Multi-epitope Antigen Research Articles

Immuno-bioinformatic approach for designing of multi-epitope merozoite surface antigen of Babesia bigemina and evaluation of its immunogenicity in inoculated calves.

Babesiosis is a parasitic disease caused by intraerythrocytic parasites of the genus Babesia, which infect both wild and domestic animals. Merozoite surface antigens (MSAs) have been identified as efficient immunogens in Babesia-infected animals. MSAs play a key role in the invasion process and have been proposed as potential targets for vaccine development. Epitope-based vaccines offer several advantages over whole protein vaccines as the immunogenic proteins are small and can induce both Th1 and Th2 immune responses, which are desirable for protection. However, the MSA, particularly gp45, is polymorphic in Babesia bigemina, posing a challenge to vaccine development. The purpose of this study was to develop a recombinant gpME (gp45-multi-epitope) for a vaccine against Babesia bigemina. B-cell, T-cell, and HLA epitope predictions were used to synthesize the gpME sequence from the consensus sequence of gp45. The gpME sequence was synthesized and cloned in the pET28α vector through the commercial biotechnology company to get pET28-gpME. The plasmid cloned with the gpME sequence comprising 1068 bp was expressed in a bacterial expression system. A band of 39 kDa of rec-gpME was obtained via SDS-PAGE and Western blotting. Rec-gpME @200ng was injected in calves 3 times at 2 weeks interval. The humoral response was evaluated through the indirect ELISA method. The ELISA with rec-gp45 protein showed a significant value of optical density. The recombinant protein containing multiple epitopes from the MSA gp45 may represent a promising candidate for a vaccine against Babesia bigemina.

Immunogenicity and protective efficacy of a multi-epitope recombinant toxin antigen of Pasteurella multocida against virulent challenge in mice.

Pasteurella multocida (P. multocida) infection frequently results in porcine atrophic rhinitis and swine plague, leading to large economic losses for the swine industry worldwide. P. multocida toxin (PMT, 146kDa) is a highly virulent key virulence factor that plays a vital role in causing lung and turbinate lesions. This study developed a multi-epitope recombinant antigen of PMT (rPMT) that showed excellent immunogenicity and protection in a mouse model. Using bioinformatics to analyse the dominant epitopes of PMT, we constructed and synthesized rPMT containing 10 B-cell epitopes, 8 peptides with multiple B-cell epitopes and 13 T-cell epitopes of PMT and a rpmt gene (1,974bp) with multiple epitopes. The rPMT protein (97kDa) was soluble and contained a GST tag protein. Immunization of mice with rPMT stimulated significantly elevated serum IgG titres and splenocyte proliferation, and serum IFN-γ and IL-12 were upregulated by 5-fold and 1.6-fold, respectively, but IL-4 was not. Furthermore, the rPMT immunization group exhibited alleviated lung tissue lesions and a significantly decreased degree of neutrophil infiltration compared with the control groups post-challenge. In the rPMT vaccination group, 57.1% (8/14) of the mice survived the challenge, similar to the bacterin HN06 group, while all the mice in the control groups succumbed to the challenge. Thus, rPMT could be a suitable candidate antigen for developing a subunit vaccine against toxigenic P. multocida infection.

Multi-Epitope Vaccine Candidates Associated with Mannosylated Chitosan and LPS Conjugated Chitosan Nanoparticles Against Brucella Infection

One promising approach to increase protection against infectious diseases is to use adjuvants that can selectively stimulate the immune responses. In this study, multi-epitope antigens associated with LPS loaded chitosan (LLC) as toll-like receptor agonist or mannosylated chitosan nanoparticle (MCN) as vaccine delivery system were evaluated for their ability to stimulate immune responses to Brucella infection in mice model. Our results indicated that the addition of MCN to our vaccine formulations significantly elicited IFN-γ and IL-2 cytokines and antibody titers, in comparison with the non-adjuvanted vaccine candidates. The present results indicated that multi-epitopes and their administration with LLC or MCN induced Th1 immune response. In addition, vaccine candidates containing MCN provided high percentage of protection against B. melitensis and B. abortus infection. Our results provided support to previous reports indicating that MCNs are attractive adjuvants and addition of this adjuvant to multi-epitopes antigens play an important role in the development of vaccine against Brucella.

A polyvalent multiepitope protein cross-protects against Vibrio cholerae infection in rabbit colonization and passive protection models

Using epitope- and structure-based multiepitope fusion antigen vaccinology platform, we constructed a polyvalent protein immunogen that presents antigenic domains (epitopes) of Vibrio cholerae toxin-coregulated pilus A, cholera toxin (CT), sialidase, hemolysin A, flagellins (B, C, and D), and peptides mimicking lipopolysaccharide O-antigen on a flagellin B backbone. Mice and rabbits immunized intramuscularly with this polyvalent protein immunogen developed antibodies to all of the virulence factors targeted by the immunogen except lipopolysaccharide. Mouse and rabbit antibodies exhibited functional activities against CT enterotoxicity, CT binding to GM1 ganglioside, bacterial motility, and in vitro adherence of V. cholerae O1, O139, and non-O1/non-O139 serogroup strains. When challenged orogastrically with V. cholerae O1 El Tor N16961 or a non-O1/non-O139 strain, rabbits IM immunized with the immunogen showed a 2-log (99%) reduction in V. cholerae colonization of small intestines. Moreover, infant rabbits born to the mother immunized with the protein immunogen acquired antibodies passively and were protected from bacterial intestinal colonization (>2-log reduction), severe diarrhea (100%), and mild diarrhea (88%) after infection with V. cholerae O1 El Tor (N16961), O1 classical (O395), O139 (Bengal), or a non-O1/non-O139 strain. This study demonstrated that this polyvalent cholera protein is broadly immunogenic and cross-protective, and an adult rabbit colonization model and an infant rabbit passive protection model fill a gap in preclinical efficacy assessment in cholera vaccine development.

Design of a polytopic construct of LACK, TSA and GP63 proteins for the diagnosis of cutaneous leishmaniasis: An in silico strategy

• The synthesized multi-epitope was successfully expressed into Escherichia coli. • rGLT increased specificity of antigen detection tests by identifying B-cell epitopes. • Our novel multi-epitope antigens represent a potential target for CL diagnostic. Cutaneous leishmaniasis (CL), which is caused by different species of the genus Leishmania, including L. major , is a significant parasitic disease worldwide. Commercial leishmaniasis diagnostic kits frequently target the entire parasite antigen, although this approach can reduce the specificity of the tests. The present study sought to increase the specificity of antigen detection tests by identifying the B-cell epitopes on the surface of the protozoa and developing an antigenic multiepitope construct to react with the system of B-cell epitopes unique to L. major . More specifically, the linear and conformational B-cell epitopes for three L. major proteins, namely, GP63, LACK, and TSA, were predicted using the ABCprd, IEDB, LBtope, CBTOPE, and DiscoTope servers. The three-dimensional structures of these proteins, as well as of the complete multiepitope, were predicted using the I-TASSER server. The multiepitope structure models were validated by means of Ramachandran plots and the Prosa and Verify3D servers. The multiepitope construct was evaluated using the ProtParam and SOLpro servers. The synthesized multiepitope was then successfully expressed into a pET28a plasmid. The expression was confirmed using SDS-PAGE and dot blot techniques. Moreover, the expressed protein was evaluated by means of Western blotting. A band of approximately 39 kDa was observed by SDS-PAGE. The rGLT-pET-28a showed a high response with the 1:1000 dilution of the anti-His-tag monoclonal antibody by Western blot. The results of this study indicated that the integrated GP63, LACK, and TSA multiepitope antigens of L. major represent a potential target for a novel diagnostic kit for CL.

In silico designing of a recombinant multi-epitope antigen for leprosy diagnosis

BackgroundLeprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Most of the affected population lives in low-income countries and may take up to 10 years to show any clinical signs, which is how physicians diagnose it. However, due to progressive cell damage, early diagnosis is very important. The best way to confirm leprosy is through bacilloscopic, which only confirms the diagnosis and has low accuracy or PCR, that requires specialized operators and is expensive. Since the bacteria are fastidious and do not grow in any culture media, therefore, diagnosing leprosy in the lab is still a challenge. In this concern, a recombinant multi-epitope protein can be a beneficial strategy in the management of the diagnosis, as diverse immunogenic epitopes are precisely selected to detect specific antibodies. Therefore, the purposes of the present study were to select immunogenic epitopes from different relevant proteins, with immunogenic properties, and then to construct a recombinant multi-epitope protein that accuses the presence of the antibodies in the early stages of the disease, making it more than appropriate to be applied as a diagnostic tool. ResultsWe selected 22 common proteins from both species and, using bioinformatics tools, predicted B and T cell epitopes. After multiple filtering and analyzing, we ended up with 29 epitopes {MHC-I (total 18) and MHC-II (total 11)} from 10 proteins, which were then merged into one construct. Its secondary and tertiary structures were also predicted and refined to comprise the amino acid residues in the best conformation possible. The multi-epitope protein construct was stable, non-host homologous, non-allergic, non-toxic, and elicit humoral and cellular responses. It has conformational B cell epitopes and potential to elicit IFN-γ, IL-4, and IL-10 secretion. ConclusionsThis novel recombinant multi-epitope protein constructed using the common epitopes from M. leprae and M. lepromatosis has a huge immunological potential, is stable, and can be lyophilized to be used in ELISA plates or even in biosensors, which are user-friendly diagnosis tools, facilitating translation into human sample tests.

Estimation of Plasmodium falciparum transmission using multiepitope chimeric antigen in the postelimination phase in Yunnan, China.

The continuous monitoring of malaria transmission intensity is still required to maintain elimination status after reaching the malaria elimination stage. In this study, serological surveillance with multiepitope artificial antigen was used to assess the transmission of Plasmodium falciparum in Yunnan, China, where malaria elimination has just been achieved, to provide data to support malaria control in the postelimination period. Samples were collected in three border counties and one inland county in Yunnan Province in 2016 using a stratified whole-group sampling method. Fingerstick blood was collected from all participants, and antibodies to Malaria Random Constructed Antigen-1 (M. RCAg-1) were detected by indirect ELISA. The transmission intensity of P. falciparum malaria was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). A total of 5566 samples were collected. There was no statistically significant difference in antibody level between the inland county and the nonendemic area, but the antibody level in border counties was significantly higher than those in the inland county and the nonendemic control area. No seropositive cases were found in Yanjin County, and the seropositivity rate increased with age in the three border counties. The highest intensity of P. falciparum malaria transmission was in Zhenkang County (SCR=0.0030, CI: 0.0029, 0.0031), followed by Gengma County (SCR=0.0013, CI: 0.0012, 0.0015) and Yingjiang County (SCR=0.00088, CI: 0.00083, 0.00090). The transmission intensity of P. falciparum malaria in Yunnan Province has obviously decreased in recent years, but for the border areas where malaria has just been eliminated, the transmission intensity will not immediately drop to zero, and it still needs to be monitored for a period of time to maintain malaria elimination status.

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya.

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Immunoinformatics Design and Assessment of a Multiepitope Antigen (OvMCBL02) for Onchocerciasis Diagnosis and Monitoring.

Onchocerciasis is a Neglected Tropical Disease that has a significant socioeconomic impact, especially in Sub-Saharan Africa. Numerous reports indicate that the Expanded Special Project for the Elimination of Neglected Tropical Diseases needs novel diagnostic tools before achieving its goal of successful elimination of onchocerciasis in Africa. The current diagnostic tests are either invasive, insensitive, or not applicable in the field and about 25% of persons infected cannot mount immune responses against the single antigen used in the only approved Ov-16 serological test. In the quest to identify novel biomarkers that can be used to certify that a patient is free from the disease, evaluate the progress of elimination programmes, and conduct post elimination surveillances, mass spectrometric analysis of Onchocerca volvulus crude extract revealed that 1392 proteins are expressed in the adult and microfilariae stages of the parasite. Computational analysis predicted six of the proteins as O. volvulus potential diagnostic targets. Linear B-epitopes were predicted from the six proteins and used to construct a multiepitope antigen (OvMCBL02). Serological analysis revealed that the OvMCBL02 test significantly differentiated between serum samples of onchocerciasis patients from the Kombone Health Area in the South West Region of Cameroon (n = 63) and control serum samples from Rwanda (n = 29) and Europe (n = 26) as well as between serum samples from the onchocerciasis hyperendemic region of Kombone Health Area (n = 63) and the hypoendemic region of Bandjoun Health District (n = 54). Interestingly, the test did not cross-react with serum samples from patients suffering from related nematode infections, thereby suggesting that further characterization of the OvMCBL02 multiepitope antigen will render it an additional member of the diagnostic toolbox for the elimination of onchocerciasis.

A multiepitope vaccine encoding four Eimeria epitopes with PLGA nanospheres: a novel vaccine candidate against coccidiosis in laying chickens

With a worldwide distribution, Eimeria spp. could result in serious economic losses to the poultry industry. Due to drug resistance and residues, there are no ideal drugs and vaccines against Eimeria spp. in food animals. In the current study, a bioinformatics approach was employed to design a multiepitope antigen, named NSLC protein, encoding antigenic epitopes of E. necatrix NA4, E. tenella SAG1, E. acervulina LDH, and E. maxima CDPK. Thereafter, the protective immunity of NSLC protein along with five adjuvants and two nanospheres in laying chickens was evaluated. Based on the humoral immunity, cellular immunity, oocyst burden, and the coefficient of growth, the optimum adjuvant was evaluated. Furthermore, the optimum immune route and dosage were also investigated according to the oocyst burden and coefficient of growth. Accompanied by promoted secretion of antibodies and enhanced CD4+ and CD8+ T lymphocyte proportions, NSLC proteins entrapped in PLGA nanospheres were more effective in stimulating protective immunity than other adjuvants or nanospheres, indicating that PLGA nanospheres were the optimum adjuvant for NSLC protein. In addition, a significantly inhibited oocyst burden and growth coefficient promotion were also observed in animals vaccinated with NSLC proteins entrapped in PLGA nanospheres, indicating that the optimum adjuvant for NSLC proteins was PLGA nanospheres. The results also suggested that the intramucosal route with PLGA nanospheres containing 300 μg of NSLC protein was the most efficient approach to induce protective immunity against the four Eimeria species. Collectively, PLGA nanospheres loaded with NSLC antigens are potential vaccine candidates against avian coccidiosis.

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children’s and travelers’ diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.

In silico Design of a Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Multi-Epitope Antigen for Vaccine Development.

There is no licensed vaccine available to prevent the severe tick-borne disease Crimean-Congo hemorrhagic fever (CCHF), caused by the CCHF virus (CCHFV). This study sought to show that a combination of computational methods and data from published literature can inform the design of a multi-epitope antigen for CCHFV that has the potential to be immunogenic. Cytotoxic and helper T-cell epitopes were evaluated on the CCHFV GPC using bioinformatic servers, and this data was combined with work from previous studies to identify potentially immunodominant regions of the GPC. Regions of the GPC were selected for generation of a model multi-epitope antigen in silico, and the percent residue identity and similarity of each region was compared across sequences representing the widespread geographical and ecological distribution of CCHFV. Eleven multi-epitope regions were joined together with flexible linkers in silico to generate a model multi-epitope antigen, termed EPIC, which included 812 (75.7%) of all predicted epitopes. EPIC was predicted to be antigenic by two independent bioinformatic servers, suggesting that multi-epitope antigens should be explored further for CCHFV vaccine development. The results presented within this manuscript provide information for potential targets within the CCHFV GPC for guiding future vaccine development.

Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli

The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti‐MEA FMNs. Optimal anti‐MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9 nm, corresponding to an 83% rate of pAb conjugation. Anti‐MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 102 colony‐forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti‐MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse‐like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture‐free detection of Campylobacter in chicken products.

Development a novel multiepitope DNA vaccine against human SARS coronavirus-2: an immunoinformatic designing study.

Human SARS coronavirus 2 (SARS-CoV-2) causes the current global COVID-19 pandemic. The production of an efficient vaccine against COVID-19 is under heavy investigation. In this study, we have designed a novel multiepitope DNA vaccine against SARS-CoV-2 using reverse vaccinology and DNA vaccine approaches. Applying these strategies led to reduce the time and costs of vaccine development and also improve the immune protective characteristics of the vaccine. For this purpose, epitopes of nucleocapsid, membrane glycoprotein, and ORF8 proteins of SARS-CoV-2 chose as targets for B and T-cell receptors. Accordingly, DNA sequences of selected epitopes have optimized for protein expression in the eukaryotic system. To this end, the Kozak and tissue plasminogen activator sequences were added into the epitope sequences for proper protein expression and secretion, respectively. Furthermore, interleukin-2 and beta-defensin 1 preproprotein sequences were incorporated to the designed DNA vaccine as an adjuvant. Modeling and refinement of fused protein composed of SARS-CoV-2 multiepitope antigens (fuspMA) have performed based on homology modeling of orthologous peptides, then constructed 3D model of fuspMA was more investigated during 50 ns of molecular dynamics simulation. Further bioinformatics predictions demonstrated that fuspMA is a stable protein with acceptable antigenic features and no allergenicity or toxicity characteristics. Finally, the affinity of fuspMA to the MHC I and II and TLRs molecules validated by the molecular docking procedure. In conclusion, it seems the designed multiepitope DNA vaccine could have a chance to be introduced as an efficient vaccine against COVID-19 after more in vivo evaluations.

Construction of a multiepitope vaccine candidate against Fasciola hepatica: an in silico design using various immunogenic excretory/secretory antigens

ABSTRACT Background Fasciola hepatica is an important pathogen that causes liver fluke disease in definitive hosts such as livestock animals and humans. Various excretory/secretory products have been used in serological diagnosis and vaccination studies targeting fasciolosis. There are no commercial vaccines against fasciolosis yet. Bioinformatic analysis based on computational methods have lower cost and provide faster output compared to conventional vaccine antigen discovery techniques. The aim of this study was to predict B- and T-cell specific epitopes of four excretory/secretory antigens (Kunitz-type serine protease inhibitor, cathepsin L1, helminth defense molecule, and glutathione S-transferase) of Fasciola hepatica and to construct a multiepitope vaccine candidate against fasciolosis. Methods and Results Initially, nonallergic and the highest antigenic B- and T- cell epitopes were selected and then, physico-chemical parameters, secondary and tertiary structures of designed multiepitope vaccine candidate were predicted. Tertiary structure was refined and validated using online bioinformatic tools. Linear and discontinuous B-cell epitopes and disulfide bonds were determined. Finally, molecular docking analysis for MHC-I and MHC-II receptors was performed. Conclusion This multi-epitope vaccine candidate antigen, with high immunological properties, can be considered as a promising vaccine candidate for animal experiments and wet lab studies.

Alum Pickering Emulsion as Effective Adjuvant to Improve Malaria Vaccine Efficacy.

Malaria is a life-threatening global epidemic disease and has caused more than 400,000 deaths in 2019. To control and prevent malaria, the development of a vaccine is a potential method. An effective malaria vaccine should either combine antigens from all stages of the malaria parasite’s life cycle, or epitopes of multiple key antigens due to the complexity of the Plasmodium parasite. Malaria’s random constructed antigen-1 (M.RCAg-1) is one of the recombinant vaccines, which was selected from a DNA library containing thousands of diverse multi-epitope chimeric antigen genes. Moreover, besides selecting an antigen, using an adjuvant is another important procedure for most vaccine development procedures. Freund’s adjuvant is considered an effective vaccine adjuvant for malaria vaccine, but it cannot be used in clinical settings because of its serious side effects. Traditional adjuvants, such as alum adjuvant, are limited by their unsatisfactory immune effects in malaria vaccines, hence there is an urgent need to develop a novel, safe and efficient adjuvant. In recent years, Pickering emulsions have attracted increasing attention as novel adjuvant. In contrast to classical emulsions, Pickering emulsions are stabilized by solid particles instead of surfactant, having pliability and lateral mobility. In this study, we selected aluminum hydroxide gel (termed as “alum”) as a stabilizer to prepare alum-stabilized Pickering emulsions (ALPE) as a malaria vaccine adjuvant. In addition, monophosphoryl lipid A (MPLA) as an immunostimulant was incorporated into the Pickering emulsion (ALMPE) to further enhance the immune response. In vitro tests showed that, compared with alum, ALPE and ALMPE showed higher antigen load rates and could be effectively endocytosed by J774a.1 cells. In vivo studies indicated that ALMPE could induce as high antibody titers as Freund’s adjuvant. The biocompatibility study also proved ALMPE with excellent biocompatibility. These results suggest that ALMPE is a potential adjuvant for a malaria vaccine.

Comparison of the multi-epitope recombinant antigen DIPOL and hydatid fluid for the diagnosis of patients with cystic echinococcosis

The use of serological tests containing multiple immunodominant antigens rather than single antigens have the potential to improve the diagnostic performance in Cystic Echinococcoses (CE) as a complement tool to clear the inconclusive imaging data. Here, we comparatively evaluated the diagnostic value of Hydatid Fluid (HF) and the recently described recombinant multi-epitope antigen DIPOL in IgG-ELISA in a clinically defined cohort of CE patients. The serum samples from 149 CE patients were collected just before surgical or Percutaneous- Aspiration- Injection- Reaspiration (PAIR) procedures. Additionally, serum samples of patients with other parasitic infections (n=49) and healthy individuals (n=21) were also included in the study as controls. To investigate the association between the genotype of the parasite and DIPOL, cyst materials from 20 CE patients were sequenced. In terms of overall sensitivity, HF was higher than DIPOL (82.55%,78.52%, respectively). However, while the sensitivity of HF was higher than DIPOL in patients with active and transitional cysts (83.3%, 75.4%, respectively), sensitivity of DIPOL in inactive cysts was higher compared to HF (95.6%, 78.3%, respectively). The sensitivity of DIPOL depending on cyst stage was statistically significant (P= 0.041). In terms of specificity, DIPOL was found to be better than HF (97.71%, 91.43%, respectively). By genotyping, the majority of 20 patients showed G1 genotype (80%). All patients harboring G3 and G1/G3 cyst genotypes were positive with both antigens, while 87.5% of patients with G1 genotype were seropositive with HF and 75% with DIPOL. The overall sensitivity and high specificity of DIPOL suggest that this recombinant protein containing immunodominant epitopes is a potential substitute for the HF by serological tests for the diagnosis of CE.

Immune Responses to Orally Administered Recombinant Lactococcus lactis Expressing Multi-Epitope Proteins Targeting M Cells of Foot-and-Mouth Disease Virus.

Foot and mouth disease virus (FMDV), whose transmission occurs through mucosal surfaces, can also be transmitted through aerosols, direct contact, and pollutants. Therefore, mucosal immunity can efficiently inhibit viral colonization. Since vaccine material delivery into immune sites is important for efficient oral mucosal vaccination, the M cell-targeting approach is important for effective vaccination given M cells are vital for luminal antigen influx into the mucosal lymph tissues. In this study, we coupled M cell-targeting ligand Co1 to multi-epitope TB1 of FMDV to obtain TB1-Co1 in order to improve delivery efficiency of the multi-epitope protein antigen TB1. Lactococcus lactis (L. lactis) was engineered to express heterologous antigens for applications as vaccine vehicles with the ability to elicit mucosal as well as systemic immune responses. We successfully constructed L. lactis (recombinant) with the ability to express multi-epitope antigen proteins (TB1 and TB1-Co1) of the FMDV serotype A (named L. lactis-TB1 and L. lactis-TB1-Co1). Then, we investigated the immunogenic potential of the constructed recombinant L. lactis in mice and guinea pigs. Orally administered L. lactis-TB1 as well as L. lactis-TB1-Co1 in mice effectively induced mucosal secretory IgA (SIgA) and IgG secretion, development of a strong cell-mediated immune reactions, substantial T lymphocyte proliferation in the spleen, and upregulated IL-2, IFN-γ, IL-10, and IL-5 levels. Orally administered ligand-conjugated TB1 promoted specific IgG as well as SIgA responses in systemic and mucosal surfaces, respectively, when compared to orally administered TB1 alone. Then, guinea pigs were orally vaccinated with L. lactis-TB1-Co1 plus adjuvant CpG-ODN at three different doses, L. lactis-TB1-Co1, and PBS. Animals that had been immunized with L. lactis-TB1-Co1 plus adjuvant CpG-ODN and L. lactis-TB1-Co1 developed elevated antigen-specific serum IgG, IgA, neutralizing antibody, and mucosal SIgA levels, when compared to control groups. Particularly, in mice, L. lactis-TB1-Co1 exhibited excellent immune effects than L. lactis-TB1. Therefore, L. lactis-TB1-Co1 can induce elevations in mucosal as well as systemic immune reactions, and to a certain extent, provide protection against FMDV. In conclusion, M cell-targeting approaches can be employed in the development of effective oral mucosa vaccines for FMDV.

Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs

BackgroundToxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits.MethodsThe synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK®Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA).ResultsMAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection).ConclusionsOur results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.Graphical abstract

Evaluation of an attenuated Listeria monocytogenes as a vaccine vector to control Helicobacter pylori infection

The increasing resistance of Helicobacter pylori (H. pylori) to antibiotics has limited the efficacy of antibiotic therapy in the treatment of H. pylori-associated gastric diseases. The vaccine as an alternative method is becoming a safe and effective way to address this problem. In previous studies, live vector vaccines have proved to be effective in controlling H. pylori infection. Attenuated Listeria monocytogenes (L. monocytogenes) is a potential candidate vector applied in clinical trials, which can deliver foreign antigens and induce a broad immune response. To further explore the effectiveness of L. monocytogenes as a vaccine vector against H. pylori, attenuated L. monocytogenes-based vaccine EGDeΔactA/inlB(EGDeAB)-MECU was constructed to secrete a multi-epitope chimeric antigen (MECU) containing multiple B cell epitopes from H. pylori antigens. EGDeAB-MECU could secrete MECU stably. After immunized by gavage and intravenous injection, both EGDeAB and EGDeAB-MECU could significantly decrease gastric H. pylori colonization and induce a high level of specific antibodies against H. pylori. In conclusion, attenuated L. monocytogenes had an immunotherapeutic effect on H. pylori-infected mice, indicating its further development as a promising candidate vaccine vector for the H. pylori vaccine.