Expression of influenza B virus hemagglutinin and its immunogenicity determination / 生物工程学报

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Distribution of HLA-DRB1 alleles in BRICS countries with a high tuberculosis burden: a systematic review and meta-analysis

Abstract INTRODUCTION: Tuberculosis (TB) is the leading cause of death worldwide caused by a single infectious disease agent. Brazil, Russia, India, China, and South Africa (BRICS) account for more than half of the world's TB cases. Bacillus Calmette-Guérin (BCG) remains the only vaccine available despite its variable efficacy. Promising antigen-based vaccines have been proposed as prophylactic and/or immunotherapeutic approaches to boost BCG vaccination. Relevant antigens must interact with the range of human leukocyte antigen (HLA) molecules present in target populations; yet this information is currently not available. METHODS: MEDLINE and EMBASE were systematically searched for articles published during 2013-2020 to measure the allelic frequencies of HLA-DRB1 in the BRICS. RESULTS: In total, 67 articles involving 3,207,861 healthy individuals were included in the meta-analysis. HLA-DRB1 alleles *03, *04, *07, *11, *13, and *15 were consistently identified at high frequencies across the BRICS, with a combined estimated frequency varying from 52% to 80%. HLA-DRB1 alleles *01, *08, *09, *10, *12, and *14 were found to be relevant in only one or two BRICS populations. CONCLUSIONS: By combining these alleles, it is possible to ensure at least 80% coverage throughout the BRICS populations.

Advances in research on aquaporins in medical helminthes / 中国血吸虫病防治杂志

Aquaporins (AQPs) are a class of membrane intrinsic proteins in medical helminthes that specifically mediate the transmembrane transport of water or other solute molecules. Previous studies have demonstrated that AQPs play a critical role in promoting the transmembrane transport of water, osmoregulation, uptake of nutrients, release of toxic metabolic products and transport of antiparasitic drugs, which may serve as promising vaccine candidates and drug targets for parasitic diseases. This review describes the structural characteristics of AQPs in medical helminthes, and discusses the feasibility of these AQPs as antihelminth vaccine candidates and drug targets, so as to provide insights into the development of novel vaccines and drugs against parasitic diseases.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Construction of a Vero cell-based high-yield H7N9 influenza vaccine candidate strain / 中华微生物学和免疫学杂志

Objective To construct an H7N9 vaccine strain by using a previously obtained Vero cell-based high-yield influenza A virus as the donor strain.Methods The recombinant virus strains, 4mut-H7N9 and PR8-H7N9, were respectively rescued with reverse genetics technique by combining the genes en-coding hemagglutinin (HA) and neuraminidase (NA) of H7N9 virus with the 6 internal genes of PR8-4mut or PR8 virus strains.The growth feature of 4mut-H7N9 virus strain was compared with that of PR8-H7N9 vi-rus strain with growth curves and plaque assays.The viral proteins produced by 4mut-H7N9 and PR8-H7N9 virus strains were measured by Western blot and Coomassie blue staining.Results The PR8-H7N9 and 4mut-H7N9 virus strains were successfully rescued.The virus titer of 4mut-H7N9 strain was about 3000 times higher than that of PR8-H7N9 strain at 72 hours after infecting Vero cells.The 4mut-H7N9 virus strain formed plaques of about 1 mm in diameter on Vero cells, while the PR8-H7N9 virus strain only formed pin-point plaques on Vero cells.The levels of viral proteins encoded by purified 4mut-H7N9 virus strain were significantly higher than that of the PR8-H7N9 virus strain as indicated by both Western blot and Coomassie blue staining.Moreover, the 4mut-H7N9 virus strain was less pathogenic than PR8-H7N9 strain in mice, and retained the trypsin dependence for infecting cells.Conclusion The reassortant 4mut-H7N9 vaccine strain as established by reverse genetics technique grew faster and better in Vero cells, suggesting the possi-bility of using it as a candidate vaccine strain whenever facing a potential epidemic of H7N9 virus.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEÄ), thymidine kinase (BoHV-5TKÄ) and both proteins (BoHV-5gEÄTKÄ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEÄ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKÄ (N = 8) or BoHV-5gEÄTKÄ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKÄ and BoHV-5gEÄTKÄ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Characterization of Immune Responses to Mycobacterium tuberculosis Rv2041c Protein

Tuberculosis, which is caused by Mycobacterium tuberculosis (M. tb), is one of the most important infectious diseases in the world. Although many functional studies have been conducted on M. tb proteins in the post-genomic era, little is known about the function of many proteins expressed specifically during latency. Previously, we reported that Rv2041c from M. tb H37Rv is highly expressed under conditions of low pH and hypoxia, which represent the in vitro mimicry of latent tuberculosis. In the present study, increased expression levels of Rv2041c under hypoxia and low pH in vitro culture was confirmed by RT-PCR. Interestingly, Rv2041c showed significantly increased expression among genes of the same operon and genes belonging to the same functional group. Finally, the immune responses elicited by the recombinant (r) Rv2041c protein were investigated using ex vivo and in vivo models of M. tb infection. A significantly high level of pro-inflammatory cytokines such as TNF-alpha, IL-6, and IL-12p40 was detected in a dose-dependent manner by treatment of murine bone marrow-derived macrophages with rRv2041c protein. In addition, IFN-gamma and TNF-alpha secretion increased after stimulation with purified Rv2041c protein to lymphocytes from latent and active TB mice in a modified Cornell model. In conclusion, our findings suggest that Rv2041c is a new T-cell antigen and could be a potential vaccine candidate against M. tb infection by inducing a strong cellular immune response.