Bosma et al. first described a simple and rapid method for BLPs preparation, which is still in use to date: Lactococcus lactis are boiled in hot acid for 30 minutes. This procedure deprives the entire intracellular contents of bacterial cells including proteins, nucleic acids and lipophosphatidic acids and extracellular macromolecules. And the degradation products and acid will be removed by washing with buffer three times (14). It has been found that hot HCl, TCA or H₂SO₄ treatment led to successful BLPs production, and the acid concentration of 10% is the best (24). After treatment, the resulting hollow particles essentially are L. lactis cell wall peptidoglycan (PGN) shell which retain their original shape and size (1–2 μm) after treatment. This method is generally applicable to gram-positive bacteria, not only for L. lactis. Apart from L. lactis, the peptidoglycan backbones of Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus rhamnosus and Corynebacterium pseudodiphtheriticum are also resistant to hot acid treatment and can also be successfully prepared into BLPs (25–27). More details regarding Lactobacillus-derived BLPs are provided in the following “Advances in Lactobacillus-derived BLPs development” section.

PA is currently the most widely used anchor in the BLPs system. With the bridging of PA, BLPs can be used as display carriers for exogenous proteins. PA derives from the peptidoglycan-binding domain of L. lactis cell-wall hydrolase AcmA which consists of 437 amino acids and contains three domains: N-terminal signal sequence region, central active region, and C-terminal domain referred to as PA. PA contains three repeating lysine motifs (LysM), which are composed of about 45 amino acids separated by spacer sequences. LysM is a very ubiquitous cell wall-binding domain (CWBD), primarily present in cell wall hydrolases for bacteria (28).Bateman and Bycroft analyzed the three-dimensional structure of LysM. The results of nuclear magnetic resonance (NMR) showed that the LysM domain derived from membrane-bound lytic murein transglycosylase D (MltD) in Escherichia coli contains two LysMs which has a βααβ secondary structure (29). Strong binding of LysM to bacteria cell wall has been previously reported in a patent (30). The specific binding of the LysM motif to the peptidoglycan backbone is by non-covalent bonding, and the binding ability is so strong that anchored protein can only be dissociated by SDS or 8M LiCl treatment. In this case, the binding of the protein of interest to BLPs can be achieved by fusing the protein with PA.

At present, the essence of molecules displayed on the BLPs surface are proteins or peptides. Most proteins displayed on the surface of BLPs are pathogenicity-related proteins of bacteria, viruses and parasites, which are used to improve the immune effect of subunit vaccines. Protein-PA fusions have been expressed successfully by prokaryotic hosts (E. coil and L. lactis) and some eukaryotic hosts (insect, yeast, tobacco, HEK, and CHO cells). Simple incubating target protein fused with PA and BLPs can realize the display of exogenous protein. The addition of protein does not seem to affect the high affinity of PA for BLPs. Notably, both in the biophysical properties (31, 32) and biological functions, various protein molecules displayed on the surface of BLPs have shown promising activity and function.

Protein linkers are crucial in enhancing binding capacity by facilitating mobility and flexibility in the fusion protein. This enables the protein to achieve an ideal orientation within the peptidoglycan structure (33). In terms of vaccine development, it has been shown that the use of flexible linker peptides “(Gly-Gly-Ser-Gly) × 2” between the PA and antigenic protein RBD could enhance the binding ability of the PA thereby improve the immunogenicity of the RBD in the MERS-CoV BLPs mucosal vaccine (34). In their subsequent work, this linker was also inserted between the PA and antigenic protein Sudan virus (SUDV) glycoprotein (eGP). The intramuscular administration to mice of adjuvanted eGP-BLPs elicited high specific IgG and neutralizing antibody titers. Likewise, another study has also applied a flexible linker “(Gly-Gly-Gly-Gly-Ser) × 3” between the PA and antigenic protein gp120 to the design of HIV BLPs vaccine which achieved good immune effects (35, 36).

Experimentally, more than 97.5% of the TCA treated staphylococcus aureus cell wall is N-Acetylglucosamine (the main component of peptidoglycan) (37). In the study of the structure and composition of BLPs, SDS-PAGE analysis denoted that L. lactis MG1363 contained its own protein ( Figure 2A , lane 2), and after 10% TCA treatment, BLPs contained almost no protein components ( Figure 2A , lane 3) (38). As shown in the Scanning electron microscopic ( Figure 2B ) (14) and Transmission electron microscopy ( Figure 2C ) (17) images, BLPs retain the original bacterial size and shape, but the cellular contents are partially degraded and the peptidoglycan backbone is exposed.

Ever since Hansson et al. pioneered the display of proteins on the surface of Staphylococcus xylosus, numerous proteins have been successfully exhibited on living bacterial surfaces. However, in all instances, the host bacteria are genetically modified organisms (GMOs), potentially carrying foreign and altered genes. This raises concerns about environmental dispersion, horizontal gene transfer to other bacteria, and associated safety risks (14). In comparison, BLPs are prepared from non-pathogenic food-grade LAB from a variety of sources. Heat acid-treated BLPs are non-living, non-genetically modified organisms and do not contain any nucleic acid material, minimizing the risk of transmission of recombinant DNA to the environment. In a preclinical GLP toxicity trial, BLPs were administered intranasally to rabbits, and no adverse events were reported (39). Furthermore, the safety of an intranasal influenza vaccine prepared by mixing seasonal trivalent inactivated influenza vaccine with BLPs has been assessed in humans in a phase I clinical trial, resulting in no adverse events reported (20).

BLPs in the absence of extracellular components bind to PA strongly on the surface, thus, loaded BLPs were stable storing in PBS or as a freeze-dried powder at room temperature, 4°C, and −80°C, and no loss or degradation of the PA fusions were observed (14). In a representative example, Yersinia pestis V immunogen was used to determine the binding force between V-PA and BLPs in AFM-based single-molecule force measurement, and the binding force at physiological pH was about 51.33 ± 5.78 pN which is notably larger than it between HIV gp120 and CD4 (about 42.25 ± 34.14 pN) (40).

Some studies have shown that the number of LysM domain in the anchor effect the stability of BLPs surface display system. As Steen et al. demonstrated, fusion protein with three LysM domains from AcmA has optimal peptidoglycan binding properties and biological activities (41). Indeed, PA with three repeats of LysM is by far the most widely used protein anchor for BLPs. In contrast, one study demonstrated that fusion proteins containing two LysM domains exhibit significantly enhanced binding activity towards BLPs compared to other numbers of domains (14). In a recent study, the binding stability of Venus-LysM proteins to BLPs at different NaCl, urea concentrations and at different temperatures and pH was tested. The result showed that under unfavorable conditions (high concentrations of urea and NaCl and low temperature), the stability of Venus-Acglu-BLPs (containing five repeats of LysM motif) was stronger than that of Venus-Pgb-BLPs (containing one LysM motif), but at different pH (4, 7.4 and 9), there was no statistical difference between them. This result indicated that the higher the number of LysM motifs, the stronger the resistance of Venus-LysM-BLPs to adverse conditions at equimolar concentrations. However, the study also showed that five LysM motifs from Acglu did not exhibit a greater binding capacity to BLPs than one LysM motif in Pgb at the same micromolar concentration (42). Therefore, we deduced that rather than the optimal number of LysM domains, it seems that the binding stability depends more on the protein from which the anchor is derived.

Compared with live L. lactis surface display system, BLPs with their peptidoglycan binding sites fully exposed have a higher PA display density which distribute over the entire surface of BLPs (43). As determined by Bosma et al., the maximum PA binding capacity is 10⁶ molecules per BLP cell compares favorably with the surface density of PA on BLPs in Staphylococcus carnosus and Bacillus subtilis spore surface display platform with 2–3 orders of magnitude enhancement, respectively (14). The single-molecule sensitive NSOM/QD based dipole mission fluorescence imaging system was employed to analyze binding density, and based on a conservative estimate, there were at least ~3000 QD-bound V-PA immunogen molecules on a single bacterium-like particles. Not only that, the density of V-PA immunogen was ~1492 molecules/mm² which is significantly higher than the density of ~866 CD4 molecules/mm² on a CD4⁺ T-cell (40). High loading capacity makes BLPs surface system an excellent platform for the large-amount and high-density loading of proteins.

The whole mucosal immune system is interconnected, indicating the vaccination of the local mucosa activates the whole mucosal system, producing antibodies in remote mucosal areas other than the site of vaccination (35). BLPs retain the size of live L. lactis, of about 1 μm, suitable for uptake by M cells located in Peyer’s patches located in gut-associated lymphoid tissue (GALT), which together with the nasal-associated lymphoid tissue (NALT) form a typical mucosa immune system (CMIS) (14). Secondly, mucosal immunization, which mimics the natural way pathogens invade the organism, activates mucosal immunity (sIgA) and induces a systemic immune response (IgG). In addition, this needle-free immunization reduces immune side effects and is suitable for mass vaccination and multiple immunizations.

Non-antigen restricted binding of protein-PA to BLP makes it possible for multiple antigenic proteins to be simultaneously anchored to the same BLP cell. Two B-cell antigenic epitopes of Plasmodium berghei malaria circumsporozoite protein were simultaneously displayed on BLPs resulting in significant levels of IgG antibodies in vaccinated mice intranasally (19). The trivalent and bivalent vaccine based on three Streptococcus pneumoniae proteins IgA1p, SlrA and PpmA provided good protection and reduced bacterial load in mice with no antigen antagonism was observed. Moreover, Moreover, when the three antigens were prepared as a bivalent vaccination or a trivalent vaccine with 1.5 times less of this antigen, the IgG antibodies generated against that antigen were equal (44). Additionally, two forms of bivalent vaccine: tHA^{H5N6 +H9N2} BLPs and a mixture of tHA^H5N6 BLPs and tHA^H9N2 BLPs both led to potent immune reactions without additional adjuvants (45).

Vaccination is one of the most effective preventative measures against pathogenic infection. According to World Health Organization estimates, it is determined that vaccines prevent 3.5–5 million deaths annually (https://www.who.int/health-topics/vaccines-and-immunization#tab=tab_1). And in the first year after the COVID-19 vaccination, it saved nearly 14.4 million lives around the world (46). While for some diseases, there is no effective vaccine available (47). Therefore, novel vaccines combined safety and immunogenicity is desperately needed. Using bacteria-based carrier as novel vaccine strategies, people set their sights on BLPs.

Acting as adjuvants, free-standing BLPs can be mixed simply with existing vaccines or purified antigens to enhance immunogenicity. In particular, the effect of BLP on enhancing the immunity of influenza vaccines has been intensively studied by Leenhouts et al. Adjuvantation of IN-administered influenza monovalent split vaccine of strain A/Beijing/262/95 (H1N1) with BLPs effectively enhanced serum IgG and HI levels and sIgA titers in nose, lung and vaginal in mice resulting in a 100% protection against homologous and heterologous influenza infection with reduction of viral titer in lungs (16). Oral immunization with monovalent subunit vaccine of strain A/Hiroshima (H3N2) mixing BLPs elicited not only systemic and local antibody responses but also a more balanced Th1/Th2 response compared to inactivated influenza vaccines added with alum (57). The administration of combined BLPs and seasonal HA (A/Wisconsin/67/2005 [H3N2]) resulted in notably elevated systemic immune responses and a more balanced Th1/Th2-type immune response compared to the administration of HA alone via the intramuscular route. The addition of BLPs HA saved 25–125 times of the antigen dose and resulted in similar HI titers (58). What’s more, just one boost is required for i.n. BLPs adjuvanted influenza monovalent subunit vaccine to reach an equal level of HI titers in serum to the conventional i.m. route. Nasal immunization with a subunit vaccine mixed with BLPs led to a decrease in the quantity of IL-4-producing cells, while significantly increasing the quantity of IFN-γ-producing cells, indicating a shift of the immune response from a balanced Th1/Th2 to a predominant Th1-type response (39). Nasal vaccination with BLPs adjuvanted split influenza vaccine of influenza A strain (A/Sydney/5/97[H3N2]) induced IAV-specific IgG2c antibody production and Th1/Th17 immunological responses (59). The safety and immunogenicity evaluation of BLPs-based vaccines in humans achieved good results in FluGEM-a phase I clinical trial (20). A/turkey/Turkey/1/2005 (H5N1) virus fatal viral challenge by the combination intranasal/intratracheal route is entirely prevented by passive inhalation of dry powder influenza vaccine formulations, and no virus was detected in choanal or cloacal swabs of vaccinated chickens (60).

Admixing low doses of BLPs with a live H120 IBV vaccine via ocular administration to chickens demonstrated the ability of rapidly boosting higher specific serum IgG titer levels, even at two weeks after hatching (61). BLPs mixed with inactivation of NDV antigens induced high levels of specific serum IgG and mucosal IgA titers. Intranasal vaccination of chickens results in serum HI titers of up to 2⁸, providing a complete protection against a challenge with a fatal dose of virulent NDV without any illness symptoms (17). Oral immunization with commercial live rotavirus vaccine with BLPs derived from immunomodulatory Lactobacillus rhamnosus resulted in enhanced levels of specific serum IgG and IgA, increased numbers of CD24⁺B220⁺ B and CD4⁺ T cells in Peyer’s patches and spleen as well as augmented production of IFN-γ (25). Similarly, oral immunization with hepatitis E virus capsid protein (ORF2) with BLPs also achieved a satisfactory immunity effect (26).

Vaccines for the prevention or treatment of viral diseases are mainly based on attenuated or inactivated viruses and recombinant viral proteins that are produced within different host cells. However, in the production process of viruses, downstream purification processing has always been a challenge due to the different structures and properties of different viruses (62). An optimal clarification process should effectively eliminate cellular debris and sizable aggregates, which may arise from either the production or processing stages (63). The usual methods of virus purification are ultracentrifugation precipitation (density gradient), ultrafiltration and size exclusion chromatography (molecular size), ion exchange chromatography (charge) and affinity chromatography (specific binding). However, these methods require for significant time and labor input, and the purity and yield of purification cannot be guaranteed. There are many limitations in applying it to vaccine production, especially heterologous substances such as foreign proteins, nucleic acids, lipids, and carbohydrates in the culture medium can easily cause immunosuppression and immune side reactions.

The addition of cell-free culture medium that contains a PA fusion to BLPs leads to a highly effective, strong, and specific surface binding of fusion protein, eliminating the requirement for supplementary purification measures. A single-step centrifugal procedure can be employed to eliminate the recombinant producer cells (14). GRFT, a lectin from the marine red alga Griftsia sp, can tightly bind to glycoproteins on the surface of many enveloped viruses. After incubation of PA-GRFT with Porcine reproductive and respiratory syndrome virus (PRRSV) and BLPs, one-step low-speed centrifugation was employed in order to isolate purified PRRSV. Further experimental results demonstrated that the PA-GRFT partnership could guide PRRSV to the BLPs surface, yielding favorable results in terms of efficiency (64). Additional research using this process to purify additional enveloped animal viruses may result in a brand-new, broadly applicable technique for viral purification (65). Another typical study connected foot-and-mouth disease virus (FMDV)-specific nanobody to BLPs through PA, named GEM-PA-nanotrap, was used to FMDV purification from cellular lysates. The recovery rate of FMDV exceeded 99% and the efficiency of removing nonviral proteins was approximately 98.3% with no effect on activity (66). This method has also been applied to purification of porcine circovirus type 2 (PCV2). They designed a bifunctional recombinant protein linker by fusing the variable heavy chain domain (VHH) antibody directed against PCV2 with PA. 100 mL of PCV2-containing culture supernatant (viral titer: 10^6.5 TCID50 mL^-1) could be purified using 1 unit (2.5×10⁹ particles) of BLPs carrying 80 μg protein linker with a recovery rate up to 99.6%. Based on the decreased total protein content after purification, this technique has an impurity removal effectiveness of about 98%. Moreover, the results of in vivo experimentation demonstrated that piglets that were immunized with purified PCV2 exhibited robust immune responses, effectively protecting them against PCV2 infection (67).

In large-scale industrial and biotechnological applications, enzymes play a growing role as effective and versatile biocatalysts. Enzyme-based catalytic processes have many advantages over traditional synthetic routes (64). Many industrially relevant enzymes are produced by microorganisms under extreme conditions, which will face two problems: enzymes still need to be purified and ensure the stability of the enzyme. Immobilizing enzymes on different types of supports is a highly appealing concept for addressing these limitations (68, 69). Appropriate immobilization systems can improve the stability of enzymes under harsh reaction conditions such as extreme pH, high temperature or the presence of organic solvents, making it easier to separate from the reaction medium. Also, certain immobilization procedures allow simultaneous purification and immobilization of enzymes from crude extracts (70).

Mediated by PA, B. licheniformis α-amylase (AmyL) and E. coli β-lactamase can be immobilized on the surface of BLPs in controlled ratios with no need for chemical treatments or extensive purification steps (14). Subtilisin QK-2 surface displayed onto BLPs was more stable in the simulated gastric juice without a loss of fibrinolytic activity (71). The BLPs could immobilize over 75% of the enzyme activity of lipases with no need to add ions to improve the activity (72).

DCs are the most critical antigen-presenting cells (APCs) with the strongest ability to activate T-naive cells. It has been shown that BLPs activate and promote the maturation of BM-derived CD11c⁺ cells of neonatal and adult mouse in vitro exhibiting an increased expression of activation and maturation cell-surface markers (CD80, CD86, CD40 and MHC-II) and secretion of proinflammatory cytokines (IL-12p70, TNF-α, IL-10, IL-6, IFN-γ, and MCP-1) (18). DCs maturation was also observed an in vivo study. An increase in CD19⁺ CD40⁺ double-positive cells nodes and CD11c⁺ MHC-II⁺ cells was observed inguinal lymph nodes early in Zika virus E protein-BLPs vaccine immunization (73).

As key executors of innate and adaptive immunity, macrophages play an important role. BLPs uptake in macrophages was observed in a previous study (74), along with the production of TNF-α. Another study showed both RAW264.7 and DC2.4 cells had a strong ability to uptake BLPs displaying CSFV E2 protein (75).

Respiratory mucosal tissues are equipped with diverse antigen uptake and presentation systems, and antigen transport across the nasal epithelial barrier is the first step in the induction of a mucosal immune response upon mucosal vaccination (76). Antigens in the nasal cavity are taken up in three forms: epithelial cells, M cells, and lamina propria DCs (77). Epithelial cells, as a non-professional APC, play a certain role in the uptake and presentation of antigens. By using an in vitro human nasal epithelial cell activation assay, it was demonstrated that the activation of in vitro human nasal epithelial cells by BLPs leads to the upregulation of IL-6 and IL-8. Additionally, the expression of chemotaxis and activation-related factors of DCs, CCL-20 and TSLP, is also induced by BLPs (78).. M cells are highly specialized epithelial cells with unique structural advantages compared to other epithelial cells, especially the deep invagination of the basolateral membrane to facilitate the encapsulation of lymphocytes (79). The distinctive capabilities of M cells allow for the targeted and effective transport of inhaled or ingested luminal antigens to APCs situated in the M cell pocket or in close proximity to the FAE of MALT. These functions are crucial for the uptake and internalization of antigens, as well as the initiation of immune responses within the mucosal system (79). In order to study the interaction of M cells with BLPs, mice were immunized intranasally with fluorescently labeled BLPs. Staining of M cells in mouse nasal mucosa after 15 min showed efficient uptake and internalization of BLPs by M cells (78). In addition, some lamina propria DCs extend their dendrites through the epithelial layer into the lumen, which can directly uptake antigens in the lumen (80).

Innate immune cells act as a bridge between innate and adaptive immunity by presenting antigens and specifically activating T cells. It has been shown that DCs pretreated with Y. pestis LcrV-attached BLPs stimulated CD4⁺ and CD8⁺ T lymphocytes proliferation significantly in vitro and induced an Th1-type response. The cytokines analysis showed that IL-2, IL-12, IL-6, TNF-α, and IL-10 levels increased in the NALT and lung cells produced IL-2, IL-6, IL-5 and IFN-γ under in vitro stimulation (18). High proportions of CD4⁺ CD69⁺ T and CD8⁺ CD69⁺ T cells and the production of IFN-γ, IL-6, IL-4, and IL-10 were observed after in vitro stimulation of splenocytes from Zika virus E protein-BLPs vaccinated mice (81). BLPs adjuvanted rotavirus vaccine and hepatitis E virus capsid protein (ORF2) both induced increased CD45⁺CD3⁺CD4⁺ T cells and levels of IFN-γ, TNF-α, and IL-4 in Peyer’s patches and spleen (25, 26). Oral immunization with Helicobacter pylori antigen CTB-UE anchored BLPs induced a Th1/Th17 memory response owing to proliferation of Th17 cells in the stomach and MLN. Oral inoculation of single-chain insulin (SCI-59) analog attached BLPs repaired Th1/Th2 imbalance and increased CD4⁺CD25⁺FoxP3⁺ Tregs in the PLN CD4⁺ T cell compartment (82).

BLPs vaccines can activation of B cells produces plasma cells, which induce high-affinity antibodies and memory cells, thereby protecting the body from pathogen attack. The study found an increased in CD69 B cells from mouse splenocytes (34), IgG ASCs (specific antibody-secreting cells) in the spleen, BM and NALT and IgA ASCs in NALT (18, 50), CD19⁺ CD40⁺ double-positive B cells in inguinal lymph (81) and CD24⁺ B220⁺ cells in Peyer’s patches (25, 26) were observed in previous studies.

Innate immune system recognizes antigens through pattern recognition receptors (PRR), of which family of toll-like receptors (TLRs) play important roles. TLRs distributed on a broad spectrum of various immune cell membranes or endosomal membranes recognize corresponding ligands is key to activate the innate immune system. Peptidoglycan, the main component of BLPs, is a ligand of TLR2. TLR2 coupled to TLR1 or TLR6 functions as a heterodimer and recognizes BLPs results in the activation of innate immune cells. It has been shown by Ramirez et al. that BLPs only interacted with HEK293 cells expressing human TLR2 leading to NF-κB activation (18). Studies on BLPs-activated TLR receptors are also being conducted in vivo, in one study using BLPs mixed split influenza vaccine. Nasally immunized TLR2KO mice showed decreased serum IgG response and sIgA levels in the nasal and lung lavages and lower numbers of IFN-γ producing T-cells and B-cells both in the local dLN and spleen, compared with wt control mice (59).

Additionally, there is a growing interest to applying BLPs to avian vaccines, to this end, explore the novel mechanism of action of BLPs in birds is needed. Yang et al. found by NF-κB reporter analysis that DF-1 cells (chicken embryo fibroblast cells) recognize BLPs through the combination of chTLR2t1 and chTLR1t1, and this result is consistent with the latest research (83). Furthermore, in HD11 cells (chicken macrophages), BLPs caused NO production and increased IFN-γ, IL-6, IL-1β and iNOS levels (17).