Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.
Humans ; Immunotherapy, Adoptive/methods* ; Receptors, Chimeric Antigen/immunology* ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics* ; Gene Editing ; CRISPR-Cas Systems/genetics* ; DNA Transposable Elements/genetics* ; T-Lymphocytes/immunology* ; Neoplasms/immunology*

Foot-and-mouth disease (FMD) is one of the major animal infectious diseases in the world. All cloven-hoofed animals are susceptible to FMD. Vaccination is still the first choice for the prevention and control of FMD. mRNA vaccines can be rapidly designed, synthesized, and produced on a large scale in vitro, and they can induce effective protective immune responses, demonstrating the advantages of rapid development, easy preparation, and low biosafety risks. The design of untranslated regions is a key to enhancing the expression and efficacy of mRNA vaccines. In order to generate an efficient FMD mRNA vaccine, we designed three FMD P12A3C expression vectors with different untranslated regions and synthesized corresponding mRNAs. By comparing expression efficiency of these vectors and their mRNAs at different time points and in different cell lines, we found that the mRNA P12A3C-UTR3 had the best expression and universality. This study laid a foundation for the development of mRNA vaccines against FMD and provided a theoretical basis for the optimal sequence design of efficient mRNA.
Foot-and-Mouth Disease Virus/genetics* ; Animals ; RNA, Messenger/biosynthesis* ; Foot-and-Mouth Disease/immunology* ; Antigens, Viral/biosynthesis* ; Viral Vaccines/biosynthesis* ; Genetic Vectors/genetics* ; Cell Line ; Vaccines, DNA/immunology*

The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has occurred in China and around the world. SARS-CoV-2-infected patients with severe pneumonia rapidly develop acute respiratory distress syndrome (ARDS) and die of multiple organ failure. Despite advances in supportive care approaches, ARDS is still associated with high mortality and morbidity. Mesenchymal stem cell (MSC)-based therapy may be an potential alternative strategy for treating ARDS by targeting the various pathophysiological events of ARDS. By releasing a variety of paracrine factors and extracellular vesicles, MSC can exert anti-inflammatory, anti-apoptotic, anti-microbial, and pro-angiogenic effects, promote bacterial and alveolar fluid clearance, disrupt the pulmonary endothelial and epithelial cell damage, eventually avoiding the lung and distal organ injuries to rescue patients with ARDS. An increasing number of experimental animal studies and early clinical studies verify the safety and efficacy of MSC therapy in ARDS. Since low cell engraftment and survival in lung limit MSC therapeutic potentials, several strategies have been developed to enhance their engraftment in the lung and their intrinsic, therapeutic properties. Here, we provide a comprehensive review of the mechanisms and optimization of MSC therapy in ARDS and highlighted the potentials and possible barriers of MSC therapy for COVID-19 patients with ARDS.
Adoptive Transfer ; Alveolar Epithelial Cells ; pathology ; Animals ; Apoptosis ; Betacoronavirus ; Body Fluids ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; Clinical Trials as Topic ; Coinfection ; prevention & control ; therapy ; Coronavirus Infections ; complications ; immunology ; Disease Models, Animal ; Endothelial Cells ; pathology ; Extracorporeal Membrane Oxygenation ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; therapeutic use ; Humans ; Immunity, Innate ; Inflammation Mediators ; metabolism ; Lung ; pathology ; physiopathology ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stem Cells ; physiology ; Multiple Organ Failure ; etiology ; prevention & control ; Pandemics ; Pneumonia, Viral ; complications ; immunology ; Respiratory Distress Syndrome, Adult ; immunology ; pathology ; therapy ; Translational Medical Research

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

Cervical cancer is the fourth most lethal women's cancer worldwide. Current treatments against cervical cancer include surgery, radiotherapy, chemotherapy, and anti-angiogenic agents. However, despite the various treatments utilized for the treatment of cervical cancer, its disease burden remains a global issue. Persistent infection of human papillomavirus (HPV) has been identified as an essential step of pathogenesis of cervical cancer and many other cancers, and nation-wide HPV screening as well as preventative HPV vaccination program have been introduced globally. However, even though the commercially available prophylactic HPV vaccines, Gardasil (Merck) and Cervarix (GlaxoSmithKline), are effective in blocking the entry of HPV into the epithelium of cervix through generation of HPV-specific neutralizing antibodies, they cannot eliminate the pre-existing HPV infection. For these reason, other immunotherapeutic options against HPV-associated diseases, including therapeutic vaccines, have been continuously explored. Therapeutic HPV vaccines enhance cell-mediated immunity targeting HPV E6 and E7 antigens by modulating primarily dendritic cells and cytotoxic T lymphocyte. Our review will cover various therapeutic vaccines in development for the treatment of HPV-associated lesions and cancers. Furthermore, we will discuss the potential of immune checkpoint inhibitors that have recently been adopted and tested for their treatment efficacy against HPV-induced cervical cancer.
Dendritic Cells/immunology ; Female ; Genetic Vectors ; Humans ; *Immunotherapy ; Papillomavirus Infections/*complications/therapy ; Papillomavirus Vaccines/therapeutic use ; *Translational Medical Research ; Uterine Cervical Neoplasms/*therapy ; Vaccines, DNA/therapeutic use ; Vaccines, Subunit/therapeutic use

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Therapeutic HIV vaccine was considered as a hopeful curative method for AIDS patients. However, there is still no suitable HIV animal model for vaccine study since the difference in the immune system between human and animals. To evaluate the therapeutic effect of combined immunization strategy with multiple vector vaccines in macaque models. Plasmid DNA, recombinant Ad5 and MVA vaccines which expressing SIV gag and env genes were constructed. Sequential and repeated immune strategy were applied to immunize mice with these three vaccines. Cellular immune responses in mice immunized with these three vaccines were measured by ELISPOT test in vitro and CTL assay in vivo. The results were analyzed and compared with different antigen combination, order of vaccines and intervals to choose a suitable immunization strategy for macaque immunization in future. It indicated that strong SIV-Gag/Env-specific cellular immune responses were induced by these three vector vaccines. It laid a foundation for evaluating the therapeutic effect of combined immunization strategy with multiple vector vaccines in SIV infected macaque models.
AIDS Vaccines ; administration & dosage ; genetics ; immunology ; Adenoviridae ; genetics ; metabolism ; Animals ; Antibodies, Viral ; immunology ; Female ; Gene Products, env ; administration & dosage ; genetics ; immunology ; Gene Products, gag ; administration & dosage ; genetics ; immunology ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Simian Immunodeficiency Virus ; genetics ; immunology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2>AAV3=AAVLK03>AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤35, 36-50, and ≥51 years old). People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.
Adult ; Aged ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Body Constitution ; Dependovirus ; classification ; immunology ; Female ; Genetic Vectors ; Humans ; Liver ; virology ; Male ; Middle Aged ; Serogroup

To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; Cross Protection ; Female ; Genetic Vectors ; biosynthesis ; genetics ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; administration & dosage ; genetics ; immunology ; Viral Core Proteins ; administration & dosage ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology