Antibodies targeting Candida albicans Als3 and Hyr1 antigens protect neonatal mice from candidiasis

Abstract

Pre-term infants in neonatal intensive care units are vulnerable to fungal sepsis. In this patient population, Candida albicans remains the predominant fungal pathogen causing high morbidity and mortality, despite antifungal therapy. Thus, new preventative/therapeutic strategies against neonatal candidiasis are needed. Previously, we have reported that vaccination with recombinant forms of the C. albicans N-termini of the cell wall proteins Als3 (rAls3p-N) and Hyr1 (rHyr1p-N) protected adult mice from disseminated candidiasis. Further, in a Phase 1b/2a NDV-3A (an rAls3p-N formulated with alum) protected women from recurrent vulvovaginal candidiasis, with anti-Als3p IgG2 isotype being a biomarker for efficacy. 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. Als3 and Hyr1 antigens when adjuvanted with complete Freund's adjuvant (CFA)/incomplete Freund's adjuvant (IFA) induced a robust antibody response with a ten-fold higher titer of IgG2, than attained by either antigen formulated with alum. Transplacental transfer of these antibodies significantly reduced fungal burden in the kidneys of mice pups, and adoptive transfer of vaccinated mothers' sera into pups displayed similar levels of protection. Neutrophils were found important for this efficacy. Finally, anti-Hyr1 antisera potentiated the activity of fluconazole in protecting from C. albicans infection. Our current studies are the first in the field to emphasize the importance of anti-Als3 and anti-Hyr1 antibodies in preventing neonatal candidiasis. Considering that Candida infections in low birthweight infants is a lethal infection, active and passive vaccination strategies using these antigens could have profound clinical relevance.

Keywords: Als3; Hyr1; antibodies; neonatal candidiasis; neonates; vaccine.

Figure 1

Active vaccination of Female BALB/c mice induce robust antibody titers that cross through placenta to induce protection in their newborn mice against C. albicans infection. Female BALB/c mice (6-weeks old) were vaccinated with Als3p-N or Hyr1p-N vaccine adjuvanted with CFA/IFA or adjuvant alone (placebo group) on day 0, 21 and 35. On day 35, mice were mated and kept in cages until the pups were delivered. Sera were obtained on day 6 post-partum both from mothers and their pups for antibody determination (A-C). Als3p-N vaccinated female BALB/c mice (A) and Hyr1p-N vaccinated mice (B) induce robust total IgG and IgG isotype and IgA antibodies, which efficiently transferred (equivalent titers) to their pups (except IgA). (C) CFA/IFA adjuvanted Hyr1p-N vaccine showed similar IgG1 and IgA isotype antibodies, but higher levels of IgG2a in CFA/IFA vaccinated mice. (D) Fungal burden in kidneys harvested from 3 day old pups delivered from mothers vaccinated with placebo, CFA/IFA or rAls3p-N or rHyr1p-N + CFA/IFA, three days after infecting with C. albicans (3 x 10⁷ cells). **P <0.01 treatment vs. placebo sera vaccinated mice.

Figure 2

Passive vaccination by rAls3p-N or rHyr1p-N antisera protect pups from candidiasis. (A) Fungal burden in kidneys harvested from pups infected with C. albicans on day 3 of birth, and treated with sera obtained from mice vaccinated with rAls3p-N, rHyr1p-N or adjuvant alone (placebo). Treatment was on day 0 (4 h post infection) and day 2 relative to infection. (B) Histopathology during systemic C. albicans infection in pups. Sections stained with PAS stain. Arrows indicate C. albicans hyphae invaded kidney tissues. Scale bar = 10 µm. (C) Kidney fungal burden in pups infected with a fluconazole (FLC) sensitive strain SC5314 and treated with control placebo sera (n= 8 pups), rHry1p-N anti-sera (n= 10 pups), or FLC alone (n= 9 pups), or a combination of rHyr1p-N+FLC (n= 12 pups). (D) Kidney fungal burden in pups infected with a FLC resistant strain CA6 and treated with control placebo sera (n= 8 pups), rHry1p-N antisera (n= 6 pups), FLC alone (n= 8 pups), or a combination of rHyr1p-N + FLC (n= 9 pups). (E) Kidney fungal burden in pups depleted of neutrophils, infected with C. albicans and treated with either the sera from placebo vaccinated mice (n= 7 pups) or rHyr1p-N anti-sera (n= 7 pups). **P <0.01 treatment vs. placebo sera vaccinated mice.