A phase III single arm, multicenter, open-label study to assess the immunogenicity and tolerability of a pentavalent DTwP–HepB–Hib vaccine in indian infants

Abstract

Compliance with recommended vaccinations for Indian infants is facilitated by using combination vaccines to minimize the number of required injections. The ready-to-use, preservative free, fully-liquid combination DTwP–HepB–Hib vaccine, Quinvaxem^®, offers convenience of administering five important vaccine antigens to infants in a single injection. This phase III, single-arm, multicenter study was designed to assess immunogenicity and safety of three doses of Quinvaxem^® to Indian infants administered at 6, 10, and 14 weeks of age. Blood samples were taken prior to the first dose and at one month post last vaccination. Infants were observed clinically for any reaction approximately 30 min following each vaccination, and parents completed subject diaries for solicited local, systemic and any adverse events (AEs) following over a 5 d period. DTwP–HepB–Hib vaccine elicited strong immune responses that exceeded seroprotection/seroconversion thresholds against all vaccine antigens. At one month after third vaccination, percentages of infants achieving predefined protective antibody levels were 99% diphtheria; 100% tetanus; 98% Hepatitis B; 100% Hib short-term (≥ 0.15 µg/mL); 95% Hib long-term (≥ 1.0 µg/mL) protection; and relevant immune response was 99% for pertussis. The vaccine was well tolerated, with no vaccine-related serious AEs. Only one case of high fever (≥ 40°C) was reported. The most frequently reported reactions were mild to moderate tenderness and erythema. Frequencies of all AEs declined with subsequent vaccinations. This study demonstrated that this convenient, fully-liquid DTwP–HepB–Hib vaccine is highly immunogenic and has a acceptable safety profile for use in Indian infants. ClinicalTrials.gov Identifier: NCT01470287. Clinical Trials Registry of India Number: CTRI/2011/11/002118

Introduction

Combination vaccines against diphtheria, tetanus and pertussis (DTP) have been in use since the 1940s and represent the core childhood vaccination program worldwide. Following the WHO recommendation, India introduced DTP vaccines under the Expanded Program of Immunization (EPI) in 1978 to reduce child mortality. The Indian Academy of Pediatrics (IAP) Committee On Immunization, has also recommended Hepatitis B (Hep B) and Hemophilus influenzae type b (Hib) vaccines be included in the national immunization program and offered to all children.¹

A potential problem of vaccination programs covering a wide range of diseases is that such programs could necessitate large numbers of injections. A useful strategy to reduce the number of injections is to use combination vaccines, in which a single injection contains more than one component vaccine, so reducing the number of required injections or the number of clinic visits to complete the schedule. Pediatric combination vaccines that include DTP with other antigens like Hep B and Hib simplify immunization delivery and provide several advantages to children, parents, and health care providers by not only reducing the number of injection and clinic visits, but also facilitating logistic requirements and minimizing discomfort, thereby increasing the rates of acceptance and compliance.^2,3

Quinvaxem^® (DTwP–HepB–Hib) is a fully-liquid, pentavalent, preservative-free, ready-to-use combination vaccine. This vaccine eliminates the need for reconstitution of the Hib component of the pentavalent vaccine, which is commonly supplied as a separate lyophilized component with other combinations, thus reducing the risk of handling errors that could occur when preparing the final mix for injection. Quinvaxem^® was licensed and prequalified by the WHO in 2006 and has been shown to be safe and immunogenic when administered in accordance with different EPI primary and booster vaccination schedules.⁴

There are only a few fully-liquid pentavalent vaccines available for immunization against DTP, Hib and Hepatitis B in India. Currently in the world market, other than Quinvaxem^® only two fully-liquid pentavalent vaccines manufactured in India have pre-qualification status, one manufactured by the Serum Institute of India Ltd. (Pentavac^™) and one by Biological E Limited.⁵ Other fully-liquid pentavalent vaccines do exist, but no longer have valid WHO prequalification status.⁴

To enhance the supply and availability of WHO-prequalified pentavalent vaccines India, the present study was conducted to obtain regulatory clearance from the Indian Health Authority, the Drugs Controller General of India, for marketing Quinvaxem^® in India. This phase III, single arm, multicenter, open-label study assessed immunogenicity, safety and tolerability of fully-liquid pentavalent DTwP–HepB–Hib vaccine administered to Indian infants at 6, 10, and 14 weeks of age. Required criteria were demonstration of acceptable reactogenicity, and achievement of established seroprotective levels of immunogenicity or seroresponses by the individual vaccine components, rather than any comparison with a control vaccine.

Results

A total of 175 eligible infants were enrolled as they presented at the clinical centers, of whom 165 received all three vaccine doses and completed the study (Fig. 1). In total, 14 infants had at least one protocol deviation, leading to their exclusion from the per-protocol (PP) immunogenicity set which therefore comprised 161 infants. Three infants had no safety data and were excluded from the safety set. The baseline characteristics are shown in Table 1. Male and female infants were enrolled in equal proportion. The mean age of infants at the time of enrollment was 48.3 ± 5.2 d with a mean weight of 4.26 ± 0.70 Kg.

Figure 1. Subject flowchart.

Table 1. Study population demographics.

Characteristics	Infants (n = 175)
Age, mean ± SD, days	48.3 ± 5.2
Male, n (%)	88 (50%)
Asian, n (%)	175 (100%)
Weight ± SD weight, kg	4.26 ± 0.70
Height ± SD height, cm	54.6 ± 2.6
Non-study vaccine#, n (%)	165 (94%)
Met Study Criteria, n (%)	175 (100%)

^# Routine non-study vaccine, BCG and OPV administered to infants as per local immunization schedule prior to study enrollment

Immunogenicity results

Immunogenicity analyses were performed on 161 infants who completed the trial and gave blood samples one month after the third vaccination and did not have any major protocol deviation during the study (i.e., PP set). The minimum seroprotective levels of antibodies considered against four of the vaccine components were for diphtheria ≥ 0.1 IU/mL; tetanus ≥ 0.1 IU/mL; Hib ≥ 0.15 µg/mL (short-term protection) and ≥ 1 µg/mL (long-term protection); and Hepatitis B ≥ 10 IU/mL. There is no accepted serologic correlate of protection for pertussis, so the required immunogenic response was seroconversion defined by percentages of infants with either a titer of ≥ 20 EIU/mL or a 4-fold increase from pre- to post-vaccination levels. Percentages of infants who developed post-immunization antibody titers above these levels are given in Figure 2.

Figure 2. Percentages of infants with antibody levels above the seroprotection or seroconversion thresholds and 95% confidence intervals at baseline and one month post third vaccine dose.

One month after the last dose, 100% of infants developed antibody levels above the pre-defined thresholds to tetanus and Hib (short-term protection > 0.15 µg/mL) antigens; 99% to diphtheria and pertussis; and 98% to Hepatitis B antigens. The anti-Hib antibody concentration indicating long-term protection (≥ 1µg/mL) was elicited in 95% of infants (Fig. 2).

Geometric means of post–vaccination antibody concentrations (GMCs) to diphtheria 1.28 (95% confidence interval: 1.08–1.52) IU/mL; tetanus 2.51 (2.18–2.89) IU/mL; Hib 15 (12–18) µg/mL and Hepatitis B 373 (296–470) IU/mL antigens, exceeded the protective levels with very high margins. For pertussis, the GMC was 54 (51–57) EIU/mL, which is higher than the limit of 20 EIU/mL broadly used as a critical threshold of clinical relevance. One month after vaccination, a highly significant rise in antibody GMCs from baseline was observed for all vaccine components except tetanus, due to the very high baseline GMC (2.68 IU/mL) against this antigen (Fig. 3), presumed to be due to maternal antibodies.

Figure 3. Geometric means of antibody concentrations and 95% confidence intervals at baseline and one month post third vaccine dose.

Safety results

172 infants who received at least one dose of the vaccine and reported some safety data were included in the reactogenicity and safety analysis. Three subjects were excluded from the safety analysis as they did not provide any safety data being lost to follow up. Solicited local (i.e., tenderness, erythema, and induration) and systemic (i.e., fever, defined as body temperature ≥ 38°C) adverse events (AEs) were recorded for 5 d after each vaccination. Overall the vaccine was well tolerated. There were fewer solicited local and systemic AEs observed after subsequent vaccinations (Table 2). The most common solicited local AEs after the first and the second vaccinations, respectively, were tenderness (29% and 18%) followed by erythema (20% and 14%) and induration (13% and 10%). Post third vaccination, erythema (11%) was the most commonly reported local reaction. Most of the local reactions were mild to moderate in intensity. Fever (≥ 38°C to 38.9°C) was also reported with decreasing frequency following subsequent vaccinations, although one infant developed a temperature ≥ 40°C after the third vaccination. Most of the unsolicited AEs were also mild to moderate in intensity. The most commonly affected system organ class was infections and infestations (6%) followed by general disorders and administrative conditions (5%).

Table 2. Percentages of infants with local and systemic reactions after each vaccination.

		First Vaccination (n = 172)	Second Vaccination (n = 169)	Third Vaccination (n = 165)
Local Reactions (%)
Erythema (mm)	Any	20	14	11
	Mild (> 5 to ≤ 20)	9	5	3
	Moderate (> 20 to ≤ 50)	2	2	2
	Severe (> 50)	0	0	0
Induration (mm)	Any	13	10	4
	Mild (> 5 to ≤ 20)	5	5	1
	Moderate (> 20 to ≤ 50)	3	2	1
	Severe (> 50)	0	0	0
Tenderness	Any	29	18	10
	Mild (light reaction to touch)	17	8	2
	Moderate (cried/protested to touch)	7	6	4
	Severe (cried, injection limb moved)	5	4	5
Systemic Reactions (%)
Temperature °C	≥ 38°C- 38.9°C	20	16	13
	≥ 39°C- 39.9°C	1	2	2
	≥ 40°C	0	0	1

There were no vaccine-related serious adverse events (SAEs) or deaths reported during the study period. Three SAEs were reported, all in one infant (anemia, bronchopneumonia, and gastresophageal reflux) leading to premature withdrawal, but these SAEs were considered unrelated to the study vaccine.

Discussion

This study echoes the results of prior studies⁴ which showed that DTwP–HepB–Hib vaccine is highly immunogenic, eliciting very high antibody levels against all five vaccine components which exceeded expected seroprotection/seroconversion thresholds.

At baseline, before vaccination, 100% of infants already had protective antibody concentrations to tetanus toxoid, with a high level of antibodies. These high baseline antibodies were presumably due to maternal immunization. High maternal anti-tetanus toxoid titers are common in countries like India where programs for the prevention of neonatal tetanus are implemented by vaccination of pregnant women. It is important to note that after DTwP–HepB–Hib vaccination, the anti-tetanus level did not decline in these infants up to 18 weeks of age, when maternal antibodies would be expected to wane, thereby indicating a response to the tetanus component of the DTwP–HepB–Hib vaccine.

At baseline there were also high antibody concentrations to Hib, such that 68% of infants were already above the short-term protective threshold (≥ 0.15µg/mL). High maternal anti-PRP concentrations were previously reported from countries with low coverage for Hib vaccination.^6-8 There was an appreciable increase in Hib antibody titers observed after vaccination, indicating a good immune response to the Hib component of the DTwP–HepB–Hib vaccine.

Overall the DTwP–HepB–Hib vaccine was well tolerated among infants and most of the local and systemic reactions were mild to moderate in severity. The AE rates on subsequent vaccination were lower than the first dose. The incidence of AEs was consistent with previous studies when Quinvaxem^® was used for vaccination in infants.^7,9-11 No study vaccine-related SAE or death was reported. The study demonstrated that Quinvaxem was immunogenic and well tolerated when administered to infants.

This trial did not have a control study population as this was the first study of Quinvaxem^® in Indian infants with the intention to assess whether the immunogenicity and reactogenicity were within acceptable parameters. This is a requirement of the regulatory authorities in India prior to granting registration of a drug/vaccine in the country. When compared with other studies on pediatric pentavalent vaccines studied in Indian infants, immunogenicity and safety profiles of Quinvaxem^® were comparable. Although direct comparisons are not possible due to differences in data collection and analysis methods, seroprotection/seroconversion rates and reactogenicity profile were found to be similar to other pentavalent vaccines studied in Indian infants.^12-17

Indian studies with different pentavalent vaccines Pentavac^® (Serum Institute of India Ltd.); Easyfive^® (Panacea Biotec), TritanrixHB^TM + Hiberix^TM (Glaxo Smithkline), and Shan 5 (Shantha Biotechnics Ltd.) reported post primary immunization seroprotection rates with ranges of 99–100% for diphtheria; 99–100% tetanus; 77–100% Pertussis; 98–100% hepatitis B; 98–100% Hib anti-PRP antibodies ≥ 0.15 μg/mL and 90–99% Hib anti PRP (≥ 1.0 mg/mL). Safety data from these studies showed pain, swelling and erythema as the most commonly reported solicited local reactions, and most frequently reported solicited systemic reactions were fever and irritability comparable with our observations.^12-17

For several decades, inactivated whole-cell pertussis vaccines (wP) have been part of national childhood vaccination programs, dramatically reducing the considerable public health impact of pertussis.¹⁸ In most emerging and developing countries, DTP vaccines containing wP are used in the EPI.¹⁹ While many of the new DTP-based combination vaccines incorporate acellular pertussis (aP), the use of aP-based vaccines is predominantly in industrialized countries. High development and production costs and limited worldwide production capacity for aP, make these vaccines less affordable than their whole-cell pertussis (wP) counterparts. Although wP vaccines are considerably cheaper, the best wP vaccines have an efficacy that is equivalent or superior to the best aP vaccines.¹⁹ Moreover, quite recently, due to the re-emergence of pertussis, the long-term protection provided by acellular pertussis (aP) vaccine has been questioned. As pertussis epidemics in adolescents have occurred in countries where the aP vaccine is used, it seems that protection offered by wP vaccines might be longer-lasting, and there is an ongoing discussion whether it makes sense to switch back to wP in aP countries.^20,21

In many developing countries vaccination campaigns are subject to the barriers and challenges encountered by the health system as a whole. The shortage of human resources, inadequate infrastructure and equipment, make it difficult to provide the necessary coverage of multiple vaccines administered separately. There is therefore a need for well characterized WHO pre-qualified pentavalent vaccines in countries like India where there are only one or two such vaccines available despite the huge pediatric population. This convenient, fully liquid ready-to-use, pentavalent DTwP–HepB–Hib combination vaccine would reduce the likelihood of missed doses of one or all of the individual vaccines which would otherwise have been administered separately, thereby potentially increasing vaccine coverage.

Methods

Study design and subjects

This phase III single arm, multicenter, open label study was conducted at three centers (Pune, Bengaluru, and Kolkata) in India between Nov 2011 and April 2012. The study was registered on clinicaltrials.gov (NCT01470287) and on Clinical Trials Registry of India (CTRI/2011/11/002118) before enrollment. Three doses of the pentavalent DTwP–HepB–Hib vaccine Quinvaxem^® were administered to infants intramuscularly in the anterolateral part of the thigh at around 6, 10, and 14 weeks of age. Each 0.5 mL dose of Quinvaxem^® (Crucell, Berna Biotech Korea Corporation) contained ≥ 30IU diphtheria toxoid, ≥ 60 IU tetanus toxoid, ≥ 4 IU (lower limit of 95% CI ≥ 2 IU) inactivated B. pertussis, 10 μg HBsAg (Hepavax-Gene^®, Crucell), and 10 μg Hib oligosaccharide conjugated to CRM197.

This study was designed to assess immunogenicity in terms of accepted seroprotection or seroconversion criteria against all five vaccine antigens one month after the third vaccination, and safety and tolerability after each vaccine dose. The study was conducted according to Good Clinical Practice and in accordance with the Ethical Guidelines for Biomedical Research on Human Subjects and Declaration of Helsinki. The protocol and associated documents were reviewed and approved by the ethics committees of the participating study centers. Written informed consent was obtained from the parents/guardians of all subjects before study entry.

Healthy infants ≥ 42 d to ≤ 64 d of age and whose parents/legal guardian gave informed consent were included in the study. Infants were excluded if: had received immunization with a vaccine containing any of the five antigen components of the investigational vaccine, including a dose of hepatitis B vaccine at birth, (oral poliovirus vaccine was allowed in line with the local EPI schedule); had planned vaccine administration during the active study period with a vaccine not foreseen by the study protocol; were immunosuppressed from any cause; received systemic immunosuppressive therapy within 1 mo prior to study entry; had a history of anaphylactic shock, urticaria or other allergic reactions after previous vaccination or known hypersensitivity to any vaccine component; met nutritional status grades of I - IV according to the IAP grading of nutritional status; or were participating in another clinical trial.

Assessment of immunogenicity

Blood samples for immunogenicity assessments were drawn at baseline prior to vaccination (day 1) and one month after the last vaccination (day 85+14). Antibodies against vaccine component antigens were measured using standard methods. Antibodies against Hepatitis B surface antigen (HBsAg) were determined at Novartis Vaccines and Diagnostics GmbH, Marburg, Germany using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Enzygnost^® Anti-HBs II, Dade Behring), with a level ≥ 10 IU/mL considered protective.²² Anti-diphtheria and anti-tetanus antibodies were determined using an indirect ELISA, with protective levels set at ≥ 0.1 IU/mL for both^23,24 and Hib ELISA, which specifically detected antibodies against type b capsular polysaccharide (PRP) of H. influenzae, was set up using ≥ 0.15 µg/mL and ≥ 1 µg/mL as indicative of short- and long-term protection, respectively.²⁵ These three assays were performed at Rochester Research Institute, 1425 Portland Avenue, Rochester, New York, USA.

A whole-cell B. pertussis antibody ELISA was used to detect IgG antibodies to B. pertussis²⁶ performed at the Department of Medical Microbiology and Immunology, Turku, Finland, with a level ≥ 20 EIU/mL being considered indicative of a satisfactory immune response in the absence of a generally accepted correlate of protection for pertussis.

Assessment of reactogenicity and safety

After each vaccination, subjects were monitored for approximately 30 min for any possible adverse reactions. Parents or legal guardians were trained to use diary cards for recording safety data and were instructed to complete the diary cards for both solicited local (i.e., tenderness, erythema, and induration) and systemic AEs (i.e., fever, defined as body temperature ≥ 38°C) for 5 d, following each vaccination. The severity of solicited local AEs, including injection-site erythema and induration were categorized as none (≤ 5 mm); mild (> 5mm to ≤ 20mm); moderate (> 20mm to ≤ 50mm); and severe (> 50mm). The severity of tenderness occurring up to 5 d after each vaccination was categorized as none; mild (minor reaction to touch); moderate (cries/protests on touch); and severe (cries when limb is moved/spontaneously painful). Axillary body temperature was categorized as < 38°C (no fever), 38–38.9°C, 39–39.9°C and ≥ 40°C (severe). Any reactions that lasted more than 5 d were recorded as unsolicited AEs. Unsolicited AEs and serious AEs (SAEs) were recorded throughout the entire study period. Completed diary cards were reviewed at each clinic visit.

Statistical methods

Sample size of 150 evaluable subjects gave reasonable bounds on the true response rate for the immunogenicity endpoints and to assess the rates of local and systemic reactions with adequate precision. Exact two-sided 95% confidence intervals (Pearson-Clopper) were calculated for seroprotection (or seroconversion for pertussis) rates for all antigens. Calculations of the geometric mean concentrations were performed on the log10 scale and the confidence intervals (CIs) were calculated based on ANOVA model with center as a factor. The GMC values and the confidence limits were reported after exponentiating the results (power 10). Descriptive statistics were generated for the safety data.

Glossary

Abbreviations:

DTP

Diphtheria, tetanus and pertussis

EPI

Expanded Program of Immunization

Hep B

Hepatitis B

Hib

Haemophilus influenza type b

IAP

Indian Academy of Pediatrics

PP

per-protocol

GMCs

geometric mean concentrations

AEs

Adverse events

SOC

system organ class

SAEs

serious adverse events

PRP

Polyribosyl phosphate

wP

whole cell pertussis

aP

acellular pertussis

ELISA

enzyme linked immunosorbent assay

HBsAg

Hepatitis B surface antigens

CIs

confidence intervals

Conflict of Interest

Hoshang Vakil, Khaleel Ahmed, Marco Costantini, and Maria Lattanzi are employees of Novartis Vaccines and Diagnostics. Drs. Adarsh Eregowda, Sanjay Lalwani, and Sukanta Chatterjee, were the study investigators and had no financial interests in the vaccine or the manufacturer but received research funding to undertake the study.

Funding

This study was funded by Novartis Vaccines and Diagnostics