Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.

Development of an M cell targeted nanocomposite system for effective oral protein delivery: preparation, in vitro and in vivo characterization.

BACKGROUND: There is a strong need for non-invasive and patient-friendly delivery systems of protein drugs for long-term therapy. However, oral delivery of protein drugs is a big challenge due to many barriers including instability in the gastrointestinal (GI) tract and low permeability. To overcome the absorption barriers in GI tract and improve the patient compliance, this study aimed to develop an M cell targeted-nanocomposite delivery system of protein drugs. RESULTS: An aminoclay-protein core complex (AC-Ins) was prepared by using insulin as a model protein and then sequentially coated with Ulex europaeus agglutinin 1 (UEA-1) for M-cell targeting and the pH sensitive polymer, Eudragit® L100 (EUAC-Ins). All nanoparticles were obtained with a high entrapment efficiency (> 90%) and their structural characteristics were confirmed by Fourier transform-infrared spectroscopy, energy dispersive X-ray spectroscopy, and circular dichroism. Among the developed nanoparticles, EUAC-Ins effectively suppressed drug release at pH 1.2, while rapidly released drugs at pH 6.8 due to dissolution of the outer coating layer. The conformational stability of insulin entrapped in EUAC-Ins was well maintained in the presence of proteolytic enzymes. Compared to free insulin, EUAC-Ins increased the membrane transport of insulin by 4.4-fold in M cells. In parallel, oral administration of EUAC-Ins in mice enhanced insulin uptake by 4.1-fold in the intestinal Peyer's patches and 2.6-fold in intestinal epithelium tissues with normal villi, compared to free insulin. Orally administered EUAC-Ins decreased significantly the blood glucose level in diabetic mice, while the effect of oral insulin solution was negligible. CONCLUSION: An M cell targeted-ternary nanocomposite system obtained by dual coating of the aminoclay-protein core complex with UEA-1 and a pH dependent polymer is promising as an effective oral protein delivery carrier.

Oral immunization with a Lactobacillus casei-based anti-porcine epidemic diarrhoea virus (PEDV) vaccine expressing microfold cell-targeting peptide Co1 fused with the COE antigen of PEDV.

AIMS: The aims of this study were to develop an effective M cell-targeting oral vaccine, involving Lactobacillus casei to deliver the porcine epidemic diarrhoea virus (PEDV) core neutralizing epitope (COE) antigen conjugated with M cell-targeting peptide Co1 as an adjuvant, against PEDV infection. METHODS AND RESULTS: Genetically engineered L. casei 393 (L393) strains expressing PEDV COE antigen only (pPG-COE/L393) or fused-expressing COE and M cell-targeting peptide Co1 (pPG-COE-Co1/L393) were constructed, and the immunogenicity upon administration as an oral vaccine was evaluated. The results showed that higher anti-PEDV serum IgG and mucosal SIgA antibody responses were induced in mice orally immunized with strain pPG-COE-Co1/L393 as compared to the mice immunized with strain L393 expressing COE alone or carrying the empty plasmid. In addition, the use of the Co1 ligand elicited a splenocyte proliferative response more effectively in comparison with the COE antigen alone and supported a skewed T helper 2 type of immune response against PEDV. CONCLUSIONS: pPG-COE-Co1/L393 can effectively induce mucosal, humoural and Th2-type cellular immune responses against PEDV infection via oral administration. Furthermore, M cell-targeting peptide ligand Co1 is a good mucosal adjuvant. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus casei delivering the COE antigen of PEDV conjugated with a M cell-targeting peptide Co1 as an immune adjuvant is a promising oral vaccine candidate for PEDV.

Structural changes of layer-by-layer self-assembled starch-based nanocapsules in the gastrointestinal tract: Implications for their M cell-targeting delivery and transport efficiency.

Characterizing the structural changes of cell-targeting delivery carriers in gastrointestinal tract (GIT) is crucial for understanding their effectiveness in cell targeting and transport. Herein, RGD peptide-grafted carboxymethyl starch (CMS) and cationic quaternary ammonium starch (QAS) were utilized to fabricate quintet-layered nanocapsules loaded with ovalbumin (OVA). The aim was to improve delivery and transportation efficiency, specifically targeting M cells. The research analyzed the impact of pH and enzyme variations in GIT on the structure of nanocapsules, interactions between carriers and the release behavior of OVA. Results showed that the size of nanocapsules increased from 229.2 to 479.8 nm and the zeta potential decreased from -1.08 to -33.33 mV during oral delivery. This was evident in TEM images, showing a more relaxed core-shell structure. Isothermal titration calorimetry and molecular dynamic simulation indicated that pH changes primarily affected the electrostatic interaction between carriers. Increasing pH led to reduced affinity constants, and around 84.42 % of OVA was successfully delivered to M cells. Moreover, the transport efficiency of nanocapsules to M cells was five times greater than that of Caco-2 cells. This suggests the feasibility of developing a nanocapsules delivery system capable of adapting to pH changes in GIT by regulating electrostatic interactions between carriers.

Aptamer-modified M cell targeting liposomes for oral delivery of macromolecules.

There is an urgent demand for non-invasive and high compliance delivery systems of macromolecules for long-term therapy. However, oral administration of macromolecules is hindered by low permeability and instability in the gastrointestinal (GI) tract. Therefore, we developed a novel aptamer-modified liposomes (Apt-Lip) with M cell targeting for oral delivery of exenatide (EXT). Firstly, we optimized aptamers to M cells by Cell-SELEX and aptamer truncations. The selected aptamer T-M3 (Apt-T-M3) with high binding affinity (Kd = 176 ± 108 nM) and specificity was modified on the surface of liposomes for targeting M cells. Liposomes were formulated by microfluidics system and characterized in terms of morphology, hydrodynamic diameter, zeta potential, and the efficiency of encapsulation. In comparison with non-targeting liposomes, cell uptake in M cells was significantly enhanced by Apt-Lip. Similarly, the transport efficiency of EXT was 2-fold increase using Apt-Lip in M cells. Additionally, the transepithelial electrical resistance (TEER) of M cell monolayers is significantly reduced. In ex vivo intestinal absorption study, Apt-Lip was proved to possess significantly high intestinal absorption in Peyer's patches (PPs) and M cells-specific targeting capacity. Consequently, Apt-Lip promoted the EXT transport could base not only on M cell mediated transport, but also on enhancement of paracellular permeability. In conclusion, the present study supported Apt-Lip as a promising M cell targeted delivery system for oral delivery of macromolecules.

Oral Immunization With a M Cell-Targeting Recombinant L. Lactis Vaccine LL-plSAM-FVpE Stimulate Protective Immunity Against H. Pylori in Mice.

There are many virulence factors of H. pylori that contribute in diverse ways to gastric disease. Therefore, designing multivalent epitope vaccines against many key virulence factors virulence factors of H. pylori is a promising strategy to control H. pylori infection. In previous studies, we constructed a multivalent epitope vaccine FVpE against four key virulence factors of H. pylori (Urease, CagA, VacA, and NAP), and oral immunization with the FVpE vaccine plus a polysaccharide adjuvant (PA) containing lycium barbarum polysaccharide and chitosan could provide protection against H. pylori infection in the Mongolian gerbil model. Oral vaccines have many advantages over injected vaccines, such as improved safety and compliance, and easier manufacturing and administration. However, the harsh gastrointestinal (GI) environment, such as gastric acid and proteolytic enzymes, limits the development of oral vaccines to some extent. Oral vaccines need a gastrointestinal delivery system with high safety, low price and promoting vaccine antigen to stimulate immune response in the gastrointestinal mucosa. Lactic acid bacteria are gastrointestinal probiotics that have unique advantages as a delivery system for oral vaccines. In this study, a M cell-targeting surface display system for L. lactis named plSAM was designed to help vaccine antigens to stimulate effective immune responses in the gastrointestinal tract, and a M cell-targeting recombinant L. lactis vaccine LL-plSAM-FVpE was constructed by using the surface display system plSAM. recombinant L. lactis vaccine LL-plSAM-FVpE could secretively express the SAM-FVpE protein and display it on the bacterial surface. Moreover, experimental results confirmed that LL-plSAM-FVpE had an enhanced M cell-targeting property. In addition, LL-plSAM-FVpE had excellent M cell-targeting property to promote the phagocytosis and transport of the antigen SAM-FVpE by gastrointestinal M cells. More importantly, oral immunization of LL-plSAM-FVpE or SAM-FVpE plus PA can stimulate IgG and sIgA antibodies and CD4+ T cell immune responses against four virulence factors of H. pylori (Urease, CagA, VacA, and NAP), thus providing protective immunity against H. pylori infection in mice. The M cell-targeting recombinant L. lactis vaccine against various key H. pylori virulence factors could be a promising vaccine candidate for controlling H. pylori infection.

Surface Modification of Biodegradable Microparticles with the Novel Host-Derived Immunostimulant CPDI-02 Significantly Increases Short-Term and Long-Term Mucosal and Systemic Antibodies against Encapsulated Protein Antigen in Young Naïve Mice after Respiratory Immunization.

Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 µm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 µL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 µm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.

Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route.

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.

Oral Vaccine Delivery for Intestinal Immunity-Biological Basis, Barriers, Delivery System, and M Cell Targeting.

Most currently available commercial vaccines are delivered by systemic injection. However, needle-free oral vaccine delivery is currently of great interest for several reasons, including the ability to elicit mucosal immune responses, ease of administration, and the relatively improved safety. This review summarizes the biological basis, various physiological and immunological barriers, current delivery systems with delivery criteria, and suggestions for strategies to enhance the delivery of oral vaccines. In oral vaccine delivery, basic requirements are the protection of antigens from the GI environment, targeting of M cells and activation of the innate immune response. Approaches to address these requirements aim to provide new vaccines and delivery systems that mimic the pathogen's properties, which are capable of eliciting a protective mucosal immune response and a systemic immune response and that make an impact on current oral vaccine development.

Incorporation of a bi-functional protein FimH enhances the immunoprotection of chitosan-pVP1 vaccine against coxsackievirus B3-induced myocarditis.

Viral myocarditis is a common clinical cardiovascular disease mainly induced by coxsackievirus B3 (CVB3) with no effective therapeutic measures. Induction of efficient mucosal immune responses is very critical against CVB3-induced myocarditis. FimH is an Escherichia coli (E. coli)-derived protein, which possesses an M cell-targeting property and functions as a TLR4 agonist. In this study, we introduced the recombinant FimH protein, into our previously developed CVB3 mucosal vaccine chitosan (CS)-pVP1, aiming to provoke more efficient mucosal immune responses and immunoprotection against CVB3-induced myocarditis. Compared with the CS-pVP1 vaccine, immunization with FimH-CS-pVP1 remarkably increased the levels and neutralizing titers of CVB3-specific protective secretory IgA (sIgA), enhanced the frequency of CVB3-specific IgA-producing B cells and amplified mucosal T-cell immune responses in mesenteric lymph nodes (MLNs), although failing to significantly amplify CVB3-specific systemic immune responses. Consistently, FimH-CS-pVP1 group showed the enhanced immunoprotection against CVB3-induced myocarditis, evidenced by the indices of limited myocardial injury, reduced viral loads and enhanced survival rate. Further study showed that this enhanced immunoprotection was not only ascribed to its M cell-targeting property, which led to the enhanced mucosal antigen VP1 expression, but also associated with the mucosal adjuvant effect of FimH, which facilitated the formation of germinal centers (GCs), production of IgA-inducing factors and maturation of antigen-presenting cells (APCs). Taken together, here we developed a bi-functional mucosal vaccine FimH-CS-pVP1, which simultaneously possessed the M cell-targeting property and mucosal adjuvant ability, and we showed that FimH-CS-pVP1 could efficiently induce the higher levels of CVB3-specific protective mucosal immune responses and provide better prophylactic effects against CVB3-induced myocarditis than CS-pVP1.

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization.

Development and application of safe and effective mucosal adjuvants are important to improve immunization efficiency in oral vaccine. Here, we report a novel mucosal adjuvant, IL-6-CKS9, a recombinant cytokine generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secrete the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreting IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting two separated mice immunization assays. In addition, we characterized that the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses by analysis of the specific anti-BmpB IgG1 and IgG2a isotypes in the sera and also investigated possible oral tolerance in our vaccine strategy. Collectively, our results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination.

M cell-targeting strategy facilitates mucosal immune response and enhances protection against CVB3-induced viral myocarditis elicited by chitosan-DNA vaccine.

Efficient delivery of antigen to mucosal associated lymphoid tissue is a first and critical step for successful induction of mucosal immunity by vaccines. Considering its potential transcytotic capability, M cell has become a more and more attractive target for mucosal vaccines. In this research, we designed an M cell-targeting strategy by which mucosal delivery system chitosan (CS) was endowed with M cell-targeting ability via conjugating with a CPE30 peptide, C terminal 30 amino acids of clostridium perfringens enterotoxin (CPE), and then evaluated its immune-enhancing ability in the context of coxsackievirus B3 (CVB3)-specific mucosal vaccine consisting of CS and a plasmid encoding CVB3 predominant antigen VP1. It had shown that similar to CS-pVP1, M cell-targeting CPE30-CS-pVP1 vaccine appeared a uniform spherical shape with about 300 nm diameter and +22 mV zeta potential, and could efficiently protect DNA from DNase I digestion. Mice were orally immunized with 4 doses of CPE30-CS-pVP1 containing 50 µg pVP1 at 2-week intervals and challenged with CVB3 4 weeks after the last immunization. Compared with CS-pVP1 vaccine, CPE30-CS-pVP1 vaccine had no obvious impact on CVB3-specific serum IgG level and splenic T cell immune responses, but significantly increased specific fecal SIgA level and augmented mucosal T cell immune responses. Consequently, much milder myocarditis and lower viral load were witnessed in CPE30-CS-pVP1 immunized group. The enhanced immunogenicity and immunoprotection were associated with the M cell-targeting ability of CPE30-CS-pVP1 which improved its mucosal uptake and transcytosis. Our findings indicated that CPE30-CS-pVP1 may represent a novel prophylactic vaccine against CVB3-induced myocarditis, and this M cell-targeting strategy indeed could be applied as a promising and universal platform for mucosal vaccine development.

M-cell targeted polymeric lipid nanoparticles containing a Toll-like receptor agonist to boost oral immunity.

Oral delivery of antigens is patient-friendly and efficient way of treating intestinal infections. However, the efficacy of oral vaccines is limited by degradation in the gastrointestinal (GI) tract and poor absorption by enterocytes and antigen-presenting cells (APC). Here we report ulex europaeus agglutinin-1 (UEA-1) conjugated poly (D,L-lactide-co-glycolide) (PLGA)-lipid nanoparticles (NP) containing a Toll-like receptor (TLR)-agonist monophosphoryl lipid A (MPL) as an oral vaccine delivery system. The uniform-sized PLGA-lipid NPs (simplified as lipid NPs) were produced by the premix membrane emulsification method. They can protect the entrapped model antigen ovalbumin (OVA) from exposure to the GI tract and release the OVA in a controlled manner. With UEA-1 and MPL modification, the UEA-MPL/lipid NPs can be effectively transported by M-cells and captured by mucosal dendritic cells (DCs). After in vivo vaccination, the OVA-UEA-MPL/lipid NPs stimulated the most effective mucosal IgA and serum IgG antibodies during the oral formulations. These results suggest that this MPL containing M-cell targeted lipid NP can potentially be used as a universally robust oral vaccine delivery system.