Multi-epitope Antigen Research Articles

A multi-epitope subunit vaccine providing broad cross-protection against diverse serotypes of Streptococcus suis.

Streptococcus suis infection represents a major challenge in pig farming and public health due to its zoonotic potential and diverse serotypes, while existing vaccines lack effective cross-protection. This study employed reverse vaccinology and immunoinformatics to identify 8 conserved proteins across 11 prevalent serotypes of S. suis. 16 candidate epitopes were selected to design three multi-epitope antigens against S. suis (designated as MEASs), which fused with a dendritic cell-targeting peptide to improve antigen presentation in host. Purified MEASs displayed favorable cross-reactogenicity against 29 serotype-specific antiserums. Robust humoral and cellular immune responses can be induced by MEAS 1 and MEAS 3 in a mouse model, which provided substantial protection against virulent strains from two different serotypes. In particular, their immune serums exhibited positive opsonization effects within bloodstream and macrophage phagocytosis. Taken together, we identified two promising MEASs with excellent cross-protection, offering potential in preventing S. suis infections in a mouse model.

Designing a novel multi-epitope antigen for diagnosing human cytomegalovirus infection: An immunoinformatics approach.

Human cytomegalovirus (HCMV) infection can lead to congenital infections and severe complications, particularly in immunocompromised individuals. Current serological tests for diagnosing HCMV infection often face limitations in sensitivity and specificity. Developing multi-epitope antigens for serological assays offers the potential for enhancing diagnostic accuracy. This study aimed to design a novel multi-epitope antigen for HCMV infection diagnosis using immunoinformatic approaches. Five tegument proteins (universal protein resource [UniProt] ID: Po8318, Po6725, F5HC97, Q6RX10, and F5HC05) were selected based on their antigenic properties and literature review. Six linear B-cell epitopes were predicted within conserved regions of each antigen sequence and linked with appropriate linkers. The designed multi-epitope antigen underwent thorough evaluation for physicochemical properties, solubility, antigenicity, and cross-reactivity. Additionally, the three-dimensional structure of the antigen was predicted, refined, and validated. The nucleotide sequence of the designed antigen was optimized for successful expression in Escherichia coli and inserted into a pET23a (+) vector. Immunoinformatic analysis revealed that the multi-epitope antigen exhibits stability, antigenicity, and lacks cross-reactivity. Our findings suggest that this multi-epitope antigen is a promising candidate for diagnosing HCMV infection. However, further validation through laboratory testing is required to confirm its diagnostic efficacy.

A novel multi-epitope peptide vaccine targeting immunogenic antigens of Ebola and monkeypox viruses with potential of immune responses provocation in silico.

The emergence or reemergence of monkeypox (Mpox) and Ebola virus (EBOV) agents causing zoonotic diseases remains a huge threat to human health. Our study aimed at designing a multi-epitope vaccine (MEV) candidate to target both the Mpox and EBOV agents using immunoinformatics tools. Viral protein sequences were retrieved, and potential nonallergenic, nontoxic, and antigenic epitopes were obtained. Next, cytotoxic and helper T-cell (CTL and HTL, respectively) and B-cell (BCL) epitopes were predicted, and those potential epitopes were fused utilizing proper linkers. The in silico cloning and expression processes were implemented using Escherichia coli K12. The immune responses were prognosticated using the C-ImmSim server. The MEV construct (29.53kDa) included four BCL, two CTL, and four HTL epitopes and adjuvant. The MEV traits were pertinent in terms of antigenicity, non-allergenicity, nontoxicity, physicochemical characters, and stability. The MEV candidate was also highly expressed in E. coli K12. The strong affinity of MEV-TLR3 was confirmed using molecular docking and molecular dynamics simulation analyses. Immune simulation analyses unraveled durable activation and responses of cellular and humoral arms alongside innate immune responses. The designed MEV candidate demonstrated appropriate traits and was promising in the prediction of immune responses against both Mpox and EBOV agents. Further experimental assessments of the MEV are required to verify its efficacy.

A novel bacterium-like particles platform displaying antigens by new anchoring proteins induces efficacious immune responses.

Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of Escherichia coli outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria Levilactobacillus brevis 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of Clostridium perfringens as the model antigen. The vaccine can induce more than 50% protection rate against C. perfringens type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.

Immunoinformatics-Based Designing of Novel and Potent Multi-Epitope PSA D15 and Cag11 Immunogens for Helicobacter pylori Immunodiagnostic Assay Development.

Helicobacter pylori (H. pylori) strain is the most genetically diverse pathogenic bacterium and now alarming serious human health concern ranging from chronic gastritis to gastric cancer and human death all over the world. Currently, the majority of commercially available diagnostic assays for H. pylori is a challenging task due to the heterogeneity of virulence factors in various geographical regions. In this concern, designing of universal multi-epitope immunogenic biomarker targeted for all H. pylori strains would be crucial to successfully immunodiagnosis assay and vaccine development for H. pylori infection. Hence, the present study aimed to explore the potential immunogenic epitopes of PSA D15 and Cag11 proteins of H. pylori, using immunoinformatics web tools in order to design novel immune-reactive multi-epitope antigens for enhanced immunodiagnosis in humans. Through an in silico immunoinformatics approach, high-ranked B-cell, MHC-I, and MHC-II epitopes of PSA D15 and Cag11 proteins were predicted, screened, and selected. Subsequently, a novel multi-epitope PSA D15 and Cag11 antigens were designed by fused the high-ranked B-cell, MHC-I, and MHC-II epitopes and 50S ribosomal protein L7/L12 adjuvant using linkers. The antigenicity, solubility, physicochemical properties, secondary and tertiary structures, 3D model refinement, and validations were carried. Furthermore, the designed multi-epitope antigens were subjected to codon adaptation and in silico cloning, immune response simulation, and molecular docking with receptor molecules. A novel, stable multi-epitope PSA D15 and Cag11 H. pylori antigens were developed and immune simulation of the designed antigens showed desirable levels of immunological response. Molecular docking of designed antigens with immune receptors (B-cell, MHC-I, MHC-II, and TLR-2/4) revealed robust interactions and stable binding affinity to the receptors. The codon optimized and in silico cloned showed that the designed antigens were successfully expressed (CAI value of 0.95 for PSA D15 and 1.0 for Cag11) after inserted into pET-32ba (+) plasmid of the E. coli K12 strain. In conclusion, this study revealed that the designed multi-epitope antigens have a huge immunological potential candidate biomarker and useful in developing immunodiagnostic assays and vaccines for H. pylori infection.

Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection

Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.

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

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

In silico design and analysis of a multiepitope vaccine against Chlamydia.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG+IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.

Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis.

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA-5' and 3' untranslated regions (UTRs)-are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein's expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5' and 3'UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5'UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5'UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5'UTR also showed higher efficiency (as compared with the 5'UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5'UTRs and 3'UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5'UTRs and 3'UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5'UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

Evaluation of the humoral and mucosal immune response of a multiepitope vaccine against COVID-19 in pigs.

This study evaluated the immune response to a multiepitope recombinant chimeric protein (CHIVAX) containing B- and T-cell epitopes of the SARS-CoV-2 spike's receptor binding domain (RBD) in a translational porcine model for pre-clinical studies. We generated a multiepitope recombinant protein engineered to include six coding conserved epitopes from the RBD domain of the SARS-CoV-2 S protein. Pigs were divided into groups and immunized with different doses of the protein, with serum samples collected over time to determine antibody responses by indirect ELISA and antibody titration. Peptide recognition was also analyzed by Western blotting. A surrogate neutralization assay with recombinant ACE2 and RBDs was performed. Intranasal doses of the immunogen were also prepared and tested on Vietnamese minipigs. When the immunogen was administered subcutaneously, it induced specific IgG antibodies in pigs, and higher doses correlated with higher antibody levels. Antibodies from immunized pigs recognized individual peptides in the multiepitope vaccine and inhibited RBD-ACE2 binding for five variants of concern (VOC). Comparative antigen delivery methods showed that both, subcutaneous and combined subcutaneous/intranasal approaches, induced specific IgG and IgA antibodies, with the subcutaneous approach having superior neutralizing activity. CHIVAX elicited systemic immunity, evidenced by specific IgG antibodies in the serum, and local mucosal immunity, indicated by IgA antibodies in saliva, nasal, and bronchoalveolar lavage secretions. Importantly, these antibodies demonstrated neutralizing activity against SARS-CoV-2 in vitro. The elicited antibodies recognized individual epitopes on the chimeric protein and demonstrated the capacity to block RBD-ACE2 binding of the ancestral SARS-CoV-2 strain and four VOCs. The findings provide proof of concept for using multiepitope recombinant antigens and a combined immunization protocol to induce a neutralizing immune response against SARS-CoV-2 in the pig translational model for preclinical studies.

Embedding of exogenous B cell epitopes on the surface of UreB structure generates a broadly reactive antibody response against Helicobacter pylori.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens has increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralising antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunisation with MECU could reduce H. pylori colonisation in the stomach and protect the stomach in BALB/c mice. This study not only provides promising immunotherapy to control H. pylori infection but also offers a reference for antigen engineering against other pathogens.

Multi-epitope Antigen for Specific Serological Detection of Dengue Viruses.

Dengue is an infectious disease of global health concern that continues to require surveillance. Serological testing has been used to investigate dengue-infected patients, but specificity is affected by the co-circulation of ZIKA virus (ZIKV), which shares extensive antigen similarities. The goal of this study was the development of a specific dengue virus (DENV) IgG ELISA based on a multi-epitope NS1-based antigen for antibody detection. The multi-epitope protein (T-ΔNS1), derived from a fragment of the NS1-protein of the four DENV serotypes, was expressed in Escherichia coli and purified via affinity chromatography. The antigenicity and specificity were evaluated with sera of mice infected with DENV-1-4 or ZIKV or after immunization with the recombinant ΔNS1 proteins. The performance of the T-ΔNS1-based IgG ELISA was also determined with human serum samples. The results demonstrate that the DENV T-ΔNS1 was specifically recognized by the serum IgG of dengue-infected mice or humans but showed no or reduced reactivity with ZIKV-infected subjects. Based on the available set of clinical samples, the ELISA based on the DENV T-ΔNS1 achieved 77.42% sensitivity and 88.57% specificity. The results indicate that the T-ΔNS1 antigen is a promising candidate for the development of specific serological analysis.

Сравнительное исследование иммуногенности полноразмерной и мультиэпитопной мРНК-вакцин против M. Tuberculosis по выраженности Т-клеточного ответа у мышей

Development of the new tuberculosis vaccines that would be effective in adults is an urgent task: worldwide, the annual death toll of this disease exceeds 1.5 million. In the recent decades, the matter has been addressed in numerous studies, but none has yielded an effective vaccine so far. There are many factors to resistance against tuberculosis; this study focuses on the T-cell response, a mechanism that enables elimination of intracellular pathogens, such as M. tuberculosis. We aimed to develop an mRNA vaccine capable of triggering a pronounced T-cell response to the M. tuberculosis antigens. The in silico analysis allowed us to select epitopes of the M. tuberculosis secreted protein ESAT6 (Rv3875) and design a multi-epitope mRNA vaccine thereon. We assessed the intensity of T-cell response in mice immunized with mRNA vaccines that encode a full-length or multi-epitope antigen. The results of this study in mice show that immunization with a multi-epitope mRNA vaccine produces twice as many IFNγ-secreting splenocytes in response to specific stimulation than immunization with an mRNA vaccine encoding the full-length protein. Thus, the developed multi-epitope mRNA vaccine can be an effective M. tuberculosis prevention agent the mode of action of which involves formation of a pronounced T-cell response.

Development and evaluation of an indirect ELISA using a multiepitope antigen for the diagnosis of intestinal schistosomiasis.

The laboratory diagnosis of intestinal schistosomiasis, carried out by detecting parasite eggs in feces, has low sensitivity when applied to individuals with low parasitic load. Serological tests can be more sensitive for the diagnosis of the disease. Therefore, the objective of this work was to develop and evaluate an ELISA-based immunoenzymatic assay, using a Schistosoma mansoni multiepitope antigen (ELISA IgG anti-SmME). For this, the amino acid sequences of S. mansoni cathepsin B and asparaginyl endopeptidase were submitted to the prediction of B cell epitopes and, together with peptide sequences obtained from earlier works, were used in the construction of a minigene. The multiepitope protein was expressed in Escherichia coli and the performance of the ELISA IgG anti-SmME for schistosomiasis was evaluated using serum samples from 107 individuals either egg positive or negative. In addition, 11 samples from individuals with other helminth infections were included. The ELISA IgG anti-SmME showed a sensitivity of 81.1% and a specificity of 46.1%. Further analysis revealed a 77.2% sensitivity in diagnosis of individuals with egg counts of ≤12 epg (eggs per gram feces) and 87.5% for individuals with 13–99 epg. It is worth mentioning that, to our knowledge, this was the first study using a multiepitope recombinant antigen in an ELISA for diagnosis of intestinal schistosomiasis, which demonstrated promising results in the diagnosis of individuals with low parasitic loads.

Developing Multi-epitope Antigen Construct from Immunodominant Proteins for Serological Diagnosis of Chlamydia trachomatis: An In Silico Approach

Background: Chlamydiasis is a widespread bacterial infection in the world. Serological tests are expensive, and in addition, intrinsic antigens can cause cross-reactions and make the diagnosis process difficult. Multi-epitope protein antigens are novel and potential diagnostic markers that have the capability of more accurate and cheaper diagnosis. Therefore, in this study, the main goal is to design a new protein vaccine, including multiple epitopes of B cells with dominant immunity from three proteins named MOMP, ompA and Pgp3D from C. trachomatis Methods: The amino acid sequences were obtained from the UniProt database. The areas with the highest antigenicity were identified using the EMBOSS server. Linear B cell epitopes were determined using BCPRED, ABCpred, and Bepipred servers. Epitopes with the highest antigenicity were connected using the EAAAK linker. Results: Two epitopes from MOMP, two from ompA, and one from Pgp3D were selected. These epitopes were connected to each other with the EAAAK linker. Three residues (0.592), 16 residues (0.76), 36 residues (0.578), and 37 residues (0.734) were obtained from the prediction of the spatial structure of the B cell multiple epitopes designed with ElliPro. Model 1 of RaptorX was selected as the best structure. In this model, the ERRAT quality, ProSA-web z-score, and Verify3D were 83.1169, - 5.17 and 84.62% with PASS score, respectively. Moreover, the Ramachandran plot showed that 86.093% of the amino acid residues were located in the favored region. To achieve the highest level of protein expression, the designed multi-epitope reverse-translated with the Genscript server and was cloned in E. coli. The highest level of expression was achieved, and a CAI score of 0.91 was reported. The gene GC content was 51.98%, and the contribution of low-frequency codons was 0%. Conclusion: The results confirmed that the designed construct could identify C. trachomatis with high sensitivity and specificity in serum samples of patients with chlamydiasis. However, further experimental studies are needed for final confirmation.

In Situ Separable Nanovaccines with Stealthy Bioadhesive Capability for Durable Cancer Immunotherapy.

Because of tumor heterogeneity and the immunosuppressive tumor microenvironment, most cancer vaccines typically do not elicit robust antitumor immunological responses in clinical trials. In this paper, we report findings about a bioadhesive nanoparticle (BNP)-based separable cancer vaccine, FeSHK@B-ovalbumin (OVA), to target multi-epitope antigens and exert effective cancer immunotherapy. After the FeSHK@B-OVA "nanorocket" initiates the "satellite-rocket separation" procedure in the acidic tumor microenvironment, the FeSHK@B "launch vehicle" can amplify intracellular oxidative stress persistently. This procedure allows for bioadhesiveness-mediated prolonged drug retention within the tumor tissue and triggers the immunogenic death of tumor cells that transforms the primary tumors into antigen depots, which acts synergistically with the OVA "satellite" to trigger robust antigen-specific antitumor immunity. The cooperation of these two immunostimulants not only efficiently inhibits the primary tumor growth and provokes durable antigen-specific immune activation in vivo but also activates a long-term and robust immune memory effect to resist tumor rechallenge and metastasis. These results highlight the enormous potential of FeSHK@B-OVA to serve as an excellent therapeutic and prophylactic cancer nanovaccine. By leveraging the antigen depots in situ and the synergistic effect among multi-epitope antigens, such a nanovaccine strategy with stealthy bioadhesion may offer a straightforward and efficient approach to developing various cancer vaccines for different types of tumors.