In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

Acinetobacter baumannii (A. Baumannii) is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A. baumannii. The bacterial load in the blood, pathological changes in the lung tissue and survival rates after challenge were evaluated. Mice immunized with OmpK/Omp22 fused protein provided significantly greater protection against A. baumannii challenge than those immunized with either of the two proteins individually. The results provide novel clues for future design of vaccines against A. baumannii.
Acinetobacter Infections ; pathology ; prevention & control ; Acinetobacter baumannii ; genetics ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Load ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Disease Models, Animal ; Female ; Mice, Inbred BALB C ; Pneumonia, Bacterial ; pathology ; prevention & control ; Recombinant Fusion Proteins ; genetics ; immunology

OBJECTIVETo generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.

METHODSThe full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.

RESULTSDNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.

CONCLUSIONRecombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.

Animals ; Antigens, Human Platelet ; immunology ; Autoantibodies ; immunology ; Base Sequence ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoassay ; methods ; Integrin beta3 ; genetics ; immunology ; metabolism ; Microspheres ; Recombinant Proteins ; immunology ; metabolism ; Reproducibility of Results

Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
Acrosome ; immunology ; Animals ; Antibodies ; analysis ; Blotting, Western ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; immunology ; Escherichia coli ; Humans ; Immunohistochemistry ; Male ; Muramidase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; Semen ; immunology ; Spermatozoa ; immunology ; Testis ; immunology

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.
Animals ; HIV Infections ; immunology ; therapy ; HIV-1 ; immunology ; Humans ; Immunity, Cellular ; Immunological Synapses ; Immunotherapy ; Killer Cells, Natural ; transplantation ; Neoplasms ; immunology ; therapy ; Receptors, Antigen, T-Cell ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; transplantation

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.
Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; immunology ; Gene Editing ; methods ; Humans ; Immunotherapy ; methods ; Receptors, Antigen, T-Cell ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or "on-target/off-tumor" toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strategies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vector designs to increase both the safety and efficacy, further T cell modification to overcome the tumor-associated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal-curing cancer with high safety, high efficacy, and low cost.
Cell Movement ; immunology ; Cell Proliferation ; Gene Expression ; Genetic Vectors ; chemistry ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Lymphocytes, Tumor-Infiltrating ; cytology ; immunology ; transplantation ; Neoplasms ; genetics ; immunology ; pathology ; therapy ; Patient Safety ; Receptors, Antigen, T-Cell ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Signal Transduction ; Single-Chain Antibodies ; chemistry ; genetics ; T-Lymphocytes ; cytology ; immunology ; transplantation ; Treatment Outcome

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.
Animals ; Antibodies, Viral ; immunology ; Ebolavirus ; genetics ; immunology ; Female ; Gene Expression ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology