Immunogenicity, Immunological Memory and Monitoring of Disease Activity Following an Anamnestic Immunization With the 13-Valent Pneumococcal Conjugate Vaccine in Children With Idiopathic Nephrotic Syndrome

Abstract

Anamnestic 13-valent pneumococcal conjugate vaccine immunization did not affect the relapse risk in pediatric idiopathic nephrotic syndrome. Pneumococcal serotype (PS)-specific antibody titers increased significantly in all groups. Children receiving immunomodulatory treatments (IMTs) displayed significantly lower levels of PS-specific antibodies for 3/8 serotypes tested. PS-specific B-cell counts significantly increased only in healthy controls and patients receiving corticosteroids.

In this work, we demonstrate the safety of 13-valent pneumococcal conjugate vaccine booster dose and the benefits of its administration in terms of protection against pneumococcal disease in children with idiopathic nephrotic syndrome. We propose a possible mechanism behind the reduced immune responses found in these patients.

BACKGROUND

Children with idiopathic nephrotic syndrome (INS) are an immunocompromised population [1]. Consequently, boosting immunity with an anamnestic dose of pneumococcal conjugate vaccine (PCV) could be beneficial [1]. We have previously reported reduced immunogenicity and duration of immunity in children with INS, especially those under immunomodulatory treatments (IMTs) [2].

In the present work, we evaluated the safety of 13-valent pneumococcal conjugate vaccine (PCV13) booster in children with INS under treatment, regarding INS relapses and investigated the effect of immunization on interleukin (IL)-5, as a biomarker of disease activity [3]. We investigated the effect of INS treatments on the PCV13 immunogenicity, PCV13-induced antibody persistence, and B-cellular responses to PCV13, by measuring pneumococcal serotype (PS)-specific antibodies and the enumeration of circulating PS-specific B-cell populations.

METHODS

This prospective cohort study was conducted in the Nephrology Department of “P&A Kyriakou” Children’s Hospital, Athens, Greece.

All children had received primary PCV vaccination series during infancy according to the Greek National Immunization Program and were categorized into 3 groups as follows: INS patients receiving IMTs (group A) those on low-dose corticosteroids (group B) and age-matched healthy controls (group C).

After enrollment, all participants received a PCV13 booster (Prevnar13; Pfizer, Pearl River, New York) intramuscularly.

Safety was evaluated through local and systemic adverse events for 7 days following PCV13 booster, INS relapses for the 6 months following booster and a case cross-over design was used to compare the counts and the odds of relapse in the 6 months following PCV13 [period 1 (P1)] to those in the previous 6 months (P2) and 6–12 months (P3). We measured the levels of IL-5 in the plasma at baseline and at 72 h [3].

For the evaluation of immunogenicity and PCV13-induced antibody persistence, blood samples were obtained at baseline, 4 weeks (4W), and 12 months (12M) after booster. The WHO ELISA protocol was used for the quantification of PS-specific Immunoglobulin G (IgG) antibodies against PS1/3/6B/7F/9V/14/19A/19F. The cut-off level of 0.35 μg/ml was used as a surrogate of protection against invasive pneumococcal disease, while we also report our results referring to PS-specific correlates of protection suggested in post-licensure real-world data [4].

PS-specific B-cell subsets [PS-specific B-cells, PS-specific memory B-cells (MBCs), and PS-specific isotype-switched Ig MBCs (sIg MBCs)] against PS1 and PS9V at baseline and 4W after PCV13 booster were evaluated in a subcohort of our study population, including 9 children with INS [4 under cyclosporine A (CsA), 5 under prednisolone] and 6 age-matched controls, using a novel flow cytometry protocol developed by our group [5].

Statistical Analysis

Statistical significance was set at a P value <.05; all tests are two-tailed. IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. and StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP and the R “rmcorr” package were used.

Materials and Methods are described in detail in Supplementary Data-Extended Methods.

RESULTS

Study Population

Forty-four children were enrolled, 27 with INS (10 group A, 17 group B) and 17 controls (group C); no loss-to-follow-up occurred. The groups did not differ regarding age, PCV product administered at infancy, and duration of INS between groups A and B (Table 1). In the B-cell subset subcohort, no differences in age were found between children under CsA [median: 5.9 years (range: 4.8–10 years)] and corticosteroids [median: 6.1 years (range: 4.9–8.3 years)] compared to the controls [median: 6.9 years (range: 6–9.9 years)] (P = .177, P = .352, respectively).

Table 1.

Study Population Characteristics

	Group A (n = 10)	Group B (n = 17)	Group C (n = 17)
Male [n (%)]	5 (50)	11 (64.7)	9 (52.9)
Age (years) [Median (range)]	8.9 (4.8–15.0)	9.8 (3.5–16.4)	10.9 (3.3–16.3)
Time since INS diagnosis (years) [Median (range)]	3.5 (0.9–11.8)	4.7 (1.8–12.5)
Dose of treatment
Immunomodulators:
CsA [c2 (ng/ml)] [Median (range)] (n = 8)	523 (430–705)
MMF (mg/m2)(n = 2)	600
Corticosteroids (mg/kg/48 h) [Median (range)]		0.21 (0.1–0.78)

Group A, children with INS under immunomodulatory treatments; Group B, children with INS under low-dose maintenance corticosteroid treatment; Group C, control group, INS, idiopathic nephrotic syndrome; CsA, cyclosporine A; c2, concentration of CsA in serum 2 h after CsA administration; MMF, mycophenololate mofetil.

Safety

No systemic side effects were reported; grade 1 local reactions (redness/swelling at injection site) occurred in 2 children with INS (7.4%) and in 2 children in the control group (11.8%) (OR = 1.67, 95% CI: 0.21–13.10). In P1, 8 relapses occurred in 7 children with INS (25.9%); in P2, 7 relapses occurred in 6 children (22.2%), and in P3, 16 relapses occurred in 13 children (48.1%). No changes in the relapse counts after booster PCV13 compared to either reference period (P2: IRR = 1.29, 95% CI: 0.59–2.81, P3: IRR = 1.76, 95% CI: 0.85–3.61); no changes in the odds of relapse were found (P2: OR = 1.2, 95% CI: 0.37–3.93, P3: OR = 0.33, 95% CI: 0.09–1.23).

Regarding the levels of IL-5, statistically significant increases were found after the PCV13 booster in all groups (P < .05 in all cases) (Supplementary Table 1); no differences were found in groups A and B compared to group C, and between patients with and without relapses.

Kinetics of PS-Specific Antibodies

At baseline, the percentage of children with protective antibody titers was lower in group A compared to groups B and C (Supplementary Table 2). All groups had achieved the universal cut-off level of 0.35 μg/ml against 6/8 serotypes evaluated at 4W (Supplementary Table 2). At 12M, all groups had retained the universal protective cutoff levels against 4/8 serotypes (Supplementary Table 2). Regarding the PS-specific minimum protective antibody concentrations [4], in group A lower percentage of children achieved the PS-specific protective titers (Supplementary Table 3).

At 4W and 12M, PS-specific antibody titers were increased significantly compared to baseline for all the serotypes evaluated in all groups (P < .05 in all cases) (Supplementary Table 2), while significantly lower PS-specific antibody titers were found in group A compared to group C against PS1/3/9V at both timepoints (P = .013, P = .015, P = .023; P = .02, P = .04, P = .004, respectively) (Supplementary Table 2).

PS-specific antibody titers at 4W were correlated to the corresponding titers at 12M (Supplementary Table 4).

PCV13-Induced Immunological Memory

At baseline, PS-specific total B-cell and MBC counts were significantly lower in children under CsA compared to controls for both PS1 and PS9V (P = .038, P = .01, respectively) (Figure 1). No differences were found between children on low-dose corticosteroids and controls. At 4W, PS-specific total B-cell, MBC, and sIg MBC counts did not increase significantly compared to baseline and were significantly lower in children under CsA compared to controls for both serotypes (PS1 and PS9V: P = .01; P = .019; P = .019, respectively) (Figure 1). In contrast, PS-specific total B-cell, MBC and sIg MBC counts increased significantly compared to baseline in children under corticosteroids (P = .043 all cases) and in healthy controls (P = .028 all cases) (Figure 1).

Figure 1.

PS1/9V-specific B-cell subset counts at baseline and at 4W after a booster dose of PCV13 in children with INS under CsA, low-dose corticosteroids, and healthy controls. PS1/9V-specific B-cell (A), MBC (B), and sIg MBC (C) counts significantly increased at 4W compared to baseline in children under low-dose corticosteroid treatment and in controls (Wilcoxon signed-rank test). Significantly lower PS1/9V-specific B-cell, MBC, and sIg counts in children under CsA compared to controls at baseline and 4W (Mann–Whitney U test). Abbreviations: PS, pneumococcal serotype; PCV13, 13-valent pneumococcal conjugate vaccine; MBC, memory B-cell; sIg MBC, immunoglobulin class-switched memory B-cells; 4W, 4 weeks.

Correlations Between Humoral and Cellular Responses

Significant within-individual correlations were found between the increase in the counts of PS1/9V-specific B-cell subsets at 4W and the increase of PS1/9V-specific antibody titers at 4W (P < .05 all cases) (Supplementary Figure 2A) and between the increase in the counts of PS1-specific B-cell subsets at 4W and the retained PS1-specific antibody titers at 12M (P < .001 all cases) (Supplementary Figure 2B). Similar trends we observed for PS9V but did not reach significance (Supplementary Figure 2B).

Discussion

This is the first study investigating the safety of PCV13 anamnestic immunization in pediatric INS regarding disease relapses monitored both by proteinuria and an immunological biomarker, as well as the effect of treatment regimens on PS-specific antibody titers and B-cell memory formation.

We show that PCV13 booster does not affect the INS activity in pediatric patients, which is further supported by the finding that changes in plasma IL-5 levels, were similar in children with and without relapses suggesting that these increases could not suffice to trigger the immunological mechanisms of INS [3].

Lower percentage of children with protective PS-specific titers are reported in children with INS under IMTs at baseline, while we report significantly lower titers against 3/8 serotypes in this group at 4W and 12M compared to controls, providing further evidence for the need for anamnestic immunization in these patients. Interestingly, unaffected responses to immunizations are described in treatment-naïve INS patients by other groups [6], which indicates that the administered treatment regimen is the main determinant of reduced responses to immunizations.

This is first study to utilize a novel flow cytometry assay [5] for the evaluation of PS-specific B-cellular responses, with the enumeration of PS-specific B-cell subsets. Parallel to the diminished immunogenicity and shorter antibody persistence in children under CsA we did not recognize a significant enrichment in the pool of PS-specific B-cells. PS1 and PS9V were utilized as proxies of the B-cellular responses to PCV13 booster: PS1 due to its unique microbiological characteristics [7] and PS9V due to the reported impaired immunogenicity and duration of protection after PCV7 in previous studies [2].

Regarding the inferior expansion of B-cell populations in children receiving CsA, traditionally, CsA-mediated T-cell help inhibition leads to the subsequent dwindling B-cell maturation and class switching, through reduced T-cell cytokine signaling [8]. Moreover, the immunological signature of INS with T-cell dysregulation [1] may exacerbate this effect. Interestingly, recent findings suggest additional direct effects of CsA on B-cell subsets, including reduced CD19+ B-cells and naïve B-cells [9].

The main limitation of this study is its limited sample size. However, we demonstrate that the PS1/9V-specific B-cell population increases are strongly correlated with the kinetics of PS1/9V-specific antibodies. Previous work underlines the importance of B-cells as novel surrogates of vaccine-induced protection [10]. Most importantly, this observation highlights the role of PS-specific B-cell memory in potent and sustained PS-specific humoral immunity.

Finally, increased valency PCVs have recently been approved for use in pediatric populations to increase the breath of protection against pathogenic serotypes [11]. Since polyvalency has been associated with reduced immunogenicity [12], further studies using novel PCVs are warranted, for potential adaptations of the immunization schemes to achieve sufficient protection.

In conclusion, we demonstrate the safety of PCV13 booster and the benefits of its administration in patients INS. Moreover, we propose a plausible mechanism behind the reduced immune responses in patients under IMTs. Finally, we provide further evidence that antigen-specific B-cellular and antibody responses are expanded in a coordinated way following vaccination and can be used as a surrogate of vaccine-induced protection.

Supplementary Data

Supplementary materials are available at the Journal of The Pediatric Infectious Diseases Society online (http://jpids.oxfordjournals.org).

Author Contributions

Study conception and design: V.S. and K.K. Data collection: K.K., V.A., and A.M. Data analysis: K.K., M.T., and I.P. Writing-original draft: K.K. Writing-revision and editing of the original draft: V.S., V.A., M.T., A.M., I.P., E.L., C.K.G., and G.M. Review and approval of the final version of the manuscript: all authors.

Ethical Considerations

The study was approved by the Institutional Review Board of the “Panagiotis and Aglaia Kyriakou” Children’s Hospital (Protocol Number: 13383/05-08-2019) and was performed under the ethical standards of the Declaration of Helsinki.

Data Availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Notes

Financial support. This work was supported by the European Society for Paediatric Infectious Diseases (ESPID), in the frameworks of an ESPID Small Grant Award 2021 to K.K. (Project Title: “Cellular immune responses to the 13-valent Pneumococcal Conjugate Vaccine (PCV13) for prediction of PCV13 immunogenicity and duration of protection in pediatric patients on immunosuppressive treatment”).

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