A single immunization with inactivated H1N1 influenza vaccine formulated with delta inulin adjuvant (Advax™) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection

Abstract

Pregnant women and neonates represent high-risk groups for influenza infection, and in general have suppressed responses to standard influenza vaccines due to pregnancy-associated immune suppression and immune system immaturity, respectively. We therefore wished to test whether addition of Advax™, a polysaccharide adjuvant based on delta inulin, to an inactivated influenza vaccine (A/H1N1/PR8) administered during pregnancy would safely enhance vaccine immunogenicity and thereby provide improved protection of pregnant mothers and their newborns. Pregnant mice received a single intramuscular injection of β-propiolactone-inactivated H1N1 antigen alone or with Advax adjuvant. Pregnant dams receiving Advax-adjuvanted vaccine exhibited significantly increased serum and breast milk anti-influenza IgG titers. This translated into higher serum anti-influenza IgG titers in the pups of these dams. Complete protection was seen in pups of dams that received Advax-adjuvanted vaccine whereas no survival was seen in pups of control mothers or mothers immunized with unadjuvanted influenza vaccine. Cross-fostering studies confirmed that enhanced protection of pups of dams that received Advax-adjuvanted vaccine was mediated by enhanced transfer of maternal IgG to the pups via breast-feeding. The delta inulin adjuvant was not associated with any reproductive or developmental adverse effects. This study shows that Advax adjuvant was safe when administered with influenza vaccine during pregnancy and provided protection of pups via enhanced breast milk transfer of anti-influenza antibodies, not seen with administration of unadjuvanted vaccine.

Keywords: Adjuvant; Breast feeding; Immunity; Influenza; Neonate; Pregnancy; Vaccine.

Figure 1. Advax adjuvant enhances immunogenicity and protection of a single influenza vaccine dose in virgin female mice

Female BALB/c mice (n = 10/group) were immunized i.m. with iPR8 100ng alone (white bars) or with Advax (black bars). Blood samples were collected 4 weeks post-immunization and mice then challenged with 1 × 10⁴ TCID₅₀ (8×LD₅₀) of PR8 virus Shown are; pre-challenge anti-PR8 antibodies measured by ELISA (mean + SEM) (A), post-challenge bodyweight changes (mean ± SEM) (B), post-challenge sickness score (mean ± SEM) (C), post-challenge survival rate (D), with number of survivors/number of challenged mice shown in parenthesis. +, no survivors; ns, not significant; *, p< 0.05; ***, p< 0.001. Data representative of 3 independent experiments.

Figure 2. Effect on protection of pups of immunization of pregnant dams

Pregnant BALB/c mice were injected on G7 with iPR8 5µg alone (white bars) or with Advax adjuvant (black bars). Serum anti-PR8 titers in postpartum mothers (A). Pups were challenged with 5000 TCID₅₀ PR8 virus at 4 weeks of age and shown are bodyweight changes of survivors (mean ± SEM) (B), mean bodyweight of animals at day 14 post-challenge (mean + SEM) (C), and survival rate (D), with number of survivors/number of challenged mice shown in parenthesis. **, p<0.01. Data representative of 3 independent experiments.

Figure 3. Enhanced passive immunity in offspring of dams receiving Advax-adjuvanted vaccine

Pregnant mice on G7 were injected with iPR8 5µg (white bars) or iPR8+Advax (black bars). Ten days postpartum sera and breast milk of dams and sera of infants were collected and anti-PR8 antibody levels determined (mean + SEM) (**, p<0.01; ***, p<0.001). Data representative of 3 independent experiments.

Figure 4. Effect of cross-fostering on acquisition of passive immunity by pups

Pregnant BALB/c mice (n = 3/group) were immunized on G7 with saline or iPR8+Advax. On the day of birth, offspring born to immune and naïve mothers were cross-fostered. At 4 weeks of age, pups were bled and then challenged with 5000 TCID₅₀ PR8 virus. Shown are pre-challenge anti-PR8 IgG1 and IgG2a antibodies (mean + SEM) for dams (white bars) and pups (black bars) (A), bodyweight changes (B), and sickness scores (C), of challenged pups and survival post-challenge (D); number of survivors/number of challenged mice shown in parenthesis. +, no survivors.

Figure 5. Delta inulin adjuvant does not exhibit reproductive or developmental toxicity

Pregnant dams immunized on G7 with either iPR8 antigen alone or iPR8+Advax had no significant differences in mean litter size (A), or body weight of their pups at 4 weeks of age (B), when compared to pups of sham immunized dams.