Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system\'s effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Mucosal vaccination presents a promising complement to parenteral vaccination. Bacterium-like particles (BLPs), peptidoglycan structures prepared from lactic acid bacteria, are explored as potential nasal vaccine adjuvants for respiratory infections. To date, studies on BLP-adjuvanted nasal vaccines against intestinal infections have remained limited. In this study, we demonstrated the efficacy of intranasal BLP-adjuvanted vaccination in controlling intestinal infections using the Citrobacter rodentium (C. rodentium) model in C57BL/6 mice. Intranasal vaccination of Intimin, an adhesin critical for intimate bacterial adhesion to colonic epithelial cells, combined with BLP (BLP+I) elicited robust Intimin-specific intestinal secretory IgA production, reduced bacterial load in feces and almost completely inhibited colonic hyperplasia, a characteristic symptom of C. rodentium infection in mice. Conversely, parenteral vaccination with Alhydrogel-adjuvanted Intimin failed to induce intestinal Intimin-specific IgA production, resulting in poor protection against C. rodentium infection. This underscores the pivotal role of mucosal IgA responses elicited by intranasal immunization in its protective efficacy. As this study did not delineate the precise protective mechanism conferred by BLP+I intranasal immunization against C. rodentium infection, further elucidation of the mechanisms underlying intranasal BLP+I immunization is required.

Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F3-GEM and H4-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F3 and H4-GEM was also designed, a mixture consisting of F3-GEM and H4-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F3-GEM, H4-GEM or F3 and H4-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F3 and H4-GEM group was higher than the other two groups. Additionally, F3 and H4-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F3 and H4-GEM induce IgG and neutralizing antibodies\' response in dogs. Conclusions: In summary, F3 and H4-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F3 and H4-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Developing novel antibacterial strategies has become an urgent requisite to overcome the increasing pervasiveness of antimicrobial-resistant bacteria and the advent of biofilms. Aggregation-induced emission-based photosensitizers (AIE PSs) are promising candidates due to their unique photodynamic and photothermal properties. Bioengineering structure-inherent AIE PSs for developing thin film coatings is still an unexplored area in the field of nanoscience. We have adopted a synergistic approach combining plasma technology and AIE PS-based photodynamic therapy to develop coatings that can eradicate bacterial infections. Here, we loaded AIE PSs within biomimetic bacterium-like particles derived from a probiotic strain, Lactobacillus fermentum. These hybrid conjugates are then immobilized on polyoxazoline-coated substrates to develop a bioinspired coating to fight against implant-associated infections. These coatings could selectively kill Gram-positive and Gram-negative bacteria, but not damage mammalian cells. The mechanistic studies revealed that the coatings can generate reactive oxygen species that can rupture the bacterial cell membranes. The mRNA gene expression of proinflammatory cytokines confirmed that they can modulate infection-related immune responses. Thus, this nature-inspired design has opened a new avenue for the fabrication of a next-generation antibacterial coating to reduce infections and associated burdens.

Canine distemper virus (CDV) poses a severe threat to both domesticated and wild animals, including multiple carnivores. With the continued expansion of its host range, there is an urgent need for the development of a safer and more effective vaccine. In this study, we developed subunit vaccines based on a bacterium-like particle (BLP) delivery platform containing BLPs-F and BLPs-H, which display the CDV F and H glycoprotein antigens, respectively, using the antigen-protein anchor fusions produced by a recombinant baculovirus insect cell expression system. The combination of BLPs-F and BLPs-H (CDV-BLPs), formulated with colloidal manganese salt [Mn jelly (MnJ)] adjuvant, triggered robust CDV-specific antibody responses and a substantial increase in the number of interferon gamma (IFN-γ)-secreting CD4+ and CD8+ T cells in mice. Dogs immunized intramuscularly with this vaccine not only produced CDV-specific IgG but also displayed elevated concentrations of IFN-γ and interleukin 6 in their serum, along with an increase of the CD3+CD4+ and CD3+CD8+ T cell subsets. Consequently, this heightened immune response provided effective protection against disease development and reduced viral shedding levels following challenge with a virulent strain. These findings suggest that this BLP-based subunit vaccine has the potential to become a novel canine distemper vaccine.
OBJECTIVE: Many sensitive species require a safe and effective distemper vaccine. Non-replicating vaccines are preferred. We constructed subunit particles displaying canine distemper virus (CDV) antigens based on a bacterium-like particle (BLP) delivery platform. The CDV-BLPs formulated with theMn jelly adjuvant induced robust humoral and cell-mediated immune responses to CDV in mice and dogs, thereby providing effective protection against a virulent virus challenge. This work is an important step in developing a CDV subunit vaccine.

Feline herpesvirus 1 (FHV-1) is a highly transmissible virus that mainly causes ocular and upper respiratory infections in cats and seriously threatens the health of domestic cats and captive or wild cats (such as tigers, cheetahs, and lions). Vaccination is crucial to reduce the incidence rate and mortality of cats infected with FHV-1. In this study, three bacterium-like particles (BLPs) displaying the gB, gC, and gD proteins of FHV-1 were constructed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Indirect immunofluorescence assay, western blot, and electron microscopy results showed that gB, gC or gD protein of FHV-1 was successfully displayed on the surface of GEM particles. Additionally, we designed one more BLPs, designated gB&gC&gD-GEM, which consisted of a mixture of gB-GEM, gC-GEM, and gD-GEM at a protein content ratio of 1:1:1. Mice were immunized with the four BLPs mixed with Gel02 adjuvant, and the results indicated that neutralizing antibody level in the gB&gC&gD-GEM group was superior than those in the other groups. Moreover, gB&gC&gD-GEM significantly increased the secretion of cytokines, as well as the activation and maturation of B cells. It also boosted the production of central memory T cells among CD4 + and CD8 + T cells. Moreover, gB&gC&gD-GEM mixed with Gel02 adjuvant provoked an antibody response in cats. In conclusion, the BLPs vaccine prepared from gB&gC&gD-GEM induced specific humoral and cellular immune responses to FHV-1 and be used as a potential vaccine candidate for the control of FHV-1 infection in cats.

Bacterium-like particles (BLPs) are hollow peptidoglycan particles obtained from food-grade Lactococcus lactis inactivated by hot acid. With the advantage of easy preparation, high safety, great stability, high loading capacity, and high mucosal delivery efficiency, BLPs can load and display proteins on the surface with the help of protein anchor (PA), making BLPs a proper delivery system. Owning to these features, BLPs are widely used in the development of adjuvants, vaccine carriers, virus/antigens purification, and enzyme immobilization. This review has attempted to gather a full understanding of the technical composition, characteristics, applications. The mechanism by which BLPs induces superior adaptive immune responses is also discussed. Besides, this review tracked the latest developments in the field of BLPs, including Lactobacillus-derived BLPs and novel anchors. Finally, the main limitations and proposed breakthrough points to further enhance the immunogenicity of BLPs vaccines were discussed, providing directions for future research. We hope that further developments in the field of antigen delivery of subunit vaccines or others will benefit from BLPs.

Porcine epidemic diarrhea virus (PEDV) causes acute watery diarrhea and high mortality in newborn piglets. Activation of intestinal mucosal immunity is crucial to anti-PEDV infection. To develop a vaccine capable of stimulating intestinal mucosal immunity, we prepared a bacterium (Lactococcus lactis)-like particle (BLP) vaccine (S1-BLPs) displaying the S1 protein, a domain of PEDV spike protein (S), based on gram-positive enhancer matrix (GEM) particle display technology. We further compared the effects of different vaccination routes on mucosal immune responses in mice induced by S1-BLPs. The specific IgG titer in serum of intramuscularly immunized mice with S1-BLPs was significantly higher than that of the intranasally administered. The specific IgA antibody was found in the serum and intestinal lavage fluid of mice vaccinated intranasally, but not intramuscularly. Moreover, the intranasally inoculated S1-BLPs induced higher levels of IFN-γ and IL-4 in serum than the intramuscularly inoculated. In addition, the ratio of serum IgG2a/IgG1 of mice inoculated intramuscularly was significantly higher with S1-BLPs compared to that of with S1 protein, suggesting that the immune responses induced by S1-BLPs was characterized by helper T (Th) cell type 1 immunity. The results indicated that S1-BLPs induced systemic and local immunity, and the immunization routes significantly affected the specific antibody classes and Th immune response types. The intranasally administered S1-BLPs could effectively stimulate intestinal mucosal specific secretory IgA response. S1-BLPs have the potential to be developed as PEDV mucosal vaccine.