The level and longevity of maternal antibodies in offspring correlate to antibody titers in pregnant mice
Adult female mice received one or two injections of seasonal trivalent influenza vaccine Inflexal which included influenza strains A/California/07/2009 (H1N1), A/Victoria/210/2009 (H3N2) and B/Brisbane/60/2008, or were mock immunized with PBS. For each of these regimens, the final immunization was given after mating, with some of the females presumed pregnant (see Fig. 1a for schematic representation). Serum samples were taken weekly from both pregnant and non-pregnant females, and pups from 1 day after delivery. At the age of 3 weeks pups were weaned and separated from their mothers (see design in Fig. 1a). The serum samples were analyzed for the presence of vaccine homologous H1 Hemagglutinin (HA) antibodies in an ELISA assay (see Additional file 1: Figure S1 for the longevity of antibodies in mothers).
In agreement with previous findings in humans [34], immune responses in pregnant females were lower than in non-pregnant female mice (p = 0.016, see Additional file 1: Figure S2). Prime/boost immunization trends towards higher titers in dams than prime only (see Additional file 1: Figure S1). This trend of influenza-specific maternal antibodies can also been seen in the pups, when dams received multiple immunizations as compared to a single immunization (Fig. 1b, no formal statistical analysis was performed on these data). The longevity of detectable maternal antibodies was correlated to level of maternal antibodies transferred, i.e. when dams received a single immunization and maternal antibodies levels were relatively low, the antibodies became undetectable within 5 weeks. In contrast, when dams received a prime-boost vaccination regimen, the maternal antibodies were still readily detectable 7 weeks after birth.
Pups were weaned at 3 weeks of age, around the time at which maternal antibodies started to decline, see Additional file 1: Figure S1, suggesting that maternal antibodies were transferred both via the milk and the placenta, as previously described [35].
To study whether maternal antibodies are able to protect offspring against influenza we challenged offspring at 7 weeks of age with a H1N1 A/Netherlands/602/09 (closely related to the H1N1 component of the vaccine). In agreement with the previous experiment, at the age of 7 weeks maternal antibodies had been cleared when dams received a single vaccination, but were detectable in all pups when dams had received a prime-boost vaccination regimen (p < 0.001 at age 7 weeks, see Fig. 1c). In addition, we tested whether the antibodies detected in the latter group would be able to neutralize virus. Remaining serum samples of five pups were tested. We found detectable neutralization titers in all these pups, see Fig. 1c. As shown in Fig. 1c, a significant proportion of mice survived challenge (65%, p < 0.001) only when mothers received two vaccinations and maternal antibodies were detectable by ELISA assay at time of challenge (p < 0.001, see Additional file 1: Figure S3 more information on change in bodyweight and clinical scoring). HAI titers were not detectable at any time prior to challenge.
Active immunization protects juvenile mice against lethal influenza challenge
Mice before the age of 6 weeks are considered immature and their immune system is not fully developed [36]. To determine from which age immunization induces a detectable IgG antibody response we vaccinated mice at the age of 1, 2 or 3 weeks, using 6 week old mice as a control with a competent immune system. Blood samples were taken 3 weeks later and analyzed for the presence of homologous influenza-specific IgG levels. We found that immunization of mice induced detectable homologous IgG titers in all pups from the age of 3 weeks, see Fig. 2a. In contrast, only 3 of the 14 pups aged 2 weeks had detectable antibody responses 3 weeks after vaccination, and none when immunized at 1 week, suggesting immune system immaturity at this age. Influenza-specific IgG titers observed in pups vaccinated at 3 weeks old were still lower than those observed in adult mice, aged 6 weeks, implying that the immune response at 3 weeks is not fully developed, or has slower kinetics.
To determine whether the antibodies induced by vaccination at 3 weeks of age can induce protection against lethal influenza challenge we inoculated the pups 4 weeks after vaccination with a lethal dose of H1N1 A/California/07/09. We observed 83% survival of pups vaccinated at the age of 3 weeks, see Fig. 2b. We found the level of homologous H1 HA ELISA antibody titers in these pups to be similar to that of pups which received maternal antibodies from dams immunized twice, see Fig. 1c. Moreover, while in actively immunized pups protection may be mediated by both cellular and humoral immunity, the level of protection we observed is also comparable to the pups from prime-boost immunized mothers (see Fig. 1c).
Maternal immunity does not lead to detectable interference with immunization of offspring
We subsequently investigated whether a level of maternal antibodies associated with direct protection would interfere with vaccine induced protection in young mice. To be able to observe whether maternal antibodies have a positive or negative impact on survival after vaccination, we first determined which dose of vaccine given to the pups elicits partial protection to influenza challenge. We vaccinated juvenile mice at the age of 3 weeks with a sub-optimal dose of vaccine and challenged 4 weeks later with a lethal dose of H1N1 A/Netherlands/602/09 influenza virus. We titrated the dose of trivalent vaccine Inflexal from 0.3 μg per HA to 0.003 μg per HA given to 3 week old pups. Overall protection decreases with a decreasing dose of vaccine, however, there is some overlap between dosages, suggesting a rather flat dose response curve (Fig. 3a). A dose of 0.01 μg/HA, which induced protection in 40% of the pups in absence of detectable HA ELISA titers, was selected for subsequent studies.
A vaccine dose of 0.01 μg/HA was given to pups at 3 weeks of age in absence or presence of maternal antibodies induced by either one or two immunizations, and subsequently challenged with influenza virus 4 weeks later, see Fig. 3b. In agreement with the previous experiment, see Fig. 3a, this dose of vaccine induced survival in approximately 40% of the pups in absence of detectable HA ELISA titers (p = 0.031).
No difference in protection of the offspring was observed when the mothers were vaccinated once during pregnancy, and maternal antibodies were present at time of vaccination of the offspring but had waned at time of challenge (42% survival in this group, see Fig. 3b).
On the other hand, two immunizations of pregnant females in combination with vaccination of offspring resulted in significant HA ELISA titers at time of influenza challenge (p < 0.001) and induced protection (as defined by survival) in 65% of the offspring (p < 0.001). This survival proportion is comparable to that seen without vaccination of the offspring (60%, see Fig. 1c), suggesting that vaccination of the offspring with a low dose of vaccine does not impact the protection transferred by maternal antibodies.
These findings confirm the ability of maternal antibodies to confer protection against lethal influenza challenge. No additional protection was gained by a suboptimal dose of vaccine given to the offspring when maternal antibodies were present at time of challenge. While these results do not formally exclude immuno-interference by maternal antibodies, they show that maternal antibodies can confer protection, even when combined with juvenile vaccination.
Three maternal immunizations induce a high level of maternal antibodies in the offspring and confer protection against influenza to all offspring
Results described up to this point indicate that maternal antibodies can confer protection against lethal influenza challenge irrespective of vaccination of the offspring with a suboptimal dose of vaccine (Fig. 3). To test whether the survival of offspring by maternal antibodies can be further increased, we repeated the experiment above and included a group which received three immunizations. As seen in Fig. 4, mothers were given either 1, 2 or 3 vaccinations of which the final immunization was given 1 week after mating. Pups were vaccinated at the age of 3 weeks with a suboptimal dose of vaccine, and challenged 4 weeks later with a lethal dose of H1N1 A/Netherlands/602/09.
In agreement with the previous experiment in Fig. 3b, vaccination of mothers during pregnancy transferred maternal antibodies to the offspring and are present during vaccination of the offspring at the age of 3 weeks, see Fig. 4b. And again, in agreement with the previous experiment, these HA binding maternal antibodies had waned in the offspring at the age of 7 weeks when the mothers had received a single vaccination but were still detectable in all the offspring when mothers received two vaccinations. When offspring was subsequently challenged with a lethal dose of influenza virus at the age of 7 weeks, the level of survival of pups from dams that were immunized twice trended to be higher than that of pups from dams that were immunized only once (85 and 55% resp.). Also, the protection of offspring observed in this experiment is comparable to that observed in the previous experiment (see Fig. 3b: 65% survival in offspring from mothers vaccinated twice and 42% survival in offspring from mothers which received one vaccination). In addition to the previous experiment we included a group of which the mothers received three vaccinations during pregnancy. Interestingly, three immunizations of the mothers, in combination with immunization of the pups at 3 weeks of age with a suboptimal dose of vaccine, further increased the level of homologous HA-binding antibodies in the pups. Even more interesting was the observation that three vaccinations of mothers during pregnancy was able to confer protection against lethal influenza virus challenge to all offspring compared to mock immunization (p < 0.001), see Fig. 4b. These results indicate that multiple immunizations of mothers prior to delivery correlate to the level of homologous antibodies found in the offspring and to passive protection transferred against lethal influenza challenge.