Approaches to assess new pneumococcal vaccines for immunogenicity, development and licensure

ABSTRACT

Pneumococcal conjugate vaccines (PCVs) have transformed the prevention of Streptococcus pneumoniae infections, yet challenges such as serotype replacement and vaccine escape demand their continual evaluation for improvement. This review outlines the core approaches used to assess pneumococcal vaccine efficacy, from early-stage development to licensure. The WHO-ELISA is the established method for quantifying serotype-specific IgG levels, while the opsonophagocytic assay assess functional antibody activity and serve as an independent correlate of protection across both vaccine and cross-reactive serotypes. Assessment of antibody avidity and memory B-cell responses offer additional insights into immune quality and durability. Together, these methodologies form a robust framework for evaluating current and next-generation PCVs, supporting evidence-based vaccine licensure, informing public health strategies, and addressing evolving challenges in pneumococcal disease prevention.

Introduction

Streptococcus pneumoniae (the pneumococcus), is a leading cause of bacterial infections worldwide, particularly among young children, the elderly, and immunocompromised individuals. Pneumococcal infections range from mild illnesses, such as otitis media and sinusitis, to severe invasive pneumococcal diseases (IPD), including bacteremia, meningitis, and sepsis.¹ These infections contribute significantly to global morbidity and mortality, particularly in low- and middle-income countries where healthcare access and pneumococcal vaccination coverage may be limited.² The World Health Organization estimates that pneumococci are responsible for more than 300,000 annual deaths among children aged under 5 years, with the highest burden observed in regions such as sub-Saharan Africa and Southeast Asia.^2,3

Pneumococcus was first identified by both Louis Pasteur⁴ and George Sternberg⁵ in 1881, and since then, extensive research has elucidated its pathogenesis, virulence factors, and epidemiology.⁶ As a Gram-positive, facultative anaerobic diplococcus, S. pneumoniae possesses a polysaccharide capsule that serves as a critical virulence factor.⁷ The capsule enables the bacteria to evade host immune responses, particularly through resistance to phagocytosis and complement-mediated opsonic killing. Capsular polysaccharides are antigenic, and more than 100 distinct serotypes have been documented to date.^8,9 However, only a subset of these serotypes is responsible for the majority of pneumococcal infections worldwide, with geographic, age-related, and vaccine-driven variations influencing serotype prevalence. Notably, certain serotypes are more frequently associated with invasive disease, while others exhibit high levels of antimicrobial resistance, posing significant challenges for treatment.¹⁰

Due to the rise of antibiotic-resistant pneumococcal strains, vaccination has become the primary strategy for preventing pneumococcal disease. Two major vaccine types are currently available: pneumococcal polysaccharide vaccines (PPSVs) and pneumococcal conjugate vaccines (PCVs). The 23-valent pneumococcal polysaccharide vaccine (PPSV23) licensed in 1983, consists of purified capsular polysaccharides from 23 serotypes and provides broad serotype-specific protection.¹¹ However, as T-cell – independent antigens, they fail to elicit immunological memory and are poorly immunogenic in infants and young children.^12–14 In contrast, PCVs – first licensed in 2000 with a 7-valent formulation using a nontoxic diphtheria toxin variant (CRM) as a carrier protein, containing polysaccharides covalently linked to protein carriers. This conjugation converts the immune response to a T-cell – dependent pathway, inducing B-cell memory and generating a more robust and durable immune response, especially in infants and young children.^15,16

While PCVs have significantly reduced vaccine-type disease and transmission via herd immunity, serotype replacement remains a persistent challenge.^17,18 To broaden serotype coverage, PCV10 and PCV13 were introduced in 2009,¹⁹ with PCV13 including six additional serotypes (1, 3, 5, 6A, 7F, and 19A).²⁰ In the U.S., PCV15 and PCV20 were licensed for adults in 2021²¹ and for children in 2022²² and 2023,²³ respectively. PCV15 expanded coverage with serotypes 22F and 33F, while PCV20 further added serotypes 8, 10A, 11A, 12F, and 15B. Most recently, PCV21 (Capvaxive) was approved in 2024 as the first PCV developed specifically for adults, incorporating eight serotypes not included in earlier PCVs.²⁴ These vaccines were approved based on noninferior immunogenicity compared to existing formulations.²⁵ Globally, novel PCVs are also being introduced, including Pneumosil – a 10-valent vaccine designed for high-burden regions such as Africa and Asia,^26,27 and three distinct PCV13s used in China.²⁸ A 24-valent PCV (VAX-24) is currently in late-stage developments.²⁹

PCVs have transformed the global landscape of pneumococcal disease prevention, significantly reducing morbidity and mortality. However, the continued expansion of serotype coverage and the introduction of newer formulations call for robust and adaptable frameworks to evaluate vaccine efficacy and effectiveness. This review has outlined the key methodologies employed in assessing vaccine performance, including their respective strengths and limitations. A comprehensive understanding of these evaluation strategies is essential to guide immunization programs, inform policy decisions, and support the rational development of next-generation PCVs tailored to evolving epidemiological needs.

Evaluation of immune responses to pneumococcal vaccines

Although large-scale clinical trials are the definitive method for evaluating vaccine efficacy, they are often difficult to conduct. Therefore, “surrogates” of vaccine efficacy – laboratory-based correlates are commonly used to estimate vaccine efficacy. These include both quantitative measures of antibody levels and qualitative assessments of protective antibody function.^30,31 Table 1 summarizes the key assays used to evaluate immune responses to pneumococcal vaccines. Among the key immunological assays, the World Health Organization-enzyme linked immunosorbent assay (WHO-ELISA) and the opsonophagocytic assay (OPA)/multiplexed opsonophagocytic assay (MOPA) are central to assessing pneumococcal vaccine responses.^32,33

Table 1.

Summary of laboratory assays used to evaluate pneumococcal vaccine immune responses.

Assay	Purpose	Advantages	Limitations	Regulatory status	Applications
Quantitative assays
WHO-ELISA	Quantifies serotype-specific IgG levels	High specificity after CWPS + 22F adsorption; WHO-adopted standard	Single serotype format; labor-intensive; large sample volumes for multivalent PCVs	Gold standard for IgG quantification and PCV licensure in infants34,37,38	Pediatric PCV trials; IgG persistence monitoring
Multiplex ELISA/Luminex (LXA)	High-throughput IgG quantification for ≥ 23 serotypes	Low serum volume; Simultaneous testing of many serotypes	Antigen coupling may alter epitope; Requires validation against WHO-ELISA	Increasingly accepted if cross-validated to WHO-ELISA41	Ideal for large-scale studies of new or high-valency PCVs
Electrochemiluminescence (ECL) Assay	Quantifies serotype-specific IgG levels	High sensitivity and wide dynamic range; Multiplex capability (≥13 serotypes); Minimal serum volume; High throughput	Requires specialized instrumentation (MSD platform); Not yet fully standardized across labs	Validated against WHO-ELISA with strong concordance (≥90% agreement for most serotypes)40	Immunogenicity assessments in pediatric PCV trials; Suitable for high-valency PCV development
Functional assays
MOPA	Measures functional opsonic killing of multiple serotypes	Minimal serum needed; High throughput; Evaluates multiple serotypes simultaneously	Technically complex and require custom-engineered target strains. Requires training.	Required for PCV licensure for both infants23,60 and adults21,25	Used in both infant and adult trials; Differentiates nonfunctional cross-reactive antibodies
Exploratory assays
Chaotropic avidity ELISA	Measures binding strength (avidity) of vaccine-induced IgG	Indicates immune maturation and response quality	No clear correlate of protection; Laborious; More variable than ELISA	Moderate; Considered supportive, not primary31–33	Useful in booster dose studies and vaccine comparisons
Memory B-cell assays (ELISPOT, flow cytometry)	Detects immunological memory (long-term immunity)	Measures persistence of vaccine response; Important for booster policy	No standard cutoff; Technically challenging and resource-intensive	Research tool; Mentioned in WHO guidance as supportive evidence31,32	Long-term immunity evaluation; Booster vaccine studies

PCV – pneumococcal conjugate vaccine; CWPS – cell wall polysaccharide; WHO – World Health Organization; MOPA – multiplexed opsonophagocytic assay; ELISPOT – enzyme-linked immunospot; PBMCs – peripheral blood mononuclear cells.

Quantitative assays

WHO-ELISA: standardized quantification of antibodies

The development of ELISA for detecting pneumococcal IgG antibodies began in the 1980s and 1990s, and has since undergone several refinements.³⁴ First generation ELISA tended to overestimate antibody levels due to cross-reactivity with non-protective antibodies, such as those targeting pneumococcal cell wall polysaccharide (CWPS). Second-generation ELISA addressed this issue by incorporating pre-adsorption of test sera with pneumococcal CWPS, although specificity remained suboptimal. The third-generation WHO-ELISA, significantly improved specificity by including pre-adsorption with CWPS and another capsular polysaccharide, type 22F.³⁴ Initially standardized against reference serum 89SF, WHO-ELISA was later validated with reference serum 007sp.³⁵ Through an international collaboration,³⁵ 007sp was assigned reference values for 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F), and later extended to seven more serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) also present in the PPSV23.³⁶ A detailed training manual for WHO-ELISA is available at https://www.vaccine.uab.edu/uploads/mdocs/ELISAProtocol(007sp).pdf (www.vaccine.uab.edu).

The WHO-ELISA is the established gold standard for quantifying serotype-specific IgG antibodies following pneumococcal vaccination and has been central to immunogenicity studies and vaccine licensure. It was instrumental in defining the 0.35 µg/mL IgG threshold – a widely accepted surrogate of protection against IPD in infants receiving PCV7.³⁷ Although this cutoff has been used across all serotypes in later pediatric trials,³⁸ it may not reflect protection against all serotypes (beyond PCV7 serotypes) or against noninvasive diseases such as otitis media, colonization, or pneumonia. Despite its specificity and reproducibility, WHO-ELISA is labor-intensive, requiring separate runs for each serotype, which limits its practicality for evaluating newer PCVs covering more serotypes. This limitation is especially challenging when sample volumes are constrained, such as in infant studies.³⁹

To address these challenges, multiplexed assays like electrochemiluminescence⁴⁰ and bead-based platforms⁴¹ have been adopted for broader and more efficient serotype-specific antibody quantification. Luminex multiple analyte (LXA) profiling, for instance, enables simultaneous measurement of antibodies against upto 23 serotypes in a single assay,^42,43 conserving sample volume and increasing throughput. Antibody concentrations measured by LXA have shown good correlation with ELISA in adult^43,44 and pediatric^45,46 sera, making it suitable for large-scale studies. However, shifting from ELISA to LXA requires careful validation, as microsphere coupling may alter antigenic structures, potentially impacting antibody binding.⁴⁵ Moreover, serological thresholds established for ELISA may not directly apply to LXA, necessitating serotype-specific standardization across laboratories.^46,47

Functional assays

Opsonophagocytosis assay: assessing functional antibody responses and its role in pneumococcal vaccine licensure

Pneumococcal antibodies confer protection primarily through opsonization of bacteria, which enables subsequent clearance by phagocytes. Unlike WHO-ELISA, which only quantifies serotype-specific IgG concentrations, OPA directly evaluates the opsonic activity of vaccine-induced pneumococcal antibodies by assessing their capacity to mediate bacterial killing by phagocytes.⁴⁸ Based on the WHO criteria,³² demonstration of functional activity of antibodies as measured by OPA is an independent endpoint when evaluating new PCVs for licensing.^31,33 However, evaluating multivalent PCVs using classical/single-plex OPA is labor-intensive, as each serotype must be tested separately, and limited serum volumes in pediatric studies further complicates the process.^49,50 To address these challenges, a “high-throughput” multiplexed opsonophagocytic assay (MOPA) was developed.^48,51,52 MOPA enables simultaneous testing of multiple serotypes using minimal serum, making it ideal for pediatric studies and evaluating multivalent vaccines like PCV15 or PCV21. A detailed protocol of UAB-MOPA is available at https://www.vaccine.uab.edu/uploads/mdocs/UAB-MOPA.pdf (www.vaccine.uab.edu), which has facilitated its worldwide adoption for large-scale pneumococcal vaccine evaluations.

OPA has been used for both children and adults.⁵³ Clinical studies in infants have suggested that an opsonophagocytic titer of ≥ 1:8 correlates with protection against IPD for all serotypes after three doses of PCV7,^37,54 although exact protective titers may vary by serotype, disease syndromes, and population. While measurement of serotype-specific IgG antibodies remains the primary approach for evaluating PCV immunogenicity, studies after PCV7 introduction revealed that anti-pneumococcal IgG levels alone may not reliably predict protection.³⁷ For instance, following the introduction of PCV7, which includes serotype 19F, high IgG levels (≥0.35 μg/mL) were detected against the structurally related but non-vaccine serotype 19A.⁵⁵ However, OPA revealed that these cross-reactive anti-19A antibodies lacked functional activity. Consequently, OPA has since been adopted as a secondary endpoint in pediatric PCV trials,²³ reinforcing its role as a biologically relevant and more direct surrogate of protective immunity.

More recent PCVs (PCV15, PCV20, and PCV21) have been licensed for adults using immunogenicity bridging studies,²⁵ which differ in key aspects from the approaches used in pediatric populations. In older adults, susceptibility to pneumococcal disease can persist despite normal serum anti-pneumococcal IgG levels, suggesting a mismatch between antibody quantity and functional capacity.^56,57 Given this discordance, OPA has been accepted as a more reliable correlate of protection and is now the primary immunogenicity endpoint in adult PCV trials.⁵⁸ Although a titer of 1:64 has been proposed as a useful threshold for adults,⁵⁹ it is based on limited data. Therefore, most adult studies instead compare geometric mean titers between groups using different vaccines or assess the proportion of subjects with ≥ 2- or ≥ 4-fold increases (pre-vaccination to post-vaccination) in OPA titers to evaluate vaccine immunogenicity.⁵³

Regulatory authorities require vaccine manufacturers to demonstrate that new PCVs elicit sufficient functional antibody responses against targeted serotypes. For instance, during the approval process of PCV13 for children, OPA data were used to show that PCV13-induced functional antibodies were non-inferior to those generated by PCV7 for shared serotypes while providing additional protection against six new serotypes, including 19A and 3, which are known for their virulence and vaccine escape potential.⁶⁰ Similarly, the development and licensing of PCV15 (Vaxneuvance),²¹ PCV20 (Prevnar 20)²¹ and PCV21 (Capvaxive)²⁵ for adults relied on OPA results to establish their immunogenicity profiles relative to existing vaccines.

Exploratory assays

Assessment of antibody avidity

Beyond antibody concentration and function, antibody avidity has been described as an important indicator of immune response quality.^32,33 Avidity refers to the binding strength between antibodies and pneumococcal capsular polysaccharides, with higher avidity reflecting immune maturation following vaccination. Avidity is commonly measured using chaotropic ELISA assays, in which antibodies with stronger binding resist dissociation by agents such as ammonium thiocyanate.⁶¹ Although avidity measurements offer qualitative insights into immune responses to different vaccines, they do not appear to be reliable predictors of protective efficacy.^31,62 Nonetheless, studies indicate that higher avidity antibodies are associated with better long-term protection, especially after booster vaccinations.^63,64 However, evidence on the correlation between avidity and OPA remains inconsistent.^59,63,65,66

Assessment of immunological memory

As per WHO, evidence of immunological memory may be useful for evaluation and licensure of new PCVs.^31,32 Long-term protection against pneumococcal disease depends not only on circulating anti-pneumococcal IgG antibodies but also on the development of immunological memory.⁶⁷ Memory B-cells play a key role in sustaining immune responses over time, allowing for rapid antibody production upon subsequent exposure to the pathogen.⁶⁸ Immunological memory can be assessed using flow cytometry or enzyme-linked immunospot (ELISPOT) assays.^69,70 These assays may be relevant for evaluating booster vaccine responses and the durability of protection.

Cross-protection correlates with OPA, not ELISA

Although pneumococcal vaccines primarily elicit serotype-specific immune responses, structural similarities among capsular polysaccharides can induce cross-reactive antibodies against non-vaccine serotypes.^71,72 However, not all cross-reactive antibodies are functionally protective. Cross-reactive antibodies may bind to structurally/biochemically related serotypes but with insufficient avidity to mediate effective opsonophagocytosis.^73,74

While ELISA and OPA results generally tend to align for vaccine serotypes, this concordance diminishes when evaluating non-vaccine cross-reactive serotypes. As described in section 2.2, PCV7-induced IgG titers against serotype 19A, due to cross-reactivity with vaccine serotype 19F, were detectable by ELISA but did not correspond to significant OPA activity, indicating limited functional cross-protection.⁷³ However, OPA immune responses after PCV13 vaccination correlated with IgG responses for both serotypes 19A and 19F.⁷³ Similarly, another study showed that PCV7 vaccination in children induced antibodies against serotype 19A detectable by ELISA, but resulted in limited OPA titers.⁵⁵ Moreover, based on cross-functional OPA responses, studies have shown that PCV13 provides some degree of protection against disease caused by serotype 6C and serotype 7A, due to cross-functional antibodies elicited by the serogroup-related conjugates 6A and 7F, respectively.^72,75

Altogether, the findings reveal that cross-reactive IgG levels alone cannot reliably indicate protective immunity and highlight OPA as the more discriminating assay. Therefore, OPA remains essential not only for evaluating cross-protection but also for guiding formulation decisions and assessing the full breadth of pneumococcal vaccine efficacy.

Operation of the WHO pneumococcal reference laboratory

To facilitate the standardization of serological assays required for the pneumococcal vaccine evaluation,^32,33 the WHO established two pneumococcal reference laboratories in 2001. These reference laboratories are located at the Institute of Child Health in London, UK, and at the Bacterial Respiratory Pathogen Reference Laboratory, The University of Alabama at Birmingham (UAB), Birmingham, AL, USA.

For over two decades, the WHO pneumococcal reference laboratory at UAB has supported global efforts in pneumococcal vaccine evaluation. It has led to the development of WHO-ELISA³⁴ and invention of MOPA,^48,51,52 which is currently the benchmark for quality testing of pneumococcal vaccines across the industry and permits the development of advanced and affordable vaccines. The laboratory supports the global vaccine community by providing validated reference materials, training resources, and disseminating validated assay protocols – which are publicly available at www.vaccine.uab.edu. The laboratory supports national and international collaborators in establishing local serological testing capacity, assay standardization, facilitating immunogenicity assessments, and training scientists. These contributions have enabled consistent and reliable evaluation of pneumococcal vaccine responses and supported the development and licensure of PCVs.

In addition, the WHO pneumococcal reference laboratory at UAB has improved the pneumococcal serotyping through the use of in-house monoclonal antibodies (Mabs).⁷⁶ Leveraging Mabs into a multidisciplinary approach (i.e., genetic, serological, and biochemical), the laboratory has discovered and characterized many novel pneumococcal capsule types,^8,77 thereby informing next-generation vaccine design. For instance, discovery of serotype 6C among “6A” isolates explained that PCV7 induces cross-protection against 6A but not 6C.^77,78

As new pneumococcal vaccines are developed and serotype landscapes shift, the WHO pneumococcal reference laboratory at UAB remains committed to support vaccine studies, public health efforts, and guiding the future of pneumococcal disease prevention.

Biographies

Feroze A. Ganaie is a Scientist at the Department of Medicine, University of Alabama at Birmingham (UAB), primarily focusing on pneumococcal capsular biology and immune responses to the capsules/vaccines. He is involved in studying— glycobiology of pneumococcus to unravel the molecular mechanisms of capsule biosynthesis, characterization and microevolution of novel pneumococcal capsule types. These studies have led to the discovery of several novel pneumococcal capsule types (like, 24C, 36B, 33E, and 20C), including the 100^th capsule type, 10D. Dr. Ganaie has received multiple awards including the “Jeanne Marrazzo Award” for research excellence by UAB and “Young Investigator Award” by International Society of Pneumonia and Pneumococcal Diseases.

Moon H. Nahm is an Endowed Professor-Emeritus in the Department of Medicine at UAB. He has been studying bacterial infections for more than 30 years with the focus on the polysaccharide capsule of Streptococcus pneumoniae and immune response to the capsule. He has studied the diversity of pneumococcal capsules using serologic, genetic and biochemical approaches, which led to the discovery of many new pneumococcal capsule types. His contributions include development of the World Health Organization (WHO) ELISA and invention of a high-throughput functional assay (MOPA) for pneumococcal antibodies. His laboratory has served as the reference laboratory of pneumococcal vaccines and respiratory pathogens for both the WHO and U.S. NIH for more than 20 years. He is a founder of SunFire Biotechnologies. His ongoing research focuses on understanding the molecular mechanism of pneumococcal capsule synthesis. https://en.wikipedia.org/wiki/Moon_Nahm#cite_note-:0-3

Funding Statement

The authors are supported with funding from the National Institutes of Health [NIH AI178586].

Disclosure statement

UAB has Intellectual Property Rights to several reagents, including monoclonal antibodies and MOPA target strains developed in M.H.N.’s laboratory. The authors (F.A.G and M.H.N.) are UAB employees. M.H.N is a founder of SunFire Biotechnologies.

Ethical approval

Preparation of this review manuscript did not involve original studies with human participants; therefore, the requirement of ethical approval was not applicable.