Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Construction and evaluation of a universal influenza mRNA vaccine / 陆军军医大学学报

Objective To construct a universal influenza mRNA vaccine and evaluate its immunogenicity.Methods The antigen sequence of hemagglutinin(HA),nucleoprotein(NP)and matrix protein 2 ectodomain(M2e)in influenza A/California/04/2009 was optimized.HA,NP and 3 tandem M2e(3M2e)were cloned into pcDNA3.1 vector,respectively.Then the mRNAs were synthesized by linearization,in vitro transcription,enzymatic capping and enzymatic tailing,and named as mRNA-HA,mRNA-NP and mRNA-3M2e,respectively.The protein expression of the 3 kinds of mRNAs in 293T cells was detected by immunofluorescence assay.Comb-mRNA vaccine was prepared by enveloped mRNA-HA,mRNA-NP and mRNA-3M2e with lipid nanoparticles,respectively,and the particle size and potential were identified.Twenty-eight 6-week-old female BALB/c mice(18～22 g)were randomly divided into LNP group(n=14)and Comb-mRNA group(n=14).Hemagglutination inhibition(HI)method and microneutralization(MN)test were used to evaluate the serum antibody titer induced by Comb-mRNA vaccines.The mice were infected by 5LD50 wild-type H1 N1 influenza virus to evaluate the protective efficacy.Results The mRNA-HA,mRNA-NP and mRNA-3M2e were successfully constructed,and the 3 mRNAs could be expressed in 293T cells.The average size of mRNA encapsulated by lipid nanoparticles was 119.53±6.5 nm,and the average potential was-8.23±1.3 mV.The geometric mean titer(GMT)of HI and MN in the Comb-mRNA group were 179.6 and 201.6,compared with the LNP group.The ratio of IFN-γ+CD4+/CD8+Tcells was increased.The Comb-mRNA group could provide protection against 5LD50 wild type influenza H1 N1 virus after 2 weeks of booster immunization.Conclusion Comb-mRNA,an influenza vaccine candidate,can induce immune responses and protect mice from influenza virus challenge.

Application of gelatin sponge-hemocoagulase plugging agent in patients with pulmonary puncture bleeding / 介入放射学杂志

Objective To discuss the application of gelatin sponge-hemocoagulase plugging agent in patients with pulmonary puncture bleeding.Methods The clinical data of 43 patients with hemorrhage caused by DSA-guided lung puncture biopsy,who received gelatin sponge-hemocoagulase plugging agent treatment at the Jining Municipal First People's Hospital of China between September 2021 and May 2023,were collected,and the hemostatic effect of gelatin sponge-hemocoagulase plugging agent was analyzed.Results Successful lung puncture needle biopsy was achieved in all the 43 patients.The puncture needle channel occlusion was accomplished by using gelatin sponge-hemocoagulase plugging agent.Five minutes after occlusion treatment,in one patient,whose moderate hemoptysis with moderate bleeding shadow before puncture needle biopsy changed to bloody sputum,the intrapulmonary bleeding shadow displayed on image became slightly enlarged when compared the size five minutes ago,while in all the remaining patients successful hemostasis was achieved,the hemoptysis disappeared and the pulmonary hemorrhage shadow was similar to that five minutes ago.No occlusion-related complications occurred in all patients.Conclusion For the treatment of pulmonary hemorrhage caused by DSA-guided lung puncture biopsy,gelatin sponge-hemocoagulase plugging agent is clinically safe and effective.

Anti-tumor Mechanism and Clinical Application of Pseudomonas Aeruginosa-Mannose-Sensitive Hemagglutinin / 医药导报

The application of Pseudomonas aeruginosa-mannose-sensitive hemagglutinin(PA-MSHA)in the field of an-titumors has been increasing.PA-MSHA has been found to promote tumor cell apoptosis,inhibit tumor cell invasion and migration,differentiation,and change drug resistance and epithelial-mesenchymal transformation(EMT)by inhibiting the EGFR pathway.Meanwhile,PA-MSHA also enhances the immune killing and inhibition of macrophages and T cells to tumor cells through toll-like receptors(TLRs).In this paper,we reviewed the reported anti-tumor mechanism and clinical application of PA-MSHA,suggesting that PA-MSHA may alter the glycosylation of EGFR and TLR proteins by acting on the regulatory process of the cellular mannosy-lation process.PA-MSHA can act on cell membrane proteins,including more receptors with high-mannosylation of signaling path-ways.Elucidating the deep relationship between PA-MSHA and mannosylation is of great significance for the mechanism research and clinical application of PA-MSHA.

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine / 中华微生物学和免疫学杂志

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Characteristics of hemagglutinin-neuraminidase gene of human parainfluenza virus type 3 among patients with acute respiratory tract infection in Henan Province / 中华微生物学和免疫学杂志

Objective:To analyze the molecular characteristics of hemagglutinin-neuraminidase (HN) gene of human parainfluenza virus type 3 (HPIV3) among the cases with acute respiratory tract infection (ARI) in Henan Province.Methods:Nasal/throat swab samples collected from patients with severe acute respiratory tract infection (SARI) in Luohe and patients with influenza-like illness (ILI) in Zhengzhou were used in this study. HPIV nucleic acids in the samples were detected using real-time fluorescent PCR. HPIV3-positive samples were subjected to RT-PCR for the amplification of HN genes and the sequences were analyzed with Sanger method. CExpress and MEGA7.0 software were used for sequences editing, evolution tree construction and gene sequence analysis.Results:A total of 374 throat swab samples collected form ARI cases in Luohe and Zhengzhou were tested and 20 (5.3%) of them were positive for HPIV3. Eighteen HPIV3 HN gene sequences were successfully amplified and all belonged to C3 subgroups, including 16 sequences of C3f genotype and two sequences of C3a genotype. The 18 HN gene sequences shared the homology of 97.6%-100.0% in nucleotide and 99.3%-100.0% in amino acid, but the differences between them and the prototype strain Wash/47885/57 were significant. There were 12 amino acid mutations shared by them, including four function-related mutations (H295Y, I391V, D556N and I53T). There were no significant differences in the nucleotide or amino acid sequences as compared with the epidemic strain of China/BCH4210A/2014.Conclusions:The C3f and C3a branches of HPIV3 were the epidemic genotypes in Henan Province in recent years and a local circulating prevalence might be established. Continuous and in-depth monitoring of HPIV3 C3 subtype would be of great significance for the prevention and control of HPIV3-associated diseases.

Phylogenetic and antigenic analysis of HA gene of influenza virus B (Victoria) in Beijing during 2021-2022 surveillance season / 中华微生物学和免疫学杂志

Objective:To investigate the phylogenetic and antigenic characteristics of hemagglutinin (HA) gene of influenza B/Victoria lineage (BV) viruses in Beijing during the 2021-2022 influenza surveillance season, and to analyze whether the circulating BV viruses match the vaccine strain.Methods:Pharyngeal swab specimens from influenza like-illness (ILI) cases in the 2021-2022 influenza surveillance season were collected from surveillance network labs in Beijing and cultured in MDCK cells and chicken embryo to isolate BV viruses. Nucleic acids of the viruses were extracted, and the HA gene was amplified and sequenced. The nucleotide and amino acid sequence identity of the HA gene was analyzed using MEGA5.0 software. A phylogenetic tree of HA gene was constructed using the maximum likelihood method. The N-glycosylation sites in HA were predicted online. Three-dimensional structure of HA was constructed using SWISS-MODEL homologous modeling. Hemagglutination inhibition (HI) test was performed to analyze the antigenicity of BV viruses.Results:A total of 402 BV viruses were collected and 58 strains with full-length HA gene sequences were chosen for further analysis. Compared with the HA gene of this year′s vaccine strain (B/Washington/02/2019), there were 27 amino acid mutations, 11 of which were located in four different antigenic determinants. The phylogenetic analysis revealed that three subgroups of 1A.3, 1A.3a1, and 1A.3a2 co-circulated in Beijing with 54 strains (54/58, 93.10%) clustered to the Clade 1A.3a2, two strains (2/58, 3.45%) clustered to the Clade 1A.3a1, and two strains (2/58, 3.45%) in the same subgroup (Clade 1A.3) as the vaccine component BV strain in 2021-2022. Compared with the vaccine strain (B/Washington/02/2019), two BV strains had an additional N-glycosylation site at residue 197, while the other 56 strains showed no change in N-glycosylation sites. Antigenic analysis showed that 35 BV strains (35/58, 60.34%) were antigenically similar to the vaccine strain and 23 strains (23/58, 39.66%) were low-response strains.Conclusions:Three subgroups of BV viruses co-circulated in Beijing during the 2021-2022 influenza surveillance season. The predominant subgroup was Clade 1A.3a2 (93.10%), showing a certain genetic distance with the vaccine strain (B/Washington/02/2019). Nearly 40% (39.66%) of the viruses were low-response strains. This study indicated that continuous monitoring of the variations of influenza epidemic strains and timely providing laboratory basis for screening vaccine component strains were the basic technical guarantee for coping with influenza pandemic.

Construction of an mRNA vaccine encoding hemagglutinin of influenza A H1N1 virus and investigation on booster immunization strategy / 上海交通大学学报（医学版）

Objective·To construct an mRNA vaccine encoding hemagglutinin(HA)of influenza A H1N1 virus,and explore the protective effects of different booster vaccination strategies.Methods·Firefly luciferase(Fluc)was used as the reporter gene to construct Fluc mRNA vaccine enveloped in lipid nanoparticles(LNP).The in vivo expression of Fluc mRNA-LNP after intramuscular injection was determined by live imaging assay in mice.Furthermore,M15-HA mRNA-LNP derived from H1N1 subtype(A/Michigan/45/2015)was constructed.Mice were immunized with 20,10,5,or 1 μg doses of M15-HA mRNA-LNP twice(with an interval of 3 weeks)through intramuscular injection.Serum antibody titers were measured by enzyme-linked immunosorbent assay(ELISA)at 2 weeks and 4 weeks after the second immunization,and functional antibody levels were detected by hemagglutination inhibition test.The third booster vaccination was performed 40 d after the second immunization in 1 μg dose group with 1 μg M15-HA mRNA-LNP or 10 μg HA subunit vaccine.The levels of specific antibody and functional antibody were detected by ELISA and hemagglutination inhibition test,respectively 2 weeks and 4 weeks later.Results·Live imaging assay showed that luciferase activity could be detected in mice 1 d after injection of Fluc mRNA-LNP.At 2 weeks and 4 weeks after the second immunization of M1 5-HA mRNA-LNP,HA-specific antibodies were significantly higher than those before the immunization in all vaccination groups at different doses(P=0.000).The hemagglutination inhibition test showed that the levels of functional antibodies in the 20 μg dose and 10 μg dose groups were significantly higher than those in the PBS control group(P＜0.05).After 1 μg dose group mice were immunized with HA protein or M15-HA mRNA-LNP,higher levels of HA-specific antibody and functional antibody were induced and maintained for a long time.There was no significant difference between the two different booster immunization strategies.Conclusion·M15-HA mRNA-LNP vaccine is constructed with immunogenicity and antibody neutralization activity.Low-dose mRNA priming vaccination followed by both homologous mRNA vaccine and heterologous protein subunit vaccine booster vaccination can induce stronger immune recall responses.

Progress in universal influenza vaccines targeting hemagglutinin and other conserved epitopes of influenza A virus / 中华微生物学和免疫学杂志

Influenza viruses are responsible for a large number of infections and deaths annually, posing a serious threat to public health. Vaccination is the most effective measure to prevent influenza virus infection. However, current seasonal influenza vaccines only protect against closely matched circulating strains. Even with extensive surveillance and annual reformulation, yearly updated vaccines are still a step behind the fast-evolving viruses, often resulting in poor matches or less effective vaccines. Due to the relatively complex evolution of influenza A viruses, it is a new idea and a new means to prevent influenza epidemics by using a series of innovative technologies to develop universal influenza vaccines that can provide extensive and long-lasting protection against influenza viruses. This review summarized the latest progress in the development of universal vaccines targeting HA in the past three years, including design methods for universal vaccines targeting HA, HA stem and other conserved epitopes, compared the advantages and disadvantages of different technologies, explored the impact of immunization programs and strategies, and discussed the potential challenges to be overcome, hoping to provide reference for the successful development of universal vaccines.

Genetic variation and evolution of influenza viruses isolated from co-infection cases in Guangdong Province / 中华微生物学和免疫学杂志

Objective:To analyze and reveal the genetic evolution and variation of influenza viruses in cases of co-infection in Guangdong Province.Methods:Throat swab samples were collected from four cases of H1N1pdm and H3N2 co-infection for viral isolation. The isolated strains were subjected to antigen analysis and neuraminidase inhibitor susceptibility test. High-throughput sequencing was used to detect the sequences of strains in three throat swab samples and one virus strain, and then genetic variations were analyzed.Results:Four influenza viruses were isolated with one strain of H1N1pdm and three of H3N2 subtype, and all of them were genetically similar to the vaccine strain in 2022-2023. The HA genes of H1N1pdm and H3N2 strains belonged to clade 6B.1A.5a.2a and 2a.3a.1, respectively. The isolated strains belonged to the same clade as the strains prevalent in Guangdong during the same period. No drug-resistant variations were detected in N1 or N2 gene, and the isolated strains were sensitive to oseltamivir and zanamivir.Conclusions:H1pdm subtype had stronger replication ability than H3 subtype in the influenza viruses isolated from co-infected cases. H1N1pdm and H3N2 subtype influenza viruses were genetically similar to the strains circulating in Guangdong at the same time. The isolated H1N1pdm and H3N2 strains were sensitive to both oseltamivir and zanamivir, indicating that they could continue to be used in the treatment of influenza virus infections caused by one or two genotypes.

Progress in antiviral strategies targeting influenza virus hemagglutinin / 中华微生物学和免疫学杂志

Influenza is a worldwide infectious disease caused by influenza virus. It has posed great challenges on public health and social stability since 1918. At present, vaccination is the most effective way to prevent and control influenza epidemics. Broad-spectrum antiviral drugs and neutralizing antibodies against influenza virus have been widely studied in recent years. Hemagglutinin (HA), which is on the surface of influenza virus, plays an important role in the stage of viral invasion into host cells. It is the main effective antigenic component of current influenza vaccines, as well as the main target of broad-spectrum neutralizing antibodies and broad-spectrum antiviral drugs. This review summarized the progress in the development of novel influenza vaccines, neutralizing antibodies, and antiviral drugs based on influenza virus HA, as well as other prevention and control measures, hoping to present new ideas for future influenza prevention and control.

Recent advances in small-molecule inhibitors targeting influenza virus glycoproteins / 药学学报

Hemagglutinin and neuraminidase, two important glycoproteins on the surface of influenza virus, play a considerable role in the entry and release stage of the viral life cycle, respectively. With in-depth investigation of influenza virus glycoproteins and the continuous innovation of drug discovery strategies, a new generation of glycoproteins inhibitors have been continuously discovered. From the point of view of medicinal chemistry, this review summarizes the current advances in seeking small-molecule inhibitors targeting influenza virus glycoproteins, hoping to provide valuable guidance for future development of novel antiviral drugs.

Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine / 中国生物制品学杂志

@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

Genetic characteristics analysis of the hemagglutinin gene of measles virus in Shanghai， 2001‒2018 / 上海预防医学

ObjectiveTo analyze the genetic characteristics of the hemagglutinin （H） gene of measles virus （MeV） in Shanghai， 2001‒2018. MethodsNasopharyngeal swab specimens were collected from suspected measles cases reported in Shanghai from 2001 to 2018， and the isolation of measles virus was conducted with Vero/hSLAM cell line. RT-PCR amplification and sequencing were conducted after RNA extraction to analyze the genetic characteristics of the complete H gene. ResultsIn total， 5 665 nasopharyngeal swab samples were collected by suspected measles case surveillance from 2001 to 2018， and 1 394 measles virus strains were isolated. The homology of nucleotide acid and amino acid among 349 representative measles virus isolates was 87.4%‒100.0% and 85.1%‒100.0%， respectively. The homology of nucleotide acid and amino acid between representative measles virus isolates and China vaccine strain （S191） was 85.7%‒100.0% and 84.1%‒100.0%， respectively. All the sub-genotype H1a MeV isolates had an amino acid substitution （Ser240Asn）， which removed a predicted N-linked glycosylation site. ConclusionMost of the MeV isolates are sub-genotype H1a analyzed based on H gene， which are identical to those of the N gene. The predicted amino acid sequences of the H protein are relatively conserved at most of the functionally significant amino acid positions.

Molecular characterisation of hemagglutinin and neuraminidase genes of the first highly pathogenic Avian Influenza H5N1 2.3.2.1c virus isolated from Sabah, Malaysia

@#Highly Pathogenic Avian Influenza (HPAI) is a highly contagious disease in poultry. The outbreaks can lead to flock mortality up to 100% in two to three days. In July 2018, high mortality in a commercial layer farm in Kauluan village, Sabah was reported. Samples were sent to Veterinary Research Institute Ipoh for diagnosis. Virus isolation and molecular detection is carried out simultaneously. The causative agent was then identified as AI H5N1 virus by real time reverse transcription-polymerase chain reaction (RT-PCR). The virus was then subjected for further nucleotide sequencing of full length hemagglutinin (HA) and neuraminidase (NA) gene. The PQRERRRKR/GLF motif at the HA cleavage site indicated that the isolate was of HPAI virus. Phylogenetic analysis of the HA gene showed that the isolate was belonged to the clade 2.3.2.1c virus. In the HA gene, besides the S133A substitution, the virus possesses conserved amino acid at most of the avian receptor binding sites including the glutamine (Q) and glycine (G) at position 222 and 224 respectively, indicating that the virus retains the avian-type receptor binding preference. As such, the zoonotic potential of the virus was relatively low. On the other hand, though the N154D and T156A substitution were detected in the same gene, the pandemic potential of this Sabah 2.3.2.1c virus is low in the absence of the Q222L, G224S, H103Y, N220K and T315I. A typical 20 amino acid deletion with loss of four corresponding glycosylation sites in the NA stalk region was visible. Though three NA resistance markers were detected, the virus was predicted to be sensitive to NA inhibitor. This is the first HPAI H5N1 outbreak in Sabah. The introduction of this virus into East Malaysia for the first time raised an alert alarm of the future epidemic potential. Strict farm biosecurity, continuous surveillance programme in poultry, wild birds, migratory birds; molecular epidemiology as well as risk assessment for the virus with pandemic potential are needed in dealing with emergence of new influenza virus in the country.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus / 生物工程学报

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

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.

Prevalence of influenza A(H1N1) viruses in Hangzhou after 2009 pandemic and characteristics in lineage changes / 中华微生物学和免疫学杂志

Objective:To study the epidemiological features of local influenza A(H1N1)pdm09 epidemic strains through analyzing the changes in lineages and to analyze how well the vaccine strains were matched to the circulating strains in Hangzhou.Methods:Of 1 112 clinical specimens from laboratory-confirmed A(H1N1)pdm09 infections in Hangzhou in consecutive seasons from 2009 to 2020, 208 (18.7%) with high viral load (Ct value <30) were randomly selected from 10 influenza epidemics for full-length hemagglutinin gene ( HA) gene sequencing. Genetic variation, evolution and lineage changes of these representative local strains were analyzed by comparison with vaccine strains and reference strains. Results:Since the 2009 pandemic, A(H1N1)pdm09 had become one of the predominant viruses causing seasonal influenza and been reported to co-circulate with influenza A(H3N2) and influenza B viruses in Hangzhou in the past decade. It caused 10 local influenza epidemics in the 12 consecutive seasons from 2009 to 2020. HA gene sequencing revealed complex sources and rapid variation of the local A(H1N1)pdm09 strains. The main epidemic strains often genetically drifted from the recommended northern hemisphere vaccine strains due to lineage changes. Conclusions:This study suggested that it was essential to update the recommended vaccine strains year by year. Besides, enhanced periodic monitoring of influenza A(H1N1)pdm09 strains circulating in the region was important for the prevention and control of influenza A(H1N1)pdm09 infection in the next epidemic season.

Preparation and identification of a broad-spectrum antibody targeting hemagglutinin protein of influenza A virus / 中华微生物学和免疫学杂志

Objective:To prepare and identify a broad-spectrum antibody FHA3 targeting influenza A virus hemagglutinin (HA).Methods:According to the single-chain antibody fragment (scFv) sequence, the heavy chain (VH) and light chain (VL) variable regions of FHA3 were amplified by PCR and a recombinant plasmid pFRT-IgG1κ-FHA3 was constructed by linking the expression vector pFRT-IgG1κ. FHA3 was expressed in the ExpiCHO system and purified by affinity purification. The binding activity of FHA3 to influenza A virus HA was detected by ELISA. The neutralizing activity of FHA3 was detected in vitro by infecting host cells with pseudovirus. Results:SDS-PAGE showed that high-purity FHA3 was obtained. FHA3 could bind to H1N1 HA, H2N2 HA, H3N2 HA, H5N1 HA, H7N9 HA and H9N2 HA in a concentration-dependent manner. FHA3 had good neutralizing activity in vitro that was it could effectively block the invasion of H5N1 and H7N9 pseudoviruses into target cells at a low concentration of 5 μg/ml and H1N1 pseudovirus at 0.012 5 μg/ml. Conclusions:A broad spectrum antibody targeting HA protein of influenza A virus with neutralizing activity in vitro was obtained.

Prokaryotic expression and serological analysis of the head domain of influenza A virus hemagglutinin / 中华微生物学和免疫学杂志

Objective:To express the head domain of influenza A virus hemagglutinin (HA) in a prokaryotic expression system and to evaluate its immunogenicity.Methods:The genes encoding the HA head domains of H1N1 and H3N2 influenza viruses were cloned into pET-22b(+ ) prokaryotic expression plasmid. After the induction with IPTG, the fusion proteins rH1N1-HA and rH3N2-HA containing HA head domain and His-tag were expressed and obtained from E. coli BL21. SDS-PAGE and Western blot was used to verify the expression of the recombinant proteins. Rabbits were immunized with multiple doses of the purified recombinant proteins to obtain polyclonal antibodies against the HA head domains of H1N1 and H3N2. The immunogenicity of the recombinant proteins was evaluated in BALB/c mice. Results:rH1N1-HA and rH3N2-HA induced protective antibodies (geometric mean titer ≥40) in mice and could be used as protective antigens. Polyclonal antibodies against rH1N1-HA and rH3N2-HA could be used as important materials for Western blot, ELISA and other immunological assays.Conclusions:The HA head domains prepared in this study could be used as protective antigens to induce protective antibodies in mice. Polyclonal antibodies against the HA head domains could be used for immunological and serological studies of influenza A viruses.