Next-generation Candida albicans vaccine VXV-01 containing recombinant Als3p and Hyr1p antigens for invasive Candida infections

Abstract

Candida species, including Candida albicans and Candida auris, represent a growing public health concern due to their increasing prevalence and resistance to antifungal agents. C. albicans is known for causing both superficial and invasive infections, while C. auris is a newly emerged, multidrug-resistant pathogen responsible for severe hospital outbreaks with a high mortality rate of ~ 60% in bloodstream infections. Vaccine candidates targeting C. albicans hyphal cell wall proteins Als3p and Hyr1p have shown protective efficacy in mice. NDV-3A, an alum-formulated Als3p-based vaccine, protected against recurrent vulvovaginal candidiasis in women. We earlier showed that both Als3p and Hyr1p have orthologs in C. auris, and that the NDV-3A vaccine, alongside an anti-Hyr1p monoclonal antibody, protected mice from multidrug resistant C. auris candidemia. Here, we optimized VXV-01, an Als3p and Hyr1p dual antigen vaccine formulated with the clinical-stage adjuvant CAF01, demonstrating robust immunity and CD4 T cell-dependent protection against lethal C. albicans and C. auris. The VXV-01 vaccine did not antagonize antifungal drug therapy and showed higher overall mouse survival than mice receiving the vaccine or antifungal drug alone, albeit this difference did not reach statistical significance. This study highlights the potential of VXV-01 in providing durable protective immunity against hematogenously disseminated C. albicans and C. auris and mucosal C. albicans infections.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-32488-8.

Introduction

Candida is the most common cause of invasive fungal infections in countries with advanced medical technologies¹. In particular, invasive Candida infections are predominant in the immunocompromised patient population admitted to intensive care units (ICU) and have invasive medical devices (e.g. catheters, ventilators, and breathing tubes)^2–34. Candida spp. (including those caused by the predominant C. albicans) are now statistically tied with Enterococcus as the third most frequent nosocomial bloodstream isolates^35–37, surpassing the incidence of bacteremia caused by Escherichia coli or Klebsiella species. Even with antifungal therapy, disseminated candidiasis has an ~ 40% mortality rate^38,39. The cost associated with hematogenously disseminated candidiasis is estimated to be $2–4 billion/year in the United States^40–42. Candida auris has emerged as a significant threat to global health, having been reported in > 50 countries. C. auris clinical isolates are highly drug-resistant, which makes them challenging to eradicate once an infection has been established leading to increased healthcare costs⁴³ and associated mortality of ~ 60%⁴⁴.

The past few decades provide evidence that the development of resistance to antibiotics is inevitable. Thus, it is essential to develop novel anti-infective approaches that do not rely solely on a drug’s antimicrobial action. In this context, boosting the patient’s immunity through vaccines and immunomodulators is a promising new adjunctive and/or alternative therapeutic concept. Vaccines are the most effective and practical strategy for eliminating certain diseases, as exemplified by smallpox⁴⁵.

C. albicans Agglutinin-like sequence-3 protein (Als3p) contributes to the adhesion and invasion of the fungus to host tissues. Als3p also contributes to the formation of drug resistant biofilm and invade host tissues^46–48. Hyphal-regulated protein (Hyr1p) helps C. albicans to evade killing by neutrophils⁴⁹. We previously demonstrated that antibodies targeting these antigens neutralize C. albicans virulence by preventing adhesion, inhibiting biofilm formation, and enhancing opsonophagocytic activities in phagocytes^49–53 Our recombinant Als3p-based alum-adjuvanted (NDV-3A) vaccine and a recombinant Hyr1p-based vaccine⁴⁹ elicited robust T- and B-cell responses and protected against murine C. albicans hematogenously disseminated candidiasis (including non-albicans spp.) and vulvovaginal candidiasis. In a Phase 1b/2a trial, a single dose of the recombinant Als3p antigen adjuvanted in Alhydrogel^® (NDV-3A) was found to be safe, immunogenic and protected women < 40 years of age from recurrent vulvovaginal candidiasis (RVVC) up to 12 months of follow up. However, this protection was modest with 42% of the vaccinated versus 22% of the placebo patients were symptom-free⁵⁴.

We recently identified three Als3p orthologs on the C. auris cell wall that share remarkable structural and functional similarities with C. albicans Als3p. NDV-3A vaccination significantly protected mice from a lethal hematogenously disseminated C. auris infection⁵⁵. C. auris also has 8 orthologs of Hyr1p, of which two proteins are present in all four clades of C. auris. These Hyr1-orthologs have high predicted structural similarity with Hyr1p and contain a central adhesive domain, N-terminal substrate-binding domain, and GPI-anchor (like Hyr1p)⁵⁶. Anti-Hyr1p monoclonal antibodies raised by us⁵⁷ and others⁵⁸, bind to C. auris surface and protect mice from C. auris disseminated infection. Furthermore, we first reported that Als3p and Hyr1p specific antibodies cross-react and bind to C. auris and prevent its adhesion to plastic and abrogate biofilm formation. These findings were confirmed by subsequent studies emphasizing the role of Als3p and Hyr1p orthologs in C. auris adhesion, aggregation, biofilm formation, and skin colonization^56,59–61.

Our next-generation fungal vaccine development efforts leverage newer adjuvant systems and combine C. albicans recombinant Als3p and Hyr1p antigens to maximize immunogenicity and protection in preclinical models of infection. The primary goal of this endeavor was to achieve a balanced, broader, and robust antibody and T-cell immune responses against each vaccine antigen. We generated over 100 vaccine formulations utilizing different ratios of Als3p and Hyr1p antigens mixing with a variety of adjuvants shown to be safe in clinical trials or approved by regulatory agencies for vaccine development. These adjuvants included alum (Adjuphos™, comparator), Cation Adjuvant formulation-01 (CAF01™, Serum Staten Institute, Denmark)^62,63, BDX100 and BDX300 (obtained from Biodextris, Laval,  QC, Canada)⁶⁴, Glucan Chitosan particles (GCP, developed by University of Massachusetts, MA, USA)⁶⁵, and MF59 (developed by Novartis) (Table S1)^66,67. We embarked on the immunogenicity evaluation of these Als3p/Hyr1p dual antigen vaccine formulations in outbred ICR CD-1 mice and compared the antigen-specific antibody and T- cell immune responses among different adjuvants. Based on immunogenicity screening, several formulations were advanced for protective efficacy testing against invasive Candida spp. infections and vulvovaginal candidiasis by C. albicans in clinically relevant animal models. Our study is the first to report the comparative immunogenicity of various approved, pre-clinical, and clinical-stage adjuvant formulations and evaluation of C. albicans Als3p and Hyr1p dual antigen vaccine against candidiasis due to C. albicans and MDR C. auris in mice.

Results

Manufacturing C. albicans Recombinant Als3p and Hyr1p vaccine antigens

The recombinant N-terminal regions of Als3p (18–450 amino acids) and Hyr1p (154–350 amino acids) were expressed in Saccharomyces cerevisiae FY03-1 and Escherichia coli BL21, respectively. For Als3p manufacturing, we used a previously developed Research Cell Bank (RCB) to establish a Working Research Cell Bank (WRCB) and conducted media optimization processes to enhance the growth and stability of the strain (Fig. 1A). For Hyr1p manufacturing, a vial of RCB stock was expanded into a 10-liter fermentation to determine the suitability of the selected clone for producing Hyr1p and plasmid stability at the end of fermentation (Fig. 1B). Finally, Als3p and Hyr1p were manufactured under non-Good Manufacturing Practice (GMP) conditions in a 10 L bioreactor scale with upstream processes (USP) and downstream processes (DSP) applicable for S. cerevisiae and E. coli cell lines, respectively. The manufacturing and protein purification processes were monitored via preliminary in-process controls and tests shown in Fig. 1 and Table S2. The purity and integrity of both Als3p and Hyr1p were verified by SDS-PAGE analysis (Fig. 1C). The purified antigens produced in Good Laboratory Practice (GLP)-compliant conditions were filled in vials and stored at −80 °C.

Fig. 1.

The manufacturing processes of C. albicans Als3p and Hyr1p antigens. The flowchart of the Upstream (Upper) and Downstream (Right) manufacturing process of recombinant Als3p (A) and Hyr1p (B) protein expressed and purified from S. cerevisiae and E.coli, respectively, is outlined in the flow diagram. SDS-PAGE analysis of recombinant Als3p and Hyr1p antigens (C). Single clean protein bands of each protein indicate high purity and integrity of the antigens. Bovin serum albumin (BSA) was included as a control.

Als3p/Hyr1p dual antigen vaccine immunogenicity is dependent on adjuvant selection

To optimize a dual antigen vaccine, we tested several Als3p/Hyr1p antigen ratios coupled with different adjuvants for their immunogenicity in mice. We used alum, CAF01, BDX100, BDX300, GCP or MF59 adjuvants to formulate the vaccine because of their prior safety profiles in vaccine development (Table S1). Alum formulations were used as a comparator since the previous generation vaccine, NDV-3A, was formulated with Als3p adjuvanted with alum. Vaccines were formulated by mixing an adjuvant with specific ratios of Als3p/Hyr1p antigen. For GCP, the antigens were mixed separately which results into the encapsulation of the Als3p and Hyr1p antigen as confirmed by SDS-PAGE analysis (Figure S1). We tested 0, 10, or 30 µg of Als3p or Hyr1p/dose, yielding 9 antigen ratios by the checkerboard method for each adjuvant (Fig. 2A). Due to the lipopolysaccharide component, BDX100 and BDX300 formulations were administered intranasally, while all other adjuvant formulations were administered subcutaneously on days 0 and 21. We evaluated the antigen-specific antibody and T-cell responses two weeks after the final immunization on day 35 (Fig. 2B). The immunogenicity profiles of BDX100 and CAF01 formulations are presented in the main figures (Figs. 2C-I), as they exhibit superior protective efficacies. Other adjuvant data are presented in the supplementary Figures S2 and S3.

Fig. 2.

Immunogenicity of Als3p/Hyr1p dual antigen vaccine formulated with alum, CAF01, or BDX100 adjuvants. (A). Combination of Als3p/Hyr1p doses by checkerboard method. (B). Schematic of the experimental design for determining vaccine-induced immunogenicity is shown. The ICR CD-1 mice (N = 5/group) vaccinated SC (Alum, CAF01) or IN (BDX100) with different vaccine formulations (Als3p and Hyr1p antigens ratio on the x-axis) on days 0 and 21. Two weeks after the final vaccination, serum antigen-specific IgG titers and T cells were evaluated using ELISA and FluroSpot assay, respectively. Comparison of (C). Anti-Als3p and (D). anti-Hyr1p IgG endpoint titers in alum, CAF01 or BDX100 adjuvant-antigen formulations. Anti-Als3p and anti-Hyr1p IgG endpoint titers in (E). alum, (F). CAF01, (G). BDX100 adjuvant-antigen formulations. (H). Heat map showing the mean frequency (n = 5 mice/cell/formulation) of Als3p or Hyr1p-specific Th1, Th2, and Th17 cells (IFN- γ, IL-4 or IL17 producing cells) in mice vaccinated with alum, CAF01 or BDX100 vaccine formulations. Each row represents data from each vaccine formulation. (I). Bar graph showing Als3p or Hyr1p-specific Th1, Th2, and Th17 cells.

The anti-Als3p and Hyr1p IgG titers depended on the adjuvant used and not on the antigen ratios. For example, GCP vaccine formulations induced the highest anti-Als3p IgG antibody titers, followed by MF59, alum, CAF01, BDX100, and BDX300 (Fig. 2C, S2A). Anti-Hyr1p IgG titers were the highest for the alum formulations, followed by CAF01, BDX100, BDX300, and MF59 adjuvant formulations (Fig. 2D, S2B). Relative antigen dosage in the vaccine formulations did not influence the anti-Als3p or anti-Hyr1p-specific antibody titers in any adjuvant except the CAF01 adjuvant, which showed reduced anti-Als3p IgG titters with higher Hyr1p dose of 30 µg in the vaccine formulation (Fig. 2E-G, S2C). Furthermore, the dual vaccine formulations induced similar or higher anti-Als3p or Hyr1p IgG titers than the mono-antigen vaccines, and this effect was not altered by the antigen dosage. Collectively, these results show that the anti-Als3p- or anti-Hyr1p-specific IgG titers were not negatively influenced by increasing the relative Hyr1p or Als3p antigen dose in the vaccine formulation. Therefore, the Als3p and Hyr1p antigens are not antagonistic to each other (Fig. 2C-D).

Alum coupled with Hyr1p mono-antigen formulation at 10 µg/dose induced higher anti-Hyr1p IgG titers compared to comparative dose mixed with either CAF01 or BDX100 adjuvants. Moreover, CAF01 and BDX100 adjuvant formulations showed an antigen dose-dependent increase in anti-Hyr1p IgG titers, which became similar to alum mono-Hyr1p antigen formulations at 30 µg/dose, indicating more antigen dose dependency with these two adjuvants (Fig. 2D). In general, anti-Hyr1p IgG titers were higher than anti-Als3p IgG titers when alum, MF59, BDX100 or BDX300 were used as adjuvants, and this trend did not change with increasing Als3p antigen relative to Hyr1p antigen dosage (Fig. 2E-G, S3A-B). In contrast, when GCP was used as an adjuvant, anti-Als3p IgG titers where noticeably higher than anti-Hyr1p IgG at all Als3p/Hyr1p antigen ratios (Figure S2C).

We also examined IFN-γ, IL4, and IL17 responses upon in vitro antigen stimulation of splenocytes using FluoroSpot assays to assess representative antigen-specific Th1 (IFN-γ), Th2 (IL4), and Th17(IL17) responses. Alum-based vaccine formulations failed to elicit CD4 + T cell responses, while all other vaccine formulations induced detectable Th1, Th2, and Th17 immune responses (Fig. 2H). Among these, CAF01 formulations generated robust and balanced Th1, Th2, and Th17 immune responses in a vaccine antigen dosage-dependent manner, targeting both Als3p and Hyr1p antigens (Fig. 2H-I). BDX100 formulations predominantly elicited Th1-biased responses that equally targeted Als3p and Hyr1p antigens (Fig. 2H-I), whereas BDX300 induced balanced Th1, Th2, and Th17 responses that were proportionally equivalent but of lower magnitude compared to CAF01 (Figure S3C). A notable difference between BDX100 and BDX300 was the stronger Th1 response observed with BDX100 (Figure S3C). GCP formulations elicited strong Th1- and Th17-skewed immune responses, favoring the Als3p antigen (Figure S3C). In contrast, MF59-based formulations generated weak Th1, Th2, and Th17 responses regardless of the Als3p and Hyr1p antigen dosage (Figure S3C). Overall, CAF01, BDX100, and BDX300 formulations produced proportionally similar Th1, Th2, and Th17 responses specific to Als3p and Hyr1p, with the magnitude of responses generally dependent on antigen dosage (Fig. 2H-I, S3C).

The CAF01 Als3p/Hyr1p dual antigen vaccine is effective against invasive candidiasis

To test the efficacy of vaccine, we vaccinated mice with dual Als3p/Hyr1p antigens at 10 µg/30 µg–30 µg/30 µg ratio formulated with alum, CAF01, GCP, or MF59 adjuvant on days 0 and 21 subcutaneously. The same antigen ratios were used with BDX100 adjuvant and administered intranasally on days 0 and 21. Two weeks following the second vaccination mice were intravenously infected with C. albicans or C. auris. The Candida strains used for infection were chosen based on earlier studies that determined the virulence of these strains in this model. Specifically, C. auris CAU-03 (Clade III) and CAU-09 (Clade I) isolates were found to be the most virulent strains (100% mortality) compared to other strains representing Clade II (CAU-01, 0% mortality) and clade IV (CAU-05, 66% mortality) (Figure S4). Thus, we used C. auris CAU-09 isolate and C. albicans SC5314, a widely used C. albicans strain in virulence and genetic studies^68–70 for efficacy testing of Als3p/Hyr1p dual antigen vaccine. Infected mice were monitored for protective efficacy by survival at day 21 as the primary endpoint.

Among all the adjuvant formulations tested, only CAF01 and BDX100 vaccine formulations showed significant protection against both hematogenously disseminated C. albicans (Figure S5A) and C. auris (Figure S5B) infections with 36 to 42% survival (12–14 days of median survival time [MST]) vs. the placebo group showing 0% survival (8–11 days of MST).

Based on the immunogenicity profile and preliminary efficacy studies, we prioritized CAF01 and BDX100 Als3p/Hyr1p dual antigen vaccine formulations to further optimize and evaluate their protective efficacy against C. albicans and C. auris infections. CAF01 or BDX100 vaccine formulations with Als3p/Hyr1p dual antigens at 10 µg/10 µg, 30 µg/10 µg, 10 µg/30 µg–30 µg/30 µg, were administered on days 0 and 21 (two vaccinations or 1 booster) or days 0, 21, and 35 (three vaccinations or 2 booster) as described above (Fig. 3A).

Fig. 3.

Survival efficacy of the Als3p/Hyr1p dual antigen vaccine against invasive candidiasis due to C. albicans and C. auris. (A). Experimental design for the in vivo survival efficacy. Mice were vaccinated two or three times with Als3p/Hyr1p dual antigens formulated with CAF01 or BDX100 adjuvant. Two weeks after the final vaccination, mice were infected intravenously with 2 × 10⁵ cells/mouse of C. albicans or 5 × 10⁷ cells/mouse of C. auris. For C. auris infection, the mice were immunosuppressed on day − 2 relative to infection. (B). Vaccine-induced survival efficacy against C. albicans infection after two booster immunizations. (C). Vaccine-induced survival efficacy against C. auris infection after one booster immunization. The number of mice, N/group indicated on graphs as color coded number. Mice survivals were compared by the Mantel-Cox test, and p < 0.5 was considered statistically significant.

Certain vaccine formulations were excluded from testing under one- and two-booster immunization regimens and across both target pathogens. In the C. albicans infection model, several formulations demonstrated improved survival outcomes with two booster doses compared to a single booster dose. However, this trend was not consistent in C. auris infection model, where no significant survival advantage was observed with the two-booster regimen. Consequently, to reduce animal use while maintaining experimental rigor, some formulations were evaluated using only one immunization schedule.

Efficacy against lethal hematogenously disseminated C. albicans infection

Two vaccinations (one booster) with CAF01 formulations showed 10–32% survival (9–12 days MST) vs. placebo with 0% survival efficacy (8 days MST) against C. albicans disseminated infection (p < 0.01) (Figure S6). Using three doses of the CAF01 vaccine formulations (two boosters) further significantly enhanced survival efficacy to 40% to 56% overall survival by day 21 and prolonged MST to 17 - >21 days (Fig. 3B). For BDX100 formulations, two vaccinations with the Als3p/Hyr1p dual antigens showed 10%−40% overall survival efficacy with 10 to 11 days MST vs. 0% survival and 8 days MST for placebo. Similarly, three immunizations further enhanced the survival efficacy demonstrated by different ratio of the Als3p/Hyr1p dual antigen vaccine, showing 29% to 50% survival efficacy with 13 to 18 days MST (Fig. 3B, Figure S6, Table S3-4).

Efficacy against lethal hematogenously disseminated C. auris infection

We also evaluated the efficacy of CAF01 and BDX100 vaccine formulations using one or two booster vaccination experiments. CAF01 vaccine formulations (Als3p/Hyr1p: 30 µg/30µg, 10 µg/30µg, 30 µg/10µg, and 10 µg/10µg) with one booster showing 30% to 50% overall survival and 13 to 21 days of MST vs. 0% survival and 9 days MST for placebo mice vaccinated with only CAF01 adjuvant. Similarly, using the same ratios of antigens coupled with BDX100 as a one booster vaccine resulted in an overall survival of 22% to 47% with 14 to 18 days of MST vs. 0% survival and 10 days of MST for placebo. Two booster vaccinations with CAF01 or BDX100 formulation did not further enhance the survival efficacy (10% to 40% with 11 to 14 days of MST) (Fig. 3C, S7A-B, Table S3-4).

The CAF01 Als3p/Hyr1p dual antigen vaccine formulations prevented weight loss and reduced tissue microbial burden

Due of the extensive clinical experience with CAF01 adjuvant, the balanced immune response afforded in mice and the significant improvement in survival with CAF01 formulation, we focused on the top two CAF01 formulations, Als3p/Hyr1p: 10 µg/10µg and 30 µg/10µg for further fungal burden studies. Mice were vaccinated and infected with C. albicans (after two boosters) or C. auris (after one booster) as described earlier and euthanized at day 4 post-infection to enumerate tissue fungal burden. Mice weight was also recorded as a measure of the overall progression of infection and health status (Fig. 4A).

Fig. 4.

Tissue fungal burden in C. albicans and C. auris infected mice vaccinated with CAF01 Als3p/Hyr1p dual antigen vaccine. (A). Experimental design for the tissue fungal studies is shown. ICR (CD-1) mice were vaccinated two or three times with Als3p/Hyr1p dual antigens at 10 µg/10 µg, 30 µg/10 µg dose formulated with CAF01 adjuvant, or CAF01 alone (0/0). Two weeks after the final vaccination, mice were infected intravenously with 2 × 10⁵ cells/mouse of C. albicans or 5 × 10⁷ cells/mouse of C. auris. For C. auris infection, the mice were immunosuppressed on day − 2 relative to infection. Four days (hematogenously disseminated infections) or five days (VVC) post-infection, the mice were euthanized, and tissue fungal burden/gram tissues in target organs of vaccinated or placebo mice were determined. After 4 days of infection, the mice’s weight was also measured to assess their health status. (B). Weight loss post-C. albicans infection, (C). Kidney fungal burden in C. albicans infected mice, (D). Vaginal C. albicans burden, (E). Weight loss post-C. auris infection, (F). Kidney, (F). Heart, and (F). Brain C. auris burden. Mice weights and tissue fungal burdens were compared between vaccinated and placebo mice (N = 9–10/group) by the Mann-Whitney Test (Median ± IQR).

For C. albicans, the fungal burden in the kidneys was determined, as this organ is the primary target organ⁶⁵. Both CAF01 Als3p/Hyr1p 10 µg/10 µg and 30 µg/10 µg vaccine formulations prevented significant weight loss compared to the placebo group in C. albicans which was accompanied by ~ 0.5–1.5-log reduction in the kidney fungal burden (Fig. 4B, C). In a subset study and since the NDV-3A was protective against murine VVC⁵³, mice vaccinated with 10 µg/10 µg vaccine formulation were infected intravaginally, and tissue fungal burden was determined in vaginal tissues on days 4 and 5 post-infection. The CAF01 vaccine formulation reduced Day 4 C. albicans burden in the vagina by ~ 0.5-log (p = 0.003) and strongly trended to reduce fungal burden on Day 5 by 1.0-log (p = 0.09) when compared to placebo mice (Fig. 4D). Similarly, the CAF01 vaccine formulations protected mice infected with C. auris from weight loss and reduced Day 4 kidney, heart, and brain fungal burden by ~ 0.5–1.5-log versus placebo-vaccinated mice (Fig. 4E-H).

CD4 T cells and to a lesser extent antibodies are required for the CAF01 Recombinant Als3p/Hyr1p dual antigen vaccine (VXV-01)-mediated protection

CAF01 formulated recombinant 10 µg (Als3p)/10 µg (Hyr1p) dual antigen vaccine consistently showed robust immunogenicity and efficacy against both C. albicans and C. auris, thus we focused on this formulation in the subsequent study and named is VXV-01 (hereafter). To investigate the role of VXV-01-mediated humoral and adaptive immunity, we conducted passive serum transfer and CD4 T cell depletion experiments. For C. auris, the mice were immunosuppressed, as described earlier, while immunocompetent mice were used for C. albicans model. For the passive serum transfer experiment, sera from 3 times (for C. albicans) or 2 times (for C. auris) vaccinated mice with VXV-01 or those vaccinated with placebo (CAF01 adjuvant alone) were administered intraperitoneally to naïve infected mice at 2 and 168 h days post-infections and survival of mice was monitored for 21 days. For the CD4 T cell depletion experiments, we vaccinated mice with VXV-01 as above and then used anti-CD4 antibodies to deplete mice from CD4 T cells prior to infecting them with either C. albicans or C. auris. Survival of mice was documented over a 21-day period. CD4 T cell depletion was confirmed by determining the number of CD4 T cell population in both spleen and lymph nodes of representative mice treated with anti-CD4 IgG-treated mice vs. or isotype-matched control IgG (Figure S8)⁵².

For C. albicans infection, adoptive transfer of sera from VXV-01 -immunized mice resulted in modest 20% survival with a MST of 9.5 days, compared to 0% survival and a MST of 8.5 days in placebo sera-transferred mice, albeit not statistically significant (p = 0.089) (Fig. 5A). Depletion of CD4 T cell completely reversed protection afforded by the VXV-01 vaccine against C. albicans infection (Fig. 5B). Collectively, these results highlight the potential role of CD4 T cells in vaccine-mediated protection against C. albicans hematogenously disseminated infection.

Fig. 5.

Mechanism of VXV-01-mediated protection. Naïve mice or vaccinated mice (n = 10 mice/group) were infected with C. albicans or C. auris. For vaccination, the VXV-01 or placebo (adjuvant only) was administered on days 0, 21, and 35 (for C. albicans infection) or days 0 and 21 (for C. auris infection). Mice were immunosuppressed with cyclophosphamide and cortisone acetate for C. auris infection. Naïve mice (n = 10 mice/group) infected with (A). C. albicans or (C). C. auris received two intraperitoneal anti-sera injections at 2 and 168 h relative to infection. Vaccinated mice (n = 10 mice/group) received anti-CD4 or isotype control antibodies to deplete the CD T cells. The mice were infected with (B). C. albicans or (D). C. auris. Mice survivals were compared by Mantel-Cox test, and p < 0.5 was considered statistically significant.

Similarly, for C. auris infection, adoptive transfer of anti-VXV-01 sera trended to confer 20% survival benefit with an MST of 9 days, compared to 0% survival and an MST of 7 days in placebo sera-treated mice (p = 0.052) (Fig. 5C). Further, the protection afforded by the VXV-01 with 30% survival and MST of 11 days was significantly diminished to 0% survival and MST of 5 days after CD4 T cell depletion (p = 0.0135) and became equivalent to placebo mice (Fig. 5D). These results confirm the critical role of CD4 T cells and point to a possible role for antibodies in the afforded protection elicited by the VXV-01 against murine C. auris candidemia.

The VXV-01-induced long-lasting protective vaccine-induced immunity

To investigate the durability of the immunity afforded by the VXV-01 vaccine, antigen-specific antibody and T cell responses were tracked over 270 days following two (days 0 and 21) or three (days 0, 21, and 35) immunizations. After the final booster vaccination, mice were euthanized on days 14, 28, 90, 180, or 270 to evaluate antibody titers by ELISA and T-cell immune responses by FluroSpot assay.

Mice vaccinated with the VXV-01 showed robust antibody responses against Als3p and Hyr1p antigens on day 14, which did not wane for up to 9 months of follow up. The two booster series induced ~ 1-log higher IgG antibody titer than the one booster series. For T-cell immunity, the one booster immunization resulted in Als3p and Hyr1p-specific T-cell responses that peaked at Day 14 and were similar in magnitude and biased towards Th1 and Th2. These T-cell specific responses declined after two weeks but were detectable for up to 9 months (Fig. 6A). For the two booster immunizations, Th1-antigen specific immune responses were similar to the one booster regimen (Fig. 6B), with two exceptions of being higher in magnitude and responses peaked at Day 28 instead of Day 14 for the one booster immunization (Fig. 6C, D). These results are concordant with data showing that mice intravenously challenged with C. auris 15 weeks following one booster regimen had a 25% 21-day survival vs. 0% for placebo and 14 days MST for vaccinated vs. 9.5 days for placebo (p = 0.0027) (Fig. 6E). These results demonstrate that durable VXV-01-induced immunity translates into durable protective efficacy.

Fig. 6.

The durability of VXV-01 vaccine-induced immunity. The ICR (CD-1) mice were vaccinated sub-cutaneously with VXV-01 or placebo (adjuvant alone) on days: (A). 0, 21; or (B). 0, 21, 35. The serum anti-Als3p and anti-Hyr1p IgG endpoint titers and T cells were evaluated using ELISA and FluroSpot assay on days 14, 28, 90, 180, and 270 post-final vaccination. (C). Anti-Als3p, and (D). Hyr1p IgG endpoint titers and T-cell responses were compared between two and three vaccination schedules over the period of 270 days post-vaccination. Data presented as mean ± SE of N = 5 mice/group. (E). VXV-01 efficacy was tested after 15 weeks of final two doses vaccination regimen (day 0, 21) against C. auris infection in immunosuppressed mice (N = 10 mice/group). Survival curves were compared by the Mantel-Cox test, and p < 0.5 was considered statistically significant.

VXV-01 vaccine did not antagonize antifungal drug therapy

We also evaluated the efficacy of the VXV-01 in combination with a sub-protective dose of antifungal drugs currently used to treat Candida infections. Briefly, we vaccinated mice as above, and two weeks after the vaccination, mice were infected with C. albicans or C. auris. We used a suboptimal dose of fluconazole or micafungin starting on day + 1 post-infection to achieve minimal protection and enable detection of potential additive/synergy with the VXV-01 vaccine. While the overall survival of mice receiving both VXV-01 and antifungal drug therapy was higher (50% survival for both C. albicans or C. auris candidemia) than antifungal drug therapy alone (30% and 0% survival for C. albicans or C. auris candidemia, respectively), or VXV-01 vaccine alone (30% for both C. albicans or C. auris candidemia), this enhanced survival in the combination arm was not statistically different than the vaccine alone treatment or antifungal monotherapy (Fig. 7A-C).

Fig. 7.

VXV-01 vaccine efficacy in combination with antifungal drug therapy. Male ICR (CD-1) mice were vaccinated with VXV-01 or placebo (adjuvant alone) on days 0, 21, and 35 (for C. albicans infection) or days 0 and 21 (for C. auris infection). After final vaccination, mice were randomly divided in four groups (n = 10 mice/group/infection): (1) Placebo, (2) Antifungal therapy alone, (3) VXV-01 alone, and (4) VXV-01 + antifungal therapy combination. Sub protective dose of fluconazole or micafungin was used as the antifungal therapy C. albicans and C. auris, respectively, and mice were immunosuppressed with cyclophosphamide and cortisone acetate prior to infection C. auris. Mice survival was compared by Mantel-Cox test, and p < 0.5 was considered statistically significant.

Discussion

Our previous efforts have focused on developing vaccine targeting C. albicans adhesin/invasion protein Als3p or neutrophil evasion factor Hyr1p^49,71,72. Alum-adjuvanted Als3p or Hyr1p recombinant protein-based vaccines have shown protection against invasive C. albicans infection in mice^71,72. NDV-3A, an alum-formulated Als3p-based vaccine, has been effective in protecting women from RVVC ⁵⁴. Additionally, the newly emerged multidrug-resistant C. auris harbors orthologs of both Als3p and Hyr1p. NDV-3A-induced cross-protective immune responses in immunosuppressed mice from C. auris candidemia⁵⁵. Further, Hyr1p-epitope-based cross-reactive monoclonal antibodies have protected immunosuppressed mice against lethal C. auris infection⁵⁷. However, because of the modest protection effect elicited in women vaccinated by a single dose of NDV-3A, further development of this vaccine has stalled. To further advance our efforts in developing the next generation of fungal vaccines, we explored combining both Als3p and Hyr1p in a dual antigen vaccine using more advanced and clinically safe adjuvant systems.

A major disadvantage of using alum as an adjuvant is the lack of induction of a Th1 and Th17 immune responses^73,74. It is widely reported that IFN-γ is a key Th1 cytokine that enhances macrophage activation and promotes fungal clearance. Also, IL-17, produced by Th17 cells and innate lymphocytes, is crucial for mucosal and dermal immunity against C. albicans and C. auris, respectively, by driving neutrophil recruitment and antimicrobial peptide production, ultimately leading to fungal clearance^75–79. IL-4, a Th2 cytokine, has a more complex role, as it can support humoral immunity; however, excessive Th2 responses may be detrimental by skewing immune responses away from protective Th1/Th17 responses. Thus, our objective was to identify adjuvant and dual-antigen vaccine formulations incorporating Als3p and Hyr1p that could elicit strong, long-lasting, and balanced immune responses. Specifically, we aimed to induce both humoral and cellular immunity, characterized by Th1 (IFN-γ) and Th17 (IL-17) responses targeting both antigens, which are known to contribute to protective immunity against fungal pathogens^49,80,81.

We used a checkerboard strategy in mixing different ratios of Als3p and Hyr1p protein antigens with clinically safe adjuvants to minimize the potential immunodominance of one antigen over the other. We screened these formulations in vivo for immunogenicity elicited by antigen-specific IgG endpoint titers and T cell responses. The Als3p/Hyr1p dual antigen vaccine formulations induced strong IgG titers, with GCP and MF59 adjuvant formulations performing particularly well. The relative antigen doses did not significantly influence the specific IgG titers for most adjuvant formulations, except for CAF01. This observation aligns with earlier research indicating that certain adjuvants may not exhibit antigen dose-dependent effects⁸². Further, the choice of adjuvant also significantly impacts the immune response. In our study, GCP formulations induced the highest anti-Als3p IgG titers, while alum formulations induced the highest anti-Hyr1p IgG titers. This finding is also consistent with previous studies, which have shown that different adjuvants influence the magnitude and type of immune response^83,84. Of note, the Als3p/Hyr1p dual antigen vaccine formulations induced similar or higher immune responses compared to responses induced by single antigen formulations. This suggests that combining Als3p and Hyr1p does not negatively impact the immunogenicity of either antigen. This observation has significant implications for developing vaccines combining multiple antigens from similar or related pathogens to target several healthcare-associated pathogens. These results also highlight the critical role of adjuvants in vaccine formulation.

We evaluated the Als3p/Hyr1p dual antigen vaccine in murine models of invasive candidiasis due to C. albicans and C. auris. SC5314 and CAU-09 isolates represent the dominant isolates of C. albicans and C. auris, respectively, both in the USA and worldwide^85–87. Thus, we used these Candida isolates to test the efficacy of the vaccine in our murine models of invasive candidiasis. Using top immunogenic Als3p/Hyr1p dual antigen vaccine formulations, we identified BDX100 and CAF01 as the most efficacious adjuvant formulations. Other adjuvant formulations, specifically GCP and MF59, failed to provide protection against both C. albicans and C. auris despite inducing robust antibody responses and with GCP also inducing a strong Th1/Th17 immune response. This suggests that a strong antibody response alone does not necessarily correlate with protective efficacy. It is possible that the antibodies induced by these two adjuvants are of limited avidity, specificity or functionality. Additionally, the role of cellular immunity, particularly the contributions of CD4 T cell subsets, is more critical in combating these fungal infections, as previously reported by us and others^88,89.

We prioritized BDX100 and CAF01 vaccine formulations and tested additional Als3p/Hyr1p antigen ratios for further optimization. Our results indicate that both CAF01 and BDX100 vaccine formulations may offer significant protection against lethal C. albicans and C. auris infections. The efficacy of the vaccine formulations was dose-dependent, and lower antigen doses provided better protective efficacy against both fungal infections (e.g. doses of 10 µg/10 µg–10 µg/30 µg of Als3p/Hyr1p had better mouse survival than 30 µg/30 µg [Table S3]). This could be potentially due to the induction of poor quality of antibody and CD4 T cells as previously reported^88,89.

In this study, the three-dose vaccination series using CAF01 or BDX100 induced higher antigen-specific IgG titers and Th1, Th2, and Th17 cells than the two-dose series and resulted in better protection against C. albicans hematogenously disseminated infection (Figs. 3 and 6). However, the three-dose vaccination regimens of these two vaccines series did not provide better protection against C. auris infections (Fig. 3). The two vaccine doses showed peak antigen-specific Th1/Th2 T cell responses at day 14, compared to three-dose vaccinations, which peaked at day 28. The fact that protective vaccination against C. albicans disseminated infection required three vaccinations, while C. auris disseminated infections required two vaccinations emphasize the differential pathogenicity of both infections. Specifically, C. albicans cause lethal infection in immunocompetent mice, while C. auris lethal infection models are only achieved in immunosuppressed or immunodeficient mice^90,91. Additionally, these results clearly show the critical role of booster doses and vaccination scheduling in the magnitude, quality, and kinetics of vaccine-induced immunity, aligning with previous studies in both mouse and human models^92–96. Finally, these results underscore the importance of optimizing both the antigen dose and vaccination schedule to achieve optimal protective outcomes. It is prudent to mention that if the vaccine is advanced into clinical testing, a single booster of the vaccine might be sufficient to elicit protection against C. albicans infections because the majority, if not all, humans are colonized with this yeast^97–99.

Based on the efficacy studies and the fact that CAF01 has been shown to be safe in several clinical trials^100–103. We further prioritized CAF01-based Als3p/Hyr1p dual antigen vaccine formulations and tested them in tissue fungal burden studies. To avoid the survival biasing effect, we opted to compare the effect of the CAF01-based vaccine formulation on the tissue fungal burden at day + 4 prior to losing mice in the placebo arm. The vaccination significantly reduced the tissue fungal burden in the kidney and vagina of C. albicans and, kidney, heart and brain of C. auris infected mice compared to placebo mice. However, CFU levels in the target organs, particularly in the kidneys of vaccinated mice remained relatively high. This partial reduction in the fungal burden is biologically significant as it resulted in statistically significant less weight loss and an increase in the survival rate of the vaccinated groups compared to placebo. These studies resulted in the optimization of Als3p/Hyr1p dual antigen-based vaccine containing 10 µg of each antigen and formulated with CAF01 adjuvant and named as VXV-01.

Further investigation showed that antibodies alone had a limited protective role, while T cells were critical in the VXV-01-induced antifungal immunity. These results are aligned with our previous studies and emphasize the importance of cellular immunity, particularly the role of CD4 T cells in orchestrating and sustaining protective immune responses against both C. albicans and C. auris^55,104. However, it is imperative to note that the antibody levels present in the adoptively transferred sera may have been insufficient to confer effective protection in this study. Also, due to the low half-life (5–7 days) of the CD4 depletion antibodies, CD4 T cells may have been repopulated after one week of infection and thus may not be reflecting a sustained CD4 T cell depletion.

Long-lasting immunity is crucial for the effectiveness of vaccines, especially in preventing infections over extended periods. Our study demonstrated that both single and two-booster immunization regimens with VXV-01 elicited robust and durable antibody and T cell responses lasting up to nine months, reducing the need for frequent booster doses. Using the C. auris hematogenously disseminated infection model, we demonstrated that this durable VXV-01-induced immunity also translated into durable protective efficacy. This proof-of-concept experiment was designed to assess whether such sustained immune responses translate into long-term protection, particularly against C. auris, given its emerging multidrug resistance and limited treatment options. Our current immunogenicity data and previous studies with Als3p-based NDV-3 A vaccine suggest that VXV-01-induced immunity will likely confer long-lasting efficacy against C. albicans mucosal infections⁵⁰. It is also prudent to point out that for hematogenously disseminated C. albicans infection, the major patient populations that will benefit from such a vaccine are immunocompetent patients who would undergo intra-abdominal surgery. These patients usually develop candidemia within 1–3 weeks from admission to ICU¹⁰⁵ and long-lasting infection is not needed.

It is imperative that any antifungal prophylactic or therapeutic approach be used in conjunction with clinically approved drugs. Thus, we investigated the potential VXV-01 as an adjunct prophylactic approach. Our results showed that the VXV-01 vaccine combined with antifungal drug therapy enhanced survival rates and median survival times compared to placebo mice with a p value that is more statistically significant than either treatment alone, suggesting a benefit of this combination treatment regimen. However, it is important to note that the difference between the combination treatment and the VXV-01 or antifungal drug alone did not reach statistical significance to demonstrate clear synergy/additive effect. This lack of clear benefit could be due to limitations of the animal model and the sample size used (e.g. more aggressive Candida infections in these experiments as highlighted by rapid early mortality post infection). Nonetheless, the observed trends in higher survival in the combination arms suggest a potential additive benefit that warrants further investigation.

Future studies should focus on elucidating the specific and more in-depth immune mechanisms that confer protection against each fungal pathogen. Additionally, exploring the reasons behind the failure of GCP and MF59 to protect despite robust immune responses could provide valuable insights into the complexities of immune protection and guide the development of more effective vaccines. Further, understanding why certain adjuvants perform better with specific antigens can lead to the development of even more effective adjuvant-antigen combinations. Further studies should focus on optimizing the dosing and timing of both the VXV-01 and the antimicrobial drug to maximize their combined efficacy. Additionally, exploring the underlying immune mechanisms that contribute to the observed survival benefits could provide valuable insights into how to enhance the protective effects of the VXV-01 and drug combination. Finally, evaluating the breadth of VXV-01-induced protection against other strains of C. albicans and C. auris as well as clinically important Candida species is crucial.

In conclusion, our data provide evidence that an improved second-generation Als3p/Hyr1p dual antigen vaccine, VXV-01, is more effective than the NDV-3 A vaccine in providing a sustained immune response and long-lasting vaccine efficacy in murine models of C. albicans and C. auris infection. Therefore, future studies focusing on evaluating the immunogenicity, safety and efficacy in clinical trials are warranted.

Materials and methods

Antigens, expression systems, cell banks, and manufacturing

The Als3p antigen is a recombinant N-terminal region of an adhesin protein from C. albicans expressed in Saccharomyces cerevisiae. The expressed protein has 416 amino acids. Hyr1p constitutes a recombinant N-terminal portion (197 amino acids) of the native cell surface protein from C. albicans, that is expressed in an insoluble inclusion body form in E. coli.

For Als3p expression, a Master Cell Bank (MCB) of S. cerevisiae strain FY03-1 maintaining the vector pTEF1-S1Als3-2 (codon optimized for S. cerevisiae) was established earlier by Althea Technologies utilizing the parent strain DY150 (Clontech). Subsequently, this strain was further optimized to develop a Research Cell Bank (RCB) designated as Fy03-1/2um-full/TEF1p. This optimization yielded a more stable cell line due to the inclusion of the full 2 μm origin sequence. A Working Research Cell Bank (WRCB) was prepared from the above RCB by Biodextris for the manufacturing process of Al3p. Briefly, one vial of Working Research Cell Bank containing S.cerevisiae FY03-1 producing Als3p was thawed and added to the primary shake flask, and then expanded into a secondary shake flask to further enlarge the working culture for bioreactor inoculation. Once the secondary shake flask reaches its set OD, the culture is used to inoculate the 10 L production bioreactor, which is run for approximately 40 h in fed-batch mode while the Als3p product is constitutively expressed. Once the harvest criterion is achieved, the biomass is separated from soluble expressed Als3p by centrifugation, with the product being clarified by depth filtration and 0.2 μm filtration. Following harvest, the filtered concentrate is loaded onto a Capto MMC column for Als3p product capture, eluted, and flowed through a Benzamidine FF column for host cell protein reduction, followed by polishing on a Butyl HP chromatography step. The eluate from the Butyl HP step is then buffer exchanges and flowed through a Sartobind Q membrane adsorber and then is 0.2µM filtered, aliquoted, and stored at ≤ −60 °C (Fig. 1).

For Hyr1p expression, an N-terminal region of the Hyr1p gene was bacterial codon-optimized, cloned in a bacterial proprietary plasmid expression vector (Nature Technologies Inc.), and transformed into a competent E. coli BL21 strain (New England Biolabs). Subsequently, the plasmid-transformed E. coli cell line underwent rounds of culture and clonal isolation to screen for promoter regulation, productivity, copy number, restriction map, and level of dimerization, resulting in the selection of a colony to establish the pre-RCB cell bank. Briefly, one vial of Research Cell Bank containing E.coli BL21 producing Hyr1p by promoter induction is expanded by shake flask to inoculate a 10 L bioreactor in chemically defined media in the presence of Kanamycin. Once inoculated, the 10 L production bioreactor is run in fed-batch mode for approximately 24 h to increase biomass and then induced with IPTG for Hyr1p product expression over 24 h. Once the harvest criterion is achieved, the biomass is harvested by centrifugation and stored at a temperature of ≤ −60 °C. A portion of the biomass is thawed, and the insoluble cell-bound Hyr1p is chemically and mechanically lysed from the cells through homogenization and centrifugation to isolate the inclusion bodies, which are then washed for impurity reduction before being subjected to tangential flow filtration for protein refolding. Once refolded, the Hyr1p is flowed through an anion exchange QFF resin and then captured and eluted from a cation exchange MegaCap II SP550 column. The eluate is then concentrated and diafiltered by TFF, flowed through a Mustang E anion exchange membrane for impurity reduction, 0.2µM filtered and frozen at ≤ −60 C (Fig. 1).

Adjuvants

We used CAF01, BDX100, BDX100, MF59, Glucan Chitosan Particles (GCPs) and alum adjuvants in this study. CAF01 is a two-component liposomal suspension composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA), and α’-trehalose-6,6’-dibehenate (TDB) and is being developed by Serum and Statens Institut, Denmark. CAF01 is prepared by forming thin lipid films containing DDA and TDB in a 5:1 (w/w) ratio, followed by hydration in a buffer solution, resulting in liquid crystalline bilayer vesicles. BDX100 (Protolin) consists of Neisseria meningitidis outer membrane protein (OMP) non-covalently associated with Shigella flexneri lipopolysaccharide (LPS) in a 1:1 ratio. BDX300 (V2 Proteosome) consists of N. meninigitidis Omp and LPS (Biodextris, Laval, QC, Canada). Alum (aluminum hydroxide, Adjuphos) and MF59 (Novartis Proprietary adjuvant) were sourced from Inovio. MF59 is an Oil-in-Water emulsion (squalene, Tween 80, and Span 85 surfactants)-based adjuvant known to induce Th2 and humoral antibody response¹⁰⁶. GCPs were prepared from fungus Rhodotorula mucilaginosa⁶⁵, loaded with Als3p or Hyr1p antigens, and administered separately subcutaneously (Table S1).

Vaccine formulations

The final formulated (Als3 + Hyr1 + adjuvant) vaccine consists of two separately purified recombinant antigens and one of the adjuvants (Alum, CAF01, BDX100, BDX300, MF59, and GCP). We used a checkerboard method to obtain different antigen ratios by combining Als3p antigen at 0, 10, or 30 µg/dose with either 0, 3, 10, or 30 µg/dose of Hyr1p. Monovalent vaccine formulations with Als3p or Hyr1p alone were used to compare antibody and T cell development to Als3p/Hyr1p dual antigen vaccine formulations and any potential immunodominance by one antigen over the other.

For each vaccine dose, different antigen ratios of Als3p and Hyr1p antigens were mixed with 200 µg of alum (Inovio), 300 µg of a two-component liposomal adjuvant system (CAF01; supplied by Croda International Plc), 100 µl of MF59, 50 µg of BDX100, or 50 µg of BDX300 adjuvant. Als3p or Hyr1p antigens were encapsulated in 200 ug (1 × 10⁸ particles) GCPs. The volume for each vaccine dose was adjusted to 0.2 ml with diluent Phosphate buffer saline (pH 7.4).

Mice vaccination

The ICR (CD-1) 4–6 weeks old mice were vaccinated with the formulated vaccine candidates on days 0 and 21 or days 0, 21, and 35 subcutaneously (SC) or intranasally (IN) (for BDX100 and BDX300 formulations only due to lipopolysaccharide component). The mice were euthanized two weeks after the final vaccination for immunogenicity determination, and sera and spleens were collected. Sera were used to evaluate anti-Als3p and Hyr1p IgG antibody endpoint titers by ELISA. Splenocytes were used for FluroSpot assay to determine the frequency of Als3p or Hyr1p antigen-specific Th1, Th2, or Th17 cells. The mice were infected two weeks after the final vaccination for infection experiments.

Antibody titer determination

Polystyrene 96-well plates were coated with 5 µg/ml of Als3p or Hyr1p in 1X PBS buffer (pH 7.4) and incubated overnight at 4 °C. The following day, the plates were washed three times with 1X wash buffer (PBS with 0.05% Tween-20) and blocked with 1% BSA solution for 2 h at room temperature. After another three washes, diluted serum samples were added in duplicates and incubated for 2 h. Post-incubation, the plates were washed three times, and 1:1000 diluted anti-mouse IgG antibodies (Jackson, Cat#115-035-164) labeled with peroxidase were added and incubated for 1 h at room temperature. Finally, the plates were washed five times with wash buffer, TMB substrate (Invitrogen, Cat#00–4201-56) was added, and color development was allowed for 5–10 min. Absorbance was measured at 450 nm after stopping the reaction with 1 N sulfuric acid (Sigma, Cat#339741)⁵⁵.

FluroSpot assay

We employed a CTL™ IFN-γ/IL-4/IL-17 triple color FluoroSpot assay kit (CTL ImmunoSpot, Cleveland, OH) to assess antigen-specific T cell immune responses. The FluoroSpot assay plates were prepared by activating the membrane with ethanol and washing with PBS. Mouse Cytokine Capture Solution was prepared according to the manufacturer’s instructions and added to the plates. The plates were incubated overnight at 4 °C and then washed with 1X PBS.

Spleens from vaccinated animals were collected and individually processed by homogenizing through a 100 μm cell strainer. RBCs were lysed using 1x RBC lysis buffer (Santa Cruz Biotech, Dallas, Cat# SC-296258) and filtered through 100 μm sterile filters. The cells were resuspended in CTL serum-free medium, counted, and plated at 3 × 10⁵ splenocytes/0.1 ml/well. The splenocytes from each mouse were either left unstimulated or stimulated with 0.1 ml/well Als3p or Hyr1p at 10 µg/ml, or with mitogen (10 ng/ml Phorbol myristate acetate [PMA]/250 ng/ml Ionomycin) along with anti-CD28 antibody.

The plates containing antigens and splenocytes were incubated for 24 h at 37 °C. After incubation, cytokine spots were developed using an anti-mouse IFN-γ/IL-4/IL-17 cytokine detection solution, followed by a tertiary solution. The developed FluoroSpots were air-dried, imaged, and counted using the CTL ImmunoSpot plate reader. FluoroSpots in the unstimulated wells of each mouse were subtracted from the antigen-stimulated spot counts and graphed.

Infectious inoculum preparation

The C. albicans reference strain SC5314 (ATCC- MYA-2876) and C. auris strain CAU-09 (South Asian clade, bronchoalveolar lavage [BAL]) were used in this study. These strains were grown in Yeast Extract Peptone Dextrose (YPD) broth overnight at 30 °C with shaking at 200 rpm. Yeast cells were washed with 1x phosphate-buffered saline (PBS, Gibco by Life Technologies) three times prior to counting blastopores with a hemocytometer. For intravenous injection, C. albicans and C. auris inoculum were adjusted to 2 × 10⁵ cells/0.2 ml and 5 × 10⁷ cells/0.2 ml, respectively. For vaginal infection, C. albicans inoculum was adjusted at 1 × 10⁸ cells/mL^{53,55,107,108}.

Mice infection and treatment

ICR (CD-1) mice were intravenously infected two weeks after vaccination with either C. albicans or C. auris. For C. albicans infection, 2 × 10⁵ cells/0.2 ml were administered via tail vein injection. For C. auris infection, mice were immunosuppressed with 200 mg/kg cyclophosphamide (i.p.) and 250 mg/kg cortisone acetate (s.c.) given on day − 2 relative to infection. To prevent bacterial superinfection, enrofloxacin (50 µg/ml) was added to the drinking water and continued until day 7 post-infection. Mice were then intravenously injected with 5 × 10⁷ cells/0.2 ml of C. auris⁵⁵.

For the antifungal and vaccine combination treatment, vaccinated and infected mice received a minimal protective dose of 2.0 mg/kg/day of Fluconazole for C. albicans and 0.5 mg/kg/day of micafungin (i.p.). for C. auris. Treatment began 24 h post-infection and continued until day + 7. Mice were monitored for survival over 21 days post-infection.

For vaginal infection, vaccinated mice received a 1.6 µg/gram mouse weight dose of β-Estradiol 17-valerate (Sigma, Cat# E1631-1G) before and during infection with C. albicans. β-Estradiol 17-valerate was administered subcutaneously at 0.1 ml/mouse in the back of the neck on days − 3, 0, and + 3 relative to infection⁵³.

To determine fungal burden, mice were euthanized on day 4 (day 5 for vaginal infection) post-infection to collect kidneys, hearts, and brains (vaginal tissues for vaginal infection). The organs were weighed, homogenized, and quantitatively cultured using 10-fold serial dilutions on YPD plates. Plates were incubated at 37 °C for 48 h before enumerating colony-forming units (CFUs)/gram of tissue^53,55.

Antibody adoptive transfer and T cell depletion studies

The mice were vaccinated as described above and grouped as depletion and control depletion arms. For CD4 + T cell depletion, 200 µg/mouse dose of rat anti-mouse CD4 IgG2b (clone GK1.5, BioXcell, Cat #BE0003-1)) or rat IgG2b isotype antibodies (Clone: LTF-2, BioXcell, Cat #BE0090) were administered intraperitoneally on day − 3 and 0 relative to infection. Mice were infected intravenously with either C. albicans or C. auris and monitored for their survival for three weeks.

Three additional mice were taken in each depletion and control depletion arm to verify the CD4 T Cell depletion 4 days after administering the second dose of the depletion drug. The mice were euthanized, and their spleen and inguinal lymph nodes were harvested and homogenized to make a single-cell suspension.

The sera were collected and pooled from the vaccinated animals for Antibody adoptive transfer. Naïve mice infected with C. albicans or C. auris as above and treated intraperitoneally with 0.4 ml/mouse with either serum from vaccinated mice or placebo mice on days 0 and 7. The mice were monitored for survival⁵⁵.

Flow cytometry

Splenocytes were stained with anti-CD3 APC (BD Pharmigen, Cat #BDB565643) and anti-CD4 Alexa Fluor 700 antibodies (Biolegend, Cat #100536). The stained cells were acquired in a BD LSR II flow cytometer, and data were analyzed in FlowJo V10 software.

Statistical analysis

The animal groups in this study were not blinded. For CD4 T cell depletion and antifungal drug combination studies placebo (adjuvant alone, 0/0) and vaccine groups were randomly sub-grouped into CD4 depletion and non-depletion, and vaccine alone and vaccine + drug combination group, respectively. Survival differences were analyzed using the non-parametric Log Rank test for overall survival and Mantel-Cox comparisons for median survival times. All other comparisons were performed using the non-parametric Mann-Whitney test. A p-value of < 0.05 was considered significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SS conceptualized and designed the study, performed experiments, collected and analyzed the data, and wrote the original draft of the manuscript. EGY and AB performed animal procedures and assisted in mouse tissue processing and fungal burden experiments. HH contributed to mouse sample processing and in vitro assays. HH, SN, TG, and SA helped with animal procedures. GO, DC, and TC contributed to the critical materials for the study. ASI conceptualized, designed, supervised, and secured the funding for this study and edited the manuscript.

Data availability

The data are available in the main text or the supplementary materials of this manuscript.

Declarations

Competing interests

A.S.I. is a founder of Vitalex Biosciences, which is developing a Candida dual antigen vaccine targeting healthcare-associated pathogens. S.S., T.G., S.A. and T.C. are shareholders of Vitalex Biosciences. The rest of the authors have no competing interests.

Ethics declaration

The IACUC of The Lundquist Institute approved all procedures involving mice (protocol #31413-02), in accordance with the NIH guidelines for animal housing and care and the ARRIVE guidelines¹⁰⁹. Moribund mice, according to detailed and well-characterized criteria, were euthanized by pentobarbital overdose, followed by cervical dislocation.