Human Als3p Antibodies are Surrogate Markers of NDV-3A Vaccine Efficacy Against Recurrent Vulvovaginal Candidiasis

A Phase 1b/2a clinical trial of NDV-3A vaccine containing a Candida albicans recombinant Als3 protein protected women <40 years old from recurrent vulvovaginal candidiasis (RVVC). We investigated the potential use of anti-Als3p sera as surrogate marker of NDV-3A efficacy. Pre- and post-vaccination sera from subjects who experienced recurrence of VVC (R) versus those who were recurrence-free (non-recurrent, NR) were evaluated. Anti-Als3p antisera obtained were evaluated for; 1) titer and subclass profile; 2) their ability to influence C. albicans virulence traits including hyphal elongation, adherence to plastic, invasion of vaginal epithelial cells, biofilm formation on plastic and catheter material, and susceptibility to neutrophil killing in vitro. Serum IgG titers in NR patients were consistently higher than in R patients, particularly for anti-Als3 subclass IgG2. Sera from vaccinated NR patients reduced hyphal elongation, adhesion to plastic, invasion of vaginal epithelial cells and biofilm formation significantly more than pre-immune sera, or sera from R- or placebo-group subjects. Pre-adsorption of sera with C. albicans germ tubes eliminated these effects, while heat inactivation did not. Finally, sera from NR subjects enhanced neutrophil-mediated killing of C. albicans relative to pre-immune sera or sera from R patients. Our results suggest that higher Als3p antibody titers are associated with protection from RVVC, attenuate C. albicans virulence and augment immune clearance of the fungus in vitro. Thus, Als3p serum IgG antibodies are likely useful markers of efficacy in RVVC patients vaccinated with NDV-3A. Abbreviations Als3p Agglutinin-like sequence 53 3 protein AUC area under the curve CFU colony forming unit ConA Concanavalin A ELISA enzyme-linked immunosorbent assay Hyr1p hyphal regulating protein 1 IRB institutional review board OPK opsonophagocytic killing NR non-recurrent NDV-3 recombinant His-tagged N-terminus of Als3p R formulated with alum NDV-3A recombinant N-terminus of Als3p R formulated with alum recurrent RVVC recurrent vulvovaginal candidiasis ROC Receiver-operating characteristic Sap2 secreted aspartyl proteinase 2 SE silicone elastomer VVC vulvovaginal candidiasis YNB yeast nitrogen base YPD yeast peptone dextrose

protein protected women <40 years old from recurrent vulvovaginal candidiasis (RVVC). We 30 investigated the potential use of anti-Als3p sera as surrogate marker of NDV-3A efficacy. Pre-and 31 post-vaccination sera from subjects who experienced recurrence of VVC (R) versus those who 32 were recurrence-free (non-recurrent, NR) were evaluated. Anti-Als3p antisera obtained were 33 evaluated for; 1) titer and subclass profile; 2) their ability to influence C. albicans virulence traits 34 including hyphal elongation, adherence to plastic, invasion of vaginal epithelial cells, biofilm 35 formation on plastic and catheter material, and susceptibility to neutrophil killing in vitro. Serum 36 IgG titers in NR patients were consistently higher than in R patients, particularly for anti-Als3 37 subclass IgG2. Sera from vaccinated NR patients reduced hyphal elongation, adhesion to plastic, 38 invasion of vaginal epithelial cells and biofilm formation significantly more than pre-immune sera, 39 or sera from R-or placebo-group subjects. Pre-adsorption of sera with C. albicans germ tubes 40 eliminated these effects, while heat inactivation did not. Finally, sera from NR subjects enhanced 41 neutrophil-mediated killing of C. albicans relative to pre-immune sera or sera from R patients. Our 42 results suggest that higher Als3p antibody titers are associated with protection from RVVC, Candida species cause distressing mucocutaneous infections of the integument, oral and 63 genitourinary tracts. Vulvovaginal candidiasis is estimated to occur in 50-75% of women in their 64 childbearing years (1-3) and recurrence of vulvovaginal candidiasis (RVVC) is common (4).

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Hematogenously disseminated candidiasis is a life-threatening condition of increasing incidence 66 in recent decades (1). Despite the use of antifungal therapy, candidemia is associated with ~40% 67 attributable mortality (5). Compounding these concerns is the alarming rise in emergence of 68 Candida species resistant to antifungal drugs (6).

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C. albicans has multiple putative virulence capabilities including avid adherence to abiotic and 71 host surfaces (7), the capacity to produce tissue-invading filaments (hyphae) (8), and the 72 development of biofilms that promote immune evasion and impede efficacy of antifungal therapy 73 (6). Targeting of these key virulence mechanisms provides opportunities for developing novel 74 therapeutic interventions with minimal effects on the host mycobiome, and reduction in selection 75 pressures that favor drug resistance (9).

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NDV-3 is a vaccine containing a His-tagged recombinant version of the C. albicans Als3 protein 78 (Als3p) N-terminus formulated with alum. Expressed on C. albicans hyphae, Als3p promotes 79 adhesion of the fungus to biotic and abiotic substrates, enables invasion of host cell tissues, and 80 facilitates biofilm formation (10, 11). Deletion of the Als3 gene significantly impairs these 81 virulence traits of C. albicans in vitro (10, 11). Consistent with these themes, NDV-3 decreases 82 disease severity caused by Candida species in mice (12-15). with RVVC in a recent exploratory Phase 1b/2a study. This immune response protected patients 89 <40 years of age with a history of RVVC from recurrence over a twelve-month study period (16).

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Specifically, post-hoc exploratory analysis revealed a statistically significant increase in the 91 percent of the symptoms-free patients at twelve months post vaccination (42% vaccinated vs. 22% 92 placebo; p=0.029) and a doubling time to first symptomatic episode (210 days vaccinated vs. 105 93 days placebo) for the subset for the patients <40 years of age (n=137).

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The objective of the current study is to investigate the role of Als3p antibodies induced by NDV-96 3A as biomarkers of vaccine efficacy by quantitative and qualitative analysis of antibody titers and 97 by evaluating the effect of these antibodies on C. albicans virulence traits.

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All sera used in this study were prepared from blood collected from NDV-3A or placebo recipients 104 in a Phase 1b/2a study in women with RVVC (ClinicalTrials.gov access number, NCT01926028) 105 (16) using previously described methods (13) and were stored at -80 o C until analyzed. Sera were 106 obtained from 64 of 66 NDV3-A recipients and 53 of 60 placebo recipients using appropriate 107 collection, processing and storage practices. In the NDV3-A group, 27 patients had no recurrence 108 of VVC during the 12-month follow-up period and were classified as "non-recurrent" (NR), while 109 37 patients had one or more recurrences of VVC and were designated "recurrent" (R). For the 110 placebo group, only 7 patients were classified as NR, while the rest were classified as R. Because

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Als3p antibody titers in sera were measured using an ELISA assay as previously described (13).

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To inactivate complement, aliquots of patient sera were independently heated at 55 o C for 1 h, 128 added to wells containing C. albicans in Yeast Nitrogen Base (YNB) medium, and incubated for 129 24 h at 37 o C to permit biofilm development. To adsorb anti-C. albicans antibodies, the sera were 130 incubated with C. albicans germ tubes for 1 h with gentle shaking at room temperature. The 131 mixture was centrifuged at 21,000 g prior to using the cell-free supernatant in the biofilm assay.

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The presence and/or extent of removal of anti-Als3 antibodies (total IgG, IgG1 and/or IgG2) was   We analyzed the antibody titers of sera from R or NR patient in an attempt to understand the partial 203 protection elicited by NDV-3A vaccine. We conducted an area under the curve (AUC) analysis of 204 the total IgG titers of sera collected from NDV-3A vaccinated subjects over the 12-month period.

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AUC of total IgG titers from NR patients was significantly higher than those in sera from R patients 206 during the early time points of collection (Day 0-90) ( Figure 1A). Similarly, the geometric mean

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Interestingly, the later recurrence corresponded with the decreased IgG levels beyond 90 days.

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In parallel, the serum antibody profiles were evaluated for anti-Als3 IgG subclasses. The IgG1 218 subclass comprised the predominant isotype in vaccinated NR and R sera (NR vs R IgG1 titer, 219 p=0.9, data not shown). Remarkably, the IgG2 titer in NR sera was much higher than in the R 220 patient sera (Figure 1D), suggesting an isotype-specific enrichment in NR immune responses.

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Titers of IgG3 and IgG4 were not significantly different in sera of R vs. NR patients (data not 222 shown). Also, we did not find any differences among IFN-Ɣ or IL-17 levels between R vs. NR Post-vaccination sera from NR subjects that received NDV-3A significantly reduced adhesion of 230 C. albicans to plastic, compared to pre-vaccination sera from the same patients (Figure 2A). In 231 contrast, post-vaccination sera obtained from R patients that received NDV-3A did not 232 significantly alter C. albicans adhesion relative to pre-vaccination sera ( Figure 2B). As expected, 233 sera from patients who received the placebo did not influence C. albicans adhesion to plastic 234 ( Figure 2C). Sera from the NR-NDV-3A cohort was the only one that significantly reduced 235 adhesion, compared to the other two groups (Figure 2D).

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Post-vaccination sera from NR subjects who received NDV-3A reduced biofilm development as 239 compared to their paired pre-vaccination sera ( Figure 3A). This reduction was not observed when 240 cells were incubated with sera from R subjects who received NDV-3A or sera from placebo 241 recipients (Figure 3B, C). As in the adhesion assay, a significantly greater reduction in biofilm 242 formation was observed in sera from NR versus R patients that received NDV-3A or placebo 243 recipients ( Figure 3D).

I n r e v i e w
Bare-plastic is the gold-standard to measure biofilm formation (20). We wanted to confirm that 245 sera samples that prevented biofilm formation on bare-plastic also prevent biofilm formation on 246 SE used in manufacturing catheters. Thus, antisera of NR patients displaying the highest extent of 247 biofilm inhibition were tested for their ability to impede biofilm formation on SE. These sera 248 reduced C. albicans biofilm formation on the SE substrate to an extent similar to that observed in 249 96 well-plates ( Figure S3 in Supplemental Material).

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Consistent with reduction in biofilm formation, wells containing post-vaccination sera from NR 252 patients also displayed reduced C. albicans adhesion by bright field microscopy, as depicted by 253 reduced density of cells in the bottom of the wells (Figure 4A). This reduction in adhesion to

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Although sera preventing adhesion and biofilm formation were predominantly from the NR group, 261 some R and placebo subject sera also impeded these C. albicans virulence functions. Therefore,

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To determine whether anti-C. albicans antibodies were the active constituent of serum, we 278 incubated the pre-and post-sera with C. albicans germ tubes to adsorb antibodies against Als3.

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This process will adsorb antibodies targeting all surface proteins that are expressed on C. albicans 280 germ tubes including those targeting Als3p. Indeed, ELISA plates coated with rAls3p for both day 281 0 and post-vaccination confirmed that the absorption process significantly reduced the anti-Als3 282 IgG titers in the samples ( Figure S6 in Supplemental Material). Next, these sera were used in C. 283 albicans biofilm assays as detailed above. Adsorption of antibodies from post-vaccination serum 284 reduced their ability to inhibit biofilm formation ( Figure 5B). 287 We questioned whether such functionally active sera influenced interactions of the fungus with 288 neutrophils from unvaccinated human volunteers ex vivo. As displayed in Figure 5C, sera from 289 NR patients enhanced neutrophil killing of fungal cells, compared to sera from R or P patients.

I n r e v i e w
We also determined whether sera from NR patients that demonstrated the highest reduction in C. 291 albicans adhesion to plastic also exhibited the highest level of neutrophil-mediated killing. We 292 found a strong correlation between the ability of sera from NR patients to reduce adhesion and 293 increased neutrophil-mediated killing (p<0.05 and R 2 of 0.66). Further, overall larger numbers of 294 NR patients (13 NR subjects) induced OPK and prevented adhesion than the R group (13 subjects) 295 ( Figure 5D and Figure S7 in Supplemental Material).

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To statistically further validate the sensitivity and specificity of each of the in vitro assays, we 299 performed an area under the ROC curve (cvAUROC) analysis for four assays comparing R to NR 300 patient sera for patients that received NDV-3A. Our analysis of IgG2 predict that NR patients have 301 higher IgG2 antibodies than 75% of the R patients (area under curve 0.75, p value 0.008). Further, 302 our data reveal that an IgG2 antibody titer cutoff of above 1680 (100% sensitivity and >63% In this study, we had the unique opportunity to compare humoral immune responses in patients 313 who derived a measurable health benefit from the NDV-3A vaccine versus those who did not, and 314 versus placebo patients. In addition, sera from R vs. NR subjects could be compared for their 315 ability to impede key virulence functions of C. albicans in vitro. The study goals were to explore 316 potential surrogate biomarkers of protection that might be useful in future studies of this and more 317 serious Candida infections and to gain insight into the potential mechanism(s) contributing to

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Based on our current data, it is possible that the IgG2 subclass antibody component could impair 337 C. albicans interactions with host tissues, and contribute to neutrophil activation leading to 338 enhanced C. albicans killing by NR antisera. However, an important alternative hypothesis is also 339 of interest, that IgG2 and IgG4 antibody are surrogates for non-inflammatory skewing of immune 340 responses, biasing against symptoms of relapse.

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Sera from NR patients that received NDV-3A significantly reduced C. albicans adherence to 343 plastic and SE, and impeded invasion of vaginal epithelial cells by C. albicans hyphae more than 344 antisera obtained from R patients that received NDV-3A or those from placebo. Biofilm formation 345 is a function of the ability of C. albicans to adhere to abiotic surfaces. Thus, it was not unexpected 346 that higher levels of anti-Als3 antibodies, as seen in antisera from NR patients but not R patients, 347 would also significantly reduced Candida biofilm formation. As determined from antibody 348 adsorption and complement inactivation studies, such abrogation of these C. albicans virulence 349 functions was due, at least in part, to anti-Als3p antibodies and did not require complement 350 fixation.

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Our group previously demonstrated that NDV-3 protects mice from VVC by a mechanism that 353 involves priming of both B cell-and T cell-mediated adaptive immune responses (12).

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Specifically, anti-Als3p antibodies enhanced the ex vivo killing of C. albicans by neutrophils 355 primed with . Although in the current study we could not detect a correlation between 356 I n r e v i e w IgG titers of NR and R vaccinated subjects and their corresponding IFN-Ɣ levels, antisera from 357 NR patients significantly enhanced the ability of human neutrophils to kill C. albicans ex vivo as 358 compared to antisera from R patients or patients administered placebo. These results are 359 concordant with the finding that RVVC is a disease in which a discordance of exacerbated 360 neutrophil influx often occurs in the face of inefficiency in clearing the infection (32-34). We 361 postulate that in NR women, the vaccine was able to induce an antibody response that; 1) protected 362 against C. albicans adherence to and invasion of mucocutaneous barriers; 2) reduced the capability 363 of the organism to form biofilm from which persistent infection occurs; 3) induced a coordinated 364 phagocyte response that is more efficacious in clearing the infection; and/or 4) modulated 365 profusive inflammatory responses of the host associated with relapse.

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The statistical robustness of the in vitro assays of IgG2, adhesion, biofilm formation and neutrophil 368 killing which were validated in our ROC analyses, revealed that any of these tests could be used of the total IgG titers over time (0-90 and 90-360 days) for each patient, in the NR and R NDV-3A-510 vaccinated subjects was plotted. In the first 3 months post-vaccination, AUC of NR patients was 511 significantly higher (p=0.046) than that of R patients (A). In months 3 -12, this difference in AUC was 512 also significant (p=0.022) (B). The decrease in AUC of the IgG titers in R patient sera in the later months 513 corresponded with the increase in recurrent episodes of VVC during this period (C). Finally, significantly 514 more number of NR patients displayed IgG2 antibodies in their sera, also mean (geometric) IgG2 antibody 515 titer was higher in NR patients, compared to R patients (p=0.003) (D). Each dot in A, B, and D represents 516 antibody titers in each analyzed serum samples from the indicated individual patients. Each dot in C 517 represent a first relapse in infection as a function of time post vaccination. Data in A, B, and D are presented 518 as geometric mean with 95% confidence interval. 519 520 Figure 2. In vitro assessment of Candida albicans adherence to plastic in presence of patient sera. Post-521 vaccination sera from 27 non-recurrent NR patients significantly (p=0.0005) reduced C. albicans adhesion 522 when compared to their respective pre-vaccination sera (A). There was no difference in the extent of 523 adhesion between pre and post vaccination sera from 37 recurrent (R) patients (p=0.33) (B), or 53 placebo 524 (P) patients (p=0.067) (C). Percent inhibition of C. albicans adhesion to plastic was significantly higher in 525 post-vaccination sera of NR patients versus that of R or P patients (D). Data in D are presented as median 526 + interquartile range. Each dot represents alteration in C. albicans adhesion due to an individual patient 527 sample. 528 529 Figure 3. In vitro assessment of Candida albicans biofilm formation in presence of patient sera. Post-530 vaccination sera from 27 non-recurrent (NR) patients significantly (p=0.003) reduced C. albicans biofilm 531 formation on 96-well microtiter plates, when compared to their respective pre-vaccination sera (A). There 532 was no difference in the extent of biofilm growth between pre-and post-vaccination sera from 37 recurrent 533 (R) patients (p=0.97) (B), or 53 placebo (P) patients (p=0.33) (C). The percent inhibition of C. albicans 534 biofilms to plastic was significantly higher in post-vaccination sera of NR patients versus that from R or P 535 patients (D). Data in D are presented as median + interquartile range. Each dot represents alteration in C. 536 albicans biofilm formation due to an individual patient sample. The open data points in D represent 6 537 placebo and 6 NDV3-A vaccinated patients (3 R and 3 NR) whose sera showed the highest reduction in 538 biofilm formation, and were chosen for the vaginal epithelial cell invasion assay presented in Figure 3B. that post-vaccination sera from NR patients that abrogated biofilm formation displayed short and wavy 544 hyphae, compared to the normal robust hyphae in the biofilms formed in pre-vaccination serum, or the 545 control commercial pooled human serum (A). Six samples from placebo or NDV-3A vaccinated patients 546 (3 R and 3 NR) were selected from Figure 2D (open symbols) for analysis in invasion of vaginal epithelial 547 cells. Post vaccination sera from NR patients inhibited invasion of vaginal epithelial cells two-fold more 548 than R patient or P patient sera (B). **P <0.01 for post-vs. pre-vaccination sera from R patients. + P <0.05 549 for post-vaccination sera vs. pre-vaccination sera from NR and vs. post-vaccination sera from R or placebo 550 patients. Data in B are presented as mean + SD. 551 552 Figure 5. Assessment of the role of antibodies in affecting virulence, and evaluation of OPK of C. 553 albicans germ tubes in the presence of patient sera. Heat treatment of post-vaccination sera from NR 554 patients does not significantly reduce its biofilm-inhibitory activity, compared to paired untreated sera (A).

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Adsorption of antibodies in post-vaccination sera from NR patients with C. albicans germ tubes 556 significantly (p=0.03) abolishes the biofilm inhibitory activity of the sera, when compared to paired 557 I n r e v i e w adsorbed pre-vaccination sera. Only post-vaccination sera from NR patients significantly (p=0.03) enhance 558 OPK and killing of C. albicans germ tubes by human neutrophils, compared to post-vaccination sera from 559 R or P patients (C). A comparison between percent increase in OPK activity and percent reduction in 560 adhesion, in post-vaccination sera from NR and R patients, resulted in significant correlation within the 561 respective subject sera (D). Each open circle represents individual NR sera, which displayed both an overall 562 greater reduction in adhesion and increase in neutrophil killing. Solid circles denote the individual R 563 patients that compared to the NR patients, show a smaller % decrease in adhesion as well as neutrophil 564 killing. Negative values on the graph represent % increase in adhesion or % decrease in neutrophil killing. 565 566 Figure 6. ROC analysis of the in vitro assays. An ROC analyses for four in vitro studies was performed 567 on GraphPad Prism software, where a graph was generated of 100% -(minus) Specificity% versus 568 Sensitivity % for each of the assays: IgG2 titers (A), adhesion (B), biofilm formation (C) and neutrophil 569 killing (D). For each graph, an Area Under the Curve (Area), standard error of the AUC under the ROC 570 curve, as well as the 95% confidence interval is reported. A p-value of <0.05 in each of the ROC curves 571 concludes that the results are significant, and robust. 572 573