Mucosal Immunity to Poliovirus in Children 0–15 Years of Age: A Community-Based Study in Karachi, Pakistan in 2019

Abstract

This study assesses poliovirus type 1 (PV1) immunity in children to inform the contribution of mucosal immunity in and prevention of poliovirus circulation. A community-based study was conducted in periurban Karachi, Pakistan. Randomly selected children (0–15 years of age) received oral poliovirus vaccine (OPV) challenge dose. Blood and stool samples were collected at several time points and evaluated for polio-neutralizing antibodies and serotype-specific poliovirus, respectively. Eighty-one of 589 (14%) children excreted PV1 7 days post-OPV challenge; 70 of 81 (86%) were seropositive at baseline. Twelve of 610 (2%) were asymptomatic wild poliovirus type 1 (WPV1) excretors. Most poliovirus excretors had humoral immunity, suggesting mucosal immunity in these children likely waned or never developed. Without mucosal immunity, they are susceptible to poliovirus infection, shedding, and transmission. Asymptomatic WPV1 excretion suggests undetected poliovirus circulation within the community.

poliovirus, mucosal immunity, viral shedding, humoral immunity, Pakistan

As of 2023, Pakistan and Afghanistan are the 2 remaining countries endemic to wild poliovirus (WPV) type 1 (WPV1). The 2 other WPV serotypes, types 2 and 3, were certified as eradicated in 2015 and 2019, respectively. The persistence of WPV1 and transmission of circulating vaccine-derived poliovirus (cVDPV) continue to challenge efforts for global poliovirus eradication. In 2019, the year of this study, there were 147 paralytic cases of WPV1 and 22 of cVDPV type 2 (cVDPV2) in Pakistan [1, 2].

Environmental surveillance, the collection and analysis of wastewater samples for poliovirus detection, is a valuable tool to provide evidence of persisting transmission, even in the absence of paralytic cases. In 2019, 379 of 786 positive environmental isolates for WPV1 were reported in Pakistan [3]. In Karachi, WPV1 isolates continued to be detected in sewage water samples despite almost monthly oral poliovirus vaccine (OPV) campaigns representing one of the most important epicenters of poliovirus circulation in Pakistan. The reasons for persistent transmission could include children’s missed vaccination, barriers to vaccine infection in the gut, or absence/waning of mucosal intestinal immunity [4]. Waning and subsequent absence of mucosal immunity enables reinfections, excretion, and further transmission of poliovirus even among those who previously received poliovirus vaccines.

To better understand why WPV1 persists in Pakistan, it is important to evaluate the proportion and demographics of individuals who do not develop mucosal intestinal immunity despite repeated OPV campaigns. While humoral immunity protects against paralysis from poliomyelitis, mucosal immunity prevents infection in the gut and transmission [5]. As such, individuals who do not develop mucosal immunity shed virus and contribute to continued poliovirus circulation in communities, as do those who do not get vaccinated. In this study, a lack of Sabin-like (SL) poliovirus excretion 7 days after Sabin-OPV challenge was used as a proxy for mucosal intestinal immunity; the presence of mucosal immunity limits viral replication in the gut and subsequent shedding [6–8].

The objectives of the study were to estimate the proportion of children in Karachi lacking intestinal mucosal immunity to type 1 poliovirus (PV1) and assess what proportion of children possess humoral immunity.

METHODS

Study Design

This was a community-based, open-label, observational trial conducted in September–November 2019 in Bin Qasim Town, Karachi, Pakistan, in the 2 periurban areas of Cattle Colony and Rehri Goth. In these areas, Aga Khan University's Department of Paediatrics and Child Health has a well-established demographic surveillance system (DSS). Children in target age groups of 0–1, 3–5, 8–10, and 13–15 years were eligible for enrollment. The age groups were sampled to ensure adequate representation, particularly since there are limited mucosal immunity data on older children from an endemic polio country. Consecutive sampling using the DSS was used to identify eligible participants and caregivers who provided informed consent for enrollment. Exclusion criteria included immunodeficiency, administration of immunosuppressive medications, or any congenital abnormalities.

At enrollment, a sociodemographic questionnaire including poliovirus vaccination history was administered to the parent/guardian of the child. A phlebotomist took 2 mL of peripheral blood and 1 stool sample to assess baseline poliovirus immunity and virus shedding, respectively. Enrolled participants received 1 dose of bivalent OPV (bOPV, containing poliovirus type 1 and 3) and were observed for immediate adverse effects. After 7 days, a second stool sample was collected to assess postvaccination viral shedding.

Sera were tested for the presence of poliovirus neutralizing antibodies using standard microneutralization assays at the US Centers for Disease Control and Prevention (CDC) [9]. Stool samples were tested for the presence of poliovirus using standard viral culture at the National Institute of Health, Islamabad, Pakistan [10].

Outcomes

The primary objective of this study was to quantify mucosal immunity to PV1. The study endpoints were (1) proportion of children excreting Sabin 1 poliovirus (SL1) in stool 7 days after bOPV challenge; (2) seroprevalence of poliovirus neutralizing antibodies; (3) association of poliovirus neutralizing antibodies and shedding; and (4) comparison of mucosal immunity in children above and below the age threshold for vaccination campaign eligibility (5 years).

The presence of poliovirus 7 days post-bOPV in stool suggests a lack of intestinal mucosal immunity. We defined the presence of humoral immunity as seropositivity with a reciprocal antibody titer of ≥8 (≥3, in log₂ scale) [9]. Dependent on their mucosal and humoral immune status, children could then be classified as immune shedders, naive shedders, immune nonshedders, or naive nonshedders.

Statistical Analysis

Assuming a PV1 prevalence in the stool of <10%, accepting 95% confidence interval (CI) spanning ±5%, the sample size per each age group was calculated to be 139 children. Accounting for <10% loss after enrollment, 150 children were enrolled in each age group, for a total sample size of 600. Data were analyzed with Stata version 11 software. Descriptive analysis was performed by generating mean and standard deviation, median and interquartile range (IQR), and frequency and percentage. The χ² test was performed for difference in proportions between age groups, excluding individuals with unknown values.

This study was approved by the Ethical Review Committee of Aga Khan University, the National Bioethics Committee of Pakistan, and the Ethical Review Committee of the World Health Organization (WHO). Permission was obtained from the Town Health Officer Bin Qasim Town and the Emergency Operation Center Polio Program of Sindh. This activity was reviewed by CDC and conducted consistent with applicable federal law and CDC policy (see eg, 45 Code of Federal Regulations [C.F.R.] part 46, 21 C.F.R. part 56; 42 US Code [U.S.C.] §241(d); 5 U.S.C. §552a; 44 U.S.C. §3501 et seq).

RESULTS

A total of 1036 children were screened, with 913 of 1036 (88.1%) eligible and 610 of 1036 (58.8%) enrolled in the study (Supplementary Figure 1). Reasons for eligible children that were not enrolled included refusals, sickness, home locked, and inadequate sample collection. Of those enrolled, 606 of 610 (99.3%) completed both study visits and were included in the analysis. There were 149 individuals aged 0–1 years, 153 aged 4–5 years, 153 aged 8–10 years, and 151 aged 13–15 years.

Baseline indicators of vaccination and nutrition status are summarized in Supplementary Table 1. Of 606 participants, 283 (46.7%) of participants were male and 346 (57.3%) had a vaccination card available. Regarding routine immunization (RI), 64.4% (390/606) of individuals reported receiving all 4 RI doses of OPV (including birth dose), while 15.0% (91/606) reported receiving zero doses. A mean of 10 total OPV doses received was reported from both RI and immunization campaigns, with significant differences between age groups: 4.0 doses for 0–1 years, 12.6 doses for 4–5 years, 11.8 doses for 8–10 years, and 13.8 doses for 13–15 years (P < .001). Wasting (low weight-for-height) and stunting (low height-for-age) were calculated as indicators of acute and chronic malnutrition, respectively. In children 0–1 and 4–5 years of age, wasting was present in 26.8% (40/149) and 15.7% (24/302) of individuals, respectively, and stunting was in 24.8% (37/149) and 51% (78/153) of individuals, respectively.

Poliovirus neutralizing serum antibodies were assessed at baseline. The overall seroprevalence of PV1 antibodies was 96.7% (95% CI, 94.9%–97.9% [586/606]), with 82.0% (95% CI, 78.7%–85.0% [497/606]) against type 2 and 90.1% (95% CI, 88.0%–92.8%) against type 3 (Supplementary Figure 2A). Seroprevalence was significantly lower for the age group 0–1 years than for older age groups for all serotypes (P < .001 for all serotypes). Median antibody titers in log₂ scale were 10.17 (IQR, 8.17–10.50) for type 1, 7.17 (IQR, 4.17–9.50) for type 2, and 8.83 (IQR, 5.587–10.50) for type 3, with the lower limit of detection being 2.50 log₂ and the upper limit of detection being 10.50 log₂ for all 3 serotypes. Antibody titers to all 3 serotypes peaked in the age group 4–5 years and waned after that; levels for type 2 flattened out with older age while type 1 and 3 antibodies continued to wane (Supplementary Figure 2B).

Polioviruses in stool samples were assessed at baseline and 7 days post-bOPV challenge dose; Figure 1 displays SL viral shedding stratified by age group. At baseline, SL1 and SL type 3 poliovirus (SL3) were detected in 5 of 606 (0.8% [95% CI, .3%–1.9%]) and 4 of 606 (0.7% [95% CI, .2%–1.7%]) individuals, respectively. One week after the bOPV challenge, shedding increased to 81 of 606 (13.4% [95% CI, 10.8%–16.3%]) and 71 of 606 (11.7% [95% CI, 9.3%–14.5%]) for SL1 and SL3, respectively. No SL type 2 poliovirus (SL2) was detected at any timepoint evaluated.

Figure 1.

The proportion of individuals shedding Sabin-like (SL) virus 7 days after bivalent oral poliovirus vaccine challenge dose with 95% binomial confidence intervals. Children excreting poliovirus at baseline were excluded from this analysis (<1%). No type 2 Sabin-like poliovirus was detected at any timepoint evaluated. Abbreviations: SL1, type 1 Sabin-like poliovirus; SL3, type 3 Sabin-like poliovirus.

Table 1 summarizes the association of SL1 shedding with baseline type 1 seroprevalence. Among individuals seronegative against PV1 at baseline, 11 of 20 were shedding SL1 after the bOPV challenge (55% [95% CI, 31.5%–76.9%]), termed “naive shedders.” In comparison, in individuals seropositive at baseline, 70 of 586 (11.9% [95% CI, 9.43%–14.9%]) shed SL1 after the bOPV challenge, termed “immune shedders.” This difference was statistically significant (Fisher exact P = .008).

Table 1.

Association of Type 1 Seroprevalence and Type 1 Sabin-like Poliovirus Shedding in Stool Samples in Children (N = 606)

Baseline Seroprevalence	SL1 in Stool 7 Days After bOPV
	Present (n = 81)	Absent (n = 525)
Seropositive (n = 586)	Immune shedders (n = 70)	Immune nonshedders (n = 516)
Seronegative (n = 20)	Naive shedders (n = 11)	Naive nonshedders (n = 9)

The difference of proportion of immune shedders and naive shedders was statistically significant (Fisher exact P = .008). Immune shedders are described as children with existing poliovirus humoral immunity, yet lack mucosal immunity as observed by poliovirus shedding after the bOPV challenge. Naive shedders are described as children without humoral and mucosal immunity. Immune nonshedders are described as children with poliovirus humoral and mucosal immunity, while naive nonshedders have no humoral immunity and do not shed virus (nonreactive to bOPV).

Abbreviations: bOPV, bivalent oral poliovirus vaccine; SL1, Sabin-like poliovirus type 1.

A subset analysis to determine associated risk factors was performed on these immune shedders (n = 70; Supplementary Table 2). The highest levels of shedding (17.7%) were observed in the youngest age group; however, comparison with the other age groups was not statistically significant (P = .125). The number of OPV doses received in RI (P = .127), doses through campaigns (P = .067), chronic malnutrition (P = .211), acute malnutrition (P = .668), or body mass index (P = .220) did not have statistically significant impacts on prevalence of immune shedders. Sex was the only factor that was revealed as a risk factor, with females being more likely to be immune shedders than males (15.2% vs 9.0%, respectively; P = .029).

Twelve of 606 (2.0% [95% CI, 1.0%–3.4%]) individuals had WPV1 identified in stool samples at either timepoint (Supplementary Table 3). Seven of these individuals were found to be excreting WPV1 at baseline; 4 stopped excreting after the bOPV challenge. WPV1-shedding individuals ranged in age from 6 months to 14 years; all individuals were seropositive against PV1, and 11 of 12 (91.6%) reported receiving the third dose of OPV in RI.

DISCUSSION

In this study, we found that 13.4% of children excreted poliovirus after the bOPV challenge, despite most having humoral immunity to poliovirus at baseline. Children who shed poliovirus despite the presence of poliovirus humoral immunity represent those whose mucosal immunity either never developed or it waned. These children would likely transmit poliovirus within their communities if replication-competent poliovirus, whether wild-type or vaccine-derived, were to be introduced.

Virus shedding was observed across all age groups including those >5 years of age; a significantly higher prevalence of SL1 shedding was detected in the youngest age group (0–1 years). The finding of excretion in the >5-year group is important because polio vaccination campaigns typically target only those <5 years of age.

There are several possibilities why some individuals had humoral immunity but no mucosal immunity against poliovirus: OPV did not successfully elicit mucosal immunity in the gut of these children, mucosal immunity induced by OPV waned over time, or humoral immunity was induced by inactivated poliovirus vaccine (IPV) given in RI [6]. It is well documented that oral vaccines (including polio, cholera, and rotavirus), have demonstrated lower immunogenicity in low-income settings [11–13]. Factors that were shown to influence OPV immunogenicity include a high incidence of enteric infections and malnutrition; however, it remains unclear why these conditions impair vaccine immunogenicity [14, 15]. Furthermore, mucosal immunity may not always prevent shedding after a high-dose bOPV challenge (10³ median tissue culture infectious dose [TCID₅₀]); however, it may be sufficient to prevent shedding during natural exposure to WPV (10 TCID₅₀) [8]. This relationship between a challenge dose and natural exposure to viral shedding is unknown and expected to vary dependent on population characteristics. As such, these children would have mucosal immunity but would be misclassified in the pretext of this study.

Asymptomatic excretors of WPV1 were detected within this study at a 2% rate (12/610). They were represented in all age groups, with 50% aged between 5 and 15 years, which is above the traditional age targeted in supplementary immunization activities (SIAs). The finding of asymptomatic excretors is unexpected since these are areas of Karachi that had not reported any acute flaccid paralysis cases caused by WPV1 in many years (informal communication with WHO utilizing data from the internal WHO Polio Information System).

We found 20 of 586 (3.4%) children without humoral immunity in our sample. These children represent those persistently missed by vaccination campaigns or whose immunity has waned.

There were several limitations to the study. There was a delay in testing the study samples, primarily due to the coronavirus disease 2019 pandemic. There were no data collected on the number of IPV doses received.

The data presented herein provide insights into the persistence of children without mucosal immunity despite repeated rounds of vaccination. These data provide valuable information to direct programmatic activities for the polio eradication program in the planning of SIAs, taking into consideration the frequency and types of vaccines and targeting specific age groups.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. We would like to thank the Population Immunity Laboratory (William Hendley, Kathryn Jones, Sharla McDonald, Deborah Moore, Ashley Smith, and Yiting Zhang) at the Centers for Disease Control and Prevention for performing poliovirus serological testing. Most of all, we would like to thank the children and their parent/legal guardian for participating in this study.

Disclaimer. The findings in this article are those of the authors and do not necessarily represent the official position of the affiliated institutions.

Financial support. This study was funded by the World Health Organization through an International Polio Plus Committee Rotary Grant.

Author notes

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.