Recombinant listeriolysin-O as a platform for next-generation vaccines: Mechanisms, immunological evidence, and translational applications in human and veterinary medicine

Abstract

Recombinant Listeriolysin O (rLLO), a cholesterol-dependent cytolysin (CDC) derived from Listeria monocytogenes, has gained increasing attention as a novel immunological adjuvant for next-generation vaccine development. rLLO enhances both innate and adaptive immune responses by facilitating controlled endosomal membrane permeabilization, promoting cytosolic antigen delivery, and enabling efficient antigen cross-presentation, leading to robust CD8⁺ T-cell activation. These immunological properties are particularly advantageous for vaccines targeting intracellular pathogens and malignancies. This narrative review synthesizes current experimental, preclinical, and translational evidence on the application of rLLO across diverse vaccine platforms in human and veterinary medicine, including DNA-, protein-, nanoparticle-, and vector-based formulations. Studies addressing infectious diseases, such as tuberculosis, HIV, dengue, and listeriosis, as well as cancer immunotherapy models, are critically examined. Emerging strategies involving fusion proteins, detoxified rLLO mutants, and recombinant delivery systems are highlighted to illustrate advances in formulation design and immunogenic optimization. Despite promising preclinical outcomes, several challenges remain, including the need for comprehensive safety evaluation, dosing and delivery strategy optimization, and regulatory pathway clarification. Addressing these limitations through standardized toxicological assessment and large-scale clinical and veterinary trials is essential for the successful translation of rLLO-based vaccine components. Overall, rLLO represents a versatile and potent immunological tool with significant potential to advance vaccine design in human and veterinary applications.

Introduction

The current global burden of infectious illnesses and the complications associated with cancer treatment make it even more important to improve vaccination performance (Yusuf et al., 2023). When it comes to complicated illnesses such as cancer and intracellular infections, traditional vaccination techniques struggle to produce robust and long-lasting immune responses from time to time. This has prompted extensive investigations into the development of novel vaccination platforms and efficient adjuvants for immune modulation. Listeria monocytogenes is a potent inducer of host cell-mediated immunity, particularly strong CD8⁺ T-cell responses, owing to its intracellular lifestyle and efficient antigen presentation (Pham et al., 2025).

Listeriolysin O (LLO) is the most important Listeria monocytogenes (LM) virulence protein (Obaidi et al., 2010; Ibrahim et al., 2017). LLO is a thiol-activated cholesterol-dependent pore-forming toxin and the major LM virulence factor (Hernández-Flores and Vivanco-Cid, 2015). This pore-forming toxin exhibits optimum activity at acidic pH levels, associating with the phagosome milieu, and is closely regulated to prevent host cell damage upon cytosolic release (Banerji et al., 2021). Although L. monocytogenes induces strong antigen-specific T-cell immune responses, its intrinsic virulence and cytotoxicity limit its direct application as a live bacterial vaccine vector. Consequently, safer alternatives are required to harness its potent immunomodulatory properties (Hamon et al., 2012; Sun and Liu, 2013). Recombinant LLO (rLLO), recombinantly expressed from L. monocytogenes, preserves essential immunostimulatory functions, including controlled pore-forming activity, while removing the broader bacterial context responsible for pathogenicity (Liang et al., 2022). Recent studies have highlighted the significance of LLO in modulating host immune responses and influencing antigen presentation pathways (Hamon et al., 2007; Hamon and Cossart, 2011; Khitam et al., 2018; Eldridge and Hamon, 2021; Cong et al., 2024), including apoptosis and pyroptosis, and by altering host gene expression through histone modifications (Hamon et al., 2007; Hamon and Cossart, 2011; Eldridge and Hamon, 2021). Furthermore, the chaperone protein PrsA2 has been recognized as vital for the proper secretion, stability, and folding of LLO during infection, underscoring the complexity of its regulation (Agbavor et al., 2024). The field of vaccine improvement is rapidly evolving, mostly with the use of recombinant proteins and innovative adjuvants (Asokan et al., 2025; Frosth et al., 2025). Improved understanding of intracellular antigen delivery and immune activation pathways has enabled the development of more effective and safer vaccine adjuvants, including pore-forming proteins such as rLLO (Rea et al., 2001). Current vaccine strategies increasingly employ live recombinant bacteria or viral vectors that mimic natural infections and exploit their intrinsic adjuvant properties to enhance immune activation (Gupta and Pellett, 2023).

Future research should focus on boosting existing adjuvants and developing new ones that can effectively stimulate a robust immune response without undesirable side effects (Ibrahim et al., 2012; Nascimento and Leite, 2012). Occurrences in mRNA vaccine formulation represent a promising avenue for future vaccine applications (Lukeman et al., 2025). The adaptability of mRNA technology facilitates rapid updates to vaccines in response to evolving infectious diseases, as demonstrated by the current success of mRNA vaccines against COVID-19 (Wang et al., 2023). These adjuvants could promote the stimulation and proliferation of cytotoxic T lymphocytes (CTLs), thereby advancing the overall efficacy of immunotherapeutic methods (Zhao et al., 2023). Moreover, recent approaches have demonstrated that incorporating double-stranded RNA or protein antigens into lipid nanoparticles can function as an effective adjuvant strategy to enhance immune responses (Nguyen et al., 2012; Wang et al., 2023; Lai et al., 2025). Furthermore, clinical trials and studies will provide valuable insights that can guide future vaccine formulations and applications, potentially revolutionizing the landscape of preventive disease and cancer treatment (Wang et al., 2023; Lukeman et al., 2025).

Recombinant LLO (rLLO) lacks the bacterial context but retains its pore-forming and immune‐stimulatory properties (Yin et al., 2010). In the last decade, interest in using rLLO as a vaccine component has grown. rLLO could act as a natural adjuvant, stimulating CD4⁺ and CD8⁺ responses (Peng et al., 2007). The use of recombinant LLO in vaccine development presents significant opportunities for improving immune responses against various pathogens. Extra recent work has intensive on manufacturing rLLO (mutated or fused forms) that can be coupled as a vaccine adjuvant or carrier, supplying a mechanism for the efficient delivery of antigens and boosting both humoral and cellular immune responses (Hernández-Flores and Vivanco-Cid, 2015).

Despite the wide-ranging investigational research on Listeriolysin-O, the immunological mechanisms, safety profile, and translational importance of rLLO as a vaccine adjuvant remain fragmented across diverse experimental platforms (Xia et al., 2025). Accordingly, this narrative review aims to critically synthesize and integrate current experimental, preclinical, and translational evidence regarding the immunological mechanisms, safety profile, and vaccine applications of rLLO. This review summarizes mechanistic insights related to innate and adaptive immune activation, delivery system strategies, and reported immunological outcomes across diverse vaccine platforms. This review compares findings from human and veterinary disease models and highlights underrepresented veterinary data to delineate the emerging role of rLLO as a versatile immunological adjuvant for next-generation vaccine development.

Materials and Methods

Study design

This narrative review synthesizes experimental, preclinical, and translational evidence on the application of rLLO as a vaccine component or immunological adjuvant in human and veterinary medicine. A comprehensive literature search was conducted across multiple scientific databases, and relevant studies were qualitatively analyzed and integrated to provide a thematic narrative synthesis. No original experimental data were generated.

Literature search strategy and data sources

A comprehensive literature search was conducted in PubMed, Scopus, Web of Science, and Google Scholar to identify peer-reviewed studies relevant to rLLO-based vaccine research. Controlled vocabulary and free-text keywords were combined, including “Listeriolysin O”, “recombinant Listeriolysin O”, “rLLO”, “Listeria-based vaccine”, and “vaccine adjuvant.” These terms were supplemented with disease-specific keywords (HIV, tuberculosis (TB), dengue, and listeriosis) to capture applications in both human and veterinary contexts. Recent publications were prioritized, and seminal studies were included where appropriate.

Inclusion and exclusion criteria

Studies that satisfied the following obligations were encompassed:

• rLLO or LLO-expressing recombinant platforms were studied.

• rLLO as a vaccine antigen, fusion component, or immunological adjuvant was evaluated.

• Reported data from in vitro studies, animal models, preclinical studies, or translational training.

• Described immunological results, such as cell-mediated immunity, cytokine responses, antigen presentation, and vaccine efficacy.

Studies were ignored if they:

• Non-English publications.

• Lacked peer-review.

• Not directly related to rLLO or LLO-based immunological applications.

• This study focused solely on unrelated virulence or microbiological aspects without vaccine relevance.

Review articles were used selectively for contextual background and conceptual frameworks but were not relied upon as primary sources for experimental outcomes unless they presented novel insights directly relevant to the review objectives.

Study selection and data extraction

The headlines and abstracts of all retrieved records were initially screened for relevance. Worthy studies then underwent full-text assessment. After the selected articles were published, key information was extracted and systematically organized, including:

• Type of rLLO construct or delivery build.

• Target disease and pathogen category (bacterial, viral, or parasitic).

• Trial model (in vitro systems, small animal models, or livestock species).

• Immunological end point estimated (CD8⁺ T-cell stimulation, cytokine secretion, antibody responses).

• Immunization parameters (dose, administration route, and formulation strategy)

• Conclusions regarding efficacy, immunogenicity, and safety.

These data formed the basis for the narrative synthesis and the comparative summary (Tables 4 and 5), which highlight representative applications of rLLO across different vaccine platforms.

Table 4. rLLO as an immunological adjuvant in experimental vaccines targeting bacterial and viral antigens in human and veterinary models.

Study ID	rLLO construct type or vaccine strategy category	Immunogenicity	Animal model	Immune response	Dose, methods of immunization, and challenge	Treatment	Conclusion	References
Recombinant BCG ΔureC::hly (rBCG)	Recombinant strains expressing hly	In preclinical trials, it provides better protection against TB than parental BCG (pBCG).	Mice	Infection with rBCG led to increased activation of AIM2 inflammasomes, caspase activation, and generation of IL-1β and IL-18. In addition, it induced autophagy that was AIM2-dependent and STING-dependent. Similarly, rBCG-immunized mice showed early elevated expression of Il-1β, Il-18, and Tmem173 (also known as STING).	10⁵–10⁶ CFU of VPM1002 were delivered intradermally (ID) or subcutaneously (SC); the challenge models with low-dose aerosol Mtb (~50–100 CFU, strain H37Rv)	M. tuberculosis	VPM1002 was tested in preclinical models (mice, guinea pigs, primates) using intradermal/subcutaneous immunization (10⁵–10⁶ CFU) and aerosol Mtb challenge to assess protection. Human trials (Phases I/II) used intradermal doses of 10⁵ CFU, confirming safety and immune activation.	Saiga et al. (2015)
A combination of non-cytolytic LLO mutant (dtLLO) mixed with E protein	Detoxified mutant (dtLLO)	Increase and modulate the immune response to the E protein of the dengue virus	Mice	A robust specific humoral response was generated along with isotype diversity in immunoglobulin (Ig)G antibodies (IgG1 and IgG2a).	On day 14, a booster injection was administered intravenously (IV) or intraperitoneally (IP) with the same dosage and method; a single combination of dtLLO (20 µg) and E protein (5 µg) from Dengue virus serotype 4 dtLLO or wtLLO	Dengue virus	Encourage the possible application of the dtLLO mutant as a secure and efficient adjuvant molecule in vaccination	Hernández‐Flores et al. (2017)
Recombinant proteins pc-NS3 (NS3 fragment [1,095–1,380 amino acids]/pLLO [5–415 amino acids of listeriolysin O (LLO) full-length protein]	Fusion Proteins	Recombinant plasmids were efficiently generated in mammalian cell lines, and co-administration of pc-NS3 and pLLO resulted in the highest total IgG titer against the NS3 antigen in vivo. Immunological responses were also investigated using total specific antibody levels, lymphocyte proliferation, cytotoxicity, and cytokine levels.	Mice	In comparison to the pc-NS3 group and control groups (PBS, pcDNA3.1, and pLLO), the total IgG level in the pc-NS3/pLLO group showed extremely significant variations in the specific antibody production (p < 0.05) (p < 0.05). Additionally, the results show a tendency in both Th1 and Th2 response profiles for both pc-NS3/pLLO and pc-NS3 vaccination regimens. In addition, mice who received pc-NS3/pLLO immunization exhibited greater numbers of IFN-γ and IL-4 secreting cells in ELISpot than mice that received pc-NS3 immunization.	100 µg of DNA vaccine per mouse (either NS3 alone or LLO-NS3), intramuscular (IM) injection, the study did not use a live HCV challenge (as HCV does not infect mice)	Hepatitis C virus	Substantial implications for using the LLO gene as a genetic adjuvant in the immune response to HCV.	Pouriayevali et al. (2019)
S1 Subunit of Pertussis Toxin (PTS1) with Listeriolysin O (LLO-PTS1)	Fusion proteins	Inducing the release of IL-17 and IL-4 This work serves as a foundation for future research into the production of booster pertussis vaccines.	Mice	Following LLO-PTS1 vaccination, the blood levels of IFN-γ, IL-4, and IL-17 cytokines (representing T-helper 1, 2, and 17 responses, respectively) were assessed.	LLO-PTS1 protein (35 µl; 0.05 mg/ml) plus 35 µl CFA for the initial injection and 35 µl IFA for subsequent injections, administered intraperitoneally (ip).	Pertussis	LLO-PTS1, an alum-free monovalent recombinant fusion protein, can promote IFN-γ production.	Forghani et al. (2020)
Brucella abortus (RB51) recombinants with pbbr1ori-LLO and pBlu–mLLO-BAX-SMAC	Recombinant strain expressing hly	Cell-mediated immune responses are crucial for resistance to intracellular bacterial infections. Thus, LLO expression of Listeria monocytogenes, BAX, and SMAC apoptotic proteins in strain RB51 may improve vaccination effectiveness and safety.	Mice	IFN-γ production is more effectively modulated. Furthermore, LDH revealed that the RB51-mLLO-BAX-SMAC and RB51-LLO strains exhibited greater cytotoxicity in J774A.1 cells than RB51. Furthermore, RB51 recombinants reduced macrophage survival while increasing apoptosis and necrosis. The RB51 recombinant incorporating mLLO-BAX-SMAC elicited an increased Th1 immune response. This increased immune response is mainly due to bacterial endosome escape and bacterial antigens, which result in better apoptosis and cross-priming.	Bacteria (RB51 strain and RB51 recombinant with mLLO-BAX-SMAC) were injected intraperitoneally at a concentration of approximately 4/5 × 108.	B. abortus	Higher TCD8+- and T cell-mediated immunity improves the safety and efficacy of the RB51 recombinant (RB51 mLLO-BAX-SMAC) vaccine candidate against B. abortus.	Sarmadi et al. (2022)
Modified strain of Listeria ivanovii LIΔilo:hly (in which ilo was replaced by hly)	Recombinant strains expressing hly	LI can be used as a viable bacterial vaccine vector. However, we have also observed in vivo that LI vector vaccine candidates survive in the immune organ (spleen) for a shorter time than Listeria monocytogenes (LM) and elicit weaker immune responses than LM.	Mice	The LIΔilo:hly group had greater levels of TNF-α, IL-6, and IFN-γ on day 9 and IL-6 and IFN-γ on day 40 than the LI group. In addition, the LI group had a 10% greater protection rate than the LI group. The LI group had a superior pathological state at day 3 compared with the LM and LI groups. This indicates that hly addition enhanced the biosafety of the strain.	Mice were given LIΔilo:hly at a dosage of 0.1 times LD50 of each strain twice every 7 days. Challenges were held 7 days following the second vaccination with 5 x LD50 of LM.	Listeriosis	The recombinant strain LIΔilo:hly demonstrated high biosafety and immunogenicity, making it a promising candidate for the development of vaccines.	Liang et al. (2022)
Recombinant LLO produced by Lactococcus lactis	Recombinant strain expressing hly	Mutated internalin A is expressed on its surface, and substantial quantities of listeriolysin O (LLO) are secreted to boost its potential as a vehicle for L. lactis DNA vaccination.	Western blot analysis (WB)	The culture supernatant exhibited the highest hemolytic activity. This hemolytic experiment indicated that LLO, a highly active pore-forming toxin, is widely produced in L. lactis.	Cell line	Vaccine carrier	The use of L. lactis, which expresses both virulent factors from L. monocytogenes LLO, as a tool for DNA plasmid delivery may be an effective technique with major applications in fundamental research and vaccine development.	De Azevedo et al. (2015)
Bacillus Calmette-Guérin (BCG) ΔureC::hly	Recombinant strains expressing hly	By considerably lowering the TB burden in mouse lungs, improving pulmonary pathology, and boosting immune responses, the BCG ΔureC::hly ΔnuoG vaccine was safer than BCG and offered better protection than the parental BCG ΔureC::hly.	Mice	Following immunization with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG, transcriptome analysis of draining lymph nodes revealed an earlier and more robust stimulation of immune responses compared with BCG SSI. It also indicated an elevation of IFN-induced GTPases and inflammasome activation.	106 CFU of BCG strains were administered subcutaneously to the tail bases of the animals. Additionally, the mice were aerosol challenged with a low dosage of 100–200 CFU of M. tuberculosis 90 days after immunization.	Mycobacterium bovis	A potential next-generation TB vaccine candidate with exceptional effectiveness and safety is BCG ΔureC::hly ΔnuoG.	Gengenbacher et al. (2016)
Recombinant hly gene in Lactobacillus via pNZ8110	Recombinant strains expressing hly	LLO-expressing probiotic bacteria, such as lactic acid bacteria, are used as delivery strains in pathobiotechnological immunization.	N/A (in vitro)	Cell-mediated immunity, stimulation of CD4⁺ and CD8⁺ T cells, secretion of LLO confirmed by western blot	Induction with 2% nisin supernatant; secreted protein detected in engineered L. plantarum culture supernatant. Not performed in this study; only molecular cloning and expression	No in vivo treatment; only bacterial transformation, plasmid induction (with nisin), and detection of LLO expression. Listeria monocytogenes	The hly gene was successfully cloned and expressed in L. plantarum. Successful cloning and expression of LLO in L. plantarum; potential as a live vaccine candidate for listerial infections or tumor therapy	Hayati et al. (2017)

Table 5. Delivery systems and formulation strategies using rLLO in experimental vaccines against listeriosis and other pathogens.

Study ID	rLLO construction type	Immunogenicity	Animal model	Immune response	Dose, methods of immunization, and challenge	Treatment	Conclusion	References
(Chitosan + rLLO) Listeriolysin O with chitosan	Fusion protein or chitosan-based	LLO and CS may support each other to provide the most beneficial effect by eliciting good cellular and humeral immunity.	Mice	The third group’s skin thickness, IgG, and IL6 concentrations were all noticeably greater.	For 4 weeks, the mice were fed a diet enriched with 1.5 g of chitosan per kilogram. At the end of the second week, 0.3 ml of LLO was injected S/C, and 14 days after the first injection, the booster dosage of LLO was administered. After 7 days of the booster dosage, 0.2 ml of 1x109 CFU/ml was injected intraperitoneally as a challenge dose of L. monocytogenes culture.	Listeriosis	By inducing strong cellular and humeral immunity against listeriosis, LLO and chitosan may have the most positive impact.	Ali and Yasssein (2021)
(Recombinant B. subtilis vector) Recombinant Bacillus subtilis vegetative cells expressing LLO adjuvant with chicken egg ovalbumin	Fusion protein	Heterologous antigens may be efficiently delivered to the cytosol of a host cell by expression in a nonpathogenic bacterial host, where they are mostly processed via the cytosolic MHC class I presentation pathway.	Mice	LLO-OVA strains may transfer heterologous antigens to the cytosol of APCs, and all LLO-producing strains (BR1S control and R_OVA1-R_OVA4 strains) have significant hemolytic activity. In addition, the ability of LLO-OVA-producing strains to trigger a cytotoxic T-cell response was assessed. The whole-length OVA gene fused to the N-terminal LLO region of the R_OVA3 strain displayed the SIINFEKL peptide in complexes with MHC class I.	Protein samples of 40 μg in western blotting	Vector vaccine	The native full-sequence OVA fused to the N-terminal segment of LLO was adequate for epitope delivery and CD8+ cell activation. The ability of the fusion antigen to enhance MHC I presentation demonstrates that LLO-producing B. subtilis may be a unique and appealing choice for a vaccine vector.	Roeske et al. (2018)
(PLGA microparticles + BMDC) Mouse bone marrow-derived dendritic cells loaded with L. monocytogenes listeriolysin O-derived	Cell-based delivery	CTL/Th-hybrid epitope long peptide was used to assess lymphocyte proliferation and IFN-γ production, while T-cell epitope short peptide requires a high amount of peptides for successful immunization because of its easy degradation in vivo.	Mice	LLO- and OVA-long peptide/PLGA vaccination resulted in considerably increased CTL and Th proliferation and IFN-γ production compared with long peptide alone or CTL and Th short peptides/PLGA vaccination.	Mice received 2 × 105 cells of antigen-loaded BMDCs subcutaneously into their flanks twice at a 2-week interval. Four weeks later, they were challenged intraperitoneally with 1 × 105 colony forming units (CFU) of L. monocytogenes.	Listeriosis	The new vaccine that combines CTL/Th-hybrid epitope long peptide and PLGA microparticles are effective against intracellular bacteria.	Tanaka et al. (2020)
(GNP + Advax) GNPs loaded with listeriolysin peptide 91–99 (LLO91–99)	Fusion protein or nanoparticle-based	GNP-LLO91–99 nanovaccines made with Advax adjuvant protect against listeriosis during pregnancy.	Mice	A magnifying lens was used to examine all puppies for clinical data, weight, length, coordination movement tests, and general and skin observations. Clinical data were normal in both the control group and mothers with GNP-LLO91-99 vaccination. As a result, systemic listeriosis immunization lowered IL-6 levels (Th2) while significantly increasing IL-12 levels (Th1). We confirmed that neonatal listeriosis induced MG to generate a high TNF-a level.	GNP-LLO91-99, compounded with AdvaxTM, was administered intravenously (i.v.) via the lateral tail vein (5 µg of nanoparticles and 250 µg of adjuvants per mouse). At E16, all animals received 100 µL of an LMWT bacterial suspension in saline (1 × 105 CFU/mL) by intravenous injection.	Listeriosis	Nanovaccines are excellent prenatal immunization strategies because they may overcome the placental barrier and reduce the morbidity of listeriosis.	Calderón-Gonzalez et al. (2016)
™) (DC Vaccine + GNP-LLO + Advax) Dendritic cells (DCs) loaded with gold glyconanoparticles (GNPs) loaded with a listeriolysin O peptide LLO 91–99 (GNP-LLO) and delta inulin (Advax)	Fusion protein	The inclusion of Advax™ adjuvant enhanced the immunogenicity of GNP-LLO-loaded DC vaccines compared with DC loaded in vitro using free LLO peptide.	Mice	Following the challenge, there was an increase in splenic CD4+ and CD8+ T cells, NK cells, and CD8α+ DC, as well as Th1 cytokine production (IL-12, IFN-γ, TNF-α, and MCP-1).	Mice were challenged intraperitoneally (i.p.) or intravenously (i.v.) with 5 × 103 CFU of Listeria microorganisms/mouse 7 days after immunization or DC vaccines (DC-LLO91–99, DC-LLO189–201, DC-GAPDH1–22, and DC-GNP-LLO91–99, all at doses of 1 × 106 DC per immunization).	Listeriosis	Immunization with GNP-LLO91–99 plus Advax™ adjuvant provided equally robust Listeria protection as the best DC vaccine strategy but without the complexity and cost	Rodriguez-Del Rio et al. (2015)

Data synthesis

As a result of the substantial heterogeneity detected across the included studies, mainly in terms of rLLO construct types, vaccine platforms, animal models, immunization routes, dosing regimens, and immunological endpoints (Tables 4 and 5), a qualitative narrative synthesis approach was accepted. Data were extracted, categorized, and thematically integrated to highlight mechanistic insights, translational relevance, and initial trends in rLLO-based vaccine development. Additionally, the statistically reported results and inferential immunological data derived from these research papers were systematically briefed in Tables 6 and 7 to support claims related to next-generation immunization, rather than performing a quantitative meta-analysis.

Table 6. Immunological and protective outcomes of rLLO-based experimental vaccine studies.

Key outcome	Effect direction	Immune metric	Statistical evidence (reported)	Study (Author, Year)
Enhanced innate immunity and protection	↑	IL-1β, IL-18, and AIM2 activation	Statistically significant	Saiga et al. (2015)
Enhanced humoral immunity	↑	IgG1 and IgG2a antibodies	Statistically significant	Hernández-Flores et al. (2017)
Balanced Th1/Th2 immune activation	↑	Total IgG, IFN-γ, IL-4	p < 0.05	Pouriayevali et al. (2019)
Multilineage cytokine induction	↑	IFN-γ, IL-4, and IL-17	Statistically significant	Forghani et al. (2020)
Th1-Biased Cellular Immunity	↑	IFN-γ, CD8⁺ T cells	Comparative statistical support	Sarmadi et al. (2022)
Enhanced inflammatory response and protective immunity	↑	TNF-α, IL-6, IFN-γ	Statistically significant	Liang et al. (2022)
Confirmation of the expression and activity of rLLO	↑	Antigen delivery/hemolysis	Functional validation	De Azevedo et al. (2015)
Improved protection versus parental BCG	↑	Immune activation and TB protection	Comparative statistical support	Gengenbacher et al. (2016)
Stable protein expression and immune activation	↑	Immune stimulation	Enhancement of functional immune	Hayati et al. (2017)
Favorable safety and immune profile	↑	Safety and immunogenicity	Statistically supported	Saiga et al. (2015), Gengenbacher et al. (2016)

Table 7. Functional and mechanistic evidence supporting rLLO-mediated antigen delivery systems.

Mechanistic outcome	Effect direction	Delivery metric	Statistical evidence (reported)	Delivery strategy	Study (Author, Year)
Enhanced inflammatory and humoral responses	↑	Skin thickness, immunoglobulin G, IL-6	Statistically significant	rLLO-based delivery	Ali and Yasssein (2021)
Improved antigen processing via MHC-I	↑	Access to cytosolic antigens	Significant increase	LLO-secreting bacteria	Roeske et al. (2018)
Enhanced cellular immune activation	↑	CTL and Th proliferation, IFN-γ	Comparative functional enhancement	LLO-mediated antigen delivery	Tanaka et al. (2020)
Th1 polarization and enhanced protection	↑	IL-12 ↑ / IL-6 ↓	Cytokine-based inferential support	Antigen + LLO delivery	Calderón-Gonzalez et al. (2016)
Improved cytosolic antigen delivery	↑	Membrane disruption	Functional validation	LLO membrane interaction	Rodriguez-Del Rio et al. (2015)

Structure of LLO

LLO belongs to the CDC family of pore-forming toxins that disrupt host cell membranes and facilitate the intracellular survival of L. monocytogenes (Fig. 1) (Dubail et al., 2001; Hernández-Flores and Vivanco-Cid, 2015; Banerji et al., 2021). The LLO molecule is organized into four distinct structural domains (D1–D4) (Köster et al., 2014). Domain 1 (D1) contains a five-stranded β-sheet and is surrounded by six α-helices) PEST-like sequence (amino acids 39–51), enriched in proline, glutamate, serine, and threonine residues, which is dispensable for hemolytic activity but essential for phagosomal escape, intracellular stability, and virulence regulation (Hamon et al., 2012; Hotze and Tweten, 2012; Hernández-Flores and Vivanco-Cid, 2015). Domain 2 (D2) functions as a flexible hinge connecting D1 and D4, which consists of four β-strands (Köster et al., 2014). It is connected to D4 through a glycine linker and contains the immunodominant CD8⁺ T-cell epitope LLO₉₁–₉₉, highlighting its importance in adaptive immune recognition (Dubail et al., 2001; Wallecha et al., 2013). Domain 3 (D3) is formed by a five-stranded antiparallel β-sheet, which is surrounded by six α-helices (Köster et al., 2014). It acts as a pH sensor, with key acidic residues (D208, E247, and D320) regulating LLO activation under acidic phagosomal conditions (Schuerch et al., 2005), thereby ensuring controlled pore formation and minimizing host cytotoxicity (Köster et al., 2014; Nguyen et al., 2019). Domain 4 (D4) has eight β-sheets, which are organized forming a β-sandwich structure that mediates binding to cholesterol-rich host membranes through a conserved undecapeptide motif, representing a critical step in oligomerization, pore formation, and cytolytic activity (Köster et al., 2014; Banerji et al., 2021). Collectively, the coordinated functions of these domains link the LLO structure to membrane disruption, immune modulation, and intracellular pathogenic mechanisms (Ilangumaran Ponmalar et al., 2021).

Fig. 1. Crystal structure of LLO (domains D1–D4). The undecapeptide in D4 is inserted into membranes. The key functional motifs (e.g., cholesterol-binding loop) are highlighted in red (Köster et al., 2014).

LLO is a potent immunogen capable of inducing robust CD4⁺ and CD8⁺ T-cell responses (Singh et al., 2005; Sun and Liu, 2013; Wallecha et al., 2013; Phelps et al., 2020). Several immunodominant epitopes, including LLO₉₁–₉₉, LLO₁₈₉–₂₀₁, and LLO₂₁₅–₂₂₆, have been shown to elicit strong antigen-specific T-cell responses in experimental models (Singh et al., 2005; Sun and Liu, 2013; Hernández-Flores and Vivanco-Cid, 2015). Notably, the immunogenicity of LLO is independent of its cytotoxicity; dtLLO, which lacks pore-forming ability, still effectively stimulates immune responses (Phelps et al., 2020). This property has been harnessed in vaccine development, where dtLLO serves as an adjuvant, enhancing antigen presentation and promoting dendritic cell maturation, thereby augmenting both innate and adaptive immunity (Wallecha et al., 2013). The structural domains of LLO are intricately associated with its role in pathogenesis and its capability to modulate the host immune response, making it a focal point in the study of bacterial virulence and vaccine design.

rLLO: mechanism of action and properties

Recombinant protein vaccines represent an important enhancement in existing vaccinology because they are designed to provoke targeted immune responses against defined antigens, particularly those related to intracellular pathogens (Huleatt et al., 2007). These vaccines depend on extremely purified recombinant proteins or pathogen-derived subunits, permitting particular immune targeting while maintaining promising safety profiles. Within this context, rLLO has attracted significant attention as a vaccine component and immunological adjuvant due to its strong immunogenic potential and capacity to augment antigen delivery and processing (Li et al., 2021).

LLO is a non-enzymatic, thiol-activated, CDC produced by L. monocytogenes. Its primary biological function is to facilitate bacterial escape from the phagosome through pore formation in host cell membranes, a process essential for intracellular survival and pathogenicity (Beauregard et al., 1997; Hamon et al., 2012; Li et al., 2022). In contrast, rLLO used in vaccine platforms is typically genetically modified, detoxified, or carefully dose-controlled to reduce cytotoxicity while preserving immunologically relevant functions. Experimental studies have demonstrated that rLLO retains the ability to transiently permeabilize endosomal or phagosomal membranes without causing extensive host cell damage, supporting its suitability for safe vaccine and adjuvant applications (Köster et al., 2014; Phelps et al., 2020).

Mechanistically, rLLO exerts its activity through pH-dependent and transient pore formation within endosomal and phagosomal membranes of APCs, including dendritic cells and macrophages (Radtke et al., 2011). This controlled membrane permeabilization enables the translocation of co-delivered antigens from endosomal compartments into the cytosol, thereby facilitating access to cytosolic processing pathways and enhancing major histocompatibility complex (MHC) class I cross-presentation (Portnoy et al., 1988; Hiltbold et al., 1996). Consequently, rLLO promotes the efficient activation of CD8⁺ T lymphocytes, which is critical for protective immunity against intracellular pathogens (Hiltbold et al., 1996).

In addition to its membrane-permeabilizing activity, LLO and rLLO can activate innate immune signaling pathways in APCs. LLO has been shown to stimulate macrophages and dendritic cells via Toll-like receptor (TLR) 4 (TLR4), inducing lipid raft aggregation, NF-κB activation, and pro-inflammatory cytokine production (Kayal et al., 1999; Nguyen et al., 2019). Furthermore, transient pore formation by rLLO induces controlled increases in intracellular Ca²⁺ levels and activates inflammasome-associated signaling without causing irreversible membrane damage or widespread cytolysis, further supporting its favorable safety profile (Tsuchiya et al., 2005; Nguyen et al., 2019). Collectively, these mechanisms underpin the capacity of rLLO to function as an effective facilitator of intracellular antigen delivery, cross-presentation, and immunostimulation (Fig. 2).

Fig. 2. AI-generated schematic illustration depicting the immunological mechanisms mediated by rLLO.

rLLO mediates immune activation

Based on the aforementioned molecular mechanisms, rLLO-containing vaccine formulations elicit a coordinated activation of innate and adaptive immune responses (Wira et al., 2005). Antigen delivery into the cytosol of antigen-presenting cells promotes dendritic cell maturation and enhances their capacity to prime T lymphocytes. rLLO has been shown to engage TLR signaling pathways, particularly TLR4, leading to the downstream activation of transcription factors, such as NF-κB and the initiation of proinflammatory gene expression (Stierschneider and Wiesner, 2023).

As a consequence of these signaling events, rLLO-based vaccines induce robust cytokine responses characterized by increased production of interleukin-12 (IL-12), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). This cytokine milieu favors a Th1-biased immune response, which is particularly desirable for vaccines targeting intracellular pathogens and malignancies (Rodriguez-Del Rio et al., 2015). Furthermore, enhanced cross-presentation resulting from rLLO-mediated antigen delivery leads to the efficient activation and expansion of CD8⁺ cytotoxic T cells, thereby strengthening cell-mediated immunity.

In addition to shaping cellular immune responses, rLLO exhibits adjuvant-like properties that amplify humoral immunity when administered in combination with vaccine antigens. By improving antigen availability and immune cell activation, rLLO enhances both the magnitude and breadth of the immune response. Antigen delivery strategies involving LLO fusion proteins have been shown to unmask subdominant epitopes, thereby broadening the repertoire of antigen-specific T-cell responses (Singh et al., 2005). Picard et al. (2015) reported efficient MHC class I antigen presentation in antigen libraries incorporating cLLO. However, the specific contribution of cLLO to MHC-I presentation cannot be definitively determined because no comparison was performed with libraries lacking cLLO. Similarly, rLLO has been successfully employed to direct DNA- and protein-based vaccine antigens into the cross-presentation pathway, effectively functioning as an endosomolytic carrier to improve cytosolic antigen delivery (Carrero et al., 2009) (Fig. 3).

Fig. 3. Enhanced cross-presentation and cytokine signaling induced by rLLO-antigen fusion proteins.

Key mechanistic features of rLLO

Membrane perforation and cytosolic antigen delivery

At acidic pH, rLLO is quickly incorporated into the host cell membrane to form pores. This crucial mechanism results in the disruption and lysis of phagosomes and endosomes, traditionally responsible for the internalization of pathogens or antigens. The immediate effect of membrane perforation is the efficient delivery of the antigen to the cell cytosol. The process significantly increases vaccine efficacy by enabling the processing and presentation of antigens through the MHC class I pathway (Del Valle et al., 2021), which primarily presents cytoplasmic, viral, and tumor-associated antigens. Antigen loading via the TAP-dependent pathway is essential for effective CD8⁺ T-cell priming and the development of CTL responses, which are critical for cellular immunity. Accordingly, rLLO-based antigen chimeras have been shown to enhance MHC class I presentation and CD8⁺ T-cell activation, supporting their potential to promote CTL responses in experimental vaccine platforms (Mantel et al., 2022).

Innate activation

Beyond antigen delivery, rLLO contributes to the activation of innate immune responses. Its pore-forming activity induces transient increases in intracellular calcium (Ca²⁺) levels, likely as a consequence of membrane permeabilization, which can contribute to downstream signaling events, including inflammasome activation (Lee et al., 2012). In addition, LLO has been reported to activate TLR4-dependent signaling pathways, leading to enhanced pro-inflammatory cytokine production in APCs (Cheng et al., 2020).

Immune activation mechanisms by rLLO: implications for innate and adaptive immunity and vaccine development

rLLO is a powerful immunological adjuvant that can improve the effectiveness of vaccines against intracellular pathogens by activating both innate and adaptive immune responses through inflammasome activation and improved antigen presentation.

Innate immune responses

Antigen-presenting cell activation

rLLO enhances the activation and functional maturation of antigen-presenting cells, particularly dendritic cells and macrophages. Pouriayevali et al. (2019) demonstrated that exposure to LLO-containing formulations resulted in increased expression of APC activation markers and increased production of pro-inflammatory cytokines, indicating enhanced innate immune stimulation. This activation state is associated with improved antigen processing and presentation capacity, thereby creating a favorable immunological environment for initiating adaptive immune responses. These effects were observed without inducing excessive cytotoxicity, supporting the suitability of rLLO as an immunostimulatory component in vaccine platforms (Pouriayevali et al., 2019).

Inflammatory cytokine induction

The liberation of key pro-inflammatory cytokines like TNF-α, IL-6, and IL-12, is induced, and these molecules act as messengers, coordinating the immune response (Pouriayevali et al., 2019).

Nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) Inflammasome activation

Through its pore-forming activity, LLO-mediated membrane perturbation has been shown to induce potassium (K⁺) efflux and reactive oxygen species (ROS) generation during L. monocytogenes infection. These events have been implicated in the activation of the NLRP3 inflammasome and subsequent IL-1β production (Ayna et al., 2012).

Phagosome escape mimicry

rLLO helps deliver antigens into the cell’s main compartment (cytosol) in cellular models. This effectively mimics how L. monocytogenes naturally infects cells, making it a powerful tool for immune activation (Wan et al., 2015).

Adaptive immune responses

rLLO also plays a pivotal role in training the adaptive immune system, providing long-lasting, specific protection (Sun and Liu, 2013).

CD4⁺ T cell activation

When processed inside endosomes, rLLO can be present on MHC II molecules, leading to helper T cell activation. These CD4⁺ T cells are essential for developing strong antibody responses and play a crucial role in preparing CTLs for action (Roche and Furuta, 2015).

CD8⁺ T cell priming

rLLO disrupts the phagosome, allowing antigens to access the cytosol. This enables cross-presentation on MHC I molecules, significantly boosting CTL priming. These "killer" T cells are vital for eliminating infected cells (Ito and Seishima, 2010).

Adjuvant properties

rLLO acts as an effective adjuvant, enhancing the immune response to co-administered antigens. It has already been used in DNA vaccines, peptide vaccines, and recombinant viral vectors (Huleatt et al., 2007; Kim et al., 2015).

Standard adjuvants, such as alum, CpG oligonucleotides, and bacterial flagellin, stimulate the immune system through well-characterized pattern recognition receptors (PRRs), such as TLR4, TLR9, and NOD-like receptors. In contrast, rLLO promotes immunity via a well-defined mechanism involving the creation of transient pores in cholesterol-rich host cell membranes. This activity initiates NLRP3 inflammasome activation via potassium efflux, ROS generation, and phagosomal disruption and enables cytosolic antigen release, promoting cross-presentation via MHC class I. This twofold role in both innate and adaptive immunity is relatively rare among protein-based adjuvants (Sun and Liu, 2013; Wallecha et al., 2013). Although the interaction of rLLO with cholesterol is well understood, the precise immune-sensing receptors remain unclear. In contrast to adjuvant proteins such as flagellin (TLR5), lipopolysaccharide (TLR4), and CpG DNA (TLR9), which occupy definite PRRs, rLLO's immune stimulation likely depends on secondary mechanisms, including damage-associated molecular patterns (DAMPs), inflammasome priming (Lamkanfi and Dixit, 2014), and cytosolic sensing pathways such as absent in melanoma-2 (AIM-2) or stimulator of interferon genes (STING), though direct indication is motionless deficient (Mandal and Lee, 2002; Wallecha et al., 2013).

Additionally, while adjuvants, such as cholera toxin B subunit (CTB) and heat shock proteins (HSPs), bind to GM1 gangliosides and TLR2/TLR4 and correspondingly encourage APC development without membrane damage, rLLO imitates ordinary intracellular bacterial invasion, disrupting membranes and facilitating cytosolic antigen delivery. This increases CD8⁺ T cell responses, which is particularly appreciated in vaccine approaches against intracellular pathogens and tumors (Sun and Liu, 2013; Wallecha et al., 2013).

Despite these advantages, rLLO remains underexplored compared with conventional adjuvants, particularly regarding its receptor specificity, immune profile in neonates, and safety in immunocompromised hosts. Defining its molecular targets and comparing them with other immune activators is essential for optimizing its use in next-generation vaccines (Peng et al., 2007). Table 1 shows the comparative characteristics of rLLO and other adjuvant proteins.

Table 1. Comparative characteristics between rLLO and other protein-based adjuvants.

Feature	rLLO	Flagellin	CTB	Group with HSP
Source	Listeria monocytogenes	Salmonella and E. coli (Smith et al., 2003)	Vibrio cholerae (Stratmann, 2015)	Bacteria or mammalian cells (Asea et al., 2002)
Main receptor	Cholesterol levels (indirect PRRs) (Mandal and Lee, 2002)	TLR5 (Smith et al., 2003)	GM1 ganglioside (Stratmann, 2015)	TLR2 and TLR4 (Asea et al., 2002)
PRR pathway	NLRP3 inflammasome (K⁺ efflux, ROS) (Wallecha et al., 2013)	NF-κB via TLR5 (Smith et al., 2003)	cAMP via GM1	TLR2/4 via MyD88 (Asea et al., 2002)
Mechanism of immune activation	Pore formation, inflammasome formation, DAMP signaling	TLR5 activation	GM1 binding and retrograde trafficking	TLR binding and APC maturation
Cross-presentation	Yes (via cytosolic delivery) (Wallecha et al., 2013)	Limited	No	No
CTL induction	Strong	Moderate	Weak	Moderate
Mucosal immunity	Possible with formulation (Wallecha et al., 2013)	Strong (Stratmann, 2015)	Strong (Stratmann, 2015)	Weak (Asea et al., 2002)
Advantages	Cross-presentation, inflammasome activation, antigen escape	Predictability, strong innate activation, and good mucosal immunity (Smith et al., 2003)	Excellent for mucosal tolerance or IgA boost (Stratmann, 2015)	Good DC activation, APC maturation, and some CTL (Asea et al., 2002)

rLLO applications in vaccine development

Human applications

Cancer immunotherapy

Recombinant LLO has been widely explored in cancer vaccine platforms, especially due to its ability to facilitate cytosolic antigen delivery and promote CD8⁺ T cell responses. Listeria-based vectors expressing LLO-tumor antigen fusion proteins, such as HPV E7 or HER2/neu—have demonstrated therapeutic efficacy in cervical, prostate, and breast cancer murine models (Peng et al., 2007). These vectors enhance MHC I presentation and cytotoxic lymphocyte priming, which are crucial for tumor clearance (Sun and Liu, 2013) (Table 2).

Table 2. Summary of rLLO applications in vaccinology.

Domain	Disease/target	Formulation	Species/model	Outcome	References
Human	Cancer (HPV, HER2/neu, and melanoma)	LLO tumor fusion in Listeria vectors	Mice	Strong CTL response and tumor regression	Peng et al. (2007), Sun and Liu (2013)
	Tuberculosis (TB)	VPM1002 rBCG insertion (hly)	Mice, Humans	Enhanced Th1/Th17 Immunity: Clinical Trial (Phase II/III)	Nieuwenhuizen et al. (2017)
	TB	rLLO + DNA/protein vaccines	Mice	Improved cytosolic delivery and protection	Phelps et al. (2020)
	HIV	LLO-fused viral antigens	Mice (preclinical)	Increased immunogenicity, enhanced CD8⁺ responses	Mata et al. (2001)
	TB, cancer	rLLO in liposomes and polymeric nanoparticles	In vitro animal models	Improved cytosolic delivery and cross-presentation	Mandal and Lee (2002), Rodriguez-Del Rio et al. (2015), Roeske et al. (2018)
Veterinary	Listeriosis in sheep	Live-attenuated Lactobacillus monocytogenes + LLO	Sheep	~78% protection, DIVA-compatible via anti-LLO serology	Meng et al. (2023)
	Bovine tuberculosis	VPM1002 (same approach as in humans)	Goats	Safe and immunogenic in juvenile goats	Figl et al. (2023)
	Brucella BVDV (theoretical)	LLO-conjugated subunit or vectored vaccines	Ruminants (potential)	Not yet studied, proposed for Th1-inducing effect	Figl et al. (2023)

Infectious disease vaccines

Tuberculosis

The most sophisticated application of rLLO is the recombinant BCG strain VPM1002, which substitutes Listeria's hly gene (encoding LLO) for ureC. Th1/Th17 immunity and antigen export are improved by LLO-mediated phagosomal disruption. VPM1002 is undergoing phase II/III clinical trials and has demonstrated improved protection in mice and immunogenicity in humans (Nieuwenhuizen et al., 2017). Additionally, rLLO enhances the presentation of antigens in DNA and protein subunit TB vaccines, thereby enhancing their protective effectiveness (Phelps et al., 2020; Imad Abd-AlAziz et al., 2023), Table 2.

Human immunodeficiency virus

LLO-fused HIV antigens have improved CD8 T cell responses in preclinical models. These results imply that rLLO may serve as a carrier or adjuvant in viral subunit vaccines intended to boost cellular immunity (Mata et al., 2001) (Table 2).

Nanoparticle and liposomal delivery systems

rLLO was successfully integrated into nanoparticles and liposomes to improve endosomal escape and antigen presentation (Table 2):

- LLO-functionalized liposomes fuse with host membranes to release cargo directly into the cytoplasm (Rodriguez-Del Rio et al., 2015; Roeske et al., 2018).

- Polymeric nanoparticles coloaded with rLLO and antigen improve cytosolic access and CTL priming (Mandal and Lee, 2002).

Veterinary applications

rLLO has also been used in livestock vaccines, particularly for listeriosis and TB.

Listeriosis in sheep

A live-attenuated L. monocytogenes strain (ΔactA, ΔplcB, ΔorfX) expressing LLO conferred 78% protection in sheep and allowed for differentiating infection from vaccinated animals (DIVAs) via anti-LLO serology (Meng et al., 2023) (Table 2).

TB in goats and cattle

The VPM1002 strategy has been translated into veterinary contexts, demonstrating safety and immunogenicity in juvenile goats, thereby supporting its potential application in bovine TB vaccination (Figl et al., 2023).

Probiotic and mucosal delivery platforms in animal models

Recombinant probiotic systems expressing LLO have also been investigated as oral or mucosal vaccine platforms in animal models. Lactococcus lactis and Lactobacillus strains engineered to express LLO demonstrated stable antigen expression, effective mucosal immune stimulation, and enhanced antigen delivery, indicating the feasibility of non-invasive livestock vaccination strategies (De Azevedo et al., 2015; Hayati et al., 2017).

Other potential applications

Although these applications remain theoretical, rLLO has been proposed as a potential adjuvant for veterinary vaccines targeting diseases that require strong Th1-biased immune responses, such as brucellosis and bovine viral diarrhea virus (BVDV), based on immunological rationale and translational evidence (Figl et al., 2023).

Future directions in rLLO-based vaccine research

These prospective applications position rLLO as a viable immunostimulant and a customizable platform for next-generation vaccines targeting cancer, infectious diseases, and animal diseases. The integration of molecular engineering, advanced delivery systems, and computational design will be essential for translating preclinical advances into clinical and field-level success (Table 3).

Table 3. Emerging directions for rLLO vaccine research.

Area	Innovation	Goal/Benefit	References
Molecular Engineering	pH-sensitive mutants and cell-targeting motifs	Reduces toxicity and increases targeting	Kim et al. (2015)
Nanoparticles	Liposomes, gold NPs, and VLPs	Enhance delivery and endosomal escape	Singh et al. (2005)
Combination Adjuvants	Co-delivery with TLR ligands and chemokines	Synergistic immune activation and stronger CTL response	Peng et al. (2007)
Nucleic acid vaccines	LLO–protamine fusion for DNA/mRNA delivery	Improved cytosolic translocation and expression	Kim et al. (2015)
Veterinary field trials	VPM1002 in livestock; new vaccines for Salmonella, Brucella, etc.	Broaden application in animal health	Meng et al. (2023; Figl et al. (2023)
Immunoinformatic	Epitope mapping and structure-based redesign	Safer and more refined rLLO analogs	Schijns et al. (2021)

Molecular engineering of rLLO

Efforts to develop engineered rLLO variants with improved safety and specificity are underway. For example, pH-sensitive LLO mutants selectively activate pore formation under acidic endosomal conditions, thereby reducing off-target effects in neutral extracellular environments. In addition, rLLO fusion with cell-targeting ligands or nanoparticle scaffolds enables localized delivery to APCs, enhancing immunological precision (Kim et al., 2015).

Nanoparticle and biomaterial integration

The incorporation of rLLO into nanoparticle-based platforms, including gold nanoparticles, liposomes, and virus-like particles (VLPs), has shown promise in improving antigen stability, endosomal escape, and cytosolic delivery (Table 3), while simultaneously harnessing rLLO’s natural membrane-disrupting properties and reducing systemic toxicity (Singh et al., 2005).

Combination of adjuvant strategies

rLLO can interact with other immunostimulants, such as TLRs ligands or cytokines, as granulocyte-macrophage colony-stimulating factor (Cao et al., 2024). For example, DNA vaccines co-delivering rLLO and chemokines have shown superior tumor immunity in murine models (Peng et al., 2007). These groupings could tailor the immune response more precisely, enhancing both humoral and cell-mediated responses (Wang and Xu, 2020).

Applications in gene and mRNA vaccine platforms

The global shift toward nucleic acid vaccine technologies, rLLO, is being explored to enhance the intracellular delivery of DNA and mRNA vaccines (Lukeman et al., 2025). In addition, an LLO–protamine fusion protein has been shown to improve DNA transfection efficiency both in vitro and in vivo, providing a foundation for future mRNA vaccine designs that exploit rLLO-mediated endosomal escape (Kim et al., 2015).

Veterinary expansion and field testing

Field trials of VPM1002-based vaccines for bovine TB are underway in veterinary medicine (Kaufmann et al., 2014). Beyond TB, rLLO could be integrated into vaccines for other economically important livestock diseases, such as Salmonella or Eimeria, in poultry, where Th1-skewed immunity is advantageous because it promotes cell-mediated immune responses and IFN-γ production that are critical for controlling intracellular pathogens (Figl et al., 2023; Yao et al., 2023). The demonstrated safety and immunogenicity of rLLO-based platforms in multiple animal systems support their potential expansion to livestock species such as swine, poultry, and ruminants for vaccines targeting intracellular pathogens (De Azevedo et al., 2015; Hayati et al., 2017; Figl et al., 2023; Yao et al., 2023). To date, no rLLO-based adjuvant has been formally approved for use in human or veterinary vaccines; however, rLLO-containing platforms, such as the recombinant BCG vaccine VPM1002, are currently undergoing clinical evaluation (Kaufmann et al., 2014).

Rational design via immunoinformatics

Advances in immunoinformatics and structural biology have enabled the rational design of rLLO variants (Ostuni et al., 2021). Computational tools are being used to optimize T cell epitopes, reduce immunopathology, and enhance stability (Suleman et al., 2022). Such epitope-guided engineering can yield safer, more effective adjuvants with tailored immune signatures (Schijns et al., 2021) (Table 3).

Experimental use of rLLO in vaccine platforms

Tables 4 and 5 summarize key studies evaluating rLLO as an adjuvant or delivery enhancer in vaccines targeting bacterial and viral pathogens across both human and veterinary models, highlighting rLLO constructs, immune responses, immunization strategies, and experimental outcomes that collectively demonstrate its role in enhancing cellular and humoral immunity and facilitating antigen delivery in DNA-, protein-, and live-attenuated vaccine platforms.

Statistical overview of rLLO-based vaccine and delivery system studies

The statistical and inferential evidence summarized in Tables 6 and 7 collectively demonstrate a consistent trend toward enhanced cellular and Th1-biased immune responses in rLLO-based vaccine strategies. Experimental vaccine studies (Table 6) frequently reported statistically significant increases in cytokine production, CD8⁺ T-cell activation, and protective efficacy compared with conventional formulations. Complementarily, delivery system–oriented studies (Table 7) provided functional and inferential support by demonstrating improved antigen delivery and immune polarization. Together, these findings substantiate the role of rLLO as a key component in next-generation immunization platforms without necessitating quantitative meta-analysis.

Safety, cytotoxicity, and regulatory considerations

Regardless of the show's potential immunological capability of rLLO, more than a few safety- and regulation-correlated challenges must be considered. Wild-type LLO is a member of the CDC family and has potent pore-forming and cytolytic activity, which initiates its function as a crucial virulence factor of L. monocytogenes (Vadia and Seveau, 2004; Hamon et al., 2012). This integral cytotoxicity promotes safety concerns for vaccine applications if membrane destruction is not tightly controlled. Although recombinant and detoxified variants of LLO have been developed to reduce cytotoxic effects, LLO-mediated membrane permeabilization and calcium influx can still induce inflammasome activation, apoptosis, and proinflammatory signaling in a dose-dependent manner (Seveau et al., 2004; Nguyen et al., 2019).

Preclinical research commonly describes suitable safety profiles for rLLO-based vaccine platforms; nevertheless, heterogeneity in delivery systems, antigen fusion strategies, dosing regimens, and administration routes confuses direct comparisons across studies and underscores the need for homogeneous toxicological estimation (Kim et al., 2015; Rodriguez-Del Rio et al., 2015). From a regulatory perspective, additional challenges include manufacturing consistency, long-term stability, and the classification of rLLO as either a functional antigen carrier or an immunological adjuvant, all of which may influence regulatory trials for agreement (Del Giudice et al., 2018). At the same time, these deliberations highlight the importance of rigorous safety evaluation and clear regulatory frameworks for the extensive implementation of rLLO-based vaccine components in clinical or veterinary settings.

Limitations of the review

Despite a comprehensive literature search across multiple databases, this narrative review has limitations. Restricting sources to peer-reviewed English-language articles may have resulted in the omission of relevant findings published in other languages or gray literature. Furthermore, heterogeneity in study designs, experimental models, and immunological endpoints precluded quantitative synthesis, necessitating a qualitative narrative approach. These constraints should be considered when interpreting the findings, and future research with standardized methodologies should be encouraged.

Conclusion

rLLO has great potential as a multifunctional adjuvant and delivery enhancer in vaccine development. rLLO induces innate and adaptive immune responses, including CD8⁺ T cell activation, to attack intracellular infections and tumors. This is accomplished by phagosomal escape, NLRP3 inflammasome activation, and cytosolic antigen delivery. Compared with distinctive adjuvants, rLLO has distinct advantages, as proven in a variety of investigational models targeting bacterial, viral, and tumor antigens in both human and animal contexts. Furthermore, its combination with delivery systems, such as nanoparticles, liposomes, and DNA vectors, boosts its immunogenic capacity. However, issues persist. More research is desirable to control the particular immunological receptors occupied in rLLO identification, its safety profile in susceptible individuals, and dosage optimization. To wholly use rLLO's capabilities, extra study into molecular engineering, combination tactics, and clinical evaluation is essential. In conclusion, rLLO is a practical approach for next-generation vaccines, with the potential to increase immunogenicity, broaden protection, and increase vaccine efficacy across species and disease conditions.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

This analysis did not obtain any specific grant from community, commercial, or not-for-profit agencies.

Authors' contributions

Thamer J. Shihab and Eman Hashim Yousif drafted the major and minor sections of this review. The authors have reviewed and approved the final version of the manuscript.

Data availability

This narrative review does not include any original datasets. The statistical results presented in Tables 6 and 7 were taken from peer-reviewed, previously published studies that were referenced throughout the text. All underlying data may be found in the cited corresponding open-access papers.