Baculovirus Expression System Research Articles

Tributyltin-binding protein type 1 (fish acid glycoprotein) is a potential gatekeeper of ethinylestradiol action in fish

Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 μM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17β-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 μM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 μM) was stronger than that for 17β-estradiol (Ki = 30.0 μM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen–estrogen balance.

Molecular Characterization and Functional Analysis of the Dipeptidyl Peptidase IV from Venom of the Ectoparasitoid Scleroderma guani.

Dipeptidyl peptidase IV (DPPIV) is a proline-specific serine peptidase that remains poorly investigated in terms of venom composition. Here, we describe the molecular characteristics and possible functions of DPPIV as a major venom component of the ant-like bethylid ectoparasitoid, Scleroderma guani, named SgVnDPPIV. The SgVnDPPIV gene was cloned, which encodes a protein with the conserved catalytic triads and substrate binding sites of mammalian DPPIV. This venom gene is highly expressed in the venom apparatus. Recombinant SgVnDPPIV, produced in Sf9 cells using the baculovirus expression system, has high enzymatic activity, which can be efficiently inhibited by vildagliptin and sitagliptin. Functional analysis revealed that SgVnDPPIV affects genes related to detoxification, lipid synthesis and metabolism, response to stimuli, and ion exchange in pupae of Tenebrio molitor, an envenomated host of S. guani. The present work contributes towards understanding the role of venom DPPIV involved in the interaction between parasitoid wasp and its host.

Abstract 5980: Identification of CDK15 activating cyclins by a recombinant co-expression approach

Abstract Cyclin dependent kinases (CDK) form a family of 20 different CDKs with an additional 6 CDK-like proteins. Members of this family have been shown to be involved in the regulation of many critical cellular processes, including the regulation of cell cycle progression, gene transcription and neuronal function. Deregulated CDK activity has been observed frequently in human cancer and other diseases. Enzymatic kinase activity of CDKs has been found to depend on the formation of complexes with regulatory proteins of the Cyclin family and requires in some cases the presence of additional complex partners, like MAT1 for CDK7/Cyclin H. To date, at least 15 different Cyclins have been described. For some CDKs it has been shown that they can also be activated by binding to proteins distinct from classical Cyclins. However, even while the 20 canonical CDKs share significant structural homology, they regulate very different functions of cell growth and development, partially due to their inherent substrate specificity but also regulated by their respective Cyclin partners. During the past 30 years several CDKs have been subject to extensive study resulting in a multitude of publications. This includes specifically CDK1, CDK2 and CDK4/6 and to a lesser extend CDK7 and CDK9. Other CDKs have been studied much less extensively and consequently the accumulated knowledge about their biological function and regulation is very limited. CDK15 (also known as PFTK2 or ALS2CR7) is one of these understudied members of the CDK family, with less than 20 publications in PubMed and the search term "CDK15" in contrast to more than 8600 hits for "CDK2". Its biological function is largely obscure even while evidence was presented that CDK15 is involved in the regulation of breast and colorectal cancer. Comparable to all other members of the CDK family, recombinantly overexpressed purified CDK15 does not exhibit detectable in-vitro activity and to date no Cyclin or other activating complex partner for CDK15 has been published. Since CDK inhibitors have been in the focus of interest at least since the approval of the first candidate (Palbociclib) for the treatment of ER+/HER+ breast cancer, testing the selectivity of other drug candidates within the complete group of CDKs is critically important to avoid unwanted off-target effects and toxicity. In order to enable in-vitro activity studies with CDK15 we evaluated different CDK15 constructs in combination with a comprehensive panel of different Cyclins using the baculo virus expression system. CDK15 and the respective Cyclins were co-expressed in insect cells, purified by affinity chromatography and tested for in-vitro activity using a panel of generic, broad-spectrum protein kinase substrates. We will present data on the identification of CDK15 activating Cyclins and the biochemical characterization of the in-vitro kinase activity of such CDK15/Cyclin complexes including a panel of kinase inhibitors. Citation Format: Daniel Mueller, Constance Ketterer, Diane Kraemer, Carolin Heidemann-Dinger, Lena Pilgermayer, Thomas Weber, Andreas Gericke, Frank Totzke, Michael H. Kubbutat. Identification of CDK15 activating cyclins by a recombinant co-expression approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5980.

Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1–266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

Development of modern immunization agent against bovine papillomavirus type 1 infection based on BPV1 L1 recombinant protein.

Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 μg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.

Baculovirus-expressed self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induce protective immunity in mice

Spike (S) protein, a homotrimeric glycoprotein, is the most important antigen target for SARS-CoV-2 vaccines. A complete simulation of the advanced structure of this homotrimer during subunit vaccine development is the most likely method to improve its immunoprotective effects. In this study, preparation strategies for the S protein receptor-binding domain, S1 region, and ectodomain trimer nanoparticles were designed using ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare three nanoparticle vaccines with high expression levels recorded in silkworms. The results in mice showed that the nanoparticle vaccine prepared using this strategy could induce immune responses when administered via both the subcutaneous administration and oral routes. Given the stability of these ferritin-based nanoparticle vaccines, an easy-to-use and low-cost oral immunization strategy can be employed in vaccine blind areas attributed to shortages of ultralow-temperature equipment and medical resources in underdeveloped areas. Oral vaccines are also promising candidates for limiting the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.

Identification of a Novel Interferon Lambda Splice Variant in Chickens.

Type III interferons (IFNLs) have critical roles in the host's innate immune system, also serving as the first line against pathogenic infections of mucosal surfaces. In mammals, several IFNLs have been reported; however, only limited data on the repertoire of IFNLs in avian species is available. Previous studies showed only one member in chicken (chIFNL3). Herein, we identified a novel chicken IFNL for the first time, termed chIFNL3a, which contains 354 bp, and encodes 118 amino acids. The predicted protein is 57.1% amino acid identity with chIFNL. Genetic, evolutionary, and sequence analyses indicated that the new open reading frame (ORF) groups with type III chicken IFNs represent a novel splice variant. Compared to IFNs from different species, the new ORF is clustered within the type III IFNs group. Further study showed that chIFNL3a could activate a panel of IFN-regulated genes and function mediated by the IFNL receptor, and chIFNL3a markedly inhibited the replication of Newcastle disease virus (NDV) and influenza virus in vitro. These data collectively shed light on the repertoire of IFNs in avian species and provide useful information that further elucidate the interaction of the chIFNLs and viral infection of poultry. IMPORTANCE Interferons (IFNs) are critical soluble factors in the immune system, and are composed of 3 types (I, II, and III) that utilize different receptor complexes (IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively). Herein, we identified IFNL from the genomic sequences of chicken and termed it chIFNL3a, located on chromosome 7 of chicken. Phylogenetically clustered with all known types of chicken IFNs, the finding of this IFN is considered a type III IFN. To further evaluate the biological properties of chIFNL3a, the target protein was prepared by the baculovirus expression system (BES), which could markedly inhibit the replication of NDV and influenza viruses. In this study, we uncovered a new interferon lambda splice variant of chicken, termed chIFNL3a, which could inhibit viral replication in cells. Importantly, these novel findings may extend to other viruses, offering a new direction for therapeutic interventions.

Virus-like Particle Vaccine Expressing the Respiratory Syncytial Virus Pre-Fusion and G Proteins Confers Protection against RSV Challenge Infection.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in children and the elderly. However, there are no effective antiviral drugs or licensed vaccines available for RSV infection. Here, RSV virus-like particle (VLP) vaccines expressing Pre-F, G, or Pre-F and G proteins on the surface of influenza virus matrix protein 1 (M1) were produced using the baculovirus expression system, and their protective efficacy was evaluated in mice. The morphology and successful assembly of VLPs were confirmed by transmission electron microscope (TEM) and Western blot. High levels of serum IgG antibody response were detected in VLP-immunized mice, and significantly higher levels of IgG2a and IgG2b were found in the Pre-F+G VLP immunization group compared to the unimmunized control. Serum-neutralizing activity was higher in the VLP immunization groups compared to the naïve group, with Pre-F+G VLPs demonstrating superior neutralizing activity to the single antigen-expressing VLP groups. Pulmonary IgA and IgG responses were generally comparable across the immunization groups, with VLPs expressing the Pre-F antigen eliciting higher IFN-γ in spleens. The frequencies of eosinophils and IL-4-producing CD4+ T cell populations were substantially lower in the lungs of VLP-immunized mice, with the PreF+G vaccine inducing a significant increase in CD4+ and CD8+ T cells. VLP immunization significantly decreased the viral titer and inflammation in the lungs of mice, with Pre-F+G VLPs conferring the best protection. In conclusion, our present study suggests that the Pre-F+G VLPs could be a potential vaccine candidate against RSV infection.

Expression and purification of MERS-CoV envelope protein, an essential viroporin, using the baculovirus expression system.

The causative agent of Middle East Respiratory Syndrome (MERS) is a zoonotic Coronavirus (MERS-CoV) identified in Saudi Arabia in 2012. The envelope (E) protein of MERS-CoV is a small viral protein which plays several essential roles during virus replication. To facilitate study of the structure and function of the E protein, recombinant MERS-CoV E protein was expressed using the baculovirus expression system. A recombinant E open reading frame including an 8-histidine tag at the amino terminus was designed and cloned into a baculovirus transfer vector. Following construction of a recombinant virus insect cells were infected and the expression of the E protein assessed by SDS-PAGE and Western blotting. Recombinant E protein, tagged at the N-terminus with a polyhistidine sequence, with a molecular mass of 10.18 kD was identified by Western blotting with an anti-His antibody. Following large scale infection E protein was released by detergent mediated lysis of infected cells and purified by Immobilized Metal Ion Affinity Chromatography (IMAC). Purified full length recombinant MERS-CoV E protein can be isolated by IMAC and is suitable for further functional, biophysical or immunological studies.

Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Resilience of transfluthrin to oxidative attack by duplicated CYP6P9 variants known to confer pyrethroid resistance in the major malaria mosquito Anopheles funestus

Resistance to common pyrethroids, such as deltamethrin and permethrin is widespread in the malaria mosquito Anopheles funestus and mainly conferred by upregulated cytochrome P450 monooxygenases (P450s). In the pyrethroid resistant laboratory strain An. funestus FUMOZ-R the duplicated genes CYP6P9a and CYP6P9b are highly upregulated and have been shown to metabolize various pyrethroids, including deltamethrin and permethrin. Here, we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus using a baculovirus expression system and evaluated the interaction of the multifluorinated benzyl pyrethroid transfluthrin with these enzymes by different approaches. First, by Michaelis-Menten kinetics in a fluorescent probe assay with the model substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC), we showed the inhibition of BOMFC metabolism by increasing concentrations of transfluthrin. Second, we tested the metabolic capacity of recombinantly expressed CYP6P9 variants to degrade transfluthrin utilizing UPLC-MS/MS analysis and detected low depletion rates, explaining the virtual lack of resistance of strain FUMOZ-R to transfluthrin observed in previous studies. However, as both approaches suggested an interaction of CYP6P9 variants with transfluthrin, we analyzed the oxidative metabolic fate and failed to detect hydroxylated transfluthrin, but low amounts of an M-2 transfluthrin metabolite. Based on the detected metabolite we hypothesize oxidative attack of the gem-dimethyl substituted cyclopropyl moiety, resulting in the formation of an allyl cation upon ring opening. In conclusion, these findings support the resilience of transfluthrin to P450-mediated pyrethroid resistance, and thus, reinforces its employment as an important resistance-breaking pyrethroid in resistance management strategies to control the major malaria vector An. funestus.

A new tool for producing and investigating recombinant sarcomeric actin.

Actin forms many cytostructural elements, including the backbone of striated muscle thin filaments. Our mechanistic understanding of most actin-dependent processes remains limited, partly due to difficulty with recombinant actin expression. While fully-functional actin cannot be produced in bacteria, it can be generated using baculovirus expression systems. Here, we created an additional tool for recombinant actin production, for hypothesis testing and for resolving the molecular properties and cellular effects of unique variants. Specifically, we engineered Drosophila that can express untagged actins exclusively in indirect flight muscles (IFMs). Act88F encodes sarcomeric IFM actin. Attempts to rescue IFM function (i.e. flight) in Act88F-nulls, by inserting transgenic Act88F (Act88FTG-WT) ectopically throughout the genome have failed, potentially due to ineffective cis/trans regulation of Act88FTG-WT expression. To circumvent these problems, we used CRISPR-editing and introduced an attP landing site directly into Act88F, to disrupt the endogenous gene and serve as a target for site-specific transgene insertion. attP-mediated disruption of Act88F yielded flightlessness and actin-free IFMs. Transformants expressing GFP or Act88FTG-WT, from the endogenous Act88F integration locus, demonstrated robust IFM-specific protein production and restored flight, respectively. Having established the expression system, we designed Drosophila mutants to test the roles of specific actin residues in tropomyosin/myosin-binding and nemaline myopathy pathogenesis. Both Act88FTG-R147Q and Act88F TG-F352Striggered IFM hypercontraction and impaired flight. Finally, we determined whether mammalian actin could be generated and observed high-level expression of human α-cardiac actin (ACTC1), which behaved indistinguishably from bovine ACTC1 in unregulated and regulated motility (IVM) assays. We are currently comparing IVM properties of IFM-purified vs. baculovirus/Sf12-purified ACTC1. Overall, our novel fly line permits transgenic actin expression in mature muscle fibers that are themselves amenable to structural and functional analyses, and serves as a cost-effective, perpetual source of ample protein for biophysical experimentation.

A lef5-deficient baculovirus expression system with no virion contamination and promoting secretion

The baculovirus expression system (BEVS) is widely used in biomanufacturing. However, massive late and very late genes are expressed during baculovirus infection of the host cell, and the protein aggregation has a significant negative impact on the intercellular environment of cells and organelle function. Baculovirus particle contamination also hinders the purification of particulate products. In this study, a novel baculovirus vector of deficient-lef5 (Ac-Δlef5) was established to prevent the production of baculovirus particles. It could transduce cells and replicate viral DNA but the expression of the late and very late genes was reduced so that no virus was packed and budded. The Ac-Δlef5 expression system was successfully used to express a secreted protein, thyroid peroxidase (TPO). Compared to wild-type baculovirus (Ac-wt), Ac-Δlef5 delayed the decline in cell viability and prolonged the harvest period from 4 to 6 days after infection. When expressed in the Ac-Δlef5 system, 98% of TPO was secreted extracellularly, which was about 1.8 times that of the Ac-wt system. Meanwhile, the transcription levels of protein folding-related genes were significantly increased. The results show that the Ac-Δlef5 system is a potential novel viral-free baculovirus expression system, which omits the virion removal operation in biomanufacturing.

Structure-based design of oligomeric receptor-binding domain (RBD) recombinant proteins as potent vaccine candidates against SARS-CoV-2

ABSTRACT The receptor-binding domain (RBD) of SARS-CoV-2 S protein is proved to be the major target of neutralizing antibodies. However, on the S protein, only a portion of epitopes in RBD can be effectively displayed with dynamic changes in spatial conformations. Using RBD fragment as antigen can better expose the neutralizing epitopes, but the immunogenicity of RBD monomer is suboptimal. Multimeric display of RBD molecules is a feasible strategy to optimize RBD-based vaccines. In this study, RBD single-chain dimer derived from Wuhan-Hu-1 was fused with a trimerization motif, and a cysteine was also introduced at the C-terminus. The resultant recombinant protein 2RBDpLC was expressed in Sf9 cells using a baculovirus expression system. Reducing/non-reducing PAGE, size-exclusion chromatography and in silico structure prediction indicated that 2RBDpLC polymerized and possibly formed RBD dodecamers through trimerization motif and intermolecular disulfide bonds. In mice, 2RBDpLC induced higher levels of RBD-specific and neutralizing antibody responses than RBD dimer, RBD trimer and prefusion-stabilized S protein (S2P). In addition, cross-neutralizing antibodies against Delta and Omicron VOC were also detected in the immune sera. Our results demonstrate that 2RBDpLC is a promising vaccine candidate, and the method of constructing dodecamers may be an effective strategy for designing RBD-based vaccines.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

A neutralizing monoclonal antibody-based blocking ELISA to detect bovine herpesvirus 1 and vaccination efficacy.

Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC50) of 37.82ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R2 = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developedbased on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests.

Steroid-Resistant Nephrotic Syndrome-Associated &lt;i&gt;MYO1E&lt;/i&gt; Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in &lt;i&gt;MYO1E&lt;/i&gt; are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the &lt;i&gt;MYO1E&lt;/i&gt; variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. EGFP-tagged human &lt;i&gt;Myo1e&lt;/i&gt; constructs were delivered into the Myo1e-KO mouse podocyte-derived cells &lt;i&gt;via&lt;/i&gt; adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated &lt;i&gt;Myo1e&lt;/i&gt; constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity &lt;i&gt;in vitro&lt;/i&gt;. Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel &lt;i&gt;MYO1E&lt;/i&gt; variants associated with SRNS.

A gold nanoparticles-based lateral flow assay utilizing baculovirus expressed recombinant nucleocapsid and receptor binding domain proteins for serodetection of IgG and IgM against SARS-CoV-2

Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.

Immunosuppressive lncRNA LINC00624 promotes tumor progression and therapy resistance through ADAR1 stabilization

BackgroundDespite the success of HER2-targeted therapy in achieving prolonged survival in approximately 50% of treated individuals, treatment resistance is still an important challenge for HER2+ breast cancer (BC) patients. The...

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.