Four million neonates are born every year in the United States. About 11.4% are born preterm (between 28 and 36 weeks of gestation), 8% have low birth weight (LBW) and 1.4% are of very low birth weight (VLBW) (1). It is recognized that 75% of infants admitted to the neonatal intensive care unit (NICU) are colonized by Candida by the first month (2). Infection is acquired by vertical transmission during vaginal delivery, postnatally from contact with maternal skin or the skin of direct care providers, or direct transmissions via contaminated equipment or intravenous catheters. Candida albicans remains the most prominent pathogen in neonates, followed by significant cases due to C. parapsilosis ( 3–5 ). Candida infections are responsible for ~10-12% of nosocomial sepsis in VLBW (<1500 g) infants, with a collective incidence of up to 4% among all NICU admissions (6). In fact, Candida is the 3rd most frequently isolated organism (after coagulase negative Staphylococcus spp. and S. aureus) in late onset sepsis in VLBW infants (2). Despite empirical antifungal therapy, mortality related to the disease remains considerably high (20-30%), with even higher rates (59-73%) of long-term neurodevelopmental impairment in survivors (2, 6).

Protection against infection in premature neonate depends upon the innate immune system, and on antibodies acquired passively from the mother (7). The transplacental transfer of maternal IgG to the fetus is a specific adaptation that minimizes deficiencies in the production of antibodies (8). Transplacental passage of antibodies via maternal immunization can reduce the risk of vaccine-preventable diseases that may occur in early weeks of life in preterm infants (9, 10) (11). For instance, vaccination against influenza, pertussis and pneumococcal infections that have been clinically used (11, 12) Furthermore, use of formulations containing hyperimmune IgG antibodies against specific antigens to treat diseases has been considered. Passive transfer of IgG promotes opsonic activity and antibody dependent cytotoxicity, activates complement, and improves neutrophilic chemotaxis (8, 13, 14). The consensus for passive immunization approaches in preterm neonates has garnered great support (13–17). Vaccine-based approaches for combating candidiasis in neonates could be a viable option because, i) neonatal candidiasis is a lethal disease in a sensitive population, despite antifungal therapy, ii) C. albicans antigens are available as targets for immunotherapeutics development, iii) Such approaches have been successful in protection against other diseases in new born infants.

Harnessing immunodominant cell wall proteins of C. albicans (Als3p and Hyr1p) for developing immunotherapeutic modalities have proven to be valuable for combating a host of Candida-associated infections (18–24). To elaborate, we have previously reported on the efficacy of NDV-3 (the N-terminus of recombinant Als3p formulated with alum) in preventing disseminated candidiasis in mice infected with C. albicans or with C. auris ( 19, 25). Vaccination with Als3p coupled with complete/incomplete Freund’s adjuvant also protects mice from hematogenously disseminated candidiasis by other Candida species including C. glabrata, C. tropicalis, C. krusei and C. parapsilosis ( 19, 26). Besides Als3, another cell surface protein Hyr1p [contributes to C. albicans virulence by resisting phagocyte killing (27)] has been the subject of investigation by our group and has shown outstanding efficacy against disseminated candidiasis (24, 27). Interestingly, Hyr1p shares striking three-dimensional structural homology with cell surface proteins of Gram-negative bacteria and active vaccination with recombinant N-terminus of Hyr1p (rHyr1p-N) or passive immunization with anti-Hyr1p antibodies protect mice from Acinetobacter infection (21, 22). Our prior successes with these novel vaccine candidates in protection against Candida infections form a robust premise for their evaluation as immunotherapeutic interventions against candidiasis in neonates. Here, we performed a proof-of-concept study to evaluate if anti-Als3p or anti-Hyr1p antibodies are important for prevention of disseminated candidiasis in neonates. We show that vaccination of mothers with rAls3p-N or rHyr1p-N formulated in CFA/IFA, generates antibodies that transfer efficiently and at high titers into neonates via the placenta. These transplacentally transferred antibodies significantly reduce fungal burden in the kidneys of neonatal mice. Moreover, adoptive transfer of sera from vaccinated mothers into pups also affords similar level of protection, including against fluconazole resistant C. albicans. Neutrophils were found to be important for the protective outcome. These studies are the first step in the direction of discovering immunotherapeutic strategies for combating life-threatening Candida infections in newborns.

Neonatal candidiasis is associated with significant morbidity and mortality in infants. Few clinical signs or laboratory assays have been validated for candidiasis in infants, and successful outcomes rely heavily on early removal of indwelling catheters, followed by prompt treatment by standard of care antifungal drugs such as fluconazole (29). Despite antifungal treatment, one in five very low birthweight neonates will succumb to candidiasis; one in two of the survivors will demonstrate severe neurodevelopmental impairment (30, 31) and suffer end-organ damage in the central nervous system, heart, and genitourinary tract (30). Clearly, alternative strategies to combat life threatening candidiasis in an immunocompromised population such as premature infants are sorely needed.

We performed a proof-of-concept study to evaluate if antibodies raised against Candida antigens have the capability of protecting against neonatal candidiasis. Previous studies from our group have shown that vaccine candidates based on C. albicans cell surface proteins such as rAls3p-N and rHyr1p-N, adjuvanted with alum, can protect against candidiasis in several animal models (19, 20, 27, 32). Furthermore, anti-rHyr1p-N antibodies have robust cross-kingdom efficacies extending to protection of mice from lethal infections also by Gram-negative bacteria (21, 22). Thus, expansion of the scope of these two antigens, for protection of neonates against disseminated Candida infections is the most logical step, in light of the high mortality rates despite antifungal treatments in VLBW infants.

The adaptive immune response in newborns is underdeveloped, having had no previous exposure to microbes in utero, and due to the impaired functions of B and T cells (33). Protection against infection in a premature neonate essentially depends upon the innate immune system, and on antibodies acquired passively from the mother (7). We found that, vaccinating female mice with rAls3p-N or rHyr1p-N adjuvanted with CFA/IFA produced a robust antibody response with titers as high as 5 logs. CFA/IFA are considered as the most effective adjuvant available for consistently producing high titer antibodies to diverse antigens, and are irreplaceable and vital for immunology research and antibody production. Importantly, we found that the quality of these antibodies were predictors of efficacy against Candida infections. In particular, IgG2 titers generated by CFA/IFA vaccination was 15-fold higher than that produced by alum. We have previously reported that IgG2 titers are biomarkers of efficacy for protection of women from recurrent vulvovaginal candidiasis (28). Our hypothesis was that in the absence of adaptive immunity, antibodies are important for protection from neonatal candidiasis. The adjuvants CFA/IFA generated a high IgG2 response prompting us to perform all our studies using these adjuvants.

Vaccination of mothers by CFA/IFA-rAls3p-N or CFA/IFA-rHyr1p-N, before conception, generated antibodies that robustly transferred via the placenta to the neonates, and accumulated in the pups at concentrations comparable to titers present in their mothers. In humans, IgG transfer from mother to fetus begins as early as 13 weeks of gestation, with a continuous rise in IgG levels in fetal circulation between 17 and 41 weeks of gestation. Fetal IgG concentrations reach 50% of the maternal concentrations at weeks 28–32, and increase rapidly thereafter (34, 35). Thus, presence of high titer antibodies in full-term delivered pups was not surprising. Furthermore, IgA is incapable of transplacental passage and hence the titers of IgA in pups was negligible. Post rAls3p-N and rHyr1p-N vaccination of mothers, the placentally transferred antibodies reduced kidney infection in pups by a 1.5 to 2 logs compared to placebo (CFA/IFA with antigen) vaccinated mice. Maternal immunization can reduce the risk of vaccine-preventable diseases that may occur in the early weeks of life, especially in the pre-term infants (11). One of the best examples is vaccination against influenza that can protect pregnant women from a life threatening disease, induce protective specific antibody levels and protect infants in the first weeks of life (11). Other examples are vaccinations against pertussis and pneumococcal infections (11, 12).

Our studies have some limitations. First, experiments were performed in full term pups, despite the fact that neonatal candidiasis is predominant in low birth weight preterm infants. However, it is impossible to keep prematurely delivered pups alive for longer than a few hours. Besides, disseminated candidiasis is a serious problem also in intubated full-term infants, whereas mucosal candidiasis in the form of oral thrush is a frequent occurrence in term babies. Hence, we do not consider this as a debilitating weakness. Second, despite several attempts, we were unsuccessful in establishing a survival model of disseminated infection in pups; hence, we performed all our efficacy studies focusing on recording changes in fungal burden in the primary target organ (kidneys), an endpoint that has been used in numerous publications as a parameter for evaluating therapeutic efficacies of antifungal drugs and vaccines alike (36–40). Third, the route of adoptive transfer in neonates was intraperitoneal (i.p), rather than intramuscular that has been the preferred route in our previous preclinical investigations and in clinical trials (18, 20, 32). The anatomy of pups makes i.p the most relevant route to evaluate efficacy of transferred antibodies, and has been extensively reported in the area of neonatal vaccinology (41, 42). In fact, i.p administration of vaccines has long been used and studied as an experimental immunization route for the induction of systemic and mucosal immunity in animal models of vaccination (43).

Adoptive transfer of antisera collected from rAls3p-N or rHyr1p-N vaccinated mice, into three-day old pups, significantly protected the infants from candidiasis by >1- to 1.5-fold, versus placebo vaccinated pups (antisera of mice treated with CFA/IFA alone). Administration of passive antibodies to protect from neonatal infections has shown tremendous promise. Passive transfer of IgG promotes opsonic activity and antibody dependent cytotoxicity, activates complement, and improves neutrophilic chemotaxis (44). Case reports from several clinical trials have indicated that antenatal administration of anti-CMV IgG may be associated with significantly favorable outcomes (a rate of 16:1) in fetuses suspected of having congenital CMV infection (45–49). Such encouraging success stories warrant further development of passive antibody transfer approaches for protection of VLBW neonates against disseminated candidiasis, the incidence of which is several fold higher than CMV.

Adoptive transfer of CFA/IFA-rHyr1p-N antisera however was not as effective in neutrophil-depleted mice. Neutrophils are a major component of the innate immune system, and the adaptive immune response in pups is absent. Thus, loss of neutrophils had a significant impact on the protective nature of the anti-rHyr1p-N antisera. We have shown previously that anti-rHyr1p-N polyclonal antibodies enhance mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro, and that neutrophils are important for protection post vaccination by Hyr1 (21, 27).

Immunotherapies are not expected to replace antibiotics, rather serve as adjunct therapies to enhance the potency of drugs. We have previously shown that anti-Hyr1 antisera synergizes with antibiotics for prevention of the Gram-negative bacteria Acinetobacter baumannii growth in vitro ( 21 ). Thus, we evaluated the potential of the CFA/IFA-rHyr1p-N sera to synergize with fluconazole, in protecting mice from candidiasis. Reassuringly, the antisera was not antagonistic when used in combination with fluconazole and could significantly abrogate infection by fluconazole resistant strains of Candida, indicating that passive vaccination strategy using anti-rHyr1p-N antibodies could be an important strategy to strengthen therapeutic attempts to combat candidiasis in neonates.

Ours is the first step in the direction of discovering immunotherapeutic strategies for combating life-threatening Candida infections in newborns. Our findings collectively highlight the potential of using rAls3p-N and/or rHyr1p-N based maternal vaccine strategies, or antibody-based immunotherapies, for protection from debilitating and lethal candidiasis in neonates. In future, FDA-approved commercially available adjuvants with profiles of antibody isotypes similar to CFA/IFA could be used in conjunction with the antigens for protection against neonatal candidiasis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

All mice procedures were approved by the IACUC of The Lundquist Institute at Harbor-UCLA Medical Center, according to the NIH guidelines for animal housing and care.

SS: Designing, implementation, troubleshooting, editing and writing parts of the manuscript; SN: Implementation, data analysis, editing manuscript; AB: Contributed to carry out in vitro ELISA assays; AI: Designing, providing proprietary antigens, discussions, writing and editing manuscript to final completion; PU: Conceptualization, designing, implementation, supervision, writing the manuscript. All authors contributed to the article and approved the submitted version.

We thank the funding support from NIH that helped enable our studies for this manuscript: NICHD R21HD097480 and NIAID R01AI141794 awarded to PU; NIAID 1R01AI141202-01 awarded to AI.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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