Recombinant Chimera Research Articles

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania donovani

Aim: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin (LTB) in the functional activity of L. donovani. Background: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease (gp63) has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival. Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani was fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. Results: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin (LTB) of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. Conclusion: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein (LTB) could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Effect of period supplementation of Saccharomyces boulardii in humoral immune response of sheep immunized with recombinant chimera of Paeniclostridium sordellii

Saccharomyces boulardii has emerged as a promising probiotic agent in bolstering the immune system. In vaccinology, its application can be explored to optimize vaccine efficacy by augmenting host immune response and consequently enhancing the immunogenicity of antigenic formulations. However, it is imperative to conduct further research to comprehend the effectiveness of this probiotic in novel vaccines targeting significant pathogens affecting animal health. Hence, this study investigated the effects of a short (3 - 5 days) or continuously (56 days) S. boulardii supplementation (3 × 108 CFU/mL) on the adaptive immunity of sheep immunized with a recombinant antigen against Paeniclostridium sordellii (rAPS). Four immunized groups (G1-G4) were evaluated, varying the regimen of S. boulardii supplementation. Elevated levels of anti-rAPS IgG immunoglobulins were detected in all vaccinated animals. Daily supplementation with S. boulardii (G1) resulted in higher IgG levels, reaching antibody titers of up to 25,600, which were 16 times higher than those observed in not supplemented group (G4) and 4 times higher than in the group supplemented for 3 days (G3) or 5 days (G2) before each immunization. These findings demonstrate that S. boulardii can enhance vaccine-induced immune responses against P. sordellii, particularly IgG-mediated immune responses in sheep. The possibility of a short supplementation for 5–3 days is a very important finding for animal husbandry, considering the supplementation and supply management cost.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D.

Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p < 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids

Tacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter- and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micromotors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.

Domain Shuffling and Site-Saturation Mutagenesis for the Enhanced Inhibitory Potential of Amaranthaceae α-Amylase Inhibitors.

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural-functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI's optimization and functional differentiation for future biotechnological applications.

Development of an immunoassay for the simultaneous detection of GADA and ZnT8A in autoimmune diabetes using a ZnT8/GAD65 chimeric molecule.

The combined presence of autoantibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) and to the islet-specific cation efflux transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss of immune tolerance and the clinical manifestation of autoimmune diabetes mellitus (DM). The screening of GADA and ZnT8A could help to reach to a correct diagnosis and to start an early and adequate treatment. The aim of the study was to develop an immunoassay for the simultaneous detection of these autoantibodies using a chimera molecule that includes the immunodominant regions of ZnT8 and GAD65, expressed by baculovirus-insect cells system. ZnT8/GAD65 was expressed using the Bac to Bac™ baculovirus expression system. The recombinant chimera was purified by an His6-tag and identified by SDS-PAGE and western blot analysis, and by an indirect ELISA using specific antibodies against ZnT8 and GAD65. A fraction of ZnT8/GAD65 was biotinylated. A bridge ELISA (b-ELISA) was developed using ZnT8/GAD65 immobilized in polystyrene microplates, human sera samples from healthy individuals (n = 51) and diabetic patients (n = 49) were then incubated, and afterwards ZnT8/GAD65-biotin was added. Immune complexes were revealed with Streptavidin-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. ZnT8/GAD65 was efficiently produced, yielding 30 mg/L culture medium, 80% pure. This recombinant chimera retains the immunoreactive conformation of the epitopes that are recognized by their specific antibodies, so it was used for the development of a high sensitivity (75.51%) and specificity (98.04%) b-ELISA for the detection of ZnT8A and/or GADA, in a one-step screening assay. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from healthy individuals and diabetic patients (AUC = 0.9488); the cut-off value was stablished at 2 SDs. This immunoassay is useful either to confirm autoimmune diabetes or for detection in routine screening of individuals at risk of autoimmune DM. As DM is a slow progress disease, remaining asymptomatic for a long preclinical period, serological testing is of importance to establish a preventive treatment.

A Recombinant Chimera Vaccine Composed of LTB and Mycoplasma hyopneumoniae Antigens P97R1, mhp390 and P46 Elicits Cellular Immunologic Response in Mice.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

Administration of soluble gp130Fc disrupts M-1 macrophage polarization, dendritic cell activation, MDSC expansion and Th-17 induction during experimental cerebral malaria

Regulatory effect of IL-6 on various immune cells plays a crucial role during experimental cerebral malaria pathogenesis. IL-6 neutralization can restore distorted ratios of myeloid dendritic cells and plasmacytoid dendritic cells as well as the balance between Th-17 and T-regulatory cells. IL-6 can also influence immune cells through classical and trans IL-6 signalling pathways. As trans IL-6 signalling is reportedly involved during malaria pathogenesis, we focused on studying the effects of trans IL-6 signalling blockade on various immune cell populations and how they regulate ECM progression. Results show that administration of sgp130Fc recombinant chimera protein lowers the parasitemia, increases the survivability of Plasmodium berghei ANKA infected mice, and restores the distorted ratios of M1/M2 macrophage, mDC/pDC, and Th-17/Treg. IL-6 trans signalling blockade has been found to affect both expansion of myeloid derived suppressor cells (MDSCs) and expression of inflammatory markers on them during Plasmodium berghei ANKA infection indicating that trans IL-6 signalling might regulate various immune cells and their function during ECM. In this work for the first time, we delineate the effect of sgp130Fc administration on influencing the immunological changes within the host secondary lymphoid organ during ECM induced by Plasmodium berghei ANKA infection.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

A recombinant rabies virus chimera expressing the DC-targeting molecular MAB2560 shows enhanced vaccine immunogenicity through activation of dendritic cells.

Rabies, caused by the rabies virus (RABV), is an ancient and neglected zoonotic disease posing a large public health threat to humans and animals in developing countries. Immunization of animals with a rabies vaccine is the most effective way to control the epidemic and the occurrence of the disease in humans. Therefore, the development of cost-effective and efficient rabies vaccines is urgently needed. The activation of dendritic cells (DCs) is known to play an important role in improving the host immune response induced by rabies vaccines. In this study, we constructed a recombinant virus, rCVS11-MAB2560, based on the reverse genetic system of the RABV CVS11 strain. The MAB2560 protein (a DC-targeting molecular) was chimeric expressed on the surface of the viral particles to help target and activate the DCs when this virus was used as inactivated vaccine. Our results demonstrated that inactivated rCVS11-MAB2560 was able to promote the recruitment and/or proliferation of DC cells, T cells and B cells in mice, and induce good immune memory after two immunizations. Moreover, the inactivated recombinant virus rCVS11-MAB2560 could produce higher levels of virus-neutralizing antibodies (VNAs) in both mice and dogs more quickly than rCVS11 post immunization. In summary, the recombinant virus rCVS11-MAB2560 chimeric-expressing the molecular adjuvant MAB2560 can stimulate high levels of humoral and cellular immune responses in vivo and can be used as an effective inactivated rabies vaccine candidate.

A multiepitope chimeric antigen from Rhipicephalus microplus-secreted salivary proteins elicits anti-tick protective antibodies in rabbit

Rhipicephalus (Boophilus) microplus, the Cattle Fever Tick, causes significant economic losses in livestock in tropical and subtropical regions of the world. As the usual control strategy based on chemical acaricides presents different drawbacks, alternative control strategies have been considered for tick control. In recent decades, several tick proteins have been evaluated as targets for the development of anti-tick vaccines. Thus, in the present work, coding sequences from three different proteins present in tick saliva were employed together to construct a recombinant chimeric protein that was evaluated as an antigen in rabbit immunization. Then, the elicited antibodies were tested in a tick artificial feeding experiment to verify the protective effect against the parasites. In addition to Rhipicephalus microplus subtilisin inhibitor 7 (RmSI-7), a serine protease inhibitor member of the TIL (Trypsin Inhibitory Like) family, an interdomain region from the Kunitz inhibitor BmTI-A, and a new cysteine-rich AMP-like microplusin, called RmSEI (previously identified as an elastase inhibitor), were selected to compose the chimeric protein. Anti-chimeric IgG antibodies were able to affect R. microplus female egg production after artificial feeding. Moreover, antibodies elicited in infested tick-resistant and tick-susceptible cattle recognized the recombinant chimera. Additionally, the functional characterization of recombinant RmSEI was performed and revealed antimicrobial activity against gram-positive bacteria. Moreover, the antimicrobial protein was also recognized by antibodies elicited in sera from cattle previously exposed to R. microplus bites. Together, these data suggest that the chimeric protein composed of three salivary antigens is suitable for anti-tick vaccine development.

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

In silico analyses and design of chimeric proteins containing epitopes of Bartonella henselae antigens for the control of cat scratch disease.

Bartonella henselae is a Gram-negative bacterium that causes cat scratch disease (CSD), as well as bacteremia, endocarditis, and other clinical presentations. CSD remains one of the most common infections caused by bacteria in the genusBartonella, and it is transmitted to humans through a scratch or cat bite. Vaccination and more efficient diagnostic methods would represent a promising and sustainable alternative measure for CSD control in humans and animals. Here, we described thein silicoanalyses and design of three recombinant chimeric proteins (rC1, rC2, and rC3), for use in the control of CSD. The chimeras were constructed with epitopes identified from the sequences of the GroEL, 17kDa, P26, BadA, Pap31, OMP 89, and OMP 43, previously described as the most important B. henselae antigens. The rC1, rC2, and rC3 were expressed and purified using a heterologous system based on Escherichia coli and reacted with antibodies present in the sera of humans naturally infected. The chimeric proteins were used to immunize mice using Freund adjuvant, and the humoral immune response was evaluated. Animals immunized with rC1 and rC3 showed a significant IgG antibodies response from the 28th day (P < 0.05), and the animals immunized with the rC2 from the 35th day (P < 0.05) remained until the 56th day of experimentation, with a titer of 1:3200 (P < 0.05), 1:1600 (P < 0.05) and 1:1600 (P < 0.05) from rC1, rC2, and rC3, respectively. Significant production of IgA and IgG1 isotype was detected in animals immunized with rC1 and rC2 proteins. Additionally, analysis using 13 serum samples from naturally infected patients showed that the proteins are recognized by antibodies present in sera, reinforcing the possibility of using these chimeras for CSD control. KEY POINTS: • The recombinant chimeras were expressed in Escherichia coli with 37kDa (rC1), 35kDa (rC2), and 38kDa (rC3). • Animals immunized with rC1, rC2, and rC3 showed significant antibody response. • The chimeras were recognized by the sera of naturally infected patients.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Characterization of Cytochrome P450s with Key Roles in Determining Herbicide Selectivity in Maize.

Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.

Immunodominant conformational and linear IgE epitopes lie in a single segment of Ara h 2

Contribution of conformational epitopes to the IgE reactivity of peanut allergens Ara h 2 and Ara h 6 is at least as important as that of the linear epitopes. However, little is known about these conformational IgE-binding epitopes. We investigated the distribution of conformational epitopes on chimeric 2S-albumins. Recombinant chimeras were generated by exchanging structural segments between Ara h 2 and Ara h 6. Well-refolded chimeras, as verified by circular dichroism analysis, were then used to determine the epitope specificity of mAbs by performing competitive inhibition of IgG binding. Furthermore, we delineated the contribution of each segment to the overall IgE reactivity of both 2S-albumins by measuring the chimeras' IgE-binding capacity with sera from 21 patients allergic to peanut. We finally assessed chimeras' capacity to trigger mast cell degranulation. Configuration of the conformational epitopes was preserved in the chimeras. Mouse IgG mAbs, raised against natural Ara h 6, and polyclonal human IgE antibodies recognized different conformational epitopes distributed all along Ara h 6. In contrast, we identified human IgG mAbs specific to different Ara h 2 linear or conformational epitopes located in all segments except the C-terminal one. The major conformational IgE-binding epitope of Ara h 2 was located in a segment located between residues 33 and 81 that also contains the major linear hydroxyproline-containing epitope. Accordingly, this segment is critical for the capacity of Ara h 2 to induce mast cell degranulation. Chimeric 2S-albumins provide new insights on the conformational IgE-binding epitopes of Ara h 2 and Ara h 6. Proximity of the immunodominant linear and conformational IgE-binding epitopes probably contributes to the high allergenicpotency of Ara h 2.

Recombinant Proteins-Based Strategies in Bone Tissue Engineering.

The increase in fracture rates and/or problems associated with missing bones due to accidents or various pathologies generates socio-health problems with a very high impact. Tissue engineering aims to offer some kind of strategy to promote the repair of damaged tissue or its restoration as close as possible to the original tissue. Among the alternatives proposed by this specialty, the development of scaffolds obtained from recombinant proteins is of special importance. Furthermore, science and technology have advanced to obtain recombinant chimera’s proteins. This review aims to offer a synthetic description of the latest and most outstanding advances made with these types of scaffolds, particularly emphasizing the main recombinant proteins that can be used to construct scaffolds in their own right, i.e., not only to impregnate them, but also to make scaffolds from their complex structure, with the purpose of being considered in bone regenerative medicine in the near future.

In Silico Design of Recombinant Chimera T Cell Peptide Epitope Vaccines for Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. Systemic VL is fatal if untreated and there are no prophylactic human vaccines available. Several studies suggest that Th1 cell-mediated immunity plays a major role in protecting against VL. In this chapter we describe a method for designing recombinant chimera vaccines in silico based on the prediction of T cell epitopes within protein antigens identified as potential protective immunogens. Development of a recombinant chimera protein (RCP) vaccine using T cell epitope peptides identified from four Leishmania proteins is used as an exemplar of this method.