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An Investigation of Japanese Neonatal and Maternal Antibody Status against Pertussis

after a cesarean section. Infant serum was collected at the routine test within one week after delivery. Gestational age (GA) was obtained from maternal medical records and determined by ultrasound during the early phase of pregnancy. Serum pertussis toxin (PT)-specific IgG antibody levels were measured by enzyme immunoassay at SRL Limited (Tokyo, Japan) using the Pertussis Antibody Enzyme Immunoassay Kit (Denka Seiken, Co., LTD., Tokyo, Japan). The antibody levels were expressed in EU/mL and subsequently log-transformed to determine the geometric mean titers (GMTs). The SUMMARY: To clarify the pertussis immune status of the Japanese population, we investigated levels of serum pertussis toxin (PT)-specific immunoglobulin G (IgG) antibody in infants and mothers between April 2016 and March 2018. A total of 206 infants ( n = 22, < 32 weeks of gestational age [wGA]; n = 70, 32–36 wGA; n = 114, ≥ 37 wGA) and 170 mothers were enrolled. The maternal seroprevalence and antibody geometric mean titer (GMT) were 52.4% and 10.7 EU/mL, respectively. The antibody GMT, seroprevalence, and mean ratio of infant to maternal antibody titers of infants at < 32 wGA were 3.2 EU/mL, 13.6%, and 42.5%, respectively, and were significantly lower than those of infants at 32–36 wGA (9.7 EU/mL, 54.3%, and 110.2%) and infants at ≥ 37 wGA (12.1 EU/ mL, 57.9%, and 112.6%). Of the 21 infants who underwent a second examination, five were positive in the first examination. Of those five, the GMT for PT had decreased by an average of 52.6% at 4.3-week intervals. In the second examination, two infants were seropositive. Approximately half of the mothers and infants were negative for anti-PT antibody. Thus, new vaccination strategies, such as the vaccination of pregnant women, are needed to prevent pertussis infection in early infancy.


INTRODUCTION
Pertussis is a vaccine-preventable disease, the resurgence of which has been recognized in many developed countries (1)(2)(3). In Japan, the number of pertussis infections in adolescents and adults has increased since 2002 and large-scale outbreaks occurred in several universities in 2007 (4,5). Moreover, adolescents and adults with pertussis are sources of infection to infants, leading to severe and lifethreatening consequences (6,7).
The protection of infants against pertussis depends on the presence of maternal antibodies during pregnancy. Although the vaccination of pregnant women has recently been implemented as a measure against infantile infection in many countries (8)(9)(10), it is not a current practice in Japan. Childhood pertussis vaccination with diphtheria-tetanus whole-cell pertussis (DTwP) vaccine began in 1958 in Japan but was discontinued in 1975 due to serious adverse events. An acellular pertussis (aP) vaccine with fewer adverse reactions was developed in Japan (11) and the DTaP vaccine was introduced in 1981. Presently, DTaP vaccination is recommended in the third, fourth, and fifth to eleventh months of life, with a booster dose at 12 to 23 months. The vaccination coverage rate has risen to over 95% and surveys since 2010 show that most infants have been inoculated four times. However, pertussis vaccines were not hitherto administered after three years until a booster vaccination was approved in 2018. This study determined the concentrations of antibodies against pertussis antigens in neonatal and maternal blood and evaluated the efficacy of transplacental antibody transfer.

MATERIALS AND METHODS
Women and infants admitted to the neonatal intensive care unit at Konan Kosei Hospital, Aichi, Japan, between April 2016 and March 2018 were recruited. Those who provided written informed consent to participate were enrolled in the study. Only the eldest of twin infants was enrolled. Mothers who did not provide consent by the time of their routine blood test were excluded from the study. Maternal serum was collected at the routine test generally performed on the third day after a vaginal delivery or on the seventh day after a cesarean section. Infant serum was collected at the routine test within one week after delivery. Gestational age (GA) was obtained from maternal medical records and determined by ultrasound during the early phase of pregnancy. Serum pertussis toxin (PT)-specific IgG antibody levels were measured by enzyme immunoassay at SRL Limited (Tokyo, Japan) using the Pertussis Antibody Enzyme Immunoassay Kit (Denka Seiken, Co., LTD., Tokyo, Japan). The antibody levels were expressed in EU/mL and subsequently log-transformed to determine the geometric mean titers (GMTs). The same measurements were performed at 4-6 weeks of age for infants hospitalized for > 1 month. Anti-PT IgG levels ≥ 10 EU/mL were defined as positive. The placental transfer ratio was defined as the ratio of infant to maternal antibody titers and was evaluated in motherinfant pairs with maternal anti-PT IgG levels > 5 EU/ mL. This study was approved by the Konan Kosei Hospital Clinical Research Review Committee. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria) (12). Fisher's exact, Mann-Whitney U, Kruskal-Wallis, Steel-Dwass, and Holm tests were used for statistical comparisons. P < 0.05 was considered statistically significant.

RESULTS
This study enrolled a total of 206 infants and 170 mothers. Thirty-six mothers did not provide consent before their routine blood test; therefore, only their infants were included in this study. The median maternal age was 33.9 years. The maternal seroprevalence and antibody GMT for PT were 52.4% and 10.7 EU/mL, respectively, and there were no significant differences in age (P > 0.05) ( Table 1). There were also no significant differences in seroprevalence and antibody GMTs between primiparous and multiparous women.
The median GA of all infants was 37.1 weeks (range 26.4-41.6 weeks) and the median birth weight was 2,373 g (range 781-4,218 g). No infants were administered immunoglobulin therapy before the blood tests. Of the 206 infants, 22 were born at < 32 weeks of gestational age (wGA), 70 at 32-36 wGA, and 114  at ≥ 37 wGA. Their median GAs were 29.3, 35.3, and 39.0 weeks, respectively. The mean age of infants when blood was collected was 0.81 ± 0.16 weeks. The infant seroprevalence and antibody GMTs for PT were 51.9% and 9.7 EU/mL, respectively. The values for infants born at < 32 wGA were 13.6% and 3.2 EU/mL, respectively, and were significantly lower than those for infants born at 32-36 wGA and ≥ 37 wGA (P < 0.01) ( Table 2). The mean placental transfer ratio in 137 motherinfant pairs with maternal anti-PT IgG levels > 5 EU/ mL was 104.1%. The ratio of infants born at < 32 wGA was 42.5% and was significantly lower than that of infants born at 32-36 wGA and ≥ 37 wGA (P < 0.01) ( Table 3).
A second measurement was carried out in 21 infants with a median GA of 30.0 weeks (range 27.0-39.8 weeks). Five of the twenty-one infants were seropositive in the first examination. Of those five, the mean duration between the two assays was 4.3 weeks (range 3.9-5.0 weeks) and the GMTs for PT were 21.1 and 10.0 EU/ mL in the first and second examinations, respectively. The GMT decrease rate between the two assays was 52.6%. Two of the five infants were seropositive in the second examination.

DISCUSSION
The results of this study showed that approximately half of the mother-infant dyads in a region within the northern part of the Aichi Prefecture in Japan were negative for antibodies against the pertussis antigen. Moreover, the antibody level in infants born at < 32 wGA was significantly lower than that in infants born at ≥ 32 wGA. Previous studies have investigated pertussis antibody levels in infants aged < 6 months in Japan (13-16), but most did not indicate the number of enrolled neonates. To our knowledge, this is the most comprehensive study evaluating neonatal antibodies against pertussis in Japan.
According to national surveys conducted every five years since 2003 that cover all age groups regardless of sex, the seroprevalence of anti-PT IgG of individuals in their 20's and 30's was 45.5% in 2003, 45.2% in 2008, and 70.7% in 2013 (15,16). The maternal seroprevalence of anti-PT IgG in the present study was lower than that recorded in the national survey conducted in 2013 but was close to the levels reported in the previous two surveys. The nationwide pertussis epidemics in Japan between 2007 and 2010 (4,5), could have led to the increased seroprevalence observed in the national survey in 2013. Between 2008 and 2012, 57 children with pertussis were treated at our hospital (17); however, our results were similar to those of the two national surveys conducted in 2003 and 2008. The anti-PT IgG titers and the seroprevalence of mothers in their thirties were lower than those of other age groups, although the differences were not statistically significant. DTwP vaccination was discontinued in Japan in 1975 due to fatal post-vaccination cases. Subsequently, the nationwide pertussis epidemics occurred and the number of pertussis-related death cases increased. A DTaP vaccination with reduced adverse reactions was introduced in 1981; however, the women in their 30's who participated in this study were born between 1982 and 1993, shortly after routine DTaP vaccination commenced. As such, the vaccination rate was low compared to that in the present period. Consequently, the vaccine policies in Japan may have influenced the relatively low pertussis antibody titers observed in this study. I n t h e p r e s e n t s t u d y, t h e o v e r a l l n e o n a t a l s e r o p r e v a l e n c e w a s s i m i l a r t o t h e m a t e r n a l seroprevalence. Maternal antibody transfer to the fetus has been reported starting between 13 and 17 wGA, being most active after 32 wGA (18,19). At birth, the reported fetomaternal ratios of PT antibodies are 0.55 before 32 wGA, 0.79 between 32 and 37 wGA, and 1.07 after 37 wGA (20). This study specifically showed significantly lower seroprevalence and antibody titers to PT in preterm infants born at < 32 wGA than those born at ≥ 32 wGA. However, the seroprevalence and antibody titers of preterm infants born at 32-36 wGA did not differ significantly from those of infants born at ≥ 37 wGA. This finding suggests similar risks of pertussis infection between relatively late preterm infants (≥ 32 wGA) and full-term infants (after 37 wGA). The birth levels of pertussis-specific IgG decline rapidly to negligible values by 2 months of age and the half-life of maternal antibody to PT is 36 days (21,22). In the present study, many of those who were tested twice were preterm infants born at < 32 wGA and only five infants were seropositive in the first examination. Although the number of cases was small, the antibody titer to PT was halved in about 4-5 weeks, an observation that supports previous findings (21). Furthermore, many infants, even seropositive cases, did not have high anti-PT IgG titers; hence, the titers became negative within a short period after birth. Therefore, measures to increase antibody titers at birth are needed to protect infants before vaccination against pertussis infection. Several countries have introduced routine Tdap vaccination to pregnant women to increase the transfer of pertussis antibodies to infants (8)(9)(10), which is effective for the prevention of infantile pertussis (23,24). The recommended schedule for maternal pertussis vaccination is during the third trimester (> 26 weeks gestation); however, this approach may not affect extremely preterm infants (8). Recent observational studies have demonstrated superior antibody transfer to both preterm and fullterm infants following maternal pertussis immunization during the second trimester compared to that during the third trimester (25,26). Since preterm infants are most vulnerable to pertussis and this study demonstrated the high risk of infection in infants born at < 32 wGA, maternal vaccination during pregnancy earlier than the recommended period may be suitable when premature birth is expected. This study had some limitations. First, the antibody titers measured in the neonates should have been lower than those at birth because the serum samples were collected nearly one week after birth. Assuming that the half-life of the infant anti-PT IgG is five weeks, the values at birth were presumed to be approximately 15% higher than those observed in this study. Second, as this study was conducted at a regional hospital, the results may be geographically biased.
In conclusion, across this region in Japan, approximately half of the mothers and their infants at birth lacked protective antibodies against pertussis. In addition, anti-PT IgG in seropositive infants rapidly decreased after birth. Thus, new vaccination strategies, such as the vaccination of pregnant women, are needed to prevent pertussis infection in the early stages of infancy.
Conflict of interest None to declare.