Use of a Neonatal-Mouse Model to Characterize Vaccines and Strategies for Overcoming the High Susceptibility and Severity of Pertussis in Early Life

Abstract

Newborns and unvaccinated infants, compared to other age groups, are more susceptible to pertussis infection, manifesting severe symptoms leading to a higher mortality. The recent increase in pertussis cases demands more effective strategies to overcome this major health problem. In parallel with maternal-immunization, neonatal-immunization (NI) is a strategy needing revision. Here, using the intranasal-challenge-mouse-model we evaluated the protective capacity of NI in both naïve-mice and those with maternally acquired immunity. We tested our acellular-vaccine-candidate based on outer-membrane-vesicles derived from Bordetella pertussis (OMVP) that induces Th2-profile but also the recommended Th-profile for protection: Th1/Th17-profile and CD4 T-memory-cells that reside in the lungs. Commercial acellular-vaccine (aP) and whole cell-vaccine (wP) inducing mainly Th2-profile and Th1-profile, respectively, were also tested. Analyzing the induced immunity and protection capability of NI included in 1- or 2-dose schedules with the same or different types of vaccine, we detected that the aP-vaccine administered in either single- or 2-dose schedules protected against sublethal B. pertussis infection. Schedules consisting of doses of aP neonatally and of OMVP or wP vaccine during infancy greatly reduced bacterial lung colonization while inducing the highest levels of high-avidity anti-pertussis toxin (PTx) IgG. That OMVP or wP neonatal dose did not interfere with the protection of transferred maternal immunity was especially encouraging. Moreover, OMVP- or wP used as a neonatal dose enhanced the quality of the humoral immune response in immunized pups. Antibodies generated by OMVP-or wP-vaccinated mice born to aP-immunized mothers were of higher avidity than those from mice that harbored only maternal immunity; but when mothers and neonates were immunized with the same aP-vaccine, the humoral response in the neonates was partially suppressed through the blunting of the level of anti-PTx IgG induced by the neonatal aP dose. These results demonstrated that neonatal immunization is a possible strategy to be considered to improve the current pertussis epidemiology. For neonates without maternal-immunity, mixed-vaccination schedules that include the aP- and OMVP-vaccines appear to be the most appropriate to induce protection in the pups. For offspring from immune mothers, to avoid blunting-effect, NI should be carried out with vaccines other than those applied during pregnancy.

Keywords: Bordetella pertussis; neonatal immunization; outer-membrane vesicles; pertussis; protection.

FIGURE 1

Effect of neonatal immunization on the protection of offspring against Bordetella pertussis infection. (A) Schematic representation of vaccination and challenge protocols. Seven-day-old neonatal mice were vaccinated with a commercial aP, OMVP or a commercial wP vaccines (n = 8 in each group). For the two-dose vaccination schedules consisting in two doses, the second dose (black horizontal arrow) was administered 14 days after the first dose. Mice immunized with 1 dose (gray horizontal arrow) or 2 doses (black arrow) were challenged with B. pertussis at 28 and 35 days after birth, respectively. Non-immunized mice of the same age (used as a negative control for protection) were also challenged with B. pertussis. (B) Protection of offspring through the vaccination schedules of (A). The number of bacteria recovered from the mouse lungs, expressed as the log₁₀(CFUs per lung), is plotted on the ordinate for the type and vaccine schedule indicated on the abscissa, with the data representing the means ± the SD. The dotted horizontal line demarcates the lower limit of detection. ^∗∗∗p < 0.001 for both the non-immunized mice versus the 1-dose–immunized mice and the aP-immunized mice versus all the other treatments performed for the 2-dose–schedule assays.

FIGURE 2

Effect of neonatal immunization on the first dose administered during the infancy. The infant doses were administered in both immunized or non-immunized mice at 21 days during the neonatal period in either the presence or the absence of a neonatal dose at 7 days of age. In the neonates immunized (upper indications on the abscissa), the type of vaccine used was the same as that administered during infancy (lower indications on the abscissa). The non-immunized mice were used as a negative control of protection. All the groups of mice (n = 8) were challenged with B. pertussis at 35 days after birth. The number of bacteria recovered from mouse lungs, expressed as the log₁₀(CFUs per lung), is plotted on the ordinate for the type and vaccine schedule indicated on the abscissa, with the data representing the means ± the SD. The dotted horizontal line indicates the lower limit of detection. ^∗∗∗p < 0.001, ^∗∗p < 0.05.

FIGURE 3

Effect of 2-dose schedules including neonatal immunization on protection of offspring against B. pertussis infection. (A) Schematic representation of vaccination and challenge protocols. Seven-day-old neonatal mice were vaccinated with a commercial aP, OMVP, or a commercial wP vaccine (n = 8 in each group). The second dose, involving a different type of vaccine from the first, was administered 14 days after the first dose. Non-immunized and aP-aP–immunized mice were used as respective negative and positive controls of protection. All the mice were then challenged with B. pertussis at 35 days after birth followed by sacrifice at 42 days. (B) Protection of offspring through the vaccination schedules of (A). The number of bacteria recovered from mouse lungs, expressed as the log₁₀ of the CFUs per lung, is plotted on the ordinate for the combination of vaccine types indicated on the abscissa, with the data representing the means ± the SD. The dotted horizontal line demarcates the lower limit of detection. ^∗∗∗p < 0.001, ^∗∗p < 0.05.

FIGURE 4

Anti-Ptx antibodies induced by 2-dose vaccination schedules. (A) Anti-PTx IgG titers along with the IgG isotypes were measured 14 days after the second vaccination dose. The titers are expressed as the geometric mean of the data from each group. The cut-off levels for IgG, IgG1, and IgG2a assays were 12.5 ± 3.6, 1.9 ± 0.9, and 5.8 ± 2.7, respectively. No values were detected in the non-immunized control animals. The values detected were significantly different from those of the non-immunized mice (p < 0.05). The avidity of IgG antibodies was also measured by ammonium thiocyanate (NH₄SCH) elution 14 days after the second dose and is expressed as the percentage of PTx-specific antibodies eluted. (B) Immunoblotting of purified PTx separated by 12.5% (w/v) SDS-PAGE and probed with polyclonal sera obtained from immunized mice. The sera are designated according to the vaccination schedule used to raise the immune response in the donor mice.

FIGURE 5

Effect of neonatal immunization on the protection of pups with maternal immunity against B. pertussis infection. (A) Schematic representation of the vaccination and challenge protocols. Seven-day-old neonatal mice born either 3 weeks or later than 2.5 months after maternal aP immunization were vaccinated with a commercial aP or wP or the OMVP vaccine (n = 6 in each group). Non-immunized mice were used as a negative control for protection. All the groups were challenged with B. pertussis at 28 days after birth. (B) Protection of offspring through the vaccination schedules of (A). The number of bacteria recovered from the mouse lungs, expressed as the log₁₀ of the CFUs per lung, is plotted on the ordinate for the treatments indicated on the abscissa, with the data representing the means ± the SD. The dotted horizontal line indicates the lower limit of detection. The left side of the panel depicts the CFU values for the different groups of mice born 3 weeks after the last maternal vaccination, while the right side contains the corresponding data for the same experimental groups born 2.5 months after that last maternal vaccination. ^∗∗∗p < 0.001 non-immunized mice versus immunized Ipups. A significant difference (p < 0.05) was also recorded between the CFU values detected in the aP-vaccinated Ipups born 2.5 months after maternal immunization and those values detected in the non-vaccinated and vaccinated Ipups born 3 weeks after the completion of maternal immunization.

FIGURE 6

Anti-Ptx antibodies in pups born to aP immunized mothers. (A) Anti-PTx IgG titers and the IgG1 isotype were measured at 21 days of life in the Ipups. The titers are expressed as the geometric mean of the data from each group. No values were detected in pups born to non-immunized mothers used as the control group (not indicated in the figure). The values detected in the aP-immunized Ipups were significantly lower (p < 0.05) than those of the non-immunized mice. The cut-off levels for IgG and IgG1 assays were 12.5 ± 3.6 and 1.99 ± 0.98, respectively. The avidity of the IgG antibodies was also measured in the Ipups at 21 days of life, as indicated by the percentages of PTx-specific antibodies eluted after treatment with increasing concentrations of ammonium thiocyanate (NH₄SCN). (B) Immunoblotting of purified PTx separated by 12.5% (w/v) SDS-PAGE and probed with the polyclonal antisera obtained from immunized mice. The sera are designated according to the vaccination schedule used to raise the immune response in mice.