Factors associated with superior antibody responses to a single dose of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine administered to Chilean infants at 2 months of age

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Vaccine, Vol. 15, No. 3, pp. 325-328, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264410X/97 $17+0.00

Factors associated with superior antibody responses to a single dose of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine administered to Chilean infants at 2 months of age Orin S. Levine*ll, Dan M. Granoffj-1, Rosanna Lagos& Bernard and Myron

FritzellllI

M. Levine*

The anticapsular antibody response of Chilean infants to a single dose of Haemophilus toxoid conjugate vaccine is substantially higher than that observed among infants of similar age from the USA. Comparison of selected demographic and environmental factors indicates that low maternal education and a greater number of persons in the home are significantly associated with the superior responder phenotype. High anticapsular antibody responses were associated with high antibody responses to tetanus toxoid, the carrier protein in this conjugate, but not to diphtheria toxoid. These data suggest that environmental factors may enhance the magnitude of the primary antibody response to PRP-T vaccine. 0 1997 Elsevier Science Ltd.

influenzae type b capsular polysaccharide-tetanus

Keywords:

Haemophilus

influenxe;

conjugate

vaccines; immunization

Between 1989 and 1991, we conducted two separate phase 2 studies of the safety and immunogenicity of diphtheria-tetanus-pertussis (DTP) vaccine and Huemophilus injknzae type b (Hib)-tetanus toxoid conjugate vaccine (PasteurlMerieuxlConnaught, referred to herein as HibPS-T) co-administered in the same syringe among 2, 4, and 6 month old infants in Santiago, Chilele3. The antibody response of Chilean infants following the first dose of HibPS-T at 2 months of age was much higher than among infants vaccinated at a similar age in the United States or elsewherek8. In Chile, 2629% of infants responded to one dose with concentrations 2 1.O lug ml-’ of anticapsular antibody’.3, compared to l-10% in the other studiesh8. The reasons for the high antibody concentrations following a single dose of vaccine at age 2 months among a subset of Chilean

*Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA. tChiron Biocine, Emeryville, CA 94608, USA. SChildren’s Hospital Oakland Research Institute, Oakland, CA 94609, USA. 5Centro para Vacunas en Desarollo-Chile, Servicio de Salud Metropolitana Norte, Santiago, Chile. nPasteur/ Merieux Serums et Vaccins, Paris, France. /Current address: Childhood and Vaccine Preventable Diseases Epidemiology Section, Centers for Disease Control and Prevention, Mailstop C-09, 1600 Clifton Road, NE, Atlanta, GA 30333, USA. *To whom correspondence should be addressed. (Received 5 March 1996; revised 8 July 1996; accepted 16 July 1996)

infants are unknown. Differences in laboratory methods do not explain it because in each study sera were analyzed using a standard reference serum specimen. In an effort to distinguish those Chilean infants who achieved high antibody concentrations ( 2 1.Opg ml _ ‘) following the first dose of vaccine from those who did not, we analyzed the data from our two studies and compared the distribution of selected characteristics among high and normal first dose responders. The exact concentration of antibody sufficient to confer protection is unknown. However, an anti-Haemophilus injuenzae type b capsular polysaccharide (anti-HibPS) concentration of 1.0 pug ml-’ is a commonly used threshold that is thought to correlate with long-lived protection’.

MATERIALS

AND METHODS

Methods for recruitment of the original study participants, specimen and data collection, and laboratory methods are described in detail in the original manuscripts’-3. Briefly, infants between 6 and 12 weeks of age were recruited during their routine well-baby visit in the local health center. Each infant received one injection in each arm. Participating infants were randomly allocated to one of three vaccine regimens: (1) co-administered DTP/HibPS-T in one arm; placebo in the other; (2) DTP in one arm; HibPS-T alone in the other; or (3) DTP only in one arm; placebo in the other arm.

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Figure 1 Differences in geometric mean anti-HibPS serum antibody by age, and type of response to the first dose of PRP-T vaccine, Santiago, Chile, 1998-90

For purposes of the present analysis, this third group was excluded. Two milliliters of blood were collected by venipuncture prior to administration of each dose, and 60 days after the third dose of vaccine. Serum anti-HibPS concentrations were measured by Farr-type radioimmunoassay. Categorical data were analyzed by the 2 test; continuous measures were analyzed by the t-test or by the Kruskal-Wallis test, when nonparametric methods were appropriate, using EPI-INFO (USD, Inc., version 5.01; Stone Mountain, GA). For multiple logistic regression analysis, the LOGISTIC procedure of SAS for the IBM mainframe was used (SAS Institute, version 6.07; Cat-y, NC). For guidance, P-values 10.05 were considered statistically significant.

RESULTS Figure I illustrates the kinetics of the anti-HibPS

response among superior first dose and normal first dose responders. While there was no observed difference in baseline antibody concentrations between groups, superior first dose responders continued to have significantly higher antibody concentrations at ages 6 and 8 months. Follow up sera obtained at 18 months of age from a subsample of the participants (N=88) indicates that the differences observed at age 8 months have disappeared. Table I presents a comparison of selected demographic and environmental factors between superior first dose responders and normal first dose responders. Among the demographic, socioeconomic, and environmental variables available for analysis, low maternal education (I 8 years) was most strongly associated with an anticapsular antibody response 2 1.O pg ml-‘. Crowding (defined as more than one person in a bed who are not a couple) was not significantly associated with a superior response (P=O.O8) yet a comparison of the mean number of persons in the house and mean number of children ~6 years of age (excluding the participant) indicates that these measures are higher among children with superior anti-HibPS responses than among the normal first dose responders (PcO.01). The two groups were similar with respect to gender, the proportion receiving co-administered HibPS-T/DTP vaccine in the same syringe as opposed to HibPS-T as a separate inoculation, the proportion participating in the

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first phase 2 study, and the average age at either the first or the second dose. The adjusted odds ratio for low maternal education and superior anti-HibPS antibody response was 1.68 (P=O.O5) in a multiple logistic regression model that included baseline anti-HibPS concentration, crowding, and the number of children ~6 years old in the household. Adjusted odds ratios for the other variables were not significant. In addition, no significant interaction was seen between crowding and low maternal education. Since the magnitude of the anticapsular antibody response may be influenced by immunologic priming to the carrier protein’Q’3, in this case tetanus toxoid, we compared tetanus antitoxin concentrations at baseline and following the first dose. Since laboratory methods for the measurement of tetanus antitoxin differed in the two studies233, we analyzed infants in each study separately. In each study, there was no difference in the baseline tetanus antitoxin level between superior and normal responders (Table 2). However, in each study, infants with anti-HibPS concentrations 2 1 pg ml- ’ also had significantly higher tetanus antitoxin concentrations than normal responders at age 4 months (Table 2). In contrast, no significant difference was observed in the diphtheria antitoxin concentrations at baseline or at age 4 months in either study (P~0.1, data not shown).

DISCUSSION The anticapsular antibody response to a single dose of HibPS-T is higher among Chilean infants than that observed among infants immunized by a similar regimen in the USA, Europe, and Israelk8. Interestingly, one other study of the immunogenicity of PRP-T in South America (Venezuela) reported a similar phenomenon14. In the two populations in the United States where antibody responses of similar magnitude as those observed in Chile have been reported’53’6, infants in those US studies were immunized by a different schedule that included prior vaccination with tetanus toxoid, the carrier protein. Possible explanations for the superior anti-HibPS responses following a single dose of HibPS-T in some of the Chilean infants include host and/or environmental factors. Because of the retrospective nature of our analysis, the data available were limited largely to the child’s environment. The observation of an association with an increased number of household members, and particularly young children in the household, may indicate that the high responses are attributable to increased exposure to Hib organisms transmitted by siblings. Infants with superior anticapsular antibody responses also had higher antibody responses to tetanus toxoid, the carrier protein in HibPS-T, but not to diphtheria toxoid. Conceivably, the superior anticapsular antibody phenotype may be a marker for inherently higher responses to the tetanus toxoid carrier protein used in this conjugate since recent data indicate that immunity to the carrier protein of a conjugate can markedly affect the magnitude of the antibody response to the polysaccharide’0-‘3. The observation that the responses of the two groups to diphtheria toxoid were not significantly different indicates the superior first dose responders to Hib and tetanus toxoid are not simply overall immunologically superior vaccine responders.

Antibody responses in infants to H&-tetanus Table 1 Comparison

of characteristics of infants vaccinated response to the first dose of HibPS-T vaccine

with HibPS-T

toxoid conjugate vaccine: OS. Levine et al.

in Santiago,

Chile,

1989-1991,

by magnitude

of anti-HibPS

Characteristic

Superior first dosea responders (MM)

Normal first dosea responders (lv=278)

P-valueb

Low maternal education (a years) Crowding (more than one person per bed)d Mean No. persons in house Mean No. children c6 yrs old” Female gender Co-administered HibPS-TIDTP Mean age at first dose (days) Mean age at second dose (days) First Phase 2 study

37 (44.0%)” 61 (72.6%) 6.7 (+2.8) 1.2 (+1.3) 40 (47.6%) 40 (47.6%) 62.7 (k5.8) 124.3 (k6.3) 35 (41.7%)

84 (30.2%) 171 (61.5%) 5.9 (e2.7) 0.8 (*O-9) 141 (50.7%) 144 (51.8%) 62.0 (23.0) 123.3 (25.9) 141 (50.7%)

0.03 0.08 0.01 0.02 0.71 0.58 0.17 0.18 0.18

aSuperior first dose responders were defined as children with serum anti-HibPS antibody concentrations 21 _O&ml two months following one dose of HibPS-T given at ca 2 months of age. Normal first dose responders were defined as children with cl.0 rg ml-’ anti-HibPS post-vaccination. bf-values correspond to x2 for categorical variables, r-test for normally distributed continuous variables. ‘Figures in parentheses correspond to the proportion of children who reported the characteristic, or the standard deviation of the mean. dDoes not include couples. g\rot including participating infant

Table 2 Geometric mean antibody concentrations among Chilean infants, 1988-1991

to tetanus toxoid, by age, Phase 2 study, and response to the first dose of PRP-T vaccine

First study”

Age (months)

Superior anti-HibPS

2 4

0.01 0.41

responder

Normal anti-HibPS

Second studyb

responder

0.01 0.19

P-valuec

Superior anti-HibPS

0.59 co.oo1

0.02 0.05

responder

Normal anti-HibPS 0.01 0.03

responder

P-value” 0.16 0.002

“Antibody concentrations, determined by radioimmunoassay, are expressed as IU ml-‘. bAntibody concentrations, determined by ELISA, are expressed as ELISA units ml-‘. Xruskal-Wallis test; P-values for contrast between superior and normal responder concentrations

These data cast doubt upon possible hypotheses that are based upon factors related to overall immune responses, such as a differential prevalence of malnutrition or vitamin A intake. Although it is possible that some mothers could have received tetanus toxoid during their pregnancy, and that this could have primed their infants to tetanus toxoid, immunization of pregnant women with tetanus toxoid is not a routine part of the Chilean immunization programme, and thus seems unlikely to explain our observations. There are a number of other bacteria with crossreacting antigens to the HibPS that have been identified from the ordinary flora of the respiratory and gastrointestinal tracts of healthy individuals (Escherichiu coli K-100, Staphylococcus aureus, Bacillus purnilis, B. subtilis, and Lactobacillus plantarum)‘7. It is conceivable that infants living in crowded conditions are more often exposed to one of these cross-reacting bacteria than infants living in less crowded environments. Potentially such exposures may prime for a higher anti-HibPS antibody response to Hib conjugate vaccination, or enhance the booster response following vaccination”. Such exposure, however, would not have been expected to enhance the tetanus antibody responses. Persons of native South American heritage in this population tend to be overrepresented in the lower socioeconomic levels. It is possible, therefore, that there are genetic phenotypes in this population, which unlike the Eskimos of North America*, respond better on average than others, and that low maternal education is a marker for native South American heritage. Further investigations of the environmental and host-specific factors related to this observed phenomenon are warranted.

Vaccines that confer high levels of protection among infants in industrialized countries have on occasion been found to be less immunogenic when administered to children in developing areas (e.g. oral polio vaccine). In the case of the unconjugated HibPS vaccine, immunogenicity varied across ethnic groups and correlated with specific genotypes”. Therefore, the unexpected observation that a subset of infants from a developing country have higher responses to an Hib conjugate vaccine than infants from industrialized countries immunized by an identical regimen is of great interest. While we are unable to explain this observation from the limited data available, it is important to consider the possibility that the immunogenicity of a vaccine may either be enhanced or diminished when evaluated in infants in developing countries. Our observation highlights the need to analyze individual subsets before pooling results from vaccine studies in different countries. Efforts to determine the factors that influence the antibody response to Hib conjugate vaccines may enable scientists and policy-makers to anticipate and capitalize on this effect.

ACKNOWLEDGEMENTS The phase 2 studies were supported by Pasteur Merieux Serum et Vaccins, the Ministry of Health of Chile, and the Servicio de Salud Metropolitana Area Norte, Santiago, Chile. The current analyses were supported in part by Grants AI 17962 and AI 21842 from the National Institute on Allergy and Infectious Diseases, National Institutes of Health.

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