Immunogenicity and safety of a 23-valent pneumococcal polysaccharide vaccine in Chinese healthy population aged >2 years: A randomized, double-blinded, active control, phase III trial

Abstract

Streptococcus pneumoniae is an important pathogen causing invasive diseases such as sepsis, meningitis, and pneumonia. Vaccines have become the most effective way to prevent pneumococcal infections. This phase III trial was designed to evaluate the immunogenicity and safety of a 23-valent pneumococcal polysaccharide vaccine in Chinese healthy population aged >2 years. We conducted a randomized, double-blinded, active-controlled, multicenter trial in which 1660 healthy population (>2 years of age) were randomly assigned in a 1 : 1 ratio to receive 2 intramuscular doses of either the treatment vaccine or the active control vaccine, PNEUMOVAX 23. The surveillance period was 30 days. The primary end point was the 2-fold increase rate of anti-pneumococcal antibody for all 23 included serotypes in each group. In the intention-to-treat cohort, the 2-fold increase rate of anti-pneumococcal antibody for 23 included serotypes varied from 62.47% to 97.01% in the treatment group, and from 51.49% to 95.77% in the control group. According to −10% non-inferiority margin and 95% confidence intervals of rate difference, almost all included serotypes of the treatment group reached non-inferiority to control group except for serotype 6B, the lower limit of rate difference of which was −10.00%, equal to the non-inferiority margin. The 2-fold increase rates of anti-pneumococcal antibody were significantly higher in the treatment group for serotype 2, 3, 4, 10A, 11A and 20. Furthermore, for all 23 serotypes, IgG geometric mean concentrations (GMCs) at day 30 were significantly higher in treatment group for serotype 2, 3, 4, 9V, 10A, 11A, 15B, 18C, 19A, 22F and 33F. Higher geometric mean fold increase (GMFI) were also observed in the treatment group correspondingly. Serious adverse events occurred in 3 of 830 participants in the treatment group (0.36%) and 2 of 830 participants in the control group (0.24%). No death occurred during the trial. The frequencies of both solicited and unsolicited adverse events (AEs) were small lower in the treatment group (34.34% vs 35.66% for solicited AEs, 4.34% vs 5.42% for unsolicited AEs). Both vaccines were well tolerated and most AEs were mild or moderate in intensity. The newly vaccine was well tolerated and immunologically non-inferior to the active control vaccine PNEUMOVAX 23 for all 23 vaccine serotypes in the Chinese population (>2 years of age).

Keywords:

• immunogenicity
• pneumococcal polysaccharide vaccine
• safety

Introduction

Streptococcus pneumoniae is a common cause of invasive disease and respiratory-tract infections in more and less developed countries. Risk groups for diseases caused by pneumococci, such as meningitis, sepsis, and pneumonia, include young children, elderly people, and patients with immunodeficiencies.¹ Despite the use of antibiotics and intensive care for over 50 years, the case fatality rate of pneumococcal bacteremia has remained at 15% to 20% in children and young adults and 30 to 40% in the elderly. Moreover, the rates of antimicrobial resistance in pneumococci have steadily increased.^2-4 Vaccines have become the most effective way to prevent pneumococcal infections.¹

The serotype distribution varied with age, year, specimen and geographic location, which played an important role in the development and formulation of the vaccines.^5,6 The first national investigation of serotype distribution of S. pneumoniae in hinterland of China was organized by WHO which involved 27 hospitals and institutes in 18 provinces and cities in 1981. The study showed that the 8 common serotypes were serotypes 5, 6, 1, 19, 2, 14, 23 and 3, which together accounted for 63.3% of all isolates, the most common serotype causing lung infection in infants were 1 and 14 while those isolated from older children and adults with lung infections were 1 and 5.^7,8 As a result of the serious abuse of antibiotics in China, the resistance of S. pneumoniae to various antibiotics is increasing. A series of studies reported after 2000 from several locations in China suggested that the distribution of common serotypes of S. pneumoniae isolated from healthy children or adults was different significantly. Serotypes 19A, 19F, 23F, 6B, 15B and 14 became common in most of the studies.^9-15

The current 7-valent pneumococcal conjugate vaccine (PCV7) covering serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F, PCV10 (PCV7 plus serotypes 1, 5, and 7F), and PCV13 (PCV10 plus serotypes 3, 6A, and 19A) have been shown to be effective in preventing invasive diseases caused by the vaccine-type strains.^16,17 However, with the increasing rates of antimicrobial resistance in pneumococci and the shifting serotypes distribution, the protective coverage of the conjugate vaccines may be lower, and replacement by non-vaccine serotypes resulting in disease is a serious threat for the near future.^18-23 The effective vaccines with more extensive coverage of serotype distribution of S.pneumoniae, included wider geographical areas and in different epidemiological/clinical settings including rural localities are most suitable for China. 23-valent PPV (PPV23), expanding serotype coverage to more than 85% of the organisms that caused invasive pneumococcal disease (IPD), and has been shown to be highly cost-effective when provided as a routine immunization.^24-32 23-valent PPV is currently recommended for persons >65 years of age and for high-risk individuals 2–64 y of age in many developed countries.^33,34

In the present large scale, double-blinded, randomized, active-controlled, phase III study, we assessed the safety and immunogenicity of a newly developed 23-valent pneumococcal polysaccharide vaccine in Chinese population, and according to the age strata of the participants.

Results

Study population

A total of 1,660 participants (>2 years of age) were enrolled and received the treatment or control vaccine. Of these, 19 participants in the treatment group and 22 in the control group were excluded from immunogenicity analysis for they did not have valid serum value before vaccination. As a result, 811(97.71%) participants in the treatment group and 808(97.35%) in the control group were included in intention-to-treat analysis (Fig. 1).

Figure 1. Trial profile.

Moreover, 9 participants in the treatment group and 4 in the control group were excluded from the per-protocol analysis due to the GMCs of post-vaccination less than pre-vaccination, which was not logical. Finally, 802(96.63%) of the treatment group and 804(96.87%) of the control group were included in the per-protocol analysis (Fig. 1).The demographic characteristics of the participants were well balanced in the two groups (Table 1).

Table 1. Baseline characteristics in safety analysis set

	Treatment vaccine	Control vaccine
Participants, No.	830	830
Age(yrs), mean ± SD	40.17 ± 25.07	39.98 ± 24.92
Male sex, No. (%)	348(41.93)	304(36.63)
Ethnic group, No. (%)
Han	350(42.17)	378(45.54)
Zhuang	413(49.76)	391(47.11)
Yao	13(1.57)	14(1.69)
Miao	13(1.61)	8(0.96)
Other	41(4.94)	39(4.70)

Efficacy end points and immunogenicity

In the treatment group, the 2-fold increase rate of anti-pneumococcal antibody varied from 62.47% to 97.01% for all 23 serotypes, serotype 2, 8, 9N, 18C and 33F increase exceeded 90%. In the control group, the rate varied from 51.49% to 95.77% for all 23 serotypes, serotype 8, 9N, 18C and 33F increase exceeded 90%.

According to −10% non-inferiority margin and 2-sided 95% confidence intervals (CIs) of rate difference, the treatment group was non-inferior to the control group except for serotype 6B. For serotype 2, 3, 4, 10A, 11A and 20, rate of 2-fold increase treatment were significantly higher in the treatment group compared to the control group (90.02% in treatment group vs. 85.82% in control group for serotype 2; 72.19% in treatment group vs. 61.69% in control group for serotype 3; 81.67% in treatment group vs. 71.89% in control group for serotype 4; 66.58% in treatment group vs. 51.49% in control group for serotype 10A; 75.69% in treatment group vs. 70.40% in control group for serotype 11A; 82.42% in treatment group vs. 77.24% in control group for serotype 20).While for serotype 6B, the lower limit of rate difference between the two groups was −10.00%, equal to the non-inferiority margin ( Fig. 2; Table S1).

Figure 2. Rate difference of 2-fold increase after vaccination in the ITT population.

For IgG geometric mean concentrations (GMCs) of participants, pre-immunization antibody levels were balanced in both groups. At day 30, the levels of the treatment group were statistically significantly higher by 2–3 folds for serotype 2, 3, 4, 9V, 10A, 11A, 15B, 18C, 19A, 22F and 33F, compared with the control recipients, with the exception of serotype 6B, which was lower in the treatment group (Table 2; Fig. S1). Similarly, geometric mean fold increase (GMFI) in participants vaccinated with the treatment vaccine were higher than those in participants vaccinated with the control vaccine for all serotypes except 6B and 19F, which were lower in the treatment group (Table 2).

Table 2. Immune response to pneumococcal vaccine in the intention-to-treat population

	Day 0 GMCs			Day 30 GMCs			Day 30 GMFI
Serotype	Treatment vaccine N = 811	Control vaccine N = 808	p value	Treatment vaccine N = 811	Control vaccine N = 808	p value	Treatment vaccine N = 811	Control vaccine N = 808	p value
1	12.17(11.35–13.06)	12.06(11.24–12.94)	0.8535	45.69(42.84–48.73)	45.16(42.22–48.29)	0.8039	3.75(3.47–4.05)	3.74(3.46–4.06)	0.9658
2	17.04(15.86–18.32)	16.66(15.56–17.84)	0.6533	121.01(111.87–130.89)	102.61(94.91–110.94)	0.0035	7.10(6.58–7.66)	6.16(5.71–6.64)	0.0091
3	30.93(27.22–35.15)	31.73(28.15–35.77)	0.7752	155.10(144.75–166.18)	117.99(110.58–125.89)	<0.0001	5.01(4.44–5.66)	3.72(3.32–4.17)	0.0004
4	21.31(20.00–22.70)	20.40(19.17–21.72)	0.3379	103.62(97.04–110.64)	76.62(71.89–81.66)	<0.0001	4.86(4.54–5.21)	3.76(3.52–4.01)	<0.0001
5	24.21(22.93–25.56)	24.28(23.05–25.57)	0.9394	79.60(74.98–84.51)	77.43(73.02–82.12)	0.5177	3.29(3.11–3.48)	3.19(3.02–3.37)	0.4487
6B	3.55(3.07–4.11)	3.53(3.05–4.09)	0.9620	17.27(15.07–19.79)	21.63(19.03–24.58)	0.0183	4.90(4.26–5.64)	6.16(5.39–7.04)	0.0192
7F	36.74(34.35–39.30)	37.00(34.73–39.42)	0.8789	154.62(145.51–164.31)	158.09(148.61–168.18)	0.6158	4.21(3.94–4.50)	4.27(4.02–4.54)	0.7436
8	7.49(6.86–8.18)	7.35(6.71–8.04)	0.7626	110.58(103.34–118.33)	110.86(103.35–118.92)	0.9595	14.76(13.34–16.34)	15.09(13.68–16.64)	0.7594
9N	6.07(5.52–6.68)	6.02(5.45–6.64)	0.8968	91.21(85.33–97.50)	88.53(82.70–94.77)	0.5387	15.02(13.78–16.38)	14.71(13.43–16.13)	0.7457
9V	12.18(10.87–13.66)	10.24(9.03–11.61)	0.0450	112.21(104.31–120.71)	89.31(82.84–96.28)	<0.0001	9.21(8.33–10.19)	8.72(7.84–9.71)	0.4689
10A	26.52(25.04–28.09)	25.69(24.36–27.10)	0.4272	96.98(89.43–105.17)	64.55(59.99–69.45)	<0.0001	3.66(3.42–3.91)	2.51(2.37–2.67)	<0.0001
11A	22.78(20.76–25.00)	23.20(21.27–25.31)	0.7778	103.84(97.90–110.14)	91.53(86.17–97.22)	0.0034	4.56(4.18–4.97)	3.94(3.64–4.27)	0.0157
12F	30.94(28.31–33.82)	32.50(30.01–35.19)	0.4207	153.70(141.80–166.60)	153.91(140.42–168.68)	0.9829	4.97(4.52–5.46)	4.74(4.31–5.21)	0.4875
14	23.36(22.02–24.78)	25.13(23.52–26.86)	0.1057	73.45(68.27–79.01)	77.34(72.08–83.00)	0.3179	3.14(2.95–3.35)	3.08(2.88–3.28)	0.6417
15B	34.97(33.10–36.94)	33.63(31.82–35.56)	0.3287	155.20(144.74–166.42)	135.45(126.63–144.87)	0.0059	4.44(4.16–4.74)	4.03(3.77–4.30)	0.0370
17F	19.07(17.87–20.34)	18.01(16.98–19.11)	0.2022	119.81(103.02–117.05)	108.94(102.40–115.90)	0.8606	5.76(5.38–6.17)	6.05(5.66–6.47)	0.3141
18C	6.56(5.75–7.48)	6.77(6.03–7.61)	0.7148	72.27(67.20–77.73)	64.11(59.76–68.78)	0.0202	11.02(9.84–12.35)	9.46(8.54–10.49)	0.0512
19A	31.22(29.22–33.36)	29.36(27.34–31.53)	0.2149	129.03(119.09–139.81)	111.73(103.15–121.03)	0.0127	4.13(3.56–4.07)	3.81(3.56–4.07)	0.1006
19F	25.80(24.11–27.60)	23.86(22.28–25.55)	0.1120	115.10(106.03–124.94)	110.70(102.02–120.12)	0.5089	4.46(4.15–4.80)	4.64(4.30–5.00)	0.4681
20	28.72(26.48–31.14)	28.26(26.25–30.42)	0.7723	152.95(142.27–164.44)	137.14(125.03–150.42)	0.0682	5.33(4.92–5.77)	4.85(4.47–5.27)	0.1091
22F	37.01(35.15–38.97)	35.66(33.92–37.49)	0.3102	118.73(111.93–125.94)	108.63(102.55–115.06)	0.0342	3.21(3.02–3.41)	3.05(2.88–3.22)	0.2167
23F	25.31(23.81–26.90)	25.01(23.54–26.57)	0.7867	85.16(79.03–91.78)	80.88(74.90–87.35)	0.3455	3.37(3.15–3.59)	3.23(3.03–3.45)	0.4012
33F	7.93(7.15–8.78)	7.58(6.89–8.35)	0.5340	114.16(106.35–122.54)	92.52(86.31–99.17)	<0.0001	14.40(13.15–15.78)	12.20(11.17–13.34)	0.0108

Subgroup analysis

According to the age strata of the participants in the study design and randomization, we analyzed the primary endpoint for the 2 subgroups (2∼6 y and >60 years) respectively. The similar trend of antibody responses were observed. In the subgroup of 2∼6 y of age, the 2-fold increase rate of all included anti-pneumococcal antibody serotype were no statistical difference except 20 (ITT: 88.43% vs. 59.17% at day30, P < 0.0001), the rate difference (95%CI) was 29.26(95%CI, 18.78 to 39.74). In the subgroup of >60 years of age, the 2-fold increase rate of serotype 2, 8, 10A and 14 showed statistical difference (p = 0.0076, 0.0306, 0.0005 and 0.025 respectively), the rate differences (95% CIs) were 6.74(95%CI, 1.82 to 11.65), −4.13(95%CI, −7.85 to −0.41), 13.97(95%CI, 6.21 to 21.73) and 9.89(95%CI, 1.29 to 18.50) respectively. The 2-fold increase rates of the other included serotypes were no statistical difference. (Fig. 3; Tables S2–5).

Figure 3. Rate difference of 2-fold increase after vaccination (Subgroups).

Adverse reactions

During the entire study period, 5 serious adverse events were reported and considered to be unrelated to vaccination: 3 in the treatment group (1 case of thyrophyma, 1 case of renal calculus, and 1 case of bronchitis) and 2 in the control group (1 case of cervical spondylopathy, and 1 case of cerebral infarction). No death occurred during the trial.

The incidence of all adverse reactions was 32.53% and 34.10% in the treatment and control groups (p = 0.5321). Within each subgroup, there was also no significant difference in the occurrence of adverse reactions between the two groups. The frequencies of both solicited and unsolicited adverse events were similar in the 2 study groups (Table 3). The most common systemic adverse reactions were fever, headache, fatigue, and nausea, the most common injection-site adverse reactions were pain, redness and swelling. A total of 20 participants in the treatment group (2.41%) and 19 participants in the control group (2.29%) had grade 3 adverse events within 7 d after an injection.

Table 3. Adverse events in the safety analysis population

Event	Treatment vaccine N = 830	Control vaccine N = 830	p value
Serious adverse events(no. of participants)
Events within 7 d after injection	1(0.12%)	0(0%)	1.0000
Events from day 8 to day 30	2 (0.24%)	2 (0.24%)	1.0000
Solicited adverse reactions within 7 d after an injection(no. of participants)
Any	285(34.34%)	296(35.66%)	0.6069
Grade 3	20(2.41%)	19(2.29%)	1.0000
Injection-site reactions
Pain
Any	201(24.22%)	202(24.34%)	1.0000
Grade 3	0(0.00%)	0(0.00%)	1.0000
Redness
Any	50(6.02%)	62(7.47%)	0.2817
Grade 3	10(1.20%)	12(1.45%)	0.9320
Swelling
Any	44(5.30%)	53(6.39%)	0.4026
Grade 3	10(1.20%)	13(1.57%)	0.8360
Pruritus
Any	20(2.41%)	15(1.81%)	0.4949
Grade 3	0(0.00%)	0(0.00%)	1.0000
Induration
Any	1(0.12%)	0(0.00%)	1.0000
Grade 3	0(0.00%)	0(0.00%)	1.0000
Systemic reactions
Fever
Any	82(9.88%)	88(10.60%)	0.6282
Grade 3	3(0.36%)	1(0.12%)	0.3549
Headache
Any	24(2.89%)	32(3.98%)	0.2808
Grade 3	0(0.00%)	0(0.00%)	1.0000
Fatigue
Any	30(3.61%)	30(3.61%)	1.0000
Grade 3	0(0.00%)	0(0.00%)	1.0000
Nausea or vomiting
Any	6(0.72%)	12(1.45%)	0.2353
Grade 3	0(0.00%)	0(0.00%)	1.0000
Diarrhea
Any	9(1.08%)	10(1.20%)	1.0000
Grade 3	1(0.12%)	1(0.12%)	1.0000
Rash
Any	5(0.60%)	7(0.84%)	0.7736
Grade 3	0(0.00%)	0(0.00%)	1.0000
Allergy
Any	2(0.24%)	1(0.12%)	1.0000
Grade 3	1(0.12%)	0(0.00%)	1.0000

Discussion

In this large scale, double blinded, randomized phase III study, the efficacy, immunogenicity and safety of a newly 23-valent pneumococcal polysaccharide vaccine developed by a Chinese local biotech manufacture have been extensively studied. The study clearly showed that both vaccines were highly immunogenic. The newly PPV23 vaccine have similar efficacy and safety with the established control vaccine PNEUMOVAX 23 (Merck & Co., Inc.) in all included serotypes.

A series of studies reported serotype 19A, 19F, 23F, 6B and 14 were common among children <5 years of age in China.^8-15 The treatment vaccine showed similar efficacy and safety with the control group, all reached the pre-defined non-inferiority margin for the serotype 19A, 19F, 23F and 14. For serotype 6B, lower efficacy was observed in the treatment group, the primary end point was equaled to the non-inferiority margin. While in the subgroup analysis of 2∼6 y of age, all efficacy endpoints for serotype 6B have no statistical difference between the two groups. A number of studies suggested that serotype 6B is just common in infants and younger children, but not in older children and adults,^8,14,19 which means their natural exposed chance is low, and resulted in the magnitude of antibody response to vaccination is not as high and sensitive as in younger children's. In addition, the sample size of younger children was just a small part of our all participants. As a consequence, the antibody response to serotype 6B in in all age groups was attenuated, while still remained highly in the subgroup.

Pneumococcal disease also remains a major cause of morbidity and mortality among elderly persons. PPV23 is currently recommended for use in elderly persons and in persons at high risk of infection. In the United States, the Advisory Committee on Immunization Practices (ACIP) recommends PPV23 vaccination for all adults aged >65 years and for individuals aged >2 years with risk factors for pneumococcal disease.^33,34 It provides approximately 50–80% protection against invasive disease in the general elderly population. The vaccine significantly reduced mortality, stay duration, and intensive care admissions among adults hospitalized with community-acquired pneumonia.^19,36,37 In our treatment vaccine, the recent studies reported prevalent serotypes (serotype 19A, 19F, 14, 3 and 23F) in older adults were observed as efficacious as the control vaccine, and totally reached the pre-defined non-inferiority margin. Furthermore, the results of all endpoints have no statistical difference between the two groups in subgroup analysis.

The immunogenicity of the treatment vaccine in our study was small superior to that of the control for 2 of 23 serotypes studied (3, and 10A). Referred to the results of several previous studies^38-40 critically, small different immune responses are accep via diverse prevalence and pre-exposure in different region or population. In addition, double-blinding principle across the whole trail ensured the same storage and administration were conducted in both groups.

With respect to the duration of PPV23 neutralizing antibody titers, vaccination with polysaccharide antigens does not establish T-cell mediated immune memory, although it may induce memory plasma cells. The duration of the immune response of PPV23 is likely limited, indicating a need for periodic revaccination. Musher et al. recently reported both primary vaccination and revaccination with PPV23 induce antibody responses that persist during 5 y of observation in middle-aged and older adults, consistent with the findings of previous studies, antibody levels 30 d after revaccination were generally modestly lower than those observed after primary vaccination. Induction of relatively long-lived memory suppressor T regulatory cells may be responsible for the observed suppression and may be intrinsic to the immune response to polysaccharide antigens.³⁹ The utility of routine pneumococcal revaccination in long-lived populations, to avoid further decline in anti-pneumococcal antibodies to levels like those among unvaccinated adults. Instead, revaccination seems to sustain antibody levels. Routine revaccination could reduce susceptibility to vaccine serotypes, to the extent that antibody levels are associated with protection. Both primary vaccination and revaccination with PPV23 in elderly persons result in significant and sustained increases in total IgG and functional antibody activity levels without evidence of significant hyporesponsiveness with revaccination.^40-43

Pneumococcal polysaccharides are T-cell independent antigens that are poorly immunogenic for important pneumococcal serotypes in infancy. The conjugation of pneumococcal capsular polysaccharide to carrier proteins results in a T-cell dependent immune response, characterized by increased antibody concentrations in infants, induction of memory cells, and a booster response upon subsequent antigenic exposures. Schedules combining PCV with 23-valent pneumococcal polysaccharide vaccine (PPV23) have been studied and proposed as a means to expand disease protection against serotypes not included in the PCVs. Several studies in children, using a variety of conjugate formulations and schedules in different countries, have shown that PPV23 as a booster dose following a priming series of PCV induces higher concentrations of antibodies than a PCV booster. The same results still observed in studies with PCV/PPV23 combined regimens have been done in patient populations for whom PPV23 is routinely recommended.^44-48

In conclusion, this newly vaccine is immunologically non-inferior and as well tolerated as the established vaccine PNEUMOVAX 23. Long-term antibody persistence and efficacy evaluations should be preferred in further studies.

Methods

Study design

This double-blinded, randomized, active-controlled non-inferiority clinical trial was designed by the Center for Disease Control and Prevention of the Guangxi Zhuang Autonomous Region (Guangxi CDC); Walvax Biotech (the study sponsor and manufacture of the vaccine) and the Department of Health Statistics of Fourth Military Medical University (FMMU). Data were collected by Guangxi CDC and monitored by Simon Record, a Contract Research Organization (CRO).This trial was performed from September 2012 to May 2013 at 2 centers (Jinchengjiang and Liucheng Counties) comprising 25 sites in Guangxi, China. The trial protocol and the informed-consent form were approved by the ethics committee of the Guangxi CDC. Before enrollment, written informed consent was obtained from each participant or legal guardian. The trial was conducted in accordance with the principles of the Declaration of Helsinki, the standards of Good Clinical Practice and Chinese regulatory requirements. The trial was registered at Clinicaltrials.gov (NCT02285036).

Each dose of treatment and control vaccine was assigned a code from a randomization list byage stratified block randomization (block size 10). The randomization list was generated by an external statistician with SAS (version 9.1). All packages of treatment and control vaccine were identical in appearance, with the code as the only identifier. Each participant was assigned a sequential number according to their sequence of enrolment and received the treatment or the control labeled with the same numbers. Thus, participants were randomly assigned to receive the treatment or the control in a 1:1 ratio. Investigators involved in randomization and masking did not participate in any other part of the trial. Allocation was masked from all participants, their guardians, and investigators.

Participants

Healthy participants (>2 years of age), with axillary temperature less than Celsius 37 degrees, not yet having received pneumococcal vaccine or other prevention biological products within 7 d were eligible for enrollment in this study. The exclusion criteria included (1) any known primary or secondary immunodeficiency, (2) allergy, (3) severe cardiovascular disease bleeding disorders, (4) receipt of immunoglobulin or blood products within one month and so on. All eligible participants were stratified by age (2∼6,7∼17, 18∼59 and >60 years), were randomly assigned in a 1:1 ratio to receive the treatment vaccine or the control. The same dose of either the treatment vaccine or the control vaccine were administered intramuscularly, with a 30 d surveillance period after an injection.

Vaccine

The treatment pneumococcal vaccine was developed by Yunnan Walvax Biotech Co. Ltd (Walvax) China, is a sterile, liquid vaccine for intramuscular or subcutaneous injection. The vaccine contained a mixture of highly purified capsular polysaccharides from the 23 most prevalent or invasive serotypes of Streptococcus pneumonia(serotype 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F). The dose of vaccine is contained 23 kinds of pneumococcal polysaccharide serotypes each 25μg in a 0.5ml dose per individual, and without any preservatives and stored at 2–8°C.

The active control, PNEUMOVAX 23, is an established US-licensed vaccine and manufactured by Merck. Each 0.5 ml dose of vaccine contains 25μg of each polysaccharide type in isotonic saline solution with 0.25% phenol as a preservative and stored at 2–8°C.

Both the treatment and control vaccines were prepared in a facility that was compliant with Good Manufacturing Practices and were tested by the National Institutes for Food and Drug Control before the start of the study.

Efficacy end points and Immunogenicity

The primary endpoint was the 2-fold increase rate of anti-pneumococcal antibody for all included 23 serotypes in each group.

The secondary endpoints included geometric mean fold increase (GMFI) and geometric mean concentration (GMCs) of IgG after vaccination in each group.

Blood samples were collected at baseline and day 30 after the injection for immunogenicity evaluation. All serum samples were assessed for anti-pneumococcal neutralizing antibody by means of enzyme-linked immunospot assay (ELISA) at the Chinese National Institute for Food and Drug Control in blindness.

Safety assessment

All participants or guardians who received the treatment or control vaccines were asked to fill out diary cards that listed injection-site adverse reactions (e.g., pain, redness, and swelling) and systemic adverse reactions (e.g., fever, headache, and diarrhea). Safety data were collected on solicited adverse events that occurred within 7 d after an injection and unsolicited adverse events (those reported spontaneously by a participant or guardian) that occurred within 30 d after an injection. Data on serious adverse events were collected throughout the trial. The grade and the relationship of the adverse event or serious adverse event with receipt of an injection were decided by investigators before unblinding.

Statistical analysis

On the assumption of the 2-fold increase rate of 92% and the non-inferiority margin of −10%, 830 participants per group were required to achieve at least the overall power of 80% with the one-sided significance level of 2.5%. The Bonferroni method was employed for the primary endpoint to control the overall type II error.

All analysis was performed on the intention-to-treat (ITT) population and per-protocol (PP) population. For the primary endpoint, the null hypothesis is rejected unless each serotype of the treatment group is non-inferior to the control group. The 95% confidence interval (CI) of the 2-fold increase rate difference for all serotypes between the two groups were calculated and the non-inferiority was considered if the lower bound of 95% CI was larger than −10%, which was determined by all investigators and recorded in the protocol before enrollment. The Student's t-test was used for the analysis of log-transformed GMI and GMC and the Chi-square test or Fisher's exact test (when data were sparse) was used for the analysis of dichotomous outcomes. All data were analyzed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA), hypothesis testing was 2-sided with an α value of 0.05.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

We deeply appreciate reviewers' helpful comments.

Funding

This work was partially supported by Research Grants from the National Natural Science Foundation of China (Program No. 81473069, 81273176, 81302509) and the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2015JM8440).

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