Fig 1.
Functional mRNA-encoded ZAC-3 IgG1 and IgA2 in serum.
(A) Cartoon depiction of experimental timeline. Adult female SW mice were intravenously administered ZAC-3 encoding mRNA-LNP (1 mg/kg) or PBS. Serum samples were collected 24, 48, 72, and 168 h later. (B) Total hIgG (µg/mL) in serum at the indicated time points, as determined by an isotype-specific ELISA. (C) V. cholerae C6706 specific IgG binding was assessed by whole-cell ELISA (serum used at a 1:50 dilution corresponding to hIgG concentrations of 3, 4, 3 and 2.5 µg/mL at 24, 48, 72 and 168 h, respectively. (D) Total hIgA (µg/mL) in serum at the indicated time points, as determined by an isotype-specific ELISA. (E) V. cholerae C6706 specific IgA binding was assessed by whole-cell ELISA (serum used at a 1:50 dilution corresponding to hIgA concentrations of 0.2, 0.2, 0.16 and 0 µg/mL at 24, 48, 72 and 168 h, respectively. For panels B, C, D, and E each symbol (gray or pink circles) represents a single mouse. The bars represent the mean ± SD per group (n = 10 mice for experimental groups; n = 4 mice for control groups). (F) Complement-dependent vibriocidal activity in corresponding pooled serum samples from indicated time points, as described in the Methods section. The dashed line represents the average CFUs recovered from control sera at equivalent dilutions to experimental samples. Panel A created in BioRender: Adewunmi, Y. (2026) https://BioRender.com/ifii944.
Fig 2.
Mouse lactation status influences mRNA-LNP encoded ZAC-3 IgG and IgA serum levels.
Lactating and non-lactating SW mice were intravenously administered LNPs (1 mg/kg) or PBS on postpartum days 3-7. (A) Cartoon depicting experimental design in which serum was collected at 24, 48, 72, and 168 h. (B) Serum hIgG levels in naïve (lavender circles), lactating (red circles) and non-lactating (gray circles) mice, as determined using an isotype-specific ELISA, at indicated time points. Each symbol indicates a single mouse. (C) Serum hIgA levels in naïve (lavender circles), lactating (red circles) and non-lactating (gray circles) mice, as determined using an isotype-specific ELISA, at indicated time points. Each symbol indicates a single mouse. (D) Complement-dependent, vibriocidal activity of serum samples within naïve (lavender circles), lactating (red circles) and non-lactating (gray circles) mice. V. cholerae C6706, expressed as Log₁₀ CFU reduction. Pooled serum samples used in the vibriocidal assay were taken at the same time points and at concentrations corresponding to the average hIgG levels shown in panel B. Each symbol represents one pooled sample per time point. Bars represent mean ± SD per group (n = 4 mice per group). Panel A created in BioRender: Adewunmi, Y. (2026) https://BioRender.com/2aphrkh.
Fig 3.
Functional ZAC-3 IgG1 and IgA in breast milk.
SW mice were intravenously administered 1 mg/kg of LNPs or PBS 3-7 days after giving birth. (A) Cartoon depicting experimental design in which milk was collected at 24, 48, 72, and 168 h. (B) Concentration of hIgG detected in mice breast milk, as determined by an isotype-specific ELISA. Milk was collected at the indicated time points from three individual lactating dams (indicated by circle, square and triangle). (C) V. cholerae-specific IgG in milk was assessed by whole-cell ELISA. Milk collected at 24, 48, 72, and 96 h was tested at a fixed 1:4 dilution, which correspond to 0.2, 0.2, 0.25, and 0.3 µg/mL of ZAC-3 IgG, respectively. (D) Concentration of hIgA2 detected in mice breast milk, as determined by an isotype-specific ELISA. Milk was collected at the indicated time points from three individual lactating dams (indicated by circle, square and triangle). (E) V. cholerae-specific IgA2 in milk was assessed by whole-cell ELISA. Milk collected at 24, 48, 72, and 96 h was tested at a fixed 1:4 dilution, which correspond to 0.1, 0.4, 0.08, and 0.1 µg/mL of ZAC-3 IgA2, respectively. (F) Vibriocidal activity of pooled milk samples from each dam. The concentration of ZAC-3 IgG in the pooled samples corresponding to the average hIgG levels shown in panel B. Each group represents the mean ± SD of 2-3 mice per group. Panel A created in BioRender: Adewunmi, Y. (2026) https://BioRender.com/ea34xua.
Fig 4.
Maternally-derived ZAC-3 IgG accumulates in the serum of suckling mice.
ZAC-3 mRNA-LNPs (1 mg/kg) or PBS was administered intravenously to dams 3-7 days after giving birth. (A) Cartoon depicting experimental design in which serum was collected from pups at 24, 48, 72, and 168 h from four different experimental litters and two naïve litters. The day of mRNA-LNP administration was considered time 0. (B) Human IgG in serum, as determined using an isotype-specific capture ELISA. For each time point, a symbol represents values from two pups. (C) Vibriocidal activity of pup sera over the course of 168 h. For each time point, sera from all litters were pooled and used in the vibriocidal assay with average concentrations corresponding to hIgG concentrations of 1.5, 2, 2, and 2.5 µg/mL at 24, 48, 72, and 96 h, as described previously. Pools were from 2 mice per litter with two naïve litters and four ZAC-3 mRNA-LNP litters. Panel A created in BioRender: Adewunmi, Y. (2026) https://BioRender.com/8g0r3s2.
Fig 5.
Capacity of ZAC-3 IgG1 and IgA to reduce V. cholerae colonization.
(A) Lactating SW mice were intravenously administered with LNPs (1 mg/kg; N = 3) or PBS (N = 2) 3-7 days after giving birth. This was designated as study time 0. 48 h post-administration, breast-fed pups were intragastrically challenged with V. cholerae (1 x 105 CFUs). Serum and milk were collected from ZAC-3 treated (n = 3) or naïve dams (n = 2) at 48 h, while serum from all pups (n = 10/group) was collected at 72 h. Pups were euthanized and small and large intestines were harvested, homogenized, and plated for CFUs (n = 5). (B) Data represents the concentration of hIgG in dam serum (pink bars with red circle symbols), milk (white bars with white circle symbols) and pup serum (pink bars with red diamond symbols), assessed using isotype-specific ELISA. Dam serum and milk samples are denoted by “D” under the appropriate bars and pup serum samples are denoted by “P”. (C) Data represents recovered CFUs of V. cholerae in the intestinal homogenates from each litter in the ZAC-3 IgG treatment and naïve groups. (D) Data represents the concentration of hIgA in dam serum (pink bars with red circle symbols), milk (white bars with white circle symbols) and pup serum (pink bars with red diamond symbols), assessed using isotype-specific ELISA. Dam serum and milk samples are denoted by “D” under the appropriate bars and pup serum samples are denoted by “P”. (E) Data represents recovered CFUs of V. cholerae in the intestinal homogenates from each litter in the ZAC-3 IgA treatment and naïve groups. Bars for dams represent an individual animal, while bars for pups show the mean ± SD of the group. Panel A was created in BioRender. Adewunmi, Y. (2026) https://BioRender.com/s16efvl.
Fig 6.
Continuous breastfeeding improves V. cholerae reduction in pup guts.
(A) Lactating SW mice were intravenously administered with LNPs (1 mg/kg; N = 3) or PBS (N = 2) 3-7 days after giving birth. This was designated as study time 0. 48 h post-administration, breast-fed pups were intragastrically challenged with V. cholerae (1 x 105 CFUs). (B) Data represents recovered CFUs of V. cholerae in the intestinal homogenates from each pup in the ZAC-3 IgG treatment (n = 46) and naïve groups (n = 11) across two independent experiments. (C) Data represents recovered CFUs of V. cholerae in the intestinal homogenates from each pup in the ZAC-3 IgA treatment (n = 34) and naïve groups (n = 11) across two independent experiments. Statistical analysis was performed using unpaired t test without Welch’s correction after confirming that variances were equal. Panel A was created in BioRender: Adewunmi, Y. (2026) https://BioRender.com/7tgdxtz.