Relative Contribution of Dengue IgG Antibodies Acquired during Gestation or Breastfeeding in Mediating Dengue Disease Enhancement and Protection in Type I Interferon Receptor-Deficient Mice

Abstract

Dengue virus (DENV) causes a spectrum of diseases ranging from self-limiting dengue fever to severe conditions such as haemorrhagic fever and dengue shock syndrome. Antibody-dependent enhancement (ADE) is thought to explain the occurrence of severe dengue whereby pre-existing binding but non-neutralising antibodies enhance DENV infection. The ADE phenomenon is supported by epidemiological findings that infants that born to dengue immune mothers are at greater risk to develop severe dengue upon primary infection. The role of maternally acquired dengue-specific antibodies in disease enhancement was recently recapitulated in a mouse model where mice born to DENV1-immune mothers experienced enhanced disease severity upon DENV2 infection. Here, this study investigates the relative contribution of maternal dengue-specific antibodies acquired during gestation and breastfeeding in dengue disease. Using a surrogate breastfeeding mother experimental approach, we showed that majority of the maternal dengue-specific antibodies were acquired during breastfeeding and conferred an extended enhancement window. On the other hand, in the context of homologous infection, breastfeeding conferred protection. Furthermore, measurement of dengue-specific antibody titres over time in mice born to dengue immune mothers revealed a biphasic pattern of antibody decay as reported in humans. Our work provides evidence of the potential contribution of breast milk-acquired dengue-specific IgG antibodies in enhancement and protection against dengue. Should such contribution be established in humans as well, it may have important implications for the development of guidelines to dengue-immune breastfeeding mothers.

Fig 1. The breastfeeding surrogate mother experiment.

Adult female A129 mice were iv. infected with 10⁶ PFU per mouse of DENV1. One week post-infection after virus clearance, the females were mated with naïve adult males. Age-matched naïve females were also mated concurrently. At birth, pups born to either DENV1-immune or naïve mothers were switched and nursed by naïve or dengue-immune mothers respectively. Control groups comprised of mice nursed by birth mothers were also included. DD: Born to and nursed by dengue-immune mother; DN: Born to dengue-immune mother, but nursed by naïve mother; ND: Born to naïve mother, but nursed by dengue-immune mother; NN: Born to and nursed by naïve mother.

Fig 2. Maternally acquired dengue-specific IgG and IgA antibody titers.

(A) DENV1-specific IgG and (B) cross-reactive DENV2-IgG titers in sera from 5-week old A129 mice (n = 4–6) born to and nursed by DENV1-immune (DD), or born to DENV1-immune mothers and nursed by naïve mothers (DN), or born to naïve mothers and nursed by DENV1-immune mothers (ND), or born to and nursed by naïve mothers (NN). **p<0.01 based on Mann-Whitney test comparing DN and ND with respect to DD control group. (C) DENV1-specific IgA titers in sera from 5-week old mice born to and nursed by DENV1-immune or naive mothers (n = 5). UD: Undetectable. (D) DENV1-specific IgG and IgA titers in pooled sera from mothers infected with DENV1 at week 2, 4, and 6 post-infection. Dotted line denotes the limit of detection of the assay.

Fig 3. DENV2 challenge of 5-week old mice born to and/or nursed by DENV1-immune mothers.

DD, DN, ND and NN groups were iv. infected with 10⁶ PFU DENV2. (A) Survival rate, (B) clinical score of individual group (0: Healthy; 1: Ruffled fur; 2: Hunched back; 3: Diarrhoea; 4: Lethargic; 5: Moribund) (n = 8) and (C) viremia measured at day 4 post-infection (n = 5). Dotted line denotes the limit of detection in plaque assay. *p<0.05; **p<0.01 based on Mann-Whitney test compared against NN control.

Fig 4. Decay of maternal DENV1-IgG antibodies over time.

(A) DENV1-specific IgG and (B) cross-reactive DENV2-IgG measurement in sera from mice born to and/or nursed by DENV1-immune mothers (n = 4–5). Dotted line denotes the limit of detection of the assay. *p<0.05; **p<0.01 based on Mann-Whitney test.

Fig 5. DENV2 sub-lethal challenge of 10-week old A129 mice born to and/or nursed by DENV1-immune mothers.

10-week old A129 mice from DD, DN, ND and NN groups were iv. infected with 10⁶ PFU DENV2. (A) Survival rate and (B) clinical score of individual group (0: Healthy; 1: Ruffled fur; 2: Hunched back; 3: Diarrhoea; 4: Lethargic; 5: Moribund) (n = 6–10).

Fig 6. DENV2-specific IgG titres in mice born to and/or nursed by DENV2-immune mothers.

DENV2-specific IgG levels were measured by ELISA in sera from 5-week old mice born to and nursed by DENV2-immune or naïve mothers (n = 4–5). Dotted line denotes the limit of detection of the assay. *p<0.05 based on Mann-Whitney test comparing DN and ND against DD control.

Fig 7. DENV2 lethal challenge of 5-week old mice born to and/or nursed by DENV2-immune mothers.

5-week old mice were infected iv. with 10⁷ PFU DENV2. (A) Survival rate, (B) clinical score of individual group (0: Healthy; 1: Ruffled fur; 2: Hunched back; 3: Diarrhoea; 4: Lethargic; 5: Moribund) (n = 8) and (C) viremia titres were measured at 4 days post-infection (n = 5–7). Dotted line denotes the limit of detection in plaque assay. *p<0.05; **p<0.01 based on Mann-Whitney test compared against NN control.