Neisseria gonorrhoeae strains for GMMA production and functional assays

Neisseria gonorrhoeae strains were routinely cultured at 37 °C in an atmosphere of 5% CO₂ on Gonococcus (GC) agar medium plates enriched with 1% v/v of Isovitalex. For liquid cultures, bacteria from a fresh overnight plate culture (16 ± 2 h) on the GC agar medium plates 1% Isovitalex were diluted to reach a starting optical density at 590 nm (OD₅₉₀) equal to 0.3–0.4 in liquid GC—1% v/v Isovitalex, supplemented if needed, and incubated at 37 °C at 160 rpm. Cytidine-5’-O-monophospho-N-acetylneuraminic acid [CMP-NANA] was not added to the broth medium used for these liquid cultures. When required, to induce pilus expression and enable transformation, GC strains were cultured on GC agar plates supplemented with 0.25 mM (IPTG).

Neisseria gonorrhoeae MS11 LOS lgt mutant strains

Neisseria gonorrhoeae MS11 LOS lgt mutant strains used in this work consist of eight isogenic N. gonorrhoeae mutant strains created in the background of N. gonorrhoeae MS11 4/3/1, a variant of MS11 VD300 with an isopropyl-D-thiogalactopyranoside (IPTG)-inducible pilE that controls pilus expression²⁰. As described in Chakraborti et al.²⁰, in this series of mutants, the glycan extensions from LOS core heptoses (Heptose-I and Heptose-II) have been modulated genetically, setting on or off (or deleting) the phase variable expression of the four LOS glycosyltransferase loci: lgtA, lgtC and lgtD genes, responsible for the variation in the α-chain extensions, and the lgtG gene, which controls the expression of the β-chain (a lactose on the Heptose-II). The resulting LOS structure of the mutant strains is reported in Fig. 2.

Generation of Neisseria gonorrhoeae knockout strains

To reduce the endotoxin activity in the strains used, the lpxL1 gene was deleted as in Spinsanti et al.¹⁰ for SK92-679 and F62, while the ΔlpxL1 cmR plasmid containing upstream and downstream regions of the gene flanking a cloramphenicol resistance cassette was used in the collection of MS11 mutant strains. The plasmid was synthesized by GeneArt (Thermo Fisher Scientific) and transformed into DH5α E. coli competent cells (Thermo Fisher Scientific). Plasmid DNA was purified using HP Plasmid Mini kit II (Omega Biotek) according to the manufacturer’s instructions, and it was used as a template for a PCR amplification using primers LpxL1 UP Fwd and LpxL1 DO Rev. All primers used in this study are reported in Supplementary Table 2. The PCR product was purified using the Wizard SV Gel and PCR clean-up system (Promega) following the manufacturer’s protocol and then used for the transformation of the eight MS11 mutant strains. Transformations were carried out by spotting a mixture of bacterial resuspension in phosphate-buffered saline (PBS) and DNA onto a GC agar + 1% isovitalex plate and by incubating it for 5–6 h. Transformants were selected onto GC agar + 1% isovitalex plate with either kanamycin (70 µg/ml for F62 and 150 µg/ml for SK92-679) or cloramphenicol 10 μg/ml. To check the correct event of double recombination, transformants were tested by PCR using the Accuprime Taq Polymerase (Thermo Fisher Scientific) and with external primers LpxL1 est Fwd and LpxL1 est Rev. Positive clones were streaked repeatedly onto selective agar plates and glycerol stocks and DNA lysates were collected at each passage to test for the presence of remaining wild-type (WT) population. PCR screenings were performed using Accuprime Taq Polymerase and with the primer LpxL1 est FW in combination with the primer NGO_lpxL1_wtcheck-Rev, specific for the WT DNA. Absence of a PCR product would indicate the proper removal of all WT cells from the mutant clone.

Bacterial fermentation

For each batch, bacteria from a fresh overnight plate culture (16 ± 2 h) on the GC agar medium plates 1% Isovitalex were diluted to reach a starting optical density at 590 nm (OD₅₉₀) equal to 0.3–0.4 in liquid GC—1% v/v Isovitalex and incubated at 37 °C at 180 rpm until an OD_600nm equal to 1.5 ± 0.5 was reached. GMMA preparations were obtained from bacteria grown in absence of CMP-NANA.

GMMA isolation and purification

The collected growth was centrifuged at 8000×g for 15 min at 4 °C. and the cell-free supernatant was then carefully recovered, filtering the solution through a 0.2 μm Sartobran P H9 filter to ensure bacteria removal. Then, MgCl₂⋅6H₂O was added to a final molarity of 10 mM and 50 U/L of benzonase was added for DNA removal and incubated at 4–8 °C while stirring overnight. A tangential flow filtration step using 200 cm2 300 kDa cut-off PESU membrane (Sartocon Slice 200 Sartorius stedim polyethersulfone 300 kDa) was used for retention of the GMMA and buffer exchange in PBS, followed by an ultracentrifugation step (150,000×g for 2 h) to pellet the GMMA, which were subsequently resuspended in PBS and sterile filtered using a 0.2 µm filter, to obtain final samples.

GMMA characterization

Total protein quantification and protein concentration were performed with Pierce Modified Lowry Protein Assay from Thermo Scientific. The purity of the GMMA preparations was assessed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and performing size exclusion chromatography module (SE-HPLC) to determine the purity using fluorescence to monitor soluble protein contaminants and UV 260 and 280 nm wavelength for DNA content determination.

LOS immunochemical and physicochemical characterization

A panel of physicochemical and immunochemical methods for the complete characterization of gonococcal LOS was combined to fully characterize this antigen on different types of biological samples, such as bacterial lysates, outer membrane vesicles and extracted LOS samples.

LOS quantification by semicarbazide derivatization and SE-HPLC analysis

The quantification of lipooligosaccharide content in GMMA samples was measured by SE-HPLC analysis by quantifying the reactive carbonyl groups of the saccharide moiety, generated after acid hydrolysis to remove the Lipid A and derivatized with semicarbazide, as previously reported^49,50. The final quantified nmol of terminal KDO corresponds to the moles of LOS in N. gonorrhoeae biological samples. The KDO content of the GMMA samples is quantified based on a calibration curve prepared starting with a standard KDO ammonium salt solution. The LOS content is expressed in nmol/mL of KDO, which matches the nmol/mL of OS, and it is lastly reported as nmol_LOS/mg protein, to normalize on the protein content.

Silver stain

From a qualitative perspective, the glycoforms of LOS structures exposed by each strain were established by normalizing the quantity of heat-inactivated bacterial lysates (according to the OD_600nm) and/or corresponding GMMA/extracted LOS samples (based on the LOS quantification titer by HPLC) and analyzing samples on 16% Tris–glycine gels using Tris–glycine 1x running buffer (Thermo Fisher Scientific). Then, after the fixation step using 5% acetic acid and 40% ethanol solution, an additional step of oxidation of the sugar with 0.7% sodium metaperiodate was added, finally staining using SilverQuest Staining kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Silver-stained gel images were acquired using the Scanner EPSON PERFECTION V700 PHOTO.

This method detects oligosaccharide-derived relative masses, thus enabling the visualization of all the predominant LOS structures in the sample. The eight MS11 lgt mutant strains expressing defined LOS structures confirmed that different LOS structures could be differentiated using this method.

Western Blot

Samples were further characterized by Western Blot, evaluating their reactivity with the combination of specific anti-LOS monoclonal antibodies. Heat-inactivated bacterial lysates, GMMA or extracted LOS were run on a 16% Tris–glycine SDS–PAGE gel using a Tris–glycine 1x buffer. LOS was transferred to nitrocellulose membranes (The iBlot Kit, Thermo Fisher Scientific), and membranes were blocked with PBS 1x + Tween20 0.05% + BSA 3% for 1 h at room temperature. Anti-LOS mAbs were incubated with membranes for 1 h at room temperature, and mAb-reactive LOS bands were visualized with anti-mouse IgG alkaline phosphatase secondary antibody, followed by reaction with the AP Conjugate Substrate kit (Biorad). Membrane images were acquired using the Scanner EPSON PERFECTION V700 PHOTO.

For the Western Blot analysis, mAb L3,7,9⁵¹, targeting the lacto-N-neotetraose structure, composed of four sugars extending from Heptose-I (Galβ1-4GlcNAcβ1-3Galβ1-4Glc, Fig. 2A, green box), and mAb 17-1-L1 (henceforth referred to as mAb L1), which binds to an alternative Heptose-I structure (Galα1-4Gal), thus recognizing the L1 meningococcal serotype, also known as the P^K structure (Galα1-4Galβ1-4Glc as α-chain, Fig. 2A, purple box), were selected. Furthermore, anti-gonococcal mAb 2C7 was implemented to detect the presence of lactose α-linked to Heptose-II (Fig. 2A, yellow box), with at least the first two sugars in the α-chain, but able to recognize structures with extensions from Heptose I beyond lactose, together with mAb 4C4, targeting highly truncated structures of LOS (Glcβ1-4Hep), were selected to finalize this panel, as reported in Fig. 2A, blue box. The anti-LOS mAb L1²⁴, 4C4²⁵, L3,7,9²⁴ and 2C7²⁶ have been described previously. Mouse mAb 2C7 has been produced internally as a recombinant mAb. Purified mAb 4C4 and supernatant of mAb L3,7,9 are commercially available (Novus Biologicals). Using these mAbs, which specifically react with different terminal sugar components of LOS, in Western Blot allowed us to precisely identify the prevalent LOS structure expressed in each strain.

Immunization study

Animal husbandry and experiments were ethically reviewed and carried out in accordance with European Directive 2010/63/EU, in compliance with relevant guidelines (Italian Legislative Decree No. 26/2014) and GSK’s policy and guidelines on the care, welfare and treatment of animals, in GSK animal facilities located in Siena, Italy (AAALAC accredited). The ethical protocol P004/26/01 was reviewed by the local GSK ethical committee. The studies refer to research projects approved by the Italian Ministry of Health.

Female CD1 mice, 7-week-old, were purchased from Charles River Laboratories and kept in a controlled environment (individually ventilated cages; 22 ± 3 °C; 12 h/12 h light/dark cycle). Animals and their housing and husbandry were checked daily, and their well-being and health status were recorded in a dedicated logbook according to the local standard operating procedures. Final bleeding was performed under general anesthesia, and animals were euthanized by cervical dislocation before recovery from anesthesia.

CD1 mice (10/group) were immunized intraperitoneally twice on day 1 and day 29 with GMMA from different strains (F62 and SK92-679 strains) at a 10 μg protein-based dose in 200 μL adsorbed to Alum hydroxide (3 mg/mL). Mouse sera collected two weeks after the second dose (day 43) were analyzed in pools in functional assays as described below. In the in vivo immunization study using GMMA from MS11 mutant strains, female 7-week-old CD1 mice (10/group) were immunized intraperitoneally twice on day 1 and day 29. Different from the other study, GMMA were normalized based on LOS content at a dose of 1.5 nmol_LOS in 200 μL, adsorbed to Alum hydroxide (3 mg/mL). Mouse sera were collected two weeks after the second dose (day 43) and analyzed in pools in functional assays as described below.

Human serum bactericidal assay (hSBA)

Bactericidal antibodies were measured by human serum bactericidal activity (hSBA) assay against the GC strains FA1090, F62, SK92-679, MS11, BG27, WHO-M, WHO-G and WHO-N using normal human serum from healthy donors as a complement source. Bacterial colonies from an overnight culture were resuspended in GC + 1% Isovitalex. Cytidine-5’-O-monophospho-N-acetylneuraminic acid [CMP-NANA] was added to the broth medium for serum-sensitive strains to render them resistant to killing by normal human serum: 0.5 µg/ml for F62 and 0.2 µg/ml for WHO-M and MS11 strains, and incubated at 37 °C with gentle shaking until the culture reached OD₆₀₀ = 0.5. The broth culture was then diluted 1:10,000 in SBA buffer (Dulbecco’s phosphate-buffered saline [dPBS] + 1% BSA + 0.1% glucose) with the exception of the BG27 strain, which was diluted 1:2500. Mouse sera, previously heat-inactivated at 56 °C for 30 min, were serially diluted in SBA buffer. The assay was assembled in a sterile 96-flat-bottom well microplate in a final volume of 32 µL/well. The serial dilutions of each test sample were allowed to react with pre-diluted bacteria and with human serum from healthy donors as a complement source (16% for FA1090, SK92-679, BG27, WHO-G, WHO-N and 10% for F62, MS11, WHO-M). The reaction mixture was incubated at 37 °C for 60 min at 160 rpm, then agar overlay medium was added and the plate was incubated overnight at 37 °C with 5% CO₂ in a humid atmosphere. One day later, colony-forming units (CFUs) were manually counted or automatically acquired with a high-throughput image analysis system (Discovery v12 Axiolab or ScanLab) and were automatically counted for each well by an image analysis system (Reading AxioVision or the internally developed software). Bactericidal titer was defined as the reciprocal of the serum dilution giving a killing >50% with respect to the average number of CFU calculated in replicates of ‘without serum’ control. The bactericidal titer for each test sample was calculated as the reciprocal of the serum dilution giving a killing >50% respect to the average number of CFU calculated on the 8 replicates without serum control at T_60min (average CFU without serum control). Where more than one serum dilution gives 50% U-test was used to statistically compare hSBA titers obtained for experimental replicates from different immunization groups.

Competitive human serum bactericidal assay

The competitive human serum bactericidal assay (hSBA) was used to measure the residual serum bactericidal activity of a pool of immunized mouse sera, known to give high hSBA titers, after incubation with different competitors. These experiments allowed us to understand the role of anti-LOS antibodies in the SBA response elicited by a GMMA-based vaccine. Due to the limited availability of sera reagents at adequate volumes, the competitive SBA experiments were performed only once. A fixed dilution of the tested pool (sera obtained from mice immunized with GMMA vaccine) was incubated 1:1 (vol/vol) with 3 different concentrations (in terms of nmol_LOS/mL) of each competitor for 1 h at 37 °C, 180 rpm. The same pool was also incubated 1:1 (vol/vol) with SBA buffer to measure the hSBA titer of “not inhibited” sample. After 1 h of incubation, the mixture serum-competitor was dispensed in a plate, diluted 1:2 for 11 dilution-steps and then bacteria and human complement were added following the hSBA assay protocol. The “no serum” control (8 wells) was included in each plate, and it comprises bacteria mixed with active human complement (AC) in the absence of a serum sample. This control is used to exclude complement toxicity and to determine 100% bacterial growth.

Bacterial Adhesion Inhibition (BAI) assay

A cell-based fluorescent BAI assay was used to assess the capacity of the tested murine pooled sera to inhibit the adhesion of FA1090 and SK92-679 strains to SV-HUC1 human urethral epithelial cells.

Cultured cells were detached from a 175 cm² flask after resuspension to avoid cell clumping. Cell number and viability were determined by an automated counter. They were then seeded into 96-well plates (3 × 10⁴ cells/well) and cultured in F-12K Nut Mix medium till confluence. GC strains were harvested from a fresh overnight plate culture into 10 ml GC + 1% Isovitalex medium. Bacteria were grown at 37 °C under shaking till OD₆₀₀ = 0.5, then resuspended in DPBS and labeled with Oregon Green dye for 15 min at 37 °C. Afterwards, bacteria were washed to remove excess dye, resuspended in DPBS–2% BSA, and combined, at final OD₆₀₀ = 0.1, with an equal volume of serially cell medium-diluted sera for 15 min at RT. Bacteria-sera complexes were added to cell plates and incubated for 1 h at 37 °C to allow bacterium-cell adhesion. After three washings with DPBS, samples were fixed for 20 min with 4% formaldehyde at room temperature and, after one washing step with DPBS, finally one volume of distilled water was added to each well. Plates were analyzed by the Opera Phenix instrument (Revvity). Due to variability in the results obtained with the negative control (Alum), a threshold of 30% was set to discriminate between positive samples and Alum itself. This 30% cutoff was defined based on over 100 observations during the setup of the BAI assay, where Alum serum showed bacterial inhibition lower than 15% in around 95% of the observations and lower than 30% in 100% of the observations.