Maternal antibodies protect offspring from severe influenza infection and do not lead to detectable interference with subsequent offspring immunization

Immunization and mating of mice

Thirteen-week-old female mice (BALB/c, Charles River) were subcutaneously (s.c.) immunized in the scruff of the neck with 100 μl of seasonal trivalent vaccine (Inflexal) containing 3 μg HA per influenza strain, at an interval of 3 weeks when receiving multiple immunizations. One day before each immunization, a pre-immunization blood sample was obtained via submandibular bleeding to assess immunization efficacy. One week prior to the last immunization the female mice were mated with unimmunized male BALB/c mice, aged 17 to 37 weeks, two females to one male in the same cage. Females were separated from the males when a mating plug was observed, or after 4 nights. Three weeks after mating most female mice gave birth and pups of both sexes were randomly distributed within treatment groups. Infant mice were subcutaneously immunized in the scruff of the neck with 50 μl PBS or seasonal trivalent vaccine (Inflexal), diluted in PBS when applicable. Infant mice were weaned and separated from their mothers at the age of 3 weeks before immunization.

Influenza challenge

At the age of 7 weeks offspring was challenged an H1N1 influenza strain. A stock of H1N1 A/Netherlands/602/2009 (Viroclinics) was grown on embryonated chicken eggs. Anaesthetized mice were challenged intranasally with 25xLD50 virus in a total volume of 50 μl, 25 μl per nostril. Groups of mice (n = 20 and n = 8) receiving either PBS or a broadly protective monoclonal antibody (CR6261, 15 mg/kg in PBS) intramuscularly were used as negative and positive controls for challenge models, respectively. Challenges were considered valid when there was a statistically significant difference in survival proportion (Fisher’s exact-test, 2-sided) between the control groups (data not shown). Bodyweight and clinical score were monitored daily for up to 21 days or until a humane endpoint to limit animal discomfort.

Humane endpoint was defined based on clinical score as is established practice [31–33]. Our experience with Influenza challenge models suggests that using alternative humane endpoints, such as bodyweight-loss would lead to underestimation of protection. The amount of bodyweight-loss before animals reach clinical score 4 is variable. We explicitly discussed this issue with our Ethical Review Board, and they agreed to allow clinical score 4 as a humane endpoint. Clinical scores were defined as: 0 = no clinical signs, 1 = rough coat, 2 = rough coat, less reactive, passive during handling, 3 = rough coat, rolled up, labored breathing, passive during handling, 4 = humane endpoint: rough coat, rolled up, labored breathing and unresponsive. All mouse studies were approved by DEC Consult (Independent ethical institutional review board).

Repeated measurements in the challenge phase (bodyweight and clinical scores) were summarized as a single outcome per animal using an area under the curve (AUC) approach where missing values for animals that died early were imputed with a last-observation-carried-forward method. See Additional file 1: Figures. S3, S4 and S5 for changes in bodyweight and observed clinical scores.

recH1 a/California/07/2009 ELISA

Multisorp (Nunc) 96-well flat bottom plates were coated overnight with rH1 A/California/07/2009 (Protein Science Inc., 0.5 μg/ml), washed and blocked. Wells were incubated with duplicate serial 2-fold dilutions with a start dilution of 1:50 of mouse serum in block buffer (2% skimmed milk in PBS) for 1 h at room temperature, and washed. Wells were incubated with anti-human IgG for the detection of the human monoclonal CR6261 in the positive control group(Jackson ImmunoResearch), or anti-mouse IgG (KPL) conjugated to HRP for 1 h at room temperature, washed and developed using OPD substrate (Thermo Fischer Scientific). OD was read at 492 nm using a Powerwave Synergy plate reader (Biotec), and compared to the standard curve of CR9114 (produced in house), for calculation of ELISA units using a slope based weighted average approach: The OD of each sample dilution was quantified against the standard curve of CR9114 included on each plate. The final concentration per sample was calculated by a weighted average, using the squared slope of the standard curve at the location of each quantification as weight. Negative samples were set at the limit of detection (LOD), defined as the lowest sample dilution multiplied by the lowest standard concentration, with an OD response above the lower asymptote of the standard curve and background. ELISA titers were expressed as log10 ELISA Units (EU) per ml, and are not intended to infer amounts in micrograms. CR9114 is a monoclonal antibody binding to a conserved epitope found on the stem region of group 1 and 2 influenza A viruses and influenza B viruses [31].

Pseudoparticle neutralization assay

The PhenoSense® neutralization assay using pseudoparticles was conducted at Monogram Biosciences, Inc. The assay uses a single infection round of HEK293cells with replication deficient retroviral vector encoding a firefly luciferase indicator gene and HA and NA A/California/07/09 expression vectors. A fourth expression vector containing a human airway serine protease used in processing the HA protein, TMPRSS2, is also transfected. The resultant pseudoviruses are harvested from culture supernatants, filtered, titered and stored at -80 °C. The pseudovirus stocks, at a concentration giving approximately 30,000–300,000 relative light units (RLU) per well, were incubated at 37 °C for one hour with 3 fold serial dilutions of test antibody (i.e. serum or control mAbs) in a 96 well plate starting at a 1:50 dilution. HEK293 cells are then added to each well and incubated at 37 °C in 5% CO2 for 3 days. Luciferase substrate and cell lysing reagents are added to the plates which are read on a luminometer. Data were provided as half-maximal inhibitory concentration (IC50) values by Monogram Biosciences and are reported as such: Inhibition curves are defined by a four-parameter sigmoidal function and are fit to the data by nonlinear least-squares and bootstrapping. The ability of antibody in the serum to neutralize Influenza infectivity is assessed by measuring luciferase activity in the culture 72 h after viral inoculation as compared to a control infection using a murine leukemia virus envelope (aMLV) pseudotyped virus. Neutralization titers are expressed as the reciprocal of the serum dilution that inhibited the virus infection by 50%. A reaction is called positive for neutralization when there is at least 50% inhibition of infection of an Influenza strain and when there is an IC50 at least 3 fold higher than the IC50, if any, of the same sample tested with the specificity control, aMLV env.

Hemagglutination inhibition assay

Nonspecific hemagglutination inhibitors were removed from sera by O/N incubation with Cholera Toxin (Sigma-Aldrich; St. Louis, MO, USA; diluted 1:25 in PBS) at 37 °C in presence of turkey red blood cells (bioTRADING Benelux B.V., Mijdrecht, the Netherlands, 0.5% in PBS). Cholera toxin was subsequently inactivated by heat at 56 °C for 30 min. Sera were tested in duplicate 2-fold serial dilutions with an initial dilution of 1:8. Diluted sera were mixed with 8 HA units of H1N1 A/California/07/09 (reassortant NYMC X-181) for 1 h at RT. Turkey red blood cells (1% in PBS) were added to the serum and incubated for 1 h at room temperature (RT). Confirmed positive and negative sera were used as assay controls. HAI titer is expressed as reciprocal of the highest dilution that completely inhibited hemagglutination.

Statistical methods

In the challenge studies, differences in survival have been analyzed by exact logistic regression while accounting for the correlation of pups from the same nest. Differences in survival duration have been tested as a rank test by Cochran-Mantel-Hänszel test with the nesting included as covariable. The clinical scores have been analyzed as cumulative logistics models f score classes with nest as random factor. The body weight has been analyzed as mixed model of the AUC of the difference in weight compared to baseline with treatment as fixed factor and nest as random factor.

In all challenge studies, first a gatekeeper test was performed: the group of which both mother and pups received mock immunization compared the positive challenge control group which received monoclonal anti-influenza antibody CR6261. The studies in Figs. 1c and 3b a step-wise test approach was used: the contrast between the group of which both mothers and pups received TIV immunization compared to both receiving a mock immunization was the primary contrast (without adjustment for multiple testing). If significant all remaining contrasts between groups were tested simultaneously with Bonferroni adjustment for testing 5 contrasts. In the studies in Figs. 3a and 4b (after the gatekeeper) a step-wise adjustment of p values was used going from the highest to the lowest dose (3A) or from the most to the least injections (4B).

The immunogenicity results were tested by t-test (2 groups) or anova (multiple groups) in case of normal distributed results or by (a set of) Mann-Whitney Rank test(s) if a considerable proportion of the results was at LOD. P values from the study in Fig. 2b have been Dunnett adjusted for multiple testing for comparison of set of test groups with one reference group. P values from the studies in Figs. 1c and 3b have been Tukey adjusted (anova) or Šidak adjusted (Mann-Whitney) for testing all pair-wise comparisons.

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.