Complement-dependent Killing Research Articles

Mechanisms of antibody-dependent enhancement of infectious disease.

Antibody-dependent enhancement (ADE) of infectious disease is a phenomenon whereby host antibodies increase the severity of an infection. It is well established in viral infections but ADE also has an underappreciated role during bacterial, fungal and parasitic infections. ADE can occur during both primary infections and re-infections with the same or a related pathogen; therefore, understanding the underlying mechanisms of ADE is critical for understanding the pathogenesis and progression of many infectious diseases. Here, we review the four distinct mechanisms by which antibodies increase disease severity during an infection. We discuss the most established mechanistic explanation for ADE, where cross-reactive, disease-enhancing antibodies bound to pathogens interact with Fc receptors, thereby enhancing pathogen entry or replication, ultimately increasing the total pathogen load. Additionally, we explore how some pathogenic antibodies can shield bacteria from complement-dependent killing, thereby enhancing bacterial survival. We interrogate the molecular mechanisms by which antibodies can amplify inflammation to drive severe disease, even in the absence of increased pathogen replication. We also examine emerging roles for autoantibodies in enhancing the pathogenesis of infectious diseases. Finally, we discuss how we can leverage these insights to improve vaccine design and future treatments for infectious diseases.

Preclinical efficacy of a cell division protein candidate gonococcal vaccine identified by artificial intelligence.

Vaccines 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.

Modeling human anti-pig xenoimmune responses in a pig artery tissue grafted humanized mouse model.

Blood vessels that contain endothelial cells (ECs) on the surface are in direct contact with host blood and are the first target of xenograft rejection. Currently, our understanding of human anti-pig vessel immune responses is primarily based on in vitro assays using pig ECs. Therefore, it is necessary to develop an animal model that permits in vivo study of human immunological rejection of pig vessels. Pig artery tissues (PAT) were transplanted into human immune system (HIS) mice or immunodeficient NSG mice (as controls). Intragraft human immune cell infiltration and antibody deposition were quantified using histology and immunohistochemistry. Donor antigen-specific immune responses were quantified using a mixed lymphocyte reaction and a complement-dependent killing assay. Pig CD31+ ECs were detected and increased 2-fold from weeks 3 to 5 in PAT xenografts from immunodeficient NSG mice. However, compared with NSG mice, PAT xenografts in HIS mice had significantly lower numbers of porcine CD31+ ECs and showed a marked reduction from week 3 to week 5. PAT xenograft rejection in HIS mice is associated with intensive infiltration of human immune cells, deposition of human IgM and IgG antibodies, and the formation of a tertiary lymphoid structure. Robust donor pig antigen-specific human T cells and antibody responses were detected in PAT-transplanted HIS mice. We have developed a humanized mouse model to evaluate human anti-pig xenoimmune responses by PAT transplantation in vivo. This model is expected to facilitate the refinement of pig gene-editing strategies (the expression on EC surface) and the testing of local immunosuppressive strategies for clinical pig organ xenotransplantation.

Clinical, biological and genome-wide comparison of carbapenem-resistant Klebsiella pneumoniae with susceptibility transformation to polymyxin B during therapy

ObjectivesThe emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major clinical concern, and polymyxin B (PMB) is a ‘last resort’ antibiotic for its treatment. Understanding the effects of drug susceptibility transformation in CRKP-infected patients undergoing PMB treatment would be beneficial to optimize PMB treatment strategies. MethodsWe retrospectively collected data from patients infected with CRKP and treated with PMB from January 2018 to December 2020. CRKPs were collected before and after PMB therapy, and patients were classified into the ‘transformation’ group (TG) and ‘non-transformation’ group (NTG) by the shift of susceptibility to PMB. We compared clinical characteristics between these groups, and further analysed the phenotypic and genome variation of CRKP after PMB susceptibility transformation. ResultsA total of 160 patients (37 in the TG and 123 in the NTG) were included in this study. The duration of PMB treatment before PMB-resistant K. pneumoniae (PRKP) appearance in TG was even longer than the whole duration of PMB treatment in NTG (8 [8] vs. 7 [6] days; p 0.0496). Compared with isogenic PMB-susceptible K. pneumoniae (PSKP), most PRKP strains had missense mutations in mgrB (12 isolates), yciC (10 isolates) and pmrB (7 isolates). The competition index of 82.4% (28/34) of PRKP/PSKP pairs was <67.6% (23/34), and 73.5% (25/34) of PRKP strains showed a higher 7-day lethality in Galleria mellonella and a greater ability to resist complement-dependent killing than their corresponding PSKP, respectively. ConclusionLow dose with longer PMB treatment durations may be associated with the emergence of polymyxin resistance. The evolution of PRKP is predominantly mediated by an accumulation of mutations, including those in mgrB, yciC, and pmrB. Lastly, PRKP exhibited reduced growth and increased virulence compared with parental PSKP.

Riemerella anatipestifer GldG is necessary for secretion of effectors by type IX secretion system

Riemerella anatipestifer secretes proteins through the type IX secretion system (T9SS). Recent studies have shown that the R. anatipestifer T9SS component proteins GldM and GldK also act as crucial virulence factors. In our previous study, the disruption of AS87_RS00460 gene, which encodes the predicted protein GldG, significantly reduced the bacterial virulence of R. anatipestifer wild-type strain Yb2, but the mechanism was unclear. In this study, we investigated the function of the GldG in bacterial virulence and protein secretion using the mutant strain Yb2ΔgldG and complementation strain cYb2ΔgldG. Our results demonstrate that the gldG gene encodes a gliding-motility-associated ABC transporter substrate-binding protein GldG, which was localized to the bacterial membrane in an immunoblotting analysis, and functions in the bacterium’s adherence to and invasion of host cells and its survival in host blood. The resistance of mutant strain Yb2ΔgldG to complement-dependent killing was significantly reduced. Yb2ΔgldG displayed reduced gliding motility and deficient protein secretion. Label-free quantification (LFQ) with liquid chromatography–mass spectrometry (LC–MS) showed that 10 proteins with a conserved T9SS C-terminal domain were differentially secreted by Yb2ΔgldG and Yb2. The secretion levels of those 10 proteins were determined with immunoblotting, and the results were consistent with the LFQ LC–MS data. All of these effects were rescued by complementation with a plasmid encoding Yb2 gldG. Our results demonstrate that the R. anatipestifer gldG gene encodes the protein GldG, which is involved in bacterial virulence and protein secretion.

Borrelia peptidoglycan interacting Protein (BpiP) contributes to the fitness of Borrelia burgdorferi against host-derived factors and influences virulence in mouse models of Lyme disease.

The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7.

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.

Confronting Tigecycline-Resistant Acinetobacter baumannii via Immunization Against Conserved Resistance Determinants.

Antimicrobial-resistant (AMR) bacterial infections, including those caused by Acinetobacter baumannii, have emerged as a clinical crisis worldwide. Immunization with AMR determinants has been suggested as a novel approach to combat AMR bacteria, but has not been validated. The present study targeted tigecycline (TGC) resistance determinants in A. baumannii to test the feasibility of this approach. Using bioinformatic tools, four candidates, AdeA, AdeI, AdeK, and TolC, belonging to the resistance-nodulation-division (RND) efflux pump were identified as highly conserved and exposed antigens from 15 A. baumannii genomes. Antisera generated from recombinant proteins showed the capability to reserve Hoechst 33342, a substrate of the efflux pump, in bacterial cells. The rTolC antisera had the highest complement-dependent killing and opsonophagocytosis effect compared to the sera from phosphate-buffered saline immunized mice. Among the antisera, anti-rAdeK-specific antisera decreased the minimal inhibitory concentration of TGC in 26.7% of the tested isolates. Immunization with rAdeK significantly potentiated TGC efficacy in treating TGC-resistant A. baumannii pneumonia in the murine model. The bacterial load (7.5 × 105 vs. 3.8 × 107, p < 0.01) and neutrophil infiltration in the peri-bronchial vasculature region of immunized mice was significantly lower compared to the PBS-immunized mice when TGC was administrated concomitantly. Collectively, these results suggest that active immunization against resistance determinants might be a feasible approach to combat multidrug-resistant pathogens in high risk population.

Complement activity and complement regulatory gene mutations are associated with thrombosis in APS and CAPS

AbstractThe antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-I (anti-β2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-β2GPI antibodies) and recurrent thrombosis. Patient-derived anti-β2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-β2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-β2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a “second hit,” leading to uncontrolled complement activation and a more severe thrombotic phenotype.

Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7.

The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

C4BP-IgM protein as a therapeutic approach to treat Neisseria gonorrhoeae infections.

Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 porin B1a (PorB1a) and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding substantially correlated with the ability to evade complement-dependent killing (r = 0.78). We designed 2 chimeric proteins that fused C4BP domains to the backbone of IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (KD C4BP-IgM = 2.4 nM; KD C4BP-IgG 980.7 nM), but only hexameric C4BP-IgM efficiently outcompeted heptameric C4BP from the bacterial surface, resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM substantially attenuated the duration and burden of colonization of 2 C4BP-binding gonococcal isolates but not a non-C4BP-binding strain in a mouse vaginal colonization model using human factor H/C4BP-transgenic mice. Our preclinical data present C4BP-IgM as an adjunct to conventional antimicrobials for the treatment of gonorrhea.

674. Pre-Clinical and Phase I Safety Data for Anti-Pseudomonas aeruginosa Human Monoclonal Antibody AR-105

BackgroundAnti-bacterial monoclonal antibodies can serve as a new treatment modality for difficult to treat infections. AR-105 is a fully human IgG1 monoclonal antibody (mAb) that binds to an extracellular polysaccharide epitope of Pseudomonas aeruginosa (PA) and was shown to mediate in vitro complement-dependent opsonophagocytic killing. AR-105 is currently being tested in a global Phase 2 clinical trial as an adjunctive treatment to standard of care antibiotics in ventilator-associated pneumonia patients. Here we present pre-clinical efficacy and clinical safety data for AR-105.MethodsEfficacy in nonclinical studies against PA pneumonia was tested in prophylactic and therapeutic mouse models, either as a stand-alone therapy or in combination with antibiotics. Mice were dosed intranasally or by intravenous infusion with AR-105 post or prior to infection with PA and survival or lung bacteriology were monitored. In a clinical Phase 1 open-label study, 16 healthy volunteers received 2, 8, or 20 mg/kg of AR-105. Adverse events, immunogenicity, and pharmacokinetic (PK) profiles were evaluated for up to 84 days following administration.ResultsIn the animal models, AR-105 reduced lung bacterial counts in a dose-dependent manner, and improved survival (80% in the treated group vs. 0% in the control group). Combination of AR-105 with antibiotics was more effective than monotherapy. In the Phase I study, no serious adverse events (AE) were observed in any cohort. Few AE were deemed related to the investigational drug, and all were mild and transient. AR-105 was found to be well tolerated in healthy volunteers with no anti-drug antibodies (ADA) detected. The PK profile was comparable with other human IgG1 mAbs, exhibiting a serum half-life of approximately 20 days.ConclusionAR-105 was confirmed to be effective in PA pneumonia animal models, either as stand-alone therapeutic or in combination with antibiotics. In the Phase 1 clinical study, AR-105 was shown to be safe and well-tolerated, with a PK profile similar to that of other IgG1 mAbs. AR-105 is a promising drug candidate for therapy of PA pneumonia. Disclosures All authors: No reported disclosures.

Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency.

SummaryVaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi‐component meningococcal serogroup B (MenB)‐4C. MenB‐4C is a multi‐component, protein‐based vaccine against MenB consisting of factor H‐binding protein, Neisserial heparin‐binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine‐mediated protection by a whole cell enzyme‐linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine‐induced antibodies and capacity to activate complement‐mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement‐mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB‐4C vaccination still induced effective complement‐dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement‐deficient children against MenB.

Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody.

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.

Bactericidal fully-human monoclonal antibodies can be cloned from patients convalescing from invasive meningococcal disease

Reverse vaccinology 2.0 (RV 2.0), in which the cloning and recombinant expression of antigen-specific antibodies is followed by determination of their functional activity, is a valuable approach that can unravel novel vaccine antigens, or reinforce the vaccine candidacy of already known antigens. Invasive meningococcal disease (IMD) remains a serious source of concern even with the availability of vaccines. Incomplete strain coverage is a limitation of current vaccines, hence efforts to identify candidate antigens that will compose supplementary or replacement vaccines are necessitated. In this proof-of-principle study, we sought to assess the applicability of RV 2.0 to anti-meningococcal vaccine antigen discovery. Antibody-secreting cells (ASCs) obtained from a patient convalescing from serogroup B (MenB) IMD, were isolated and sorted singly using FACS. The specificity and functionality of each antibody produced by individual ASCs were assessed in ELISA and bactericidal assays, respectively. Eight cross-reactive anti-meningococcal antibodies were successfully cloned; three of these mediated complement-dependent killing of antigenically-heterologous MenB strains. Western blot data shows binding of these three bactericidal antibodies to a ∼35 kDa antigen. None of the three bactericidal antibodies were reactive with a target in current vaccine formulations strongly suggesting that the ∼35 kDa antigen does not compose available vaccines. Unequivocal determination of the identity of the ∼35 kDa antigen is ongoing. Given the need for antigens that would compose improved or novel anti-meningococcal vaccines, this study shows that the RV 2.0 approach has the potential to be a powerful tool in the identification of functionally-immunogenic anti-meningococcal antigens.

Candida albicans Factor H Binding Molecule Hgt1p - A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils.

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans.

EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies.

Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41−65, EF-Tu161−185, EF-Tu221−245, and EF-Tu281−305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.

Human Factor H Domains 6 and 7 Fused to IgG1 Fc Are Immunotherapeutic against Neisseria gonorrhoeae.

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.

Mechanism by which Regulatory Rheumatoid Factor Prevents Experimental Autoimmune Encephalomyelitis.

One mechanism that underlies protection from autoimmunity and avoidance of uncontrolled inflammation is the controlled contraction of lymphocyte expansion during the immune response. We identified regulatory rheumatoid factor (regRF), the production of which is associated with resistance to and remission of experimental autoimmune diseases. RegRF is anti-idiotypic antibodies to lymphocyte receptors against autoimmune disease-inducing antigens; at the same time, it is specific to epitopes in the hinge Fc fragments of IgG. The aim of this study is to test the hypothesis that regRF prevents autoimmunity by limiting the expansion of lymphocytes. To test this hypothesis, we used a model of experimental autoimmune encephalitis. We found that in the lymph nodes that drain the injection site in rats producing regRF in response to immunization with myelin basic protein (MBP) the proportion of CD4+lymphocytes was lower than in rats in which MBP-immunization did not induce higher regRF levels. RegRF-containing plasma obtained from MBP-immunized rats induces complement-dependent killing of MBP-activated lymphocytes. Activated MBP-specific lymphocytes are not sensitive to the regRF-containing plasma of intact rats. The regRF produced during the immune response is a specific control factor for the expansion of antigen-activated CD4+lymphocytes.