Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.     

Generation and Characterization of Thymidine/d-Alanine Auxotrophic Recombinant Lactococcus lactis subsp. lactis IL1403 Expressing BmpB

Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA ⁻ alr ⁺ bmpB ⁺), IAD (thyA ⁺ alr ⁻ bmpB ⁺), and ITDAD (thyA ⁻ alr ⁻ bmpB ⁺). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed thymidine auxotrophy of ITD and ITDAD and d-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited. In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in M17G supplemented with thymidine and d-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis.     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> as a live vehicle for oral immunization against chicken anemia virus

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens.     

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate‐binding domain (VP8*)

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate‐binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus‐cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*_64‐224 of canine rotavirus strain K9 interacts with N‐acetylneuraminic and N‐glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase‐sensitive animal rotaviruses from pigs (CRW‐8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N‐glycolyl‐ over the N‐acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.     

Loss of IrpT Function in Lactococcus lactis subsp. lactis N8 Results in Increased Nisin Resistance

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-l-rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Rotavirus capsid protein VP5* permeabilizes membranes

Proteolytic cleavage of the VP4 outer capsid spike protein into VP8* and VP5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. In this study we addressed the function of the VP5* cleavage fragment in permeabilizing membranes. Expressed VP5* and truncated VP5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (LUVs) preloaded with carboxyfluorescein (CF). VP5* and VP5* truncations, but not VP4 or VP8*, permeabilized LUVs as measured by fluorescence dequenching of released CF. Similar to virus-induced CF release, VP5*-induced CF release was concentration and temperature dependent, with a pH optimum of 7.35 at 37 degrees C, but independent of the presence of divalent cations or cholesterol. VP5*-induced permeability was completely inhibited by VP5*-specific neutralizing monoclonal antibodies (2G4, M2, or M7) which recognize conformational epitopes on VP5* but was not inhibited by VP8*-specific neutralizing antibodies. In addition, N-terminal and C-terminal VP5* truncations including residues 265 to 474 are capable of permeabilizing LUVs. These findings demonstrate that VP5* permeabilizes membranes in the absence of other rotavirus proteins and that membrane-permeabilizing VP5* truncations contain the putative fusion region within predicted virion surface domains. The ability of recombinant expressed VP5* to permeabilize membranes should permit us to functionally define requirements for VP5*-membrane interactions. These findings indicate that VP5* is a specific membrane-permeabilizing capsid protein which is likely to play a role in the cellular entry of rotaviruses.     

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H⁺ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H⁺ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H⁺ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H⁺ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.     

Construction and Application of a Food-Grade Expression System for Lactococcus lactis

A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000Δalr by double-crossover recombination using temperature-sensitive integration plasmid pG⁺host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry.     

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).     

Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.     

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.     

Rotavirus vp7 antigen produced by Lactococcus lactis induces neutralizing antibodies in mice

AIMS: To determine if live recombinant Lactococcus lactis strains expressing rotavirus VP7 antigen are immunogenic in mice. METHODS AND RESULTS: Using the food-grade lactic acid bacterium L. lactis as a carrier, we expressed VP7, the major rotavirus outer shell protein and one of the main components of the infective particle, as a cytoplasmic, secreted or cell wall anchored forms. Our results showed that recombinant L. lactis strains secreting VP7 proved to be more immunogenic than strains containing the antigen in the cytoplasm or anchored to the cell wall. CONCLUSIONS: This is the first demonstration that recombinant L. lactis producing VP7 can induce the production of a neutralizing antibody response against rotavirus by the intragastric route. SIGNIFICANCE AND IMPACT OF THE STUDY: Rotaviruses are the single most important aetiological agents of severe diarrhoea of infants and young children worldwide and have been estimated to be responsible for 650 000-800 000 deaths per year of children younger than 5 years old in development countries. Thus, the development of a safe and effective vaccine has been a global public health goal. Although two of five mice orally inoculated with L. lactis strains secreting VP7 elicited a specific-antibody response, these strains could be very useful to be used as a prototype to develop a new generation of protective rotavirus vaccines.     

VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection.

We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.     

Enhanced expression of heterologous inulinase in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> by disruption of <Emphasis Type="Italic">hap1</Emphasis> gene

Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml⁻¹ after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis.     

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of <Emphasis Type="Italic">Lonomia obliqua</Emphasis>

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system.     

Synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50–55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.     

Cell surface display system for Lactococcus lactis: a novel development for oral vaccine

The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.     

M cell–targeting strategy enhances systemic and mucosal immune responses induced by oral administration of nuclease-producing L. lactis

Efficient delivery of antigens to the gut-associated lymphoid tissue (GALT) is the most critical step for the induction of mucosal immunity by oral vaccines. As M cells are the main portal for luminal antigens into the GALT, the M cell-targeting of antigens affords a promising strategy toward the development of effective oral vaccines. Lactococcus lactis is a fascinating recombinant host for oral vaccines, as they survive and produce antigens in the gut and have a particularly safe profile for human use. In this study, we developed and evaluated an M cell–targeting oral immunization system using recombinant L. lactis strains. For the purpose, we generated an L. lactis strain that secretes a model antigen fused with the OmpH β1α1 domain of Yersinia enterocolitica, which has been shown to bind to a complement C5a receptor on the M cell surface. As the model antigen, Staphylococcus aureus nuclease was used for fusion, resulting in L. lactis–expressing Nuc-OmpH (LL/Nuc-OmpH). Ex vivo intestinal loop assays showed that the amount of Nuc-OmpH taken up into Peyer’s patches was more than that of the unfused nuclease (Nuc). In addition, oral administration of the recombinant L. lactis strains to mice demonstrated that LL/Nuc-OmpH-induced nuclease-specific fecal IgA and serum IgG titers were significantly higher than those induced by LL/Nuc. These results indicate that OmpH works as an M cell–targeting molecule when fused with antigens secreted from L. lactis and that the M cell–targeting strategy affords a promising platform for L. lactis–based mucosal immunization.