{'fraction_non_alphanumeric': 0.05249332340965807, 'fraction_numerical': 0.023422792347095135, 'mean_word_length': 5.0364693446088795, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Lot-to-lot consistency of a hexavalent DTwP-IPV-HB-PRP∼T vaccine and non-inferiority to separate DTwP-HB-PRP∼T and IPV antigen-matching vaccines at 6–8, 10–12, and 14–16 weeks of age co-administered with oral rotavirus vaccine in healthy infants in India: A multi-center, randomized, controlled study

Background Combination vaccines reduce the number of pediatric injections but must be as safe, immunogenic, and effective as each of the individual vaccines given separately. Additionally, consistency in manufacturing lots is essential for WHO prequalification. This study aimed to establish the lot-to-lot consistency of a fully liquid, hexavalent diphtheria (D)-tetanus (T)-whole-cell pertussis (wP)-inactivated poliovirus (IPV)-hepatitis B (HB)-Haemophilus influenzae b (PRP-T) (DTwP-IPV-HB-PRP∼T) vaccine and to demonstrate non-inferiority to licensed DTwP-HB-PRP∼T and IPV vaccines. Methods A Phase III, randomized, active-controlled, and open-label study was conducted at multiple centers across India. Healthy infants who had received a birth dose of oral poliovirus vaccine and hepatitis B vaccine received one of three lots of DTwP-IPV-HB-PRP∼T or separate DTwP-HB-PRP∼T and IPV vaccines at 6–8, 10–12, and 14–16 weeks of age. Oral rotavirus vaccine was co-administered at 6–8 weeks of age and 10–12/14–16 weeks of age. DTwP-IPV-HB-PRP∼T lot-to-lot consistency and non-inferiority (pooled DTwP-IPV-HB-PRP∼T) versus DTwP-HB-PRP∼T and IPV post-third dose were assessed using seroprotection rates (anti-D, anti-T, anti-HBs, anti-PRP, anti-polio 1, 2, 3) and adjusted geometric mean concentrations (anti-PT, anti-FIM). Safety was assessed by parental reports. Results Lot-to-lot consistency was demonstrated for DTwP-IPV-HB-PRP∼T and non-inferiority versus DTwP-HB-PRP∼T and IPV was confirmed with 95% CIs for seroprotection rate differences and adjusted geometric mean concentration ratios within pre-defined clinical margins. Pooled seroprotection rate was ≥ 99.7% for anti-D ≥ 0.01 IU/mL, anti-T ≥ 0.01 IU/mL, anti-HBs ≥ 10 mIU/mL, anti-PRP ≥ 0.15 µg/mL, and anti-polio 1, 2, and 3 ≥ 8 (1/dil) and vaccine response rate was 83.9% for anti-PT and 97.7% for anti-FIM. There were no safety concerns. Conclusions Immunogenicity of three lots of the fully liquid DTwP-IPV-HB-PRP∼T vaccine was consistent and non-inferior to licensed comparators following vaccination at 6–8, 10–12, and 14–16 weeks of age. There were no safety concerns and no evidence of any effect of co-administration with rotavirus vaccine.


Introduction
Pediatric combination vaccines are an important tool that have led to current low incidences of childhood diseases including diphtheria (D), tetanus (T), pertussis, hepatitis B (HB), Haemophilus influenzae type b (Hib) infection, and polio [1,2]. Such vaccines have consistently been shown to be safe and immunogenic, and add value by improving compliance to pediatric vaccination schedules [1].
Following the successful development in India of a pentavalent D, T, whole cell pertussis (wP), HB, and Hib vaccine (SHAN5 TM ), and an inactivated poliovirus (IPV) vaccine (SHANIPV TM ), Sanofi Healthcare India Private Limited (SHIPL) developed a fully liquid, readyto-use, DTwP-IPV-HB-PRP$T vaccine (SHAN6 TM ) which combines established SHAN5 TM and SHANIPV TM antigens. Combining antigens in a fully liquid presentation reduces the time for vaccine preparation, further improving compliance to increasingly crowded vaccination schedules and helping to support the Global Polio Eradication Initiative outlined in its most recent Endgame Strategic Plan (2022-2026) [3].
The World Health Organization (WHO) guidelines for vaccine prequalification indicate that the consistency of manufacturing for vaccine lots used in clinical trials should be demonstrated. Ideally, at least three lots with the same formulation should be used, manufactured at full production scale, and the study should be designed and analyzed as an equivalence trial with pre-defined criteria to conclude comparability [4,5]. The new DTwP-IPV-HB-PRP$T vaccine has been evaluated as safe and immunogenic in randomized, controlled, early phase clinical studies [6,7]. This study was conducted to establish that the hexavalent vaccine was safe and immunologically non-inferior to the administration of the licensed pentavalent and injectable polio vaccines. Additionally, the lot-to-lot consistency of the hexavalent vaccine was evaluated to demonstrate that different vaccine lots were consistent in terms of immunogenicity as per the WHO requirement for vaccine development and pre-qualification [4,5].

Study design and participants
A Phase III, randomized, active-controlled, open-label study was carried out at 13 sites in India (Clinical Trials Registry India Number CTRI/2019/01/017155). The study protocol and one amendment were approved by institutional ethics committees, and the study was performed according to local and national regulations consistent with the standards established by the Declaration of Helsinki and compliant with the International Council for Harmonization guidelines for Good Clinical Practice. Before enrolment, an informed consent form was signed by at least one parent or legally acceptable representative following an audio-visual consenting process as mandated by the Drugs Controller General of India (DCGI). Vaccinations occurred from February 2019 through December 2019.
Healthy infants aged 6-8 weeks, born at full term (!37 weeks), with birth weight ! 2.5 kg, and having received a birth dose of oral poliovirus vaccine (OPV) and hepatitis B (HB) vaccine, and Bacillus Calmette-Guérin (BCG) vaccine at least 4 weeks before the first study vaccination were eligible. The main exclusion criteria were previous vaccination or planned receipt of any vaccine against D, T, P, HB, Hib, poliovirus, or rotavirus (except OPV at any time or HB vaccine at birth) in the 4 weeks following study vaccination or history of any such infection; receipt of any other vaccine in the 4 weeks preceding the first study vaccination or planned receipt within 7 days before and after each study vaccination; known hypersensitivity to any vaccine component; any chronic illness that could interfere with study conduct or completion; personal or maternal history of human immunodeficiency virus, HB surface antigen (HBsAg), or hepatitis C seropositivity or receipt of immunosuppressive therapy; previous or planned participation in another clinical study; receipt of blood products in the 30 days prior to inclusion or planned during the study; known thrombocytopenia, bleeding disorder, or receipt of anticoagulants in the 3 weeks prior to inclusion; acute illness or febrile illness on the day of vaccination; history of seizure, encephalopathy, or intussusception; natural or adopted child of anyone with direct study involvement.
Infants were randomly allocated to receive one of three lots of the investigational vaccine (DTwP-IPV-HB-PRP$T) (Lot A, B, or C) or separately administered DTwP-HB-PRP$T and IPV vaccines at 6-8, 10-12, and 14-16 weeks of age. A permuted block method was used for the randomization to guarantee a similar ratio of participants between groups at any time. All vaccines were coadministered with oral rotavirus vaccine at 6-8 weeks of age and either 10-12 or 14-16 weeks of age.

Study vaccines
The The commercial rotavirus vaccine (Rotarix TM ) (lot number A41FB629A, expiry August 2020) was manufactured by GlaxoSmithKline Biologicals and supplied as a lyophilized powder and solvent for oral suspension. After reconstitution, each 1 mL dose contained ! 10 6.0 50% cell culture infective doses (CCID 50 ) (viral infectious units) live, attenuated human rotavirus RIX4414 strain. In addition, subjects enrolled in the study received 1 to 4 bOPV 1&3 doses as part of routine national immunization campaigns in India, in addition to the study vaccine doses, administered either before or concomitantly with the study vaccines.
All assays except the anti-rotavirus EIA were performed at the Sponsor's Global Clinical Immunology (GCI) laboratory (Swiftwater, PA, USA). Anti-rotavirus testing was done at the Cincinnati Children's Hospital Medical Center.

Reactogenicity and safety
Participants were monitored at the study site for immediate unsolicited adverse events (AEs) for 30 min after each vaccination. Subsequently, for 7 days parent(s)/legal representative(s) used diary cards to record the duration and intensity of pre-defined (solicited) injection site reactions (tenderness, erythema, swelling) and systemic reactions (fever, vomiting, crying abnormal, drowsiness, appetite lost, irritability). Axillary temperature was preferred. Solicited reactions were automatically considered to be vaccinerelated.
Unsolicited AEs were recorded using diary cards for 28 days after each vaccination. For each unsolicited AE the investigator assigned an intensity (Grade 1 [no interference with activity], Grade 2 [some interference with activity], or Grade 3 [prevents daily activity]) and assessed the relationship to vaccination. Details of serious adverse events (SAEs) were collected throughout the study until 28 days after the last vaccination, and the investigator assessed their relationship to vaccination.

Statistical analyses
The primary statistical objectives were (i) to demonstrate equivalence in terms of immunogenicity of three lots of DTwP-IPV-HB-PRP$T (ii) to demonstrate non-inferiority of DTwP-IPV-HB-PRP$T to DTwP-HB-PRP$T and IPV vaccines. Secondary objectives were to describe the immune responses for DTwP-IPV-HB-PRP$T, DTwP-HB-PRP$T and IPV, and rotavirus vaccines, and to describe the safety profile in each group.
Antibody thresholds and criteria used to define seroprotection (SP) and vaccine response (VR) rates are presented in Table 1, Table 2, and Table 3. In additional to SP and VR rates, geometric mean titers (GMTs) (IPV) and geometric mean concentrations (GMCs) (D, T, HB, PRP, PT, FIM, PRN, FHA) are also presented. Rotavirus GMCs and seroconversion rate (!4-fold rise in anti-rotavirus IgA) are presented. Data are shown with 95% confidence intervals (CIs), calculated using the exact binomial distribution (Clopper-Pearson) [14] for proportions and the normal approximation method for GMCs/GMTs. An equivalence testing approach was used to test lot-to-lot consistency using SP rates for anti-D, anti-T, anti-HBs, anti-PRP, and anti-polio 1, 2, 3 based on the use of the two-sided 95% CI of the difference between two pairs of batches. The 95% CI was calculated using Wilson score method without continuity correction [14] and equivalence was demonstrated if the 95% CI of the paired difference lay entirely with À10% to 10%. For the pertussis antigens, three paired equivalence tests on adjusted GMCs (aGMCs) were performed to test lot-to-lot consistency for anti-PT and anti-FIM responses based on the use of the two-sided 95% CI of the ratio between two pairs of batches. The 95% CI of the ratio between aGMCs was calculated using normal approximation of the log10 of the concentration, and equivalence between lots was demonstrated if the 2-sided 95% CI fell entirely within 0.5-2.0. Of note, adjusted GMCs were computed to adjust for baseline disparities and consider the correlation between pre-and postconcentration, through an analysis of covariance model using the pre-vaccination log-transformed concentration as a covariate for adjustment to account for the associated variability. Overall equivalence between lots was concluded if equivalence for both SP rates and aGMCs was demonstrated.
Following demonstration of lot-to-lot consistency for DTwP-IPV-HB-PRP$T, the three lots were pooled for non-inferiority comparison to DTwP-HB-PRP$T and IPV based on the 95% CI of the difference (DTwP-IPV-HB-PRP$T minus DTwP-HB-PRP$T and IPV). For SP rates non-inferiority was concluded if the lower limit of the 95 %CI of the difference was greater than À10%, and for aGMCs non-inferiority was concluded if the lower limit of the 95% CI for the ratio was > 0.5. Overall non-inferiority was concluded if noninferiority was demonstrated for both SP rates and aGMCs.
For pertussis, equivalence and non-inferiority were also assessed using non-adjusted GMCs for anti-PT and anti-FIM endpoints as a post hoc analysis.
A total of 1280 participants was planned (320 participants in each group), providing an overall power of 90.2% for the equivalence analysis and 99.9% for the non-inferiority analysis, assuming an attrition rate of 15%. A subset of 640 participants (160 per group) was randomized for the assessment of rotavirus vaccine immunogenicity.
The immunogenicity analyses, including the equivalence and non-inferiority testing, used the per protocol (PP) population (participants with no protocol violation that could have interfered with the evaluation criteria, and analyzed according to the vaccine received). Data from the full analysis set (FAS) (those who received at least one vaccination, and analyzed according to the randomization) supported the evaluation done using the PP population. The safety evaluation used the safety analysis set (SafAS) (participants who received at least one vaccination).
Statistical analyses were done under the responsibility of Sanofi Pasteur's statistical group using SAS software, Version 9.4 (SAS Institute, Cary, NC, USA).

Participants studied
A total of 1280 participants received at least one vaccination, with 959 randomized to DTwP-IPV-HB-PRP$T (318, 321, and 320 participants for Lots A, B, and C, respectively) and 321 randomized to DTwP-HB-PRP$T and IPV. Overall, 890 participants (DTwP-IPV-HB-PRP$T: 296, 295, and 299 participants for Lots A, B, and C) and 297 participants (DTwP-HB-PRP$T and IPV) completed the study. Participant disposition, including reasons for withdrawal and reasons for exclusion from the PP population, is presented in Fig. 1. Demographic characteristics were similar in each group with a comparable proportion of male and female participants (overall 47.6% female).

Immunogenicity
The three DTwP-IPV-HB-PRP$T lots were equivalent as the 95% CIs for the SP rate differences (for anti-D, anti-T, anti-HBs, antipolio 1, 2, 3, and anti-PRP) and aGMC ratios (for anti-PT and anti-FIM) were within the pre-defined clinical margins (Table 1). Therefore, immunogenicity data were pooled for the three DTwP-IPV-HB-PRP$T lots and the 95% CIs for the SP rate differences and aGMC ratios between DTwP-IPV-HB-PRP$T and DTwP-HB-PRP$T and IPV confirmed overall non-inferiority ( Table 2). The post hoc evaluation using non-adjusted GMCs confirmed the equivalence and non-inferiority demonstrated using the aGMC data.
There were no clinically important differences between groups pre-first vaccination or post-third vaccination for antibodies to the D, T, pertussis, HB, IPV (anti-polio 1, 2, and 3), or PRP antigens in terms of the thresholds evaluated or for GMCs and GMTs, although differences were noted post-third vaccination for anti-polio GMTs (all types) (higher for DTwP-IPV-HB-PRP$T) and anti-FHA GMC (higher for DTwP-HB-PRP$T and IPV) ( Table 3) (Table 3).

Safety and tolerability
There were no clinically important differences in the safety profile of the three DTwP-IPV-HB-PRP$T lots (79.4%, 81.0%, and 81.9% of participants reporting at least one solicited reaction and 12.9%, 15.9%, and 16.3% of participants reporting at least one unsolicited Table 1 Equivalence of seroprotection rate (anti-D, anti-T, anti-HBs, anti-polio 1, 2, 3, and anti-PRP) and geometric mean concentration (anti-PT and anti-FIM) between three lots of DTwP-IPV-HB-PRP$T at 28 days after 3-dose vaccination series at 6-8, 10-14, and 16-18 weeks of age (Per Protocol population).    AE for Lot A, B, and C, respectively) and so only the pooled data are presented (Table 4). There were no immediate AEs (within 30 min after vaccination). The overall incidence of participants with at least one injection site reaction was similar for DTwP-IPV-HB-PRP$T (80.8%) and concomitant DTwP-HB-PRP$T and IPV (81.3%), and in each group the most common injection site reaction was tenderness (64.9% and 69.4% of participants) ( Table 4). Grade 3 solicited injection site reactions were reported by 15.0% (DTwP-IPV-HB-PRP$T) and 12.2% (DTwP-HB-PRP$T and IPV) of participants. For DTwP-HB-PRP$T and IPV, respectively, 65.3% and 30.3% of participants reported tenderness, 17.5% and 6.9% reported erythema, and 34.7% and 11.9% reported swelling almost exclusively at the DTwP-HB-PRP$T injection site. For solicited systemic reactions, the overall incidence was similar in each group (69.0% and 62.8% of participants), with 5.0% and 4.4% of participants reporting a Grade 3 reaction, the most common in each group being fever (43.8% and 37.8% of participants) (Table 4). Generally, the incidence of solicited injection site and systemic reactions either reduced or similar after each subsequent vaccination.
The overall incidence of participants reporting at least one unsolicited AE was similar for DTwP-IPV-HB-PRP$T (15.0%) and DTwP-HB-PRP$T and IPV (12.1%). Overall, the incidence after each vaccination was similar, most were Grade 1 in intensity with no Table 3 Seroprotection rates, vaccine response rates, geometric mean concentrations, and geometric mean titers for DTwP-IPV-HB-PRP$T and DTwP-HB-PRP$T and IPV pre-first vaccination and post-third vaccination at 6-8, 10-14, and 16-18 weeks of age (Per Protocol population).

DTwP-IPV-HB-PRP$T* (N = 864 y )
DTwP-HB-PRP$T and IPV (N = 285 y ) Data are % (95% CI) participants with titer or concentration above threshold, VR, 4-fold rise from pre-first vaccination, GMT, or GMC.   [15][16][17][18][19]. Furthermore, anti-PT antibodies are considered important for protection against clinical symptoms, and WHO recommendations include the documentation of anti-PT responses [20,21]. For these reasons, anti-PT and anti-FIM were selected as the primary endpoints for the evaluation of the pertussis response using a robust qualified assay [8], and were included in the main statistical analyses. A descriptive comparison of all immune response thresholds, GMCs, and GMTs confirmed the robust response. Despite isolated differences, such as higher anti-polio GMTs in the DTwP-IPV-HB-PRP$T group and higher anti-FHA GMC in the DTwP-HB-PRP$T and IPV group, no differences between the two groups were considered to be clinically important and such isolated differences were expected for this type of vaccine [22][23][24]. These data are comparable to previous data for the new hexavalent vaccine [6,7] as well as other vaccines of this type [25][26][27][28][29][30][31][32] suggesting no interaction due to the co-administration with rotavirus vaccine. In particular, the high anti-polio antibody titers for the DTwP-IPV-HB-PRP$T vaccine, which were almost twice as high as for standalone IPV and expected due to the adjuvant effect of aluminum contained in the vaccine [33][34][35][36] and consistent with previous studies, ensure alignment with the polio endgame strategy and withdrawal of OPV [3,37]. The literature for poliovirus vaccines is extensive, and documents various vaccination regimens that can be used in associa-tion with DTwP-containing vaccines, eg, bivalent OPV1&3-only, IPV-only, OPV followed by IPV, IPV followed by OPV, or mixed IPV/OPV [24,38]. These data demonstrate that SHAN6 can be administered following any regimen that uses IPV-containing vaccines and OPV. Additionally, the immune response to routine rotavirus vaccination was similar in each group and in the expected range [39,40].
The use of the ECL assay for the evaluation of the pertussis response in our study provides data that are precise, accurate, and reproducible [8]. Commercially available assays developed for routine pertussis diagnostic purposes that have been used in the evaluation of pertussis immunogenicity in many other clinical studies of wP-containing vaccines [28][29][30][31][32][41][42][43] have unknown specificity and are less well-suited to the robust evaluation that is required in clinical trials. Additionally, the pertussis component of the DTwP-IPV-HB-PRP$T vaccine is similar to that contained in an established range of vaccines (DTCoq, Tetracoq, Pentacoq) that have been used extensively and have shown efficacy and field effectiveness against pertussis [44][45][46].
There were no safety concerns associated with administration of the DTwP-IPV-HB-PRP$T vaccine in this study, consistent with other wP-containing multivalent vaccines in India and elsewhere [30,31,42,47,48]. There were very few unsolicited vaccine-related AEs, no vaccine-related SAEs, and no episodes of convulsions, hyporesponsive state, or encephalopathy. There was an imbalance in SAEs that were not related to vaccination, which could have been due to the imbalanced sample size between the DTwP-IPV-HB-PRP$T and DTwP-HB-PRP$T and IPV groups or possibly due to unconscious bias in the collection of safety data in the investigational vaccine group in this open-label study. No differences between groups were considered to be of clinical importance, and overall reactogenicity was similar in each group. One participant died of SIDS before the third dose of DTwP-IPV-HB-PRP$T was due, and this was not considered to be related to vaccine administration. The DTwP-IPV-HB-PRP$T safety profile was comparable to previous studies with this vaccine [6,7] and its wPantigen matching predecessor [48][49][50].
Limitations of the study included the open-label design, which could have introduced bias in the reporting of safety, and the lack of a control group without rotavirus vaccination although comparison to historical data, described earlier, was not indicative of any interaction.
In conclusion, three lots of the fully liquid DTwP-IPV-HB-PRP$T vaccine showed consistency of manufacturing by showing consistent immunogenicity which was comparable to licensed vaccines following vaccination of infants at 6-8, 10-12, and 14-16 weeks of age in India. There was no evidence of any effect of coadministration with rotavirus vaccine, and there were no safety concerns. Hexavalent vaccines containing IPV are expected to increase compliance to vaccination schedules, favor access to multi-dosing IPV regimens, and help to support switching to IPV use globally.
Financial support: the study was funded by Sanofi Healthcare India Private Ltd (SHIPL).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: Clinical investigators involved in these studies received fees from Sanofi Pasteur through their institutions for the conduct of these clinical studies, but did not receive any direct payment from Sanofi Pasteur in this regard.