Multi-epitope Protein Research Articles

B and T Cell Bi-Cistronic Multiepitopic Vaccine Induces Broad Immunogenicity and Provides Protection Against SARS-CoV-2

Background: The COVID-19 pandemic, caused by SARS-CoV-2, has highlighted the need for vaccines targeting both neutralizing antibodies (NAbs) and long-lasting cross-reactive T cells covering multiple viral proteins to provide broad and durable protection against emerging variants. Methods: To address this, here we developed two vaccine candidates, namely (i) DNA-CoV2-TMEP, expressing the multiepitopic CoV2-TMEP protein containing immunodominant and conserved T cell regions from SARS-CoV-2 structural proteins, and (ii) MVA-CoV2-B2AT, encoding a bi-cistronic multiepitopic construct that combines conserved B and T cell overlapping regions from SARS-CoV-2 structural proteins. Results: Both candidates were assessed in vitro and in vivo demonstrating their ability to induce robust immune responses. In C57BL/6 mice, DNA-CoV2-TMEP enhanced the recruitment of innate immune cells and stimulated SARS-CoV-2-specific polyfunctional T cells targeting multiple viral proteins. MVA-CoV2-B2AT elicited NAbs against various SARS-CoV-2 variants of concern (VoCs) and reduced viral replication and viral yields against the Beta variant in susceptible K18-hACE2 mice. The combination of MVA-CoV2-B2AT with a mutated ISG15 form as an adjuvant further increased the magnitude, breadth and polyfunctional profile of the response. Conclusion: These findings underscore the potential of these multiepitopic proteins when expressed from DNA or MVA vectors to provide protection against SARS-CoV-2 and its variants, supporting their further development as next-generation COVID-19 vaccines.

Development and evaluation of a recombinant multi-epitopic protein (TsMEP) as an antigen candidate for Tityus serrulatus antivenom production

Scorpionism is Brazil's most prevalent envenomation. Treatment typically involves the use of heterologous antivenoms derived from the immunization of horses with crude T. serrulatus venom (TsV). Due to the high toxicity of this immunogen, as well as the particular challenges associated with the use of venoms as antigens, there is interest in exploring new alternatives for reducing their use in antivenom production. In this study, ten linear B-cell epitopes from hyaluronidase and voltage-gated sodium channel toxins, previously identified using the SPOT-synthesis method, were selected to construct a recombinant chimeric MultiEpitopic Protein from T. serrulatus scorpion venom (TsMEP). We demonstrated that TsMEP is non-toxic, and antibodies elicited in rabbits against this antigen exhibited reactivity in ELISA with Brazilian T. serrulatus, T. bahiensis, T. obscurus, and T. stigmurus venoms, as well as with Peruvian Hadruroides lunatus and North African Androctonus australis hector scorpion venoms. In vivo and in vitro neutralization assays revealed that rabbit anti-TsMEP antibodies partially neutralize the hyaluronidase activity of T. serrulatus venom and its lethality. Data presented here suggests that this multiepitopic protein could be a promising candidate in experimental immunization approaches for antivenom production for clinical use against Tityus spp. venoms.

A multi-epitope protein vaccine encapsulated in alginate nanoparticles as a candidate vaccine against Shigella sonnei

Shigellosis, caused by the Gram-negative bacterium Shigella, is a major global health challenge. Despite extensive research over the past two decades, no commercial vaccine is available to prevent Shigella infection. Developing multi-epitope vaccines offers a promising and innovative approach to tackling infectious diseases. In this study, we produced a multi-epitope vaccine candidate using E. coli BL21 (DE3) plysS bacteria and purified the vaccine protein with Ni-NTA affinity chromatography. We then prepared alginate nanoparticles containing the vaccine protein, with a particle size of 122 ± 6 nm, PDI 0.17, SPAN 0.83, and zeta potential of -27 ± 2 mV. Successful protein loading was confirmed through nanodrop and ATR-FTIR analyses. To evaluate the immunogenicity of the encapsulated vaccine, mice were orally vaccinated, and their serum was analyzed for IgG, IL-4, and IFN-γ levels cytokines. The results showed a significant increase in IgG level in the vaccinated group compared to controls. Additionally, the vaccinated group exhibited a notable increase in IL-4 and IFN-γ cytokines, indicating a robust Th-cell-mediated immune response essential for combating Shigella. Our nano-vaccine demonstrated high efficacy in activating both humoral and cellular immunity, effectively protecting against the bacteria. The alginate-based oral vaccine candidate thus emerges as a promising strategy for developing a multi-epitope vaccine candidate against Shigella.

A Multi-Epitope Protein for High-Performance Serodiagnosis of Chronic Chagas Disease in ELISA and Lateral Flow Platforms.

We developed a protein to rapidly and accurately diagnose Chagas disease, a life-threatening illness identified by the WHO as a critical worldwide public health risk. Limitations in present day serological tests are complicating the current health situation and contributing to most infected persons being unaware of their condition and therefore untreated. To improve diagnostic testing, we developed an immunological mimic of the etiological agent, Trypanosoma cruzi, by combining ten pathogen-specific epitopes within the beta-barrel protein structure of Thermal Green Protein. The resulting multi-epitope protein, DxCruziV3, displayed high specificity and sensitivity as the antibody capture reagent in an ELISA platform with an analytical sensitivity that exceeds WHO recommendations. Within an immunochromatographic platform, DxCruziV3 showed excellent performance for the point of application diagnosis in a region endemic for multiple diseases, the municipality of Barcelos in the state of Amazonas, Brazil. In total, 167 individuals were rapidly tested using whole blood from a finger stick. As recommended by the Brazilian Ministry of Health, venous blood samples were laboratory tested by conventional assays for comparison. Test results suggest utilizing DxCruziV3 in different assay platforms can confidently diagnose chronic infections by T. cruzi. Rapid and more accurate results will benefit everyone but will have the most noticeable impact in resource-limited rural areas where the disease is endemic.

Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP.

Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.

In silico design of Mycobacterium tuberculosis multi-epitope adhesin protein vaccines

Mycobacterium tuberculosis (Mtb) adhesin proteins are promising candidates for subunit vaccine design. Multi-epitope Mtb vaccine and diagnostic candidates were designed using immunoinformatic tools. The antigenic potential of 26 adhesin proteins were determined using VaxiJen 2.0. The truncated heat shock protein 70 (tnHSP70), 19 kDa antigen lipoprotein (lpqH), Mtb curli pili (MTP), and Phosphate transport protein S1 (PstS1) were selected based on the number of known epitopes on the Immune Epitope Database (IEDB). B- and T-cell epitopes were identified using BepiPred2.0, ABCpred, SVMTriP, and IEDB, respectively. Population coverage was analysed using prominent South African specific alleles on the IEDB. The allergenicity, physicochemical characteristics and tertiary structure of the tri-fusion proteins were determined. The in silico immune simulation was performed using C-ImmSim. Three truncated sequences, with predicted B and T cell epitopes, and without allergenicity or signal peptides were linked by three glycine-serine residues, resulting in the stable, hydrophilic molecules, tnlpqH-tnPstS1-tnHSP70 (64,86 kDa) and tnMTP-tnPstS1-tnHSP70 (63,96 kDa). Restriction endonuclease recognition sequences incorporated at the N- and C-terminal ends of each construct, facilitated virtual cloning using Snapgene, into pGEX6P-1, resulting in novel, highly immunogenic vaccine candidates (0,912–0,985). Future studies will involve the cloning, recombinant protein expression and purification of these constructs for downstream applications.

Single Cell Expression Systems for the Production of Recombinant Proteins for Immunodiagnosis and Immunoprophylaxis of Toxoplasmosis.

Toxoplasmosis represents a significant public health and veterinary concern due to its widespread distribution, zoonotic transmission, and potential for severe health impacts in susceptible individuals and animal populations. The ability to design and produce recombinant proteins with precise antigenic properties is fundamental, as they serve as tools for accurate disease detection and effective immunization strategies, contributing to improved healthcare outcomes and disease control. Most commonly, a prokaryotic expression system is employed for the production of both single antigens and multi-epitope chimeric proteins; however, the cloning strategies, bacterial strain, vector, and expression conditions vary. Moreover, literature reports show the use of alternative microbial systems such as yeast or Leishmania tarentolae. This review provides an overview of the methods and strategies employed for the production of recombinant Toxoplasma gondii antigenic proteins for the serological detection of T. gondii infection and vaccine development.

Immunization of BALB/c Mice against Shigella sonnei Using a Multiepitope Protein Vaccine through Intranasal and Subcutaneous Administration.

As the most common cause of bacillary dysentery or shigellosis, Shigella sonnei (S nonnei) has spread throughout the world. Invasion of the colorectal epithelial cells by this facultative intracellular bacterium occurs via various virulence factors. The increase in the resistance rate highlights the need for novel interventions, particularly increasing the urgency of the development of Shigellavaccines that may offer an effective solution. A multiepitope protein vaccine (MEPV) construct previously designed using bioinformatics tools against Shigella species, was applied in vivo in BALB/C mice. The designed vaccine construct was expressed in a bacterial host, purified, and finally confirmed by Western blot analysis. The immunogenicity of the purified MEPV was assessed against S sonnei via intranasal and subcutaneous administration routes, followed by evaluating its protective efficiency. We observed that interferon-gamma, interleukin-4, and immunoglobulin G levels were increased in all experimental groups. Therefore, The MEPV effectively protected the mice against S sonnei.

Novel prophylactic and therapeutic multi-epitope vaccine based on Ag85A, Ag85B, ESAT-6, and CFP-10 of Mycobacterium tuberculosis using an immunoinformatics approach.

Current tuberculosis (TB) control strategies face limitations, such as low antibiotic treatment compliance and a rise in multidrug resistance. Furthermore, the lack of a safe and effective vaccine compounds these challenges. The limited efficacy of existing vaccines against TB underscores the urgency for innovative strategies, such as immunoinformatics. Consequently, this study aimed to design a targeted multi-epitope vaccine against TB infection utilizing an immunoinformatics approach. The multi-epitope vaccine targeted Ag85A, Ag85B, ESAT-6, and CFP-10 proteins. The design adopted various immunoinformatics tools for cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and linear B lymphocyte (LBL) epitope prediction, the assessment of vaccine characteristics, structure modeling, population coverage analysis, disulfide engineering, solubility prediction, molecular docking/dynamics with toll-like receptors (TLRs), codon optimization/cloning, and immune simulation. The multi-epitope vaccine, which was assembled using 12 CTL, 25 HTL, and 21 LBL epitopes associated with CpG adjuvants, showed promising characteristics. The immunoinformatics analysis confirmed the antigenicity, immunogenicity, and lack of allergenicity. Physicochemical evaluations indicated that the proteins were stable, thermostable, hydrophilic, and highly soluble. Docking simulations suggested high-affinity binding to TLRs, including TLR2, TLR4, and TLR9. In silico immune simulation predicted strong T cell (cytokine release) and B cell (immunoglobulin release) responses. This immunoinformatics-designed multi-epitope vaccine targeting Ag85A, Ag85B, ESAT-6, and CFP-10 proteins showed promising characteristics in terms of stability, immunogenicity, antigenicity, solubility, and predicted induction of humoral and adaptive immune responses. This suggests its potential as a prophylactic and therapeutic vaccine against TB.

Recombinant proteins as promising antigens applied to the immunodiagnosis of Chagas disease: a scoping review.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important public health problem, occurring mainly in Latin America. The disease has a major social and economical effect, negatively impacting the life of the infected individuals, and bringing great costs to public health. An early and accurate diagnosis is essential for administration of early treatment. In addition, prognostic tests may aid disease management, decreasing hospitalization costs. However, the serological diagnostic scenario for CD still faces several challenges, making the development of new diagnostic kits a pressing matter. Facing this scenario, several researchers have expanded efforts in developing and testing new antigens, such as recombinant proteins and recombinant multiepitope proteins, with promising results. These recombinant antigens offer several advantages, such as improved sensitivity and specificity, in addition to facilitated scaling. Also, it has been possible to observe a rising number of studies using ELISA and point-of-care platforms, employing these antigens in the past few years. Among them, recombinant proteins were the most applied antigens, demonstrating great capacity to discriminate between positive and negative samples. Although fewer in number, recombinant multiepitope proteins also demonstrated an improved diagnostic performance. Indeed, a great number of studies employing these antigens showed sensitivity and specificity values above 90%, greatly impacting diagnostic accuracy. Nevertheless, despite the good results found, it is still possible to observe some bottlenecks in the development of new antigens, such as the scarcity of tests with sera from the acute phase and the variability of results in different geographic areas. In this sense, aiming to contribute to control and health programs, the continuous search for a more accurate serological diagnosis is essential, both for the acute and chronic phases of the disease.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P<0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P<0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Development of recombinant proteins for vaccine candidates against serotypes O and A of Foot-and-Mouth Disease virus in Bangladesh.

Frequent vaccine failure leading to recurrent outbreaks of Foot-and-Mouth Disease (FMD) in livestock populations necessitates the development of a customizable vaccine platform comprising potential antigenic determinants of circulating lineages of FMD viruses. Artificially designed, chimaeric protein-based recombinant vaccines are novel approaches to combat the phylogenetically diverse FMD Virus (FMDV) strains. Among seven recognized serotypes, only serotypes O and A are dominantly circulating in Bangladesh and neighbouring countries of Asia, where transboundary transmission, recurrent outbreaks and emergence of novel lineages of FMDV are highly prevalent. The objective of this study was to develop multi-epitope recombinant proteins, procuring immunogenicity against circulating diverse genotypes of FMDV serotypes O and A. Two chimaeric proteins, named B1 (41.0 kDa) and B3 (39.3 kDa), have been designed to incorporate potential B-cell and T-cell epitopes selected from multiple FMDV strains, including previously reported and newly emerged sub-lineages. After expression, characterization and immunization of guinea pigs with a considerable antigen load of B1 and B3 followed by serological assays revealed the significant protective immunogenicity, developed from the higher (100 µg) doses of both antigens, against most of the currently prevalent serotype O and A strains of FMDV. The efficient expression, antigenic stability, and multivalent immunogenic potency of the chimaeric proteins strongly indicate their credibility as novel vaccine candidates for existing serotypes O and A of FMDV in Bangladesh and surrounding territories.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis

BackgroundOpisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis.MethodsThe Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique.ResultsThe recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively.ConclusionsThe high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.Graphical

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.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

Toward Consensus Epitopes B and T of Tropomyosin Involved in Cross-Reactivity across Diverse Allergens: An In Silico Study.

Tropomyosin (TM) is a pan-allergen with cross-reactivity to arthropods, insects, and nematodes in tropical regions. While IgE epitopes of TM contribute to sensitization, T-cell (MHC-II) epitopes polarize the Th2 immune response. This study aimed to identify linear B and T consensus epitopes among house dust mites, cockroaches, Ascaris lumbricoides, shrimp, and mosquitoes, exploring the molecular basis of cross-reactivity in allergic diseases. Amino acid sequences of Der p 10, Der f 10, Blo t 10, Lit v 1, Pen a 1, Pen m 1, rAsc l 3, Per a 7, Bla g 7, and Aed a 10 were collected from Allergen Nomenclature and UniProt. B epitopes were predicted using AlgPred 2.0 and BepiPred 3.0. T epitopes were predicted with NetMHCIIpan 4.1 against 10 HLA-II alleles. Consensus epitopes were obtained through analysis and Epitope Cluster Analysis in the Immune Epitope Database. We found 7 B-cell epitopes and 28 linear T-cell epitopes binding to MHC II. A unique peptide (residues 160-174) exhibited overlap between linear B-cell and T-cell epitopes, highly conserved across tropomyosin sequences. These findings shed light on IgE cross-reactivity among the tested species. The described immuno-informatics pipeline and epitopes can inform in vitro research and guide synthetic multi-epitope proteins' design for potential allergology immunotherapies. Further in silico studies are warranted to confirm epitope accuracy and guide future experimental protocols.

Immunoinformatics Design and In Vivo Immunogenicity Evaluation of a Conserved CTL Multi-Epitope Vaccine Targeting HPV16 E5, E6, and E7 Proteins.

Human papillomavirus type 16 (HPV16) infection is responsible for more than 50% of global cervical cancer cases. The development of a vaccine based on cytotoxic T-lymphocyte (CTL) epitopes is a promising strategy for eliminating pre-existing HPV infections and treating patients with cervical cancer. In this study, an immunoinformatics approach was used to predict HLA-I-restricted CTL epitopes in HPV16 E5, E6, and E7 proteins, and a set of conserved CTL epitopes co-restricted by human/murine MHCs was screened and characterized, with the set containing three E5, four E6, and four E7 epitopes. Subsequently, the immunogenicity of the epitope combination was assessed in mice, and the anti-tumor effects of the multi-epitope peptide vaccine E5E6E7pep11 and the recombinant protein vaccine CTB-Epi11E567 were evaluated in the TC-1 mouse tumor model. The results demonstrated that mixed epitope peptides could induce antigen-specific IFN-γ secretion in mice. Prophylactic immunization with E5E6E7pep11 and CTB-Epi11E567 was found to provide 100% protection against tumor growth in mice. Moreover, both types of the multi-epitope vaccine significantly inhibited tumor growth and prolonged mouse survival. In conclusion, in this study, a multi-epitope vaccine targeting HPV16 E5, E6, and E7 proteins was successfully designed and evaluated, demonstrating potential immunogenicity and anti-tumor effects and providing a promising strategy for immunotherapy against HPV-associated tumors.

A Review on the use of Synthetic and Recombinant Antigens for the Immunodiagnosis of Tegumentary Leishmaniasis.

Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment.

Designing a Novel Multi-epitope Peptide as a Potential Serodiagnosis Marker for the Diagnosis of Acinetobacter baumannii: An In silico Approach

Background: Acinetobacter baumannii is an opportunistic pathogen that causes many infections, including nosocomial infections; this bacterium has a high mortality rate among other bacteria. A. baumannii has an elastic genome that changes rapidly when exposed to harsh environmental conditions, leading to widespread bacterial resistance to various disinfectants and antibiotics. The high ability of bacteria to bind to all surfaces and survive in different conditions has caused the spread of bacteria in various environments. Rapid detection is very important in preventing the spread and even treatment of the infection. Methods: Currently, the Polymerase Chain Reaction (PCR) method is the only effective method used for diagnosis, which has some pros and cons. Results and Conclusion: This study aimed to design a new recombinant multi-epitope protein from Acinetobacter baumannii that can be used in ELISA for rapid diagnosis. The unique feature of this study from others is the use of patient serum for antibody monitoring.

Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy.

High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20-40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).