crossmark Research © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea*□S Ryszard A. Zielke‡, Igor H. Wierzbicki‡, Benjamin I. Baarda‡, Philip R. Gafken§, Olusegun O. Soge¶, King K. Holmes¶ʈ, Ann E. Jerse**, Magnus Unemo‡‡, and Aleksandra E. Sikora‡§§ Expanding efforts to develop preventive gonorrhea vac- exposed, secreted via naturally released membrane ves- cines is critical because of the dire possibility of untreat- icles, and elicited bactericidal antibodies that cross-re- able gonococcal infections. Reverse vaccinology, which acted with a panel of temporally and geographically di- includes genome and proteome mining, has proven very verse isolates. In addition, analysis of polymorphisms at successful in the discovery of vaccine candidates against the nucleotide and amino acid levels showed that these many pathogenic bacteria. However, progress with this vaccine candidates are highly conserved among N. gon- approach for a gonorrhea vaccine remains in its infancy. orrhoeae strains. Finally, depletion of BamA caused a loss Accordingly, we applied a comprehensive proteomic plat- of N. gonorrhoeae viability, suggesting it may be an es- form—isobaric tagging for absolute quantification cou- sential target. Together, our data strongly support the use pled with two-dimensional liquid chromatography and of proteomics-driven discovery of potential vaccine tar- mass spectrometry—to identify potential gonococcal gets as a sound approach for identifying promising gono- vaccine antigens. Our previous analyses focused on cell coccal antigens. Molecular & Cellular Proteomics 15: envelopes and naturally released membrane vesicles de- 10.1074/mcp.M116.058800, 2338–2355, 2016. rived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously ex- pressed and specifically induced by physiologically rele- Despite current prevention and management strategies, in- vant environmental stimuli: oxygen availability, iron dep- fections caused by sexually transmitted pathogens affect rivation, and the presence of human serum. Together, hundreds of millions of women and men in both resource- these studies enabled the identification of numerous po- constrained and developed countries (1). Gonorrhea remains tential gonorrhea vaccine targets. Initial characterization a leading public health burden with an estimated 78 million of five novel vaccine candidate antigens that were ubiq- new cases annually worldwide (2). Major challenges in erad- uitously expressed under these different growth condi- icating this ancient human disease include increasing multi- tions demonstrated that homologs of BamA (NGO1801), drug-resistance among Neisseria gonorrhoeae strains and the LptD (NGO1715), and TamA (NGO1956), and two unchar- acterized proteins, NGO2054 and NGO2139, were surface high incidence of asymptomatic infections that contributes to the spread of gonorrhea (3, 4). Gonococcal infections often From the ‡Department of Pharmaceutical Sciences, College of have devastating sequelae in women including pelvic inflam- Pharmacy, Oregon State University, Corvallis, Oregon; §Proteomics matory disease, ectopic pregnancy, and infertility (5, 6). In Facility, Fred Hutchinson Cancer Research Center, Seattle, Washing- addition, gonorrhea during pregnancy causes chorioamnion- ton; ¶Neisseria Reference Laboratory, Department of Global Health, itis, which can be complicated further by a septic abortion, ʈ University of Washington, Seattle, Washington; Departments of premature membrane rupture, and preterm delivery. Infants Medicine and Global Health, University of Washington, Seattle, Wash- ington; **Department of Microbiology and Immunology, F. Edward born to mothers with cervical gonorrhea have an increased Herbert School of Medicine, Uniformed Services University of the risk of neonatal gonococcal conjunctivitis, which can lead to Health Sciences, Bethesda, Maryland; ‡‡WHO Collaborating Centre corneal scarring and blindness. In men, untreated urethritis for Gonorrhoea and other Sexually Transmitted Infections, National may evolve into penile edema, urethral stricture and epidid- Reference Laboratory for Pathogenic Neisseria, Department of Lab- ymitis (7, 8). Furthermore, gonorrhea facilitates the transmis- oratory Medicine, Microbiology, Örebro University Hospital, Örebro, Sweden sion and acquisition of HIV (9). For these reasons, it is critical Received February 3, 2016, and in revised form, April 25, 2016 to develop effective interventions against gonorrhea. Cur- Published, MCP Papers in Press, May 2, 2016, DOI rently, strains resistant to the last effective option for empiric 10.1074/mcp.M116.058800 monotherapy—third-generation cephalosporins—are emerg- Author contributions: A.E.S. designed research; R.A.Z., I.H.W., ing and clinical treatment failures have been documented in B.I.B., P.R.G., O.O.S., and M.U. performed research; K.K.H. and M.U. contributed new reagents or analytic tools; R.A.Z. and A.E.S. ana- several countries (4, 10). History indicates that the incremental lyzed data; A.E.J. and A.E.S. wrote the paper; A.E.S. prepared all development of antibiotic resistance in N. gonorrhoeae inev- figures. itably continues to challenge antibiotic therapy (4). The World 2338 Molecular & Cellular Proteomics 15.7 Proteomics-driven Reverse Vaccinology for Gonorrhea Health Organization recently recognized the escalating prob- relevant to infection including different oxygen tensions (aer- lem of the spread of antimicrobial resistance in N. gonor- obic, anaerobic), iron deprivation, and the presence of normal rhoeae and highlighted the importance of novel approaches to human serum. In addition, a subset of five identified proteins identify alternative strategies for the treatment and prevention was evaluated for the capacity to induce antibodies that rec- of N. gonorrhoeae infections, including a gonorrhea vaccine ognize a collection of diverse clinical isolates and have bac- (11). tericidal activity against serum resistant and serum sensitive Pioneered by Rappuoli and colleagues, reverse vaccinol- N. gonorrhoeae strains. ogy—which initially included genome and later proteome min- ing—has proven to be very successful in the discovery of EXPERIMENTAL PROCEDURES vaccine candidates against many pathogenic bacteria (12– Bacterial Strains and Growth Conditions—The following N. gonor- 15). In particular, these methodologies paved the way for the rhoeae strains were used in this study: FA1090 (22), MS11 (23), 1291 newly developed group B meningococcal vaccine, which was (24), F62 (25), FA19 (26), the clinical isolates LGB1, LG14, LG20, and LG26, which were collected from two public health clinics in Balti- a formidable effort for many years. Among the 28 antigens more from 1991 to 1994 and differ in porB variable region type and that were discovered and that elicited bactericidal antibodies pulsed gel electrophoresis patterns (21, 27), 13 isolates from patients against group B meningococci in vitro were the Neisserial attending the Public Health-Seattle & King County Sexually Transmit- heparin-binding antigen (NHBA), factor H-binding protein ted Disease clinic from 2011 to 2013 (supplemental Table S1), and the (fHbp), and the Neisserial adhesin A (NadA). These three WHO 2015 reference strains (10, 28, 29). N. gonorrhoeae isolates were cultured from frozen stocks stored at proteins are formulated as part of the Bexsero meningococcal Ϫ80 °C onto gonococcal base agar solid medium (GCB1, Difco, group B vaccine approved by the European commission in Sparks, MD) containing Kellogg’s supplements I (1:100) and II (1: 2013 and the United States in February of 2015 (12, 15, 16). 1000) (30). After incubation for 18–20 h at 37 °C in a humid, 5% CO2 Unfortunately, N. gonorrhoeae homologs of these proteins are atmosphere, transparent colonies with piliated or nonpiliated colony not suitable vaccine targets (17). Further, in contrast to me- morphologies were sub-cultured onto GCB plates. Piliated N. gonor- rhoeae variants were used for transformation, whereas nonpiliated ningococcal vaccines, progress on gonococcal vaccines has bacteria were used in all other experiments. Following ϳ18hof been hampered primarily by the absence of a vaccine-tar- incubation as described above, bacteria were harvested from the getable surface capsule, exceptional variability of several sur- solid media using a polyester-tipped applicator (Puritan, Guilford, face antigens, a poor understanding of protective responses, ME), and suspended to a final OD600 of 0.1 in gonococcal base liquid (GCBL) medium containing Kellogg’s supplements I and II and so- and until relatively recently, the lack of a small laboratory dium bicarbonate (at a final concentration of 0.042%) (30 ,31). Sub- animal model to systematically test potential vaccine candi- sequently, gonococci were propagated as stated in the text: in GCBL dates (16). However, recent potential breakthroughs—such as at 37 °C or aerobically on GCB; GCB with Kellogg’s supplement I and the availability of transgenic mice to alleviate some host re- with deferoxamine mesylate salt (Desferral, Sigma, St. Louis, MO) at strictions, new insights into immunosuppression mechanisms 5 ␮M final concentration [iron limited conditions (32)]; GCB with the addition of 7.5% normal human serum [NHS (33)]; and anaerobically