Pneumococcal serotype-specific IgG and opsonophagocytic activity in young Japanese patients with asplenia

ABSTRACT

Patients with asplenia are at high risks of severe infections caused by encapsulated bacteria, particularly Streptococcus pneumoniae. Thirteen-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPSV23) are recommended for invasive pneumococcal disease prevention; however, little is known about the immunity to pneumococci in young patients with asplenia. We measured pneumococcal serotype-specific IgG (Pn-IgG) levels and pneumococcal opsonophagocytic activity (Pn-OPA) against some PCV13-contained serotypes (1, 3, 5, 6A, 7 F, 19A) in 23 young patients with asplenia using surplus serum samples. In this study, 5 and 13 patients had received PCV13 during routine immunizations and PPSV23, respectively; however, >5 years had passed since the last dose in most cases. The geometric mean concentrations (GMCs) of Pn-IgG in all study patients were not under the cutoff level against six serotypes, but they were lower than the those of age-matched healthy controls, as we have previously published. The patients who had received only PPSV23 had significantly lower GMCs against four serotypes (serotypes 1, 6A, 7 F, and 19A) than that of the patients who had received at least one PCV13 vaccination. The patients who had received only PPSV23 also had significantly lower geometric mean titers (GMTs) of Pn-OPA against all three serotypes we measured (serotypes 3, 5, and 19A) than that of the patients who had received at least one PCV13 vaccination. Our findings are useful data that can indicate insufficient immunity in young patients with asplenia against some PCV13 pneumococci serotypes and suggest the need for appropriate vaccinations in the post-PCV13 era.

KEYWORDS:

• Asplenia
• serotype specific IgG level
• opsonophagocytic activity
• 13-valent pneumococcal conjugate vaccine
• 23-valent pneumococcal polysaccharide vaccine

Introduction

Asplenia occurs as a congenital anomaly or as a result of splenectomy due to hematologic disease or trauma. Patients with asplenia are at high risk of severe infections and sometimes die, even after aggressive treatment.^1,2 These infections are mostly caused by encapsulated bacteria, Streptococcus pneumoniae in particular. Although antibiotic prophylaxis is recommended, it is not always efficient. Immunization is important for preventing infection, and the Infectious Diseases Society of America has recommended pneumococcal vaccinations for patients with asplenia and sickle cell diseases for the prevention of invasive pneumococcal disease (IPD).³

In Japan, the heptavalent pneumococcal conjugate vaccine (PCV7) was licensed in February 2010 and was approved as a routine immunization in children aged <5 years in April 2013. PCV7 was replaced in November 2013 by the 13-valent pneumococcal conjugate vaccine (PCV13), and the recommended immunization for PCV13 has been a 3 + 1 schedule. Following the introduction of PCV in Japan, the incidence of IPD in children aged <5 years had decreased; however, the incidence of IPD in children aged 5–15 years had not decreased, and half of these IPDs occurred in children with underlying diseases.^4,5

As a pneumococcal immunization for high-risk patients with IPD in Japan, 23-valent pneumococcal polysaccharide vaccine (PPSV23) has been covered by health insurance for patients aged ≥2 years. In May 2020, PCV13 was approved as a voluntary vaccine for high-risk patients with pneumococcal infections regardless of age. PPSV23 induces a T cell-independent immune response that is the reason for recommendation of revaccination every 5 years. On the other hand, PCV13 induces a T cell-dependent immune response resulting in long-lasting protection against pneumococcal infection.⁶ Both PPSV23 and PCV13 can help prevent IPD in asplenic patients;⁷ however, the compliance of patients with these immunizations is poor.⁸ As a result of the limited evidence regarding the immunity to pneumococci in young patients with asplenia, the necessity of immunization has not been fully clarified. Therefore, we conducted a serological study on young Japanese patients with asplenia to determine their immunity against pneumococci and evaluated the necessity of pneumococcal immunization for such patients to prevent IPD.

Patients and methods

We collected surplus serum samples from routine blood examinations between March 2016 and March 2018 from asplenic patients without any other comorbid immunodeficient disease who were treated in Chiba Children’s Hospital, Chiba prefecture, Japan. Collected samples were stored in a − 80°C deep freezer, and were then transferred to the Medical Mycology Research Center, Chiba University, and analyzed. We measured pneumococcal serotype-specific immunoglobulin G (Pn-IgG) levels and pneumococcal opsonophagocytic activity (Pn-OPA) against some PCV13-contained serotypes.

This study was approved by the research ethics committee of Chiba Children’s Hospital (No. 2016–01-13). Written informed consent was obtained from the patients and/or their legal guardians.

The statistical analyses were performed with JMP 15 (SAS Institute, Cary, NC, USA). Chi-squared and Mann–Whitney U-tests were performed, and differences with a p value ≤0.05 were considered statistically significant.

Pneumococcal serotype-specific IgG

We measured Pn-IgG with a World Health Organization-approved enzyme linked immunosorbent assay, employing the standard reference serum (007sp), C-polysaccharide, and 22 F polysaccharide absorptions (http://www.vaccine.uab.edu), as previously reported.⁹ We assessed the concentrations of antibodies against 6 serotypes that were contained in PCV13 and were not contained PCV7 (serotypes 1, 3, 5, 6A, 7 F, and 19A). A positive Pn-IgG concentration for IPD prevention was defined as ≥0.2 μg/mL, as previously reported.¹⁰ In comparison with the healthy controls, we used our previously published data; Pn-IgGs of healthy 100 people aged 6–23 years who had not received PCV13 before.¹¹

Pneumococcal serotype-specific opsonophagocytic activity

We measured Pn-OPA to investigate the functional activity of a specific antibody employing a multiplexed opsonophagocytic killing assay (MOPA) procedure, as previously described.¹² Since the study samples were insufficient to investigate Pn-OPA against all six serotypes, as we could with Pn-IgG, we chose three serotypes (serotype 3, 5, and 19A) that were included in PCV13. Serotype 3 and 19A are the key serotypes for prevention of IPD in Japan and other countries,^13,14 and these serotypes, as well as serotype 5, were the best combination to measure using MOPA. A positive Pn-OPA titer for IPD prevention was defined as ≥8.¹⁵

Results

Tables 1 and 2 shows the patient characteristics and vaccine status of all 23 patients included in this study. The study patients consisted of 17 (74%) males and 6 (26%) females, with a median age of 11 (range, 0–22). Most had congenital asplenia with cardiac anomalies (91%), except for 2 patients post-splenectomy due to hereditary spherocytosis (9%). Regarding immunization against pneumococci, 7, 5, and 14 patients had received PCV7, PCV13, and PPSV23, respectively; 5 had never received any pneumococcal vaccines. Four patients had received only PCV13 (never received PPSV23) and all of them had received it during routine immunizations (two patients received four doses, and others received three doses). One patient received three doses of PCV7 and an additional dose of PCV13. The patients who had received PPSV23 comprised approximately 60% of the study patients; however, more than 5 years had passed from their last dose for most of these patients. Antibiotic prophylaxis by oral sulfamethoxazole-trimethoprim had been administered to 2 patients.

Table 1. Patient characteristics (n = 23)

Median age [range]	11 [0–22]
Male, number (%)	17 (74)
Reason for asplenia, number (%)
Congenital asplenia	21(91)
Post splenectomy	2(9)

Table 2. Vaccine status in study patients

PCV13	PPSV23 (<5 years*)	PPSV23 (≥5 years*)	n (%)	Median age [range]
+	-	-	4 (17)	1.5 [0–3]
+	+	-	1 (4) ^#	5
+	-	+	0 (0)
-	+	-	1 (4) ^#	5
-	-	+	12 (52) ^#	15.5 [10–22]
-	-	-	5 (22) ^#	10 [6–16]

PCV13, 13-valent pneumococcal conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine.

*<5 years: within 5 years from the last dose; ≥5 years: 5 years and more from the last dose.

^#Including the patients who had received PCV7 (total 7 patients).

Table 3 shows geometric mean concentrations (GMCs) and positive rates of Pn-IgG against six serotypes. The GMCs of all the study patients were not below the cutoff level: 0.2 µg/mL against 6 serotypes, and the positive rates were almost high, except for serotype 3. However, comparing these GMCs with that of the age-matched healthy controls that we had previously published¹¹ (the bottom line of Table 3), the GMCs of the study patients were lower against all serotypes except 7 F, 3 of which were statistically significant (serotype 1, 3, and 6A; as shown by * in Table 3). Comparing GMCs based on their vaccination status, patients who had never received a pneumococcal vaccine (neither PCV13 nor PPSV23) had no significantly lower GMCs against six serotypes than those of patients who had received at least one pneumococcal vaccine. In the 18 patients who had vaccine history, the patients who had received only PPSV23 had significantly lower GMCs against 4 serotypes (serotype 1, 6A, 7 F, and 19A; as shown by^# in Table 3) than that of the patients who had received at least 1 PCV13 vaccination. Moreover, these results were significantly lower against five serotypes (serotype 1, 3, 5, 6A, and 19A; as shown by * in Table 3) than that of age-matched healthy controls.

Table 3. Geometric mean concentrations and positive rates of pneumococcal serotype-specific IgG against 6 serotypes

Group	Serotype 1	Serotype 3	Serotype 5	Serotype 6A	Serotype 7F	Serotype 19A
All (n = 23)	0.78* [0.22–5.67]	0.31* [0.07–6.28]	1.57 [0.45–5.31]	1.00* [0.13–3.32]	1.07 [0.15–3.87]	1.91 [0.36–15.1]
	100	70*	100	96	96	100
Vaccine status
PCV13 at least once (n = 5)	1.41 [0.35–3.92]	0.48* [0.14–6.28]	2.27 [1.2–5.31]	1.48 [0.92–3.32]	2.45 [1.54–3.87]	4.03 [0.94–15.1]
	100	80.0*	100	100	100	100
PPSV only (n = 13)	0.60*^# [0.22–5.67]	0.26* [0.07–0.63]	1.25* [0.45–4.07]	0.76*^# [0.13–3.26]	0.65^# [0.15–1.80]	1.26*^# [0.36–3.57]
	100	69.2*	100	92.3*	92.3	100
None (n = 5)	0.87 [0.66–1.08]	0.32* [0.13–1.05]	1.96 [1.5–3.45]	1.39 [0.90–2.81]	1.66 [0.44–2.85]	2.7 [0.70–8.89]
	100	60.0*	100	100	100	100
Control[13] (n = 100)	1.5 [0.36–15.66]	1.38 [0.38–10.18]	1.95 [0.50–10.91]	2.07 [0.42–9.59]	0.89 [0.16–12.49]	2.66 [0.46–23.48]
	100	100	100	100	95	100

In order from the upper line: GMC (μg/mL), range, and positive rate (%) of Pn-IgG against each serotype were shown in each cell. PCV13, 13-valent pneumococcal conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine.

*Significantly lower than that of age-matched healthy controls (p < 0.05). ^#Significantly lower than that of the patients who had received at least one PCV13 vaccination.

Table 4 shows geometric mean titers (GMTs) and positive rates of Pn-OPA against three serotypes. In the 18 patients with a vaccine history, those who had received only PPSV23 had significantly lower GMTs against 3 serotypes (as shown by ^# in Table 4) than those of the patients who had received PCV13 vaccination at least once. We could not perform a comparison with control data because we had no OPA data on healthy controls as we did in the Pn-IgG analysis, and there are no published data on healthy Japanese people at these ages.

Table 4. Geometric mean titers and positive rates of pneumococcal serotype-specific opsonophagocytic activity against three serotypes

Group	Serotype 3	Serotype 5	Serotype 19A
All (n = 23)	50 [4–380]	11 [2–156]	281 [4–8765]
	91	48	87
Vaccine status
PCV13 at least once (n = 5)	165 [63–344]	57 [11–156]	1955 [891–8765]
	100	100	100
PPSV23 only (n = 13)	39^# [4–147]	7^# [2–127]	111^# [4–797]
	92	31	77
None (n = 5)	31 [7–380]	9 [4–67]	450 [34–4129]
	80	40	100

In order from the upper line: GMT (Titer), range, and positive rate (%) of Pn-OPA against each serotype were shown in each cell. PCV13, 13-valent pneumococcal conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine.

^#Significantly lower than those of the patients who had received at least one PCV13 vaccination.

Discussion

Several studies have shown the risk of pneumococcal infection in patients with asplenia;^16–18 however, few studies have evaluated humoral immunity in these patients. Wang et al. reported that the amount of total IgG in patients with asplenia was not less than that in healthy controls.¹⁹ Regarding specific immunity to pneumococci, Rosado et al. showed that Pn-IgGs did not decline after splenectomy in post-splenectomy patients.²⁰ In our study, the positive rates of Pn-IgGs were almost 100% except for serotype 3, even in the 5 non-vaccinated patients, and might have been caused by natural exposure of pneumococcus. However, the GMCs of Pn-IgGs were lower than those of age-matched healthy controls, which were slightly different from the results of previous studies. We speculate this low Pn-IgG might be caused by the loss of splenic function, such as the place of antigen presentation and survival and/or generation of memory B cells.^20,21 This situation is similar to that of other high-risk patients with underlying diseases, as we previously reported.²² We considered that these levels of Pn-IgGs were not always sufficient to protect against IPD, given that the Pn-IgG levels of the infecting serotype in children with IPD were sometimes over the positive cutoff level (≥0.2 µg/mL).²³ Considering their predisposition for pneumococcal infection, immunizations would be important to keep the antibody levels high.

The immune response to pneumococcal vaccines in patients with asplenia and post-splenectomy has been reported in other serological studies. Mikoluc et al. investigated Pn-IgG against PCV7-contained serotypes in children with asplenia and showed that a dose of PCV7 was safe and immunogenic.²⁴ They also showed that the patients who had received a single PPSV23 vaccination approximately 5 years previously did not have sufficient Pn-IgG levels and needed an additional dose of PPSV23 or PCV7, and they speculated this outcome could be due to T cell-independent immune response to PPSV23. Other similar studies have reported that PCV7 revaccination was safe and immunogenic in children with asplenia who were previously vaccinated with PPSV23.^25,26 The immunogenicity of nontypeable Haemophilus influenzae protein D conjugate vaccine and PCV13 has also been reported.^7,27 In our study, the patients who received only PPSV23 had lower GMCs of Pn-IgG than that of the patients who received at least one PCV13 vaccination. Although this result could be influenced by the age distribution of each group, it could also be due to a good T cell-dependent immune response after receiving PCV13 and insufficient antibody persistence after receiving PPSV23 in patients, as the previous studies had reported.

Limited evidences can be found about pneumococcal opsonophagocytic activity in asplenia. Two studies had reported about the immunogenicity of pneumococcal vaccines in asplenia through employing Pn-OPA.^27,28 In both studies, Pn-OPA responses after vaccinations were as good as those in the healthy controls. Our study did not investigate Pn-OPA before and after vaccinations; however, we compared the GMTs of Pn-OPA by the patients’ vaccination status. Although the patients who had never received pneumococcal vaccine had lower GMTs, the difference was not statistically significant (probably because of the small sample size). We compared the GMTs of Pn-OPA in the patients who had received PCV13 with that of the patients who had previously received only PPSV23 (the last dose of which had been administered long ago in most cases), and we found statistically significantly lower GMTs in the patients who had received only PPSV23. Just as in Pn-IgG, this outcome could be due to a good antibody response after receiving PCV13 and insufficient antibody persistence after receiving PPSV23 in asplenic patients. Eighteen of the patients in the study had not received the PCV13 vaccination, because PCV13 was not applied for routine immunizations of patients within the target age (before November 2013), and it was not applied for voluntary immunizations when we collected their study samples (before May 2020). An additional PCV13 vaccination is needed for such patients.

This study has some limitations. First, the sample size was small because there are not many patients with asplenia in a single center; thus, a larger multicenter study is needed. Second, we could not investigate Pn-IgG and Pn-OPA against all vaccine serotypes. We were unable to collect additional samples for our study, and thus performed as much analysis as possible using our limited surplus serum samples. Finally, we did not collect and investigate the patient sample and that of the normal healthy controls simultaneously. Instead, we referred to previous study data on age-matched healthy controls regarding Pn-IgG; however, there are no data on Japanese age-matched healthy controls regarding Pn-OPA.

This is the first study not only about Pn-IgG but also about Pn-OPA in young patients with asplenia in the post-PCV13 era, which we hope will provide valuable evidence regarding immunity.

In conclusion, we have investigated young Japanese patients with asplenia and found that some did not have sufficient vaccine status (more than 5 years had passed from the last PPSV23 dose in most cases), nor did they have sufficient immunity against PCV13 excluding PCV7 serotype pneumococci. These findings suggest the need for young patients with asplenia to receive appropriate pneumococcal vaccinations.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the patients and families involved in the study, as well as the pediatric oncologists and cardiologists who cared for the patients.

Additional information

Funding

This study was supported in part by the Medical Mycology Research Center, Chiba University under joint research funding [Accepted No. 19-9].