Streptococcus pneumoniae (Spn) remains a leading cause of global morbidity and mortality, responsible for both invasive and non-invasive diseases across diverse populations. While it is a frequent cause of community-acquired pneumonia, its capacity to trigger invasive sequelae like sepsis and meningitis drives its high fatality rates. This pathogenicity results in huge annual mortality figures, particularly in low-to middle-income countries where the burden of infection is exacerbated by limited access to vaccination and healthcare [1]. This high burden is complicated by the fact that Spn possesses over 100 known capsular serotypes, each expressing a structurally and antigenically distinct polysaccharide capsule [2]. While not all serotypes cause disease, the global diversity of pathogenic serotypes presents a major challenge for vaccine development, necessitating vaccines that can provide broad coverage.

In response to this burden, decades of research have culminated in the development of two major classes of vaccines that have reshaped pneumococcal prevention: pneumococcal polysaccharide vaccines (PPVs) and pneumococcal conjugate vaccines (PCVs) [1, 3]. The PPV vaccine, licensed in the United States in 1983, includes 23 serotypes (namely serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) and is recommended for older adults (≥ 65 years) and individuals aged 2–65 with specific medical conditions [4]. PPVs elicit a T-cell-independent immune response, in which the repeating polysaccharide subunits directly stimulate B cells to produce serotype-specific IgG antibodies [4]. These antibodies enhance opsonization and complement activation, facilitating bacterial clearance, and in some cases provide cross-reactive protection against related serotypes with similar capsule structures [3].

Falkenhorst et al. [5] demonstrated that PPVs offer moderate protection against invasive pneumococcal disease (IPD) in adults aged 60 and above, with vaccine efficacy ranging from 45 to 73% across study designs. Similarly, Latifi-Navid et al. [6] confirmed PPV23’s effectiveness in reducing IPD incidence in randomized controlled trials. Their meta-analysis, which included observational studies, found no significant reduction in pneumococcal pneumonia or related mortality [7]. Nevertheless, subsequent evaluations have questioned the impact of PPV23 on non-invasive disease outcomes [8]. Public Health England reported an overall effectiveness of 24% [9], which declined from 48% within two years post-vaccination to only 15% after five years [10]. Protection dropped to as low as 5% in individuals aged 75 years and older [11]. This waning immunity reflects the T-cell-independent mechanism of PPVs, which elicits serotype-specific IgG responses without generating durable immunological memory [11–15]. Moreover, PPV23 has a limited impact on non-invasive pneumonia, remains costly and thus, inaccessible in many low- and middle-income countries [16, 17].

PCVs were engineered by covalently linking capsular polysaccharides (CPSs) to carrier proteins to address the suboptimal immunogenicity of pure polysaccharide vaccines in pediatric populations [18]. This chemical conjugation transforms the immune response from T-cell-independent to T-cell-dependent, enabling the generation of robust immunological memory and long-term protection [18, 19]. PCV7, licensed in 2000–2001 with seven serotypes (4, 6B, 9V, 14, 18C, 19F, 23F), led to more than 70% decline in IPD among children under two years in the United States [20, 21] and an approximately 20% reduction in acute otitis media [22]. Unlike PPVs, PCVs elicit T-cell-dependent responses through polysaccharide-protein conjugation, which enables class switching, affinity maturation, and durable immunological memory [23, 24]. Subsequent expanded formulations PCV10 and PCV13, which include the addition of serotypes 1, 5, 7F, 3, 6A, and 19A [25], reduce nasopharyngeal colonization and induce herd immunity [26]. The selection of serotypes in PCV10 and PCV13 was guided not only by their disease burden but also by their association with antibiotic resistance [27]. Multiple studies have confirmed that these formulations are effective in reducing infections caused by drug-resistant pneumococcal strains [28].

More recently, PCV15 (Vaxneuvance, Merck) and PCV20 (Prevnar 20, Pfizer) extended the vaccine coverage to serotypes 22F, 33F, and seven additional serotypes [29–31], and showed promise in reducing non-invasive illnesses such as otitis media and pneumonia [32]. PCVs remain advantageous in infants [33] and have reduced vaccine-serotype disease in both vaccinated and unvaccinated populations. However, PCVs still cover only a subset of serotypes and face challenges, including serotype replacement through capsular switching [34], genetic recombination that drives resistance and virulence [35], and the indirect promotion of non-vaccine serotypes [36]. Beyond restricted serotype coverage, the production of polysaccharide-based conjugate vaccines presents significant technical and economic challenges [37]. Manufacturing requires costly containment for toxic bacteria, extensive purification steps that result in material losses, and yields heterogeneous products that may include free polysaccharides, which can impair immune responses [38]. The complexity of scaling up production while maintaining consistent vaccine characteristics further increases costs [39]. Consequently, the high manufacturing costs of multivalent PCVs remain a significant barrier to widespread adoption, particularly in low- and middle-income countries [40, 41].

Despite the substantial impact of PPVs and PCVs in reducing IPD, important limitations remain. Both vaccine classes depend on serotype-specific immunity, leaving populations vulnerable to non-vaccine strains and the phenomenon of serotype replacement [42]. The T-cell-independent response elicited by PPVs results in limited immunological memory and waning protection in older adults [9], while PCVs, though effective in infants and young children [10], cover only a subset of circulating serotypes and are associated with high production costs [38]. Moreover, neither vaccine type induces strong mucosal immunity, which is critical for preventing colonization and transmission [43].

Given the limitations of existing pneumococcal vaccine platforms, live attenuated vaccines (LAVs) have emerged as a promising alternative, offering the potential to elicit both systemic and mucosal immune responses, broaden serotype coverage, and thereby reduce the costs of manufacturing. This review aims to synthesize and critically evaluate current evidence on LAVs, highlighting their distinctive immunological advantages, strategies for enhancing safety and genomic stability, and progress toward clinical translation while outlining the challenges and opportunities that will shape their future development.

Pneumococcal vaccine guidelines continue to evolve in response to emerging serotypes, variable effectiveness, and the need to optimize protection across age groups and risk populations. For older adults, immunization strategies have shifted from a primary reliance on PPSV23 to a more integrated framework following the introduction of PCV13 for pediatric and subsequently adult use [40, 41]. In 2014, the Advisory Committee on Immunisation Practices (ACIP) recommended sequential administration of PCV13 and PPSV23 for adults aged ≥ 65 years [42], with dosing intervals refined in 2015 to optimize immunogenicity [43]. The widespread success of pediatric PCV13 programs generated substantial indirect protection for older adults, leading ACIP in 2019 to conclude that routine PCV13 offered only marginal additional benefit for most healthy seniors [44]. Consequently, current guidelines emphasize ‘shared clinical decision-making’, reserving PCV13 primarily for high-risk cohorts rather than the general population aged ≥ 65 years [44]. This policy shift is further supported by immunological evidence linking vaccine-induced antibody concentrations to protection against pneumococcal colonization [45].

Several supplementary factors complicate the interpretation and application of pneumococcal vaccination recommendations. Firstly, the prevalence of pneumococcal disease varies regionally due to differences in surveillance, epidemiology, and PCV implementation, hindering direct geographical comparisons [46]. Secondly, the prevalence of risk factors for IPD, such as smoking, HIV infection, and other comorbidities, also exhibits regional variation, influencing susceptibility to the disease [47]. Additionally, the extent of herd protection and the remaining disease burden can differ significantly across regions. For instance, a cost-effectiveness analysis in South Africa favoured PCV13 over PPV23 for adult immunization in both public and private sectors, including individuals with HIV [48]. Moreover, herd protection remains incomplete in some European countries and South Africa, potentially allowing specific serotypes to cause infections in older children and adults even with existing vaccination programs [49, 50].

Pelton et al. [51] demonstrated that comorbidities and advanced age diminish the effectiveness of childhood PCV in older US adults, underscoring the need for updated adult recommendations with PCV15 and PCV20. Debate persists over whether adult vaccination should rely primarily on herd immunity from childhood programs or incorporate newer conjugate vaccines directly [52–55]. Additionally, key considerations in this debate include the persistence of vaccine-type diseases and the growing challenge posed by emerging non-vaccine serotypes [52, 53]. The residual disease burden and the degree of herd protection also vary across regions [46]; while serotype replacement is a notable concern in Europe and other parts of the world, it is not currently regarded as a significant issue in the United States [56]. Additionally, environmental factors play a significant role in shaping vaccine responses and influencing the overall burden of pneumococcal disease [56].

These challenges underscore the importance of robust surveillance and the exploration of serotype-independent strategies. Novel approaches based on conserved pneumococcal protein antigens [57–61] and LAVs [60–62] represent pivotal research directions, aiming to deliver broad and durable protection across diverse populations.

LAVs elicit stronger and more durable immune responses than conventional formulations. Early pneumococcal LAV candidates have shown the ability to reduce colonization and protect against lethal challenges across serotypes [63]. These genetically modified strains, rendered safe by deletion of pathogenic genes, provide broader antigen exposure and superior mucosal immunity compared to standard polysaccharide or conjugate vaccines [64]. To facilitate direct comparison across studies, Table 1 summarizes the attenuation strategies, protection outcomes, preclinical findings, and limitations with recommendations.

An early effort by Ogunniyi et al. [65] examined the colonization and attenuation of ply, pspA, and pspC with single, double, and triple knockout variants. The colonization ability of single and double mutations was lower compared to the wild-type strain. However, the triple mutations exhibited colonization potential similar to the wild-type strain. According to Ogunniyi et al. [65], the pspA knockout showed significant differences from the pspC and ply knockouts in single knockouts and provides a compelling rationale for further enhancing the current reservoir of information about the involvement of characterized pneumococcal virulence proteins in both colonization and systemic disease produced by Spn. Additionally, the authors emphasized that the critical complexity of the dynamics of these processes requires further investigation. Regardless of the mutated genes, it appears that the colonization potential of the mutants remained variable [65].

A subsequent in-depth study was conducted by Roche et al. [63], who created Spn mutants lacking the CPS and/or key virulence factors Ply and PspA. The authors generated mutants significantly attenuated for disease transmission but capable of colonizing sufficiently to produce protective immunity by eliminating these determinants alone or in combination, as previously identified pneumococcal virulence factors [66, 67]. Their findings revealed that single mutants varied in attenuation, and combining deletions of virulence factors proved more promising. Interestingly, colonization density tests showed that while capsule-deficient and wild-type strains initially had lower colonization, mutants in the ply and pspA genes showed increased colonization later. Crucially, double mutants lacking CPS and Ply, or Ply and PspA, induced significant mucosal and systemic protection against the same and different pneumococcal strains. However, the protection also depended on both antibodies and CD4⁺ T cells, highlighting the potential of these rationally attenuated strains to elicit a comprehensive immune response [63].

Knocking out the caxP gene (encoding a calcium/magnesium transporter) and the ftsY gene (involved in protein transport) disrupts nutrient uptake and proper protein delivery, hindering bacterial colonization and invasive illness [68, 69], resulting in similar levels of attenuation but differing colonization capacities across serotypes. Rosch et al. [70] found that ftsY mutants exhibited prolonged colonization in the upper nasopharynx and enhanced expression of cbpA and pspA, while caxP mutants were rapidly cleared and did not cause invasive disease. Notably, the ftsY mutant demonstrated superior protection against otitis media and sinusitis in mice compared to heat-killed whole-cell pneumococci, highlighting that even with prolonged colonization, specific attenuating mutations can still elicit a strong protective immune response [70].

Kim et al. [71] established the role of pneumococcal LytA in autolysis and identified Pep27 as a LytA-dependent inducer of this autolysis process. The Spn pep27 knockout mutant conferred protection against lethal pneumococcal challenge in mice, suggesting a potential comparable to CPS mutants in antibody production and resistance. However, Kim et al. [71] noted a key difference: the pep27 mutant’s rapid clearance from the nasopharynx, potentially due to its propensity for aggregation, which hinders mucus survival and may contribute to its lower virulence. Further work demonstrated the pep27 mutant’s inability to adhere to or colonize key organs. Intranasal immunization without adjuvant provided long-lasting protection against heterologous strains, even preventing secondary colonization [72]. Nevertheless, the erythromycin resistance gene that was introduced in the pep27 mutant could be of concern as it may be passed to other commensal microbes in the nasopharynx through immunization [71].

Recognizing a safety concern with the erythromycin resistance gene in their initial mutant, Choi et al. [73] developed a marker-free pep27 mutant. Their findings were similar to those previously reported by Kim et al. [71], confirming the inactivated pep27 mutant’s ability to protect against a lethal challenge and elicit cross-reactive antisera, highlighting its rapid clearance within 48 hours and the induction of adjuvant-independent mucosal immunity. Additionally, vaccination against secondary pneumococcal infections was conferred by the pep27 mutant pneumococcus (designated Δpep27) [71], providing compelling evidence for the efficacy of the mutant as a potentially safe and effective mucosal vaccine strategy, with the marker-free approach used by Choi et al. [73] directly addressing a key safety hurdle for potential clinical translation.

Building upon their previous research, Kim et al. [71] refined their Δpep27 live attenuated pneumococcal vaccine candidate by deleting the comD gene to prevent reversion to virulence [72]. This double mutant (designated Δpep27ΔcomD) enhanced safety in healthy and immunocompromised mice while eliciting significant IgG responses against Spn serotype D39 and PspA-specific antibodies [71, 72, 74]. Immunization with this double mutant provided substantial protection (> 80% survival) against challenge with serotypes D39 and 6B, though sustained protection against the 6B strain remained limited [71, 72, 74]. The study suggests the Δpep27ΔcomD strain holds promise as a safe and effective pneumococcal vaccine, warranting further investigation into its efficacy against type 6B in humans and the impact of the host environment and nasopharyngeal microbiota on challenge strain dynamics [75, 76]. The robust long-term immunity offered by current commercial vaccines underscores the potential value of this double-mutant approach if these limitations can be addressed.

Ibrahim et al. [77] investigated the potential of a live-attenuated Spn strain deficient in the HtrA as a vaccine candidate. Deleting the htrA gene, which is essential for bacterial survival under environmental stresses such as oxidative stress and high temperatures [78–81], resulted in a significantly attenuated mutant. The rationale was that this attenuated strain, capable of nasopharyngeal colonization, could induce protective immunity. Indeed, intranasal immunization with the htrA-deficient strain elicited enhanced specific humoral immune responses at both systemic and mucosal levels [75]. Furthermore, immunized mice exhibited substantial protection against nasopharyngeal colonization and challenge with multiple pneumococcal strains at both mucosal and systemic sites [77, 81], highlighting the potential of targeting stress response pathways for live attenuation and offering a strategy to induce broad protection by leveraging mucosal immunity.

Another live attenuated pneumococcal vaccine development strategy involves deleting genes encoding essential proteins. Wu et al. [82] created the capsule-negative Spn strain SPY1 by deleting the SPD_1672 gene, which rendered defects in three key virulence factors: the capsule, teichoic acids, and pneumolysin [83]. Intranasal immunization with SPY1 protected mice against colonization and lethal challenge [82, 84]. Subsequent research demonstrated that mucosal and systemic immunization with SPY1 elicited protective humoral and cellular responses, highlighting its potential as a candidate vaccine [81]. SPY1 induced detectable cytokine levels in mice, including IFN-γ, IL-17A, IL-10, and IL-4, but the specific roles of different CD4⁺ T cell subtypes in SPY1-mediated protection remained under investigation [85, 86]. This approach of targeting essential genes for attenuation, exemplified by SPY1, offers a promising avenue for developing LAVs that elicit broad protective immunity.

Xu et al. [87] further elucidated the protective mechanisms elicited by the SPY1 candidate LAV using an immunodeficient mouse model. Their findings revealed a collaborative role between T-cell and humoral immunity in mediating vaccine-specific protection. Specifically, protection against lethal pneumococcal challenge and colonization was attributed to Th2 immunological subsets and Th17-mediated phagocyte recruitment. Experiments in B-cell-deficient mice demonstrated the necessity of antibody-mediated immunity for the protective effects afforded by SPY1. Furthermore, studies in T cell-deficient mice highlighted the crucial role of T cell-mediated immunity, particularly the IL-17 response, in protecting SPY1-vaccinated mice [87]. The vaccine-specific Th17 cells facilitated neutrophil recruitment and activation against colonization, indicating that a Th2 response was required for protection against a lethal challenge. Notably, T regulatory (Treg) cells were found to counteract both pneumococcal colonization and lethal challenge. Consequently, Xu et al. [87] emphasized the importance of designing novel pneumococcal vaccines that can induce protective Treg cell responses, as strong Th1 or Th17 responses without Treg cell balance could lead to detrimental pathological consequences during both disease and colonization.

Jang et al. [88] adopted a different live attenuation strategy by deleting the lgt gene, responsible for lipoprotein synthesis, in the Spn strain TIGR4, which is crucial for various bacterial processes, including virulence and immune evasion. The TIGR4Δlgt mutant exhibited reduced virulence and inflammatory activity. Nasopharyngeal colonization with this strain in mice elicited robust mucosal IgA and systemic IgG2b-dominant antibody responses that showed cross-reactivity against other pneumococcal serotypes. Intranasal immunization with TIGR4Δlgt conferred protection against pneumococcal infection and challenge with heterologous strains. This immunogenicity profile suggests that targeting lipoprotein synthesis for live attenuation can generate a broad-spectrum pneumococcal vaccine candidate by inducing cross-reactive immunity.

Recent surveillance highlights Spn serotype 1 as a significant cause of invasive disease in sub-Saharan Africa, despite its infrequent presence in asymptomatic individuals [89]. Terra et al. [90] engineered a serotype 1 strain (519/43Δply) with a specific mutation inactivating the ply gene, thereby abolishing its haemolytic activity. While intraperitoneal immunization of mice with this mutant reduced bacterial load in the bloodstream following challenge, it did not confer complete protection against pneumonia, suggesting that the ply gene in this serotype 1 strain may be a virulence factor with limited impact. The authors advocate for continued research to better understand the transformability of various serotype 1 strains, enabling the targeted genetic manipulation of relevant genes [90].

Our research group engineered a live attenuated pneumococcal vaccine candidate by creating a double mutant of Spn serotype 2 (strain D39) lacking genes for cpsE and endA. Mutation of cpsE is known to abolish capsule formation, reduce virulence, and enhance host immune responses [91]. Deletion of endA, strongly associated with natural transformation, aimed to minimize the risk of cpsE gene reintegration and capsule restoration [91]. Immunization of mice with this ΔcpsEΔendA double mutant resulted in a 56% survival rate following intranasal challenge, comparable to the 50% survival observed in mice challenged with the wild-type strain. Furthermore, mice immunized with the double mutant showed higher serum levels of D39-specific IgG and IgM antibodies than those exposed to the wild-type [91]. This dual-gene deletion strategy demonstrates a promising approach for live attenuation by combining reduced virulence and enhanced immunogenicity with a potential safety mechanism against reversion.

In another study, Ramos-Sevillano et al. [92] investigated the protective effects of nasal colonization in mice using two Spn serotype 6B mutant strains with disrupted cps genes, consistent with previous findings. Importantly, each strain also carried an additional mutation in either pspA or proABC, both of which are established pneumococcal virulence factors. Following a two-dose intranasal colonization regimen, the mice developed increased serum IgG antibodies against Spn antigens at lower levels than those observed in wild-type mice. While this colonization provided resistance against septicaemia, it failed to prevent subsequent recolonization by the wild-type strain [92, 93]. This study highlights the potential of using cps-deficient mutants to induce systemic protection. However, it suggests that additional modifications or strategies may be needed to achieve robust and persistent protection against colonization.

Building upon the promising safety and immunogenicity profiles demonstrated in numerous preclinical models, the development of pneumococcal LAVs has recently achieved a significant milestone. The first randomized controlled clinical trial was conducted by Hill et al. [94] in response to the discrepancy in their previous preclinical data, as reported by Ramos-Sevillano et al. [92]. Earlier research demonstrated that two double-mutant Spn strains (ΔproABC/piaA and Δfhs/piaA), which had mutations affecting virulence-related metabolic functions, were suitable vaccine candidates. In a mouse model, these strains induced significant protective immunity against subsequent colonization, pneumonia, and sepsis from the homologous wild-type strain [93]. Hill et al. [94] reported that 148 participants were randomly assigned to receive nasal inoculation of either wild-type Spn 6B (BHN418 strain, SpnWT), one of two specific double mutant attenuated strains [SpnA1 (Δfhs/piaA) or SpnA3 (ΔproABC/piaA)], and immune responses were monitored through regular nasal wash and blood sample collection. The Experimental Human Pneumococcal Challenge (EHPC) model was used to test whether the nasopharyngeal administration of the Spn6B Δfhs/piaA or Spn6B ΔproABC/piaA strain could prevent subsequent recolonization with wild-type Spn [94].

Collectively, these studies highlight the potential of LAVs in eliciting broad and durable immune responses, encompassing both systemic and mucosal protection. Preclinical work has provided valuable insights into genetic attenuation strategies and their immunological effects, though comparisons with established conjugate vaccines in real-world settings remain limited. The evidence highlights the need to refine attenuation approaches to ensure safety, stability, and broad-spectrum coverage. Building on this summary, the following section examines specific strategies deemed most promising, explaining why prior approaches introduced by different researchers are sensible and hold strong scientific potential.

Recent research has highlighted several promising approaches that not only enhance immunogenicity but also address critical concerns regarding immune-protective mechanisms induced by LAV, genetic stability, safety, and clinical applicability. These strategies include rational attenuation through multi-gene deletion or irreversible genomic modifications, optimization of immune-protective mechanisms to achieve broad-spectrum and durable responses, and careful evaluation of delivery routes and safety profiles in early human trials. Together, these advances provide a framework for developing next-generation LAVs, and the following subsections discuss each strategy in detail, supported by relevant scientific evidence from previous studies.

Despite encouraging progress in pneumococcal LAV development, several interrelated challenges continue to constrain their translation into clinical practice. A critical synthesis of current evidence highlights five key domains that collectively define the future research agenda. First, safety profiles must be rigorously evaluated across diverse demographics to ensure tolerability and public confidence. Second, the variability in colonization outcomes and the absence of reliable immunological correlates of protection necessitate a deeper investigation into host and pathogen factors to better predict vaccine efficacy. Third, advancements in human challenge models offer opportunities to accelerate development; however, their inherent limitations, such as ethical considerations and the difficulty in mimicking natural disease progression, must be carefully addressed. Fourth, precision genome editing technologies like CRISPR provide novel attenuation strategies; yet, their potential is tempered by safety, regulatory, and ethical considerations. Finally, addressing concerns related to vaccine stability, manufacturing standards, and securing sustainable funding remains essential for ensuring the global feasibility and long-term implementation of pneumococcal LAVs. By critically assessing current evidence and highlighting these five priorities, this section provides a roadmap for advancing pneumococcal LAVs toward clinical reality.

The past two decades have firmly established the tantalizing promise of pneumococcal LAVs as a promising strategy to address the limitations of current vaccine options, offering the potential for improved efficacy and cost-effectiveness. Advances in attenuation methodologies, the identification of promising knockout mutants, and refined techniques for introducing multiple genetic modifications have significantly strengthened the scientific foundation for LAV development. Importantly, foundational preclinical work, now complemented by emerging clinical trial data, offers critical insights into the safety and efficacy of LAVs, signalling considerable progress in overcoming challenges related to attenuation safety, manufacturing, and the induction of broad immune protection. Despite this promising trajectory, significant challenges persist that must be actively addressed to ensure feasibility and scalability. These include optimizing manufacturing processes, securing sustainable funding, and refining predictive models of the immune response to guarantee consistent protection. On a primary impact level, ensuring robust safety across diverse populations and achieving durable, population-wide protection remain the pivotal hurdles that will ultimately determine the success of LAVs. The final realization of pneumococcal LAVs will depend on a collaborative and integrated effort among researchers, clinicians, industry partners, and key global health stakeholders. By overcoming these obstacles, LAVs are poised to play a crucial role in reducing the global burden of pneumococcal disease, providing effective and affordable protection, particularly in low-resource settings where current vaccine strategies are often insufficient.