NDV-3 protects mice from vulvovaginal candidiasis through T- and B-cell immune response

Abstract

We have previously reported that vaccination with rAls3p-N protein of Candida albicans, formulated with alum adjuvant (also designated as NDV-3) protects immunocompetent mice from, lethal disseminated candidiasis and mucosal oropharyngeal candidiasis. NDV-3 vaccine was recently, tested in a Phase 1 clinical trial and found to be safe, well-tolerated, and induced robust humoral and, cellular immune responses with increased interferon (IFN)-gamma and interleukin (IL)-17 secretion. In preparation for a Phase 2 clinical trial against vulvovaginal candidiasis (VVC), we evaluated NDV-3, efficacy in a murine VVC model. Here, NDV-3 induced a strong immune response characterized by high, anti-rAls3p-N serum IgG and vaginal IgA titers. Furthermore, moderate doses of the vaccine (a range of 1-30μg given subcutaneously [SQ] or 0.3-10μg given intramuscularly [IM]) elicited a 10-1000 fold, decrease in vaginal fungal burden vs. control (mice injected with alum adjuvant alone) in both inbred, and outbred mice infected with different clinical C. albicans isolates. Additionally, NDV-3 required both, T and B lymphocytes for efficacy in reducing C. albicans tissue burden, which is followed by a reduction, in neutrophil influx to the affected site. Finally, anti-rAls3p-N antibodies enhanced the ex vivo killing, of C. albicans by neutrophils primed with IFN-gamma. These data indicate that NDV-3 protects mice, from VVC by a mechanism that involves the concerted priming of both humoral and adaptive immune, responses.

Keywords: Als3; Candida albicans; Murine; NDV-3; VVC.

Figure 1. NDV-3 induces strong serum antibody response through intramuscular or subcutaneous vaccination

BALB/c female mice (n=10/group) were vaccinated through intramuscular (IM) or subcutaneous (SQ) injection with indicated dose of NDV-3 on Day 0. Mice receiving alum alone were used as controls. Mice were boosted with the same dose on Day 21. Serum samples were collected on Day 33 and anti-rAls3p-N IgG were evaluated with ELISA plates coated with rAls3p-N. Data are displayed as IgG geomean dilution corrected OD for each mouse [32]. *P < 0.0001 vs. 0.0 dose (alum without rAls3p-N). ^¥P=0.03 vs. 3.7 μg dose. ^#P=0.09 vs. 3.7 μg dose.

Figure 2. Intermediate doses of the NDV-3 vaccine protect mice against VVC

BALB/c female mice were vaccinated with indicated dose of NDV-3 on Day 0. Mice receiving alum alone were used as control. Mice were boosted with the same dose of NDV-3 on Day 21 and then challenged on day 35 with 1×10⁶ CFU of C. albicans strain SC5314. Vaginas were harvested on day 5 post infection for fungal burden determination. (A) Mice were vaccinated subcutaneously. *P <0.03 vs. mice receiving alum alone (0). n=20 mice/group except for 300 μg dose in which n=10. (B) Mice were vaccinated intramuscularly. n= 20 mice/group except for 1, 10, and 100 μg in which n=10 mice per group. *P <0.02 vs. mice receiving alum alone (0).

Figure 3. NDV-3 efficacy requires B and T lymphocytes cells

B-cell-deficient, T-cell-deficient nude, or congenic BALB/c wild-type control mice were immunized with 3 μg of NDV-3 vaccine or given alum alone as control on study days 0 and 21. Eleven days post boost (day 32), mice were treated with β-estradiol. (A) Two days after estradiol administration (day 34), 20 μL of PBS was used to collect vaginal lavage to determine the titers of IgA by ELISA plates coated with rAls3p-N. (B) Vaccinated mice were challenged with 1×10⁶ CFU C. albicans SC5314 on day 35 then vaginas were harvested on day 5 post infection for fungal burden. Data are displayed as medians ± interquartile ranges of 10 mice per group. *P<0.001 compared to all other arms. **P <0.04 vs. wild-type alum-vaccinated mice.

Figure 4. NDV-3 vaccination protects ICR mice against a high colonizer oral isolate of C. albicans

ICR female mice (n=10) were vaccinated with the indicated dose of NDV-3 on day 0, boosted with the same dose on day 21, and then infected with 1×10⁶ CFU C. albicans strain 529L on day 35. Mice that received alum alone were used as controls. Vaginas were harvested on day 5 post infection for fungal burden. Data are displayed as medians ± interquartile ranges. *P≤0.03 vs. alum.

Figure 5. NDV-3 vaccination results in fewer phagocytes being recruited to the vagina in the later time points

ICR female mice (n = 9 or 10 per group) were vaccinated SQ with NDV-3 (3 μg) or alum alone. Two weeks after the boost, mice were infected intravaginally with 1×10⁶ of C. albicans 529L. At the indicated time point, individually marked vagina was harvested, homogenized, and quantitatively cultured (A) and the MPO levels in the organ homogenates were measured by ELISA (B) Data are displayed as medians ± interquartile ranges. * P=0.03 vs. alum. Immunohistochemistry was performed on fixed sections of vagina using rat anti-mouse CEA antibody (neutrophils are stained in brown) (C) Magnification is 200x.

Figure 5. NDV-3 vaccination results in fewer phagocytes being recruited to the vagina in the later time points

ICR female mice (n = 9 or 10 per group) were vaccinated SQ with NDV-3 (3 μg) or alum alone. Two weeks after the boost, mice were infected intravaginally with 1×10⁶ of C. albicans 529L. At the indicated time point, individually marked vagina was harvested, homogenized, and quantitatively cultured (A) and the MPO levels in the organ homogenates were measured by ELISA (B) Data are displayed as medians ± interquartile ranges. * P=0.03 vs. alum. Immunohistochemistry was performed on fixed sections of vagina using rat anti-mouse CEA antibody (neutrophils are stained in brown) (C) Magnification is 200x.

Figure 6. NDV-3 protects mice from VVC by enhancing IFN-γ primed neutrophil killing via opsonophagocytosis

To study the role of neutrophils in the efficacy of NDV-3, ICR female mice (n=10 per arm) were vaccinated SQ with NDV-3 (3 μg) or alum alone on day 0, boosted with another dose on day 21 made neutropenic by cyclophosphamide-treatment on day −2 and +3 relative to infection. Mice were infected intravaginally with 1×10⁶ of C. albicans 529L. Normal mice (n ≥ 18) were used as a control (A). ^¥P =0.04 vs. alum sham-vaccinated mice. To study the effect to anti-rAls3p-N antibodies, serum from mice vaccinated with alum (n=25) or NDV-3 (n=25) were pooled and adoptively transferred to naïve mice (n=10 per arm) at a low concentration (0.1 mL) or high concentration (0.5 mL) 2 hours prior to infecting with C. albicans 529L (B). To investigate the effect of IFN-γ (C) and IL-17 (D) priming of neutrophil on the anti-rAls3p-N antibodies opsonophagocytosis killing of C. albicans, mouse neutrophils were primed with different concentrations of IFN-γ or IL-17 for 1 hour prior to co-culturing with C. albicans that has been incubated with serum (5% v/v) from alum- or NDV-3-vaccinated mice for 1 hour. N=12 from three independent experiments. *P < 0.05 vs. IFN-γ alone or IFN-γ + alum-vaccinated serum. Data in all experiments are expressed as the median ± interquartile range.