Neisseria gonorrhoeae (Ng) is the causative agent of the sexually transmitted infection (STI), gonorrhea. Worldwide, >100 million new cases occur annually. Ng has become resistant to almost every antibiotic that has been used and has now achieved """"""""superbug"""""""" status. The emergence and spread of ceftriaxone-resistant strains, in particular, has heralded an era of untreatable gonorrhea and represents a global public health crisis. Novel therapeutics against this pathogen are urgently needed. Complement (C2) is a critical arm of innate immune defenses against Ng. Ng evade C2 using several mechanisms, including scavenging the human C2 inhibitor, factor H (fH). fH comprises 20 domains - domains 18-20 interact with porin B and binding is augmented by lipooligosaccharide sialylation, while domains 6-7 bind to neisserial surface protein A. All Ng we have tested bind to one or both of these regions in fH. Chimeric molecules created by fusing fH domains 6-7 or 18-20 to the Fc region of IgG can effect C2-dependent killing of Ng. fH domains 18-20 in the context of full-length native fH binds to and serves to limit pathological injury to host cells, which limits the therapeutic use of 'unmodified'domains 18-20 in fH/Fc. We have overcome this limitation by introducing a point mutation in domain 19 (D1119G) of fH18-20/Fc that eliminates C2-mediated lysis of host cells without altering anti-Ng efficacy, thus providing us with a lead therapeutic molecule.
In Aim 1, we will maximize the ability of the human IgG1 Fc region of fHD1119G/Fc to mediate opsonophagocytosis and C2-dependent killing of multi-drug resistant Ng isolates by introducing 3 amino acid point mutations in the Fc and eliminating the core fucose of Fc glycans. Similarly, in Aim 2a, we will optimize the effector functions of the Fc that is fused to human fH domains 6 and 7 (fH6,7/Fc) and evaluate its ability to mediate C2-dependent killing and opsonophagocytosis of drug-resistant Ng. The ability of fHD111G/Fc and fH67/Fc, which recognize distinct epitopes, to synergize and stimulate bactericidal activity against Ng will be evaluated in Aim 2b. The milestones achieved in Aims 1 and 2 (the R21 phase) will be followed by studies in vivo (R33 phase) to determine the safety, efficacy and mechanism of action of fH/Fc. Renal, ocular and hematologic toxicity (target organs for C2-mediated injury) will be assessed in Aim 3. Introducing the D1119G mutation could create a neo-epitope and elicit auto-Abs;this possibility will be addressed using our human fH transgenic (Tg) mice.
In Aim 4 we will determine efficacy of the fH/Fc fusion proteins against a panel of drug-resistant Ng in the mouse model of Ng infection using our novel human fH/C4BP (C4BP;classical pathway C2 inhibitor) """"""""double"""""""" Tg mice (Ng bind only human, but not mouse C2 inhibitors). Using mice that lack complement C3, C5, Fc receptors, PMNs or macrophages we will define how fH/Fc clear Ng in vivo. Many medically important microbes bind human fH through domains 6-7 and/or 18-20. While we will use Ng as a model pathogen, successful completion of these studies will provide a strong platform to assess these fH/Fc molecules against other emerging drug-resistant pathogens.

Public Health Relevance

Gonorrhea is a sexually transmitted infection that affects over 100 million worldwide, annually. Neisseria gonorrhoeae, the bacterium that causes gonorrhea, has become resistant to almost every antibiotic and novel approaches to treat this disease are urgently needed. We have capitalized on a mechanism that this bacteria uses to escape killing by complement, an arm of innate immunity, to design novel molecules that can mediate killing of the gonococcus. Because several other medically important and drug-resistance pathogens also use similar complement evasion mechanisms, this novel immunotherapeutic could have broad utility.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZAI1-SM-M (J1))
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David, Hagit S
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University of Massachusetts Medical School Worcester
Internal Medicine/Medicine
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Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika et al. (2016) A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae. J Immunol 196:1732-40
Wong, Sandy M; Shaughnessy, Jutamas; Ram, Sanjay et al. (2016) Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae. Front Cell Infect Microbiol 6:40
Chakraborti, Srinjoy; Lewis, Lisa A; Cox, Andrew D et al. (2016) Phase-Variable Heptose I Glycan Extensions Modulate Efficacy of 2C7 Vaccine Antibody Directed against Neisseria gonorrhoeae Lipooligosaccharide. J Immunol 196:4576-86
Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B et al. (2016) Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae. Immunobiology 221:1110-23
Gulati, Sunita; Schoenhofen, Ian C; Whitfield, Dennis M et al. (2015) Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics. PLoS Pathog 11:e1005290
Gulati, Sunita; Mu, Xin; Zheng, Bo et al. (2015) Antibody to reduction modifiable protein increases the bacterial burden and the duration of gonococcal infection in a mouse model. J Infect Dis 212:311-5
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Shaughnessy, Jutamas; Vu, David M; Punjabi, Rahi et al. (2014) Fusion protein comprising factor H domains 6 and 7 and human IgG1 Fc as an antibacterial immunotherapeutic. Clin Vaccine Immunol 21:1452-9
Lewis, Lisa A; Ram, Sanjay (2014) Meningococcal disease and the complement system. Virulence 5:98-126
Beernink, Peter T; Shaughnessy, Jutamas; Stefek, Heather et al. (2014) Heterogeneity in rhesus macaque complement factor H binding to meningococcal factor H binding protein (FHbp) informs selection of primates to assess immunogenicity of FHbp-based vaccines. Clin Vaccine Immunol 21:1505-11

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