ABSTRACT Gonorrhea affects over 87 million people annually globally. In 2021, over 700,000 cases of gonorrhea were reported to the CDC, a 118% increase since the historic low in the US in 2009. However, the true incidence of cases annually in the US is estimated to be ~1.5 million. The spread of antimicrobial resistance has severely limited treatment options – currently, ceftriaxone is the only approved first line of treatment. A safe and effective therapeutic against gonorrhea is urgently needed. Gonococci (Ng) possess several mechanisms to evade killing by complement (C’), including binding of factor H (FH) and C4b-binding protein (C4BP), key host inhibitors of the alternative and classical pathway respectively. We showed previously that a chimeric protein comprising human IgG3 Fc fused to FH domains 18-20 (containing a point mutation in domain 19 to prevent lysis of host cells) (Fc3/FH*) killed Ng in a complement-dependent manner and reduced the duration and bacterial burden in the mouse vaginal colonization model of gonorrhea. Ng can be broadly classified into two serovars based on the sequence of the outer membrane protein (PorB): PorB1A and PorB1B. PorB1B strains account for 70-90% of gonococcal infections. PorB1A strains are strongly associated with disseminated gonococcal infection (DGI) and have caused serious sequelae including dermatitis, arthritis and endocarditis. Fc3/FH* is effective in vitro against all PorB1B Ng strains tested, but it killed only 13% of tested PorB1A strains. We have developed and tested 2 additional C’ based immunotherapeutic molecules with better activities against PorB1A strains: FH (6-7)/Fc3 and C4BP-IgM. We have shown that FH(6-7)/Fc3 binds to PorB1A strains and killed 60% of PorB1A strains tested. C4BP/IgM binds 90% of PorB1A strains and killed 92% PorB1A Ng. The main goals of this project are to develop a FH6-7 or a C4BP based-Fc fusion that is effective against PorB1A Ng to be part of a combination therapeutic with Fc3/FH* in order to provide broad protection against both PorB1A and PorB1B strains. In Aim 1, we will design and produce novel C4BP/Fc3 fusions with improved characteristics. Instead of using C4BP-IgM (which is impractical to produce commercially), we will develop and produce multimeric C4BP/IgG fusion proteins in tobacco plants . In Aim 2, we will evaluate in vitro efficacy of FH(6-7)/Fc3and C4BP/Fc3, alone and in combination with Fc3/FH*. We will use a panel of 60 diverse Ng, consisting of 40 PorB1A and 20 PorB1B strains for testing. We will test the binding of the fusion proteins to Ng by flow cytometry, and their bactericidal activity in the presence of serum. The lead molecules will be evaluated under conditions mimicking those in the vagina and we will test for any in vitro cytotoxicity. In Aim 3, we will test the efficacy of lead FH(6-7)/Fc3and C4BP-based/Fc3 immunotherapeutic molecules in human FH/C4BP transgenic mice. The murine Ng vaginal colonization model is currently the only non-h...