Durable Anti-Vi IgG and IgA Antibody Responses in 15-Month-Old Children Vaccinated With Typhoid Conjugate Vaccine in Burkina Faso

Abstract We assessed anti-Vi IgG/IgA responses to typhoid conjugate vaccine (TCV) in children enrolled in a double-blind randomized controlled, phase 2 trial in Burkina Faso. Anti-Vi IgG seroconversion and anti-Vi IgA titers were higher in TCV than control recipients at 30–35 months post-vaccination. TCV induces durable immunity in Burkinabe children vaccinated at 15 months.


INTRODUCTION
In 2017, the World Health Organization prequalified the first typhoid conjugate vaccine (Typbar TCV) and deems anti-Vi IgG an appropriate measure of vaccine immunogenicity [1].In adults in the UK, TCV induced high anti-Vi IgG and IgA responses [2,3].In addition, anti-Vi IgA antibodies generated by TCV were associated with protection against controlled human infection with Salmonella Typhi (S Typhi) [3].Vi IgA and IgG responses together predicted protection from typhoid [4].In Burkina Faso, TCV was safe and immunogenic when coadministered with measles-rubella (MR) and yellow fever vaccines at 9-11 months [5], and with MR and meningococcal serogroup A conjugate vaccine (MCV-A) at 15-23 months in a double-blind, randomized, controlled Phase 2 clinical trial [6].
We evaluated anti-Vi IgA antibodies 28 days after vaccination, and anti-Vi IgG and IgA antibodies 30-35 months post-vaccination in participants from a clinical trial where TCV was coadministered with routine MR vaccine, with and without MCV-A, at the 15-month vaccination visit [6].The study was approved by ethics committees in Burkina Faso and Maryland, USA.Parents/guardians of study participants provided informed consent (Clinicaltrials.govNCT03614533).

METHODS
Detailed methods of this randomized, double-blinded, controlled, Phase 2 trial were published previously [6].Briefly, TCV or inactivated polio vaccine (IPV) was administered intramuscularly to children 15-23 months of age.Children were randomized 1:1:1 to Group 1 (TCV and IPV, MCV-A 28 days later), Group 2 (TCV and MCV-A), or Group 3 (MCV-A and IPV).All children received their second dose of MR vaccine.Adverse events were recorded for 7 days (solicited), 28 days (unsolicited), and 180 days (serious) during the clinical trial [6].Children 15-23 months of age were eligible if their parent/ guardian consented.Participants were excluded if they received blood products in the last 6 months and/or had any other condition likely to interfere with immunity or pose a health risk.The extended immunogenicity study protocol is provided in Supplementary Information.
Families were informed about the study and provided written informed consent in a private area before enrollment and venipuncture.Blood was drawn at baseline (Day 0), 28 days, and for participants whose parent/guardian could be recontacted and provided consent for an additional collection, 30-35 months after vaccination.Serum was tested for anti-Vi IgG and IgA antibodies using a commercially available enzyme-linked immunosorbent assay (ELISA) kit, VaccZyme S Typhi Vi immunoglobulin IgG (The Binding Site Group Ltd, Birmingham, UK) at the University of Maryland, Baltimore, MD.

Outcomes
Primary study outcomes were anti-Vi IgA antibody titers at 28 days, and both anti-Vi IgA and IgG antibody titers at 30-35 months post-vaccination.Participants with a fourfold rise in titer from baseline were considered seroconverted.

Statistical Analysis
Baseline demographics were compared between groups receiving TCV (Groups 1 and 2) and those that received the control vaccine (Group 3), and compared between participants who returned for their 30-35 month visit and participants who did not, using Chi-square test for differences in sex, Mann-Whitney U test for age, and two-sample t-test with unequal variances for height and weight.
Geometric mean anti-Vi titers were compared between groups using a two-sample t-test on log 10 -transformed titer values.According to standard practice when computing geometric means, zeros and values below the detection limit were replaced by one-half the limit of detection.Individual results below the assay detection limit for IgG (7.4 EU/mL) and IgA (3.0 EU/mL) were replaced with one-half this threshold, 3.7 EU/mL and 1.5 EU/mL, respectively.Differences in percent seroconversion were compared using Pearson's chi-square test or Fisher's exact test.A p-value of ≤0.05 was considered statistically significant.Results were analyzed using SAS software version 9.4 (Copyright 2016; SAS Institute Inc., Cary, NC).

Baseline Characteristics
Of the 150 participants enrolled and vaccinated, blood was collected at 28 days from 148 children and at 30-35 months from 115 children (Table 1 and Figure 1).Among participants who received TCV (Groups 1 and 2), there were more males and slightly older than participants who received the control vaccine (Group 3), p = .03and .01,respectively.Height and weight were similar between the TCV and control groups, p = .66and .88,respectively.Participants who did not return for their 30-35 month visit had similar baseline characteristics in sex, height, and weight as participants who returned.Those who were lost to follow-up at the 30-35 month visit were slightly older at baseline (Supplementary Table A1).

IgG Geometric Titer and Seroconversion at 30-35 Months After Vaccination
Baseline (day 0) anti-Vi IgG antibody geometric mean titer (GMT) was similar for all three groups.As reported previously, titers increased at 28 days after TCV immunization (Table 1) [6].At 30-35 months post-vaccination, GMT was at least 13-fold greater in TCV recipients versus controls and did not differ between MCV-A or IPV coadministration groups (Figure 2 and Table 1).Anti-Vi IgG seroconversion at 30-35 months postvaccination was ≥88% in TCV recipients compared to 7% in controls (Table 1).Results did not differ by sex (Supplementary Table A2).

IgA GMT at 28 Days and 30-35 Months After Vaccination
Day 28 anti-Vi IgA antibody GMT was at least 20-fold higher in children who received TCV versus controls (Table 1).Concomitant versus MCV-A administration 1 month later did not influence Day 28 anti-Vi IgA responses (p = .32).By months 30-35, GMT declined for TCV recipients, though remained significantly higher than controls (p < .0001; Figure 2).Results did not differ by sex (Supplementary Table A3).

DISCUSSION
TCV safety, immunogenicity, and efficacy have been demonstrated in adults and children as young as 6 months of age in typhoid-endemic areas [5][6][7][8][9][10]  antibody titers at baseline suggests a lack of environmental exto typhoid before 15 months of age and probably the disappearance of pre-existing maternal antibodies that could protect against disease.Increased anti-Vi IgG and anti-Vi IgA antibodies at 30-35 months post-vaccination in TCV recipients supports TCV's broad and durable immune response for at least 2.5 years.These data support findings from India where GMTs in children 6-23 months vaccinated with TCV were 21-fold, 8-fold, and 5-fold above baseline at 3-, 5-, and 7-years post-vaccination and seroconversion rates were 72% and 44%, at 5-and 7-years post-vaccination, respectively [11].A study from Malawi also reported favorable safety and durable immunogenicity for up to 3 years after TCV was coadministered with MR vaccine at 9-11 months and when given alone to children up to 12 years of age [12].TCV was introduced by Liberia, Pakistan, Nepal, Samoa, Zimbabwe, and Malawi.Routine TCV use in these countries will provide additional opportunities to assess TCV immunogenicity in similar settings.TCV recipients in Burkina Faso, vaccinated at the 15-month immunization visit, reported least 94% seroconversion 28 days after vaccination in a previous analysis of anti-Vi IgG antibody [6].Similar robust anti-Vi IgG immune responses were reported in children from Vietnam, Nepal, India, and Malawi [7,10,12,13].Studies conducted in these countries also document high vaccine efficacy for single-dose TCV, including 80% vaccine efficacy for 18-24 months after vaccination in Malawian children [9].Our finding of sustained anti-Vi IgG seroconversion of at least 88% at 30-35 months demonstrates the persistence of immunological responses to a single-dose vaccination of TCV and supports that the vaccine is likely protective.Although IgG titers decreased from 28 days to 30-35 months after vaccination, at least 88% of participants remained above the seroconversion threshold.This longer-term seroconversion rate is higher than the 60% documented in India at 25 months [7] and the 80% documented in Malawi at 2-3 years [12], and similar to the 95% documented in Nepal at 18 months [10].Anti-Vi IgA results from this cohort at 30-35 months are comparable to responses in Nepalese children <5 years of age at 18 months post-vaccination, for whom vaccine efficacy of 79% was demonstrated [10].Sustained vaccine immunogenicity demonstrated in this study is important because it confirms coadministration of routine MCV-A does not diminish longer-term immune responses to TCV, and that children from distinct geography, climate, and ethnic background maintain a vaccine-induced immune response similar to children from other typhoid-endemic areas that is consistent with protective efficacy against typhoid fever.
In the absence of an ideal assay for functional antibody assessment and its correlation with vaccine efficacy, anti-Vi IgG and IgA antibody estimates are accepted measures of TCV immunogenicity [1].Although no international standard exists, a potential seroprotective threshold was defined as a fourfold or greater rise in anti-Vi antibody titers from baseline, based on results from field trials in children in Bangladesh [8], Nepal [10], and Malawi [9] that show 79%-85% efficacy.
A single dose of TCV is highly immunogenic in Burkinabe children vaccinated at the 15-month visit, providing both robust anti-Vi IgA and IgG responses at 28 days and durable immunity for 30-35 months after vaccination.Sustained immunogenicity for over 2.5 years after vaccination at 15 months informs vaccination schedule planning in endemic areas and suggests a booster dose, if needed, can likely wait until school entry.Future studies should continue to assess the longevity of vaccine-induced responses and the potential need for booster dosing in target populations.

Figure 1 .
Figure 1.Flow chart showing enrollment, randomization, and loss to follow-up.

Figure 2 .
Figure 2. Anti-Vi antibody titers.(A) Anti-Vi IgG antibody titers before vaccination (Day 0), 28 days and 30-35 months after vaccination.(B) Anti-Vi IgA antibody titers 28 days and 30-35 months after vaccination.Circles and triangles represent the log 10 transformed antibody titer result for each individual participant in each corresponding vaccine group.Bar in the middle of individual participant results for each time point represents the mean antibody titer value on log 10 scale with 95% confidence interval.*Using two-sample t-test with unequal variances on log 10 transformed data.‡ Previously published, Sirima et al. [6].

Table 1 . Demographics and Anti-Vi IgG and IgA Antibody Responses
Data are n (%), mean (±standard deviation) or mean (95% CI).n = number of participants.N = total number.CI, confidence interval; IPV, inactivated polio vaccine; MCV-A, group A meningococcal conjugate vaccine; TCV, typhoid conjugate vaccine.a Previously published Sirima et al. [6].b Sirima et al. [6].c One participant not vaccinated.d One participant excluded in per-protocol immunogenicity analysis for late Day 28 visit and one participant missing titer, n = 47.e Day 0 anti-Vi IgA not assessed.