Antimicrobial Functionalization of Surfaces by a Chimeric Adhesive Protein.

The main aim of this work is to account for the prevention and control of microbial growth on surfaces of interest in medical technology. Surface modification is often achieved by physiotherapy or chemical treatments that can involve time-consuming steps, hazardous reagents, and harsh conditions. One of the ways to overcome these drawbacks is the use of surface-active proteins such as hydrophobins. They can form stable protein layers on different surfaces, serving as anchoring points for other molecules of interest. The fungal hydrophobin Vmh2, already exploited for its adhesive ability, has been fused with the antimicrobial peptide GKY20, forming the chimeric protein used herein for functionalizing polystyrene (PS) and bacterial cellulose (BC). As a natural biomass, BC has multiple advantages, including biodegradability, low cost, renewability, high purity, and excellent mechanical properties. The chimeric protein has been proven to successfully adhere to both surfaces. A strong decrease in biofilm formation on PS and a good bactericidal effect of BC have been demonstrated. These findings provide evidence of an alternative strategy to obtain functional composites using a green, easy process.

Investigation of therapeutic potential of the Il24-p20 fusion protein against breast cancer: an in-silico approach.

Targeted delivery of therapeutic anticancer chimeric molecules enhances drug efficacy. Numerous studies have focused on developing novel treatments by employing cytokines, particularly interleukins, to inhibit the growth of cancer cells. In the present study, we fused interleukin 24 with the tumor-targeting peptide P20 through a rigid linker to selectively target cancer cells. The secondary structure, tertiary structure, and physicochemical characteristics of the constructed chimeric IL-24-P20 protein were predicted by using bioinformatics tools. In-silico analysis revealed that the fusion construct has a basic nature with 175 amino acids and a molecular weight of 20 kDa. By using the Rampage and ERRAT2 servers, the validity and quality of the fusion protein were evaluated. The results indicated that 93% of the chimeric proteins contained 90.1% of the residues in the favoured region, resulting in a reliable structure. Finally, docking and simulation studies were conducted via ClusPro and Desmond Schrödinger, respectively. Our results indicate that the constructed fusion protein exhibits excellent quality, interaction capabilities, validity, and stability. These findings suggest that the fusion protein is a promising candidate for targeted cancer therapy.

Hybrid Exb/Mot stators require substitutions distant from the chimeric pore to power flagellar rotation.

Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion-powered motors with more distant homology. Here, we synthesized chimeras of MotAB, PomAPotB, and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimized by directed evolution. Whole-genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with the rational design of chimeric IRMs. IMPORTANCE: Ion-powered rotary motors (IRMs) underpin the rotation of one of nature's oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors.

Broadly cross-reactive immune responses in chickens immunized with chimeric virus-like particles of nodavirus displaying the M2e originated from avian and human influenza A viruses.

Avian influenza A viruses (IAVs) pose a persistent threat to poultry industry worldwide, despite the presence of vaccines. Additionally, reverse-zoonosis transmission potentially introduces human-originated IAVs into poultry and complicates the efforts to control the spread of influenza. Current avian influenza vaccines are primarily based upon the rapidly mutating hemagglutinin (HA) and neuraminidase (NA) glycoproteins, which limit their efficacy against diverse strains of IAVs. Hence, the highly conserved ectodomains of matrix 2 protein (M2e) of IAVs are widely studied as alternatives to the HA and NA. However, the differences in the M2e amino acid sequences between avian and human IAVs generate antibodies that do not cross-react reciprocally with IAVs from other origins. To broaden and enhance the immunogenicity of M2e, we fused two copies each of the M2e derived from avian and human IAVs at the C-terminal end of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (NvC). Transmission electron microscopic and dynamic light scattering analyses revealed that the chimeric protein self-assembled into virus-like particles (VLPs). Immunization of chickens with the chimeric VLPs demonstrated a robust induction of broadly reactive immune responses against both the M2e of avian and human IAVs. Additionally, the chimeric VLPs elicited the production of cytotoxic T lymphocytes (CTL), macrophages, as well as a well-balanced Th1 and Th2 population, indicating their potential in activating cell-mediated immune responses in chickens. Furthermore, the chimeric VLPs triggered the production of both Th1- and Th2-cytokines, attesting their potential in mounting a robust and balanced immune response in avian species. This study demonstrated the potential of these chimeric VLPs in stimulating and broadening cross-reactive immune responses in chickens against both avian and human IAVs.

Collagen-binding bone morphogenetic protein-2 designed for use in bone tissue engineering.

Bone tissue engineering using biodegradable porous scaffolds is a promising approach for restoring oral and maxillofacial bone defects. Recently, attempts have been made to incorporate proteins such as growth factors to create bioactive scaffolds that can engage cells to promote tissue formation. Collagen-based scaffolds containing bone morphogenetic protein-2 (BMP2) have been studied for bone formation. However, controlling the initial burst of BMP2 remains difficult. Here we designed a functional chimeric protein composed of BMP2 and a collagen-binding domain (CBD), specifically the A3 domain of von Willebrand factor, to sustain BMP2 release from collagen-based scaffolds. Based on the results of computer-based structural prediction, we prepared a chimeric protein consisting of CBD and BMP2 in this order with a peptide tag for affinity purification. The chimeric protein had a collagen-binding capacity and enhanced osteogenic differentiation of human mesenchymal stem cells. These results are consistent with insights from in silico structural prediction.

CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

Variations of VEGFR2 Chemical Space: Stimulator and Inhibitory Peptides.

The kinase pathway plays a crucial role in blood vessel function. Particular attention is paid to VEGFR type 2 angiogenesis and vascular morphogenesis as the tyrosine kinase pathway is preferentially activated. In silico studies were performed on several peptides that affect VEGFR2 in both stimulating and inhibitory ways. This investigation aims to examine the molecular properties of VEGFR2, a molecule primarily involved in the processes of vasculogenesis and angiogenesis. These relationships were defined by the interactions between Vascular Endothelial Growth Factor receptor 2 (VEGFR2) and the structural features of the systems. The chemical space of the inhibitory peptides and stimulators was described using topological and energetic properties. Furthermore, chimeric models of stimulating and inhibitory proteins (for VEGFR2) were computed using the protein system structures. The interaction between the chimeric proteins and VEGFR was computed. The chemical space was further characterized using complex manifolds and high-dimensional data visualization. The results show that a slightly similar chemical area is shared by VEGFR2 and stimulating and inhibitory proteins. On the other hand, the stimulator peptides and the inhibitors have distinct chemical spaces.

Construction and mouse antibody response evaluation of juvenile stage-specific chimeric protein from Fasciola gigantica.

Fasciolosis, caused by the liver fluke Fasciola gigantica, is a major parasitic disease that affects livestock and therefore causes significant economic losses in tropical countries. Although anthelminthic drugs can kill the parasite, drug-resistant liver fluke populations are increasing. In this study, a recombinant F. gigantica chimeric protein (rFgCHI) consisting of cathepsin L1H (FgCL1H), cathepsin B3 (FgCB3), and Saposin-like protein 1 (FgSAP1) was designed and expressed in Escherichia coli (BL21). The molecular weight of rFgCHI was 61 kDa. To study the antibody response, male BALB/c mice were immunized via the subcutaneous injection of rFgCHI combined with Quil A. Immunization with rFgCHI showed the induction of IgG1 and IgG2a with a higher IgG1 isotype level, indicating the potential of mixed Th1/Th2 immune responses, with Th2 predominating. However, the results showed high levels of IgG against the single proteins, except for rFgSAP1. Through Western blotting, mouse anti-rFgCHI polyclonal antibodies could be detected to the native proteins obtained from the parasite at all stages. Immunolocalization also revealed that the anti-rFgCHI antibodies could detect targeted antigens in the cecal epithelium of the parasite. These results demonstrated that rFgCHI is immunogenic to the mouse immune system and may potentially be a protein candidate for the development of a fasciolosis vaccine.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Evaluation of the Humoral Response after Immunization with a Chimeric Subunit Vaccine against Shiga Toxin-Producing Escherichia coli in Pregnant Sows and Their Offspring.

Shiga toxin-producing Escherichia coli (STEC) poses a significant public health risk due to its zoonotic potential and association with severe human diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. Ruminants are recognized as primary reservoirs for STEC, but swine also contribute to the epidemiology of this pathogen, highlighting the need for effective prevention strategies across species. Notably, a subgroup of STEC that produces Shiga toxin type 2e (Stx2e) causes edema disease (ED) in newborn piglets, economically affecting pig production. This study evaluates the immunogenicity of a chimeric protein-based vaccine candidate against STEC in pregnant sows and the subsequent transfer of immunity to their offspring. This vaccine candidate, which includes chimeric proteins displaying selected epitopes from the proteins Cah, OmpT, and Hes, was previously proven to be immunogenic in pregnant cows. Our analysis revealed a broad diversity of STEC serotypes within swine populations, with the cah and ompT genes being prevalent, validating them as suitable antigens for vaccine development. Although the hes gene was detected less frequently, the presence of at least one of these three genes in a significant proportion of STEC suggests the potential of this vaccine to target a wide range of strains. The vaccination of pregnant sows led to an increase in specific IgG and IgA antibodies against the chimeric proteins, indicating successful immunization. Additionally, our results demonstrated the effective passive transfer of maternal antibodies to piglets, providing them with immediate, albeit temporary, humoral immunity against STEC. These humoral responses demonstrate the immunogenicity of the vaccine candidate and are preliminary indicators of its potential efficacy. However, further research is needed to conclusively evaluate its impact on STEC colonization and shedding. This study highlights the potential of maternal vaccination to protect piglets from ED and contributes to the development of vaccination strategies to reduce the prevalence of STEC in various animal reservoirs.

KD-409, a Respiratory Syncytial Virus FG Chimeric Protein without the CX3C Chemokine Motif, Is an Efficient Respiratory Syncytial Virus Vaccine Preparation for Passive and Active Immunization in Mice.

Although respiratory syncytial virus (RSV) vaccine development initiatives have existed for half a century, no candidate has been approved for application at all ages from neonates to children. Developing an effective and safe RSV vaccine for pediatric use is challenging owing to RSV-associated disease and vaccine-enhanced disease (VED). We aimed to design an RSV vaccine, KD-409, by structurally incorporating the F ectodomain and G protein central conserved domain without the CX3C chemokine motif and test its efficacy and safety. KD-409 formed rosette particles or trimmers. KD-409 immunization of mice mainly induced anti-RSV F protein IgG. The induced anti-F antibodies had a higher IgG2a/IgG1 ratio than pre-fusion F, suggesting that they induced Th1-dominant immunity. Active and passive immunities were assessed by analyzing the viral titers in BALB/c mice intranasally challenged with RSV after intramuscular KD-409 immunization and pups derived from mothers who were intramuscularly vaccinated with KD-409 twice, respectively. KD-409 was more effective than post-fusion F and had a lower minimum effective dose than pre-fusion F. Thus, KD-409 demonstrated great potential as a novel RSV vaccine candidate, outperforming existing RSV F-based candidates. Our findings provide a promising strategy to overcome RSV-associated acute lower respiratory infections without the risk of VED associated with traditional approaches.

A Nasal Vaccine Candidate, Containing Three Antigenic Regions from SARS-CoV-2, to Induce a Broader Response.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

Protective Efficacy of the Epitope-Conjugated Antigen N-Tc52/TSkb20 in Mitigating Trypanosoma cruzi Infection through CD8+ T-Cells and IFNγ Responses.

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.

A recombinant chimeric antigen constructed with B-cell epitopes from Mycobacterium leprae hypothetical proteins is effective for the diagnosis of leprosy.

The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins.

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Structure and function of engineered stromal cell-derived factor-1α.

To design biologically active, collagen-based scaffolds for bone tissue engineering, we have synthesized chimeric proteins consisting of stromal cell-derived factor-1α (SDF) and the von Willebrand factor A3 collagen-binding domain (CBD). The chimeric proteins were used to evaluate the effect of domain linkage and its order on the structure and function of the SDF and CBD. The structure of the chimeric proteins was analyzed by circular dichroism spectroscopy, while functional analysis was performed by a cell migration assay for the SDF domain and a collagen-binding assay for the CBD domain. Furthermore, computational structural prediction was conducted for the chimeric proteins to examine the consistency with the results of structural and functional analyses. Our structural and functional analyses as well as structural prediction revealed that linking two domains can affect their functions. However, their order had minor effects on the three-dimensional structure of CBD and SDF in the chimeric proteins.

Advancing PEDV Vaccination: Comparison between Inactivated and Flagellin N-Terminus-Adjuvanted Subunit Vaccines.

Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC99-mCOE, which combines the N-terminus of flagellin (FliC99) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct's non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC99-mCOE's strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC99-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC99-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC99-mCOE fusion protein may serve as a promising vaccine candidate against PEDV.