Safety and immunogenicity of an indigenously developed tetanus toxoid, diphtheria toxoid, and acellular pertussis vaccine (Tdap) in adults, adolescents, and children in India

ABSTRACT

Background

This study assessed safety and immunogenicity of Serum Institute of India Pvt Ltd (SIIPL)’s tetanus toxoid (TT), diphtheria toxoid (DT), and acellular pertussis booster vaccine (Tdap).

Research Design and Methods

In this Phase II/III, multicenter, randomized, active-controlled, open-label study, 1500 healthy individuals, aged 4–65 years, were randomized to receive a single dose of SIIPL Tdap or comparator Tdap vaccine (Boostrix®; GlaxoSmithKlines, India). Adverse events (AEs) during initial 30 minutes, 7-day, 30-day post-vaccination were assessed. Blood samples were taken before and 30 days post-vaccination for immunogenicity assessment.

Results

No significant differences in incidence of local and systemic solicited AEs were observed between the two groups; no vaccine-related serious AEs were reported. SIIPL Tdap was non-inferior to comparator Tdap in achieving booster responses to TT and DT in 75.2% and 70.8% of the participants, respectively, and to pertussis toxoid (PT), pertactin (PRN), and filamentous hemagglutinin (FHA) in 94.3%, 92.6%, and 95.0% of the participants, respectively. Anti-PT, anti-PRN, and anti-FHA antibody geometric mean titers in both the groups, were significantly higher post-vaccination compared to pre-vaccination.

Conclusions

Booster vaccination with SIIPL Tdap was non-inferior to comparator Tdap with respect to immunogenicity against tetanus, diphtheria, and pertussis and was well tolerated.

KEYWORDS:

• Acellular pertussis
• booster
• diphtheria
• tetanus
• vaccine

1. Introduction

A highly infectious disease, pertussis remains a significant public health concern globally [1]. Despite established infant and childhood immunization programs, which have reduced the burden of pertussis in childhood, in the last 30 years a resurgence of pertussis has been noted not only in high-income countries but also in many low- and middle-income countries [2]. Whether after vaccination against or infection with Bordetella pertussis, immunity can wane [3,4]. Waning immunity against pertussis enables increased susceptibility and a reservoir for further transmission and disease, sometimes with severe complications in those unvaccinated or incompletely vaccinated vulnerable populations. In 2021, there were 28,871 pertussis cases globally and 593 were reported in India, which is a less number as compared to those reported in the year 2020 (69,552 cases globally and 12,566 cases in India) [5]. However, the true burden of pertussis is often underestimated, and there is much support for the expansion of routine vaccination to include booster doses for children at school entry, adolescents, and older adults [3,6,7].

Vaccination is the most effective intervention to reduce the burden of disease, and while vaccine effectiveness (VE) after boosting with reduced antigen content, acellular pertussis-containing tetanus-diphtheria-acellular pertussis (Tdap) vaccine is initially high and wanes over time. It has been estimated that the initial absolute VE of 91% and 85% observed after childhood series vaccination and adolescent booster vaccination has been decreased at 9.6% and 11.7% annually, respectively [8]. In addition to childhood booster doses, a single booster vaccination with Tdap is recommended in adults in several European countries and Canada, while it is recommended for all adults every 10 years in the United States, Austria, Belgium, and Italy including for adults working with infants and young children and for all healthcare workers in Australia to improve community protection in several countries [9–14]. A recent economic evaluation and systematic review found favorable cost-effectiveness ratios for adolescents’ and adults’ vaccination, particularly for adolescents’ vaccination [15]. As per the WHO's position paper on pertussis vaccine, national programs currently using wP vaccination should continue to use wP vaccines for primary vaccination series. While national programs currently using aP vaccine may continue using aP-based combination vaccines but should consider the need for additional booster doses and strategies to prevent early childhood mortality in case of pertussis resurgence [16]. Thus, from the demand-supply perspective, availability of cost-effective Tdap vaccine as compared to currently available high priced vaccines will be beneficial.

To meet the large and increasing global demand for pertussis vaccines, a Tdap vaccine indigenously developed and manufactured by Serum Institute of India Pvt Ltd (SIIPL) demonstrated a good tolerability and safety profile in healthy adults in a Phase I trial [17]. Thereafter, the current Phase II/III clinical trial [CTRI/2018/06/014617] was conducted to assess the safety and immunogenicity of SIIPL Tdap in subjects aged 4 to 65 years in comparison to comparator Tdap vaccine (Boostrix®; GlaxoSmithKlines, India).

2. Patients and methods

2.1. Study design

The study was prospective, open-label, active-controlled, and randomized with two study groups. Enrollment was age-stratified into three age cohorts: 1. adults, ≥18 to 65 years; 2. adolescents, 10 to <18 years; and 3. children 4 to <10 years. Centralized, computer-generated, block randomization lists (block size of 2), stratified based on age cohorts, were developed to ensure equal numbers of participants enrolled in each age cohort. Eligible participants were randomized to one of the two vaccine groups in a 1:1 ratio to receive a single dose of either SIIPL Tdap or comparator vaccine. Independent oversight of the Phase II safety study was provided by a data and safety monitoring board (DSMB) to assess the safety data to permit age de-escalation from cohorts 1 and 2 to cohort 3, and to the Phase III immunogenicity study. The duration of each participant’s study participation was 30 (+14) days. Safety and immunogenicity data of Phase II participants were included in the Phase III analyses.

The study was conducted in accordance with the International Conference on Harmonization – Good Clinical Practice (ICH-GCP) guidelines, the principles of Declaration of Helsinki, Drugs and Cosmetics Rules, 2013, 122-DAB, and New Drugs and Clinical trial Rules-2019, Government of India. Permission to conduct this study was granted by the National Regulatory Authority of India, i.e. Drugs Controller General India (DCGI). The protocol, protocol amendments, and associated documents were reviewed and approved by ethics committees at each study site. Prior to the study entry, written informed consent was obtained from each study participant (≥18 years) and from either parent of each study participant aged <18 years. For study participants aged 7 to <12 years, verbal informed assent also was obtained and for study participants aged 12 to <18 years, written informed assent also was obtained.

2.2. Study participants

Healthy individuals aged 4 to 65 years were invited to participate in the study, which was conducted in 6 (Phase II) and 11 (Phase III) sites across India between September 2018 and June 2020. Sexually active participants were required to have used an effective method of contraception for the previous 30 days and throughout the study period. Prior to enrollment, a urine pregnancy test (UPT) was performed for female participants of childbearing potential. Participants were excluded from participation if they had received any tetanus, diphtheria, or pertussis vaccine (except tetanus-prone wound management for adults and/or tetanus vaccination in pregnant women) within the previous 5 years, a history of tetanus, diphtheria, or pertussis infection, or received any other vaccine within the previous 30 days or planned to receive during study participation. Other exclusion criteria were history of known hypersensitivity to any study vaccine component; a history of anaphylactic shock; a history of neurological complications following an earlier vaccination against tetanus and/or diphtheria; a history of encephalopathy within 7 days of a previous dose of pertussis vaccine; a history of any major systemic illness; a history of any cancer, HIV infection, organ transplant, or any other immune system disorder; received immunoglobulins and/or any blood products within the previous 3 months, or planned administration during the study; or received chronic administration (>14 days in total) of immunosuppressant within the previous 6 months. Pregnant or lactating women were also ineligible.

2.3. Study vaccines

Based on the prior clinical Phase I SIIPL Tdap vaccine study, a single 0.5 mL dose of SIIPL Tdap vaccine was selected for the study and contained ≥20 international units (IU), tetanus toxoid (TT), ≥2 IU diphtheria toxoid (DT), ≥8 mcg pertussis toxoid (PT), ≥2.5 mcg pertactin (PRN), and ≥8 mcg filamentous hemagglutinin (FHA), with ≤1.25 mg aluminum hydroxide. TT, DT, and PT in SIIPL Tdap are detoxified using formaldehyde. A single 0.5 mL dose of comparator Tdap vaccine contained TT not <20 IU (5 LF), DT not <2 IU (2.5 LF), 8 mcg PT, 2.5 mcg PRN, and 8 mcg FHA, adsorbed on aluminum hydroxide hydrate (0.3 milligrams Al³⁺) and aluminum phosphate (0.2 milligrams Al³⁺). In the comparator Tdap, TT and DT are detoxified using formaldehyde, while PT is detoxified using both glutaraldehyde and formaldehyde. Both the vaccines contain sodium chloride and water for injections as excipients. The content of each study vaccine is included in the protocol (Supplementary material Appendix). Study vaccines administered intramuscularly into the deltoid or in the anterolateral aspect of the thigh using a 23-gauge needle of 1-inch length.

2.4. Assessment of reactogenicity and safety

Post-vaccination, study participants were observed for at least 30 minutes for occurrence of any immediate adverse events, and physical examination with vital signs assessment was performed. Participants were given a digital thermometer and Part 1 diary card with careful instructions to record daily their temperature, any solicited local (pain, redness, and/or swelling at the injection site) and general (fever, headache, dizziness, fatigue, malaise, and/or nausea) adverse events (AEs) occurring during the 7-day follow-up period after vaccination. Unsolicited AEs and concomitant medications were also recorded. On the Day 7 visit, Part 1 diary cards were reviewed, and a physical examination with vital signs assessment performed. Then participants were given a Part 2 diary card to record any unsolicited AEs, serious AEs (SAEs) and any concomitant medications taken, after the Day 7 visit. On Day 30 (+14 days) visit, Part 2 diary cards were reviewed, a physical examination with vital signs assessment performed, and a UPT completed for female study participants of childbearing potential. All AEs and SAEs were classified according to the Medical Dictionary for Regulatory Activities (MedDRA version 21.1) System, Organ, and Class. Concomitant medications were coded using the WHO Drug Dictionary (September 2020). The severity of all AEs/SAEs was graded (Grade 1 to 4) as per the clinical judgment of the Investigator taking into account information provided by subject/subject’s parents based on the guidance provided in the protocol.

2.5. Assessment of immunogenicity

Blood samples were collected from all study participants prior to and 30 (+14) days after study vaccination. The sera analysis was conducted at Vimta Labs Ltd, India, using Multiplex Luminex assays with standardized and validated procedures, and with adequate controls. The development and validation of Luminex assays for simultaneous quantification of antibodies against D, T, PT, FHA, and PRN antigens using mice sera samples has been previously reported [18]. The assay was adapted to human serum samples and calibrated using the National Institute for Biological Standards and Control (NIBSC, UK) standards and was validated using a panel of human serum samples as per ICH and US FDA guidance on validation of bioanalytical methods [19,20]. Antibody concentrations ≥0.1 IU/mL against TT and DT were considered indicative of seroprotection. For pertussis antigens PT, PRN, and FHA, the lower limits of quantitation (LLOQ) values based on bioanalytical method validation were 0.132 IU/mL, 0.772 IU/mL, and 0.468 IU/mL, respectively, and participants with antibody concentrations above the respective LLOQs were considered seropositive. The assay sensitivity and LLOQ values are in line with other studies, which used similar multiplex assays for screening antibodies against the three pertussis antigens [21,22].

2.6. Statistical analysis

The immunogenicity analysis was performed on the per-protocol study population that included all participants who received a study vaccine and had 30-day immunogenicity data available with no major protocol deviations. The co-primary study objectives were demonstration of the non-inferiority of SIIPL Tdap compared to comparator, 30 days after vaccination in terms of the percentages of participants achieving booster responses to TT, DT, PT, PRN, and FHA and with respect to geometric mean concentrations (GMCs) for antibodies against PT, PRN, and FHA. Secondary endpoints included percentages of participants seroprotective against TT and DT, seropositive against pertussis (PT, PRN, and FHA), and anti-TT and anti-DT GMCs 30 days after vaccination.

Booster responses for TT and DT were defined as 30 days after vaccination, a post-vaccination antibody concentration ≥0.4 IU/mL in participants with a pre-vaccination concentration <0.1 IU/mL, or a post-vaccination increase of at least four times the pre-vaccination antibody concentration in participants with an initial concentration ≥0.1 IU/mL and <1 IU/mL, or a post-vaccination increase of at least two times the pre-vaccination antibody concentration in participants with an initial concentration ≥1 IU/mL. Booster responses to pertussis (PT, PRN, and FHA antigens) were defined as 30 days after vaccination, a post-vaccination antibody concentration of at least four times the pre-vaccination concentration for initially seronegative participants, or a post-vaccination antibody concentration of at least two times the pre-vaccination concentration for initially seropositive participants.

The proportions of study participants with a booster response rate in each Tdap vaccine group were presented for anti-TT, anti-DT, anti-PT, anti-PRN, and anti-FHA. To assess the differences, 95% confidence intervals (CIs) were calculated for the differences in proportions. A two-group Farrington-Manning test was used to test a one-sided hypothesis of non-inferiority in proportions. For each study endpoint, non-inferiority was found if the lower bound of the two-sided 95% CI for the vaccine group difference in proportions was ≥-10%. The GMC ratio (SIIPLTdap/comparator) of antibodies against PT, PRN, and FHA and the associated two-sided 95% CI were calculated by assuming the log normality of concentrations and using the ANCOVA model on the logarithm transformation of the concentrations with age and vaccine group as factors and baseline concentration as covariate. If the lower limit of the 95% CI of the GMC ratio of each antibody was ≥0.67, then the SIIPL Tdap vaccine’s response was considered non-inferior to that of comparator. Although the study was not planned to demonstrate non-inferiority in each of the age cohorts, a post-hoc subgroup analysis of the primary objectives was performed.

The safety analysis was performed on the total vaccinated Phase II/III cohort that included all vaccinated study participants for whom post-vaccination safety data were available with the objective to assess the frequency of reactogenicity and AEs through 30 days after vaccination with SIIPL Tdap or comparator. Differences between groups were calculated along with the p-value using Fisher’s exact test or Chi-squared test and two-sided 95% CI based on the asymptotic standardized method for the difference of incidence rates. A p-value <0.05 was considered statistically significant but not necessarily clinically relevant.

The sample size of 1350 study participants, 675 in each vaccine group, would achieve 93.4% power to detect a non-inferiority margin difference between the group proportions (booster responses) of −0.10 with an actual difference in proportion assumed to be 0.0%. A one-sided Farrington-Manning score test was used with a one-sided alpha of 0.025. With 675 evaluable participants per vaccine group, a non-inferiority margin assumed to be 0.67 and the true GMC ratio assumed to be 1, 99.5% power to detect non-inferiority using a one-sided, two-sample t-test was calculated. Thus, the overall power of the study to evaluate the non-inferiority hypotheses of booster response for DT, TT, pertussis antigens, and GMC for three pertussis antigens was 92.9%. Allowing for a 10% drop-out rate, 750 subjects per vaccine group were randomized. The sample size was powered for each of the primary endpoints separately; no alpha adjustment for multiplicity of primary endpoints was made. Statistical analyses were performed using the Statistical Analysis System (SAS)® software Version 9.4.

3. Results

3.1. Study population

A total of 1500 healthy participants were enrolled, randomized, and vaccinated (Figure 1), of whom 1439 (96%), 719 (250 adults, 239 adolescents, and 230 children) in the SIIPL Tdap vaccine group, and 720 (249 adults, 243 adolescents, and 228 children) in the comparator vaccine group, were included in the per-protocol immunogenicity analysis. The two vaccine groups were similar to each other with respect to baseline characteristics including age, sex, weight, and height (Table 1).

Figure 1. Participant flowchart.

Table 1. Demographics characteristics – ITT population (N = 1500).

Characteristics	Parameter/Categories	Cohort 1(≥ 18 to 65 years)		Cohort 2(10 to < 18 years)		Cohort 3(4 to < 10 years)		Overall
		SIIPL Tdap (N = 250)	Comparator (N = 250)	SIIPL Tdap (N = 251)	Comparator (N = 249)	SIIPL Tdap (N = 249)	Comparator (N = 251)	SIIPL Tdap (N = 750)	Comparator (N = 750)
Age in years (at vaccination)	Mean	33.6	33.9	12.6	13.0	6.3	6.2	17.5	17.7
	SD	9.96	9.54	2.14	2.20	1.93	1.85	13.12	13.16
	Median	32.0	32.0	12.0	13.0	6.0	5.0	12.0	13.0
	Range	(18:62)	(18:63)	(10:17)	(10:17)	(4:9)	(4:9)	(4:62)	(4:63)
Gender, n (%)	Male	110 (44.00%)	110 (44.00%)	134 (53.39%)	133 (53.41%)	137 (55.02%)	149 (59.36%)	381 (50.80%)	392 (52.27%)
	Female	140 (56.00%)	140 (56.00%)	117 (46.61%)	116 (46.59%)	112 (44.98%)	102 (40.64%)	369 (49.20%)	358 (47.73%)
Height (cm)	Mean	160.2	159.7	148.8	149.6	116.4	115.3	141.8	141.5
	SD	9.14	10.06	11.24	11.79	13.08	12.33	21.68	22.21
	Median	160.0	159.0	148.5	150.0	115.0	114.0	147.0	147.0
Weight (kg)	Mean	60.7	61.9	39.5	40.7	20.7	19.7	40.3	40.8
	SD	13.03	12.22	11.72	10.96	6.60	5.64	19.59	19.95
	Median	58.9	61.0	38.0	39.3	19.0	19.0	38.7	39.4

n: Subject Count, N: Sample Size.

3.2. Safety

In the 1500 participants, 690 AEs were reported in 356 (47.5%) participants in the SIIPL Tdap group, and 664 AEs were reported in 376 (50.1%) participants in the comparator group (p-value = 0.302); all participants with reported AEs recovered without sequelae. There were also no statistically significant differences between vaccine groups in terms of numbers of AEs in individual participants (p-value = 0.800), numbers of solicited (p-value = 0.379) vs unsolicited AEs (p-value≥0.999), or AE severity (p-value (Grade 1, mild) = 0.324; p-value (Grade 2, moderate) = 0.866; p-value (Grade 3, severe) = 0.624). The most commonly reported local and systemic solicited AEs in both vaccine groups were injection site pain and headache, respectively, with no statistically significant differences between vaccine groups (Table 2). No potentially life-threatening AEs or deaths (Grade 4) were reported in either vaccine group.

Table 2. Summary of solicited adverse events after vaccination intention to treat population (N = 1500).

Adverse Event	SIIPL Tdap (N = 750)		Comparator Tdap (N = 750)		p-value^b
	n (%) [E]	95% CI^a	n (%) [E]	95% CI^a
Local adverse event
Injection site pain	308 (41.1%) [315]	[37.52:44.68]	322 (42.9%) [329]	[39.36:46.56]	0.464
Injection site erythema	17 (2.3%) [17]	[1.33:3.6]	8 (1.1%) [8]	[0.46:2.09]	0.069
Injection site swelling	46 (6.1%) [47]	[4.52:8.1]	42 (5.6%) [42]	[4.07:7.49]	0.660
Systemic adverse event
Fever	32 (4.3%) [38]	[2.94:5.97]	28 (3.7%) [36]	[2.49:5.35]	0.598
Dizziness	15 (2.0%) [16]	[1.12:3.28]	19 (2.5%) [20]	[1.53:3.93]	0.488
Fatigue	43 (5.7%) [44]	[4.18:7.65]	41 (5.5%) [42]	[3.95:7.34]	0.822
Headache	73 (9.7%) [76]	[7.71:12.08]	66 (8.8%) [70]	[6.87:11.06]	0.533
Malaise	21 (2.8%) [21]	[1.74:4.25]	18 (2.4%) [18]	[1.43:3.77]	0.626
Nausea	20 (2.7%) [23]	[1.64:4.09]	23 (3.1%) [25]	[1.95:4.57]	0.643

n (%), E: where n = number of participants with at least one adverse event (counted only once), % = (number of participants with at least one adverse event/number of participants in respective study vaccine group of safety population)*100 and E = number of adverse events, CI = confidence interval

a 95% CI were calculated by Clopper–Pearson Method.

b p-value was calculated using using Chi-square test. p-value below 0.05 was considered statistically significant.

In the SIIPL Tdap group, two SAEs (one SAE of appendicitis in one subject and one SAE of lower respiratory tract infection in another subject) were reported (both judged as not related to the study vaccine), whereas no SAEs were reported in the comparator group.

3.3. Immunogenicity

3.3.1. Response to tetanus and diphtheria toxoids

Post-vaccination, anti-TT and anti-DT antibody levels consistent with seroprotection were achieved in 719 (100%) and 675 (93.9%) participants, respectively, in the SIIPL Tdap group and 719 (99.9%) and 678 (94.2%) participants, respectively, in the comparator group (Table 3). Booster responses were also comparable between the SIIPL Tdap and the comparator, meeting the pre-defined statistical non-inferiority criteria: 75.2% for anti-TT and 70.8% for anti-DT in the SIIPL Tdap vaccine group, and 72.8% for anti-TT and 76.5% for anti-DT in the comparator group (Table 4). Pre-, post-vaccination, and GMC values for anti-TT and anti-DT antibodies were similar between Tdap vaccine groups. Post-vaccination and GMC values for anti-TT and anti-DT and were 13.41 IU/ml and 1.34 IU/ml, respectively, in the SIIPL Tdap vaccine group and 12.22 IU/ml and 1.82 IU/ml, respectively, in the comparator group. Reverse cumulative distribution curves demonstrate similar distributions for anti-TT and anti-DT antibody concentrations pre- and post-vaccination between the SIIPL Tdap and comparator vaccine group (Figure 2).

Figure 2. Reverse cumulative distribution curves showing IgG antibody concentrations at Pre-vaccination (Pre) and 30 days after post-vaccination (Post) in per protocol population for anti- Diphtheria toxoid (DT) and anti-Tetanus Toxoid (TT). The X-axis is logarithmic. Y-axis is percentage of subjects. The units used in the multiplex immunoassay to measure IgG antibodies are International Units/ml (IU/ml).

Table 3. Seroprotection/Seropositivity in per protocol population (N = 1439) one month after vaccination with the SIIPL Tdap (N = 719) or Comparator Tdap (N = 720) vaccines.

Seroprotection/Seropositivity
Antibody	SIIPL Tdap			Comparator Tdap			Difference^b (%)	(95% CI)^c
	N	n	%^a	N	n	%		LL	UL
Anti-TT	719	719	100.00	720	719	99.86	0.14	−1.71	1.99
Anti-DT	719	675	93.88	720	678	94.17	−0.29	−2.68	2.11
Anti-PT	719	719	100.00	720	720	100.00	0.00	NE	NE
Anti-FHA	719	717	99.72	720	720	100.00	−0.28	−2.12	1.56
Anti-PRN	719	719	100.00	720	720	100.00	0.00	NE	NE

N = number of available participants for each of the antigen, n = number of participants achieving seroprotection/seropositivity, CI = confidence interval, LL = lower limit, UL = upper limit, NE = not estimable, TT = Tetanus Toxoid, DT = Diphtheria Toxoid, PT = Pertussis Toxiod, PRN = pertactin, FHA = filamentous hemagglutinin. N = number of subjects with pre- and post-vaccination results available

a Percentages were calculated using respective column header group count as denominator.

b Proportion in SIIPL Tdap group – Proportion in Comparator Tdap group

c Two-sided 95% CI for difference in proportion of subjects between groups were calculated using

Farrington-Manning Test

cutoff value for seroprotection against diphtheria and tetanus is ≥ 0.1 IU/ml. For pertussis, antigens cut off values for PT, FHA, and PRN, the values are 0.132 IU/ml, 0.468 IU/ml, and 0.772 IU/ml respectively.

Noninferiority was declared if the lower bound of the two-sided 95% CI for the treatment difference (SIIPL Tdap – Comparator Tdap) in proportions were greater than ≥ −10%.

Table 4. Booster responses in the per protocol population (N = 1439) one month after vaccination with the SIIPL Tdap (N = 719) or comparator Tdap (N = 720) vaccines.

Booster Response^a
Antibody	SIIPL Tdap			Comparator Tdap			Difference^c (%)	95% CI^d
	N	n	%^b	N	n	%^b		LL	UL
Anti-TT	719	541	75.2	720	524	72.8	2.47	−1.35	6.28
Anti-DT	719	509	70.8	720	551	76.5	−5.74	−9.54	−1.93
Anti-PT	719	678	94.3	720	659	91.5	2.77	0.20	5.34
Anti-FHA	719	683	95.0	720	686	95.3	−0.28	−2.57	2.00
Anti-PRN	719	666	92.6	720	662	91.9	0.68	−1.90	3.26

N = number of available participants for each of the antigen, n = number of participants achieving booster response, CI = confidence interval, LL = lower limit, UL = upper limit, TT = Tetanus Toxoid, DT = Diphtheria Toxoid, PT = Pertussis Toxiod, PRN = pertactin, FHA = filamentous hemagglutinin

a Booster responses were defined as below

For anti-D and anti-T:

• Initially, seronegative subjects (pre-vaccination concentration below cutoff: <0.1 IU/mL)

should have a concentration at least four times the cutoff, days after vaccination (post- vaccination concentration ≥ 0.4 IU/mL).

• Initially seropositive subjects (pre-vaccination concentration ≥0.1 IU/mL and <1 IU/mL)

Should have an increase of at least four times the pre-vaccination concentration, 30 days after vaccination.

• Initially seropositive subjects (pre-vaccination concentration ≥1 IU/mL) should have an

increase of at least two times the pre-vaccination concentration, 30 days after vaccination.

For pertussis antigens (PT, FHA, and PRN):

• For initially seronegative subjects with an increase in antibody concentrations of at least

four times the pre-vaccination concentration, 30 days after vaccination.

• For initially seropositive subjects, an increase in antibody concentrations of at least two times the

pre-vaccination concentration, 30 days after vaccination.

b Percentages were calculated as n/N x 100

C 95% CI difference in proportion of subjects between groups will be calculated using Farrington- Manning Test

d Proportion difference is the difference of booster response between groups.

Two-sided 95% CI for difference in proportion of subjects between groups were calculated using Farrington-Manning Test.

Noninferiority was declared if the lower bound of the two-sided 95% CI for the treatment difference (SIIPL Tdap – Comparator Tdap) in proportions will be greater than ≥ −10%.

3.3.2. Response to acellular pertussis antigens

Post-vaccination, anti-PT, anti-PRN, and anti-FHA antibody levels consistent with seropositivity were observed in 719 (100%), 719 (100%), and 717 (99.7%) participants, respectively, in the SIIPL Tdap group and 720 (100%) in the comparator group (Table 3). Booster responses were also comparable between the SIIPL Tdap and the comparator vaccine, meeting the pre-defined statistical non-inferiority criteria: 94.3% for anti-PT, 92.6% for anti-PRN, and 95.0% for anti-FHA in the SIIPL Tdap group, and 91.5% for anti-PT, 91.9% for anti-PRN, and 95.3% for anti-FHA in the comparator group (Table 4). Prior to vaccination, GMC values for anti-pertussis antibodies were similar between Tdap vaccine groups. Post vaccination, GMC values for anti-PT, anti-PRN, and anti-FHA were 197.68 IU/ml, 172.23 IU/ml, and 313.33 IU/ml, respectively, in the SIIPL Tdap group and 120.01 IU/ml, 156.69 IU/ml, and 441.92 IU/ml, respectively, in the comparator group. Non-inferiority criteria for anti-PT and anti-PRN antibody GMC ratios were met; however, for the anti-FHA antibody GMC ratio, the lower limit of the CI (0.63) was below the predefined threshold of 0.67 (Table 5). Reverse cumulative distribution curves demonstrate similar distributions for anti-PT, anti-PRN, and anti-FHA antibody concentrations pre- and post-vaccination between the SIIPL Tdap and comparator group (Figure 3).

Figure 3. Reverse cumulative distribution curves showing IgG antibodies concentration at Pre-vaccination (Pre)and 30-days post-vaccination (Post) IgG in per protocol population against PT (a), FHA (b), PRN (c) antigen. The X-axis is logarithmic. Y-axis is percentage of subjects. The units used in the multiplex immunoassay to measure IgG antibodies are International Units/ml (IU/ml).

Table 5. Non-inferiority assessment for anti-PT, anti-FHA, and anti-PRN GMCs one month after vaccination – per protocol population (N = 1439).

Pertussis antigen	SIIPL-Tdap (N = 719)		Comparator Tdap (N = 720)		Post-vaccination GMC ratio	95% CI^b
	Pre-vaccination GMC (IU/ml)	Post-vaccination GMC^a (IU/ml)	Pre-vaccination GMC^a (IU/ml)	Post-vaccination GMC (IU/ml)	SIIPL-Tdap /Comparator Tdap	LL	UL
Anti-PT	8.11	197.68	7.79	120.01	1.65	1.46	1.86
Anti-FHA	11.04	313.33	11.38	441.92	0.71	0.63	0.80
ANTI-PRN	5.72	172.23	4.76	156.69	1.1	0.95	1.28

Abbreviations: N = number of subjects with post-vaccination results available; GMC = Geometric Mean Concentration, PT = Pertussis Toxiod, PRN = pertactin, FHA = filamentous hemagglutinin, CI = confidence interval, LL = lower limit, UL = upper limit, IU = International Unit, ml = milliliter.

a GMC one month post-vaccination

b 95% CI were estimated from the ANCOVA

Non-inferiority was declared if the lower limit of the 95% CI of the GMC ratio (SIIPL Tdap/ Comparator Tdap) for each of the antigen was ≥ 0.67

The results of post-hoc subgroup analyses for each age cohort for the primary objectives of immunogenic non-inferiority of the SIIPL Tdap to comparator group showed more than 90% power for all the endpoints except for anti-T booster response in cohort 2, anti-D booster responses, and FHA GMC comparison (Supplementary Tables S1 and S2). Post-hoc subgroup analyses showed that the seroprotection/seropositivity rates in the SIIPL Tdap are non-inferior to the comparator group in each age cohort (Supplementary Table S3). Since the subgroup analyses were not pre-specified they should be interpreted with caution.

4. Discussion

A single booster dose of SIIPL Tdap was shown to be well tolerated, safe, and immunogenic in healthy individuals aged 4 to 65 years. There were no significant differences in the incidence of local and systemic solicited AEs observed between SIIPL Tdap and comparator. Furthermore, no grade 4 AEs or vaccine-related SAEs were reported. With respect to the percentage of participants with seroprotective levels of anti-TT and anti-DT antibodies as well as the percentage of participants with seropositive pertussis antibody levels to all three pertussis antigens, PT, PRN, and FHA, the SIIPL Tdap was comparable to comparator. Study immunogenicity endpoints, including booster responder rates for all Tdap vaccine antigens and assessment of the magnitude of response, demonstrated that non-inferiority criteria were met.

While the GMCs for anti-PT and anti-PRN were higher in the SIIPL Tdap group than the comparator group, the GMC for anti-FHA was not, and the non-inferiority threshold for FHA GMC was marginally missed.

The multiplex-bead-based Luminex assay, which provides excellent specificity and sensitivity as compared to conventional ELISA assays, was used for quantification of antibodies against D, T, PT, FHA, and PRN [22,23]. Whereas immunological correlates of protection (ICP) are available for tetanus and diphtheria, no such immunological correlates of protection have been identified for pertussis [24]. Designed to determine the antibody values at the time of household exposure to pertussis infection, two multicomponent acellular pertussis vaccine efficacy trials evaluated whether the presence of specific antibodies was correlated with protection from pertussis [25,26]. Both studies demonstrated the presence of antibodies to PT, PRN, and fimbriae correlated with protection; however, neither study found a correlation with antibody to FHA. It has been concluded that anti-PT and anti-PRN may be utilized as surrogate markers of protection for multicomponent acellular pertussis vaccines [24]. In the absence of ICP for pertussis, the clinical relevance of antibody GMCs is unknown; booster responses may better reflect the actual immune responses [27]. Hence, failure to meet non-inferiority for FHA GMC is of limited clinical significance and unlikely to affect the effectiveness of the SIIPL Tdap vaccine to provide protection against pertussis given the booster response to all three pertussis antigens. The SIIPL Tdap vaccine’s immunogenicity results demonstrating seroprotection/seropositivity against diphtheria, tetanus, pertussis in a majority of participants are consistent with previous studies for licensed Tdap vaccine [28,29].

The open-label design of our study may be viewed as a limitation with respect to the interpretation of final safety results; however, vaccine allocation was centrally randomized for this trial conducted at multiple sites across India. Conclusions are limited to the study population, i.e., generally healthy population, with a list of exclusion criteria. Strengths of this study were the inclusion of different age cohorts in equal proportions and the use of a validated multiplex immunogenicity assay calibrated against NIBSC reference standards.

5. Conclusion

In summary, the SIIPL Tdap vaccine was non-inferior to the comparator Tdap vaccine for all five included antigens, showing similar booster responder rates against all three acellular pertussis antigens when compared to the comparator and inducing a higher magnitude of response against PT and PRN. Furthermore, the SIIPL Tdap vaccine’s AE profile was generally consistent with the known AE profile for comparators. Thus, these data support that when administered to healthy individuals aged 4 to 65 years, the SIIPL Tdap booster vaccine is comparable to the comparator Tdap vaccine with respect to reactogenicity and safety and to immunogenicity for all components of the vaccine.

Declaration of interests

The authors H Sharma, S Parekh, P Pujari, S Shewale, S Desai, H Rao, M Gautam, S Gairola, and U Shaligram are employees of Serum Institute of India Pvt. Ltd., Pune India. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

Hitt J Sharma, Sameer Parekh, Pramod Pujari, and Sunil Shewale were involved in the conception and design, or analysis and interpretation of the data; the drafting of the paper. Anand Kawade, Mandyam Ravi, Jitendra Oswal, Saji James, N. Mahantashetti, Renuka Munshi, Apurba Ghosh, Venkateshwar Rao, Sundaram Balsubramaniam, P. Varughese, A. Somshekhar were the principal investigators for this trial and were involved in the analysis and interpretation of the data. Shivani Desai, Amy Sarah Ginsburg, Harish Rao, Manish Gautam, Sunil Gairola, and Umesh Shaligram contributed in revising the manuscript critically for intellectual content. All authors approved the submitted version to be published; and agreed to be accountable for all aspects of the work.

Ethical approval

The study was conducted in accordance with the International Conference on Harmonization – Good Clinical Practice (ICH-GCP) guidelines, the principles of Declaration of Helsinki, Drugs, and Cosmetics (First Amendment) Rules, 2013, 122-DAB, and New Drugs and Clinical trial Rules-2019, Ministry of Health and Family Welfare, Government of India. Permission to conduct this study was granted by the National Regulatory Authority of India, i.e., DCGI. All the 11 Institutional Ethical Committees (IECs) were duly registered with DCGI. The protocol, protocol amendments, and other relevant documents were submitted to the IEC by the investigator and reviewed and approved by the IEC before the study was initiated, at their respective sites.

Clinical Trial Registration

Trial is registered at the Clinical Trials Registry-India (CTRI/2018/06/014617)

Acknowledgments

We thank the trial participants and their caregivers for their participation and support. We thank the dedicated study staff at the participating hospitals for implementing the study and providing patient care. We also thank the DSMB members, Vimta Labs Ltd, and JSS Medical Research India Pvt Ltd for their contributions to the study.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14760584.2023.2188942

Additional information

Funding

This study was funded by the Serum Institute of India Pvt. Ltd. Pune, India, and Global Health Investment Fund, USA.