Serum Antibodies to Diphtheria-Tetanus-Pertussis Vaccine ... · (tetanus toxoid) or Td (adult type tetanus-diphtheria vac- cine which contains a low amount of diphtheria toxoid to

Originalia

M. Pase t t i , P. E r i k s s o n , E F e r r e r o , M. M a n g h i

Serum Antibodies to Diphtheria-Tetanus-Pertussis Vaccine Components in Argentine Children

Summary: The Argentine vaccination schedule against diphtheria, tetanus and pertussis (DTP) recommends three doses of DTP vaccine at 2, 4 and 6 months of age, two boosters at 18 months and 6 years, a booster dose of tetanus vaccine every 10 years and two doses during pregnancy. To evaluate the effect of this schedule, antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) and against tetanus and diphtheria toxoids were determined by ELISA in serum samples from children (1 month to 6 years) who received different doses of DPT vaccine: 0 dose (n=50), I dose (n=25), 2 doses (n=25), 3 doses (n=55), first and second booster (n--25); 25 pregnant women and their offspring, and 45 adults. High antibody levels against PT (>140 EU/ml) and FHA (>80 EU/ml) were recorded in mothers and in the newborn. Antibody titers against PT increased with the number of doses given and decreased with time. Full protection against tetanus (titers >0.1 IU/ml) was observed in the group of adults (0.37 IU/mi), in mothers (4.4 IU/ml) and their newborn offspring (5.5 IU/ml), and in children after receiving the second dose of DTP vaccine (1.86 IU/ml). The immune status for diphtheria was far lower, as most of the groups lacked adequate protection. After the third dose of DTP vaccine, only 78% of the children had antibody titers above the protective level (0.1 IU/ml). Since antibody levels considered to provide full protection were only achieved after the first booster dose of DTP vaccine, the primary three-dose schedule seems to be insut~cient to confer adequate immunity in all vac- cinees. Because of the high proportion of non-protected adults, a booster dose of Td vaccine should be considered for this group.

Introduction

Tetanus, diphtheria and whooping cough are entirely pre- ventable, vaccination proving to be the most practical and cost-effective means of control [1-3]. Active immunization with diphtheria-tetanus-pertussis (DTP) vaccine has been routinely performed in children and has led to a marked reduction in the incidence of cases and deaths [1-4]. However, they still remain health problems, espe- cially in developing countries [1-3, 5, 6]. The classic DTP vaccine combines both tetanus and diphtheria toxoids with inactivated whole-cell Bordetella pertussis. Protec- tion against tetanus and diphtheria relies on the produc- tion of toxin neutralizing antibodies [1, 2], while pertussis immunity is rather complex, involving not only antibodies against several antigens such as filamentous hemagglutinin (FHA), pertussis toxin (PT) and other surface proteins [7-10], but also T-cell-mediated responses [11-13]. The recommended vaccination schedule in Argentina re- quires the administration of three doses of DTP vaccine at 2, 4 and 6 months of age as primary immunization. Rein- forcement doses are given at 18 months and to school children at 5-6 years [14]. Childhood vaccination is compulso- ry and certificates of vaccination are required at school entry. Booster doses of tetanus vaccine also proved to be effective for protection of adults who, according to this program, should be vaccinated every 10 years with either TT (tetanus toxoid) or Td (adult type tetanus-diphtheria vaccine which contains a low amount of diphtheria toxoid to

avoid reactogenicity). In order to reduce the rates of neonatal tetanus two booster doses of tetanus toxoid are recommended for pregnant women at the fifth and seventh month of pregnancy [14, 15]. Although this schedule follows international guidelines, and has proven to be highly effective worldwide [4], the reliability and the level of protection achieved in our child population had not yet been accurately evaluated. There are several factors related to the population under study, such as the prevalence of these illnesses and the percentage of healthy carriers, as well as socioeconomic factors, among others, that may exert an influence on protective immunity acquired after vaccination. Since numerous cases and outbreaks of these diseases have been observed even despite vaccination [5, 6, 16-18], it is essential to evaluate the immune status of a given population. For that purpose, serological surveys have been strongly encour- aged [19, 20]. Such studies are also useful to appraise the

Received: 13 March 1997/Revision accepted: 27 July 1997

Marcela Pasetti, Ph, D., Patricia Eriksson, Ph.D., Marcela Manghi, Ph.D., IDEHU, Instituto de Estudios de la Inmunidad Humoral (CONICET- UBA), Facultad de Farmacia y Bioqufmica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires; F. Ferrero, M. D., Ph.D., Hospital General de Nifios "Pedro de Elizalde", Avenida Montes de Oca 40, 1270 Buenos Aires, Argentina. Dr. M. Pasetti's present address: Center for Vaccine Development, Uni- versity of Maryland at Baltimore, 685 West Baltimore St., Room 480, Baltimore, MD 21201, USA.

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impact of vaccinat ion p rograms and to improve immuni- zat ion policies [21-23]. T he aim of the present work was to evaluate, in different groups o f children in our country , the effect of D T P vaccinat ion and the degree of pro tec t ion by measur ing an t ibody levels achieved with this t radit ional schedule.

Materials and Methods

Ethics: This was a collaborative study carried out by the Univer- sity of Buenos Aires, School of Pharmacy and Biochemistry, and the Hospital General de Nifios "Pedro de Etizalde" in Buenos Aires, and was approved by their respective Scientific Research Committees. Informed consent was obtained from the adults and parents of the children asked to participate in the study after de- tailed explanation of its purposes. Subjects and vaccine schedule: The participating children attend- ed the Department of Pediatrics of the Hospital de Nifios "Pe- dro de Elizalde" in Buenos Aires, for routine control. None of them had any underlying disease which could be suspected of impairing antibody response to vaccination. All pregnant mothers and newborns were assisted at the Department of Ob- stetrics of the same hospital. Participants were randomly select- ed from healthy patients who had received the recommended doses of DTP or TT vaccines outlined by our National Vaccina- tion Program, according to their age [14]. As mentioned above, this schedule recommends three doses of DTP vaccine at 2, 4 and 6 months and two booster doses at 18 months and at 6 years, before school entry. Reinforce doses with either Td or TT are recommended at 16 years and every 10 years. During pregnancy women receive two doses of TT, at 5 and 7 months. Verifia- ble vaccination records were required. Other vaccines they received, according to this schedule, were: one dose of BCG vaccine during the first month of age and a second dose before school entry, three doses of polio vaccine at 2, 4 and 6 months, followed by two boosters at 18 months and at school entry, and one dose of measles vaccine at 12 month of age [14]. All the participants were Caucasians, the prevalent ethnic group in our population. a) Children: The study population comprised 205 children, 90 boys and 115 girls, from 1 month to 6 years of age, divided into four groups according to the number of DTP vaccine doses received: 50 non-vaccinated children (aged 1-1,4 months): 25 children (aged 3-3.3 months) who received the first dose; 25 children (aged 5-5.2 months) who received the second dose; and 55 children (aged 7-7.8 months) who received the third dose. Serum antibodies were invariably measured 20 to 30 days after each vaccine dose. Antibody titers were also measured in 25 children (aged 18-26 months) before and after receiving the first booster and in another group of 25 children (aged 5.2-6.3 years) before and after receiving the second booster dose. Titers were determined 20-30 days before and after each booster dose. b) Mothers and newborns: Serum antibody levels were assessed in paired maternal and cord-blood samples from 25 healthy women (aged 18-38 years) who delivered healthy term babies. All women had been fully immunized during childhood and given two doses of tetanus vaccine at the 5th and 7th month of pregnancy [14]. Within this group, ten women were primiparae, the other 15 had had previous pregnancies in the last 5 years, having received vaccination accordingly. Sera were separated from 1-3 ml of venous blood samples or cord-blood samples, in the case of the newborn, and stored at -20°C until assayed.

c) Adults': A group of 45 healthy adults (20 non-pregnant women and 25 men) of different ages (25-42 years) who volunteered to participate in the study, some of them members of our laborato- ry staff, was also included. All of them had received complete childhood vaccination and TT boosters at least 5 years previously. Sera were separated from 1-3 ml of venous blood samples. Vaccines: Children received one immunizing dose (0.5 ml) of DTP vaccine (Pasteur M6rieux, Serums & Vaccines, Lyon, France) containing at least 10 and 25 Lf/ml of purified tetanus and diphtheria toxoids, respectively, a suspension of killed B. pertussis and hydrated aluminium hydroxide. All their components satisfied the minimum potency requirements (4 IU/dose for pertussis, 30 IU/dose for diphtheria and 60 IU/dose for tetanus) according to international recommendations [24]. ELISAs: Serum antibodies against tetanus (anti-T) and diphtheria toxoid (anti-D) were measured with enzyme-linked immunosorbent assay (ELISA), as previously described [25, 26]. Tetanus or diphtheria toxoids (Statens SerumInstitut, Copenhagen, Den- mark) at 5 p,g/ml were used as coating antigens. Two preparations of human immunoglobulins previously calibrated against the International Standards for tetanus and diphtheria antitoxin by in vivo toxin neutralization tests [27, 28] were included in each assay. Antibody titers were calculated relative to these reference preparations and expressed in International Units/ml (IU/ml). Total IgG antibodies to PT and FHA were measured by ELISA, as previously described [29], using PT (Sigma Chem. Co., St. Louis, MO) and FHA as coating antigens at t ~g/ml. FHA was purified as described by Sato et al. [30]. Antibody titers were calculated by comparison with an Internal Reference Serum, which displayed the same level of anti-PT and anti-FHA antibodies (200 EU/ml), calibrated against the US Reference Human Per- tussis Antiserum. Statistics: All antibody data were log transformed to calculate the geometric mean titers. Comparison among the different groups of immunized children was performed using Kruskal-Wallis non- parametric ANOVA. Chi-square p values are specified in each case. Mother and newborn antibody fiters were compared as paired samples by two-tailed Student's t-test or alternate Welsh t-test, and also analyzed by linear regression. All these statistical studies were carried out using InStat GraphPad software (Instant Statistic, San Diego, CA).

Results

Tetanus and Diphtheria Antibodies

The immune status of a person or a popula t ion against tetanus and diphther ia may be inferred f rom serum anti toxin titers. Accordingly , three categories o f pro tec t ion have been p roposed [31, 32] and current ly adop ted in serological surveys [22, 33-35]: fully protect ive (titers >0.1 IU/mt) , part ly protect ive (titer range 0.01-0.1 IU/ml) or non-protective (titers <0.01 IU/ml). Serum IgG anti-T ant ibody titers in 50 non-vacc ina ted infants (aged 1-1.4 month) and in infants (aged 3-7.8 months) who received one (n=25), two (n=25) or three (n=55) doses of D T P vaccine, as well as the percentages of pro tec t ion within each group are shown in Table 1. High an t ibody levels were found a m o n g non-vaccinated infants, a lmost all o f w h o m were fully protected. Even though the g roup receiving the first dose showed a significantly lower mean an t ibody ti ter (p<0.01), the same

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Table 1: Serum IgG antibody titers against tetanus and diphtheria toxoids corresponding to the three primary immunization course and booster doses. Percentage of children falling into each category of protection are also indicated. A: protected ( > 0.1 IU/ml), B: partially protected (0.01-0.1 IU/ml) and C: non-protected (< 0.01 IU/ml).

None (1 - 1.4 months) 1.8 92 8 - 0.074 44 50 6 n = 50 (1.1 - 2.8) (0.05 - 0.11) First (3 - 3.3 months) 0.38 b 92 8 - 0.038 28 56 16 n -- 25 (0.2 - 0.6) (0.02 - 0.06) Second (5 - 5.2 months) 1.86 100 - - 0.26 a 68 32 - n = 25 (1.2 - 2.8) (0.13 - 0.51) Third (7 - 7.8 months) 3.0 a 100 - - 0.32 78 22 - n = 55 (2.2 - 4.1) (0.21 =- 0.47) First booster (n = 25)

Before (18 - 24 months) 1.20 96 4 - 0.11 72 20 8 (0.7 - 1.8) (0.06 - 0.22)

After (19 - 26 months) 3.90 c 100 - 0.28 100 - - (3.4 - 4.4) (0.22 - 0.36)

Second booster (n = 25) Before (5.2 - 6 years)

After (5.3 - 6.3 years)

0.52 a 100 - - 0.065 52 36 12 (0 .35-0.80) (0 .03-0 .14)

3.13 b 100 - - 0.45 a 96 4 - (2.6 - 3.7) (0.36 - 0.57)

*GM values (95% confidence interval); a p < 0.001, b p < 0.01, c p < 0.05.

p e r c e n t a g e of ful ly p r o t e c t e d c h i l d r e n was o b s e r v e d . A f t e r t he s e c o n d dose , a p rog re s s ive i n c r e a s e in t i ters was ob- se rved , all c h i l d r e n p r o v i n g ful ly p r o t e c t e d , b u t this in- c rease was n o t s ign i f i can t u n t i l t he th i rd dose (p<0.001) . T h e effect o f b o o s t e r doses was also s t u d i e d a n d resu l t s a re s h o w n in T a b l e 1. A l t h o u g h t h e a n t i b o d y t i te rs a c h i e v e d wi th the t h r e e p r i m a r y doses pe r s i s t ed u n t i l t h e first b o o s t - er dose (p>0.05) , 4 % of the c h i l d r e n were p a r t i a l l y p r o - t e c t e d at t ha t t ime. F o l l o w i n g this b o o s t e r dose , m e a n an - t i b o d y t i ters rose s ign i f ican t ly (p<0.05) , d e c r e a s i n g b e f o r e the s e c o n d b o o s t e r dose (p<0.001) b u t i n c r e a s i n g t he re - a f te r (p<0.01) . In sp i te o f v a r i a t i o n s in a n t i b o d y levels , full p r o t e c t i o n aga ins t t e t a n u s was a c h i e v e d a n d m a i n t a i n e d

t h r o u g h o u t a f te r the first b o o s t e r dose. F u l l p r o t e c t i o n was also o b s e r v e d in t he g r o u p of adul t s , a l t h o u g h m e a n t i ters w e r e s ign i f i can t ly l o w e r (p<0.001) ( T a b l e 2). P a i r e d m o t h - ers a n d the i r n e w b o r n p r o v e d to be ful ly p r o t e c t e d aga ins t t e t a n u s w i th a m e a n a n t i b o d y t i t e r 40- fo ld h i g h e r t h a n t he p r o t e c t i v e level (0.1 I U / m l ) . A n t i b o d y t i ters m e a s u r e d in c o r d b l o o d w e r e f o u n d to b e s ign i f i can t ly h i g h e r t h a n m a - t e r n a l m e a n t i t e r (p<0.05) ( T a b l e 2). A s ign i f i can t co r re l a - t i o n b e t w e e n m a t e r n a l a n d n e w b o r n t i te rs was d e m o n - s t r a t ed by l i n e a r r e g r e s s i o n ana lys i s (r=0.86, p<0.001) . D e - sp i te h a v i n g r e c e i v e d v a c c i n a t i o n d u r i n g p r e v i o u s p reg - nanc i e s , m u l t i p a r o u s m o t h e r s fa i led to s h o w a n t i - T t i te rs h i g h e r t h a n those o f p r i m i p a r o u s m o t h e r s (p>0.05) ( i nd i -

Table 2: Serum IgG antibody titers against tetanus, diphtheria, pertussis (PT) and filamentous hemagglutinin (FHA) corresponding to the group of paired mothers and newborns and the group of adults.

Mothers (18 - 38 years) 4.4 0 . t l 83.2 152.2 n = 25 (3.6 - 5.5) (0.08 - 0.16) (65.2 - 1.06.2) (106.2 - 218.3) Newborn 5.5 c 0.13 c 80.4 140.1 n = 25 (4.5 - 6.6) (0.09 - 0.18) (62.4 - 103.5) (94.8 - 207.7) Adults (25 - 42 years) 0.37 a 0.064 147.2 123.0 n -- 45 (0.22 - 0.62) (0.045 - 0.089 (128.2 - 169.0) (103.5 - 146.6)

*GM values (95% confidence interval); a p < 0.001, c p < 0.05; EU = ELISA units.

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Table 3: Serum IgG antibody titers against FHA and TP corresponding to different doses of primary immunization and booster doses.

None (1 - 1.4 months) 51.9 68.1 n = 50 (39.2 - 68.7) (51.6 - 89.9) First (3 - 3.3 months) 10.2 a 14.7 n = 25 (6.6 - 15.8) (8.6 - 25.2) Second (5 - 5.2 months) 14.6 253.0 a n = 25 (10.6 - 20.2) (164.8 - 389.0) Third (7 - 7.8 months) 30.1 221.0 n = 55 (23.8 - 38.1) (161.4 - 302.7) First booster (n = 25)

Before (18 - 24 months) 19.5 44.4 a (14.9 - 25.5) (39.1 - 50.5)

After (19 - 26 months) 82.4 a 410.2 a (70.0 - 97.1) (342.8 - 419.8)

Second booster (n = 25) Before (5.2 - 6 years)

After (5.3 - 6.3 years)

26.3 c 79.5 a (21.7 - 32.0) (69.5 - 91.0)

158.7 a 418.7 a (140.0-179.9) (371.5- 472.1)

*GM values (95% confidence interval); a p < 0.001, C p < 0.05; EU = ELISA units.

vidual data not shown). Maternal anti-T titers invariably proved significantly higher (p<0.001) when compared with those of the adult group. This effect was not observed for the other three specificities.

Regarding diphtheria, the immune status of our population proved to be far below that observed for tetanus (Ta- ble 1). Both non-vaccinated and first-dose groups showed mean titers falling into the partial protect ion category. Non-vaccinated mean titer declined to half its val- ue in children who had already received the first dose, although this difference was not significant (p>0.05). In agreement , the percentage of fully protected children decreased from 44% to 28%, whereas the percentage of non-protected infants increased from 6% to 16% after the first dose (Table 1 ). The second vaccine dose led to a significant increase in titers (p<0.001). Although many children achieved full protect ion in this group, 32% remained partially protected. Children who had received the third dose had somewhat, but not significantly higher titers (p>0.05). Even when a 10% increase in the percentage of protect ion was achieved, 22% of the children remained partially protected. It was only after the first booster dose that all the children became fully protected against diphtheria (Table 1 ). Before the second booster dose, the mean titer decreased, though not significantly (p>0.05), with 12% of the children proving to be non-protected and 36% partially protected. The second booster dose induced a significant increase in antibody titers (p<0.001), reaching 96% protection. However , antibody

levels dropped significantly in adults (p<0.001) (Table 2), with only 60% becoming fully protec ted (individual data not shown). Fifty percent of the mothers and newborn were fully protected, while the rest were partially protected (individual data not shown). Mean antibody titer determined in cord blood was significantly higher (p<0.02) than the maternal titer (Table 2). A very close correlat ion was observed between maternal and cord blood titers (r--0.95, p<0.001).

Pertussis Toxin and FHA Antibodies

All non-vaccinated infants had anti-PT and ant i-FHA antibodies. After the first dose, a significant decrease was observed for F H A (p<0.001) but not for PT titers (p>0.05) (Table 3). Anti-PT titers increased after the second dose (p<0.001), and persisted after the third dose (p>0.05). In contrast, neither the second nor third dose induced a significant increase in ant i-FHA titers (p>0.05). A long-term decrease was observed for PT antibodies, when tested before booster doses (p<0.001); however, high antibody levels were achieved after each one (p<0.001). A significant increase in F H A antibody levels was also recorded follow- ing the first and second booster doses (p<0.001) (Table 3), and titers remained high in adults (p>0.05). On the other hand, PT antibody titers failed to persist in this group (p<0.001), thus reflecting the long-term decrease already observed (Table 2). Regarding mothers and their newborn, there was no significant difference between mean maternal and cord blood antibody titers (p>0.05), with a close statistical correlation (r;0.89, p<0.001).

Discussion

Routine immunization policies for D P T vaccines must be based on reliable data with adequate determination of protective immunity [20-22]. It is widely recognized that serological studies are very useful to evaluate the immune status of a population and the impact of immunization programs [19-23]. Unfortunately, there is little such information available from developing countries [23]. In this study, ELISA techniques were employed to deter- mine antibody levels, as they are suitable for analyzing large numbers of samples and provide reliable results [25, 26, 35-37]. Unfortunately, ELISA does not discriminate between neutralizing and non-neutralizing antibodies; only toxin neutralization (TN) tests reflect the true serum protective capacity, particularly for tetanus and diphtheria antibodies. However, several independent studies have shown that ELISA'S titers above 0.1 IU/ml are predictive of neutralizing activity [38-40]. The high anti-T antibody titers found in the newborn, conferring full protection, clearly reflect vaccination during pregnancy, indeed, the close correlation observed between maternal and cord blood antibody titers for all the antigens tested was not unexpected, but the higher titers for tetanus and diphtheria antibodies observed in cord

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blood versus maternal serum are noteworthy. Such findings can be explained by the fact that anti-T and anti-D antibodies are mainly of the IgG 1 subclass [41], which is pref- erentially transported across the placenta compared to other IgG subclasses [42]. Interestingly, mothers who received multiple doses of ~IW vaccines during previous pregnancies did not show anti-T titers significantly higher than those from primiparous women, indicating that multiple vaccination of pregnant women does not increase the antibody response in those who already have high titers. Others have also found that multiple vaccination during pregnancy does not necessar- ily increase the concentration or avidity of antibodies [43, 44]. Considering that hyperimmunization may produce adverse reactions [22, 45], and also that placental factors may influence not only the level but also the quality of the antibodies produced [44, 46], the potential risks of unnec- essary vaccination during consecutive pregnancies should be evaluated. The current national vaccination schedule proved to be effective at conferring immunity against tetanus, since all children became fully protected after the second dose of DTP vaccine and high protective levels were maintained by booster doses. Similar results have been reported by other authors [23, 35, 47]. The situation was quite different regarding diphtheria, as evidently most of the groups studied lacked adequate protection. In agreement with other reports, we also found a high proportion of non-protected adults [5, 6, 34, 40, 48-52]. Indeed, the alarming percentage of non-protected mothers and children is of considerable concern. Only 50% of the mothers had protective antibody levels and this inadequate level of immunity was reflected in the low titers found in newborns. The primary three-dose schedule seems to be insufficient to confer adequate immunity, since full protection was only achieved after the first booster dose. Widespread vaccination against diphtheria resulted in a considerable decrease in its incidence, leading to its disap- pearance in many parts of the world [5, 6, 40]. Regrettably, it has re-emerged in several European countries, and this is a cause of global concern [5, 6, 20, 40, 48, 52]. Since outbreaks of diphtheria among non-protected adults would provide the source of infection for poorly protected infants, we believe, in agreement with other authors, that administration of a booster dose with Td vaccine in adult- hood should be considered [5, 6, 20, 34, 40, 48~ 52, 53]. As applied in other countries, our vaccination schedule in- cludes the option of DT vaccine for adult boosters [6, 1@ but in practice, only TT vaccine is administered. While childhood vaccination is feasible in schools, immunization of adults is difficult to monitor [5, 6]. The non-protected females of child-bearing age represent another group which needs to be targeted for immunization to provide protection for their children in the first months of life [5]. Even though it is known that several antigen components of whole-cell pertussis vaccine are involved in the gener-

ation of a protective immune response by the host, our study only evaluated immune response against two of them, PT and FHA. Results show that the immunization schedule with whole vaccine applied in Argentina induc- es much higher levels of anti-PT than those of anti-FHA. While the reason for this difference is poorly understood, it is most likely due to the quantity of each one of these antigens present in the cellular vaccine, in agreement with findings reported by other authors who demonstrated wide variations in antibody levels induced by diverse cellular vaccines [54, 55]. Regarding the persistence of antibodies against these two antigens, results obtained in the adult group show that anti-PT levels decline over time, whereas those of anti-FHA remain high, similar to findings from other parts of the world [56, 57]. Presumably, exposure to Bordetella parapertussis, which also express- es FHA, or to cross-reacting antigens of other bacteria, may explain the persistence of raised anti-FHA levels in adults. PT, on the other hand, is unique to B. pertussis. In contrast with tetanus and diphtheria antitoxin, where threshold titers in serum clearly correlate with protection, no similar protective levels have been established for any of the antigens of the whole-cell pertussis vaccines; there- fore, it is difficult to evaluate the significance of our pertussis results. Ideally, a large number of samples should be evaluated in serological studies in order to achieve representative results. However, parents are often reluctant to consent that their children participate. The inclusion of randomly se- lected patients who routinely attend a public hospital provides a reliable group in terms of vaccination history and medical records, which is clearly representative of the general population. However, the follow up of outpatients be- comes difficult. For these reasons only a limited number of participants could be studied. Finally, while the serological surveys can be useful in as- sessing the immune status of a population and identifying susceptible groups, epidemiological studies on the incidence of these diseases are crucial in determining vaccination policies. The total number of cases (per 100,000 in- habitants) reported during 1995 and 1996 in Argentina were: six for pertussis; 0.03 for diphtheria and 0.3 for tetanus. Regarding neonatal tetanus, eight cases in total were reported during 1995 and 1996, within a population of 700,000 live newborns per year [58]. It is important to stress that the number of cases officially reported may indeed be much less than those actually occurring, mainly due to the absence of health facilities, clinical data and good epidemiological surveillance in areas where the diseases are most common. This study has clearly identified populations at risk in Ar- gentina and provides a basis for improving the national vaccination policy. Bearing in mind that the avoidance of outbreaks of these diseases relies on the maintenance of a high vaccination coverage rate, achieved by effective im- munizations schedules, we hope that this study may provide a useful contribution.

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A c k n o w l e d g e m e n t s

The au thors want to t h a n k Dr. Ricardo Margni, I D E H U , B u e n o s Aires , Argen t ina ; and Drs. Christoph Tang and Tim Wyant, Cen- ter for Vacc ine D e v e l o p m e n t , Ba l t imore , U S A , for the i r critical comments . This inves t iga t ion was suppo r t ed by grants f rom the Na t iona l R e s e a r c h Counci l , C O N I C E T , B u e n o s Aires, Argen t i - na and the Univers i ty of Buenos Aires.

R e f e r e n c e s

1. Orenstein, W. A., Wassilak, S. G. F.: Tetanus. In: Evans, A. S, Feld- man, H. A. (eds.): Bacterial infectious of humans, Epidemiology and control. 2nd ed., Plenum, New York 1991, pp. 707-726.

2. Wehrle, P. F.: Diphtheria. In: Evans, A. S., Feldman, H. A. (eds.): Bacterial infection of humans: Epidemiology and control. 2nd ed., Plenum, New York 1991, pp. 227-237.

3. Blaek, S.:Epidemiologyonpertussis. Pediatr. Infect, Dis, J, 16(1997) $85-89.

4. Immunization Practices Advisory Committee (ACIP): Diphtheria, tetanus and pertussis: guidelines for vaccine prophylaxis and other preventive measures. MMWR 34 (1985) 405-426.

5. Galazka, A. M., Robertson, S. E.: Diphtheria: changing patterns in the developing world and the industrialized world, Eur. J. Epidemi- ol. 11 (1995) 107-117.

6. Galazka, A. M., Robertson, S. E., Oblapenko, G. P.: Resurgence of diphtheria. Eur. J. Epidemiol. 11 (1995) 95-105.

7. Hewlett, E. L.: Pertussis: current concepts of pathogenesis and pre- vention. Pediatr. Infect. Dis. J. 16 (1997) $78-84.

8. Schmitt, H. J., Wagner, S.: Pertussis vaccines - 1993. Eur. J. Pediatr. 152 (1993) 462-466.

9. Thomas, M. G., Ashworth, L. A. E , Miller, E., Lambert, P. H.: Se- rum IgG, IgA and IgM reponses to pertussis toxin, filamentous hemagglutinin and agglutinogens 2 and 3 after infection with Bordetel- la pertussis and immunization with whole-cell pertussis vaccine. J. In- fect. Dis. 160 (1989) 838-845.

10. Ibsen, P. H., Petersen, J. W., Heron, I.: Quantification of pertussis toxin, filamentous haemagglutinin, 69 kDa outer membrane protein, agglutinogens 2 and 3 and lipopolysaccharide in the Danish whole- cell pertussis vaccine. Vaccine 11 (1993) 318-322.

11. Petersen, J. W., Andersen, P., Ibsen, P. H., Capiau, C., Waehmann, C. H., Haslov, K., Heron, I.: Proliferative responses to purified and fractioned Bordetella pertussis antigens in mice immunized with whole-cell pertussis vaccine. Vaccine 11 (1993) 463-472.

12. Redhead, K., Watkins, J., Barnard, A., Mills, K. H. G.: Effective immunization against Bordetella pertussis respiratory infection in mice is dependent on cell-mediated immunity. Infect. Immun. 61 (1993) 3190-3198.

1'3. Tamizifar, H., Jennings, S., Aft, S. A., Potter, C. W.: Induction of IL-2 and IFN-~/ in BALB/c mice immunised with subunit influenza A vaccine in combination with whole cell or acellular DPT vaccine. J. Med. Microbiol. 46 (1997) 61-66.

14. Normas de Vacnnacion (Aetualizaci6n 1985). Ministerio de Satud y Acci6n Social. Direcci6n Nacional de Promoci6n y Protecci6n de la Salud. Area de Vigilancia Epidemiol6gica e Immunizaciones.

15. Gasse, F.: Neonatal tetanus elimination initiative: progress report and recommendations. Geneva: World Health Organization 1990. WHO document EPI/MCH/NNT/GEN/90.t.

16. Noah, N. D.: Attack rates of notified whooping cough in immunized and unimmunized children. Br. Med. J. 1 (1976) 128-132.

17. Luisto, M., Iivanainen, M.: Tetanus of immunized children. Dev. Med. Child Neurol. 35 (1993) 346-358.

18. de Moraes Pinto, M. I., Oruamabo, R. S., Igbagiri, F. P., Chan, M. C. K., Prado, S. M. A., Vancetto, M. D. C., Johnson, P. M., Hart, C. A.: Neonatal tetanus despite immunization and protective antitoxin antibody. J. Infect. Dis. 171 (1995) 1076-1077.

19: Report on a WHO Meeting. The control of diphtheria in Europe; 1990 Apr.; Geneva. Switzerland. Report EUR/ICP/EPI 024 588 2r.

20. Popovic, T., Wharton, M., Wenger, J. D., Mclntyre, L., Wachsmuth, L K.: Are we ready for diphtheria? A report from the diphtheria diagnostic workshop, Atlanta, 11 and 12 July 1994. J. Infect. Dis. 171 (1995) 765-767.

21. Cutts F, Begg, N.: Diphtheria, pertussis and tetanus vaccination. Lancet 339 (1992) 1355-1356.

22. Schriiefler, J. P , Kuldmann, W. D.: Avoidance of hyperergic reactions after booster tetanus toxoid vaccination. Lancet 340 (1992) 379.

23. Briissow, H., Sidoti, 3[, Freire, W. B.: Tetanus and diphtheria immunization coverage in Ecuadorian children after a national vaccination campaign. J. Infect. Dis. 168 (1993) 479-483.

24. British Pharmacopoeia: Adsorbed diphtheria, tetanus and pertussis vaccine. Her Majesty's Stationery Office, London (1988) p. 1052.

25. Manghi, M. A., Pasetti, M. F., Brero, M. L., Deluchi, S., di Paola, G., Mathet, V., Eriksson, P.: Development of an alternative method for testing the immunogenicity of diphtheria vaccines. Vaccine 13 (1995) 597-601.

26. Manghi, M. A., Pasetti, M. F., Brero, M. L., Deluchi, S., di Paola, G., Mathet, V., Eriksson, P.: Development of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice. J. ImmunoL Methods 168 (1994) 17-24.

27. European Pharmacopoeia. Immunoserum antidiphthericum. Maison- neuve, Paris 1971, p. 268.

28. European Pharmacopoeia. Tetanus vaccine adsorbed monograph. Immunoserum antitetanicum, Maisoneuve, Paris 1971, p. 274.

29. Dellepiane, N. I., Manghi, M. A., Eriksson, P. V., di Paola, G., Can- gelosi, A.: Pertussis whole cell vaccines: relation between intracere- bral protection in mice and antibody response to pertussis toxin, filamentous hemagglutinin and adenylate cyclase. Zbl. Bacteriol. 277 (1992) 65-73.

30. Salo, Y., Cowell, J. L., Sato, H., Burstyn, D. G., Manclark, C. R.: Separation and purification of the hemagglutinins from Bordetetla pem~ssis. Infect. Immun. 41 (1983) 313-320.

31. Edsall, G.: Immunization of adults against diphtheria and tetanus. Am. J. Public Health 42 (1952) 393-400.

32. Edward, D., Allison, V.: Diphtheria in the immunized with observa- tions on a diphtheria-like disease associated with non-toxigenic strains of Corynebacter[um diphtheriae, J. Hyg. 49 (1951)205-219.

33. B6ttiger, M., Pettersson, G.: Vaccine immunity to diphtheria: a 20- year follow-up study. Scand. J. Infect. Dis. 24 (1992) 753-758.

34. Junum, P. A., Skogen, V., Danilova, E., Eskild, A., Sjursen, H.: Im- munity to diphtheria in northern Norway and northwestern Russia. Eur. J. Clin. Microbiol. Infect. Dis. 14 (1995) 794-798.

35. Christenson, B., B6ttiger, M.: Immunity and immunization of children against tetanus in Sweden. Scan& J. Infect. Dis. 23 (1991) 643-647.

36. Meade, B. D., Lynn, F., Reed, G. F., Mink, C. M., Romani, T. A., De- forest, A., Deloria, M.: Relationship between functional assays and enzyme-immunoassays as measurements of responses to acellular and whole-cell pertussis vaccines. Pediatrics 96 (Suppl.) (1995) 595-600.

37. Lagerg~rd, T., Trollfors, B., Ciaesson, B. A., Karlberg, J., Taranger, J.: Determination of neutralizing antibodies and specific immuno- globulin isotype levels in infants after vaccination against diphtheria. Eur. J. Clin. Microbiol. Infect. Dis. 11 (1992) 341-345.

38. Sesardie, D., Corbel, M. J.: Testing for neutralizing potential of serum antibodies to tetanus and diphtheria toxin. Lancet 340 (1992) 737-738.

39. Gupta, R. K., Griffin, P. Jr., Xu, J., Rivera, R., Thompson, C., Siber, G.: Diphtheria antitoxin levels in US blood and plasma donors, J. In- fect. Dis. 173 (1996) 1493-1497.

40. Galazka, A.: Diphtheria: the immunological basis for immunization; module 2: diphtheria. Geneva: World Health Organization, 1993. WHO document WHO/EPI/GEN/93.12.

41. Shackelford, P. G.: IgG subclasses: importance in pediatric practice. Pediatr. Rev. 14 (1993) 291-296.

42. Einhorn, M. S., Granoff, D. M., Nahm, M. H., Quinn, A., Shackel- ford, P. G.: Concentrations of antibodies in paired maternal and infant sera: relationship to IgG subclass. J. Pediatr. 111 (1987) 783-788.

20 / 344 Infection 25 (1997) No. 6 © MMV Medizin Verlag GmbH Mttnchen, Mtinchen 1997


43. Abacio~lu, Y. H., Eski~ar, T., Yulu~, N.: Concentration and avidity of anti-tetanus antibodies in mother-infant pairs: relation to immunization time. FEMS Immunol. Med. Microbiol. 11 (1995) 273-278.

44. Pasetti, M., Dokmetjian, J., Brero, M. L, Eriksson, P. V., Ferrero, F., Manghi, M.: Structure and protective capacity of tetanus and diphtheria antibodies produced during human pregnancy and trans- ferred to new-born. Am. J. Reprod, Immunol. 37 (1997) 250-256.

45. Sutter, R. W.: Adverse reactions to tetanus toxoid. JAMA 271 (1994) 1629.

46. Kelemen, K., Bognar, L, Paal, M., Szekeres-Bartho, J.: A progeste- rone-induced protein increases the synthesis of asymmetric antibodies. Cell Immunol. 167 (1996) 129-134.

47. Christenson, B., B6ttiger, M.: Epidemiology and immunity to tetanus in Sweden. Scand. J. Infect. Dis. 19 (1987) 429-435.

48. Sutter, R. W., Hadler, S. C., McQuiilan, G., Gergen, P. J.: Protection against tetanus (reply). N. Engl. J. Med. 333 (1995) 599~500.

49. Ceilesi, C., Zanchi, A., Miehclangeli, C., Giovannoni, F., Sansoni, A., Rossilini, G. M.: Immunity to diphtheria in a sample of adult population from central Italy. Vaccine 7 (1989) 417-420.

50. Koblin, B. A., Townsend, T. R.: Immunity to diphtheria and tetanus in inner-city women of child-bearing age. Am. J. Public Health 79 (1989) 1297-1298.

51. Maple, P. A., Efstratiou, A., George, R. C., Andrews, N. J., Sesardic, D.: Diphtheria immunity in UK blood donors. Lancet 345 (1995) 963-965.

52. Centers for Disease Control: Diphtheria epidemic - new independent states of the former Soviet Union, 1990-1994. MMWR 44 (1995) 17%181.

53. Simonsen, O.: Vaccination against tetanus and diphtheria: evalua- tion of immunity in the Danish population, guidelines for revaccina- tion, and methods for control of vaccination programs. Dan. Med. Bull, 36 (1989) 24-47.

54. Trollfors, B.: Bordetella pertussis whole cell vaccines - efficacy and toxicity. Acta Paediatr. 73 (1984) 417-425.

55. Edwards, K. M., Decker, M. D., Halsey, N. A., Koblin, B. A., Tow- send, T., Auerbach, B., Karzon, D. T.: Differences in antibody response to whole-cell pertussis vaccines. Pediatrics 88 (1991) 1019-1023.

56. Granstr6m, M., Lindberg, A., Askel6f, P., Hederstedt, B.: Detection of antibodies in human serum against fimbrial hemagglutinin of Bor- deteIla pertussis by enzyme-linked immunosorbent assay. J. Med. Mi- crobiol. 15 (1982) 85-96.

57. Torbicka, E., Lagerg~,rd, B., Trollfors, B.: Serum antibodies to the components of diphtheria-tetanus-pertussis vaccine in Polish children related to vaccination status. Infection 93 (1995) 212-215.

58. Bolet~n epidemiologico semanal de patologias de vigilancia intensi- ficada: (1996) Direccidn de Epidemiologfa, Ministerio de Salud y Accidn Social, Argentina.

Book Review

T. J. Harrison, A. J. Zuckerman (eds.) The Molecular Medicine of Viral Hepatitis 282 pages, several figures and tables John Wiley & Sons, Chichester-NewYork-Weinheim-Bris- bane-Toronto, 1997 Price: £ 40.00 The last two decades have witnessed the identification, molecular expression and characterization of five distinct human hepatitis viruses that are transmitted either parenterally or enterally. These five viruses differ from each other in terms of organization and nucleotide sequence of their respective genomes, and are classified in separate viral families. Viral hepatitis is a cause of considerable morbidity and mortality in the human population, affecting several hundreds of millions of people in the world. The editors and 24 other experts in the field, all of them re- nowned specialists, have organized in 15 chapters a wealth of information on several points of the molecular medicine of viral hepatitis. This book provides a comprehensive overview in vari- ous fields but is not a complete summary of all relevant information on the molecular medicine of viral hepatitis. The authors fo-

cus on themes that are of particular interest in the areas of research for the different viruses to date. Special chapters include, for example, the molecular biology of the attenuation of hepatitis A virus, development of vaccines against hepatitis E virus infection, the molecular role of hepatitis B virus in the development of hepatocellular carcinoma and the genotypes and genet- ic variation of hepatitis C virus. Each chapter is followed by important references for further reading. It is not possible to cover the entire field of the clinical and diagnostic aspects of viral hepatitis in a book of this size. But the authors succeeded in includ- ing information on subjects where advances are currently being made. The last chapter, "Conclusions and Perspectives,'" summa- rizes a number of findings concerning the different viral hepatitis infections. Information on the newly discovered GBV-C/ hepatitis G virus is also provided in this chapter. Finally, this book may be recommended for all readers who want to gain insight into the molecular medicine of viral hepatitis in a brief but in-depth publication.

A. Berger, H. W. Doerr Frankfurt/Main

Infection 25 (1997) No. 6 © MMV Medizin Verlag GmbH Mtinchen, Mfinchen 1997 3 4 5 / 2 1

Documents

Serum Antibodies to Diphtheria-Tetanus-Pertussis Vaccine ... · (tetanus toxoid) or Td (adult type tetanus-diphtheria vac- cine which contains a low amount of diphtheria toxoid to