Vaccine 32 (2014) 4620–4624

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Antibodies against Haemophilus influenzae type b in The Gambia: Investigating the extent of protection across age groups O.T. Idoko a,∗ , E. Roberts a , M. Cox a , J. Jafali a , J. Njie-Jobe a , G. Mackenzie a,b , M.O. Ota c , B. Kampmann a,d a

Medical Research Council Unit, The Gambia Murdoch Children’s Research Institute, Melbourne, Australia c WHO Regional Office for Africa, Brazzaville, Congo d Academic Department of Paediatrics, Imperial College London, London W2 1NY, UK b

a r t i c l e

i n f o

Article history: Received 17 March 2014 Received in revised form 16 June 2014 Accepted 17 June 2014 Available online 25 June 2014 Keywords: Hib vaccine Antibody Protection Gambia

a b s t r a c t Following a landmark clinical trial, the vaccine against Haemophilus influenzae type b (Hib) was introduced in The Gambia in 1997. Whilst the immunogenicity of this vaccine is well established subsequent to the doses administered under the EPI schedule, little data exists assessing longevity of protection, using serology. Such data are needed however to predict the susceptibility to Hib at the population level. To determine antibody persistence in 5–6 year old fully vaccinated Gambian children compared with older children, adolescents and young adults, 427 serum samples from healthy 5–37 year old participants were tested for Hib antibodies using VaccZyme Human Anti-Hib ELISA kits. 86% of the children who had received 3 doses of Hib vaccine in infancy had Hib antibody concentrations ≥0.15 mg/l at the age of 5–6 years. This proportion was 76% for adolescents who had also largely been vaccinated and 90% for adults who had never received Hib vaccine. Although most participants had anti-Hib antibody above concentrations putatively defined as protective, significantly fewer had concentrations thought to confer long-term protection. This suggests a population with insufficient or waning antibody that may be susceptible to breakthrough disease and transmission. © 2014 Elsevier Ltd. All rights reserved.

Prior to the availability of a vaccine, the incidence of invasive disease caused by Haemophilus influenzae was around 250 per 100, 000 person years in The Gambia [1]. Following surveillance [2] and an efficacy trial [3,4] in The Gambia, the country’s Expanded Programme of immunization (EPI) was the first in Africa to introduce Haemophilus influenzae type b (Hib) vaccine into the schedule in 1997 [5]. Extensive surveillance studies carried out by the Medical Research Council (MRC) Unit The Gambia before and after introduction of the vaccine confirmed excellent disease control with very low incidence of invasive Hib disease [6,7] compared to previous incidence rates [1]. Initially introduced as a monovalent preparation, since April 2009 the Hib vaccine has been used as a component of a pentavalent vaccine containing Hib, Diphtheria, whole cell Pertussis and Tetanus (DPwT) and Hepatitis B vaccines. Unlike the 4 dose schedule (2, 3, 4 and 12-18 month doses) used in many

∗ Corresponding author. Tel.: +220 4495442/6 x 3031. E-mail address: [email protected] (O.T. Idoko). http://dx.doi.org/10.1016/j.vaccine.2014.06.078 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

developed nations, The Gambian EPI uses 3 doses of Hib vaccine administered at 2, 3 and 4 months. The World Health Organization (WHO) recommendations suggest that in countries where the peak burden of disease occurs in young infants, administration of 3 doses in early life may confer greater benefit. Thus, most developing countries schedule 3 doses of Hib vaccine in early infancy. The recommendation also includes the need to maintain high quality surveillance to monitor impact and changes in disease epidemiology so as to determine need for addition of a booster dose [8]. Since its introduction, the vaccination coverage for Hib has been generally high with over 90% of children under 5 receiving 3 doses of Hib. Between 2005 and 2006, after cessation of formal disease surveillance, 5 cases of Hib meningitis were detected from routine hospital samples in children with a median age of 15 months, higher than previously noted for invasive Hib disease [9]. Ongoing hospital-based surveillance between 2007 and 2010 also reported a low incidence of 5 cases per 100,000 in children under 5 years of age [10]. The data from this report show that 5 of the 6 children with Hib disease had received 2 or more doses of vaccine. Seven

O.T. Idoko et al. / Vaccine 32 (2014) 4620–4624

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Table 1 Gambian expanded programme on immunization schedule. Age/vaccine

Birth

BCG Hepatitis B OPV PCV 13 Rotavirus DPwT/HepB/Hib Vitamin A supplement Measles Yellow fever

× × ×

2 mths

3 mths

4 mths

× × × ×

× × × ×

× × × ×

6 mths

9 mths

12 mths

×

×

×

× × ×

18 mths

× (DPwT only) × ×

KEY: BCG, Bacillus Calmette-Guerin; OPV, oral polio vaccine; DPwT/HepB/Hib, Diphtheria, Tetanus, whole cell Pertussis, Hepatitis B, Haemophilus influenzae type b; PCV13, 13 valent Pneumococcal conjugate vaccine; mths, months.

cases were reported in children less than 14 months of age, with 3 of these having received 2 or more vaccine doses [10]. The HIV status of these cases was largely unknown, but the potential for primary vaccine failure and/or waning of anti-Hib antibody must be considered in view of the evidence of re-emergence of Hib cases in The Gambia [9,10]. To assess the longevity of immune responses to vaccines contained in the EPI programme is important, as findings of waning immunity might have implications for vaccine schedules, including indicating the potential need for booster doses. Such seroepidemiological studies should include adolescents and adults, and in particular women of child-bearing age, since protection of neonates prior to receiving the EPI vaccines themselves is primarily correlated with titres of transplacental maternal antibody. The recent measles epidemics in children below vaccination age and adults strongly support the needs for assessment of protective immunity at the population level [11–13]. We took advantage of a study originally set up to investigate the longevity of protection from one dose of the MenAfrivac vaccine to additionally measure the antibody concentration against Hib in the same cohort who had been enrolled in clinical trials conducted between 2006 and 2009 [14]. This follow-up study was conducted in 2011. Here we report anti-Hib antibody concentrations in 5–6 year old Gambian children who had completed the primary Hib vaccine schedule in comparison to older children, adolescents and young adults, some of whom had never received the Hib vaccine (Table 1).

1. Subjects and methods The study was performed in the Upper River Region of The Gambia which is the most eastern and isolated part of the country. The original multi-site trial was funded by Program for Appropriate Technology in Health (PATH) and WHO under the Meningitis Vaccine Project (MVP) and enrolled 601 participants. Full details and results have previously been published [14]. In 2011/2012, 473 (80%) of these participants were available for re-evaluation for persistence of their Meningococcal A (MenA) antibodies 4–5 years after receiving the original MenA vaccine now aged 5–37 years. An attempt was made to contact the entire initial cohort of 601 who had received at least one dose of MenA containing vaccine. Samples were drawn from all of these who were still within the study area and from whom written consent/assent was obtained. Following ethical and sponsors approval, we preserved an additional aliquot of serum for analysis of other antibodies, where sufficient blood sample was obtained. We also collected details of the immunization history and demographic details from the entire cohort. In summary, all 12–23 month old children enrolled in the original trials had received 3 doses of Hib vaccine, which was verified in their immunization records as an entry criterion for the initial MVP trial,

and were 5–6 years old at the time of the follow-up study. None of the individuals 15 years of age and above at the time of the followup study had ever received Hib vaccine, as the vaccine had not yet been introduced in The Gambia when they were infants. 90% of the children in the remaining age groups were apparently vaccinated in infancy, but this figure was estimated from recollection and could not be verified through documented immunization records. The 473 serum samples collected from these healthy 5–37 year old participants were stored at −70◦ until assayed for meningococcal A antibody persistence. Of this number 433 aliquots were available for this ancillary analysis as priority was given to the Meningococcal A antibody depending on the quantity of serum collected. 6 of these samples were excluded as serum was insufficient. 1.1. Laboratory methods Specific IgG antibodies against Haemophilus influenzae type b (Hib) capsular polysaccharide was measured using VaccZyme Human Anti-Haemophilus influenzae Type b (Hib) Enzyme Immunoassay Kit (Binding Site Birmingham, UK), following the manufacturer’s protocol. Microwell plates were pre-coated with Hib capsular polysaccharide antigen conjugated to human serum albumin. A high and low positive control of known concentrations and five Hib calibrators/standards (0.11 ␮g/ml, 0.33 ␮g/ml, 1.0 ␮g/ml. 3.0 ␮g/ml and 9.0 ␮g/ml) were included in each assay batch. Samples (diluted 1:100), Controls and Calibrators/Standards (100 ␮L) were each added to appropriate wells and incubated at room temperature for 30 min. Plates were washed 3 times with wash buffer. Conjugate (100 ␮L) was then added to each well and incubated at room temperature for 30 min. Following 4 washes, 100 ␮L of TMB substrate was added into each well and incubated for 30 min at room temperature. Stop solution (100 ␮L) was added to each well and optical density values were read at 450 nm using microplate reader MULTISKAN 354 PENTIUM (Thermo Electron Corporation Dell Inc.). The anti-Hib antibody concentrations were directly calculated from the standard/calibration curve using the Ascent software 2.6. The anti-Hib IgG antibody concentration for the controls fell within the QC certificate ranges. Anti-Hib antibody titres of ≥0.15 ␮g/ml were regarded as protective, while values ≥1 ␮g/ml were considered indicative of long term protection in line with international recommendations [15–18]. 1.2. Statistical analysis 5–6 year old Gambians were compared with older individuals classed in age groups 7–8 years, 9–11 years, 12–14 years, 15–17 years and 18 years and above in The Gambia. The 2 test was used to test differences in proportions of individuals attaining defined cutoffs for significance. A p-value of 1.0 ␮g/ml) varied, though the differences were not statistically significant (p = 0.407) (Table 3). The mean concentration of antiHib antibody >1.0 ␮g/ml in the population aged 5–6 years (N = 223) was 3.75 ␮g/ml compared to 3.33 ␮g/ml in individuals ≥ 15 years of age who had never been vaccinated (N = 109), (p = 0.353). The proportion of participants with anti-Hib antibody ≥0.15 ␮g/ml and 1.0 ␮g/ml respectively, in the 2 groups were not significantly different. (86.10% vs 87.16%; p = 0.476 and 69.51% vs 63.30%, p = 0.484, respectively). However, less than 70% of the children under 5 and less than 65% of the adolescents and adults had serological evidence of long-term protection (Fig. 2). 3. Discussion Our data show that the majority of participants had concentrations of anti-Hib antibodies well above the defined putative threshold for protection against disease, independent of their age. Interesting to note is the fact that even unvaccinated individuals

100 90 80

6

GMC An-Hib IgG anbody

Mean an-Hib anbody μg/ml

7

5 4 3 2 1 0

70 60 50

% ≥ 0.15 mg/l

40

% ≥ 1.0 mg/l

30 20 10

5-6 yrs (N = 223)

7-8 yrs (N = 37)

9-11 yrs (N = 38)

12-14 yrs (N = 20)

15-17 yrs (N = 26)

≥ 18 yrs (N = 83)

0 5-6 yrs (N = 223)

Age (yrs) Error bars represent 95% confidence intervals. Yrs – years N = number of subjects

Fig. 1. Mean GMCs by age.

≥ 15 yrs (N = 109) Age (yrs)

Yrs – years N = number of subjects

Fig. 2. Proportions of participants with protective GMCs.

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who were 15 years of age and above, had mean anti-Hib antibody concentrations of 3.33 ␮g/ml, not significantly different from fully vaccinated young children. This is most likely due to carriage and/or exposure to natural infection implying that there is continuing circulation within the population. Data from our previous studies in HIV-negative pregnant women showed that 54% of women in the study cohort in the UK did not have anti-Hib antibody concentrations associated with longterm protection, compared with 40% in South Africa [16,19]. Our data shows similar proportions with 36.1% of unvaccinated adults without antibody concentrations associated with long term protection. The level of antibody induced by carriage alone needs to be further defined. Independent of age, over 75% of participants in The Gambia had anti-Hib antibody concentrations associated with putative protection, while over 50% had antibody concentrations associated with long term protection. The differences between the age-groups were not significant, but in some fell below the 73% considered to be required to interrupt transmission [20]. Whilst the examination of the immunization records point to an effective EPI programme with good vaccine uptake in the younger age groups, there was also a significant fraction of young children who lacked sufficient antibody. Our data show that 14% of 5–6 year olds did not have concentrations of anti-Hib antibodies associated with short- or long-term protection, despite of being fully vaccinated with 3 doses in infancy. As demonstrated in previous Gambian reports, this may represent primary vaccine failure or rapid waning of immunity [9,10]. A recent report from Israel reports that 40% of Hib disease in the country is due to vaccine failure [21]. Other recent unpublished data from The Gambia show that over 30% of recent Hib cases in The Gambia had received at least 2 doses of vaccine. Taken together, these finding suggests that potential vaccine failure may play a major role in driving disease and needs to be further explored. Data on Hib serology from within sub-Saharan Africa in vaccinated and unvaccinated cohorts appears limited. A study carried out in Mali demonstrated that 90.0% of fully vaccinated (3 doses at 6, 10 and 14 weeks of life) 6 month old infants had anti-Hib antibodies ≥1 ␮g/ml, significantly different from 13.3% of unvaccinated children of the same age (p < 0.0001). 10% of fully vaccinated infants did not attain concentrations ≥1 ␮g/ml [22]. Although the groups are very small and the vaccination information was provided by parents, our data indicate possible waning of immunity in the 12–14 year old children, especially for long term protection. A similar phenomenon was observed in the UK after the introduction of Hib vaccine in a schedule with 3 primary doses alone [8,23,24]. It is possible that waning immunity in adolescence may allow breakthrough disease and transmissions. An additional dose of Hib vaccine at an appropriate time in childhood could boost the immune response derived in infancy and mitigate against this waning trend in immunity. More detailed studies of this age group are required to determine the threshold in which a booster would have most impact. Our study has important limitations, due to the design of the original study, which had enrolled specific age groups in unequal numbers. Although there was representation of key age groups, numbers in these groups varied considerably with small numbers of samples available from children between 6 and 17 years of age. The oldest adults included were only 37 years old and again, numbers were smaller than for the younger children and did not include significant numbers of women of child bearing age. We did not test for HIV, since the prevalence of HIV in The Gambia is estimated at less than 2%. It is therefore unlikely that the antibody concentrations were influenced by underlying HIV infection. We do not have carriage data from the study group. All samples were collected in one particular geographical area of The Gambia, which might not be truly reflective of the situation in the rest of the country.

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Nonetheless the trends of waning antibody against Hib in adolescence observed in our population are consistent with data reported from some industrialized nations where booster doses have been found to be of additional benefit [8]. In the absence of boosters, sub-optimal concentrations of Hib antibody may in the future persist in women of child-bearing age and also likely to impact on the level of protection afforded to newborns, prior to EPI vaccination: low levels of transferred maternal antibodies would leave this age group particularly susceptible. We have previously reported similar findings in our studies in South Africa and the UK [19]. The number of adolescents in our sero survey is however small and further sero surveillance studies including larger numbers of individuals and an even wider age range are indicated to extend our observations to additional age groups. In conclusion, although most participants had anti-Hib antibody above concentrations putatively defined as protective, significantly fewer had concentrations thought to confer long term protection. This suggests a population with insufficient or waning antibody that may be susceptible to breakthrough disease and transmission, much like has been observed in other populations for measles or Pertussis [11,12,25]. Unvaccinated adults however also demonstrated high anti-Hib antibody concentrations, suggesting that carriage is ongoing in the population. Comprehensive carriage studies, further surveillance for cases of invasive Hib disease and sero-surveillance are therefore required to fully understand how to eradicate Hib disease in the longer term in The Gambia and other similar settings. Such information will contribute to determining possible reservoirs of disease and identify susceptible age groups with further strategies possibly involving booster vaccination or catch-up campaigns. Acknowledgements The authors acknowledge the Meningitis Vaccine Project (MVP) and Serum Institute of India (SIIL) for providing the framework within which data was collected for this manuscript. We specifically acknowledge MVP director Dr. Marie-Pierre Preziosi for her contributions and for reviewing the manuscript. We also thank all field and lab staff and the study communities. We are grateful to Medical Research Council (MRC) United Kingdom for funding, grant number MR/K007602/1. References [1] Adegbola RA, Usen SO, Weber M, Lloyd-Evans N, Jobe K, Mulholland K, et al. Haemophilus influenzae type b meningitis in The Gambia after introduction of a conjugate vaccine. Lancet 1999;354(September (9184)):1091–2. [2] Adegbola RA, Mulholland EK, Falade AG, Secka O, Sarge-Njai R, Corrah T, et al. Haemophilus influenzae type b disease in the western region of The Gambia: background surveillance for a vaccine efficacy trial. Ann Trop Paediatr 1996;16(June (2)):103–11. [3] Mulholland K, Hilton S, Adegbola R, Usen S, Oparaugo A, Omosigho C, et al. Randomised trial of Haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in Gambian infants. Lancet 1997;349(April (9060)):1191–7. [4] Mulholland EK, Byass P, Campbell H, Fritzell B, Greenwood AM, Todd J, et al. The immunogenicity and safety of Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in Gambian infants. Ann Trop Paediatr 1994;14(3):183–8. [5] Wenger J. Hib vaccine introduced in The Gambia. Afr Health 1997;20(November (1)), 13, 5. [6] Adegbola RA, Secka O, Lahai G, Lloyd-Evans N, Njie A, Usen S, et al. Elimination of Haemophilus influenzae type b (Hib) disease from The Gambia after the introduction of routine immunisation with a Hib conjugate vaccine: a prospective study. Lancet 2005;366(July (9480)):144–50. [7] Howie SR, Oluwalana C, Secka O, Scott S, Ideh RC, Ebruke BE, et al. The effectiveness of conjugate Haemophilus influenzae type B vaccine in The Gambia 14 years after introduction. Clin Infect Dis 2013;57(December (11)):1527–34. [8] WHO position paper on Haemophilus influenzae type b conjugate vaccines (Replaces WHO position paper on Hib vaccines previously published in the Weekly Epidemiological Record). Wkly Epidemiol Rec 81(November (47));2013:445–452.

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