In Vitro Pre-Clinical Evaluation of a Gonococcal Trivalent Candidate Vaccine Identified by Transcriptomics

Abstract

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, poses a significant global public health threat. Infection in women can be asymptomatic and may result in severe reproductive complications. Escalating antibiotic resistance underscores the need for an effective vaccine. Approaches being explored include subunit vaccines and outer membrane vesicles (OMVs), but an ideal candidate remains elusive. Meningococcal OMV-based vaccines have been associated with reduced rates of gonorrhea in retrospective epidemiologic studies, and with accelerated gonococcal clearance in mouse vaginal colonization models. Cross-protection is attributed to shared antigens and possibly cross-reactive, bactericidal antibodies. Using a Candidate Antigen Selection Strategy (CASS) based on the gonococcal transcriptome during human mucosal infection, we identified new potential vaccine targets that, when used to immunize mice, induced the production of antibodies with bactericidal activity against N. gonorrhoeae strains. The current study determined antigen recognition by human sera from N. gonorrhoeae-infected subjects, evaluated their potential as a multi-antigen (combination) vaccine in mice and examined the impact of different adjuvants (Alum or Alum+MPLA) on functional antibody responses to N. gonorrhoeae. Our results indicated that a stronger Th1 immune response component induced by Alum+MPLA led to antibodies with improved bactericidal activity. In conclusion, a combination of CASS-derived antigens may be promising for developing effective gonococcal vaccines.

Keywords: Th1/Th2 responses; adjuvants; bactericidal antibodies; gonorrhea; vaccine.

Figure 1

IgG against NGO0690, NGO0948 and NGO1701 in human sera from N. gonorrhoeae-infected subjects. Total IgG antibody ELISA of banked de-identified sera from: (A) women with uncomplicated acute gonococcal infection (collected 10–30 days following diagnosis) (n = 25); (B) women convalescing from uncomplicated infection (collected 180–240 days following diagnosis) (n = 25); (C) men with uncomplicated acute gonococcal infection as above (n = 25); and (D) men convalescing from uncomplicated infection as above (n = 25) against purified recombinant NGO0690 (dotted bars), NGO0948 (dashed bars), NGO1701 (striped bars) and N. gonorrhoeae F62 (black bars). Sera (1:100 dilution) were tested in triplicate or quadruplicate, and IgG levels are expressed as the mean of the IgG O.D.₄₀₅ minus the blank ± SD for each set of sera against each antigen. *, **, ***, ****—p value is significant according to two-way ANOVA with Tukey’s multiple comparisons test. (E) Banked de-identified sera from women with disseminated gonococcal infection (DGI) (n = 7). Individual data points are shown by different symbols. *, ***, ****—p value is significant according to two-way ANOVA with Tukey’s multiple comparisons test. (F) Commercially available pooled whole normal human serum (NHS) as above. **, ***, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 2

Mouse serum IgG antibodies against purified antigens. Total IgG (μg/mL ± SD) in sera from mice immunized with NGO0690+NGO0948+NGO1701 and Alum or Alum+MPLA as an adjuvant measured by ELISA against (A) NGO0690 (dotted bars), (B) NGO0948 (dashed bars) and (C) NGO1701 (striped bars). Preimmune sera, white bars; sera from mice immunized with adjuvant only, gray bars. Sera were tested in triplicate or quadruplicate. ***, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test. Note the different scale in (A) and (C) vs. (B).

Figure 3

Mouse vaginal lavage IgG antibodies against purified antigens. Total IgG (μg/mL ± SD) in vaginal lavages from mice immunized with NGO0690+NGO0948+NGO1701 and Alum or Alum+MPLA as an adjuvant measured by ELISA against (A) NGO0690 (dotted bars) and (B) NGO1701 (striped bars). Lavages from mice immunized with adjuvant only, gray bars. Lavages were tested in quadruplicate. *, ***, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

Mouse serum IgG antibodies against whole N. gonorrhoeae. Total IgG (μg/mL ± SD) in sera from mice immunized with NGO0690+NGO0948+NGO1701 and Alum or Alum+MPLA, measured by whole-cell ELISA against (A) N. gonorrhoeae F62 (black bars) and (B) N. gonorrhoeae MS11 (black bars). Preimmune sera, white bars; sera from mice immunized with adjuvant only, gray bars. (C,D) Total IgG in vaginal lavages from mice immunized with NGO0690+NGO0948+NGO1701 and Alum or Alum+MPLA, measured as above. Lavages from mice immunized with adjuvant only, gray bars. Sera and lavages were tested in triplicate or quadruplicate. *, **, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 5

Mouse serum IgG antibody subclasses against whole N. gonorrhoeae. IgG1, IgG2a and IgG3 (μg/mL ± SD) measured by whole-cell ELISA in sera from mice immunized with NGO0690+NGO0948+NGO1701 with Alum (A,B) or with Alum+MPLA (C,D) as adjuvants against (A,C) N. gonorrhoeae F62 (black bars) and (B,D) N. gonorrhoeae MS11 (black bars). Preimmune sera, white bars; adjuvant-only sera, gray bars. Sera were tested in triplicate or quadruplicate. *, **, ***, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 6

Mouse serum cytokine profile. Th2 cytokines (A) IL-10 and (B) IL-4, and Th1 cytokines (C) IFN-γ and (D) IL-12p70 measured by ELISA. Adjuvant-only sera (gray bars), NGO0690+NGO0948+NGO1701 and Alum sera and NGO0690+NGO0948+NGO1701 and Alum+MPLA (black bars) were tested in quadruplicate and cytokine levels are expressed in pg/mL ± SD. *, ***, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test. (E) IL-12p70/IL-10 ratio and IFN-γ/IL-4 ratio. * p < 0.05 according to Mann–Whitney test. (F,G) IL-6 and TNF-α measured as above. ***—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 7

Mouse serum IgM antibodies against whole N. gonorrhoeae. IgM (μg/mL ± SD) in sera from mice immunized with NGO0690+NGO0948+NGO1701 and Alum or Alum+MPLA, measured by whole-cell ELISA against (A) N. gonorrhoeae F62 (black bars) and (B) N. gonorrhoeae MS11 (black bars). Preimmune sera, white bars; adjuvant-only sera, gray bars. Sera were tested in triplicate or quadruplicate. *, **, ****—p value is significant according to one-way ANOVA with Tukey’s multiple comparisons test.

Figure 8

Serum IgM and IgG antibody avidity against N. gonorrhoeae F62. (A) IgM (O.D.₄₀₅ minus the blank ± SD) determined with a modified ELISA in the presence (open symbols) or absence (closed symbols) of 8M urea treatment. NGO0690+NGO0948+NGO1701 and Alum sera (triangles) or Alum-alone sera (circles) and (B) NGO0690+NGO0948+NGO1701 and Alum+MPLA sera (triangles) or Alum+MPLA-alone sera (circles). (C) IgG antibody avidity as above. Alum-alone sera (circles) and NGO0690+NGO0949+NGO1701 with Alum sera (squares) and (D) Alum+MPLA-alone sera (circles) and NGO0690+NGO0949+NGO1701 with Alum+MPLA sera (squares). Sera were tested in triplicate. *, ***, ****—p value is significant according to two-way ANOVA with Tukey’s multiple comparisons test.

Figure 9

Serum bactericidal activity (SBA). N. gonorrhoeae F62 survival (% CFU at T30/T0 ± SD). IgM-depleted sera from mice immunized with Alum alone (gray bar) and NGO0690+NGO0948+NGO1701 with Alum (white bars). Serum dilutions are indicated on the X-axis. ^{#, ##, ####}—p < 0.05, 0.003 and 0.0001 according to one-way ANOVA with Dunnett’s multiple comparison test vs. the Alum-only sera. *, **, ***, ****—p = 0.04 to <0.0001 according to one-way ANOVA with Tukey’s multiple comparisons test among sera dilutions.

Figure 10

Serum bactericidal activity (SBA) in the presence of 2% BSA. N. gonorrhoeae F62 survival (% CFU T30/T0 ± SD). Sera from mice immunized with (A) Alum alone (gray bar) or with NGO0690+NGO0948+NGO1701 and Alum (white bars), and (B) Alum+MPLA-alone (gray bar) or NGO0690+NGO0948+NGO1701 and Alum+MPLA (white bars). Serum dilutions are indicated on the X-axis. *, **, ***, ****—p = 0.04 to <0.0001 according to one-way ANOVA with Tukey’s multiple comparisons test among sera dilutions. ^##, ^####—p = 0.002 and <0.0001 according to one-way ANOVA with Tukey’s multiple comparisons test vs. adjuvant alone.

Figure 11

Images of the predicted structure (side view) of (A) NGO0690, (B) NGO0948 and (C) NGO1701, modeled with Alpha Fold [61], based on the protein sequence and colored by confidence (dark blue, highest; orange/yellow, lowest). (b) NGO0948, front view. (A_I–C_I) Surface model images of NGO0690, NGO0948 and NGO1701 (light gray) rendered with PyMol [65], showing the predicted linear B cell epitopes (LE) (ElliPro and Bepipred [63,64]) in different colors. LE in regions with low structure model confidence as in (A–C) is shown by transparent colors, and LE in regions with high confidence in solid colors. (A_II–C_II) Predicted conformational epitopes (CE) obtained and rendered as above. CE in regions of low confidence are shown in transparent dark gray, and CE in regions with high confidence in solid dark gray. In (B_II), CE6 and CE7 are at the back of the image, indicated by an arrow, and are not visible.