Lactiplantibacillus plantarum as a potential adjuvant and delivery system for the development of sars-cov-2 oral vaccines

The most important characteristics regarding the mucosal infection and immune responses against the Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) as well as the current vaccines against coronavirus disease 2019 (COVID-19) in development or use are revised to emphasize the opportunity for lactic acid bacteria (LAB)-based vaccines to offer a valid alternative in the fight against this disease. In addition, this article revises the knowledge on: (a) the cellular and molecular mechanisms involved in the improvement of mucosal antiviral defenses by beneficial Lactiplantibacil-lus plantarum strains, (b) the systems for the expression of heterologous proteins in L. plantarum and (c) the successful expressions of viral antigens in L. plantarum that were capable of inducing protective immune responses in the gut and the respiratory tract after their oral administration. The ability of L. plantarum to express viral antigens, including the spike protein of SARS-CoV-2 and its capacity to differentially modulate the innate and adaptive immune responses in both the intestinal and respiratory mucosa after its oral administration, indicates the potential of this LAB to be used in the development of a mucosal COVID-19 vaccine.Fil: Villena, Julio Cesar. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Li, Chang. Chinese Academy of Sciences; República de ChinaFil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino; ArgentinaFil: Sacur, Jacinto Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino; ArgentinaFil: Ren, Linzhu. Jilin University; ChinaFil: Kitazawa, Haruki. Tohoku University; Japó

Protection of DNase in the shell of a pH-responsive, antibiotic-loaded micelle for biofilm targeting, dispersal and eradication

DNase can break down the extracellular matrix that keeps infectious bacterial biofilm together through cleavage of eDNA. Herewith, biofilm bacteria can become dispersed to assist antibiotic eradication but this has hitherto remained an in vitro possibility. In vivo DNase is rapidly broken down in blood, impeding blood-injection of DNase combined with antibiotics to cure bacterial infections. Herein, we report the synthesis of pH-responsive, self-targeting micelles self-assembled from a solution of poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) and poly(ε-caprolactone)-block-poly(amino ester) (PCL-b-PAE) with DNase conjugated to PAE-blocks. At physiological pH, this conjugation protected DNase inside the micellar shell, while PEG prevented adsorption of blood-borne proteins to the micelles. PAE became positively-charged below pH 6.4 facilitating self-targeting to an infectious biofilm. Simultaneously, PAE became hydrophilic and stretched to expose DNase upon accumulation in an infectious S. aureus biofilm where it degraded the biofilm matrix. PEG/PAE-DNase micelles internally core-loaded with ciprofloxacin significantly better eradicated murine pneumonia after blood-injection than ciprofloxacin-loaded PEG/PAE micelles without conjugated DNase or ciprofloxacin free in solution. Considering that DNase is clinically approved for use in cystic fibrosis patients, this paves the way for clinical translation of ciprofloxacin-loaded, PEG/PAE-DNase micelles for the treatment of pneumonia and other infections that can be reached through self-targeting after blood-injection

Synergy between pH- and hypoxia-responsiveness in antibiotic-loaded micelles for eradicating mature, infectious biofilms

Antibiotic-loaded PEG/PAE-based micelles are frequently considered for eradicating infectious biofilms. At physiological pH, PEG facilitates transport through blood. Near an acidic infection-site, PAE becomes protonated causing micellar targeting to a biofilm. However, micellar penetration and accumulation is confined to the surface region of a biofilm. Especially matured biofilms also possess hypoxic regions. We here designed dual-responsive PEG/PAE-b-P(Lys-NBCF) micelles, responding to both acidity and low oxygen-saturation level in matured biofilms. Dual, pH- and hypoxia-responsive micelles targeted and accumulated evenly over the depth of 7- to 14-days old biofilms. Delineation demonstrated that pH-responsiveness was responsible for targeting of the infection-site and accumulation of micelles in the surface region of the biofilm. Hypoxia-responsiveness caused deep penetration in the biofilm. Dual, pH- and hypoxia-responsive micelles loaded with ciprofloxacin yielded more effective, synergistic eradication of 10-days old, matured Staphylococcus aureus biofilms underneath an abdominal imaging-window in living mice than achieved by ciprofloxacin in solution or single, pH- or hypoxia responsive micelles loaded with ciprofloxacin. Also, wound-healing after removal of window and its frame proceeded fastest after tail-vein injection of ciprofloxacin-loaded, dual, pH- and hypoxia-responsive micelles. Concluding, pH- and hypoxia-responsiveness are both required for eradicating mature biofilms and advancing responsive antibiotic nanocarriers to clinical application. Statement of significance: pH-responsive antibiotic nanocarriers have emerged as a possible new strategy to prevent antimicrobial-resistant bacterial infections from becoming the leading cause of death. In this paper, we show that commonly studied, pH-responsive micellar nanocarriers merely allow self-targeting to an infectious biofilm, but do not penetrate deeply into the biofilm. The dual-responsive (acidic pH- and hypoxia) antibiotic-loaded micelles designed here not only self-target to an infectious biofilm, but also penetrate deeply. The in vitro and in vivo advantages of dual-responsive nanocarriers are most obvious when studied in infectious biofilms grown for 10 viz a viz the 2 days, usually applied in the literature. Significantly, clinical treatment of bacterial infection usually starts more than 2 days after appearance of the first symptoms

A Guanosine-Quadruplex Hydrogel as Cascade Reaction Container Consuming Endogenous Glucose for Infected Wound Treatment-A Study in Diabetic Mice

Diabetic foot ulcers infected with antibiotic‐resistant bacteria form a severe complication of diabetes. Antimicrobial‐loaded hydrogels are used as a dressing for infected wounds, but the ongoing rise in the number of antimicrobial‐resistant infections necessitates new, nonantibiotic based designs. Here, a guanosine‐quadruplex (G(4))‐hydrogel composed of guanosine, 2‐formylphenylboronic acid, and putrescine is designed and used as a cascade‐reaction container. The G(4)‐hydrogel is loaded with glucose‐oxidase and hemin. The first cascade‐reaction, initiated by glucose‐oxidase, transforms glucose and O(2) into gluconic acid and H(2)O(2). In vitro, this reaction is most influential on killing Staphylococcus aureus or Pseudomonas aeruginosa in suspension, but showed limited killing of bacteria in biofilm‐modes of growth. The second cascade‐reaction, however, transforming H(2)O(2) into reactive‐oxygen‐species (ROS), also enhances killing of biofilm bacteria due to hemin penetration into biofilms and interaction with eDNA G‐quadruplexes in the biofilm matrix. Therewith, the second cascade‐reaction generates ROS close to the target bacteria, facilitating killing despite the short life‐time of ROS. Healing of infected wounds in diabetic mice proceeds faster upon coverage by these G(4)‐hydrogels than by clinically common ciprofloxacin irrigation. Moreover, local glucose concentrations around infected wounds decrease. Concluding, a G(4)‐hydrogel loaded with glucose‐oxidase and hemin is a good candidate for infected wound dressings, particularly in diabetic patients

Self-targeting of zwitterion-based platforms for nano-antimicrobials and nanocarriers

Self-targeting antimicrobial platforms have yielded new possibilities for the treatment of infectious biofilms. Self-targeting involves stealth transport through the blood circulation towards an infectious biofilm, where the antimicrobial platform penetrates and accumulates in a biofilm in response to a change in environmental conditions, such as local pH. In a final step, nano-antimicrobials need to be activated or the antimicrobial cargo of nanocarriers released. Zwitterions possess both cationic and anionic groups, allowing full reversal in zeta potential from below to above zero in response to a change in environmental conditions. Electrolyte-based platforms generally do not have the ability to change their zeta potentials from below to above zero. Zwitterions for use in self-targeting platforms are usually hydrophilic and have a negative charge under physiological conditions (pH 7.4) providing low adsorption of proteins and assisting blood circulation. However, near or in the acidic environment of a biofilm, they become positively-charged yielding targeting, penetration and accumulation in the biofilm through electrostatic double-layer attraction to negatively-charged bacteria. Response-times to pH changes vary, depending on the way the zwitterion or electrolyte is built in a platform. Self-targeting zwitterion-based platforms with a short response-time in vitro yield different accumulation kinetics in abdominal biofilms in living mice than platforms with a longer response-time. In vivo experiments in mice also proved that self-targeting, pH-responsive zwitterion-based platforms provide a feasible approach for clinical control of bacterial infections. Clinically however, also other conditions than infection may yield an acidic environment. Therefore, it remains to be seen whether pH is a sufficiently unique recognition sign to direct self-targeting platforms to an infectious biofilm or whether (additional) external targeting through e.g. near-infrared irradiation or magnetic field application is needed

Encapsulation of photothermal nanoparticles in stealth and ph-responsive micelles for eradication of infectious biofilms in vitro and in vivo

Photothermal nanoparticles can be used for non-antibiotic-based eradication of infectious biofilms, but this may cause collateral damage to tissue surrounding an infection site. In order to prevent collateral tissue damage, we encapsulated photothermal polydopamine-nanoparticles (PDA-NPs) in mixed shell polymeric micelles, composed of stealth polyethylene glycol (PEG) and pH-sensitive poly(β-amino ester) (PAE). To achieve encapsulation, PDA-NPs were made hydrophobic by electrostatic binding of indocyanine green (ICG). Coupling of ICG enhanced the photothermal conversion efficacy of PDA-NPs from 33% to 47%. Photothermal conversion was not affected by micellar encapsulation. No cytotoxicity or hemolytic effects of PEG-PAE encapsulated PDA-ICG-NPs were observed. PEG-PAE encapsulated PDA-ICG-NPs showed good penetration and accumulation in a Staphylococcus aureus biofilm. Penetration and accumulation were absent when nanoparticles were encapsulated in PEG-micelles without a pH-responsive moiety. PDA-ICG-NPs encapsulated in PEG-PAE-micelles found their way through the blood circulation to a sub-cutaneous infection site after tail-vein injection in mice, yielding faster eradication of infections upon near-infrared (NIR) irradiation than could be achieved after encapsulation in PEG-micelles. Moreover, staphylococcal counts in surrounding tissue were reduced facilitating faster wound healing. Thus, the combined effect of targeting and localized NIR irradiation prevented collateral tissue damage while eradicating an infectious biofilm

Self-targeting, zwitterionic micellar dispersants enhance antibiotic killing of infectious biofilms:An intravital imaging study in mice

Extracellular polymeric substances (EPS) hold infectious biofilms together and limit antimicrobial penetration and clinical infection control. Here, we present zwitterionic micelles as a previously unexplored, synthetic self-targeting dispersant. First, a pH-responsive poly(ε-caprolactone)-block-poly(quaternary-amino-ester) was synthesized and self-assembled with poly(ethylene glycol)-block-poly(ε-caprolactone) to form zwitterionic, mixed-shell polymeric micelles (ZW-MSPMs). In the acidic environment of staphylococcal biofilms, ZW-MSPMs became positively charged because of conversion of the zwitterionic poly(quaternary-amino-ester) to a cationic lactone ring. This allowed ZW-MSPMs to self-target, penetrate, and accumulate in staphylococcal biofilms in vitro. In vivo biofilm targeting by ZW-MSPMs was confirmed for staphylococcal biofilms grown underneath an implanted abdominal imaging window through direct imaging in living mice. ZW-MSPMs interacted strongly with important EPS components such as eDNA and protein to disperse biofilm and enhance ciprofloxacin efficacy toward remaining biofilm, both in vitro and in vivo. Zwitterionic micellar dispersants may aid infection control and enhance efficacy of existing antibiotics against remaining biofilm

Coating of a Novel Antimicrobial Nanoparticle with a Macrophage Membrane for the Selective Entry into Infected Macrophages and Killing of Intracellular Staphylococci

Internalization of Staphylococcus aureus by macrophages can inactivate bacterial killing mechanisms, allowing intracellular residence and dissemination of infection. Concurrently, these staphylococci can evade antibiotics that are frequently unable to pass mammalian cell membranes. A binary, amphiphilic conjugate composed of triclosan and ciprofloxacin is synthesized that self-assemble through micelle formation into antimicrobial nanoparticles (ANPs). These novel ANPs are stabilized through encapsulation in macrophage membranes, providing membrane-encapsulated, antimicrobial-conjugated NPs (Me-ANPs) with similar protein activity, Toll-like receptor expression and negative surface charge as their precursor murine macrophage/human monocyte cell lines. The combination of Toll-like receptors and negative surface charge allows uptake of Me-ANPs by infected macrophages/monocytes through positively charged, lysozyme-rich membrane scars created during staphylococcal engulfment. Me-ANPs are not engulfed by more negatively charged sterile cells possessing less lysozyme at their surface. The Me-ANPs kill staphylococci internalized in macrophages in vitro. Me-ANPs likewise kill staphylococci more effectively than ANPs without membrane-encapsulation or clinically used ciprofloxacin in a mouse peritoneal infection model. Similarly, organ infections in mice created by dissemination of infected macrophages through circulation in the blood are better eradicated by Me-ANPs than by ciprofloxacin. These unique antimicrobial properties of macrophage-monocyte Me-ANPs provide a promising direction for human clinical application to combat persistent infections

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic