Streptococcus pneumoniae is a principal causative agent of bacterial pneumonia, meningitis and sepsis in children. There is a very high disease burden observed in resource-limited countries.1,2 The pneumococcal conjugate vaccine (PCV) has been shown to have a substantial impact against these diseases, directly by inducing protective immunity in vaccinated infants and indirectly by inducing herd immunity through the reduction of nasopharyngeal carriage.3–5 Long-term surveillance of invasive pneumococcal disease (IPD) at Patan Hospital, Nepal has shown pneumococcal serotypes 1, 5 and 14 to be the cause of the majority of IPD.6 All of these serotypes are covered by available PCVs.7,8 The incidence of childhood pneumococcal pneumonia is high up to the age of 5 years.9,10 Thus, the persistence of immunity beyond the first 2 years of life is of paramount importance in the direct protection against pneumococcal pneumonia. The persistence of vaccine-related immunity also contributes to the development of herd immunity, which indirectly protects unvaccinated children and adults. In 2018, a study in Israel reported that decline in IPD rates among adults was most closely associated with reduced nasopharyngeal colonization and increased vaccination coverage among children at 3–5 years of age.11
There is some variation in the PCV vaccine schedules which have been implemented in different regions.12 In 2010, our assessment of the immunogenicity of PCV10 at Patan Hospital demonstrated that a 2-dose PCV primary schedule(6 and 14 weeks) with a 9-month booster (2 + 1) was noninferior to the conventional 3-dose priming schedule (3 + 0), without a boost, for serotype-specific IgG concentrations at 18 weeks. Added to this, the 2-dose prime-boost schedule resulted in higher IgG concentrations at 10 months and 2–4 years of age than the 3-dose prime schedule.13
In 2015, PCV10 vaccine was introduced into the routine immunization schedule in Nepal, supported by the evidence we had generated, but with a unique 2 + 1 dosing schedule using 6-week and 10-week priming schedule with a booster at 9 months of age.14 The scheduling was selected to avoid multiple injections at 14 weeks in the national immunization schedule, as implementation happened around the time that a single dose of an inactivated poliomyelitis virus vaccine was introduced at 14 weeks of age. The use of more than 2 injections at an immunization visit was believed to be a concern for parents and vaccinators. A 2 + 1 schedule with only a 1-month interval between the first 2 PCV10 doses has not previously been evaluated in this setting. In 2010, Goldblatt et al trialed a schedule with priming doses of PCV7 at 2 and 3 months. However, this study found poor immunogenicity of this schedule in an interim analysis.15 We previously reported an open-label randomized noninferiority trial of PCV10 in 304 Nepalese infants to assess the immunogenicity of the novel Nepali schedule, when compared with the standard Expanded Programme on Immunization schedule. The study demonstrated that the schedule with 2 priming doses at 6 and 10 weeks of age and a 9-month booster were inferior to the schedule with 2 priming doses at 6 and 14 weeks of age and a 9-month booster for immunogenicity against some serotypes at 9 months (before boosting), but there were no significant differences at 10 months of age (1-month post-boosting).14 To our knowledge, there are no prior studies that have directly compared the persistence of immunity, beyond the second year of life, in these 2 PCV schedules with different prime dose timings. We, therefore, undertook this study to examine the persistence of immunity in healthy Nepalese infants at 2–3 years of age, following PCV10 immunization at 6 and 10 weeks or 6 and 14 weeks of age, both with a booster at 9 months of age.
Study Design and Study Participants
We conducted a cross-sectional follow-up study at Patan Hospital among participants of our previous trial comparing different 2-dose priming schedules (6 + 10 weeks vs. 6 + 14 weeks) for PCV10.14 Oxford Tropical Research Ethics Committee (OxTREC Ref no: 34-17) and Nepal Health Research Council (NHRC Reg no: 459/2017) approved this study.
We approached by telephone, 287 participants who completed the original study, and explained the details of the study to them. We invited interested participants to visit the Paediatric Research Unit at Patan Hospital, where we provided them with an information sheet and discussed the study in more detail. The children of those parents/guardians who gave written consent to participate in the study were enrolled.
Participants attended a single visit for both baseline assessment and sample collection. Blood samples and nasopharyngeal swabs were collected and the participant’s demographics, medical history and information regarding the use of concomitant medication were recorded in the case record form.
Blood samples were centrifuged within 12 hours of collection and serum stored at −80°C before being shipped on dry ice to the World Health Organization (WHO) pneumococcal serology reference laboratory at the University College London, UK. Serotype-specific IgG antibody concentrations were measured for the 10 vaccine serotypes by enzyme-linked immunosorbent assay using 22F adsorption.16
Trained members of the research team collected the nasopharyngeal swab from each participant, according to WHO guidelines17 and samples were placed in a tube of 0.5–1 mL skim-milk-tryptone-glucose-glycerine and transferred to the laboratory in an insulated box within 4 hours of collection. The swabs were cultured within 12 hours of collection on Columbia blood agar containing 5% sheep blood and gentamicin and incubated overnight at 35–37°C in 5% carbon dioxide. Morphologically distinct colonies were confirmed by optochin sensitivity test and then serotyped by Quellung reaction. All pneumococcal isolates were stored at −80°C. Quality control of the serotyping was performed at the University of Oxford, UK.
The primary outcome was the proportion of infants with serotype-specific IgG ≥0.35 μg/mL against PCV10 serotypes at 2–3 years of age. The secondary outcomes were the geometric mean concentrations (GMCs) of PCV10 serotype-specific IgG at 2–3 years of age for each of the 2 study groups, and serotype-specific pneumococcal carriage rates at 2–3 years of age.
Serotype-specific IgG values were log-transformed and summarized as geometric means with associated 2-sided 95% confidence intervals (CIs). Observations below the threshold of detection for the assay were assigned a value of half the lower limit of detection before log transformation. The 2 groups were compared using the t test.
The geometric mean change in IgG between the post-booster visit at 10 months of age and the follow-up at 2–3 years of age was derived from the exponent of the difference of log-transformed serotype-specific IgG for each group.
The proportion of children with serotype-specific IgG levels greater than or equal to 0.35 µg/mL was calculated within each group and the CIs were computed using the binomial exact test. Chi-square test was used to compare the 2 groups.
Analyses were performed using SAS version 9.4, STATA version 14, and R Version 3.5.3.
Role of the Funding Source
The sponsor of the study was the University of Oxford. Investigators in Oxford and Nepal designed, conducted, analyzed and interpreted the study. Gavi, The Vaccine Alliance, the funder of the research had no role in study design, data collection, data interpretation, or writing of the report and data analyses. The corresponding author had complete access to all data of the study and the authors had final responsibility for the decision to submit for publication.
In the initial trial in 2015, a total of 304 children were enrolled and randomized 1:1 into either the 6 + 10 group or the 6 + 14 group, of which 287 completed the study. Both groups received a PCV10 booster at 9 months of age. For this follow-up study, between January 2018 and April 2018, we recruited 220 participants of the total 287 (76.7%) who completed the primary study. Reasons for nonenrolment were: 12 participants had moved away, 11 were lost to follow-up and 44 refused (see Figure, Supplemental Digital Content 1; https://links.lww.com/INF/E435). Children in the 2 arms of the study were similar in terms of age and sex distribution. The baseline characteristics of the participants at the time of enrolment for the follow-up study are shown in Table 1.
TABLE 1. -
Descriptive Characteristics of the Study Groups, Means (SD) or n (%)
||Group 6 + 10 (n = 108)
||Group 6 + 14 (n = 112)
|History of antibiotic taken in the last 4 weeks
|Hospitalization history within last 12 months
PCV10, 10-valent pneumococcal conjugate vaccine.
At 2–3 years of age, the proportion of children who had serum pneumococcal serotype-specific IgG greater than or equal to 0.35 µg/mL was comparable for all PCV10 serotypes between the 6 + 10 and 6 + 14 groups (Fig. 1A). At the same time point, the GMC of serum pneumococcal serotype-specific IgG levels in the 6 + 10 and 6 + 14 groups were similar for all serotypes except for serotype 19F. Antibody against serotype 19F was 32% lower in the 6 + 10 group than the 6 + 14 group (GMR 0.676, 95% CI, 0.50–0.92, P = 0.013) (Fig. 1B). Antibody levels induced by vaccination were expected to decay from the post-booster visit at 10 months to the follow-up at 2–3 years of age. However, some participants had antibody increases between these 2 visits (Fig. 2), in the absence of documented infection, indicating that there may have been an antibody response to intercurrent nasopharyngeal carriage. Using a rise in antibody as a marker of pneumococcal exposure/carriage, we found that the proportion of possible carriage acquisitions was highest for serotype 23F at 12.75% (Table 2). More than 5% of participants also saw a rise in antibody for serotypes 5, 6B, 9V and 14.
TABLE 2. -
Proportions of Participants With at least a 2-fold Increase in Antibody Level Between the Post-booster Visit at 10 Months of Age and Follow-up at 2–3 Years of Age
||6 + 10 Week Group
||6 + 14 Week Group
||n/N With 2-fold Rise (%)
||n/N With 2-fold Rise (%)
||n/N With 2-fold Rise (%)
n/N: number of participants with at least 2-fold increase by vaccine groups/Total participants with results for each serotype at both time points.
PCV10, 10-valent pneumococcal conjugate vaccine.
The overall rate of pneumococcal carriage was 73.6% at 2–3 years of age. Two (1.9%) of the 108 swabbed children in 6 + 10 group were carrying PCV10 serotypes compared with 15 (13.4%) of the 112 swabbed children in 6 + 14 group.
At 2–3 years of age, the proportion of children with nasopharyngeal carriage of any PCV10 serotype in the 6 + 14 group was significantly higher than in the 6 + 10 group (difference 11.5%, 95% CI 4.7% –18.3%; P = 0.0025). The most commonly carried serotype at 2–3 years of age was serotype 23F, detected in 6 (5.36%) of the 112 children in the 6 + 14 group. Serotypes 6B, 9V, 14 and 19F were also detected. Serotypes 1 and 18C were not detected at any visit.
There was no difference between the groups in overall carriage of nonvaccine types (Table 3).
TABLE 3. -
PCV10 Serotype Distribution of Nasopharyngeal Carriage of S. pneumoniae
||6 + 10 Week Group
||6 + 14 Week Group
|6 Weeks (N = 152) (%)
||10 Months (N = 143) (%)
||2–3 Years (N = 108) (%)
||6 Weeks (N = 152) (%)
||10 Months (N = 144) (%)
||2–3 Years (N = 112) (%)
|Any PCV10 serotype
PCV10, 10-valent pneumococcal conjugate vaccine.
This is the first clinical trial to examine the persistence of immunity following immunization with PCV10 according to Nepal’s unique 2 + 1 dosing schedule of 6-week and 10-week priming doses with a booster at 9 months, compared with the WHO standard 2 + 1 schedule of 6-week and 14-week priming doses with a 9-month booster. An important finding is that the immunogenicity of the Nepali 2 + 1 dosing schedule, with a 4-week interval between the priming doses, is similarly immunogenic to the WHO standard 2 + 1 schedule, with an 8-week interval between the priming doses at 2–3 years of age.
The rate of nasopharyngeal carriage of PCV10 serotypes was higher in the 6 + 14 week group than the 6 + 10 week at 2–3 years of age. The biggest difference at this time point was seen in the most commonly carried serotype – 23F – which was detected in 5.36% of children in the 6 + 14 group and not detected in the 6 + 10 group. Serotype 23F was also the most common serotype for which a rise in antibody was detected between the post-booster time point at 10 months of age and the follow-up at 2–3 years. However, no difference was seen between the 6 + 10 and 6 + 14 groups in the proportion of children with a rise in antibody for this serotype. This suggests the differences observed in nasopharyngeal carriage may be a chance finding due to small numbers.
In general, the serotypes with a rise in antibody were also those detected most commonly in nasopharyngeal swabs. The main exception to this was serotype 5, which was detected in only 1 swab across all visits for both groups, yet 10.6% of children had a rise in antibody to this serotype. This aligns with previous observations that serotypes 1 and 5 may circulate more widely than might be understood from nasopharyngeal swabbing alone.18
The overall GMC of the antibodies against most of the serotypes was similar in both groups. No significant difference was seen when proportion of children with IgG levels of at least 0.35 µg/mL were compared between the 2 groups. The antibody levels had substantially decayed by the time of the follow-up visit, with levels for serotypes 1, 4 and 18C showing the greatest decline (GMCs less than 10% of the post-booster levels). Our data provide support for the WHO recommendation that, in situations where the standard 2 + 1 schedule cannot be implemented, a short interval priming schedule at 6 and 10 weeks can be used as an alternative.19
In 2017, the WHO Strategic Advisory Group of Experts (SAGE) on Immunizations PCV Working Group reviewed the dosing schedule to examine how PCV administered to healthy children in a 2 + 1 schedule compared with the 3 + 0 schedule, with respect to immune response in vaccinated children and impact on clinical outcomes. The review showed higher GMCs in children administered the 2 + 1 schedule compared with the children administered the 3 + 0 schedule when assessed after the third dose in each schedule. However, the proportions of participants who had an antibody response above the correlate of protection were similar in the 2 schedule groups. Studies have shown no significant difference in immunogenicity and response after the third dose between 2 + 1 and 3 + 0 schedule when assessed after the third dose in each schedule. Hence, SAGE has recommended the use of 3 doses of PCV in infants in either a 2 + 1 or 3 + 0 schedule.20 In Nepal’s case, surveillance of IPD at Patan Hospital, Kathmandu indicates the majority of IPD occurs in late infancy and early childhood.7,8 For this reason, the National Immunisation Technical Advisory Group of Nepal preferred to use the 2 + 1 schedule, with a booster dose at 9 months, to provide better persistence of immunity beyond infancy in Nepali children, and to potentially provide improved protection against IPD.21
To our knowledge, the schedule with priming doses at 6 and 10 weeks of age, are used in Nepal and Bhutan only.22 The majority of developed countries use a 2 + 1 schedule with an 8-week gap between priming doses, and a booster administered at 9 months of age or later.12,23
In 2016, a study was conducted in Poland to evaluate the long-term persistence of antibody after PCV10 administration in a 3 + 1 schedule, with vaccinations at 2, 3, 4 and 12–18 months of age. The study findings showed the importance of booster for persistence of immunogenicity. The study showed that the antibody persistence of PCV 10 may extend until at least 4 years after booster vaccination.24
Zimmerman et al studied persistence of immunity in children, at 13 months of age, immunized with PCV13 in a 3 + 0 schedule. The study findings showed a drop in GMC levels of antibody from 7 to 13 months of age, with many 13-month-old infants having antibody levels below the correlate of protection threshold, with the lowest responses against serotypes 4, 19A, 3, 6B and 23F.25 The authors suggested that booster doses might be necessary in immunization programs for PCV to optimize protection against pneumococcal diseases.25
In 2016, Trück et al studied the antibody response in children primed with PCV13 who were boosted with either PCV10 or PCV13. The geometric mean IgG concentrations and opsonophagocytic assay titers for most PCV13 serotypes were significantly superior in recipients of a PCV13 booster compared with a PCV10 booster. However, similar or inferior responses were seen for serotypes 4, 18C and 19F. Nevertheless, the clinical significance of these differences is unknown.26
In 2018, Goldblatt et al demonstrated equivalent or superior post-booster responses among UK infants primed with a single dose compared with the standard 2 priming dosing schedule for 9 of the 13 serotypes in PCV13 and hence schedules with fewer doses, might be considered where in a mature program, and may also induce similar immune responses.27
In general, we expect the antibody induced by vaccination to decay progressively post-vaccination, before reaching a steady state. A late infancy booster dose appears to improve the persistence of immunity.
This study adds to a small number of studies providing information on persistence of immunity after PCV immunization and is the only study to demonstrate the medium-term immunity of the Nepal PCV10 vaccine schedule. The fact that this study is a follow-up of a randomized trial adds to the strength of the study. However, the persistence of immunity beyond the third year of life after vaccination remains to be studied.
This study has shown that PCV10 immunization at 6 + 10 weeks or 6 + 14 weeks, with a booster at 9 months in each case, results in similar persistence of serotype-specific antibody at 2–3 years of age. From the results, we would anticipate that direct protection from disease at this age, and herd protection of the unvaccinated population, would be similar if either schedule is used. This suggests that a 2 + 1 PCV schedule, with a 1-month interval between priming doses, is a valid alternative to the current WHO PCV schedule.
We would like to acknowledge funding from Gavi the Vaccine Alliance. We would also like to acknowledge the participants and their families who participated in the study and Dr. Bhishma Pokhrel for the clinical support; Bidhur Khatri and Rajkumar Dangol for assisting on recruitment; Krishna Govinda Prajapati for microbiologic support and Jita Nepal for office assistance.
1. O’Brien KL, Wolfson LJ, Watt JP, et al.; Hib and Pneumococcal Global Burden of Disease Study Team. Burden of disease caused by Streptococcus pneumoniae
in children younger than 5 years: global estimates. Lancet. 2009;374:893–902.
2. Wahl B, O’Brien KL, Greenbaum A, et al. Burden of Streptococcus pneumoniae
and Haemophilus influenzae
type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000–15. Lancet Glob Health. 2018;6:e744–e757.
3. Control CfD, Prevention. Direct and indirect effects of routine vaccination of children with 7-valent pneumococcal conjugate vaccine on incidence of invasive pneumococcal disease – United States, 1998-2003. MMWR Morb Mortal Wkly Rep
4. Dagan R, Givon-Lavi N, Zamir O, et al. Reduction of nasopharyngeal carriage of Streptococcus pneumoniae
after administration of a 9-valent pneumococcal conjugate vaccine to toddlers attending day care centers. J Infect Dis. 2002;185:927–936.
5. Flasche S, Van Hoek AJ, Sheasby E, et al. Effect of pneumococcal conjugate vaccination on serotype-specific carriage and invasive disease in England: a cross-sectional study. PLoS Med. 2011;8:e1001017.
6. Chhetri U, Shrestha S, Pradhan R, et al. Clinical profile of invasive pneumococcal disease in Patan Hospital, Nepal. Kathmandu Univ Med J. 2011;9:45–49.
7. Kelly DF, Thorson S, Maskey M, et al. The burden of vaccine-preventable invasive bacterial infections and pneumonia in children admitted to hospital in urban Nepal. Int J Infect Dis. 2011;15:e17–e23.
8. Shah AS, Knoll MD, Sharma PR, et al. Invasive pneumococcal disease in Kanti Children’s Hospital, Nepal, as observed by the South Asian Pneumococcal Alliance network. Clin Infect Dis. 2009;48(suppl 2):S123–S128.
9. Jauneikaite E, Jefferies JM, Hibberd ML, et al. Prevalence of Streptococcus pneumoniae
serotypes causing invasive and non-invasive disease in South East Asia: a review. Vaccine. 2012;30:3503–3514.
10. Harris M, Clark J, Coote N, et al.; British Thoracic Society Standards of Care Committee. British Thoracic Society guidelines for the management of community acquired pneumonia in children: update 2011. Thorax. 2011;66 Suppl 2:ii1–i23.
11. Weinberger DM, Pitzer VE, Regev-Yochay G, et al. Association between the decline in pneumococcal disease in unimmunized adults and vaccine-derived protection against colonization in toddlers and preschool-aged children. Am J Epidemiol. 2019;188:160–168.
12. Whitney CG, Goldblatt D, O’Brien KL. Dosing schedules for pneumococcal conjugate vaccine: considerations for policy makers. Pediatr Infect Dis J. 2014;33(suppl 2):S172–S181.
13. Hamaluba M, Kandasamy R, Upreti SR, et al. Comparison of two-dose priming plus 9-month booster with a standard three-dose priming schedule for a ten-valent pneumococcal conjugate vaccine in Nepalese infants: a randomised, controlled, open-label, non-inferiority trial. Lancet Infect Dis. 2015;15:405–414.
14. Kandasamy R, Gurung M, Thorson S, et al. Comparison of two schedules of two-dose priming with the ten-valent pneumococcal conjugate vaccine in Nepalese children: an open-label, randomised non-inferiority controlled trial. Lancet Infect Dis. 2019;19:156–164.
15. Goldblatt D, Southern J, Ashton L, et al. Immunogenicity
of a reduced schedule of pneumococcal conjugate vaccine in healthy infants and correlates of protection for serotype 6B in the United Kingdom. Pediatr Infect Dis J. 2010;29:401–405.
16. Poolman JT, Frasch CE, Käyhty H, et al. Evaluation of pneumococcal polysaccharide immunoassays using a 22F adsorption step with serum samples from infants vaccinated with conjugate vaccines. Clin Vaccine Immunol. 2010;17:134–142.
17. Satzke C, Turner P, Virolainen-Julkunen A, et al.; WHO Pneumococcal Carriage Working Group. Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae
: updated recommendations from the World Health Organization Pneumococcal Carriage Working Group. Vaccine. 2013;32:165–179.
18. Voysey M, Fanshawe TR, Kelly DF, et al. Serotype-specific correlates of protection for pneumococcal carriage: an analysis of immunity in 19 countries. Clin Infect Dis. 2018;66:913–920.
19. WHO. Summary – WHO Position Paper on Pneumococcal conjugate vaccines in infants and children under 5 years of age – February 2019. 2019. Available at: https://www.who.int/immunization/policy/position_papers/who_pp_pcv_2019_presentation.pdf
. Accessed December 25, 2019.
20. WHO. Executive Summary SAGE October 2017, Pneumococcal Conjugate Vaccine Session 2017. Available at: https://www.who.int/immunization/sage/meetings/2017/october/1_Hosangadi_PCV_ExecutiveSummary_SAGE_PCV_WG_Oct2017.pdf
. Accessed December 25, 2019.
21. Paudel K. PCV 10 introduction in National Immunization Program of Nepal. Pediatr Infect Dis
22. Choden S. Health ministry introduces Pneumococcal Conjugative Vaccine [BBSC web site]. 2019. Available at: http://www.bbs.bt/news/?p=109479
. Accessed December 28, 2019.
23. WHO. WHO vaccine-preventable diseases: monitoring system. 2019 global summary 2019. Available at: https://apps.who.int/immunization_monitoring/globalsummary/
. Accessed December 28, 2019.
24. Wysocki J, Brzostek J, Konior R, et al. Antibody persistence and immunologic memory in children vaccinated with 4 doses of pneumococcal conjugate vaccines: results from 2 long-term follow-up studies. Hum Vaccin Immunother. 2017;13:661–675.
25. Zimmermann P, Perrett KP, Berbers G, et al. Persistence of pneumococcal antibodies after primary immunisation with a polysaccharide-protein conjugate vaccine. Arch Dis Child. 2019;104:680–684.
26. Trück J, Jawad S, Goldblatt D, et al. The antibody response following a booster with either a 10- or 13-valent pneumococcal conjugate vaccine in toddlers primed with a 13-valent pneumococcal conjugate vaccine in early infancy. Pediatr Infect Dis J. 2016;35:787–793.
27. Goldblatt D, Southern J, Andrews NJ, et al. Pneumococcal conjugate vaccine 13 delivered as one primary and one booster dose (1 + 1) compared with two primary doses and a booster (2 + 1) in UK infants: a multicentre, parallel group randomised controlled trial. Lancet Infect Dis. 2018;18:171–179.