Oral Vaccine Carrier Research Articles

Engineering probiotic bacteria to express tcdB antigen as an oral vaccine carrier against Clostridium difficile infection

Background Clostridium (now known as Clostridioides) difficile (C. difficile) is a spore-forming, gram-positive organism that can pose serious public health complications. The elderly are especially vulnerable to C. difficile infections, which can be fatal. C. difficile strains cause symptomatic diseases via the release of two toxins; tcdA and tcdB, that induce inflammation and tissue damage. Vaccines targeting any of these toxins may offer an effective strategy against C. difficile infections. Objective This study aimed to use live probiotics as oral vaccines to express the C. difficile toxin B gene. Oral vaccination has many advantages over intramuscular injection, as it has higher compliance, feasibility, and simpler administration. In addition, oral vaccines can stimulate both mucosal and systemic immunity against the target antigen. Probiotic bacteria were chosen as they present ideal candidates for this goal in terms of safety and health promotion. Materials and methods We chose two probiotic strains: Lactobacillus gasseri ATCC 33323 in addition to Enterococcus faecium NM1015, which had previously been identified in our lab and is capable of suppressing C. difficile colonization. The C-terminal of the tcdB gene was amplified by polymerase chain reaction (PCR) from C. difficile, cloned, and transformed into an E. coli EZ strain. The tcdB fragment was digested with BamHI and XhoI (NEB, UK) and subcloned into the bile salt-inducible expression plasmid pLB210 (obtained from INRA, France). The transformation and electroporation procedures were employed to insert cloning and expression plasmids into the target bacteria. Colony PCR was used to confirm the engineering strains. Reverse transcription PCR was used to confirm the expression of the C. difficile tcdB fragment. Results and conclusion The expression vector ‘p210-tcdB’ was constructed, then introduced into the selected probiotic strains by electroporation and confirmed by colony PCR and plasmid extraction. The reverse transcription (RT)-PCR confirmed the expression of the gene by the engineered strains. No significant difference in the survival rate was observed between the engineered strains and their parental types at pH 2.00 and 1% oxygen. Moreover, the strains showed satisfactory plasmid stability for 210 generations. Future work will involve the in vivo evaluation of the engineered probiotic strains as oral vaccines against C. difficile using an animal model.

Using <i>Bacillus subtilis</i> as an oral vaccine carrier against <i>Streptococcus suis</i>

Due to the progressive growth of the bacteria caused by the widespread use of antibiotics, treatment of streptococcosis is becoming increasingly difficult. Reliable vaccination against Streptococcus suis is necessary. Modern molecular diagnostic and genetic engineering capabilities create prospects for direct cloning of the protective epitopes of the Lmb gene of the local S. suis strain into the proposed delivery system of the pig immune system antigen. Among oral vaccine carriers, Bacillus subtilis is recognized as a relatively environmentally friendly carrier with an efficient protein secretion system and adaptive metabolism capable of spore production under relatively harsh conditions. This spore property can be used to increase the stability and reusability of vaccines. The possibility of using the protective Lmb epitopes of S. suis in B. subtilis as a carrier of an oral recombinant vaccine against Streptococcus suis was studied. The nucleotide sequences of S. suis were obtained from the GenBank database after a preliminary analysis of literature data on the known protective antigens of S. suis of various serotypes. Nucleotide sequence analysis was performed using Unipro UGENE v. 43.0. The Immune Epitope Database (IEDB) was used to search for T (CTL and Th) and B dependent epitopes of the Lmb gene. A computer-designed vaccine in which localization of CTL, B, and Th epitopes is predicted is described. The results of cloning the sequence of the antigenically active epitope of the S. suis Lmb protein in B. subtilis for subsequent oral administration and study of changes in immunological reactions and adverse reactions in animals are described. The possibility to clone the epitopes of recombinant S. suis Lmb protein into the pBE-S polylinker vector was revealed. In the long term, it seems possible to create a new inexpensive and easy-to-use vaccine against S. suis that does not require injection.

Nano and Microparticles as Potential Oral Vaccine Carriers and Adjuvants Against Infectious Diseases.

Mucosal surfaces are the first site of infection for most infectious diseases and oral vaccination can provide protection as the first line of defense. Unlike systemic administration, oral immunization can stimulate cellular and humoral immune responses at both systemic and mucosal levels to induce broad-spectrum and long-lasting immunity. Therefore, to design a successful vaccine, it is essential to stimulate the mucosal as well as systemic immune responses. Successful oral vaccines need to overcome the harsh gastrointestinal environment such as the extremely low pH, proteolytic enzymes, bile salts as well as low permeability and the low immunogenicity of vaccines. In recent years, several delivery systems and adjuvants have been developed for improving oral vaccine delivery and immunogenicity. Formulation of vaccines with nanoparticles and microparticles have been shown to improve antigen stability, availability and adjuvanticity as well as immunostimulatory capacity, target delivery and specific release. This review discusses how nanoparticles (NPs) and microparticles (MPs) as oral carriers with adjuvant characteristics can be beneficial in oral vaccine development.

An oral biologics carrier from modified halloysite nanotubes

Herein, we report the use of surface-modified halloysite as an effective oral vaccine carrier for Nile tilapia.

Immune modulatory capacity of probiotic lactic acid bacteria and applications in vaccine development.

Vaccination is one of the most important prevention tools providing protection against infectious diseases especially in children below the age of five. According to estimates, more than 5 million lives are saved annually by the implementation of six standard vaccines, including diphtheria, hepatitis B, Haemophilus influenza type b, polio, tetanus and yellow fever. Despite these efforts, we are faced with challenges in developing countries where increasing population and increasing disease burden and difficulties in vaccine coverage and delivery cause significant morbidity and mortality. Additionally, the high cost of these vaccines is also one of the causes for inappropriate and inadequate vaccinations in these regions. Thus, developing cost-effective vaccine strategies that could provide a stronger immune response with reduced vaccination schedules and maximum coverage is of critical importance. In last decade, different approaches have been investigated; among which live bacterial vaccines have been the focus of attention. In this regard, probiotic lactic acid bacteria have been extensively studied as safe and effective vaccine candidates. These microorganisms represent the largest group of probiotic bacteria in the intestine and are generally recognised as safe (GRAS) bacteria. They have also attracted attention due to their immunomodulatory actions and their effective role as novel vaccine adjuvants. A significant property of these bacteria is their ability to mimic natural infections, while intrinsically possessing mucosal adjuvant properties. Additionally, as live bacterial vaccines are administered orally or nasally, they have higher acceptance and better safety, but also avoid the risk of contamination due to needles and syringes. In this review, we emphasise the role of probiotic Lactobacillus strains as putative oral vaccine carriers and novel vaccine adjuvants.

Enhanced paracellular delivery of vaccine by hydrogel microparticles-mediated reversible tight junction opening for effective oral immunization

The current conventional injectable vaccines face several drawbacks such as inconvenience and ineffectiveness in mucosal immunization. Therefore, the current development of effective oral vaccines is vital to enable the generation of dual systemic and mucosal immunity. In the present study, we examine the potential of pH-responsive bacterial nanocellulose/polyacrylic acid (BNC/PAA) hydrogel microparticles (MPs) as an oral vaccine carrier. In-vitro entrapment efficiency and release study of Ovalbumin (Ova) demonstrated that as high as 72% of Ova were entrapped in the hydrogel, and the release of loaded Ova was pH-dependent. The released Ova remained structurally conserved as evident by Western blot and circular dichroism. Hydrogel MPs reduced the TEER measurement of HT29MTX/Caco2/Raji B triple co-culture monolayer by reversibly opening the tight junctions (TJs) as shown in the TEM images. The ligated ileal loop assay revealed that hydrogel MPs could facilitate the penetration of FITC-Ova into the Peyer's patches in small intestine. Ova and cholera toxin B (CTB) were utilized in in-vivo oral immunization as model antigen and mucosal adjuvant. The in-vivo immunization revealed mice orally administered with Ova and CTB-loaded hydrogel MPs generated significantly higher level of serum anti-Ova IgG and mucosal anti-Ova IgA in the intestinal washes, compared to intramuscular administrated Ova. These results conclude that BNC/PAA hydrogel MPs is a potential oral vaccine carrier for effective oral immunization.

Molecular Evaluation of Oral Immunogenicity of Hepatitis B Antigen Delivered by Hydrogel Microparticles.

The development of oral vaccine formulation is crucial to facilitate an effective mass immunization program for various vaccine-preventable diseases. In this work, the efficacy of hepatitis B antigen delivered by bacterial nanocellulose/poly(acrylic acid) composite hydrogel microparticles (MPs) as oral vaccine carriers was assessed to induce both local and systemic immunity. Optimal pH-responsive swelling, mucoadhesiveness, protein drug loading, and drug permeability were characterized by MPs formulated with minimal irradiation doses and acrylic acid concentration. The composite hydrogel materials of bacterial nanocellulose and poly(acrylic acid) showed significantly greater antigen release in simulated intestinal fluid while ensuring the integrity of antigen. In in vivo study, mice orally vaccinated with antigen-loaded hydrogel MPs showed enhanced vaccine immunogenicity with significantly higher secretion of mucosal immunoglobulin A, compared to intramuscular vaccinated control. The splenocytes from the same group demonstrated lymphoproliferation and significant increased secretion of interleukin-2 cytokines upon stimulation with hepatitis B antigen. Expression of CD69 in CD4+ T lymphocytes and CD19+ B lymphocytes in splenocytes from mice orally vaccinated with antigen-loaded hydrogel MPs was comparable to that of the intramuscular vaccinated control, indicating early activation of lymphocytes elicited by our oral vaccine formulation in just two doses. These results demonstrated the potential of antigen-loaded hydrogel MPs as an oral vaccination method for hepatitis B.

From allergen to oral vaccine carrier: A new face of ragweed pollen

Oral delivery of vaccines is highly desirable, yet it has met with limited success. Previously we developed allergen-free pollen grains as a novel approach for oral vaccination. We showed that spores of Lycopodium clavatum can be used for oral vaccination. However, it is unknown if pollens of other species can be similarly used as an oral vaccine carrier. Therefore, in this study, we evaluated common ragweed (RW) pollen (Ambrosia elatior) for its oral vaccination potential. Allergen-free RW pollens were prepared from natural pollens through chemical treatment. Eight weekly oral doses of ovalbumin (OVA) formulated with treated RW generated strong systemic (anti-OVA IgG, IgG1, IgG2a, and IgA) and mucosal (anti-OVA IgA) immune responses that sustained for at least three months after vaccination. Mucosal IgA against OVA was found in the lung lavage, feces, saliva, and vaginal secretion. Moreover, three and half months after the last immunization OVA-specific plasma cells were found in the bone marrow that actively secreted IgG and IgG1 antibodies. No IgE against RW-specific proteins was detected in the serum. Overall, RW pollen demonstrated potential for oral vaccine delivery.

Complexation hydrogels as potential carriers in oral vaccine delivery systems

Most current vaccine preparations are in injectable forms, which are inconvenient to patients and ineffective in mucosal immunization. Therefore, most research in this field has been directed at developing ideal oral vaccines enabling the induction of both systemic and mucosal immune responses. In the present study, we examined the utility of a pH-responsive polymeric carrier, poly (methacrylic acid-g-ethylene glycol) [P (MAA-g-EG)] hydrogel, as a potential oral vaccine carrier that can protect cargo proteins in the gastrointestinal tract. Ovalbumin (OVA) and cholera toxin (CT) were first used as the model antigen and mucosal adjuvant, respectively. In vitro incorporation and releasing studies demonstrated that approximately 30% of both OVA and CT were entrapped in the P(MAA-g-EG) hydrogel, and the release of such proteins from the hydrogel to the media was pH-dependent. In vivo oral administration of an OVA-loaded hydrogel (OVA-LP) with either CT or a CT-loaded hydrogel (CT-LP) to rats increased the levels of anti-OVA IgG in the plasma. Furthermore, when CT-LP was orally administered to mice as an antigen, both anti-CT IgG in the plasma and IgA in the fecal extract were detected. These results indicated that the P(MAA-g-EG) hydrogel is a promising and useful carrier for developing oral vaccine delivery systems.

Yersinia enterocolitica YopH-Deficient Strain Activates Neutrophil Recruitment to Peyer's Patches and Promotes Clearance of the Virulent Strain.

Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

Key messageProlamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

Investigation of 3D ordered macroporous carbon with different polymer coatings and their application as an oral vaccine carrier

3-D ordered macroporous carbon with different polymer coatings were developed as new oral vaccine immunological systems. Poly dimethyl diallyl ammonium (PDDA), polyethyleneimine (PEI) and chitosan (CTS), three different polymers with electropositive or adsorption-promoting properties, were chosen as the coating materials to endow the vaccine delivery systems with different surface properties. The bovine serum albumin (BSA) was used as a model vaccine. The three different polymer coated systems exhibited similar release rate which minimized the influence of release rate. The measured value of immunoglobulin G (IgG) titers suggested that the sustained release rate of BSA from polymer coated systems exhibited no strengthened effect on the immune response but could delay the appearance of the peak of the IgG titers compared with uncoated system. The electrostatic attraction between the mucosal and positively charged carrier would be useful during the whole immune experiment. In addition, using the coating material with the ability of enhancing mucosal adsorption was important in the mid-late period of immune. The immunoglobulin A (IgA) titers induced by the polymer coated systems were significantly higher than that induced by the oral BSA solution or i.m. BSA with Freund’s complete adjuvant (FCA) which suggested the successful mucosal immune response of the three different coated systems. Overall, this work provides valuable information for the development of oral vaccine delivery system.

Targeted oral delivery of BmpB vaccine using porous PLGA microparticles coated with M cell homing peptide-coupled chitosan

M cells, the key players of the mucosal immunity induction, are one of the intestinal barriers for the efficient delivery of vaccines to mucosal immune tissues. To overcome the barrier, we have developed an efficient oral vaccine carrier that constitutes poly (lactic-co-glycolic acid) (PLGA) microparticle coated with M cell targeting peptide. In this study, a membrane protein B of Brachyspira hyodysenteriae (BmpB) as a model vaccine against swine dysentery was loaded into porous PLGA microparticles (MPs). The PLGA MPs were further coated with the water-soluble chitosan (WSC) conjugated with M cell homing peptide (CKS9) to prepare BmpB-CKS9-WSC-PLGA MPs. Oral immunization of BmpB vaccine with CKS9-WSC-PLGA MPs in mice showed elevated secretory IgA responses in the mucosal tissues and systemic IgG antibody responses, providing a complete immune response. Specifically, the immunization with these MPs demonstrated to induce both Th1- and Th2-type responses based on elevated IgG1 and IgG2a titers. The elevated immune responses were attributed to the enhanced M cell targeting and transcytosis ability of CKS9-WSC-PLGA MPs to Peyer's patch regions. The high binding affinity of CKS9-WSC-PLGA MPs with the M cells to enter into the Peyer's patch regions of mouse small intestine was investigated by closed ileal loop assay and it was further confirmed by confocal laser scanning microscopy. These results suggest that the M cell targeting approach used in this study is a promising tool for targeted oral vaccine delivery.

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.

Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review

Chitosan, a natural biodegradable polymer, is of great interest in biomedical research due to its excellent properties including bioavailability, nontoxicity, high charge density, and mucoadhesivity, which creates immense potential for various pharmaceutical applications. It has gelling properties when it interacts with counterions such as sulfates or polyphosphates and when it crosslinks with glutaraldehyde. This characteristic facilitates its usefulness in the coating or entrapment of biochemicals, drugs, antigenic molecules as a vaccine candidate, and microorganisms. Therefore, chitosan together with the advance of nanotechnology can be effectively applied as a carrier system for vaccine delivery. In fact, chitosan microspheres have been studied as a promising carrier system for mucosal vaccination, especially via the oral and nasal route to induce enhanced immune responses. Moreover, the thiolated form of chitosan is of considerable interest due to its improved mucoadhesivity, permeability, stability, and controlled/extended release profile. This review describes the various methods used to design and synthesize chitosan microspheres and recent updates on their potential applications for oral and nasal delivery of vaccines. The potential use of thiolated chitosan microspheres as next-generation mucosal vaccine carriers is also discussed.

Preparation and investigation of Ulex europaeus agglutinin I-conjugated liposomes as potential oral vaccine carriers

We prepared and optimized Ulex europaeus agglutinin I (UEAI)-modified Bovine serum albumin (BSA)-encapsulating liposomes (UEAI-LIP) as oral vaccine carriers and examined the feasibility of inducing systemic and mucosal immune responses by oral administration of UEAILIP. The prepared systems were characterized in vitro for their average size, zeta potential, encapsulation efficiency (EE%) and conjugation efficiency (CE%). In vitro release studies indicated that the presence of UEAI around the optimized liposomes was able to prevent a burst release of loaded BSA and provide sustained release of the encapsulated protein. In vivo immune-stimulating results in KM mice showed that BSA given intramuscularly generated systemic response only but both systemic and mucosal immune responses could be induced simultaneously in the groups in which BSA-loaded liposomes (LIP) and UEAI-LIP were administered intragastrically. Furthermore, the modification of UEAI on the surface of liposomes could further enhance the IgA and IgG levels obviously. In conclusion, this study demonstrated the high potential of lectin-modified liposomes containing the antigen as carriers for oral vaccine.

Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cell-homing peptide selected by phage display technique

This study is aimed to develop an efficient oral vaccine carrier which specifically targets the follicle-associated epithelium region of Peyer’s patch (PP). M cell-homing peptide was selected by the phase display technique and its targeting efficiency was validated using chitosan nanoparticles (CNs) conjugated with the discovered peptide. A phage clone encoding CKSTHPLSC (CKS9) peptide sequence was selected by analysis of comparative superiority in transcytosis efficacy across the M cell layer in vitro and in vivo among the candidates. CKS9 was chemically conjugated to water-soluble chitosan (WSC) and the CKS9-immobilized chitosan nanoparticles (CKS9-CNs) were prepared by ionic gelation of CKS9-WSC with tripolyphosphate, yielding spherical nanoparticles around 226.2 ± 41.9 nm. The targeting ability of CKS9-CNs to the M cell and to the PP regions of rat small intestine was investigated by in vitro transcytosis assay and closed ileal loop assay, respectively, and was visualized by fluorescence-microscopy analysis. CKS9-CNs were transported more effectively across the M cell model and accumulated more specifically into PP regions in comparison with CNs, indicating that CKS9 peptide prominently enhanced the targeting and transcytosis ability of CNs to PP regions. These results suggest that the CKS9-CNs could be used as a new carrier for oral vaccine delivery.

IgA response by oral infection with an attenuated Yersinia enterocolitica mutant: Implications for its use as oral carrier vaccine

Yersinia enterocolitica ( Ye) mutant strain ( sycH-) is unable to secrete the virulence protein YopH. Mucosal vaccination is often required to induce protection, but stimulating strong IgA response is frequently difficult. Here, we addressed whether Ye sycH- might induce IgA response, and investigated its attenuation in TNFRp55−/−, IL-12p40−/− and IL-4−/− mice. We found that Ye sycH- colonizes Peyer’s patches, and induces higher Yersinia-specific IgA levels in feces and in serum compared with Ye wild type. The Ye sycH- mutant proved to be attenuated and induced IgA in both wild-type and immunodeficient mice. These lines of evidence show the attenuation of Ye sycH- and its ability to stimulate an IgA response. This mutant might be useful as an oral vaccine carrier.

Yersinia enterocolitica Yop mutants as oral live carrier vaccines

Attenuated enteropathogenic yersiniae that translocate heterologous antigens into the cytosol of antigen presenting cells via their type three secretion system (TTSS) are considered promising candidates for the development of live oral vaccine carrier strains that induce CD8 T cell responses. Wild type Yersinia enterocolitica of serotype O:8 however efficiently suppresses the immune response of the host by translocating effector proteins called Yersinia outer proteins (Yops) into the cytosol of immune cells. We therefore tested immunogenicity, protective efficacy, and virulence of yop mutants that translocate the model antigen Listeriolysin (LLO) of Listeria monocytogenes in a mouse model. A Δ yopP mutant-based vaccine carrier strain induced the highest numbers of LLO 91–99-specific CD8 T cells and effectively protected mice against a lethal challenge with Listeria whereas Δ yopPT, Δ yopPV K42Q , and Δy opPO mutants of Y. enterocolitica induced fewer CD8 T cells and conferred only partial protection. The Δ yopPH, Δ yopPE, Δ yopPM, and Δ yopPQ mutants induced the weakest CD8 T cell response and did not significantly protect mice against Listeria presumably due to the strong attenuation of these strains in the mouse model. Even though a Y. enterocolitica strain WA-C(pTTSS), which translocated only LLO (but not Yops), induced superior MHC class I-restricted antigen presentation in DC compared to the Δ yopP mutants in vitro, this strain was not able to significantly colonize mouse tissue or to induce CD8 T cell responses in vivo. The success in designing a Yersinia oral vaccine carrier is therefore dependent to a great extent on the subtle balance between immunogenicity and attenuation.

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis ( S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially Δ phoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and Δ phoP also in pigs. In addition, Δ rpoS and Δ phoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the Δ rpoS and Δ phoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.