Therapeutic Antibody Against Neisseria gonorrhoeae Lipooligosaccharide, a Phase-variable Virulence Factor
Faculty AdvisorSanjay Ram
Academic ProgramMolecular Genetics and Microbiology
UMass Chan AffiliationsInfectious Diseases and Immunology
Document TypeDoctoral Dissertation
Keywordsmonoclonal antibody therapy
classical complement pathway
vaccines for gonorrhea
treatments for gonorrhea
complement activating antibody
complement activating IgG
Amino Acids, Peptides, and Proteins
Immunology of Infectious Disease
Immunoprophylaxis and Therapy
MetadataShow full item record
AbstractNeisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/32285
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Outer membrane protein P5 is required for resistance of nontypeable Haemophilus influenzae to both the classical and alternative complement pathwaysRosadini, Charles V.; Ram, Sanjay; Akerley, Brian J. (2014-02-01)The complement system is an important first line of defense against the human pathogen Haemophilus influenzae. To survive and propagate in vivo, H. influenzae has evolved mechanisms for subverting this host defense, most of which have been shown to involve outer surface structures, including lipooligosaccharide glycans and outer surface proteins. Bacterial defense against complement acts at multiple steps in the pathway by mechanisms that are not fully understood. Here we identify outer membrane protein P5 as an essential factor in serum resistance of both H. influenzae strain Rd and nontypeable H. influenzae (NTHi) clinical isolate NT127. P5 was essential for resistance of Rd and NT127 to complement in pooled human serum. Further investigation determined that P5 expression decreased cell surface binding of IgM, a potent activator of the classical pathway of complement, to both Rd and NT127. Additionally, P5 expression was required for NT127 to bind factor H (fH), an important inhibitor of alternative pathway (AP) activation. Collectively, the results obtained in this work highlight the ability of H. influenzae to utilize a single protein to perform multiple protective functions for evading host immunity.
Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibodyGulati, Sunita; Beurskens, Frank J.; Zheng, Bo; DeOliveira, Rosane B.; Shaughnessy, Jutamas; Nowak, Nancy; Rice, Peter A.; Ram, Sanjay (2019-06-19)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 mug 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 (FcgammaR) 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.
Phase-Variable Heptose I Glycan Extensions Modulate Efficacy of 2C7 Vaccine Antibody Directed against Neisseria gonorrhoeae LipooligosaccharideChakraborti, Srinjoy; Lewis, Lisa A.; Cox, Andrew D.; St. Michael, Frank; Li, Jianjun; Rice, Peter A.; Ram, Sanjay (2016-06-01)Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, has developed resistance to most conventional antibiotics. Safe and effective vaccines against gonorrhea are needed urgently. A candidate vaccine that targets a lipooligosaccharide (LOS) epitope recognized mAb 2C7 attenuates gonococcal burden in the mouse vaginal colonization model. Glycan extensions from the LOS core heptoses (HepI and HepII) are controlled by phase-variable LOS glycosyltransferase (lgt) genes; we sought to define how HepI glycan extensions affect mAb 2C7 function. Isogenic gonococcal mutants in which the lgt required for mAb 2C7 reactivity (lgtG) was genetically locked on and the lgt loci required for HepI variation (lgtA, lgtC, and lgtD) were genetically locked on or off in different combinations were created. We observed 100% complement-dependent killing by mAb 2C7 of a mutant that expressed lactose (Gal-Glc) from HepI, whereas a mutant that expressed Gal-Gal-Glc-HepI fully resisted killing (>100% survival). Mutants that elaborated 4- (Gal-GlcNAc-Gal-Glc-HepI) and 5-glycan (GalNAc-Gal-GlcNAc-Gal-Glc-HepI) structures displayed intermediate phenotypes (95% killing with 4 μg/ml mAb 2C7). The contrasting phenotypes of the lactose-HepI and the Gal-Gal-Glc-HepI LOS structures were recapitulated with phase variants of a recently isolated clinical strain. Despite lack of killing of the Gal-Gal-Glc-HepI mutants, mAb 2C7 deposited sufficient C3 on these bacteria for opsonophagocytic killing by human neutrophils. In conclusion, mAb 2C7 showed functional activity against all gonococcal HepI LOS structures defined by various lgtA/C/D on/off combinations, thereby providing further impetus for use of the 2C7 epitope in a gonococcal vaccine.