RESUMO
A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Animais , Vacinas Bacterianas , Proteínas do Sistema Complemento , Modelos Animais de Doenças , Feminino , Gonorreia/prevenção & controle , Lipopolissacarídeos , CamundongosRESUMO
Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 µg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.
Assuntos
Ativação do Complemento/imunologia , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Bactérias , Proteína de Ligação ao Complemento C4b/imunologia , Fator H do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Epitopos/imunologia , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neisseria gonorrhoeae/patogenicidadeRESUMO
Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
Assuntos
Sequência Rica em At/genética , DNA de Protozoário/genética , Malária Falciparum/imunologia , Oligonucleotídeos/genética , Plasmodium falciparum/fisiologia , Animais , DNA de Protozoário/metabolismo , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Malária Falciparum/parasitologia , Malária Falciparum/fisiopatologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Oligonucleotídeos/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genéticaRESUMO
Novel therapeutics against multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococcal lipooligosaccharide often expresses lacto-N-neotetraose (LNnT), which becomes sialylated in vivo, enhancing factor H (FH) binding and contributing to the organism's ability to resist killing by complement. We previously showed that FH domains 18-20 (with a D-to-G mutation at position 1119 in domain 19) fused to Fc (FHD1119G/Fc) displayed complement-dependent bactericidal activity in vitro and attenuated gonococcal vaginal colonization of mice. Gonococcal lipooligosaccharide phase variation can result in loss of LNnT expression. Loss of sialylated LNnT, although associated with a considerable fitness cost, could decrease efficacy of FHD1119G/Fc. Similar to N. meningitidis, gonococci also bind FH domains 6 and 7 through Neisserial surface protein A (NspA). In this study, we show that a fusion protein comprising FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/Fc) bound to 15 wild-type antimicrobial resistant isolates of N. gonorrhoeae and to each of six lgtA gonococcal deletion mutants. FH6,7/Fc mediated complement-dependent killing of 8 of the 15 wild-type gonococcal isolates and effectively reduced the duration and burden of vaginal colonization of three gonococcal strains tested in wild-type mice, including two strains that resisted complement-dependent killing but on which FH6,7/Fc enhanced C3 deposition. FH/Fc lost efficacy when Fc was mutated to abrogate C1q binding and in C1q-/- mice, highlighting the requirement of the classical pathway for its activity. Targeting gonococci with FH6,7/Fc provides an additional immunotherapeutic approach against multidrug-resistant gonorrhea.
Assuntos
Gonorreia , Fragmentos Fc das Imunoglobulinas , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Fator H do Complemento , Humanos , Imunoglobulina G , Camundongos , Neisseria gonorrhoeae/imunologiaRESUMO
Sialylation of lacto-N-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody.
Assuntos
Gonorreia/microbiologia , Heptoses/metabolismo , Lactose/metabolismo , Lipopolissacarídeos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/patogenicidade , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , China , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Lipopolissacarídeos/química , Masculino , Espectrometria de Massas , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Ácido N-Acetilneuramínico/análise , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/isolamento & purificaçãoRESUMO
Almost all invasive Neisseria meningitidis isolates express capsular polysaccharide. Ab is required for complement-dependent killing of meningococci. Although alternative pathway evasion has received considerable attention, little is known about classical pathway (CP) inhibition by meningococci, which forms the basis of this study. We engineered capsulated and unencapsulated isogenic mutant strains of groups A, B, C, W, and Y meningococci to express similar amounts of the same factor H-binding protein (fHbp; a key component of group B meningococcal vaccines) molecule. Despite similar anti-fHbp mAb binding, significantly less C4b was deposited on all five encapsulated mutants compared with their unencapsulated counterparts (p < 0.01) when purified C1 and C4 were used to deposit C4b. Reduced C4b deposition was the result of capsule-mediated inhibition of C1q engagement by Ab. C4b deposition correlated linearly with C1q engagement by anti-fHbp. Whereas B, C, W, and Y capsules limited CP-mediated killing by anti-fHbp, the unencapsulated group A mutant paradoxically was more resistant than its encapsulated counterpart. Strains varied considerably in their susceptibility to anti-fHbp and complement despite similar Ab binding, which may have implications for the activity of fHbp-based vaccines. Capsule also limited C4b deposition by anti-porin A mAbs. Capsule expression decreased binding of an anti-lipooligosaccharide IgM mAb (â¼ 1.2- to 2-fold reduction in fluorescence). Akin to observations with IgG, capsule also decreased IgM-mediated C4b deposition when IgM binding to the mutant strain pairs was normalized. In conclusion, we show that capsular polysaccharide, a critical meningococcal virulence factor, inhibits the CP of complement.
Assuntos
Cápsulas Bacterianas/imunologia , Complemento C1q/imunologia , Complemento C4b/imunologia , Via Clássica do Complemento/imunologia , Evasão da Resposta Imune , Neisseria meningitidis/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Lipopolissacarídeos/imunologia , Neisseria meningitidis/classificação , Porinas/imunologiaRESUMO
Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A ß-(1â6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.
Assuntos
Acetilglucosamina/imunologia , Anticorpos Antibacterianos/imunologia , Infecções Bacterianas/imunologia , Malária/imunologia , Micoses/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , Fungos/imunologia , Fungos/fisiologia , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/imunologia , Bactérias Gram-Positivas/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Malária/parasitologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Micoses/microbiologia , Micoses/prevenção & controle , Proteínas Opsonizantes/imunologia , Plasmodium berghei/imunologia , Plasmodium berghei/fisiologia , Ligação Proteica/imunologia , Staphylococcus aureus/metabolismo , Análise de Sobrevida , Fatores de TempoRESUMO
The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Hemolisinas/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , RNA Bacteriano/metabolismo , Streptococcus agalactiae/fisiologia , Animais , Humanos , Interleucina-1beta/metabolismo , Lisossomos/metabolismo , Lisossomos/microbiologia , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagossomos/metabolismo , Fagossomos/microbiologia , Streptococcus agalactiae/metabolismoRESUMO
A safe and effective vaccine is urgently needed to combat the global threat of multidrug-resistant (MDR) Neisseria gonorrhoeae. We screened 26 gonococcal proteins discovered by an artificial intelligence-driven platform called Efficacy Discriminative Educated Network (EDEN) trained to identify novel, protective vaccine antigens against pathogenic bacteria for efficacy in the mouse vaginal colonization model of gonorrhea. Combinations of two to three antigens adjuvanted with GLA-SE (induces TH1 responses) yielded 11 groups that were used to vaccinate mice. An inverse correlation was noted between the complement-dependent bactericidal activity of antisera from each of the 11 groups and the burden of gonococcal colonization. The combination of NGO1549 (FtsN; cell divisome protein) and NGO0265 (predicted cell division protein) most substantially reduced the burden of colonization by MDR strain WHO X. The EDEN prediction score for each group of antigens correlated positively with reductions in overall bacterial burden, providing evidence for its predictive potential. FtsN and NGO0265 administered either individually, in combination, or as a chimeric protein significantly attenuated gonococcal vaginal colonization by all three test strains. IgG in antisera from mice immunized with the chimeric NGO0265-FtsN protein supported the complement-dependent killing of all 50 (100%) gonococcal isolates tested. The efficacy of the chimeric NGO0265-FtsN vaccine required the membrane attack complex (C5b-9) of complement, evidenced by loss of efficacy in complement C9-/- mice. In conclusion, a chimeric molecule comprising NGO0265 and FtsN adjuvanted with GLA-SE elicits IgG with broad anti-gonococcal bactericidal activity, attenuates gonococcal colonization in a complement-dependent manner, and represents a promising gonococcal vaccine candidate.IMPORTANCEVaccines to curb the global spread of multidrug-resistant gonorrhea are urgently needed. Here, 26 vaccine candidates identified by an artificial intelligence-driven platform (Efficacy Discriminative Educated Network[EDEN]) were screened for efficacy in the mouse vaginal colonization model. Complement-dependent bactericidal activity of antisera and the EDEN protective scores both correlated positively with the reduction in overall bacterial colonization burden. NGO1549 (FtsN) and NGO0265, both involved in cell division, displayed the best activity and were selected for further development. Both antigens, when fused to create a chimeric protein, elicited bactericidal antibodies against a wide array of gonococcal isolates and significantly attenuated the duration and burden of gonococcal colonization of mouse vaginas. Protection was abrogated in mice that lacked complement C9, the last step in the formation of the membrane attack complex pore, suggesting complement-dependent bactericidal activity as a mechanistic correlate of protection of the vaccine. FtsN and NGO0265 represent promising vaccine candidates against gonorrhea.
RESUMO
Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci evade killing by complement by binding factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized as a single chain. Gonococci bind FH through domains 6 and 7, and C-terminal domains 18 through 20. Previously, we showed that a chimeric protein comprising (from the N- to C-terminus) FH domains 18-20 (containing a point mutation in domain 19 to prevent lysis of host cells) fused to human IgG1 Fc (called FH*/Fc1) killed gonococci in a complement-dependent manner and reduced the duration and bacterial burden in the mouse vaginal colonization model of gonorrhea. Considering the N. gonorrhoeae-binding FH domains 18-20 are C-terminal in native FH, we reasoned that positioning Fc N-terminal to FH* (Fc1/FH*) would improve binding and bactericidal activity. Although both molecules bound gonococci similarly, Fc1/FH* displayed a 5-fold lower IC50 (the concentration required for 50% killing in complement-dependent bactericidal assays) than FH*/Fc1. To further increase complement activation, we replaced human IgG1 Fc in Fc1/FH* with Fc from human IgG3, the most potent complement-activating IgG subclass, to obtain Fc3/FH*. Bactericidal activity was further increased ~2.3-fold in Fc3/FH* compared to Fc1/FH*. Fc3/FH* killed (defined by <50% survival) 45/45 (100%) diverse PorB1B-expessing gonococci, but only 2/15 PorB1A-expressing isolates, in a complement-dependent manner. Decreased Fc3/FH* binding accounted for the limited activity against PorB1A strains. Fc3/FH* was efficacious against all four tested PorB1B gonococcal strains in the mouse vaginal colonization model when administered at a dose of 5 µg intravaginally, daily. Furthermore, Fc3/FH* retained bactericidal activity when reconstituted following lyophilization or spray-drying, suggesting feasibility for formulation into intravaginal rings. In conclusion, Fc3/FH* represents a promising prophylactic immunotherapeutic against multidrug-resistant gonococci.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Animais , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Feminino , Gonorreia/tratamento farmacológico , Humanos , Imunoglobulina G/metabolismo , Camundongos , Neisseria gonorrhoeae/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci possess several mechanisms to evade killing by human complement, including binding of factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized in a head-to-tail manner as a single chain. N. gonorrhoeae binds two regions in FH; domains 6 and 7 and domains 18 through 20. We designed a novel anti-infective immunotherapeutic molecule that fuses domains 18-20 of FH containing a D-to-G mutation in domain 19 at position 1119 (called FH*) with human IgG1 Fc. FH*/Fc retained binding to gonococci but did not lyse human erythrocytes. Expression of FH*/Fc in tobacco plants was undertaken as an alternative, economical production platform. FH*/Fc was expressed in high yields in tobacco plants (300-600 mg/kg biomass). The activities of plant- and CHO-cell produced FH*/Fc against gonococci were similar in vitro and in the mouse vaginal colonization model of gonorrhea. The addition of flexible linkers [e.g., (GGGGS)2 or (GGGGS)3] between FH* and Fc improved the bactericidal efficacy of FH*/Fc 2.7-fold. The linkers also improved PMN-mediated opsonophagocytosis about 11-fold. FH*/Fc with linker also effectively reduced the duration and burden of colonization of two gonococcal strains tested in mice. FH*/Fc lost efficacy: i) in C6-/- mice (no terminal complement) and ii) when Fc was mutated to abrogate complement activation, suggesting that an intact complement was necessary for FH*/Fc function in vivo. In summary, plant-produced FH*/Fc represent promising prophylactic or adjunctive immunotherapeutics against multidrug-resistant gonococci.
Assuntos
Resistência a Múltiplos Medicamentos/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Neisseria gonorrhoeae/imunologia , Nicotiana/genética , Plantas Geneticamente Modificadas , Animais , Antibacterianos/farmacologia , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Gonorreia , Humanos , Imunoglobulina G , Imunoterapia , Camundongos , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Terminal complement pathway inhibition at the level of C5 alleviates symptoms of several diseases associated with complement overactivation. However, C5 blockade is associated with an increased risk of invasive meningococcal disease despite immunization. Targeting specific complement pathways proximal to C5 provides the theoretical advantage of leaving the other pathways (including the terminal pathway) intact for immune surveillance. We aimed to address the risk of Neisseria meningitidis and Streptococcus pneumoniae infections when inhibiting the classical pathway (CP) using a specific C1s inhibitor (TNT005). Addition of TNT005 to 20% normal human serum that contained anti-meningococcal capsular Ab decreased C4 deposition 8-fold and abrogated killing of N. meningitidis, despite leaving C3 deposition intact. TNT005 impaired killing of N. meningitidis in 78% nonimmune human plasma and 78% whole blood but permitted killing in both when specific anti-capsular Ab was added. Simultaneously inhibiting both the CP and alternative pathway (AP) blocked killing of Ab-coated N. meningitidis in whole blood. Blocking the AP alone abrogated C3 deposition, whereas TNT005 only partially inhibited (â¼40% decrease) C3 deposition on S. pneumoniae coated with anti-capsular Ab. Blocking either the CP or AP alone did not impair killing of pneumococci in whole blood containing specific Ab (<10% survival at 3 h); however, blocking both pathways resulted in â¼35% bacterial survival. These data suggest that killing of N. meningitidis or S. pneumoniae in whole blood containing specific anti-capsular Abs is unimpeded by TNT005. Meningococcal and pneumococcal capsular conjugate vaccines may mitigate risk of these infections in patients receiving C1s inhibitors.
Assuntos
Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Complemento C1/antagonistas & inibidores , Neisseria meningitidis/imunologia , Streptococcus pneumoniae/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Complemento C4/imunologia , Via Clássica do Complemento/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologiaRESUMO
The global spread of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a public health emergency. With limited antibiotic treatment options, there is an urgent need for development of a safe and effective vaccine against gonorrhea. Previously, we constructed a prototype vaccine candidate comprising a peptide mimic (mimitope) of a glycan epitope on gonococcal lipooligosaccharide (LOS), recognized by monoclonal antibody 2C7. The 2C7 epitope is (i) broadly expressed as a gonococcal antigenic target in human infection, (ii) a critical requirement for gonococcal colonization in the experimental setting, and (iii) a virulence determinant that is maintained and expressed by gonococci. Here, we have synthesized to >95% purity through a relatively facile and economical process a tetrapeptide derivative of the mimitope that was cyclized through a nonreducible thioether bond, thereby rendering the compound homogeneous and stable. This vaccine candidate, called TMCP2, when administered at 0, 3, and 6 weeks to BALB/c mice at either 50, 100 or 200 µg/dose in combination with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE; a Toll-like receptor 4 and TH1-promoting adjuvant), elicited bactericidal IgG and reduced colonization levels of gonococci in experimentally infected mice while accelerating clearance by each of two different gonococcal strains. Similarly, a 3-dose biweekly schedule (50 µg TMCP2/dose) was also effective in mice. We have developed a gonococcal vaccine candidate that can be scaled up and produced economically to a high degree of purity. The candidate elicits bactericidal antibodies and is efficacious in a preclinical experimental infection model.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics. The incidence of gonorrhea is also sharply increasing. A safe and effective antigonococcal vaccine is urgently needed. Lipooligosaccharide (LOS), the most abundant outer membrane molecule, is indispensable for gonococcal pathogenesis. A glycan epitope on LOS that is recognized by monoclonal antibody (MAb) 2C7 (called the 2C7 epitope) is expressed almost universally by gonococci in vivo Previously, we identified a peptide mimic (mimitope) of the 2C7 epitope, which when configured as an octamer and used as an immunogen, attenuated colonization of mice by gonococci. Here, a homogenous, stable tetrameric derivative of the mimitope, when combined with a TH1-promoting adjuvant and used as an immunogen, also effectively attenuates gonococcal colonization of mice. This candidate peptide vaccine can be produced economically, an important consideration for gonorrhea, which affects socioeconomically underprivileged populations disproportionately, and represents an important advance in the development of a gonorrhea vaccine.
Assuntos
Vacinas Bacterianas/imunologia , Lipopolissacarídeos/imunologia , Neisseria gonorrhoeae/imunologia , Peptídeos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Epitopos/imunologia , Feminino , Gonorreia/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.
Assuntos
Antibacterianos/farmacologia , Bactérias/imunologia , Infecções Bacterianas/imunologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Desenho de Fármacos , Fatores Imunológicos/farmacologia , Animais , Antibacterianos/uso terapêutico , Antígenos de Bactérias/imunologia , Bactérias/química , Bactérias/metabolismo , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fatores Imunológicos/uso terapêutico , Mimetismo Molecular/imunologia , Neisseria/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Ácidos Siálicos/imunologiaRESUMO
The emergence of antimicrobial resistance among several medically important pathogens represents a serious threat to human health globally and necessitates the development of novel therapeutics. Complement forms a key arm of innate immune defenses against invading pathogens. A mechanism of complement evasion employed by many pathogens is binding of complement inhibitors, including factor H (FH), a key downregulator of the alternative pathway. Most FH-binding bacteria engage FH through regions in FH spanned by domains 6 and 7 and/or 18 through 20. We created a chimeric protein that comprised human FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/HuFc) and tested its activity as an immunotherapeutic against Neisseria meningitidis, which binds FH through domains 6 and 7. FH6,7/HuFc bound to meningococci and effectively blocked FH binding to bacteria. FH6,7/HuFc enhanced human C3 and C4 deposition and facilitated complement-mediated killing in a dose-responsive manner; complement activation and killing were classical pathway dependent. To investigate in vivo efficacy, infant Wistar rats were treated intraperitoneally (IP) with different doses of FH6,7/HuFc and challenged 2 h later with serogroup C strain 4243 given IP. At 8 to 9 h after the challenge, the FH6,7/HuFc-treated rats had >100-fold fewer CFU per ml of blood than control animals pretreated with phosphate-buffered saline (PBS) or FH18-20/HuFc, which does not bind to meningococci (P < 0.0001). These data provide proof of concept of the utility of FH/Fc fusion proteins as anti-infective immunotherapeutics. Because many microbes share a common binding region(s) in FH, FH/Fc chimeric proteins may be a promising candidate for adjunctive therapy against drug-resistant pathogens.
Assuntos
Antibacterianos/farmacologia , Fator H do Complemento/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Imunoterapia/métodos , Infecções Meningocócicas/terapia , Neisseria meningitidis Sorogrupo C/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Sangue/microbiologia , Contagem de Colônia Microbiana , Fator H do Complemento/administração & dosagem , Fator H do Complemento/genética , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/administração & dosagem , Imunoglobulina G/genética , Viabilidade Microbiana , Neisseria meningitidis Sorogrupo C/fisiologia , Ratos Wistar , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Resultado do TratamentoRESUMO
Hemozoin (Hz) is the crystalline detoxification product of hemoglobin in Plasmodium-infected erythrocytes. We previously proposed that Hz can carry plasmodial DNA into a subcellular compartment that is accessible to Toll-like receptor 9 (TLR9), inducing an inflammatory signal. Hz also activates the NLRP3 inflammasome in primed cells. We found that Hz appears to colocalize with DNA in infected erythrocytes, even before RBC rupture or phagolysosomal digestion. Using synthetic Hz coated in vitro with plasmodial genomic DNA (gDNA) or CpG oligodeoxynucleotides, we observed that DNA-complexed Hz induced TLR9 translocation, providing a priming and an activation signal for inflammasomes. After phagocytosis, Hz and DNA dissociate. Hz subsequently induces phagolysosomal destabilization, allowing phagolysosomal contents access to the cytosol, where DNA receptors become activated. Similar observations were made with Plasmodium-infected RBCs. Finally, infected erythrocytes activated both the NLRP3 and AIM2 inflammasomes. These observations suggest that Hz and DNA work together to induce systemic inflammation during malaria.