β-Glucan Chitosan Particle Provides Cross-Protection Against Multi-Drug- Resistant Candida auris

β-Glucan Chitosan Particle Provides Cross-Protection Against Multi-Drug- Resistant Candida auris | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article β-Glucan Chitosan Particle Provides Cross-Protection Against Multi-Drug- Resistant Candida auris Shakti Singh, Ashley Barbarino, Eman G. Youssef, Kaustav Das Gupta, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7942966/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Candida auris is a multidrug-resistant fungal pathogen that can survive outside the host, easily spread, and colonize the healthcare environment, medical devices, and human skin. C. auris causes serious, life-threatening infections with mortality rates of ~ 60% in immunosuppressed patients. Some isolates of C. auris are resistant to virtually all clinically available antifungal drugs. Therefore, alternative therapeutic approaches are urgently needed. C. auris cell wall contains β-glucan similar to non-pathogenic yeasts such as Saccharomyces cerevisiae . Moreover, cellular proteins are also heavily glycosylated with β-1,3 and β-1,6 glycan structures. Recently, a monoclonal antibody raised against β-glucan was shown to recognize C. albicans hyphal-regulated cell wall protein (Hyr1p), which has a closely related family of proteins in C auris . We tested β-glucan chitosan particles (GCP) as a vaccine candidate and evaluated the humoral and cellular immune responses. Interestingly, GCP induced robust IgG antibody and Th1/Th2/ Th17 immune responses that cross-reacted with purified C. auris cell wall. Anti-GCP antibodies recognized the cell walls of C. auris isolates from four major clades through binding to cell wall glycoproteins. Mice vaccinated with GCP were protected from disseminated C. auris infection compared to placebo mice (40% vs. 0% survival, p = < 0.0001). GCP-vaccinated mice had significantly lower fungal burden than placebo in target organs (kidney, heart, and brain) and fewer fungal abscesses. The mechanism of protection appeared to require antibodies and T-cell activation. These data represent an important step forward toward developing an effective vaccine strategy against multidrug-resistant C. auris . Biological sciences/Drug discovery Biological sciences/Immunology Biological sciences/Microbiology Candida auris candidiasis vaccine β-glucan particle cross-reactive Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Discovered over a decade ago, C. auris has emerged rapidly in over 40 countries worldwide 1 – 3 . C. auris has independently evolved in six distinct clades (I-VI) isolated from different geographic regions: South Asia, East Asia, Africa, South America, Iran, and Bangladesh and Singapore 4 – 8 . One of the intriguing features of the C. auris is its ability to spread and colonize both inanimate objects and human skin within healthcare settings. This feature poses a significant risk to immunosuppressed patients in contaminated healthcare facilities 9 , 10 , where C. auris can infect the patients through attached invasive medical devices and surgical procedures 11 – 14 . In vivo studies show that hematogenously disseminated C. auris infection can result in the invasion of vital organs such as the kidneys, heart, and brain 15 , 16 . Bloodstream infections caused by C. auris are exceptionally challenging to treat and have a staggering mortality rate of approximately 60% 4 . Recently, the widespread use of corticosteroid therapy to manage immunopathological effects related to COVID-19 has led to an increase in COVID-19-associated fungal infections, including C. auris 17 . Multidrug resistance is commonly associated with bacterial rather than fungal infections. However, this perception is changing due to the emergence of drug resistance in fungal pathogens like C. auris 18 , 19 . Almost all C. auris clinical isolates are reported to be azole-resistant. The prevalence of polyene resistance among C. auris is high and estimated to be 90% 4 . In general, resistance to echinocandins among C. auris remains to be low with 7% of all clinical isolates reported to be echinocandin resistant 4 . However, a study from India found out that 37% of the tested clinical isolates had reduced susceptibility to caspofungin acetate (minimum inhibitory concentration [MIC] of ≥ 1 µg/ml) 20 . Regardless of the current frequency of resistance among C. aruis , there is an ever-increasing number of clinical isolates demonstrating resistance to all available antifungal drug classes (pan-resistance), rendering their infections untreatable 18 , 19 . Thus, the U.S. Centers for Disease Control and Prevention (CDC) 20 and the World Health Organization (WHO) have declared C. auris an "urgent threat” to public health 21 . Therefore, clearly alternative strategies for managing C. auris infections are needed. Our research on Candida albicans has yielded significant success in developing vaccine-based approaches to control fungal infections. We have identified two C. albicans cell surface proteins as potential vaccine candidates: Agglutinin-like sequence-3 protein (Als3p), an adhesin/invasin, and Hyphal-regulated protein-1 (Hyr1p), a neutrophil evasion factor 22 – 25 . Vaccines containing recombinant N-terminal regions of Als3p or Hyr1p were shown to protect mice against systemic C. albicans infections 25 – 28 . Furthermore, NDV-3A, a vaccine based on the N-terminus of Als3p adjuvanted with Alum, was shown to be safe and protected women against vulvovaginal candidiasis in a phase II clinical trial 29 , 30 . We identified Agglutinin-like sequence (Als) orthologs and Hyr1/Iff family proteins in C. auris , and both NDV-3A and mAb targeting a shared epitope in C. albicans Hyr1 protein provided protection against lethal murine infections with C. auris 16 , 31 . Glucan and chitosan are major components of the fungal cell wall, sharing common structural features among various fungi 32 , 33 . Monoclonal antibodies raised against glucan have been shown to recognize multiple fungi, including C. albicans and C. auris 34 , 35 . Interestingly, these antibodies can cross-react with cell walls, including highly glycosylated Als3p and Hyr1p 35 . Fungal β-glucans are well-known pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) on the host cells, stimulating an immune response 36 . Due to this reason, Fungal β-glucans has been exploited as novel vaccine adjuvants 37 – 39 , that induce a robust Th-17 response critically needed for protection against fungal infections. Currently, GCP is used as an adjuvant for an experimental Coccidioides posadasii vaccine candidate 38 . Interestingly, while investigating potential adjuvants for the protein subunit vaccine against invasive candidiasis (IC), mice vaccinated with GCP alone (adjuvant-only control) were protected against lethal disseminated C. auris infection. Further investigation showed that the GCP induced strong cross-reactive antibody and T-cell responses against C. auris cell wall, protecting against C. auris infection in mice. We further explored the mechanism of protection of GCP vaccination against C. auris . RESULTS β-glucan particles and glucan-chitosan particles induced robust humoral and cellular immunity. Mice were vaccinated with β-glucan particles (GP) or β-glucan chitosan particles (GCP) through the subcutaneous (S.C.) route on days 0 and 21. Two weeks after the final vaccination, GP, GCP, and C. auris cell wall proteins (CWP)-specific IgG and T cell responses were determined using ELISA and FluroSpot, respectively. For the T cell responses, we determined antigen-specific Th1, Th2, and Th17 immune responses by quantifying IFN-γ, IL-4, and IL17-producing immune cells, respectively. Splenocytes from placebo, GP, or GCP-vaccinated mice were stimulated with GP (for GP-vaccinated mice) or GCP (for GCP-vaccinated mice) or C. auris CWP for 24 hours in a FluroSpot plate coated with specific cytokine capture antibodies (Fig. 1 A). Mean IgG titers ( ± standard error [SE]) and the mean fluoro spot counts ( ± SE) were plotted and compared. GP and GCP induced robust anti-GP and anti-GCP IgG antibody responses (p < 0.05). GP-vaccinated mice showed 1920 ± 320 (mean ± SE) anti-GP and anti-GCP IgG titers, whereas GCP-vaccinated mice had 1440 ± 466 anti-GP and 2400 ± 1012 anti-GCP IgG titers. IgG antibodies induced by GP and GCP vaccination were also quantified against C. auris cell wall proteins (CWP). Strikingly, both GP and GCP-vaccinated mice induced robust cross-reactive anti- C. auris CWP IgG antibodies (3840 ± 640 and 2400 ± 506, respectively) (Fig. 1 B). Furthermore, GP and GCP vaccination induced robust GP and GCP-specific Th1, Th2, and Th17 immune responses (p < 0.05). In contrast, the placebo mice did not induce detectable GP, GCP, or CWP-specific T-cell responses. Specifically, GP and GCP vaccinations induced significant antigen-specific Th1 responses vs. placebo (p < 0.0001). Interestingly, GCP vaccination induced two times more CWP-specific Th1 responses than GP. Also, both GP and GCP-vaccinated mice produced significantly higher IL-4 and Th17 responses, even without ex vivo antigen stimulation, potentially due to non-specific T-cell activation in these mice. The induction of Th2 and Th17 responses from splenocytes of GP and GCP vaccinated mice was further enhanced when the cells were ex vivo stimulated with GP, GCP, or CWP. Finally, GCP vaccination showed consistently higher antigen-specific Th1, Th2, and Th17 responses compared to GP vaccination. T cell responses in GP and GCP mice were biased towards Th2 and Th17 types. (Figs. 1 C and 1 D). Collectively, these data show that both GP and GCP induce robust C. auris CWP-specific immunity. Anti-GCP IgG antibodies recognize C. auris isolates from all major clades, reduce biofilm formation, and enhance macrophage OPK activity. Considering vaccination with GCP induced superior C. auris -CWP-specific immune responses, we further explored the C. auris cross-reactive potential of anti-GCP IgG. We studied the binding capacity of anti-GCP IgG antibodies to the C. auris isolates (CAU-01, CAU-03, CAU-05, CAU-07, CAU-09) representing different clades by incubating the cells with serum from GCP or placebo-vaccinated mice. Bound IgG antibodies were detected by mouse anti-IgG antibodies labeled with Alexa-Fluor 480. This was followed by imaging of the stained yeast cells and quantification of the extent of binding by flow cytometry. The anti-GCP IgG antibodies recognized the cell surface of C. auris , while sera obtained from placebo mice did not bind to the yeast cells (Fig. 2 A). Further, flow cytometry analysis of the stained yeast cells showed that anti-GCP IgG antibodies bound clinical isolates of C. auris from four major clades as represented by a shift in the peaks towards the right side vs. placebo and significantly higher mean fluorescent intensities of yeast with anti-GCP IgG vs. placebo (Fig. 2 B). To determine whether anti-GCP IgG antibodies recognize only glucan structures or also require protein components for binding to the C. auris cell wall, we isolated cell wall fractions from strains CAU-03 and CAU-09. A portion of each preparation was treated with 1 N NaOH at 80°C to remove alkaline-soluble polysaccharides from proteins 40 . These samples were then analyzed by SDS-PAGE followed by Western blotting using anti-GCP or placebo sera. The anti-GCP sera detected multiple bands in both CAU-03 and CAU-09 cell wall preparations, but not in bovine serum albumin (BSA), which served as a non-specific protein control. Notably, this reactivity was completely lost in the NaOH-treated samples, indicating that the anti-GCP antibodies specifically recognize carbohydrate moiety of protein structures (glycans) of the C. auris cell wall. Additionally, the distinct banding patterns observed between CAU-03 and CAU-09 suggest heterogeneity in the target glycoproteins across these two different clades (Fig. 2 C-D). Many cell surface proteins of Candida are involved in cell adhesion and biofilm formation 41 – 44 . Hence, we tested whether the anti-GCP IgG antibody that bound to C. auris also influenced biofilm formation, a trait that is essential for the yeast survival during infection. We incubated C. auris CAU-09 in the presence of anti-GCP or placebo containing murine sera and allowed biofilm formation for 24 h prior to comparing the % biofilm formation of C. auris without any added serum. While sera obtained from placebo mice resulted in ~ 35% inhibition of biofilm formation, sera obtained from mice vaccinated with GCP resulted in ~ 60% inhibition of biofilm formation (i.e. >25% increase in biofilm inhibition) (p < 0.005) (Fig. 2 E). We also determined the opsonophagocytic capability of the anti-GCP sera. C. auris was incubated with sera obtained from mice vaccinated with GCP or placebo for opsonization, followed by incubation with the murine-derived primary macrophages. The anti-GCP sera resulted in > 20-fold increase in macrophage killing when compared to sera obtained from placebo (2% OPK activity for sera obtained from placebo mice vs. 40% OPK activity for sera obtained from GCP vaccinated mice, p < 0.005) (Fig. 2 F). Vaccinating with GCP protects mice against lethal hematogenously disseminated C. auris infection. GP and GCP vaccination induced robust antibody and T-cell responses targeting C. auris cell wall, inhibited C. auris biofilm formation, and increased OPK activity of macrophages in vitro . Thus, we investigated whether vaccination with either GCP or GP can protect mice against lethal hematogenously disseminated C. auris infection. Four-six-week-old ICR CD-1 mice (n ≥ 9) were subcutaneously vaccinated with two doses of GP, GCP, or diluent (Placebo) administered on days 0 and 21. On day 33, mice were immunosuppressed with cyclophosphamide + cortisone acetate, then infected on day 35 with intravenous administration of 5x10 7 cells of C. auris (CAU-09)/mouse. Mice were monitored for moribundity up to 21 days with moribund mice humanely euthanized (Fig. 3 A). Mice vaccinated with GCP were significantly protected against lethal C. auris infection, with 40% survival and ~ 14 days of median survival time (MST) vs. 0% survival and 6 days of MST of placebo mice (Fig. 3 B). The GP vaccination modestly prolonged time to complete moribundity with 100% mortality of vaccinated mice reaching on day 15 vs. 100% mortality of placebo mice on Day 6 (p = 0.0259) (Fig. 3 C). Next, we investigated whether GCP vaccine-mediated protection is specific to C. auris or is effective against lethal hematogenously disseminated C. albicans infection. We infected immunocompetent mice, that have been vaccinated as above, with a lethal inoculum of C. albicans (2 × 10 5 cells/mouse) intravenously after two weeks of the final and second dose of vaccination. In this model, GCP vaccination did not provide any survival benefits against C. albicans infection, suggesting that the GCP vaccine-mediated protection is C. auris- specific ( Figure S1 ). Finally, because GCP-containing adjuvants have innate immune activation properties, we investigated whether the therapeutic administration of GCP benefits acute C. auris infection. We infected immunosuppressed mice with a lethal C. auris inoculum and then vaccinated the mice with one dose (day 1 post-infection) or two doses (day 1 and 8 post-infection) of GCP administered subcutaneously. Although the single or dual therapeutic administration of GCP vaccine did not provide significant protection against C. auris , 30% and 10% of the mice that have been subjected to dual and single vaccination, respectively, survived the infection by day 21, versus 0% for placebo (unvaccinated mice) ( Figure S2 ). Vaccination with GCP reduces C. auris tissue fungal burden and attenuates fungal-mediated damage in target organs. We investigated whether vaccination with GCP reduces C. auris burden in the kidney (primary target organ), and the heart and brain (secondary target organs) (Fig. 4 A). Infected mice experienced a significant loss in body weight after four days of infection with C. auris compared to placebo-treated mice (p < 0.0005), highlighting the severity of the infection. Interestingly, mice that received the GCP vaccine demonstrated a markedly lower degree of weight loss than their placebo counterparts, indicating that GCP vaccination effectively reduced the severity of C. auris infection (Fig. 4 B; p < 0.0005). Furthermore, GCP-vaccinated mice showed a significant reduction in fungal burden, with approximately a 1.0-log, 0.5-log, and 0.3-log decrease in fungal load observed in the kidney, heart, and brain tissues, respectively (p < 0.005) (Fig. 4 C-E ) . This reduction in fungal burden was further supported by histopathological analyses, which revealed that vaccinated mice had fewer and smaller fungal lesions across these target organs. Moreover, the tissue architecture in GCP-vaccinated mice was better preserved, with less evidence of tissue damage compared to the placebo group. Histological sections showed more organized and intact structures in vaccinated animals, while placebo-treated mice displayed disrupted and damaged tissues (Fig. 4 C-E ) . Collectively, these findings indicate that vaccinating mice with GCP not only limits fungal proliferation but also protects against tissue damage, thereby significantly mitigating the impact of C. auris infection. Humoral immunity and T helper cells are essential for GCP vaccine-mediated protection against lethal hematogenous C. auris infection To determine the role of antibodies in GCP-vaccine-mediated protection against lethal C. auris infection, we conducted passive immunization experiments. We collected serum from GCP- or PBS-vaccinated mice, then pooled and passively transferred the anti-GCP or placebo sera intraperitoneally into immunosuppressed ICR CD-1 mice infected with a lethal dose of C. auris. Anti-GCP sera was given 1 hour and a repeat dose 7 days post-infection. Survival of mice was followed for 21 days. Mice that received anti-GCP sera showed a significant 40% survival rate with 18.5 days of MST vs. mice that received placebo sera, which had 0% survival with 8 days MST (P = 0.0022, Fig. 5 A). To evaluate the role of cellular immunity, we vaccinated mice with GCP or placebo on days 0 and 21, depleted their CD4⁺ T cells on day 32. CD4 + T cell depleted mice were then immunosuppressed with cyclophosphamide and cortisone acetate on day 33 prior to infecting then with C. auris on day 35. We compared the survival of these CD4⁺ T cell depleted mice to the survival of normal mice with GCP- or placebo-vaccinated mice without. As expected, GCP vaccination provided significant protection against mortality as earlier (55% survival with > 21 days of MST) compared to placebo group. In contrast, mice lacking CD4⁺ T cells lost the protective benefit of GCP vaccination and showed significantly lower survival than GCP-vaccinated mice with intact CD4⁺ T cells (P = 0.0350; Fig. 5 B). Furthermore, we observed no significant difference in survival between the CD4⁺-depleted GCP-vaccinated mice and the placebo group (P = 0.5299; Fig. 5 B). Collectively, these data highlight the critical role of antibodies and CD4 + T helper cells in GCP-vaccine-mediated protection against lethal hematogenously disseminated C. auris infection. DISCUSSION C. auris exhibits a broad spectrum of drug resistance mechanisms (including mutations in ERG genes involved in ergosterol synthesis and upregulation of efflux pumps) present across all clades, making it challenging to develop new drug variants within existing antifungal classes 5 , 45 – 48 . Thus, novel antifungal interventions with distinct mechanisms of action or alternative immune-based prophylactic or therapeutic strategies appear to be the most effective approach. β-glucan has been shown to induce protective effects against fungal infections, such as Pneumocystis pneumonia 49 and Mycobacterium bovis 50 , through mechanisms involving macrophage activation and antibody cross-reactivity with fungal cell wall carbohydrates. Due to their ability to activate macrophages and drive humoral and Th2/Th17 immune responses, β-glucan is being developed as a vaccine adjuvant, especially for fungal infections 37 – 39 . In our C. albicans Als3p and Hyr1p dual antigen-based vaccine approach against Candida infections, we explored the potential use of GCP and GP particles as adjuvants and unexpectedly discovered a protective effect of GCP particles (without Candida antigens) against C. auris . This unexpected result prompted us to further explore GCP-induced cross-protection against C. auris lethal infection. Our study demonstrates that β-glucan particles (GP) and glucan-chitosan particles (GCP) serve as effective vaccine platforms that induce robust humoral and cellular immunity against C. auris , a multidrug-resistant fungal pathogen of global concern. Notably, GCP vaccination consistently outperformed GP across multiple immunological and protective endpoints, highlighting the combined effect of chitosan and β-glucan. We show that GP and GCP vaccinations elicit strong antigen-specific IgG responses, including cross-reactive antibodies against C. auris cell wall proteins (CWPs). The significantly higher IgG titers observed in GCP-vaccinated mice compared to GP alone underscore the enhanced immunogenicity imparted by the chitosan component. Moreover, these antibodies were capable of recognizing C. auris isolates from all four major clades, indicating broad-spectrum reactivity, a crucial feature for a globally relevant vaccine. In addition to antibody production, both GP and GCP vaccinations elicited strong antigen-specific Th1, Th2, and Th17 responses, which was skewed toward Th2 and Th17 polarization. GCP consistently induced higher cytokine responses, even without ex vivo antigen stimulation. This suggests that GCP formulations not only stimulate adaptive immunity but also potentiate a heightened state of innate immune activation. Importantly, CWP-specific Th1 responses, critical for fungal clearance 51 – 53 , were significantly stronger in GCP-vaccinated mice. This Th1 dominance correlated with enhanced protection in vivo , further underscoring the functional relevance of this response profile. Our findings demonstrate that anti-GCP IgG antibodies robustly recognize C. auris cells across multiple clinical isolates representing four major clades, as evidenced by both confocal microscopy and flow cytometry. Further SDS-PAGE analysis and NaOH hydrolyzation studies revealed that anti-GCP antibodies highly likely recognize glycosylated CWP. Of importance, the anti-GCP antibodies recognized heterogenic glycans among C. auris strains, potentially due to different patterns and densities of the glycosylation. Further investigations are required to identify these cell wall proteins and their expression profile across different C. auris clades, which may have implications for novel diagnostic and therapeutic strategies targeting this emerging pathogen. The functionality of anti-GCP antibodies was evidenced by their ability to inhibit C. auris biofilm formation and enhance opsonophagocytic killing by macrophages, key mechanisms implicated in fungal clearance and attenuation of virulence. This functional immune response translated into robust in vivo protection, Notably, this protection was specific to C. auris , as it did not extend to C. albicans , reinforcing the antigen-specific nature of the immune response. Further mechanistic insights were gained through passive immunization and CD4⁺ T cell depletion studies. Transfer of anti-GCP sera conferred significant protection, highlighting the pivotal role of humoral immunity. Similarly, CD4⁺ T cells were indispensable for GCP-mediated protection, as their depletion completely abrogated the survival benefit. This observation is consistent with prior studies emphasizing the protective role of CD4⁺ T helper cells, particularly the Th17 subset, in immunity against C. auris 54 . Notably, while Th17 responses are associated with fungal clearance, a Th1-skewed CD4⁺ T cell response has been shown to exacerbate C. auris skin infection, likely due to IFN-γ–mediated suppression of IL-17–driven protective mechanisms 55 . This pathogenic Th1 bias contrasts with the immune profile induced by GCP vaccination, which appears to favor Th2/Th17 polarization over Th1, potentially contributing to its protective efficacy. Together, these findings confirm that both arms of adaptive immunity antibody-mediated and CD4⁺ T cell-dependent are essential for vaccine efficacy. Interestingly, while GCP showed modest survival trends when administered therapeutically post-infection, the lack of statistical significance suggests that prophylactic rather than therapeutic use is likely the most effective approach for this formulation. However, future studies should also investigate the potential use of GCP + antifungal in a therapeutic administration. In conclusion, our results suggest that GCP could serve as a standalone vaccine or a potent adjuvant in a broader vaccine strategy against C. auris . GCP can induce durable and functional humoral and cellular immunity with broad clade coverage that translates into significant protection against lethal C. auris infection. MATERIALS AND METHODS Candida culture and strains C. auris strains CAU-01 (Clade II), CAU-03 (Clade III), CAU-05 (Clade IV), CAU-07 (Clade I), and CAU-09 (Clade I) were obtained from the Centers for Disease Control and Prevention (CDC). C. albicans (SC5314) and C. auris strains were cultured overnight in Yeast Extract Peptone Dextrose (YPD) broth in a shaker incubator at 30°C and 200 rpm. The following day, the yeast cells were centrifuged at 4000 RPM for 10 minutes at 4°C, followed by a triple wash with 1X phosphate-buffered saline (PBS). Subsequently, the yeast cells were resuspended in 1X PBS and counted using a hemocytometer. In serum antibody binding experiments, 5 × 10 6 cells/ml C. auris cells were cultured under physiological conditions in RPMI-1640 medium, which was enriched with L-glutamine and 10% fetal bovine serum at 37°C on a shaker for 75 minutes. Vaccine and immunization GP and GCP were developed as adjuvants derived from the cell walls of nonpathogenic yeasts, specifically Saccharomyces cerevisiae for GP and Rhodotorula mucilaginosa for GCP 37,39 . GPs and GCPs are hollow structures of fungal cell walls, predominantly consisting of β-1,3-D-glucans. GP and GCP were prepared as per the methods described earlier 37 . The outbred male ICR CD-1 aged 4-6 weeks mice (n=5 mice/group) were immunized with 200 µg/0.1 ml /mouse of GP or GCP on days 0 and 21. Vaccine diluent (1X Phosphate buffer saline, pH 7.2) was used as placebo. Two weeks after the final vaccination (day 35), mice were euthanized to collect sera and spleens for analysis or infected for the vaccine efficacy determination 31 . IgG antibody titer determination Sera were used to assess the IgG endpoint titers against GP, GCP, or C. auris cell wall proteins (CWP) using ELISA. The plates were coated with 5 µg/ml of GP, GCP, or CWP extracted from C. auris CAU-09 56 in 1X PBS and incubated overnight at 4 o C. The next day, the plates were washed with 1X wash buffer containing 1X PBS and 0.05% Tween-20 three times. The serum samples were serially diluted in 1X wash buffer containing 1% bovine serum albumin (BSA) and added to the wells in duplicates. After 1 hour of incubation at room temperature, the plates were washed, and anti-mouse IgG labeled with HRP was added to each well at a 1:1000 dilution in 1X wash buffer containing 1% BSA. After 1 hour of incubation, the plates were washed as above, and TMB blue substrate solution was added to each well. After 10-30 minutes of incubation, the HRP-substrate reaction was stopped by 1N sulfuric acid, and the absorbance was measured at 450 nm. The endpoint titer was determined by the reciprocal of the highest dilution having OD450 greater than blank + 2*standard deviation 57 . FluroSpot Assay Mouse spleens were individually processed to obtain splenocyte cell suspension, as described earlier 31 . The splenocytes were counted, and cell density was adjusted to 0.3 × 10 6 cells/ 0.1 ml with serum-free culture media (CTL Serum-free media). One day before the experiment, the assay plates were coated with IFN-g, IL-4, or IL-17 capture antibodies and incubated at 4 o C overnight. The next day, the plates were washed, and GP, GCP, and CWP antigen suspension was added at a final 10 µg/ml prepared in serum-free CTL media. No antigens and mitogens cocktail containing PMA/Ionomycin (Cell Stimulation Cocktail, eBioscience™) was used as a negative and positive stimulation controls. Splenocytes were added at 0.3 x 10 6 cells/well and the plates were incubated at 37 o C for 24 hours in stationary condition. After the incubation, the plates were washed with 1X PBS containing 0.05% Tween-20 and developed by CTL Triple Color FluroSpot assay reagents, including detection antibodies with green, red or yellow dyes, as per the kit manual (Catlog# mT3004F, mT02, mT38, mT31, ImmunoSpot, Cleavland, OH). The frequencies of antigen-specific T cells were determined by subtracting the counts from unstimulated wells (no antigen) from the counts in the GP, GCP or CWP-stimulated wells 58 . Flow cytometry C. auris yeast cells were cultured as described above. Yeast cells at 2 x 10 6 cells/ tube were incubated with the 1: 200 diluted sera in 1X PBS with 1% BSA solution and incubated for 1 hour at room temperature. After the incubation, the cells were washed three times with 1x PBS containing 0.05% Tween-20, and then 0.1 ml of Alexa Fluor 488 labeled anti-mouse IgG detection antibodies were added at 1:100 dilution. After 1 hour of incubation at room temperature, the cells were washed three times and resuspended in 300 μl of PBS. All procedures were performed at 4 o C to prevent C. auris replication. The stained cell suspension was imaged under confocal microscopy or transferred to tubes for flow cytometry analysis. Twenty thousand events/samples were acquired using BD FACSymphony (BD Sciences, Franklin Lakes, NJ, USA), and data was analyzed using FlowJo software (Version 10) 16,31 . Extraction of Cell Wall Proteins (CWP) from Candida auris An overnight culture of Candida auris was washed three times with cold PBS. After the final wash, the cell pellet was resuspended in Tris-HCl (ph 7.5) containing protease inhibitor cocktail. Fungal cells were lysed using Lysing Matrix Y (MPBio, Cat: SKU:1169600-CF) in a bead beater (15 times for 40 sec with 1min intervals in between, with cells on ice). Cell wall fraction (pellet) was separated by centrifuging the lysate at 3000 g for 10 min. The cell wall fraction was washed four times with ice cold water followed by an additional four times wash with ice-cold saline solution (1M NaCl, 1 mM PMSF). The cell wall fraction was resuspended in protein extraction buffer (50 mM Tris-HCl, pH 8, 2% SDS, 100 mM EDTA, 10 mM DTT and 40 mM b-mercaptoethanol). Resuspended cell wall fraction was incubated at 100 0 C for 5 min. The fraction was again centrifuged twice at 3000 g for 10 min, while the supernatant containing intracellular fraction was discarded each time. The cell wall fraction was washed three times with distilled water and resuspended in PBS containing 0.1% Tween-20 and protease inhibitor cocktail. The cell wall fraction was again subjected to bead beating (10 times for 20 sec with 30 sec intervals in between, with cells on ice). Resultant cell wall proteins were quantified using BCA assay (ThermoFisher Scientific, Cat: 23235). SDS-PAGE and Immunoblotting Approximately 20 mg protein ( Candida auris CWP, BSA and GCP) were treated with 1N NaOH and heated at 80 0 C for 10 min. The pH was neutralized the resultant hydrolyzed proteins were mixed with Laemmli buffer (ThermoFisher Scientific, Cat: J61337.AD) containing reducing agent. Native proteins and hydrolysed proteins were then separated using SDS-PAGE and the polyacrylamide gel was stained with Coomassie Brilliant Blue. For immunoblotting, the proteins were transferred on to a nitrocellulose membrane and probed with sera from mice injected with GCP and infected with Candida auris . HRP-conjugated mouse IgG (ThermoFisher Scientific, Cat: 31430) was used as the secondary antibody for signal detection using chemiluminescent ECL substrate (ThermoFisher Scientific, Cat: 34577). Biofilm formation assay Biofilms were developed in 96-well polystyrene microtiter plates, as previously described 16,57 . Briefly, C. auris cells were added at 2 x 10 5 cells/50 µl/well (n=6/test group) to 50 μl of 1:200 diluted anti-GCP mouse serum or isotype matched control IgG1 containing wells and incubated at 37°C for 24 hours to allow the adhesion and biofilm formation. The next day, the wells were gently washed twice with 1X PBS, and the extent of biofilm formation was quantified by XTT assay (450 nm). Data are presented as % biofilm reduction ([1-OD450 of wells with anti-GCP sera/OD450 of wells with placebo sera] *100) 16,57,59 . OPK assay. To assess the OPK activity of anti-GCP antibodies, C. auris cells were incubated with 1:200 diluted mouse anti-GCP or placebo sera in a round-bottom 96-well assay plate at 4°C for 1 hour to allow opsonization. Murine macrophages were isolated from the intraperitoneal cavity of naïve mice and adjusted to a concentration of 2.5 × 10⁶ cells/mL. After the initial incubation, the assay plate was transferred to 37°C for 10 minutes, followed by the addition of macrophages at a 1:2.5 yeast to phagocytes ratio to each well. The plate was then incubated for 2 hours at 37°C in a CO₂ incubator to facilitate phagocytosis. Following incubation, 0.1 ml of 1:100 diluted cell mixtures from the above plates were plated on YPD agar for viable C. auris enumeration after overnight incubation at 37 °C. C. auris cells without macrophages and C. auris with macrophages but without antibody served as non-OPK controls. The percent killing of C. auris was calculated using the following formula: {1- [CFUs from wells with (sera + C. auris + macrophages) /average CFU in tubes with ( C. auris + macrophage)]}*100 16,57,59 . Mice infection and treatment For in vivo efficacy evaluation, the vaccinated male ICR CD-1 mice (on days 0 and 21) were immunosuppressed with 200 mg/kg cyclophosphamide intraperitoneal and 250 mg/kg cortisone acetate subcutaneous injections on days -2 relative to infection (day 33). To prevent bacterial superinfection, enrofloxacin (at 50 μg/ml) was added to the drinking water. These mice were infected intravenously with 5 × 10 7 cells of C. auris (CAU-09)/mouse/0.2 mL. For C. albicans infection, the immunocompetent male ICR CD-1 mice (n=10/group) were infected with 2 × 10 5 yeast cells intravenously. Both infections were performed two weeks after the final vaccination (day 35) 31 . For fungal burden determination, mice were infected as above, weighed, and euthanized on day 4 post-infection. The kidneys, hearts, and brains of mice were used for fungal enumeration. The homogenized tissues were 10-fold diluted and quantitatively cultured on YPD plates. Plates were incubated at 37°C for 48 hours before enumerating CFU/gram of tissue. The representative mouse organs were fixed in 10% zinc-buffered formalin, embedded in paraffin, sectioned, and stained with the Periodic Acid Schiff (PAS) stain. Stained tissue sections were imaged on an Olympus microscope 16,31 . For the GCP therapeutic treatment study, the 4-6 weeks old naïve immunosuppressed male ICR CD-1 mice (n=10/group) were infected with C. auris as above and treated with 0.1 mg GCP after 1 and 8 days of infection. For these studies survival followed for 21 days post-infection, served as an endpoint. Adoptive sera transfer studies Naive 4-6 weeks old naive immunosuppressed ICR CD-1 mice (n=10/group) were infected with C. auris as previously described. After 1 hour and 7 days of infection, mice were intraperitoneally injected with 0.1 ml of pooled anti-GCP or placebo sera collected from previously vaccinated mice. Mice were monitored for their survival 21 days post-infection 31 . CD4 depletion studies ICR CD-1 mice (n=10/group) were vaccinated with GCP- or placebo on day 0 and 21, followed by CD4 T cell depletion by administering 0.2 mg/mouse anti-CD4 IgG2b (clone GK1.5, Bio X Cell) or isotype-matched control (LTF-2, Bio X Cell) antibodies on day 32 and 35, and administration of 200 mg/kg cyclophosphamide and 250 mg/kg cortisone acetate on day 33. The mice were infected on day 35 with lethal intravenous C. auris (CAU-09) inoculum as described earlier. The CD4 T cell depletion was verified on day 39 by staining splenocytes and lymph node cells with anti-CD3 APC (BD Pharmigen, Cat #BDB565643) and anti-CD4 Alexa Fluor 700 antibodies (Biolegend, Cat #100536) and analyzing the frequency of CD4+ T cell using flow cytometry (BD FACS Symphony). Infected mice were monitored for their survival 21 days post-infection 31 . Statistical analysis Differences in survival studies were analyzed by the Log-Rank test for overall survival and with Mantel-Cox comparisons for median survival times. All other comparisons were conducted with the Mann-Whitney test. P values <0.05 were considered statistically significant. Declarations Ethical statement All animal procedures were conducted according to the NIH guidelines for animal housing and care and were approved by the IACUC (protocol #31413-02) of The Lundquist Institute at Harbor-UCLA Medical Center. Conflict of Interest ASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose. Competing Interests ASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose. Funding We acknowledge the NIH NIAID 1R01AI141202 grant to ASI, American Heart Association award number 938451, and NIH National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant Number KL2TR001882 to SS. Author Contribution SS conceptualized and designed the study, performed experiments, collected and analyzed the data, and wrote and edited the manuscript. AB performed experiments and recorded the data. EGY performed animal procedures and assisted in fungal burden experiments. K.D.G. contributed to *in vitro* assays. S.N., TK, and SA helped in animal procedures. PU was involved in the design of the experiments. G.O. provided the Glucan and Glucan chitosan particles and edited the manuscript. TK helped in animal procedures. ASI conceptualized, designed, supervised the project, provided funds and edited the manuscript. Data Availability The data are available in the main text or the supplementary materials of this manuscript. References Tracking Candida auris. Centers for Disease Control and Prevention vol. 2018 https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html (2019). Lone, S. A. & Ahmad, A. Candida auris—the growing menace to global health. Mycoses 62 , 620–637 (2019). Colombo, A. L., Júnior, J. N. D. A. & Guinea, J. Emerging multidrug-resistant Candida species. Current Opinion in Infectious Diseases vol. 30 528–538 Preprint at https://doi.org/10.1097/QCO.0000000000000411 (2017). Lockhart, S. R. et al. Simultaneous emergence of multidrug-resistant candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clinical Infectious Diseases 64 , 134–140 (2017). Muñoz, J. F. et al. Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species. Nat Commun 9 , (2018). Chow, N. A. et al. Tracing the evolutionary history and global expansion of candida auris using population genomic analyses. mBio 11 , (2020). Khan, T. et al. Emergence of the novel sixth Candida auris Clade VI in Bangladesh. Microbiol Spectr 12 , (2024). Suphavilai, C. et al. Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study. Lancet Microbe 5 , (2024). Proctor, D. M. et al. Integrated genomic, epidemiologic investigation of Candida auris skin colonization in a skilled nursing facility. Nat Med 27 , 1401–1409 (2021). Centers for Disease Control and Prevention. Screening for Candida auris Colonization | Candida auris | Fungal Diseases | CDC. Centers for Disease Control and Prevention https://www.cdc.gov/fungal/candida-auris/c-auris-screening.html (2019). Rudramurthy, S. M. et al. Candida auris candidaemia in Indian ICUs: analysis of risk factors. Journal of Antimicrobial Chemotherapy 72 , 1794–1801 (2017). de Cássia Orlandi Sardi, J., Silva, D. R., Soares Mendes-Giannini, M. J. & Rosalen, P. L. Candida auris: Epidemiology, risk factors, virulence, resistance, and therapeutic options. Microb Pathog 125 , 116–121 (2018). Sayeed, M. A., Farooqi, J., Jabeen, K. & Mahmood, S. F. Comparison of risk factors and outcomes of Candida auris candidemia with non-Candida auris candidemia: A retrospective study from Pakistan. Med Mycol 58 , 721–729 (2020). Shastri, P. S. et al. Candida auris candidaemia in an intensive care unit – Prospective observational study to evaluate epidemiology, risk factors, and outcome. J Crit Care 57 , 42–48 (2020). Chadwick, C., De Jesus, M., Ginty, F. & Martinez, J. S. Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis. PLoS One 19 , e0293011 (2024). Singh, S. et al. Protective Efficacy of Anti-Hyr1p Monoclonal Antibody against Systemic Candidiasis Due to Multi-Drug-Resistant Candida auris. J Fungi (Basel) 9 , (2023). Hoenigl, M. et al. COVID-19-associated fungal infections. Nature Microbiology 2022 7:8 7 , 1127–1140 (2022). Forsberg, K. et al. Candida auris: The recent emergence of a multidrug-resistant fungal pathogen. Med Mycol 57 , 1–12 (2019). Clancy, C. J. & Nguyen, M. H. Emergence of candida auris: An international call to arms. Clinical Infectious Diseases vol. 64 141–143 Preprint at https://doi.org/10.1093/cid/ciw696 (2017). CDC. 2019 Antibiotic Resistance Threats Report | CDC. Center for Disease Control https://www.cdc.gov/drugresistance/Biggest-Threats.html%0Ahttps://www.cdc.gov/drugresistance//biggest-threats.html (2021). WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action . https://www.who.int/publications/i/item/9789240060241 (2022). Liu, Y. & Filler, S. G. Candida albicans Als3, a multifunctional adhesin and invasin. Eukaryot Cell 10 , 168–173 (2011). Fu, Y. et al. Investigation of the function of Candida albicans Als3 by heterologous expression in Candida glabrata. Infect Immun 81 , 2528–2535 (2013). Phan, Q. T. et al. Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. PLoS Biol 5 , 0543–0557 (2007). Luo, G. et al. Candida albicans Hyr1p confers resistance to neutrophil killing and is a potential vaccine target. Journal of Infectious Diseases 201 , 1718–1728 (2010). Ibrahim, A. S., Spellberg, B. J., Avanesian, V., Fu, Y. & Edwards, J. E. The anti-candida vaccine based on the recombinant N-terminal domain of Als1p is broadly active against disseminated candidiasis. Infect Immun 74 , 3039–3041 (2006). Spellberg, B. J. et al. The anti-Candida albicans vaccine composed of the recombinant N terminus of Als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice. Infect Immun 73 , 6191–6193 (2005). Luo, G., Ibrahim, A. S., French, S. W., Edwards, J. E. & Fu, Y. Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis. PLoS One 6 , (2011). Uppuluri, P. et al. Human Anti-Als3p antibodies are surrogate markers of NDV-3A vaccine efficacy against recurrent vulvovaginal candidiasis. Front Immunol 9 , 1349 (2018). Edwards, J. E. et al. A Fungal Immunotherapeutic Vaccine (NDV-3A) for Treatment of Recurrent Vulvovaginal Candidiasis-A Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial. Clinical Infectious Diseases 66 , 1928–1936 (2018). Singh, S. et al. The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection. PLoS Pathog 15 , e1007460 (2019). Latgé, J. P. The cell wall: A carbohydrate armour for the fungal cell. Molecular Microbiology vol. 66 279–290 Preprint at https://doi.org/10.1111/j.1365-2958.2007.05872.x (2007). Gow, N. A. R. & Lenardon, M. D. Architecture of the dynamic fungal cell wall. Nature Reviews Microbiology 2022 21:4 21 , 248–259 (2022). Di Mambro, T. et al. A new humanized antibody is effective against pathogenic fungi in vitro. Scientific Reports 2021 11:1 11 , 1–17 (2021). Rudkin, F. M. et al. Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis. Nature Communications 2018 9:1 9 , 1–16 (2018). Li, S. S. et al. Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida. Nature Communications 2018 9:1 9 , 1–13 (2018). Huang, H., Ostroff, G. R., Lee, C. K., Specht, C. A. & Levitz, S. M. Characterization and optimization of the glucan particle-based vaccine platform. Clin Vaccine Immunol 20 , 1585–1591 (2013). Hung, C. Y. et al. Glucan-chitin particles enhance Th17 response and improve protective efficacy of a multivalent antigen (rCpa1) against pulmonary Coccidioides posadasii infection. Infect Immun 86 , (2018). Abraham, A., Ostroff, G., Levitz, S. M. & Oyston, P. C. F. A novel vaccine platform using glucan particles for induction of protective responses against Francisella tularensis and other pathogens. Clin Exp Immunol 198 , 143 (2019). Latgé, J. P., Beauvais, A. & Chamilos, G. The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence. Annu Rev Microbiol 71 , 99–116 (2017). Nobile, C. J., Nett, J. E., Andes, D. R. & Mitchell, A. P. Function of Candida albicans adhesin hwp1 in biofilm formation. Eukaryot Cell 5 , 1604–1610 (2006). Zordan, R. & Cormack, B. Adhesins in Opportunistic Fungal Pathogens. Candida and Candidiasis 243-P2 (2014) doi:10.1128/9781555817176.CH16. Ene, I. V. & Bennett, R. J. Hwp1 and related adhesins contribute to both mating and biofilm formation in Candida albicans. Eukaryot Cell 8 , 1909–1913 (2009). Horton, M. V. & Nett, J. E. Candida auris Infection and Biofilm Formation: Going Beyond the Surface. Curr Clin Microbiol Rep 7 , 51–56 (2020). Sarma, S. & Upadhyay, S. Current perspective on emergence, diagnosis and drug resistance in Candida auris. Infect Drug Resist 10 , 155–165 (2017). Chowdhary, A., Voss, A. & Meis, J. F. Multidrug-resistant Candida auris: ‘new kid on the block’ in hospital-associated infections? J Hosp Infect 94 , 209–212 (2016). Kean, R. & Ramage, G. Combined Antifungal Resistance and Biofilm Tolerance: the Global Threat of Candida auris. mSphere 4 , (2019). Shivarathri, R. et al. Comparative Transcriptomics Reveal Possible Mechanisms of Amphotericin B Resistance in Candida auris. Antimicrob Agents Chemother 66 , (2022). Rapaka, R. R., Dai, G., Zheng, M. & Kolls, J. K. CD4 T Cell regulation of antibodies cross-reactive with fungal cell wall-associated carbohydrates after Pneumocystis murina infection. Infect Immun 87 , (2019). Hetland, G., Løvik, M. & Wiker, H. G. Protective Effect of β-Glucan Against Mycobacterium bovis, BCG Infection in BALB/c Mice. Scand J Immunol 47 , 548–553 (1998). Romani, L. Immunity to fungal infections. Nature Reviews Immunology vol. 11 275–288 Preprint at https://doi.org/10.1038/nri2939 (2011). Spellberg, B. et al. Antibody titer threshold predicts anti-candidal vaccine efficacy even though the mechanism of protection is induction of cell-mediated immunity. Journal of Infectious Diseases 197 , 967–971 (2008). de Oliveira, L. L., Coltri, K. C., Barros Cardoso, C. R., Roque-Barreira, M. C. & Panunto-Castelo, A. T Helper 1–Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis. PLoS Negl Trop Dis 2 , e183 (2008). Huang, X. et al. Murine model of colonization with fungal pathogen Candida auris to explore skin tropism, host risk factors and therapeutic strategies. Cell Host Microbe 29 , 210-221.e6 (2021). Das, D., Ganesh, S. M., Mishra, A., Netea, M. G. & Thangamani, S. The Emerging Fungal Pathogen Candida auris Induces IFNγ to Colonize the Skin. PLoS Pathog 21 , e1013114 (2025). Pitarch, A., Nombela, C. & Gil, C. Cell Wall Fractionation for Yeast and Fungal Proteomics. Methods Mol Biol 425 , 217–239 (2008). Singh, S. et al. The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection. PLoS Pathog 15 , (2019). Singh, S. et al. 97. A Cross-kingdom Vaccine Protects against Multiple Healthcare-associated Infections. Open Forum Infect Dis 9 , (2022). Uppuluri, P. et al. Human Anti-Als3p Antibodies Are Surrogate Markers of NDV-3A Vaccine Efficacy Against Recurrent Vulvovaginal Candidiasis. Front Immunol 9 , 1349 (2018). Additional Declarations Competing interest reported. ASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose. Supplementary Files SFig2.tiff SFig1.tiff GCPCAUSupplimentaryFile1.211.04.2025Final.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviewers agreed at journal 08 Dec, 2025 Reviews received at journal 21 Nov, 2025 Reviews received at journal 20 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers invited by journal 12 Nov, 2025 Editor assigned by journal 12 Nov, 2025 Submission checks completed at journal 07 Nov, 2025 First submitted to journal 24 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7942966","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":548906359,"identity":"2db21b33-6be0-4ff6-b56c-7ec9257dc5c3","order_by":0,"name":"Shakti Singh","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Shakti","middleName":"","lastName":"Singh","suffix":""},{"id":548906360,"identity":"e26c0ba0-becb-4324-836e-cdc4166d3c9f","order_by":1,"name":"Ashley Barbarino","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ashley","middleName":"","lastName":"Barbarino","suffix":""},{"id":548906361,"identity":"065bbc94-a8dd-49ed-9bf7-cdf5972579cc","order_by":2,"name":"Eman G. Youssef","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Eman","middleName":"G.","lastName":"Youssef","suffix":""},{"id":548906362,"identity":"5e2e640e-7bf3-44a5-8a85-74936f1bd03b","order_by":3,"name":"Kaustav Das Gupta","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Kaustav","middleName":"Das","lastName":"Gupta","suffix":""},{"id":548906363,"identity":"a3b54132-5144-44d5-9771-05536e2032c6","order_by":4,"name":"Sunna Nabeela","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sunna","middleName":"","lastName":"Nabeela","suffix":""},{"id":548906364,"identity":"e9689b95-0087-4633-923e-ee74b67c8d3d","order_by":5,"name":"Teclegiorgis Gebremariam","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Teclegiorgis","middleName":"","lastName":"Gebremariam","suffix":""},{"id":548906365,"identity":"562441d2-6a94-4aa5-bbfb-43a0796c2e25","order_by":6,"name":"Sondus Alkhazraji","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Sondus","middleName":"","lastName":"Alkhazraji","suffix":""},{"id":548906366,"identity":"259f3057-23e9-4c39-8757-faed84be1e71","order_by":7,"name":"Priya Uppuluri","email":"","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Priya","middleName":"","lastName":"Uppuluri","suffix":""},{"id":548906367,"identity":"ee5c4b9f-8471-42ce-9c7d-b4fc26fe0105","order_by":8,"name":"Garry Ostroff","email":"","orcid":"","institution":"University of Massachusetts Chan Medical School","correspondingAuthor":false,"prefix":"","firstName":"Garry","middleName":"","lastName":"Ostroff","suffix":""},{"id":548906368,"identity":"53f979d2-b9ff-4902-a776-6db7225ad43d","order_by":9,"name":"Ashraf S. Ibrahim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACNgkGBmYGAwY5PiBHAoyI1WLMBtPCQ1APWAsDQ2IbA9QKglr4pHsffi4o2JbeJpF78MbbNgt7ewbmYx+/4HOYzHFj6RkGt3PbJPKSLee2SST2MLAlz5bB65c0BmkesJYcM2neNokEHgYeY2Z8oQDUwvwbqCWdDarFnhgtbCBbEmBaGHuAWhg/4PXLMTZroBbDNp43xpZzzgH9cpgtmRmPDgb52W3Mt3n+3JbnZ88xvPGmrM6evb35MOMPfHpQADhOgFYwE45PFC1AQIIto2AUjIJRMAIAAG6XOf/kHAaDAAAAAElFTkSuQmCC","orcid":"","institution":"The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Ashraf","middleName":"S.","lastName":"Ibrahim","suffix":""}],"badges":[],"createdAt":"2025-10-26 06:55:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7942966/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7942966/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96708324,"identity":"73db45a2-9edc-4256-a0ec-d4584eab2978","added_by":"auto","created_at":"2025-11-25 10:01:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11162476,"visible":true,"origin":"","legend":"","description":"","filename":"GCPCAUmanuscriptDraft1.211.04.2025Final.docx","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/c5a202d4acd395d2378e02d9.docx"},{"id":96616140,"identity":"c3b0e563-2499-444b-b548-01eb21ee7905","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11319,"visible":true,"origin":"","legend":"","description":"","filename":"935e1b06e95045ee9549fcff173ad4cb.json","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/45bf227f2fc092cafb11cf94.json"},{"id":96616136,"identity":"2bcb2cfa-595b-4cb2-ae9a-460210976d1f","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188331,"visible":true,"origin":"","legend":"","description":"","filename":"GCPCAUSupplimentaryFile1.211.04.2025Final.docx","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/3670d66dc1fffacc4e4221b3.docx"},{"id":96616158,"identity":"eaecdc39-3fbd-4500-bc66-c8ce321d698d","added_by":"auto","created_at":"2025-11-24 10:22:31","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":480012,"visible":true,"origin":"","legend":"","description":"","filename":"SFig1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/4e3b5bba1f2afa22727c5266.tiff"},{"id":96616142,"identity":"7e274b71-af32-4845-8eee-80a524e05f92","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":486946,"visible":true,"origin":"","legend":"","description":"","filename":"SFig2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/4146610724dc6ef23495e4e2.tiff"},{"id":96616143,"identity":"1a315781-1ebd-4fbf-b26e-1c12d0b5ee28","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"xml","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137845,"visible":true,"origin":"","legend":"","description":"","filename":"935e1b06e95045ee9549fcff173ad4cb1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/0139b2ac7ab58f40c93838cf.xml"},{"id":96616157,"identity":"bc0942fc-1330-4a43-8263-ec0dda09a6c4","added_by":"auto","created_at":"2025-11-24 10:22:31","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":554246,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/1f44558eff28a5aa4d43a406.jpeg"},{"id":96708211,"identity":"e781e9dc-9062-4d2a-a546-b53a2b61bd6f","added_by":"auto","created_at":"2025-11-25 09:59:16","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":783158,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/044852066eec99355fd5fd12.jpeg"},{"id":96708178,"identity":"024d79de-7aa2-4c68-ad40-caa9172bd687","added_by":"auto","created_at":"2025-11-25 09:58:42","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4670136,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/087d18566c9fbfb517e67e47.jpeg"},{"id":96616151,"identity":"2483edee-5913-428d-afbb-c796fd509662","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4670136,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/a65c569616817879308110ea.jpeg"},{"id":96708975,"identity":"23dcabf2-7bac-4715-95fc-2157c16d011d","added_by":"auto","created_at":"2025-11-25 10:06:46","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106674,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/a31890f81597b430fc1c7482.png"},{"id":96616145,"identity":"1d0d7261-e5f0-400c-8bc2-b29722d4da51","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78868,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/f3c58d95634aff876f38cbc7.png"},{"id":96616148,"identity":"e130edb0-da84-4d41-98c8-40917b9355a9","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99909,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/5b7432d21f081a8988b3422c.png"},{"id":96616146,"identity":"eb2baf85-940d-471d-88c3-adeb6e788d71","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":36709,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/7fd74104896677a20ced4fa9.png"},{"id":96708332,"identity":"0e248d84-384a-4bab-9d27-dc0b93fa0a14","added_by":"auto","created_at":"2025-11-25 10:01:14","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165101,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/33b858bd22c6eb15e3dc4d5a.png"},{"id":96708608,"identity":"7d34724f-dbc5-47c8-ae27-21597ca8e02d","added_by":"auto","created_at":"2025-11-25 10:04:48","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18038,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/8101a1195d0921be7e44630a.png"},{"id":96616152,"identity":"1778f8e9-7973-4e63-b0e6-df65d4e32bfa","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137673,"visible":true,"origin":"","legend":"","description":"","filename":"935e1b06e95045ee9549fcff173ad4cb1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/95747bd3a9b073d13e3d11c9.xml"},{"id":96616155,"identity":"975d2f13-9690-44f0-870b-d6d724663e6c","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155265,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/133069d10cf5dae22c9d9a7d.html"},{"id":96616133,"identity":"b53ef8ad-8467-48d2-871f-27d5586e9506","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":937501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGCP and GP vaccination induces robust humoral and cellular immunity. (A)\u003c/strong\u003e 4-6 weeks old ICR CD-1 mice (n=5/group) were vaccinated with GP, GCP or placebo (PBS, diluent) on day 0 and 21. Two weeks after the booster immunization, blood and spleen were collected to evaluate the immune responses. (\u003cstrong\u003eB)\u003cbr\u003e\n\u003c/strong\u003eIgG antibody titers were determined using ELISA plates coated with vaccine antigens (GP and GCP) and \u003cem\u003eC. auris\u003c/em\u003e cell wall protein (CWP). Serial dilutions of serum samples were added to the ELISA plates to determine the end-point titers. Data are presented as Mean\u0026nbsp;\u003cu\u003e+\u0026nbsp;\u003c/u\u003eSEM titers.\u0026nbsp; (\u003cstrong\u003eC-D)\u003c/strong\u003e\u0026nbsp;Spleen from each mouse was homogenized to get splenocyte single-cell suspension. RBCs were removed by RBC lysis, and 3x10\u003csup\u003e5\u003c/sup\u003e splenocytes were added to each well of the FluroSpot assay plate coated with a mixture of anti-mouse IFN-g (blue), IL-4 (red), and IL-17 (green) capture antibodies. The splenocytes were stimulated with a final 10 ug/ ml of the following antigens: GP, GCP, \u003cem\u003eC. auris\u003c/em\u003e CWP PMA/Ionamycin (assay positive control, not shown). After 24 hours of incubation with antigens, the plates were developed for FluroSpots using detection antibodies, per the manufacturer's instructions. Assay plates were scanned, and spots were counted using CTL analyzer. Mean\u003cu\u003e+\u003c/u\u003eSE or mean antigen-specific spot-forming units (SFU) for each mice group (n=5 mice) are presented in graphs (\u003cstrong\u003eC\u003c/strong\u003e)\u0026nbsp; or heat maps (\u003cstrong\u003eD\u003c/strong\u003e). *P\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, ****p\u0026lt;0.00005.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/87d1b5d71db238c67d40b1fe.png"},{"id":96708254,"identity":"e9b23673-3863-43bb-9f14-044ff8500e20","added_by":"auto","created_at":"2025-11-25 09:59:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":567894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of anti-GCP IgG antibodies to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates from different clades and their functional activity. \u003c/strong\u003eThe binding capacity of anti-GCP IgG antibodies to \u003cem\u003eC. auris\u003c/em\u003e isolates (CAU-01, CAU-03, CAU-05, CAU-07, and CAU-09), representing different clades, was assessed by incubating yeast cells with serum from GCP- or placebo-vaccinated mice. Bound IgG was detected using Alexa Fluor 480-labeled anti-mouse IgG antibodies. This was followed by: (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescence imaging of the stained yeast cells, in which \u003cem\u003eC. auris\u003c/em\u003e cells exhibited green fluorescence, indicated successful binding of anti-GCP IgG to the cell surface. In contrast, cells incubated with placebo sera showed no such fluorescence. (\u003cstrong\u003eB\u003c/strong\u003e) Quantitative analysis by flow cytometry, in which binding of anti-GCP IgG to clinical \u003cem\u003eC. auris\u003c/em\u003e isolates from the four major clades was demonstrated by a rightward shift in fluorescence intensity peaks compared to the placebo group, along with significantly higher mean fluorescence intensity (MFI) values. (\u003cstrong\u003eC-D\u003c/strong\u003e) Cell wall proteins (CWP) extracted from \u003cem\u003eC. auris \u003c/em\u003e(CAU03 (Clade III) and CAU09 (Clade I)), Bovine serum albumin (BSA) or β-glucan chitosan particles (GCP) were either left untreated or were hydrolyzed using NaOH (1N, 80\u003csup\u003e0\u003c/sup\u003eC, 10 mins). Resultant proteins/peptides were separated by SDS-PAGE and either stained with Coomassie Brilliant Blue (\u003cstrong\u003eC\u003c/strong\u003e) or immunoblotted using sera from mice injected with GCP and infected with \u003cem\u003eCandida auris \u003c/em\u003e(\u003cstrong\u003eD\u003c/strong\u003e). \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e are representative immunoblot from two independent experiments.\u0026nbsp; (\u003cstrong\u003eE\u003c/strong\u003e) Anti-biofilm activity of anti-GCP and placebo sera was assayed in 96-well plates using mouse serum from GCP- or placebo-vaccinated mice. Biofilm was quantified by XTT assay after 24 hours of incubation. (\u003cstrong\u003eF\u003c/strong\u003e) Effect of anti-GCP-antibodies on mouse macrophages OPK of \u003cem\u003eC. auris\u003c/em\u003e (CAU-09) (1:2.5 target: effector) for 2 hours. Statistical significance was determined by the Mann-Whitney Test. The p-values \u0026lt;0.05 were considered significant (**p\u0026lt;0.005).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/83a468e31a2619da471221ad.png"},{"id":96616137,"identity":"f74b751e-7896-4eac-be0e-1723da6a7a6f","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":326306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGCP vaccination protects mice against hematogenously disseminated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection.\u003c/strong\u003e 4-6 weeks old male ICR CD-1 mice were vaccinated with 200 µg/mouse\u0026nbsp; of GCP or GP placebo (PBS,\u0026nbsp;diluent) on days 0 and 21. The mice were immunosuppressed with 200 mg/kg cyclophosphamide (I.P.) and 250 mg/kg cortisone acetate (S.C.) on day 33. The mice were infected with a lethal dose (2 × 10\u003csup\u003e7\u003c/sup\u003e cells/mouse) of \u003cem\u003eC. auris\u003c/em\u003e through tail vein injections on day 35 and observed for 21 days for their survival (\u003cstrong\u003eA\u003c/strong\u003e). Survival analysis of GCP vaccinated mice (n=20/group) (\u003cstrong\u003eB\u003c/strong\u003e). Survival analysis of GP vaccinated mice (n=10/group) (\u003cstrong\u003eC\u003c/strong\u003e). Survival data were plotted and analyzed by the Mantel-Cox survival analysis. The p-values \u0026lt;0.05 were considered significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/9eb0f974155665f79264b37e.png"},{"id":96708192,"identity":"a835f3c9-05d1-458c-83d8-f1915ec1d53c","added_by":"auto","created_at":"2025-11-25 09:58:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":702536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGCP vaccination reduces \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. auris\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e burden and improved overall health.\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e4-6 weeks old ICR CD-1 mice (n=9-10/group) were vaccinated with 200 µg/mouse of GP, GCP, or placebo (PBS,\u0026nbsp;diluent) on days 0 and 21 and infected on day 35 (two days after the final vaccination). The mice were immunosuppressed two days before infection with a lethal dose (5 x 10\u003csup\u003e7\u003c/sup\u003e cells/mouse) of \u003cem\u003eC. auris\u003c/em\u003e through tail vein injections. After day 4 post-infection, mice were weighed (\u003cstrong\u003eB\u003c/strong\u003e), and euthanized to determine fungal burden in kidney (\u003cstrong\u003eC)\u003c/strong\u003e, hear(\u003cstrong\u003eD\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003et, and brain (\u003cstrong\u003eE)\u003c/strong\u003e. Fungal burden was represented as CFU/gram of tissue\u0026nbsp;and analyzed using the Mann-Whitney test to determine the difference in fungal burden. P values \u0026lt;0.05 were considered statistically significant (**p\u0026lt;0.005, ***p\u0026lt;0.0005, ****p\u0026lt;0.00005). Organs from each group were also analyzed for histopathology by sectioning and staining with Periodic Acid-Schiff (PAS) and imaged by Olympus bright-field microscopy at 2X and 10X (\u003cstrong\u003eC-E\u003c/strong\u003e, right panels). \u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/18ad4871ff41b4100081c322.png"},{"id":96616159,"identity":"0ee404a2-6331-49ac-8a6e-c599128b09c4","added_by":"auto","created_at":"2025-11-24 10:22:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":123392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune mechanism of vaccine-mediated protection. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e.) Naïve 4-6 weeks ICR CD-1 mice were infected with a lethal dose (target inoculum 5 x 10\u003csup\u003e7\u003c/sup\u003e cells/mouse) of \u003cem\u003eC. auris\u003c/em\u003e through intravenous injection, while given two doses at 1 hour and 7 days post-infection of i.p. injection of anti-GCP IgG antibodies isolated from previously GCP vaccinated mice or placebo control mice sera. \u003cstrong\u003e(B.\u003c/strong\u003e) 4-6 weeks old ICR CD-1 mice were vaccinated with GCP or placebo (PBS,\u0026nbsp;diluent) on days 0 and 21, followed by injection of anti-CD4 or isotype-matched antibodies (0.2 mg / mouse) intraperitoneally on day 32 and 35, and 200 mg/kg cyclophosphamide (I.P.) and 250 mg/kg cortisone acetate (S.C.) on day 33. All mice were infected with 5 x 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eC. auris\u003c/em\u003e yeast cells/mouse intravenously on day 35. Survival data were plotted and analyzed by the Mantel-Cox survival analysis. P values \u0026lt;0.05 were considered statistically significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/600e2fa4e3e61a3d8a0edf95.png"},{"id":96712210,"identity":"f3946de5-4c20-4d47-b646-20d84acf8f03","added_by":"auto","created_at":"2025-11-25 10:15:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3921104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/cea3805a-0203-4a22-8b04-0cc2e7c526ef.pdf"},{"id":96616135,"identity":"68514f67-495b-490b-9f64-e6db9d61b0cc","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"tiff","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":486946,"visible":true,"origin":"","legend":"","description":"","filename":"SFig2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/6b127295deb4ff713a1b1c7b.tiff"},{"id":96616134,"identity":"4c6330a3-0cd0-4e1f-9f24-2c861cf3202f","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":480012,"visible":true,"origin":"","legend":"","description":"","filename":"SFig1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/5c4c7e14308abd6ea6962c1a.tiff"},{"id":96616138,"identity":"af500492-c22a-4e75-9377-61678f60fd90","added_by":"auto","created_at":"2025-11-24 10:22:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":188331,"visible":true,"origin":"","legend":"","description":"","filename":"GCPCAUSupplimentaryFile1.211.04.2025Final.docx","url":"https://assets-eu.researchsquare.com/files/rs-7942966/v1/612fde8ad11a5228abf74ebb.docx"}],"financialInterests":"Competing interest reported. ASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose.","formattedTitle":"β-Glucan Chitosan Particle Provides Cross-Protection Against Multi-Drug- Resistant Candida auris","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDiscovered over a decade ago, \u003cem\u003eC. auris\u003c/em\u003e has emerged rapidly in over 40 countries worldwide\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC. auris\u003c/em\u003e has independently evolved in six distinct clades (I-VI) isolated from different geographic regions: South Asia, East Asia, Africa, South America, Iran, and Bangladesh and Singapore\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. One of the intriguing features of the \u003cem\u003eC. auris\u003c/em\u003e is its ability to spread and colonize both inanimate objects and human skin within healthcare settings. This feature poses a significant risk to immunosuppressed patients in contaminated healthcare facilities\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, where \u003cem\u003eC. auris\u003c/em\u003e can infect the patients through attached invasive medical devices and surgical procedures\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies show that hematogenously disseminated \u003cem\u003eC. auris\u003c/em\u003e infection can result in the invasion of vital organs such as the kidneys, heart, and brain\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Bloodstream infections caused by \u003cem\u003eC. auris\u003c/em\u003e are exceptionally challenging to treat and have a staggering mortality rate of approximately 60%\u003csup\u003e4\u003c/sup\u003e. Recently, the widespread use of corticosteroid therapy to manage immunopathological effects related to COVID-19 has led to an increase in COVID-19-associated fungal infections, including \u003cem\u003eC. auris\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMultidrug resistance is commonly associated with bacterial rather than fungal infections. However, this perception is changing due to the emergence of drug resistance in fungal pathogens like \u003cem\u003eC. auris\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Almost all \u003cem\u003eC. auris\u003c/em\u003e clinical isolates are reported to be azole-resistant. The prevalence of polyene resistance among \u003cem\u003eC. auris\u003c/em\u003e is high and estimated to be 90%\u003csup\u003e4\u003c/sup\u003e. In general, resistance to echinocandins among \u003cem\u003eC. auris\u003c/em\u003e remains to be low with 7% of all clinical isolates reported to be echinocandin resistant\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, a study from India found out that 37% of the tested clinical isolates had reduced susceptibility to caspofungin acetate (minimum inhibitory concentration [MIC] of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;1 \u0026micro;g/ml)\u003csup\u003e20\u003c/sup\u003e. Regardless of the current frequency of resistance among \u003cem\u003eC. aruis\u003c/em\u003e, there is an ever-increasing number of clinical isolates demonstrating resistance to all available antifungal drug classes (pan-resistance), rendering their infections untreatable\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Thus, the U.S. Centers for Disease Control and Prevention (CDC)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and the World Health Organization (WHO) have declared \u003cem\u003eC. auris\u003c/em\u003e an \"urgent threat\u0026rdquo; to public health\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, clearly alternative strategies for managing \u003cem\u003eC. auris\u003c/em\u003e infections are needed.\u003c/p\u003e\u003cp\u003eOur research on \u003cem\u003eCandida albicans\u003c/em\u003e has yielded significant success in developing vaccine-based approaches to control fungal infections. We have identified two \u003cem\u003eC. albicans\u003c/em\u003e cell surface proteins as potential vaccine candidates: Agglutinin-like sequence-3 protein (Als3p), an adhesin/invasin, and Hyphal-regulated protein-1 (Hyr1p), a neutrophil evasion factor\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Vaccines containing recombinant N-terminal regions of Als3p or Hyr1p were shown to protect mice against systemic \u003cem\u003eC. albicans\u003c/em\u003e infections\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Furthermore, NDV-3A, a vaccine based on the N-terminus of Als3p adjuvanted with Alum, was shown to be safe and protected women against vulvovaginal candidiasis in a phase II clinical trial\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We identified Agglutinin-like sequence (Als) orthologs and Hyr1/Iff family proteins in \u003cem\u003eC. auris\u003c/em\u003e, and both NDV-3A and mAb targeting a shared epitope in \u003cem\u003eC. albicans\u003c/em\u003e Hyr1 protein provided protection against lethal murine infections with \u003cem\u003eC. auris\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGlucan and chitosan are major components of the fungal cell wall, sharing common structural features among various fungi\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Monoclonal antibodies raised against glucan have been shown to recognize multiple fungi, including \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. auris\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Interestingly, these antibodies can cross-react with cell walls, including highly glycosylated Als3p and Hyr1p\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Fungal β-glucans are well-known pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) on the host cells, stimulating an immune response\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Due to this reason, Fungal β-glucans has been exploited as novel vaccine adjuvants\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, that induce a robust Th-17 response critically needed for protection against fungal infections. Currently, GCP is used as an adjuvant for an experimental \u003cem\u003eCoccidioides posadasii\u003c/em\u003e vaccine candidate\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInterestingly, while investigating potential adjuvants for the protein subunit vaccine against invasive candidiasis (IC), mice vaccinated with GCP alone (adjuvant-only control) were protected against lethal disseminated \u003cem\u003eC. auris\u003c/em\u003e infection. Further investigation showed that the GCP induced strong cross-reactive antibody and T-cell responses against \u003cem\u003eC. auris\u003c/em\u003e cell wall, protecting against \u003cem\u003eC. auris\u003c/em\u003e infection in mice. We further explored the mechanism of protection of GCP vaccination against \u003cem\u003eC. auris\u003c/em\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eβ-glucan particles and glucan-chitosan particles induced robust humoral and cellular immunity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMice were vaccinated with β-glucan particles (GP) or β-glucan chitosan particles (GCP) through the subcutaneous (S.C.) route on days 0 and 21. Two weeks after the final vaccination, GP, GCP, and \u003cem\u003eC. auris\u003c/em\u003e cell wall proteins (CWP)-specific IgG and T cell responses were determined using ELISA and FluroSpot, respectively. For the T cell responses, we determined antigen-specific Th1, Th2, and Th17 immune responses by quantifying IFN-γ, IL-4, and IL17-producing immune cells, respectively. Splenocytes from placebo, GP, or GCP-vaccinated mice were stimulated with GP (for GP-vaccinated mice) or GCP (for GCP-vaccinated mice) or \u003cem\u003eC. auris\u003c/em\u003e CWP for 24 hours in a FluroSpot plate coated with specific cytokine capture antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Mean IgG titers (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;standard error [SE]) and the mean fluoro spot counts (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SE) were plotted and compared.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGP and GCP induced robust anti-GP and anti-GCP IgG antibody responses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GP-vaccinated mice showed 1920\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;320 (mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SE) anti-GP and anti-GCP IgG titers, whereas GCP-vaccinated mice had 1440\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;466 anti-GP and 2400\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1012 anti-GCP IgG titers. IgG antibodies induced by GP and GCP vaccination were also quantified against \u003cem\u003eC. auris\u003c/em\u003e cell wall proteins (CWP). Strikingly, both GP and GCP-vaccinated mice induced robust cross-reactive anti-\u003cem\u003eC. auris\u003c/em\u003e CWP IgG antibodies (3840\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;640 and 2400\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;506, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFurthermore, GP and GCP vaccination induced robust GP and GCP-specific Th1, Th2, and Th17 immune responses (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the placebo mice did not induce detectable GP, GCP, or CWP-specific T-cell responses. Specifically, GP and GCP vaccinations induced significant antigen-specific Th1 responses vs. placebo (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Interestingly, GCP vaccination induced two times more CWP-specific Th1 responses than GP. Also, both GP and GCP-vaccinated mice produced significantly higher IL-4 and Th17 responses, even without \u003cem\u003eex vivo\u003c/em\u003e antigen stimulation, potentially due to non-specific T-cell activation in these mice. The induction of Th2 and Th17 responses from splenocytes of GP and GCP vaccinated mice was further enhanced when the cells were ex vivo stimulated with GP, GCP, or CWP. Finally, GCP vaccination showed consistently higher antigen-specific Th1, Th2, and Th17 responses compared to GP vaccination. T cell responses in GP and GCP mice were biased towards Th2 and Th17 types. (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Collectively, these data show that both GP and GCP induce robust \u003cem\u003eC. auris\u003c/em\u003e CWP-specific immunity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnti-GCP IgG antibodies recognize\u003c/b\u003e \u003cb\u003eC. auris\u003c/b\u003e \u003cb\u003eisolates from all major clades, reduce biofilm formation, and enhance macrophage OPK activity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsidering vaccination with GCP induced superior \u003cem\u003eC. auris\u003c/em\u003e-CWP-specific immune responses, we further explored the \u003cem\u003eC. auris\u003c/em\u003e cross-reactive potential of anti-GCP IgG. We studied the binding capacity of anti-GCP IgG antibodies to the \u003cem\u003eC. auris\u003c/em\u003e isolates (CAU-01, CAU-03, CAU-05, CAU-07, CAU-09) representing different clades by incubating the cells with serum from GCP or placebo-vaccinated mice. Bound IgG antibodies were detected by mouse anti-IgG antibodies labeled with Alexa-Fluor 480. This was followed by imaging of the stained yeast cells and quantification of the extent of binding by flow cytometry. The anti-GCP IgG antibodies recognized the cell surface of \u003cem\u003eC. auris\u003c/em\u003e, while sera obtained from placebo mice did not bind to the yeast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further, flow cytometry analysis of the stained yeast cells showed that anti-GCP IgG antibodies bound clinical isolates of \u003cem\u003eC. auris\u003c/em\u003e from four major clades as represented by a shift in the peaks towards the right side vs. placebo and significantly higher mean fluorescent intensities of yeast with anti-GCP IgG vs. placebo (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether anti-GCP IgG antibodies recognize only glucan structures or also require protein components for binding to the \u003cem\u003eC. auris\u003c/em\u003e cell wall, we isolated cell wall fractions from strains CAU-03 and CAU-09. A portion of each preparation was treated with 1 N NaOH at 80\u0026deg;C to remove alkaline-soluble polysaccharides from proteins \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These samples were then analyzed by SDS-PAGE followed by Western blotting using anti-GCP or placebo sera. The anti-GCP sera detected multiple bands in both CAU-03 and CAU-09 cell wall preparations, but not in bovine serum albumin (BSA), which served as a non-specific protein control. Notably, this reactivity was completely lost in the NaOH-treated samples, indicating that the anti-GCP antibodies specifically recognize carbohydrate moiety of protein structures (glycans) of the \u003cem\u003eC. auris\u003c/em\u003e cell wall. Additionally, the distinct banding patterns observed between CAU-03 and CAU-09 suggest heterogeneity in the target glycoproteins across these two different clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003eMany cell surface proteins of \u003cem\u003eCandida\u003c/em\u003e are involved in cell adhesion and biofilm formation\u003csup\u003e\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Hence, we tested whether the anti-GCP IgG antibody that bound to \u003cem\u003eC. auris\u003c/em\u003e also influenced biofilm formation, a trait that is essential for the yeast survival during infection. We incubated \u003cem\u003eC. auris\u003c/em\u003e CAU-09 in the presence of anti-GCP or placebo containing murine sera and allowed biofilm formation for 24 h prior to comparing the % biofilm formation of \u003cem\u003eC. auris\u003c/em\u003e without any added serum. While sera obtained from placebo mice resulted in ~\u0026thinsp;35% inhibition of biofilm formation, sera obtained from mice vaccinated with GCP resulted in ~\u0026thinsp;60% inhibition of biofilm formation (i.e. \u0026gt;25% increase in biofilm inhibition) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eWe also determined the opsonophagocytic capability of the anti-GCP sera. \u003cem\u003eC. auris\u003c/em\u003e was incubated with sera obtained from mice vaccinated with GCP or placebo for opsonization, followed by incubation with the murine-derived primary macrophages. The anti-GCP sera resulted in \u0026gt;\u0026thinsp;20-fold increase in macrophage killing when compared to sera obtained from placebo (2% OPK activity for sera obtained from placebo mice vs. 40% OPK activity for sera obtained from GCP vaccinated mice, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eVaccinating with GCP protects mice against lethal hematogenously disseminated\u003c/b\u003e \u003cb\u003eC. auris\u003c/b\u003e \u003cb\u003einfection.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGP and GCP vaccination induced robust antibody and T-cell responses targeting \u003cem\u003eC. auris\u003c/em\u003e cell wall, inhibited \u003cem\u003eC. auris\u003c/em\u003e biofilm formation, and increased OPK activity of macrophages \u003cem\u003ein vitro\u003c/em\u003e. Thus, we investigated whether vaccination with either GCP or GP can protect mice against lethal hematogenously disseminated \u003cem\u003eC. auris\u003c/em\u003e infection. Four-six-week-old ICR CD-1 mice (n\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;9) were subcutaneously vaccinated with two doses of GP, GCP, or diluent (Placebo) administered on days 0 and 21. On day 33, mice were immunosuppressed with cyclophosphamide\u0026thinsp;+\u0026thinsp;cortisone acetate, then infected on day 35 with intravenous administration of 5x10\u003csup\u003e7\u003c/sup\u003e cells of \u003cem\u003eC. auris\u003c/em\u003e (CAU-09)/mouse. Mice were monitored for moribundity up to 21 days with moribund mice humanely euthanized (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMice vaccinated with GCP were significantly protected against lethal \u003cem\u003eC. auris\u003c/em\u003e infection, with 40% survival and ~\u0026thinsp;14 days of median survival time (MST) vs. 0% survival and 6 days of MST of placebo mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The GP vaccination modestly prolonged time to complete moribundity with 100% mortality of vaccinated mice reaching on day 15 vs. 100% mortality of placebo mice on Day 6 (p\u0026thinsp;=\u0026thinsp;0.0259) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eNext, we investigated whether GCP vaccine-mediated protection is specific to \u003cem\u003eC. auris\u003c/em\u003e or is effective against lethal hematogenously disseminated \u003cem\u003eC. albicans\u003c/em\u003e infection. We infected immunocompetent mice, that have been vaccinated as above, with a lethal inoculum of \u003cem\u003eC. albicans\u003c/em\u003e (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mouse) intravenously after two weeks of the final and second dose of vaccination. In this model, GCP vaccination did not provide any survival benefits against \u003cem\u003eC. albicans\u003c/em\u003e infection, suggesting that the GCP vaccine-mediated protection is \u003cem\u003eC. auris-\u003c/em\u003especific (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFinally, because GCP-containing adjuvants have innate immune activation properties, we investigated whether the therapeutic administration of GCP benefits acute \u003cem\u003eC. auris\u003c/em\u003e infection. We infected immunosuppressed mice with a lethal \u003cem\u003eC. auris\u003c/em\u003e inoculum and then vaccinated the mice with one dose (day 1 post-infection) or two doses (day 1 and 8 post-infection) of GCP administered subcutaneously. Although the single or dual therapeutic administration of GCP vaccine did not provide significant protection against \u003cem\u003eC. auris\u003c/em\u003e, 30% and 10% of the mice that have been subjected to dual and single vaccination, respectively, survived the infection by day 21, versus 0% for placebo (unvaccinated mice) (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eVaccination with GCP reduces\u003c/b\u003e \u003cb\u003eC. auris\u003c/b\u003e \u003cb\u003etissue fungal burden and attenuates fungal-mediated damage in target organs.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe investigated whether vaccination with GCP reduces \u003cem\u003eC. auris\u003c/em\u003e burden in the kidney (primary target organ), and the heart and brain (secondary target organs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Infected mice experienced a significant loss in body weight after four days of infection with \u003cem\u003eC. auris\u003c/em\u003e compared to placebo-treated mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005), highlighting the severity of the infection. Interestingly, mice that received the GCP vaccine demonstrated a markedly lower degree of weight loss than their placebo counterparts, indicating that GCP vaccination effectively reduced the severity of \u003cem\u003eC. auris\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005). Furthermore, GCP-vaccinated mice showed a significant reduction in fungal burden, with approximately a 1.0-log, 0.5-log, and 0.3-log decrease in fungal load observed in the kidney, heart, and brain tissues, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E\u003cb\u003e)\u003c/b\u003e. This reduction in fungal burden was further supported by histopathological analyses, which revealed that vaccinated mice had fewer and smaller fungal lesions across these target organs. Moreover, the tissue architecture in GCP-vaccinated mice was better preserved, with less evidence of tissue damage compared to the placebo group. Histological sections showed more organized and intact structures in vaccinated animals, while placebo-treated mice displayed disrupted and damaged tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E\u003cb\u003e)\u003c/b\u003e. Collectively, these findings indicate that vaccinating mice with GCP not only limits fungal proliferation but also protects against tissue damage, thereby significantly mitigating the impact of \u003cem\u003eC. auris\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHumoral immunity and T helper cells are essential for GCP vaccine-mediated protection against lethal hematogenous\u003c/b\u003e \u003cb\u003eC. auris\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the role of antibodies in GCP-vaccine-mediated protection against lethal \u003cem\u003eC. auris\u003c/em\u003e infection, we conducted passive immunization experiments. We collected serum from GCP- or PBS-vaccinated mice, then pooled and passively transferred the anti-GCP or placebo sera intraperitoneally into immunosuppressed ICR CD-1 mice infected with a lethal dose of \u003cem\u003eC. auris.\u003c/em\u003e Anti-GCP sera was given 1 hour and a repeat dose 7 days post-infection. Survival of mice was followed for 21 days. Mice that received anti-GCP sera showed a significant 40% survival rate with 18.5 days of MST vs. mice that received placebo sera, which had 0% survival with 8 days MST (P\u0026thinsp;=\u0026thinsp;0.0022, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the role of cellular immunity, we vaccinated mice with GCP or placebo on days 0 and 21, depleted their CD4⁺ T cells on day 32. CD4\u0026thinsp;+\u0026thinsp;T cell depleted mice were then immunosuppressed with cyclophosphamide and cortisone acetate on day 33 prior to infecting then with \u003cem\u003eC. auris\u003c/em\u003e on day 35. We compared the survival of these CD4⁺ T cell depleted mice to the survival of normal mice with GCP- or placebo-vaccinated mice without. As expected, GCP vaccination provided significant protection against mortality as earlier (55% survival with \u0026gt;\u0026thinsp;21 days of MST) compared to placebo group. In contrast, mice lacking CD4⁺ T cells lost the protective benefit of GCP vaccination and showed significantly lower survival than GCP-vaccinated mice with intact CD4⁺ T cells (P\u0026thinsp;=\u0026thinsp;0.0350; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, we observed no significant difference in survival between the CD4⁺-depleted GCP-vaccinated mice and the placebo group (P\u0026thinsp;=\u0026thinsp;0.5299; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Collectively, these data highlight the critical role of antibodies and CD4\u003csup\u003e+\u003c/sup\u003e T helper cells in GCP-vaccine-mediated protection against lethal hematogenously disseminated \u003cem\u003eC. auris\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cem\u003eC. auris\u003c/em\u003e exhibits a broad spectrum of drug resistance mechanisms (including mutations in ERG genes involved in ergosterol synthesis and upregulation of efflux pumps) present across all clades, making it challenging to develop new drug variants within existing antifungal classes\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Thus, novel antifungal interventions with distinct mechanisms of action or alternative immune-based prophylactic or therapeutic strategies appear to be the most effective approach.\u003c/p\u003e\u003cp\u003eβ-glucan has been shown to induce protective effects against fungal infections, such as \u003cem\u003ePneumocystis pneumonia\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMycobacterium bovis\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, through mechanisms involving macrophage activation and antibody cross-reactivity with fungal cell wall carbohydrates. Due to their ability to activate macrophages and drive humoral and Th2/Th17 immune responses, β-glucan is being developed as a vaccine adjuvant, especially for fungal infections\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In our \u003cem\u003eC. albicans\u003c/em\u003e Als3p and Hyr1p dual antigen-based vaccine approach against \u003cem\u003eCandida\u003c/em\u003e infections, we explored the potential use of GCP and GP particles as adjuvants and unexpectedly discovered a protective effect of GCP particles (without \u003cem\u003eCandida\u003c/em\u003e antigens) against \u003cem\u003eC. auris\u003c/em\u003e. This unexpected result prompted us to further explore GCP-induced cross-protection against \u003cem\u003eC. auris\u003c/em\u003e lethal infection.\u003c/p\u003e\u003cp\u003eOur study demonstrates that β-glucan particles (GP) and glucan-chitosan particles (GCP) serve as effective vaccine platforms that induce robust humoral and cellular immunity against \u003cem\u003eC. auris\u003c/em\u003e, a multidrug-resistant fungal pathogen of global concern. Notably, GCP vaccination consistently outperformed GP across multiple immunological and protective endpoints, highlighting the combined effect of chitosan and β-glucan.\u003c/p\u003e\u003cp\u003eWe show that GP and GCP vaccinations elicit strong antigen-specific IgG responses, including cross-reactive antibodies against \u003cem\u003eC. auris\u003c/em\u003e cell wall proteins (CWPs). The significantly higher IgG titers observed in GCP-vaccinated mice compared to GP alone underscore the enhanced immunogenicity imparted by the chitosan component. Moreover, these antibodies were capable of recognizing \u003cem\u003eC. auris\u003c/em\u003e isolates from all four major clades, indicating broad-spectrum reactivity, a crucial feature for a globally relevant vaccine.\u003c/p\u003e\u003cp\u003eIn addition to antibody production, both GP and GCP vaccinations elicited strong antigen-specific Th1, Th2, and Th17 responses, which was skewed toward Th2 and Th17 polarization. GCP consistently induced higher cytokine responses, even without \u003cem\u003eex vivo\u003c/em\u003e antigen stimulation. This suggests that GCP formulations not only stimulate adaptive immunity but also potentiate a heightened state of innate immune activation. Importantly, CWP-specific Th1 responses, critical for fungal clearance\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, were significantly stronger in GCP-vaccinated mice. This Th1 dominance correlated with enhanced protection \u003cem\u003ein vivo\u003c/em\u003e, further underscoring the functional relevance of this response profile.\u003c/p\u003e\u003cp\u003eOur findings demonstrate that anti-GCP IgG antibodies robustly recognize \u003cem\u003eC. auris\u003c/em\u003e cells across multiple clinical isolates representing four major clades, as evidenced by both confocal microscopy and flow cytometry. Further SDS-PAGE analysis and NaOH hydrolyzation studies revealed that anti-GCP antibodies highly likely recognize glycosylated CWP. Of importance, the anti-GCP antibodies recognized heterogenic glycans among \u003cem\u003eC. auris\u003c/em\u003e strains, potentially due to different patterns and densities of the glycosylation. Further investigations are required to identify these cell wall proteins and their expression profile across different \u003cem\u003eC. auris\u003c/em\u003e clades, which may have implications for novel diagnostic and therapeutic strategies targeting this emerging pathogen.\u003c/p\u003e\u003cp\u003eThe functionality of anti-GCP antibodies was evidenced by their ability to inhibit \u003cem\u003eC. auris\u003c/em\u003e biofilm formation and enhance opsonophagocytic killing by macrophages, key mechanisms implicated in fungal clearance and attenuation of virulence. This functional immune response translated into robust \u003cem\u003ein vivo\u003c/em\u003e protection, Notably, this protection was specific to \u003cem\u003eC. auris\u003c/em\u003e, as it did not extend to \u003cem\u003eC. albicans\u003c/em\u003e, reinforcing the antigen-specific nature of the immune response.\u003c/p\u003e\u003cp\u003eFurther mechanistic insights were gained through passive immunization and CD4⁺ T cell depletion studies. Transfer of anti-GCP sera conferred significant protection, highlighting the pivotal role of humoral immunity. Similarly, CD4⁺ T cells were indispensable for GCP-mediated protection, as their depletion completely abrogated the survival benefit. This observation is consistent with prior studies emphasizing the protective role of CD4⁺ T helper cells, particularly the Th17 subset, in immunity against \u003cem\u003eC. auris\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Notably, while Th17 responses are associated with fungal clearance, a Th1-skewed CD4⁺ T cell response has been shown to exacerbate \u003cem\u003eC. auris\u003c/em\u003e skin infection, likely due to IFN-γ\u0026ndash;mediated suppression of IL-17\u0026ndash;driven protective mechanisms\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. This pathogenic Th1 bias contrasts with the immune profile induced by GCP vaccination, which appears to favor Th2/Th17 polarization over Th1, potentially contributing to its protective efficacy. Together, these findings confirm that both arms of adaptive immunity antibody-mediated and CD4⁺ T cell-dependent are essential for vaccine efficacy. Interestingly, while GCP showed modest survival trends when administered therapeutically post-infection, the lack of statistical significance suggests that prophylactic rather than therapeutic use is likely the most effective approach for this formulation. However, future studies should also investigate the potential use of GCP\u0026thinsp;+\u0026thinsp;antifungal in a therapeutic administration.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn conclusion, our results suggest that GCP could serve as a standalone vaccine or a potent adjuvant in a broader vaccine strategy against \u003cem\u003eC. auris\u003c/em\u003e. GCP can induce durable and functional humoral and cellular immunity with broad clade coverage that translates into significant protection against lethal \u003cem\u003eC. auris\u003c/em\u003e infection.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCandida\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;culture and strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. auris\u003c/em\u003e strains CAU-01 (Clade II), CAU-03 (Clade III), CAU-05 (Clade IV), CAU-07 (Clade I), and CAU-09 (Clade I) were obtained from the Centers for Disease Control and Prevention (CDC). \u003cem\u003eC. albicans\u003c/em\u003e (SC5314) and \u003cem\u003eC. auris\u003c/em\u003e strains were cultured overnight in Yeast Extract Peptone Dextrose (YPD) broth in a shaker incubator at 30\u0026deg;C and 200 rpm. The following day, the yeast cells were centrifuged at 4000 RPM for 10 minutes at 4\u0026deg;C, followed by a triple wash with 1X phosphate-buffered saline (PBS). Subsequently, the yeast cells were resuspended in 1X PBS and counted using a hemocytometer.\u003c/p\u003e\n\u003cp\u003eIn serum antibody binding experiments, 5 \u0026times; 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells/ml \u003cem\u003eC. auris\u003c/em\u003e cells were cultured under physiological conditions in RPMI-1640 medium, which was enriched with L-glutamine and 10% fetal bovine serum at 37\u0026deg;C on a shaker for 75 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVaccine and immunization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGP and GCP were developed as adjuvants derived from the cell walls of nonpathogenic yeasts, specifically \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for GP and \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e for GCP\u003csup\u003e37,39\u003c/sup\u003e. GPs and GCPs are hollow structures of fungal cell walls, predominantly consisting of \u0026beta;-1,3-D-glucans. GP and GCP were prepared as per the methods described earlier\u003csup\u003e37\u003c/sup\u003e. The outbred male ICR CD-1 aged 4-6 weeks mice (n=5 mice/group) were immunized with\u0026nbsp;200 \u0026micro;g/0.1 ml /mouse of GP or GCP on days 0 and 21. Vaccine diluent (1X Phosphate buffer saline, pH 7.2) was used as placebo. Two weeks after the final vaccination (day 35), mice were euthanized to collect sera and spleens for analysis or infected for the vaccine efficacy determination\u003csup\u003e31\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIgG antibody titer determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSera were used to assess the IgG endpoint titers against GP, GCP, or \u003cem\u003eC. auris\u003c/em\u003e cell wall proteins (CWP) using ELISA. The plates were coated with 5\u0026nbsp;\u0026micro;g/ml of GP, GCP, or CWP extracted from \u003cem\u003eC. auris\u0026nbsp;\u003c/em\u003eCAU-09\u003csup\u003e56\u003c/sup\u003e in 1X PBS and incubated overnight at 4\u003csup\u003eo\u003c/sup\u003eC. The next day, the plates were washed with 1X wash buffer containing 1X PBS and 0.05% Tween-20 three times. The serum samples were serially diluted in 1X wash buffer containing 1% bovine serum albumin (BSA) and added to the wells in duplicates. After 1 hour of incubation at room temperature, the plates were washed, and anti-mouse IgG labeled with HRP was added to each well at a 1:1000 dilution in 1X wash buffer containing 1% BSA. \u0026nbsp; After 1 hour of incubation, the plates were washed as above, and TMB blue substrate solution was added to each well. After 10-30 minutes of incubation, the HRP-substrate reaction was stopped by 1N sulfuric acid, and the absorbance was measured at 450 nm. The endpoint titer was determined by the reciprocal of the highest dilution having OD450 greater than blank \u003cu\u003e+\u003c/u\u003e 2*standard deviation\u003csup\u003e57\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluroSpot Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse spleens were individually processed to obtain splenocyte cell suspension, as described earlier\u003csup\u003e31\u003c/sup\u003e. The splenocytes were counted, and cell density was adjusted to 0.3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ 0.1 ml with serum-free culture media (CTL Serum-free media). One day before the experiment, the assay plates were coated with IFN-g, IL-4, or IL-17 capture antibodies and incubated at 4\u003csup\u003eo\u003c/sup\u003eC overnight. The next day, the plates were washed, and GP, GCP, and CWP antigen suspension was added at a final 10 \u0026micro;g/ml prepared in serum-free CTL media. No antigens and mitogens cocktail containing PMA/Ionomycin (Cell Stimulation Cocktail, eBioscience\u0026trade;) was used as a negative and positive stimulation controls. Splenocytes were added at 0.3 x 10\u003csup\u003e6\u003c/sup\u003e cells/well and the plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours in stationary condition. After the incubation, the plates were washed with 1X PBS containing 0.05% Tween-20 and developed by CTL Triple Color FluroSpot assay reagents, including detection antibodies with green, red or yellow dyes, as per the kit manual (Catlog# mT3004F, mT02, mT38, mT31, ImmunoSpot, Cleavland, OH). \u0026nbsp;The frequencies of antigen-specific T cells were determined by subtracting the counts from unstimulated wells (no antigen) from the counts in the GP, GCP or CWP-stimulated wells\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. auris\u003c/em\u003e yeast cells were cultured as described above. Yeast cells at 2 x 10\u003csup\u003e6\u003c/sup\u003e cells/ tube were incubated with the 1: 200 diluted sera in 1X PBS with 1% BSA solution and incubated for 1 hour at room temperature. After the incubation, the cells were washed three times with 1x PBS containing 0.05% Tween-20, and then 0.1 ml of Alexa Fluor 488 labeled anti-mouse IgG detection antibodies were added at 1:100 dilution. After 1 hour of incubation at room temperature, the cells were washed three times and resuspended in 300 \u0026mu;l of PBS. All procedures were performed at 4\u003csup\u003eo\u003c/sup\u003eC to prevent \u003cem\u003eC. auris\u003c/em\u003e replication. The stained cell suspension was imaged under confocal microscopy or transferred to tubes for flow cytometry analysis. Twenty thousand events/samples were acquired using\u0026nbsp;BD FACSymphony (BD Sciences, Franklin Lakes, NJ, USA), and data was analyzed using FlowJo software (Version 10)\u003csup\u003e16,31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of Cell Wall Proteins (CWP) from \u003cem\u003eCandida auris\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn overnight culture of \u003cem\u003eCandida auris\u0026nbsp;\u003c/em\u003ewas washed three times with cold PBS. After the final wash, the cell pellet was resuspended in Tris-HCl (ph 7.5) containing protease inhibitor cocktail. Fungal cells were lysed using Lysing Matrix Y (MPBio, Cat: SKU:1169600-CF) in a bead beater (15 times for 40 sec with 1min intervals in between, with cells on ice). Cell wall fraction (pellet) was separated by centrifuging the lysate at 3000 g for 10 min. The cell wall fraction was washed four times with ice cold water followed by an additional four times wash with ice-cold saline solution (1M NaCl, 1 mM PMSF). The cell wall fraction was resuspended in protein extraction buffer (50 mM Tris-HCl, pH 8, 2% SDS, 100 mM EDTA, 10 mM DTT and 40 mM b-mercaptoethanol). Resuspended cell wall fraction was incubated at 100\u003csup\u003e0\u003c/sup\u003eC for 5 min. The fraction was again centrifuged twice at 3000 g for 10 min, while the supernatant containing intracellular fraction was discarded each time. The cell wall fraction was washed three times with distilled water and resuspended in PBS containing 0.1% Tween-20 and protease inhibitor cocktail. The cell wall fraction was again subjected to bead beating (10 times for 20 sec with 30 sec intervals in between, with cells on ice). Resultant cell wall proteins were quantified using BCA assay (ThermoFisher Scientific, Cat: 23235).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE and Immunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 20 mg protein (\u003cem\u003eCandida auris\u0026nbsp;\u003c/em\u003eCWP, BSA and GCP) were treated with 1N NaOH and heated at 80\u003csup\u003e0\u003c/sup\u003eC for 10 min. The pH was neutralized the resultant hydrolyzed proteins were mixed with Laemmli buffer (ThermoFisher Scientific, Cat: J61337.AD) containing reducing agent. Native proteins and hydrolysed proteins were then separated using SDS-PAGE and the polyacrylamide gel was stained with Coomassie Brilliant Blue. For immunoblotting, the proteins were transferred on to a nitrocellulose membrane and probed with sera from mice injected with GCP and infected with \u003cem\u003eCandida auris\u003c/em\u003e. HRP-conjugated mouse IgG (ThermoFisher Scientific, Cat: 31430) was used as the secondary antibody for signal detection using chemiluminescent ECL substrate (ThermoFisher Scientific, Cat: 34577).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiofilms were developed in 96-well polystyrene microtiter plates, as previously described\u003csup\u003e16,57\u003c/sup\u003e. Briefly, \u003cem\u003eC. auris\u003c/em\u003e cells were added at 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/50 \u0026micro;l/well (n=6/test group) to 50 \u0026mu;l of 1:200 diluted anti-GCP mouse serum or isotype matched control IgG1 containing wells and incubated at 37\u0026deg;C for 24 hours to allow the adhesion and biofilm formation. The next day, the wells were gently washed twice with 1X PBS, and the extent of biofilm formation was quantified by XTT assay (450 nm). Data are presented as % biofilm reduction ([1-OD450 of wells with anti-GCP sera/OD450 of wells with placebo sera] *100)\u003csup\u003e16,57,59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOPK assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the OPK activity of anti-GCP antibodies, \u003cem\u003eC. auris\u003c/em\u003e cells were incubated with 1:200 diluted mouse anti-GCP or placebo sera in a round-bottom 96-well assay plate at 4\u0026deg;C for 1 hour to allow opsonization. Murine macrophages were isolated from the intraperitoneal cavity of na\u0026iuml;ve mice and adjusted to a concentration of 2.5 \u0026times; 10⁶ cells/mL. After the initial incubation, the assay plate was transferred to 37\u0026deg;C for 10 minutes, followed by the addition of macrophages at a 1:2.5 yeast to phagocytes ratio to each well. The plate was then incubated for 2 hours at 37\u0026deg;C in a CO₂\u0026nbsp;incubator to facilitate phagocytosis. Following incubation, 0.1 ml of 1:100 diluted cell mixtures from the above plates were plated on YPD agar for viable \u003cem\u003eC. auris\u003c/em\u003e enumeration after overnight incubation at 37 \u0026deg;C. \u003cem\u003eC. auris\u003c/em\u003e cells without macrophages and \u003cem\u003eC. auris\u003c/em\u003e with macrophages but without antibody served as non-OPK controls. The percent killing of \u003cem\u003eC. auris\u003c/em\u003e was calculated using the following formula: {1- [CFUs from wells with (sera + \u003cem\u003eC. auris\u003c/em\u003e + macrophages) /average CFU in tubes with (\u003cem\u003eC. auris\u003c/em\u003e + macrophage)]}*100\u003csup\u003e16,57,59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice infection and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e efficacy evaluation, the vaccinated male ICR CD-1 mice (on days 0 and 21) were immunosuppressed with 200 mg/kg cyclophosphamide intraperitoneal and 250 mg/kg cortisone acetate subcutaneous injections on days -2 relative to infection (day 33). To prevent bacterial superinfection, enrofloxacin (at 50 \u0026mu;g/ml) was added to the drinking water. These mice were infected intravenously with 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells of \u003cem\u003eC. auris\u003c/em\u003e (CAU-09)/mouse/0.2 mL. For \u003cem\u003eC. albicans\u003c/em\u003e infection, the immunocompetent male ICR CD-1 mice (n=10/group) were infected with 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e yeast cells intravenously. Both infections were performed two weeks after the final vaccination (day 35)\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor fungal burden determination, mice were infected as above, weighed, and euthanized on day 4 post-infection. \u0026nbsp;The kidneys, hearts, and brains of mice were used for fungal enumeration. \u0026nbsp;The homogenized tissues were 10-fold diluted and quantitatively cultured on YPD plates. Plates were incubated at 37\u0026deg;C for 48 hours before enumerating CFU/gram of tissue. The representative mouse organs were fixed in 10% zinc-buffered formalin, embedded in paraffin, sectioned, and stained with the Periodic Acid Schiff (PAS) stain. Stained tissue sections were imaged on an Olympus microscope\u003csup\u003e16,31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor the GCP therapeutic treatment study, the 4-6 weeks old na\u0026iuml;ve immunosuppressed male ICR CD-1 mice (n=10/group) were infected with \u003cem\u003eC. auris\u003c/em\u003e as above and treated with 0.1 mg GCP after 1 and 8 days of infection. For these studies survival followed for 21 days post-infection, served as an endpoint. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdoptive sera transfer studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNaive 4-6 weeks old naive immunosuppressed ICR CD-1 mice (n=10/group) were infected with \u003cem\u003eC. auris\u003c/em\u003e as previously described. After 1 hour and 7 days of infection, mice were intraperitoneally injected with 0.1 ml of pooled anti-GCP or placebo sera collected from previously vaccinated mice. Mice were monitored for their survival 21 days post-infection\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD4 depletion studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICR CD-1 mice (n=10/group) were vaccinated with GCP- or placebo on day 0 and 21, followed by CD4 T cell depletion by administering 0.2 mg/mouse anti-CD4 IgG2b (clone GK1.5, Bio X Cell) or isotype-matched control (LTF-2, Bio X Cell) antibodies on day 32 and 35, and administration of \u0026nbsp;200 mg/kg cyclophosphamide and 250 mg/kg cortisone acetate on day 33. The mice were infected on day 35 with lethal intravenous \u003cem\u003eC. auris\u003c/em\u003e (CAU-09) inoculum as described earlier. The CD4 T cell depletion was verified on day 39 by staining splenocytes and lymph node cells with anti-CD3 APC\u0026nbsp;(BD Pharmigen, Cat #BDB565643) and anti-CD4 Alexa Fluor 700 antibodies (Biolegend, Cat #100536) and analyzing the frequency of CD4+ T cell using flow cytometry (BD FACS Symphony). Infected mice were monitored for their survival 21 days post-infection\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferences in survival studies were analyzed by the Log-Rank test for overall survival and with Mantel-Cox comparisons for median survival times. All other comparisons were conducted with the Mann-Whitney test. P values \u0026lt;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical statement\u003c/h2\u003e\n\u003cp\u003eAll animal procedures were conducted according to the NIH guidelines for animal housing and care and were approved by the IACUC (protocol #31413-02) of The Lundquist Institute at Harbor-UCLA Medical Center.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eASI is the founder of Vitalex Biosciences, which is developing novel antifungal immunotherapies. SS, TG, SA, and PU own shares in Vitalex Biosciences. All other co-authors have no conflict of interest to disclose.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eWe acknowledge the NIH NIAID 1R01AI141202 grant to ASI, American Heart Association award number 938451, and NIH National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant Number KL2TR001882 to SS.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eSS conceptualized and designed the study, performed experiments, collected and analyzed the data, and wrote and edited the manuscript. AB performed experiments and recorded the data. EGY performed animal procedures and assisted in fungal burden experiments. K.D.G. contributed to *in vitro* assays. S.N., TK, and SA helped in animal procedures. PU was involved in the design of the experiments. G.O. provided the Glucan and Glucan chitosan particles and edited the manuscript. TK helped in animal procedures. ASI conceptualized, designed, supervised the project, provided funds and edited the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data are available in the main text or the supplementary materials of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTracking Candida auris. \u003cem\u003eCenters for Disease Control and Prevention\u003c/em\u003e vol. 2018 https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html (2019).\u003c/li\u003e\n\u003cli\u003eLone, S. A. \u0026amp; Ahmad, A. Candida auris\u0026mdash;the growing menace to global health. \u003cem\u003eMycoses\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 620\u0026ndash;637 (2019).\u003c/li\u003e\n\u003cli\u003eColombo, A. L., J\u0026uacute;nior, J. N. D. A. \u0026amp; Guinea, J. Emerging multidrug-resistant Candida species. \u003cem\u003eCurrent Opinion in Infectious Diseases\u003c/em\u003e vol. 30 528\u0026ndash;538 Preprint at https://doi.org/10.1097/QCO.0000000000000411 (2017).\u003c/li\u003e\n\u003cli\u003eLockhart, S. R. \u003cem\u003eet al.\u003c/em\u003e Simultaneous emergence of multidrug-resistant candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. \u003cem\u003eClinical Infectious Diseases\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 134\u0026ndash;140 (2017).\u003c/li\u003e\n\u003cli\u003eMu\u0026ntilde;oz, J. F. \u003cem\u003eet al.\u003c/em\u003e Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eChow, N. A. \u003cem\u003eet al.\u003c/em\u003e Tracing the evolutionary history and global expansion of candida auris using population genomic analyses. \u003cem\u003emBio\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eKhan, T. \u003cem\u003eet al.\u003c/em\u003e Emergence of the novel sixth Candida auris Clade VI in Bangladesh. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eSuphavilai, C. \u003cem\u003eet al.\u003c/em\u003e Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study. \u003cem\u003eLancet Microbe\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, (2024).\u003c/li\u003e\n\u003cli\u003eProctor, D. M. \u003cem\u003eet al.\u003c/em\u003e Integrated genomic, epidemiologic investigation of Candida auris skin colonization in a skilled nursing facility. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1401\u0026ndash;1409 (2021).\u003c/li\u003e\n\u003cli\u003eCenters for Disease Control and Prevention. Screening for Candida auris Colonization | Candida auris | Fungal Diseases | CDC. \u003cem\u003eCenters for Disease Control and Prevention\u003c/em\u003e https://www.cdc.gov/fungal/candida-auris/c-auris-screening.html (2019).\u003c/li\u003e\n\u003cli\u003eRudramurthy, S. M. \u003cem\u003eet al.\u003c/em\u003e Candida auris candidaemia in Indian ICUs: analysis of risk factors. \u003cem\u003eJournal of Antimicrobial Chemotherapy\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 1794\u0026ndash;1801 (2017).\u003c/li\u003e\n\u003cli\u003ede C\u0026aacute;ssia Orlandi Sardi, J., Silva, D. R., Soares Mendes-Giannini, M. J. \u0026amp; Rosalen, P. L. Candida auris: Epidemiology, risk factors, virulence, resistance, and therapeutic options. \u003cem\u003eMicrob Pathog\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 116\u0026ndash;121 (2018).\u003c/li\u003e\n\u003cli\u003eSayeed, M. A., Farooqi, J., Jabeen, K. \u0026amp; Mahmood, S. F. Comparison of risk factors and outcomes of Candida auris candidemia with non-Candida auris candidemia: A retrospective study from Pakistan. \u003cem\u003eMed Mycol\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 721\u0026ndash;729 (2020).\u003c/li\u003e\n\u003cli\u003eShastri, P. S. \u003cem\u003eet al.\u003c/em\u003e Candida auris candidaemia in an intensive care unit \u0026ndash; Prospective observational study to evaluate epidemiology, risk factors, and outcome. \u003cem\u003eJ Crit Care\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 42\u0026ndash;48 (2020).\u003c/li\u003e\n\u003cli\u003eChadwick, C., De Jesus, M., Ginty, F. \u0026amp; Martinez, J. S. Pathobiology of Candida auris infection analyzed by multiplexed imaging and single cell analysis. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, e0293011 (2024).\u003c/li\u003e\n\u003cli\u003eSingh, S. \u003cem\u003eet al.\u003c/em\u003e Protective Efficacy of Anti-Hyr1p Monoclonal Antibody against Systemic Candidiasis Due to Multi-Drug-Resistant Candida auris. \u003cem\u003eJ Fungi (Basel)\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eHoenigl, M. \u003cem\u003eet al.\u003c/em\u003e COVID-19-associated fungal infections. \u003cem\u003eNature Microbiology 2022 7:8\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1127\u0026ndash;1140 (2022).\u003c/li\u003e\n\u003cli\u003eForsberg, K. \u003cem\u003eet al.\u003c/em\u003e Candida auris: The recent emergence of a multidrug-resistant fungal pathogen. \u003cem\u003eMed Mycol\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 1\u0026ndash;12 (2019).\u003c/li\u003e\n\u003cli\u003eClancy, C. J. \u0026amp; Nguyen, M. H. Emergence of candida auris: An international call to arms. \u003cem\u003eClinical Infectious Diseases\u003c/em\u003e vol. 64 141\u0026ndash;143 Preprint at https://doi.org/10.1093/cid/ciw696 (2017).\u003c/li\u003e\n\u003cli\u003eCDC. 2019 Antibiotic Resistance Threats Report | CDC. \u003cem\u003eCenter for Disease Control\u003c/em\u003e https://www.cdc.gov/drugresistance/Biggest-Threats.html%0Ahttps://www.cdc.gov/drugresistance//biggest-threats.html (2021).\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eWHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action\u003c/em\u003e. https://www.who.int/publications/i/item/9789240060241 (2022).\u003c/li\u003e\n\u003cli\u003eLiu, Y. \u0026amp; Filler, S. G. Candida albicans Als3, a multifunctional adhesin and invasin. \u003cem\u003eEukaryot Cell\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 168\u0026ndash;173 (2011).\u003c/li\u003e\n\u003cli\u003eFu, Y. \u003cem\u003eet al.\u003c/em\u003e Investigation of the function of Candida albicans Als3 by heterologous expression in Candida glabrata. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 2528\u0026ndash;2535 (2013).\u003c/li\u003e\n\u003cli\u003ePhan, Q. T. \u003cem\u003eet al.\u003c/em\u003e Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. \u003cem\u003ePLoS Biol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 0543\u0026ndash;0557 (2007).\u003c/li\u003e\n\u003cli\u003eLuo, G. \u003cem\u003eet al.\u003c/em\u003e Candida albicans Hyr1p confers resistance to neutrophil killing and is a potential vaccine target. \u003cem\u003eJournal of Infectious Diseases\u003c/em\u003e \u003cstrong\u003e201\u003c/strong\u003e, 1718\u0026ndash;1728 (2010).\u003c/li\u003e\n\u003cli\u003eIbrahim, A. S., Spellberg, B. J., Avanesian, V., Fu, Y. \u0026amp; Edwards, J. E. The anti-candida vaccine based on the recombinant N-terminal domain of Als1p is broadly active against disseminated candidiasis. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 3039\u0026ndash;3041 (2006).\u003c/li\u003e\n\u003cli\u003eSpellberg, B. J. \u003cem\u003eet al.\u003c/em\u003e The anti-Candida albicans vaccine composed of the recombinant N terminus of Als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 6191\u0026ndash;6193 (2005).\u003c/li\u003e\n\u003cli\u003eLuo, G., Ibrahim, A. S., French, S. W., Edwards, J. E. \u0026amp; Fu, Y. Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, (2011).\u003c/li\u003e\n\u003cli\u003eUppuluri, P. \u003cem\u003eet al.\u003c/em\u003e Human Anti-Als3p antibodies are surrogate markers of NDV-3A vaccine efficacy against recurrent vulvovaginal candidiasis. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1349 (2018).\u003c/li\u003e\n\u003cli\u003eEdwards, J. E. \u003cem\u003eet al.\u003c/em\u003e A Fungal Immunotherapeutic Vaccine (NDV-3A) for Treatment of Recurrent Vulvovaginal Candidiasis-A Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial. \u003cem\u003eClinical Infectious Diseases\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 1928\u0026ndash;1936 (2018).\u003c/li\u003e\n\u003cli\u003eSingh, S. \u003cem\u003eet al.\u003c/em\u003e The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e1007460 (2019).\u003c/li\u003e\n\u003cli\u003eLatg\u0026eacute;, J. P. The cell wall: A carbohydrate armour for the fungal cell. \u003cem\u003eMolecular Microbiology\u003c/em\u003e vol. 66 279\u0026ndash;290 Preprint at https://doi.org/10.1111/j.1365-2958.2007.05872.x (2007).\u003c/li\u003e\n\u003cli\u003eGow, N. A. R. \u0026amp; Lenardon, M. D. Architecture of the dynamic fungal cell wall. \u003cem\u003eNature Reviews Microbiology 2022 21:4\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 248\u0026ndash;259 (2022).\u003c/li\u003e\n\u003cli\u003eDi Mambro, T. \u003cem\u003eet al.\u003c/em\u003e A new humanized antibody is effective against pathogenic fungi in vitro. \u003cem\u003eScientific Reports 2021 11:1\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;17 (2021).\u003c/li\u003e\n\u003cli\u003eRudkin, F. M. \u003cem\u003eet al.\u003c/em\u003e Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis. \u003cem\u003eNature Communications 2018 9:1\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1\u0026ndash;16 (2018).\u003c/li\u003e\n\u003cli\u003eLi, S. S. \u003cem\u003eet al.\u003c/em\u003e Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida. \u003cem\u003eNature Communications 2018 9:1\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1\u0026ndash;13 (2018).\u003c/li\u003e\n\u003cli\u003eHuang, H., Ostroff, G. R., Lee, C. K., Specht, C. A. \u0026amp; Levitz, S. M. Characterization and optimization of the glucan particle-based vaccine platform. \u003cem\u003eClin Vaccine Immunol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1585\u0026ndash;1591 (2013).\u003c/li\u003e\n\u003cli\u003eHung, C. Y. \u003cem\u003eet al.\u003c/em\u003e Glucan-chitin particles enhance Th17 response and improve protective efficacy of a multivalent antigen (rCpa1) against pulmonary Coccidioides posadasii infection. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eAbraham, A., Ostroff, G., Levitz, S. M. \u0026amp; Oyston, P. C. F. A novel vaccine platform using glucan particles for induction of protective responses against Francisella tularensis and other pathogens. \u003cem\u003eClin Exp Immunol\u003c/em\u003e \u003cstrong\u003e198\u003c/strong\u003e, 143 (2019).\u003c/li\u003e\n\u003cli\u003eLatg\u0026eacute;, J. P., Beauvais, A. \u0026amp; Chamilos, G. The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence. \u003cem\u003eAnnu Rev Microbiol\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 99\u0026ndash;116 (2017).\u003c/li\u003e\n\u003cli\u003eNobile, C. J., Nett, J. E., Andes, D. R. \u0026amp; Mitchell, A. P. Function of Candida albicans adhesin hwp1 in biofilm formation. \u003cem\u003eEukaryot Cell\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1604\u0026ndash;1610 (2006).\u003c/li\u003e\n\u003cli\u003eZordan, R. \u0026amp; Cormack, B. Adhesins in Opportunistic Fungal Pathogens. \u003cem\u003eCandida and Candidiasis \u003c/em\u003e243-P2 (2014) doi:10.1128/9781555817176.CH16.\u003c/li\u003e\n\u003cli\u003eEne, I. V. \u0026amp; Bennett, R. J. Hwp1 and related adhesins contribute to both mating and biofilm formation in Candida albicans. \u003cem\u003eEukaryot Cell\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1909\u0026ndash;1913 (2009).\u003c/li\u003e\n\u003cli\u003eHorton, M. V. \u0026amp; Nett, J. E. Candida auris Infection and Biofilm Formation: Going Beyond the Surface. \u003cem\u003eCurr Clin Microbiol Rep\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 51\u0026ndash;56 (2020).\u003c/li\u003e\n\u003cli\u003eSarma, S. \u0026amp; Upadhyay, S. Current perspective on emergence, diagnosis and drug resistance in Candida auris. \u003cem\u003eInfect Drug Resist\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 155\u0026ndash;165 (2017).\u003c/li\u003e\n\u003cli\u003eChowdhary, A., Voss, A. \u0026amp; Meis, J. F. Multidrug-resistant Candida auris: \u0026lsquo;new kid on the block\u0026rsquo; in hospital-associated infections? \u003cem\u003eJ Hosp Infect\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 209\u0026ndash;212 (2016).\u003c/li\u003e\n\u003cli\u003eKean, R. \u0026amp; Ramage, G. Combined Antifungal Resistance and Biofilm Tolerance: the Global Threat of Candida auris. \u003cem\u003emSphere\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eShivarathri, R. \u003cem\u003eet al.\u003c/em\u003e Comparative Transcriptomics Reveal Possible Mechanisms of Amphotericin B Resistance in Candida auris. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eRapaka, R. R., Dai, G., Zheng, M. \u0026amp; Kolls, J. K. CD4 T Cell regulation of antibodies cross-reactive with fungal cell wall-associated carbohydrates after Pneumocystis murina infection. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eHetland, G., L\u0026oslash;vik, M. \u0026amp; Wiker, H. G. Protective Effect of \u0026beta;-Glucan Against Mycobacterium bovis, BCG Infection in BALB/c Mice. \u003cem\u003eScand J Immunol\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 548\u0026ndash;553 (1998).\u003c/li\u003e\n\u003cli\u003eRomani, L. Immunity to fungal infections. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e vol. 11 275\u0026ndash;288 Preprint at https://doi.org/10.1038/nri2939 (2011).\u003c/li\u003e\n\u003cli\u003eSpellberg, B. \u003cem\u003eet al.\u003c/em\u003e Antibody titer threshold predicts anti-candidal vaccine efficacy even though the mechanism of protection is induction of cell-mediated immunity. \u003cem\u003eJournal of Infectious Diseases\u003c/em\u003e \u003cstrong\u003e197\u003c/strong\u003e, 967\u0026ndash;971 (2008).\u003c/li\u003e\n\u003cli\u003ede Oliveira, L. L., Coltri, K. C., Barros Cardoso, C. R., Roque-Barreira, M. C. \u0026amp; Panunto-Castelo, A. T Helper 1\u0026ndash;Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e183 (2008).\u003c/li\u003e\n\u003cli\u003eHuang, X. \u003cem\u003eet al.\u003c/em\u003e Murine model of colonization with fungal pathogen Candida auris to explore skin tropism, host risk factors and therapeutic strategies. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 210-221.e6 (2021).\u003c/li\u003e\n\u003cli\u003eDas, D., Ganesh, S. M., Mishra, A., Netea, M. G. \u0026amp; Thangamani, S. The Emerging Fungal Pathogen Candida auris Induces IFN\u0026gamma; to Colonize the Skin. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, e1013114 (2025).\u003c/li\u003e\n\u003cli\u003ePitarch, A., Nombela, C. \u0026amp; Gil, C. Cell Wall Fractionation for Yeast and Fungal Proteomics. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e425\u003c/strong\u003e, 217\u0026ndash;239 (2008).\u003c/li\u003e\n\u003cli\u003eSingh, S. \u003cem\u003eet al.\u003c/em\u003e The NDV-3A vaccine protects mice from multidrug resistant Candida auris infection. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eSingh, S. \u003cem\u003eet al.\u003c/em\u003e 97. A Cross-kingdom Vaccine Protects against Multiple Healthcare-associated Infections. \u003cem\u003eOpen Forum Infect Dis\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eUppuluri, P. \u003cem\u003eet al.\u003c/em\u003e Human Anti-Als3p Antibodies Are Surrogate Markers of NDV-3A Vaccine Efficacy Against Recurrent Vulvovaginal Candidiasis. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1349 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Candida auris, candidiasis, vaccine, β-glucan particle, cross-reactive","lastPublishedDoi":"10.21203/rs.3.rs-7942966/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7942966/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCandida auris\u003c/em\u003e is a multidrug-resistant fungal pathogen that can survive outside the host, easily spread, and colonize the healthcare environment, medical devices, and human skin. \u003cem\u003eC. auris\u003c/em\u003e causes serious, life-threatening infections with mortality rates of ~\u0026thinsp;60% in immunosuppressed patients. Some isolates of \u003cem\u003eC. auris\u003c/em\u003e are resistant to virtually all clinically available antifungal drugs. Therefore, alternative therapeutic approaches are urgently needed. \u003cem\u003eC. auris\u003c/em\u003e cell wall contains β-glucan similar to non-pathogenic yeasts such as \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Moreover, cellular proteins are also heavily glycosylated with β-1,3 and β-1,6 glycan structures. Recently, a monoclonal antibody raised against β-glucan was shown to recognize \u003cem\u003eC. albicans\u003c/em\u003e hyphal-regulated cell wall protein (Hyr1p), which has a closely related family of proteins in \u003cem\u003eC auris\u003c/em\u003e. We tested β-glucan chitosan particles (GCP) as a vaccine candidate and evaluated the humoral and cellular immune responses. Interestingly, GCP induced robust IgG antibody and Th1/Th2/ Th17 immune responses that cross-reacted with purified \u003cem\u003eC. auris\u003c/em\u003e cell wall. Anti-GCP antibodies recognized the cell walls of \u003cem\u003eC. auris\u003c/em\u003e isolates from four major clades through binding to cell wall glycoproteins. Mice vaccinated with GCP were protected from disseminated \u003cem\u003eC. auris\u003c/em\u003e infection compared to placebo mice (40% vs. 0% survival, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). GCP-vaccinated mice had significantly lower fungal burden than placebo in target organs (kidney, heart, and brain) and fewer fungal abscesses. The mechanism of protection appeared to require antibodies and T-cell activation. These data represent an important step forward toward developing an effective vaccine strategy against multidrug-resistant \u003cem\u003eC. auris\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"β-Glucan Chitosan Particle Provides Cross-Protection Against Multi-Drug- Resistant Candida auris","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 10:22:25","doi":"10.21203/rs.3.rs-7942966/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T16:23:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T14:18:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323117482907303910180166034851107051210","date":"2025-12-08T10:50:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T18:53:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T04:52:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87371301711060045418630928591282622343","date":"2025-11-13T14:03:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283721541905355590380991436592254626797","date":"2025-11-13T00:44:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162588297953592982014636781447296108296","date":"2025-11-12T15:26:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T10:10:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-12T10:07:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T06:38:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-10-24T19:19:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e950ace8-18f8-4bf9-82fd-1802aa742c8e","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58413201,"name":"Biological sciences/Drug discovery"},{"id":58413202,"name":"Biological sciences/Immunology"},{"id":58413203,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-07T09:27:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 10:22:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7942966","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7942966","identity":"rs-7942966","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}