Assessment of naturally acquired IgG antibodies to pneumococcal vaccine serotypes in healthy and type 2 diabetic adults in India

Authors: Mettingal Ramakrishnan Shincy, Manheri Mavupadi Akhila, Govindan Vandana, Nagaraj Geetha, Vani Rajashekaraiah, Kadahalli Lingegowda Ravikumar

Abstract

Purpose

Streptococcus pneumoniae is a leading global cause of morbidity and mortality, particularly in individuals with type 2 diabetes, who are at increased risk due to altered immunity. Vaccination is crucial for preventing pneumococcal disease in this population; however, its effectiveness depends on sufficient baseline immunity and the ability to generate protective antibodies.

Materials and Methods

This study aimed to measure baseline immunoglobulin G (IgG) antibody levels against 23 vaccine serotypes in 56 type 2 diabetic and 56 healthy Indian adults using the World Health Organization-enzyme-linked immunosorbent assay protocol.

Results

Protective IgG levels (≥1.3 µg/mL) were observed for 78% of serotypes (18/23) in healthy adults but only 35% (8/23) in diabetics. Significant differences were noted in 13 of 23 serotypes (56.2%), including 1, 6B, 7F (p=0.008), 8 (p=0.01), 9V, 11A (p=0.009), 12F, 17F, 18C (p=0.002), 19A (p=0.0006), 19F (p=0.004), 22F (p=0.0003), and 33F (p=0.04). Serotype 14 showed the highest IgG levels, while serotype 3 had the lowest. Seroprevalence ranged from 17.8% to 98.21% in non-diabetics and 10.7% to 96.4% in diabetics.

Conclusion

The findings highlight differences in baseline immunity and provide insights into pneumococcal immunogenicity in Indian adults with and without diabetes.

INTRODUCTION

Type 2 diabetes is a chronic condition, associated with immune system deficiencies, causing a global burden in the perspectives of clinical medicine and public health [1]. Among these, Streptococcus pneumoniae infections, which can lead to pneumonia, meningitis, and bacteremia, are of major concern [2]. Substantial evidence indicates that the risk ratio of pneumonia-related hospitalization increases by 25%–75% in individuals with diabetes [3]. Pneumococcal carriage has been found to stimulate an immune response that protects against further colonization and infection by S. pneumoniae [4]. However, protective immunity against these infections is mediated through naturally acquired antibodies, primarily of the immunoglobulin G (IgG) class, which recognize and neutralize serotype-specific capsular polysaccharides (cPS) [5]. The levels of these antibodies in different populations are crucial for evaluating susceptibility to pneumococcal infections and determining the need for vaccination.

The International Diabetes Federation reports a rapid escalation in the severity of diabetes. Currently, 537 million people aged 20–79 are living with diabetes, while about 90% of them with type 2 diabetes [6]. An estimated 77 million adults above the age of 18 in India have type 2 diabetes, which increases their risk of pneumonia-related hospitalization by 1.2 times [7]. Diabetes is correlated with various immune system dysfunctions, including impaired phagocytic activity, reduced cytokine production, and poor antibody responses, all of which contribute to the heightened risk of pneumococcal infections [8]. The pneumococcal vaccines (pneumococcal conjugate vaccine-PCV, pneumococcal polysaccharide vaccine-PPSV) are crucial for preventing pneumonia in diabetic individuals and other high-risk groups [910]. The American Diabetes Association’s 2024 report recommends that individuals aged 19 and older receive one dose of either PCV20 or PCV15, succeeded by a dose of PPSV23 at least after 1 year [11]. In India, a single dose of PCV13 followed by PPSV23 ≥8 weeks later is recommended for individuals with diabetes mellitus [12].

The baseline levels of pneumococcal antibodies have important implications for naturally acquired immunity, vaccine design, and evaluation. Establishing protective pneumococcal IgG levels, however, poses challenges due to differences between post-immunization and naturally attained antibodies, as well as factors such as lifestyle, ethnicity, and immune condition [13]. Naturally acquired IgG antibodies to S. pneumoniae serotypes can develop following asymptomatic carriage or infection with the bacteria [14]. These antibodies are crucial for immune protection, as they facilitate the recognition and clearance of pneumococcal bacteria through opsonization, neutralization, and promotion of phagocytosis [15]. In healthy adults, exposure to S. pneumoniae serotypes through natural infection often leads to developing protective antibody levels. However, in people with diabetes, the capacity to generate and maintain these protective antibodies may be compromised due to chronic hyperglycemia and other immune-related complications associated with diabetes [1]. This can result in a reduced ability to respond effectively to infections or vaccinations, placing diabetic individuals at a higher vulnerability to invasive pneumococcal disease (IPD).

The fold-rise in antibody levels compared to pre-vaccination levels is used to evaluate the immune response to pneumococcal vaccinations [16]. While there are no universally accepted baseline antibody cut-offs to definitively measure protective antibody responses in adults, it is generally suggested that an antibody level of at least 1.3 µg/mL against approximately 70% or more of the tested serotypes indicates protection in this population [171819]. The primary objective of this pilot study was to assess baseline concentrations of IgG antibodies against 23 pneumococcal vaccine serotypes in healthy and type 2 diabetic Indian adults, given the limited data on naturally acquired IgG titers in India. This study provides valuable insight into the immune status of these populations by comparing antibody profiles of healthy and diabetic adults to identify potential disparities in immune protection and their implications for pneumococcal vaccination strategies in India.

MATERIALS AND METHODS

Study participants

The retrospective study was conducted at the National Unit of Vaccine Immunogenicity Evaluation and Research, Central Research Laboratory, Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India, between 2021 and 2022. A total of 112 participants aged 35–65 years were included, comprising 56 type 2 diabetic adults and 56 non-diabetic adults. Eligibility criteria included individuals aged >18 years, without prior history of pneumococcal polysaccharide or conjugate vaccination, and without any recent or ongoing coronavirus infection.

Sample collection

Approximately 5 mL of blood sample was taken from each enrolled subject for analysis. The collected samples were centrifuged at 5,000 rpm for 10 minutes, and serum was stored at −80°C until further analysis.

Study materials

The standard human anti-pneumococcal reference serum, 007sp was sourced from the US Food and Drug Administration. The 23 serotype-specific polysaccharide antigens (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell wall polysaccharide (CWPS)-multi was obtained from Statens Serum Institut (SSI, Copenhagen, Denmark) and used as an absorbent to neutralize nonspecific reactivity. Pneumococcal quality control serum 96/772 (NIBSC 12/278) was obtained from the National Institute for Biological Standards and Control, Hertfordshire, UK, and used as control [20].

Serotype-specific IgG detection by enzyme-linked immunosorbent assay (ELISA)

[INSERT FIGURE 001]IgG antibodies to 23 S. pneumoniae serotypes were quantified by the ELISA according to the WHO Training Manual (http://www.vaccine.uab) [21]. Briefly, medium-binding 96-well microtiter plates (Greiner Bio-One-655001; Greiner Bio-One GmbH, Frickenhausen, Germany) were coated with the serotype-specific pneumococcal polysaccharide antigen at 5 µg/mL concentration in 1× coating buffer and incubated at 37°C for 5 hours in a humidified chamber. The serum samples, standard serum (007sp), and QC serum were pre-diluted and double pre-absorbed by the absorption solution containing 5µg/mL CWPS and 5 µg/mL 22F capsular PS or 10 µg/mL Multi-C-PS in antibody buffer (1× phosphate-buffered saline/0.2% NaN3/0.5% Tween-20). The pre-absorbed sera, QC sera, and standard serum (007sp), were diluted using a 2.5-fold serial dilution in an absorption solution. Subsequently, the diluted samples were incubated for 30 minutes at room temperature. The antigen-coated microtiter plates were washed five times using a Tris-buffered solution containing 0.1% Brij™ 35. Then, 50 µL of adsorption solution-depleted sera were transferred to a coated micro-titre plate and incubated for 2 hours at room temperature. After washing the microtiter plate, 100 µL of alkaline phosphatase conjugate anti-human goat IgG antibody (SouthernBiotech, Homewood, AL, USA) was added at 5,000 dilutions and incubated for 2 hours at room temperature. After another washing procedure, 100 µL of 1 mg/mL concentration of p-nitrophenyl phosphate (sigma) in the diethanolamine substrate buffer, pH 9.8, was added to all wells and then incubated for 30 minutes at dark. The 50 µL of 3 M NaOH was added to all wells to stop the enzyme reaction. Read the plates at 405 and 690 nm with a microplate reader (Spectramax ABS Plus; Molecular Devices, San Jose, CA, USA).

Ethical approval

The study protocol was approved by the Institutional Review Board (IRB) of KIMS (approval KIMSIEC/S04-2020). All individuals consented to respective IRB protocols before sample collection.

Statistical analysis

The Statistical analysis was conducted using GraphPad Prism 8.4.2 software (GraphPad Software Inc., San Diego, CA, USA). The antibody concentration was found to be normally distributed and symmetric. Comparisons within/between the groups were performed using the paired/unpaired t-test. Antibody concentration was reported as the geometric mean concentration/titer along with the 95% confidence interval. The concentration of cPS-specific IgG antibodies was determined using SoftMax Pro 7.1.1, with the ELISA standard curve generated through a 4-parameter logistic model. Seroprevalence was defined as the proportion of participants with serotype-specific IgG antibody concentrations ≥1.3 µg/mL. For each analysis, a p-value of <0.05 was considered statistically significant.

RESULTS

Participant demographics

A total of 112 adults participated in this study (56 with type 2 diabetes and 56 without). The median age (years) of these participants was 50 (35–66). Females represented 51% (n=57), while males were 49% (n=55) of the total recruited participants (p=0.500) (Table 1).

Baseline IgG antibody levels

At baseline, only 35% (8/23) of pneumococcal serotypes in adults with type 2 diabetes had protective IgG antibody levels (≥1.3 µg/mL), compared to 78.2% (18/23) in non-diabetic participants. The baseline anti-pneumococcal IgG concentrations were generally higher in individuals without diabetes. A significant difference in IgG levels was observed for 13 1, 6B, 7F, 8, 9V, 11A, 12F, 17F, 18C, 19A, 19F, 22F, and 33F between the 2 groups (p<0.05) (Fig. 1). The highest baseline cPS-specific IgG titers were observed against type 14 (geometric mean concentration [GMC], 8.67 μg/mL), followed by type 19A (GMC, 6.19 μg/mL) and type 33F (GMC, 4.6 μg/mL) in non-diabetic individuals. The lowest levels were against types 3, 4, and 5 (GMC, 0.51, 0.89, and 0.95 μg/mL, respectively). For individuals with type 2 diabetes, the highest baseline IgG levels were also against type 14 (GMC, 8.15 μg/mL), followed by types 33F and 19A (GMC, 2.21 μg/mL for both). The lowest levels were against types 3 (0.41 μg/mL), 5 (0.57 μg/mL), and 4 (0.64 μg/mL) (Table 2).

Gender-based serotype-specific IgG levels

We observed significant differences in anti-capsular IgG titers between males and females. Among non-diabetic individuals, significant differences were noted for serotypes 2, 3,11A, 12F, and 17F (p<0.05) (Fig. 2A). In diabetic individuals, only serotype 14 showed a significant gender-based difference (p<0.05) (Fig. 2B). Generally, females exhibited higher IgG levels than males across both study groups.

Seroprevalence rates

Non-diabetic individuals achieved higher seroprevalence rates (IgG ≥1.3 μg/mL) across most serotypes. For instance, 98.21% (n=55) of non-diabetics had protective levels for serotype 14, compared to 96.43% (n=54) of diabetics. Serotype 3 showed the lowest 17.86% (n=10) in non-diabetics and 10.71% (n=6) in diabetics. Overall, individuals without diabetes had higher seroprevalence than those with diabetes (p=0.75), except for serotype 4 (Table 3).

DISCUSSION

This pilot study represents the first systematic clinical investigation conducted in India to assess baseline IgG antibody levels in adults with type 2 diabetes compared to non-diabetic individuals, targeting the 23 pneumococcal capsular polysaccharides included in the PPSV23 vaccine. Given that individuals with diabetes are more susceptible to respiratory tract infections, this study addresses the dual burden of pneumonia and diabetes [22]. The demographic profile of participants—primarily middle-aged adults—aligns with the typical onset age for type 2 diabetes and reflects the heightened risk of pneumococcal infections in this population [23].

Protection against pneumococcal carriage and invasive infections fundamentally depends on the robust generation of protective antibody titers targeting pneumococcal capsular polysaccharides [5]. These antibodies play a critical role in the immune response, ensuring effective pathogen clearance and safeguarding against disease [1524]. In our study, naturally acquired protective antibody levels for pneumococcal serotypes were higher in healthy adults compared to those with diabetes highlighting the compromised immune response in diabetic individuals. These findings align with those reported in previous studies by Ahmad et al. [4], which also indicated that diabetic individuals exhibited diminished antibody responses compared to healthy individuals. The baseline anti-pneumococcal IgG concentrations were generally higher in individuals without diabetes. The lower baseline protective IgG antibody levels in type 2 diabetic adults compared to non-diabetic adults can be correlated with the immune dysregulation commonly associated with diabetes [25]. Chronic hyperglycemia in type 2 diabetes adversely affects multiple components of the immune system, leading to impairments in both innate and adaptive immune mechanisms [26]. This dysregulation results in reduced phagocytic activity, compromised leukocyte function, and altered cytokine production, collectively diminishing the body’s ability to react to pathogens effectively [27]. As a result, people with type 2 diabetes may exhibit a weakened immune response, making them more vulnerable to infections and less capable of achieving adequate antibody levels following vaccination or exposure to pathogens [25].

Although titers to all serotypes were comparable between individuals with and without diabetes, we observed a higher titer to capsule type 14, while the lowest IgG titers were found against capsule type 3 in both study groups. A low level of baseline IgG concentration against serotype 3 may be due to its poor immunogenicity or its more frequently associated invasive disease rather than nasopharyngeal carriage [28]. Analog to our study, they unveiled that serotype 14 has the highest antibody titer [1]. This evidence supports that frequent episodes of nasopharyngeal colonization may contribute to increased anti-pneumococcal antibody concentrations [29].

Gender and age significantly influence the immune system and can alter the immune response to vaccination. Numerous studies have highlighted the influence of gender and age on responses to pneumococcal vaccines; however, the results have remained unchanged, and antibody concentration has increased or decreased with age [3031]. We observed a considerable difference in anti-capsular IgG titers between males and females. In general, females exhibited higher IgG levels compared to males across both study groups, which is concordant with Gaultier et al. [14]. Other studies have similarly reported that IgG levels are higher in females compared to men. Additionally, studies have indicated that males have higher baseline antibody concentrations than females [32]. Consistent with our findings, previous studies also reported that females may be associated with low immune response [33]. Higher IgG levels in females than in males may be because of estrogen immune-boosting effects, the presence of 2 X chromosomes carrying immune-related genes, and generally higher immune responses in females, potentially linked to evolutionary adaptations for pregnancy. In contrast, testosterone in males may have an immunosuppressive effect contributing to observed sex-based differences in IgG levels [3435].

The baseline seroprevalence rates noted in our study further emphasize the immunogenic disparity among the 2 groups. Individuals without diabetes had higher seroprevalence rates than those with diabetes (p=0.75), except for serotype 4. These findings resonate with previous research that indicated reduced seroprevalence rates in diabetic populations [36]. Although the protective threshold is serotype-dependent and may differ for naturally acquired antibodies compared to post-vaccination antibodies. It can be hypothesized that the reduced antibody titer observed in diabetes may result from hyperglycemic conditions, which lead to increased antibody glycation [37]. This may impair the antibodies’ ability to effectively bind to antigens, thus reducing their overall efficacy [38].

In conclusion, this pilot study provides insights into the baseline IgG antibody titers to pneumococcal vaccine serotypes in Indian adults, those with and without type 2 diabetes. Our findings highlight the compromised immune response in diabetic individuals, with lower protective antibody levels observed than in non-diabetics. This disparity underscores the heightened vulnerability of those with diabetes to pneumococcal infections, emphasizing the critical need for effective vaccination strategies tailored to high-risk populations.

Looking ahead, we plan to conduct a vaccine efficacy study to evaluate further the immunogenicity and protection offered by the pneumococcal vaccines in this population.

This study is limited by its single-center design and modest sample size, which may restrict the broader applicability. We measured total IgG antibodies using ELISA, further assessments using the opsonophagocytic activity assay are needed to evaluate functional activity. Considering the dual burden of diabetes and respiratory infections, public health policies should prioritize pneumococcal vaccination for diabetic adults, ultimately aiming to reduce the incidence of pneumonia and improve health outcomes in this vulnerable group.