which human fH bound to the wild-type fHbp but not to the R41S fHbp, the NOMV vaccine with the mutant fHbp elicited 5-fold higher serum IgG anti-fHbp titers (Pâ=â0.002), and 19-fold higher bactericidal titers than the NOMV vaccine with wild-type fHbp (Pâ=â0.001). Thus, in mice that differed only by the presence of human fH, the respective results with the two vaccines were opposite. The enhanced bactericidal activity elicited by the mutant fHbp vaccine in the presence of human fH far outweighed the loss of immunogenicity of the mutant protein in wild-type animals. Engineering fHbp not to bind to its cognate complement inhibitor, therefore, may increase vaccine immunogenicity in humans.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Vacunas Meningococicas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Factor H de Complemento/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidad , Unión Proteica/genética , Proteínas Recombinantes/inmunología , Determinación de Anticuerpos Séricos BactericidasAsunto(s)
Antibacterianos/farmacocinética , Anticuerpos Monoclonales Humanizados/efectos adversos , Inactivadores del Complemento/efectos adversos , Infecciones Meningocócicas/diagnóstico , Infecciones Meningocócicas/etiología , Neisseria meningitidis/aislamiento & purificación , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Inactivadores del Complemento/uso terapéutico , Susceptibilidad a Enfermedades , Antagonismo de Drogas , Humanos , Infecciones Meningocócicas/tratamiento farmacológico , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Vacunas Meningococicas/inmunología , Neisseria meningitidis/clasificaciónRESUMEN
BACKGROUND: Factor H (fH) binding protein (fHbp) is part of vaccines developed for prevention of meningococcal serogroup B disease. More than 610 fHbp amino acid sequence variants have been identified, which can be classified into 2 subfamilies. The extent of cross-protection within a subfamily has been difficult to assess because of strain variation in fHbp expression. METHODS: Using isogenic mutant strains, we compared cross-protective serum antibody responses of mice immunized with 7 divergent fHbp variants in subfamily B, including identification numbers (ID) 1 and 55, which were chosen for vaccine development. RESULTS AND CONCLUSIONS: In the presence of the human complement downregulator fH, the ability of the anti-fHbp antibodies to deposit sufficient complement C3b on the bacterial surface to elicit bactericidal activity required inhibition of binding of fH by the anti-fHbp antibodies. With less bound fH, the bacteria became more susceptible to complement-mediated bactericidal activity. Among the different fHbp sequence variants, those more central in a phylogenic network than ID 1 or 55 elicited anti-fHbp antibodies with broader inhibition of fH binding and broader bactericidal activity. Thus, the more central variants show promise of extending protection to strains with divergent fHbp sequences that are covered poorly by fHbp variants in clinical development.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Actividad Bactericida de la Sangre , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Vacunas Meningococicas/inmunología , Adulto , Animales , Proteínas del Sistema Complemento/inmunología , Protección Cruzada , Modelos Animales de Enfermedad , Femenino , Humanos , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Ratones , Viabilidad MicrobianaRESUMEN
Certain pathogens recruit host complement inhibitors such as factor H (fH) to evade the immune system. Microbial complement inhibitor-binding molecules can be promising vaccine targets by eliciting Abs that neutralize this microbial defense mechanism. One such Ag, meningococcal factor H-binding protein (fHbp), was used in clinical trials before the protein was discovered to bind fH. The potential effect of fH binding on vaccine immunogenicity had not been assessed in experimental animals because fHbp binds human fH specifically. In this study, we developed a human fH transgenic mouse model. Transgenic mice immunized with fHbp vaccine had 4- to 8-fold lower serum bactericidal Ab responses than those of control mice whose native fH did not bind the vaccine. In contrast, Ab responses were unimpaired in transgenic mice immunized with a control meningococcal group C polysaccharide-protein conjugate vaccine. In transgenic mice, immunization with an fH nonbinding mutant of fHbp elicited Abs with higher bactericidal activity than that of fHbp vaccination itself. Abs elicited by the mutant fHbp more effectively blocked fH binding to wild-type fHbp than Abs elicited by fHbp that bound fH. Thus, a mutant fHbp vaccine that does not bind fH but that retains immunogenicity is predicted to be superior in humans to an fHbp vaccine that binds human fH. In the case of mutant fHbp vaccination, the resultant Ab responses may be directed more at epitopes in or near the fH binding site, which result in greater complement-mediated serum bactericidal activity; these epitopes may be obscured when human fH is bound to the wild-type fHbp vaccine.
Asunto(s)
Sustitución de Aminoácidos/genética , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Meningococicas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Adulto , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva/inmunología , Actividad Bactericida de la Sangre/inmunología , Factor H de Complemento/metabolismo , Femenino , Humanos , Vacunas Meningococicas/administración & dosificación , Vacunas Meningococicas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/genética , Vacunas Conjugadas/inmunologíaRESUMEN
Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Actividad Bactericida de la Sangre , Activación de Complemento , Vía Alternativa del Complemento/inmunología , Epítopos/inmunología , Neisseria meningitidis/inmunología , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Humanos , Inmunoglobulina G/inmunología , Ratones , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Meningococcal factor H binding protein (fHbp) is a human species-specific ligand for the complement regulator, factor H (fH). In recent studies, fHbp vaccines in which arginine at position 41 was replaced by serine (R41S) had impaired fH binding. The mutant vaccines elicited bactericidal responses in human fH transgenic mice superior to those elicited by control fHbp vaccines that bound human fH. Based on sequence similarity, fHbp has been classified into three variant groups. Here we report that although R41 is present in fHbp from variant groups 1 and 2, the R41S substitution eliminated fH binding only in variant group 1 proteins. To identify mutants in variant group 2 with impaired fH binding, we generated fHbp structural models and predicted 63 residues influencing fH binding. From these, we created 11 mutants with one or two amino acid substitutions in a variant group 2 protein and identified six that decreased fH binding. Three of these six mutants retained conformational epitopes recognized by all six anti-fHbp monoclonal antibodies (MAbs) tested and elicited serum complement-mediated bactericidal antibody titers in wild-type mice that were not significantly different from those obtained with the control vaccine. Thus, fHbp amino acid residues that affect human fH binding differ across variant groups. This result suggests that fHbp sequence variation induced by immune selection also affects fH binding motifs via coevolution. The three new fHbp mutants from variant group 2, which do not bind human fH, retained important epitopes for eliciting bactericidal antibodies and may be promising vaccine candidates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Neisseria meningitidis/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina , Proteínas Bacterianas/genética , Factor H de Complemento/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Vacunas Meningococicas/metabolismo , Ratones , Modelos Moleculares , Mutación , Neisseria meningitidis/genética , Unión Proteica , Conformación Proteica , Serina , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/metabolismoRESUMEN
Neisseria meningitidis binds the complement downregulating protein, factor H (fH), which enables the organism to evade host defenses. Two fH ligands, fHbp and NspA, are known to bind specifically to human fH. We developed a human fH transgenic infant rat model to investigate the effect of human fH on meningococcal bacteremia. At 18 h after intraperitoneal challenge with 560 CFU of group B strain H44/76, all 19 human fH-positive rats had positive blood cultures compared to 0 of 7 human fH-negative control littermates (P < 0.0001). Human fH-positive infant rats also developed bacteremia after challenge with isogenic mutants of H44/76 in which genes encoding fHbp and NspA (ΔfHbp ΔNspA mutant) or the lipooligosaccharide sialyltransferase (Δlst mutant) had been inactivated. A fully encapsulated ΔfHbp ΔNspA Δlst mutant unable to sialylate lipooligosaccharide or bind human fH via the known fH ligands did not cause bacteremia, which argued against global susceptibility to bacteremia resulting from random integration of the transgene into the rat genome. In vitro, the wild-type and ΔfHbp ΔNspA mutant strains were killed by as little as 20% wild-type infant rat serum. The addition of 3 µg of human fH/ml permitted survival of the wild-type strain in up to 60% infant rat serum, whereas ≥33 µg of human fH/ml was required to rescue the ΔfHbp ΔNspA mutant. The ability of meningococci lacking expression of fHbp and NspA to cause invasive disease in human fH transgenic rats and to survive in wild-type infant rat serum supplemented with human fH indicates an additional human fH-dependent mechanism of evasion of innate immunity.
Asunto(s)
Bacteriemia/inmunología , Factor H de Complemento/genética , Infecciones Meningocócicas/inmunología , Neisseria meningitidis/genética , Animales , Animales Modificados Genéticamente , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Bacteriemia/microbiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factor H de Complemento/efectos de los fármacos , Factor H de Complemento/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Inmunidad Innata , Infecciones Meningocócicas/genética , RatasRESUMEN
Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Factor H de Complemento/metabolismo , Proteínas del Sistema Complemento/inmunología , Neisseria meningitidis/inmunología , Animales , Afinidad de Anticuerpos , Bacteriólisis , Células CHO , Activación de Complemento , Factor H de Complemento/inmunología , Cricetinae , Cricetulus , Citometría de Flujo , Humanos , Regiones Constantes de Inmunoglobulina , Inmunoglobulina G/inmunología , Ratones , Neisseria meningitidis/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHbp from an epidemic Norwegian strain (ID 1). By surface plasmon resonance, association rate constants (k(a)) for fHbp ID 14 and 15 were similar to those for ID 1, but dissociation rate constants (k(d)) were 4- to 10-fold higher than those for ID 1. To determine the effect of fH affinity on fHbp fitness, we prepared isogenic mutants of strain H44/76 that expressed fHbp ID 1, 14, or 15. By flow cytometry, mutants expressing fHbp ID 14 or 15 had lower fH binding than ID 1. When incubated in plasma or blood of nonimmune donors, all three mutants showed similar increases in CFU/ml. In contrast, an isogenic fHbp knockout mutant, which grew well in broth, was rapidly killed in plasma or blood. Thus, although fHbp expression was required for survival of strain H44/76 in blood or plasma, expression of two natural fHbp sequence variants with lower fH affinity had minimal or no effect on nonimmune clearance. One reason may be the high fH concentrations in normal serum, which favor saturation of fH binding to fHbp, even when dissociation rates varied over 10-fold.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Sangre/microbiología , Neisseria meningitidis/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Clonación Molecular , Citometría de Flujo , Regulación Bacteriana de la Expresión Génica , Humanos , Mutación , Filogenia , Unión ProteicaRESUMEN
Since serogroup B meningococcal (MenB) vaccines became available in the United States, six serogroup B meningococcal disease cases have been reported in MenB-4C (n = 4) or MenB-FHbp (n = 2) recipients. Cases were identified and characterized through surveillance and health record review. All five available isolates were characterized using whole genome sequencing; four isolates (from MenB-4C recipients) were further characterized using flow cytometry, MenB-4C-induced serum bactericidal activity (SBA), and genetic Meningococcal Antigen Typing System (gMATS). Three patients were at increased meningococcal disease risk because of an outbreak or underlying medical conditions, and only four of the six patients had completed a full 2-dose MenB series. Isolates were available from 5 patients, and all contained sub-family A FHbp. The four isolates from MenB-4C recipients expressed NhbA but were mismatched for the other MenB-4C vaccine antigens. These four isolates were relatively resistant to MenB-4C-induced SBA, but predicted by gMATS to be covered. Overall, patient risk factors, incomplete vaccine series completion, waning immunity, and strain resistance to SBA likely contributed to disease in these six patients.