From:  Pneumococcal live attenuated vaccine: strategies, challenges and future perspectives

 Pneumococcal live attenuated vaccine candidates: a comprehensive overview of attenuation strategies, animal models, protection, key findings, limitations, and recommendations.

Attenuation strategy/Strain backgroundAnimal model and immunizationProtectionKey findingsLimitations and recommendationsReference(s)
Single and combined mutations of the Δply, ΔpspA, and ΔpspC (cbpA) genes (serotype 2).

  • Mouse model: CD1 and CBA/N mice.

  • Immunization: 107 CFU.

  • Challenge: 106 CFU via i.p. and 107 CFU via i.n. route.

Reduced colonization in single and double mutants; triple mutant comparable to WT.The single pspA knockout showed distinct effects compared with pspC and ply knockouts.The pspA mutant is partially attenuated, retaining the ability to colonize and cause lung infection/bacteremia. Further studies are needed to clarify how pneumococcal virulence proteins contribute to colonization and systemic disease.Ogunniyi et al., 2007 [65]
Targeted mutations in pspA, ply, and the cps locus (D39, type 4, 6A).

  • Mouse strain: C57BL/6.

  • Dose: 1–5 × 107 CFU.

  • Route: two-dose, i.n.

The cps mutant conferred independence for mucosal and systemic protection; the ply/pspA double mutant and pspA single mutant showed considerable attenuation, while the ply single mutant maintained virulence.A two-dose regimen of combined cps and ply mutations is as effective as a single cps mutation, eliciting a strong immune response efficiently.Cross-protection remains limited, and incomplete attenuation for ply/pspA. A combination of more than one attenuating mutation provides better safety and broad immunity.Roche et al., 2007 [63]
Δpep27 gene mediates both LytA-dependent and LytA-independent lysis (lyt) (D39, type 4, type 6).

  • Mouse model: CD1 mice.

  • Route: i.p.: 106 to 107 CFU; i.n.: 107 to 108 CFU.

  • Co-colonization: D39 WT 106 CFU/10 µL.

The Δpep27 gene protects against heterologous strains.Induced antibody production and resistance to lethal challenge comparable to the cps mutant; unable to colonize the lungs, blood, and brain, prevented systemic disease.The erythromycin resistance gene found in the pep27 mutant may be transferred to other commensal microbes in the nasopharynx. Further investigation is required to confirm the long-term safety and persistence.Kim et al., 2012 [71]
Δpep27 without markers (D39).

  • Mouse model: CD1 mice.

  • Immunization: three i.n. doses 1.5 × 107 CFU.

  • Challenge: i.p. 1010 CFU.

Antisera cross-reactive; increased IgG titers; protected against lethal challenge; provided adequate protection.Rapidly cleared colonization in vivo; cross-reactive with other serotypes; elicited mucosal immunity; shows inexpensive vaccine potential.Inactivated THpep27 did not increase IgG or IgA; protection appears to be primarily cell-mediated and requires further clarification of immune mechanisms.Choi et al., 2013 [73]
ΔHtrA protein (WT D39, WT TIGR4, D39 htrA–/htrA+).

  • Mouse model: female outbred MF1 mice.

  • Immunization: i.n. 106 CFU/mouse.

The HtrA mutant retained its ability to colonize the nasopharynx, and this colonization significantly prolonged the survival of mice in a systemic bacteremia model.Mutant colonization induced mucosal immunity and a strong humoral response, characterized by higher IgG titers, supporting the nasal route as a promising vaccination strategy.The effectiveness and safety in humans remain uncertain; further studies are needed to investigate long-term immune persistence.Ibrahim et al., 2013 [77]
ΔftsY and caxP genes [TIGR4 (serotype 4), D39, BHN54 (serotype 7F), ST191 (serotype 6A), BHN97 (serotype 19F)].

  • Mouse model: BALB/c mice.

  • Immunization: 105 CFU/mouse i.n. and i.p.

The live vaccine candidate provided robust, serotype-independent protection against AOM, sinusitis, bacteremia, and pneumonia, including co-infection.ftsY- and caxP- highlighted features of an optimal mucosal vaccine; BHN97ΔftsY showed prolonged colonization, higher pneumococcal-specific antibody titers, and a CD4+ T-cell-dependent isotype response.Mucosal IgA levels were not assessed; further studies are required before human trials, including the deletion of the competence system to prevent recombination and reversion, as well as the evaluation of additional safety issues.Neef et al., 2011 [68]; Rosch et al., 2008 [69]; Rosch et al., 2014 [70]
ΔSPY1 (erm cassette replacement) with deletion ply, teichoic acids, and capsule [SPY1 (WT D39), TIGR4, R6, 6B, 19F, 14, and 3].

  • Mouse model: BALB/c mice.

  • Colonization: TIGR4 (108 CFU); 19F (108 CFU).

  • Challenge: i.n. 6B at 107, 5 × 108, 7 × 106 CFU.

SPY1 long-term study showed i.n. immunization with 107 CFU D39 remained protective after three months, inducing both mucosal and systemic protection through antibody and cell-mediated immune responses.SPY1 exhibited a stable capsular phenotype resulting from a cps locus mutation; mucosal and systemic immunization elicited antibody and cell-mediated protection, supporting it as a pneumococcal vaccine candidate. The adjuvants enhanced responses except with heat-inactivated SPY1.SPY1 safety is supported by impaired reversion via phosphocholine-dependent competence. While heat-inactivated SPY1 conferred reduced protection, likely due to lower IgG and the absence of IgA titers.Wu et al., 2014 [82]
A double mutant (Δpep27ΔcomD) (D39 and 6B).

  • Mouse model: CD1 and SCID mice.

  • Inoculum: 108 CFU for i.n., i.p., or i.c.v.

Δpep27ΔcomD immunization provided long-lasting protection (up to 2 months) against type 2 and non-typeable NCC1 strains; the mutant eliminated transformability while maintaining protective efficacy.Modified Δpep27ΔcomD strain persisted across infection routes; protection was associated with elevated IgG and reduced bacterial load; considered a feasible, cost-effective mucosal vaccine candidate.A double mutant is unable to provide long-term protection against the type 6B strain only. Human trials are needed to assess efficacy changes in the challenge strain and its competition with nasopharyngeal commensals.Kim et al., 2019 [72]
Δlgt gene [serotype TIGR4, ST2 (D39), ST3 (wu2), ST6B, ST9V, ST19F, and ST23F].

  • Mouse model: C57BL/6 (i.n.) with 107 CFU.

  • For invasiveness studies, TIGR4Δlgt (105, 106, or 107 CFU).

Prolonged TIGR4Δlgt colonization promoted a Th1-biased response with the live vaccine, which conferred superior protection over the killed parental strain.TIGR4Δlgt colonization induced robust mucosal and systemic immunity with IgG2b and Th1 dominance, cross-reactive across serotypes; strain safely colonized without significant inflammation or systemic spread, even at > 1,000× parental LD50.Further studies are needed to clarify how lipoprotein-deficient pneumococci influence Th1-mediated host defense.Jang et al., 2019 [88]
Serotype one strain (519/43) with recombinant new DNA into its genome [serotype 1, (519/43)].

  • Mouse model: CD1 mice.

  • Immunization: 50 μL of i.n. at 5 × 107; 100 μL of i.p. at 3.8 × 104 CFU.

Haemolytic pneumolysin of strain 519/43 contributed to invasive disease; the Δply mutant of strain 519/43 showed reduced early bacteraemia and significantly lower blood bacterial loads.Genetic modification of this serotype required a strain-specific, plasmid-based method. The pneumolysin D380N mutation did not increase red blood cell lysis; the strain maintained growth in lab media but exhibited impaired growth in serum compared to the WT.Reduced early bacteraemia but did not prevent invasive disease due to Δply and WT strains showed similar burden; non-haemolytic pneumolysin did not abolish invasive potential of serotype 1 Spn; strain 519/43 disease capacity appeared independent of haemolytic activity; further studies are needed to clarify pneumolysin’s role in invasion.Terra et al., 2020 [90]
Gene knockouts: endA and cpsE (D39).

  • Mouse model: BALB/c mice.

  • Immunization: i.n. 5 × 108 CFU of D39.

SPEC strain immunization conferred the highest protection, with the most incredible survival rate and duration after lethal challenge, showing a 23-fold reduction in virulence compared to the WT.cpsE knockout (SPC) reduced growth, colonization density, and duration but increased biofilm formation, while endA knockout (SPE) showed no effect on biofilm or growth, yet elicited the highest anti-pneumococcal IgG levels in mice; double knock-out (SPEC) gave better protection.SPE strain elicited the highest anti-pneumococcal IgG but did not improve survival, indicating IgG alone is insufficient for protection. The single attenuation may reduce immunogenicity. The study lacked a heterologous challenge, and future work should compare immune responses with heat-inactivated bacteria and existing vaccines as well.Amonov et al., 2020 [91]
Δcps/psaA: ΔpsaA gene; Δcps/proABC: ΔproABC gene (6B from clinical Spn isolate).

  • Mouse model: CD1 mice.

  • Immunization: i.n. 107 CFU in 50 µL PBS and i.p. injection of 5 × 106 CFU.

Induced sufficient anti-protein antibodies to protect and prevent septicemia after pneumonia rechallenge.Induced sufficient antiprotein antibodies to prevent septicemia after pneumonia rechallenge.Mutant strains elicited weaker serological responses than WT but were rapidly cleared, indicating high attenuation. The immune assessment was limited to a few antigens, with no CD4+ data or heterologous challenge, suggesting that CPS locus targeting alone may be insufficient for broad-spectrum vaccine design.Ramos-Sevillano et al., 2021 [92]
The SpnA1 (Δfhs/piaA) and SpnA3 (ΔproABC/piaA) (Serotype 6B).

  • RCT human challenge in healthy adults (18–50 years).

  • Participants received nasal spray WT Spn6B or mutant strains SpnA1 and SpnA3.

Clinical trial ISRCTN22467293: SpnA1 and SpnA3 conferred partial protection against recolonization (30% and 50% vs. 47% control) with protection assessed at 6 months by WT Spn challenge.SpnA1 and SpnA3 live attenuated nasal vaccines were safe. The nasal IgG levels were similar across groups, while serum IgG was higher in SpnWT and SpnA1 than in SpnA3.The study did not assess protection against heterologous strains or efficacy in vulnerable populations; a two-dose regimen poses a practical limitation. However, SpnA1 safety requires additional mutation to prevent reversion to virulence.Hill et al., 2023 [94]
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The attenuation strategy for live attenuated vaccines, based on their major route of immunization, is whether it is intranasal (i.n.), intraperitoneal (i.p.), or intracerebroventricular (i.c.v.) injection. Spn: Streptococcus pneumoniae; WT: wild-type; CFU: colony-forming unit; PBS: phosphate buffered saline; cps: capsular; ply: pneumolysin; pspA: pneumococcal surface protein A; pspC or cbpA: pneumococcal surface protein C; pep27: LytA-dependent and LytA-independent lysis; lyt: lipoprotein diacylglycerol transferase; HtrA: high-temperature requirement A protein; ftsY: disrupts nutrient uptake; caxP: proper protein delivery and hindering bacterial colonization; comD: protein essential for competence activation; endA: endonuclease A; cpsE: capsule synthesis gene; psaA: manganese uptake gene; proABC: proline biosynthesis gene; piaA: iron transporter required for systemic virulence; fhs/piaA: mutations affecting metabolic functions; AOM: acute otitis media; CPS: capsular polysaccharide.