Potential Oral Vaccine Research Articles

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus)

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1β, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Genetic-engineered Schizochytrium sp. expressing a multiepitopic protein based on Vibrio parahaemolyticus toxins triggers immune responses in mice

Vibrio parahaemolyticus is a bacterium that naturally occurs in coastal waters and can multiply in warmer temperatures. Infection with these bacteria can inflict acute gastroenteritis by consuming contaminated seafood. Antibiotics must be prescribed. However, some antibiotics are no longer effective and no vaccine is available yet. Herein a microalgae-made immunogenic protein made up of epitopes from V. parahaemolyticus hemolysin toxins named Epi_Vp is reported. Epitopes from the proteins TDH, TRH, and TLH were selected for the Epi_Vp vaccine design by in silico studies, revealing an antigenic but not allergenic multiepitopic protein. The expression of Epi_Vp in Schizochytrium sp. was confirmed by Western blot after induction. Yields measured by ELISA reach 218. 63 μg g−1 dry-weight microalgae 48 h post-induction. Schizochytrium sp.-expressing Epi_Vp was found immunogenic in mice upon oral whole-cell administration in terms of specific serum IgG mucosa IgA. In addition, an increase of IFN-γ, IL-10, and IL-4 cytokine levels in splenocytes was detected. In conclusion, Epi_Vp protein expressed and delivered within the microalgae Schizochytrium sp. is capable of triggering humoral responses in an animal model and represents a potential oral vaccine prototype to combat infectious gastroenteritis caused by Vibrio parahaemolyticus.

Oral vaccination with recombinant Saccharomyces cerevisiae expressing Micropterus salmoides rhabdovirus G protein elicits protective immunity in largemouth bass

Micropterus salmoides rhabdovirus (MSRV) is one of the main pathogens of largemouth bass, leading to serious economic losses. The G protein, as the only envelope protein present on the surface of MSRV virion, contains immune-related antigenic determinants, thereby becoming the primary target for the design of MSRV vaccines. Here, we displayed the G protein on the surface of yeast cells (named EBY100/pYD1-G) and conducted a preliminary assessment of the protective efficacy of the recombinant yeast vaccine. Upon oral vaccination, a robust immune response was observed in systemic and mucosal tissue. Remarkably, following the MSRV challenge, the relative percent survival of EBY100/pYD1-G treated largemouth bass significantly increased to 66.7 %. In addition, oral administration inhibited viral replication and alleviated the pathological symptoms of MSRV-infected largemouth bass. These results suggest that EBY100/pYD1-G could be used as a potential oral vaccine against MSRV infection.

TonB-Dependent Receptor Protein Displayed on Spores of Bacillus subtilis Stimulates Protective Immune Responses against Acinetobacter baumannii.

The emergence of antibiotic-resistant Acinetobacter baumannii strains with limited treatment options has become a significant global health concern. Efforts to develop vaccines against the bacteria have centred on several potential protein targets, including the TonB-dependent receptors (TBDRs). In the present study, TBDRs from A. baumannii were displayed on the surface of Bacillus subtilis spores. The immunogenicity of the recombinant spores was evaluated in orally vaccinated mice. None of the immunized mice demonstrated signs of illness and were observed to be healthy throughout the study. Sera and the intestinal secretions from the recombinant spores-treated mice demonstrated mucosal and humoral antibody responses to the vaccine antigen. In addition, bactericidal activities of the sera against A. baumannii clinical isolates were demonstrated. These observations suggest that the B. subtilis spore-displayed TBDRs should be further explored as much-needed potential oral vaccine candidates against A. baumannii.

Safety and immunogenicity of orally administered poxvirus vectored constructs in the white-footed mouse (Peromyscus leucopus).

Globally, zoonotic spillover is becoming more frequent and represents a growing public health concern. Reservoir-targeted vaccination offers an intriguing alternative to traditional vaccine practices by establishing protection in wild populations that maintain the natural pathogen cycle. As an important pathogen reservoir, Peromyscus leucopus Rafinesque or the white-footed mouse has been the target of several experimental vaccines. However, strategies are limited by the method of administration, need for repeated dosing, or safety of constructs in the field. To address these concerns, we evaluated two highly attenuated poxviruses, raccoonpox virus (RCN) and modified vaccinia Ankara (MVA) virus as potential oral vaccine vectors in white-footed mice. Following oral administration, P. leucopus showed no adverse signs. A single oral dose elicited robust immune responses in mice to the foreign influenza hemagglutinin protein expressed by poxvirus vaccine vectors. Serum hemagglutinin inhibition antibody titers were detected by day 7 post immunization and persisted until study termination (77days post immunization). This study establishes the safety and immunogenicity of recombinant MVA and RCN poxviruses in P. leucopus and demonstrates the suitability of these vectors as part of a reservoir-targeted vaccine strategy for white-footed mice.

Oral Vaccination of Recombinant Saccharomyces cerevisiae Expressing ORF132 Induces Protective Immunity against Cyprinid Herpesvirus-2.

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis (HVHN) disease, which causes serious economic losses in the crucian carp culture industry. In this study, by displaying ORF132 on the surface of Saccharomyces cerevisiae cells (named EBY100/pYD1-ORF132), we evaluated the protective efficacy of oral administration against CyHV-2 infection. Intense innate and adaptive immune responses were evoked in both mucosal and systemic tissues after oral vaccination with EBY100/pYD1-ORF132. Importantly, oral vaccination provided significant protection for crucian carp post CyHV-2 infection, resulting in a relative percent survival (RPS) of 64%. In addition, oral administration suppressed the virus load and relieved histological damage in selected tissues. Our results indicated that surface-displayed ORF132 on S. cerevisiae could be used as potential oral vaccine against CyHV-2 infection.

Surface functionalization of PLGA nanoparticles for potential oral vaccine delivery targeting intestinal immune cells

This study aimed to develop surface modified PLGA nanocarriers protecting a protein-based antigen in the stomach to enable potential release of the antigen at target intestinal sites. PLGA nanoparticles (NPs) were prepared by double emulsion and solvent evaporation techniques while surface functionalization was performed using polyethylene glycol (PEG), sodium alginate (ALG) and Eudragit L100 (EUD) with ovalbumin (OVA) as a model protein antigen. Nanoparticles were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), and stability in simulated gastric fluid (SGF)/simulated intestinal fluid (SIF). Structural integrity of released OVA was analyzed by circular dichroism (CD) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while cytotoxicity against Jurkat cells was determined using MTT assay. Surface functionalized PLGA NPs protected the protein in SGF and SIF better than the non-functionalized NPs. Average size of OVA encapsulated NPs was between 235 and 326 nm and were spherical. FTIR band change was observed after surface modification and the surface modified NPs showed sustained OVA release compared with the uncoated NPs. The secondary structure of OVA released after 96 h remained intact and MTT assay showed >80 % cell viability after 72 h while unmodified and surface modified NPs achieved 17 % and 48 % mucin binding respectively. In conclusion, surface modified PLGA NPs have been shown to be safe for potential oral protein-based vaccine delivery.

Evaluation of feed utilization, immune response and disease resistance in striped catfish, Pangasianodon hypophthalmus (Sauvage 1878) fed with a novel Aeromonas hydrophila biofilm vaccine

Striped catfish, Pangasianodon hypophthalmus was immunized with Biofilm (BF) and Free cell (FC) of Aeromonas hydrophila vaccine at 1010 CFU g−1 for 20 days and monitored for growth parameters, immune responses and disease resistance up to 60 day post vaccination (dpv). Pangasius catfish in the BF vaccinated group had considerably higher growth and feed utilization than the FC vaccinated and unvaccinated groups (p < 0.05). Biofilm vaccinated group showed a significant increase (p < 0.05) in the mean weight gain (46.91 ± 0.59) than the FC (35.94 ± 0.21) and unvaccinated group (34.92 ± 0.35). The vaccinated fishes were challenged with A. hydrophila at 107 CFU/ml. Significant higher relative percentage survival (RPS) was recorded with BF (84.21 ± 1.49%) compared to that with FC (33.33 ± 1.21%). Polyclonal antibody-based ELISA was used to quantify the antibody titre. BF vaccinated group showed significantly higher antibody titer compared to other treatments (p < 0.05). Moreover, higher haematological parameters recorded in the present study were differentially stimulated by the oral administration of A. hydrophila biofilm vaccine. The mean total protein, albumin, and globulin levels of the BF vaccine groups were significantly higher (p < 0.05) than the mean total protein, albumin, and globulin contents of the unvaccinated group. Furthermore, biochemical stress parameters (SGPT, SGOT) in the vaccinated groups showed an incremental trend in the early days of the experimental period. However, the values were significantly lower (p < 0.05) in the biofilm group on 20 dpv onwards indicating improved health condition. Vaccinated BF fishes showed gut associated lymphoid tissues (GALT) within the laminar propria of mid gut. But in FC group fishes showed less aggregation of lymphoid cells. The unvaccinated control fish had no lymphoid cell aggregation in their intestines. The findings of the current research suggested that biofilm vaccine has the capability to be one of the potential oral vaccines in striped catfish against A. hydrophila infection.

Enhancing Stability and Mucoadhesive Properties of Chitosan Nanoparticles by Surface Modification with Sodium Alginate and Polyethylene Glycol for Potential Oral Mucosa Vaccine Delivery.

Background: The present study aimed to fabricate surface-modified chitosan nanoparticles with two mucoadhesive polymers (sodium alginate and polyethylene glycol) to optimize their protein encapsulation efficiency, improve their mucoadhesion properties, and increase their stability in biological fluids. Method: Ionotropic gelation was employed to formulate chitosan nanoparticles and surface modification was performed at five different concentrations (0.05, 0.1, 0.2, 0.3, 0.4% w/v) of sodium alginate (ALG) and polyethylene glycol (PEG), with ovalbumin (OVA) used as a model protein antigen. The functional characteristics were examined by dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)/scanning transmission electron microscopy (STEM). Stability was examined in the presence of simulated gastric and intestinal fluids, while mucoadhesive properties were evaluated by in vitro mucin binding and ex vivo adhesion on pig oral mucosa tissue. The impact of the formulation and dissolution process on the OVA structure was investigated by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD). Results: The nanoparticles showed a uniform spherical morphology with a maximum protein encapsulation efficiency of 81%, size after OVA loading of between 200 and 400 nm and zeta potential from 10 to 29 mV. An in vitro drug release study suggested successful nanoparticle surface modification by ALG and PEG, showing gastric fluid stability (4 h) and a 96 h sustained OVA release in intestinal fluid, with the nanoparticles maintaining their conformational stability (SDS-PAGE and CD analyses) after release in the intestinal fluid. An in vitro mucin binding study indicated a significant increase in mucin binding from 41 to 63% in ALG-modified nanoparticles and a 27–49% increase in PEG-modified nanoparticles. The ex vivo mucoadhesion showed that the powdered particles adhered to the pig oral mucosa. Conclusion: The ALG and PEG surface modification of chitosan nanoparticles improved the particle stability in both simulated gastric and intestinal fluids and improved the mucoadhesive properties, therefore constituting a potential nanocarrier platform for mucosal protein vaccine delivery.

Encapsulation of Cochleates Derived from Salmonella Infantis with Biopolymers to Develop a Potential Oral Poultry Vaccine

The aim of this study was to develop and characterize Salmonella enterica serovar Infantis (S. Infantis) cochleates protected by encapsulation technology as a potential vaccine and to determine its safety in pullets. Cochleates were encapsulated by two technologies, spray drying and ionotropic gelation at different concentrations (0–15% v/v), and were characterized by physicochemical properties, protein content and Fourier Transform Infrared Spectroscopy (FTIR). The cochleates were white liquid suspensions with tubular shapes and a protein content of 1.0–2.1 mg/mL. After encapsulation by spray drying, microparticles ranged in size from 10.4–16.9 µm, were spherical in shape, and the protein content was 0.7–1.8 mg/g. After encapsulation by ionotropic gelation, beads ranged in size from 1620–1950 µm and were spherical in shape with a protein content of 1.0–2.5 mg/g. FTIR analysis indicated that both encapsulation processes were efficient. The cochleates encapsulated by ionotropic gelation were then tested for safety in pullets. No ill effect on the health of animals was observed upon physical or postmortem examination. In conclusion, this study was the first step in developing a potential oral S. Infantis vaccine safe for poultry using a novel cochleate encapsulation technology. Future studies are needed to determine the effectiveness of the vaccine.

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.

Humoral Immune Response of Thai Dogs after Oral Vaccination against Rabies with the SPBN GASGAS Vaccine Strain.

Applied research is crucial in pushing the boundaries and finding a solution to the age-old problem of dog-mediated rabies. Although oral vaccination of dogs is considered to have great potential in mass dog vaccination campaigns and could have far-reaching benefits, it is perhaps the most ignored of all available tools in efforts to eliminate dog-mediated rabies, not least because of limited data on immunogenicity, efficacy, and safety of potential oral rabies vaccine candidates. In this study, the long-term immunogenicity in local Thai dogs after oral administration of the highly attenuated 3rd generation rabies virus vaccine strain SPBN GASGAS was assessed. The oral rabies vaccine was administered to dogs by either direct oral administration (n = 10) or by offering a vaccine loaded intestine bait (n = 15). The humoral immune response was then compared to three groups of dogs; a group that received a parenteral delivered inactivated rabies vaccine (n = 10), a group offered a placebo intestine bait (n = 7), and a control group (n = 4) for an observation period of 365 days. There was no significant difference in the immune response of dogs that received oral and parenteral vaccine in terms of magnitude, kinetics, and persistence of both rabies virus (RABV) neutralizing (RFFIT) and binding (ELISA) antibodies. Although the single parenteral injection of an inactivated rabies vaccine mounted a slightly higher humoral immune response than the orally delivered live vaccine, RABV specific antibodies of both types were still detectable after one year in most animals for all treatment groups and resulted in no difference in seropositivity. Characterization of rabies specific antibodies revealed two main classes of antibodies involved in the immune response of dogs vaccinated. While IgM antibodies were the first to appear, the succeeding IgG response was mainly IgG2 dominated independent of the vaccine type used. The results support the view that SPBN GASGAS induces a sustained detectable immune response in local dogs both after direct oral administration and via bait application.

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.

Comparison and process optimization of PLGA, chitosan and silica nanoparticles for potential oral vaccine delivery.

Aim: The study compared performance of nanoparticles prepared from synthetic organic, natural organic and inorganic materials as vaccine delivery platforms. Materials& methods: Various formulation (concentration, polymer/silica:surfactant ratio, solvent) and process parameters (homogenization speed and time, ultrasonication) affecting functional performance characteristics of poly(lactic-co-glycolic acid) (PLGA), chitosan and silica-based nanoparticles containing bovine serum albumin were investigated. Nanoparticles were characterized using dynamic light scattering, x-ray diffraction, scanning/transmission electron microscopy, Fourier transform infrared spectroscopy and in vitro protein release. Results: Critical formulation parameters were surfactant concentration (PLGA, silica) and polymer concentration (chitosan). Optimized nanoparticles were spherical in shape with narrow size distribution and size ranges of 100-300nm (blank) and 150-400nm (protein loaded). Protein encapsulation efficiency was 26-75% and released within 48 h in a sustained manner. Conclusion: Critical formulation and process parameters affected size of PLGA, chitosan and silica nanoparticles and protein encapsulation, while silica produced the smallest and most stable nanoparticles.

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Preparation of virus-like particles for porcine circovirus type 2 by YeastFab Assembly.

Porcine circovirus type 2 (PCV2) poses a genuine threat to pig industry. An effective vaccine production against the pandemic is desirable. The aim of this study was to construct recombination yeast strains with PCV2 Cap protein. We adopt to YeastFab Assembly method to synthesize transcriptional units in a single tube by piecing up promoter, open reading frame, and terminator in S. cerevisiae. Two yeast recombinants were successfully constructed using GPD and TEF2 promoters, respectively, to express PCV2 by secreting Cap protein in vitro. Electronic microscope observation demonstrated that the yeast-derived PCV2 Cap protein could self-assembles into 18-nm-diameter virus-like particles (VLPs). The yield of two different recombination yeasts containing GPD and TEF2 promoters were 12, 25μg/ml, respectively. Our results showed that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles. This indicated that there is possibility of obtaining PCV2 VLP vaccine by homologous recombination in yeast genome, and Cap protein was secreted into the cultural supernatant which can be used as a potential oral vaccine to protect pigs from PCV2-infection.

Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment. Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the three-dimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids. HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

An efficient and simple method to increase the level of displayed protein on the yeast cell surface

BackgroundThe development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. MethodsTo address this issue, we established an efficient and simple method of increasing the level of displayed protein on the yeast cell surface. We used the single chain variable fragment (scFv) of an antibody against the infectious hematopoietic necrosis virus isolate Sn1203 as a target display protein. The yeast-derived scFv was first displayed on the yeast surface by galactose induction, and then Escherichia coli-derived scFv was also displayed on the same yeast via an artificial anchoring condition to increase the total scFv level on the yeast surface. ResultsThe levels of yeast- and E. coli-derived scFv displayed on the yeast cell surface were analyzed by flow cytometry, western blotting, and fluorescent microscopy. The flow cytometry results indicated that when the cells were suspended in phosphate-buffered saline with 1mmol/L glutathione, 0.2mmol/L oxidized glutathione, and 5% dimethyl sulfoxide at 4°C for 6h, the E. coli-derived scFv protein was stably anchored to the yeast cell surface. The mean fluorescence intensity in these experiments, which is an indirect quantitative representation of the surface scFv expression, was three times higher in the treated cells than that in control cells. The western blotting results show two specific protein bands, the smaller of which was identified as the E. coli-derived scFv that was displayed on the yeast cell surface. Cell immunofluorescence is a more direct way to detect differentially produced proteins that are displayed on the yeast cell surface. The fluorescence microscopy results show that both fluorescence corresponding to the yeast-derived scFv and fluorescence corresponding to the E. coli-derived scFv can exist on the cell surface of same yeast cell. This confirms that the E. coli-derived scFv protein was successfully displayed on the yeast cell surface. ConclusionsThis method provides a rapid, simple, and high-efficiency strategy to increase the level of displayed protein on the yeast cell surface. Application of this technique may allow the yeast surface display system to be used to generate potential oral vaccines.

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.

Human Enterovirus 71 Protein Displayed on the Surface of Saccharomyces cerevisiae as an Oral Vaccine.

Human enterovirus 71 (EV-A71), a major agent of hand, foot, and mouth disease, has become an important public health issue in recent years. No effective antiviral or vaccines against EV-A71 infection are currently available. EV-A71 infection intrudes bodies through the gastric mucosal surface and it is necessary to enhance mucosal immune response to protect children from these pathogens. Recently, the majority of EV-A71 vaccine candidates have been developed for parenteral immunization. However, parenteral vaccine candidates often induce poor mucosal responses. On the other hand, oral vaccines could induce effective mucosal and systemic immunity, and could be easily and safely administered. Thus, proper oral vaccines have attached more interest compared with parenteral vaccine. In this study, the major immunogenic capsid protein of EV-A71 was displayed on the surface of Saccharomyces cerevisiae. Oral immunization of mice with surface-displayed VP1 S. cerevisiae induced systemic humoral and mucosal immune responses, including virus-neutralizing titers, VP1-specific antibody, and the induction of Th1 immune responses in the spleen. Furthermore, oral immunization of mother mice with surface-displayed VP1 S. cerevisiae conferred protection to neonatal mice against the lethal EV-A71 infection. Furthermore, we observed that multiple boost immunization as well as higher immunization dosage could induce higher EV-A71-specific immune response. Our results demonstrated that surface-displayed VP1 S. cerevisiae could be used as potential oral vaccine against EV-A71 infection.