A review of the safety and efficacy of current COVID-19 vaccines / 医学前沿

Vaccination is the most effective and feasible way to contain the Coronavirus disease 2019 (COVID-19) pandemic. The rapid development of effective COVID-19 vaccines is an extraordinary achievement. This study reviewed the efficacy/effectiveness, immunogenicity, and safety profile of the 12 most progressed COVID-19 vaccines and discussed the challenges and prospects of the vaccine-based approaches in a global crisis. Overall, most of the current vaccines have shown safety and efficacy/effectiveness during actual clinical trials or in the real-world studies, indicating a development of pandemic control. However, many challenges are faced by pandemic control in terms of maximizing the effect of vaccines, such as rapid vaccine coverage, strategies to address variants with immune escape capability, and surveillance of vaccine safety in the medium- and long-terms.

Progress in vaccine development based on baculovirus expression vector system / 生物工程学报

Baculovirus expression vector system (BEVS) has been successfully applied to the over-expression of various proteins, thus providing sufficient materials for vaccine research. Compared to other systems, BEVS has many advantages: baculovirus solely being parasitic in invertebrates, the resultant products conferring high safety to mammalian, high expression level of recombinant proteins, preferable folding for eukaryotic protein, proper post-translational modification required for biological function, suitable for multiple genes co-expression and large-scale production with serum-free culture media. To better understand the advantages and prospective of BEVS for the vaccine research, this article will review the development of BEVS and its application on vaccine research.

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay / 中华预防医学杂志

Objective@#To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.@*Methods@#HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.@*Results@#The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05).@*Conclusion@#We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

Progress of bispecific antibody in cancer treatment / 肿瘤研究与临床

Bispecific antibodies can target two different targets simultaneously with a wide application prospect and rapid development in tumor treatment. The main function is to recruit effector cells selectively in order to kill tumor cells or bind tumor-associated growth factor receptor to inhibit cell proliferation. This paper reviews the current situation of bispecific antibodies in design, mechanism of action and the research progress in the clinical cancer treatment.

Preparation and identification of antibodies against respiratory syncytial virus nucleoprotein / 中华微生物学和免疫学杂志

Objective To prepare and identify the monoclonal antibodies against respiratory syn-cytial virus nucleoprotein(RSV N protein). Methods A prokaryotic expression system was used to express recombinant RSV N protein in Escherichia coli (E.coli). BALB/c mice were immunized with the recombi-nant N protein after purification. Monoclonal antibodies (McAbs) against the N protein were sorted from these BALB/c mice and then were further characterized by Western blot, ELISA and immunofluorescence. Furthermore,this study used orthogonal experiment to identify McAbs pairs,which could be used for diagno-sis. Results This study succeeded in obtaining 24 hybridoma cells that stably secreted monoclonal antibod-ies against RSV N protein. These antibodies showed good reactivity in ELISA,of which eight had strong spe-cificity in Western blot and 13 could be used in immunofluorescence. This study obtained two McAbs pairs (12F2/11H8-HRP and 12F2/15A8-HRP) that could be used in RSV detection. Conclusion This study succeeded in screening and preparation of McAbs against RSV N protein and obtaining two potential McAbs pairs for rapid detection of RSV.

Expression and self-assembly of HIV-1 CAP2NC protein / 生物工程学报

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

The potential use of serum HBV RNA to guide the functional cure of chronic hepatitis B / 中华肝脏病杂志

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in infected hepatocytes is the main cause of off-therapy viral rebound. The half-life of cccDNA is only 33-50 days, so the conversion of newly synthesized rcDNA to cccDNA in the nucleus is essential for the maintenance of cccDNA pool in infected hepatocytes. Though not directly targeting the existing cccDNA, current nucleos(t)ide analogues (NAs) may exhaust the cccDNA reservoir by blocking the rcDNA formation. Indeed, a prolonged consolidation therapy post loss of serum HBV DNA can achieve sustained remission and thus safe drug discontinuation in a small proportion of chronic hepatitis B (CHB) patients. In recent studies, we and others have demonstrated that it is the serum HBV RNA that reflects the cccDNA activity in infected hepatocytes, particularly among the patients on NAs. Here we suggest that instead of measuring serum HBV DNA only, simultaneous measurement of both viral DNA and RNA would improve the accuracy to reflect the cccDNA activity; therefore, the virological response should be redefined as consistent loss (less than the lower limit of detection) of both serum HBV DNA and RNA, which indicates the safety of drug discontinuation. Accumulating evidence has suggested that for the CHB patients with lower serum HBsAg, switch-to or add-on pegylated interferon (Peg-IFN) treatment would result in loss of serum HBsAg in a relatively large proportion of CHB patients. Since serum HBV RNA is an ideal biomarker to reflect the intrahepatic cccDNA activity, for the patients with a serum HBsAg level lower than 1 500 IU/ml after long-term NAs treatment, the serum HBV RNA should be measured. If serum HBV RNA is detected, peg-IFN should be added on; if serum HBV RNA is not detected, NAs treatment should be switched to peg-IFN treatment. We believe the therapy based on serum HBV RNA would make the functional cure of CHB (serum HBsAg loss or even conversion to anti-HBs) more efficient.

Preparation of monoclonal antibodies against 3D protein of EV71 based on HBc particles as expression vector / 中国免疫学杂志

Objective:To prepare and preliminarily identify the monoclonal antibodies(mAbs) specifically against 3D protein of Enterovirus 71(EV71),using bioinformatics to predict the epitopes of 3D,with HBc protein as a carrier.Methods: Artificial screening of 3D protein epitope sequences by bioinformatic method,inserted into the major immunodominant region(MIR) area of Hepatitis B virus core protein(HBc),to construct the recombinant protein.BALB/c mice were immunized with the recombinant virus like particles(VLPs),to prepare the mAbs against 3D protein of EV71.Affinity chromatography technology was used to purify the mAb.The indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry staining methods were used to identify the characteristic of the mAb.Results: We displayed 3D(aa34-43),3D(aa61-76) and 3D(aa151-164) epitopes by constructing fusion protein using HBc VLPs as a vector,after hybridization,one positive hybridoma cell line(3E1) was selected by ELISA.The isotype of 3E1 was IgG2a.The results of immunofluorescence and immunohistochemistry staining assay showed that the mAb 3E1 could specifically recognize EV71.Conclusion: The prepared mAb 3E1 can specifically recognizes the EV71,which laid the foundation for the detection of virus and further study on 3D protein,and verified the bioinformatics technology combined with HBc carrier displaying peptides could prepare mAb quickly and efficiently.

Clinical significance of quantitative level of hepatitis B core antibody / 中华肝脏病杂志

Hepatitis B core antibody (anti-HBc) targets viral core protein and is produced in patients with hepatitis B virus (HBV) infection, and seroconversion occurs in the early stage of infection and often lasts for a lifetime. Qualitative detection of anti-HBc has been used in clinical practice for many years, while the clinical significance of its quantitative level remains unclear. A novel anti-HBc immunoassay based on double-antigen sandwich ELISA has been developed in recent years and lays a foundation for illustrating the change in the quantitative level of anti-HBc (qAnti-HBc) in HBV infection and its clinical significance. Several recent studies have revealed that qAnti-HBc is associated with the degree of hepatitis activity and response to pharmacotherapy and may become an important basis for selecting antiviral drugs, optimizing therapeutic regimen, and predicting treatment outcome.

Review for progress and current status of human papillomavirus prophylactic vaccine / 中国生化药物杂志

Persistant infection of high-risk human papillomavirus (HPV) is the primary cause leading to cervical cancer, which is ranked as second cancer threatening the health of women following breast cancer.Development of HPV vaccine is very important because there is no effective therapeutics for cervical cancer.Three currently licensed HPV vaccines based on major capsid protein L1 in the foreign market confered good safety and efficacy in clinical trials, but the current price is expensive due to high cost, which limits the wide application in developing countries.So far, the vaccines have not been launced in China market.Here, we review the progress and the current status of the HPV vaccine, which will attract the readers’ interest on the forthcoming emergence of HPV vaccine in China.

Development of monoclonal antibodies against PD-L1 and preliminary investigation on potential application in treatment of chronic hepatitis B virus in-fection / 中国免疫学杂志

Objective:To get specific monoclonal antibodies ( mAbs) against PD-L1 which can block PD-1/PD-L1 binding, and explore the feasibility of its application on the treatment of chronic HBV infection preliminarily by in vitro and in vivo model. Methods:E. coli expression and series chromatography purification system were employed to get human and mouse PD-1/PD-L1 that had binding activity in vitro. By immunizing BALB/c mouse with purified recombination proteins of PD-L1,mAb hybridoma cell lines against PD-L1 were obtained. The reactivity with human/mice PD-L1 of individual antibody and the interaction blocking activity of the mAbs to PD-1/PD-L1 in vitro were examined by indirect chemiluminescence immune assay. Results: 8 cell lines against PD-L1 were obtained and 2 Anti-PDL1 mAbs (Ab5 &Ab6) performed strong immune activity to human/mice PD-L1 and blocking activity to PD-1/PD-L1. In the PBMC stimulation experiment of chronic HBV patient,Ab5 and Ab6 could promote theγ-IFN levels. With HBV in-fecting mice model,intravenous injections of these mAbs induced dramatically decrease of HBV DNA copies about 20 times, HBsAg levels in serum reduced to 30% of the baseline level. Conclusion:We obtained 2 PD-L1 mAbs with the reactivity to human/mice PD-L1 and blocking activity to PD-1/PD-L1. The 2 mAbs can promote T cell function in PBMC stimulation culture of chronic HBV patient, have significant antiviral effect in HBV transgenic mice and can be candidates for immunotherapy applications.

Monoclonal antibodies against HPV11 virus-like particles：functional characteristics and application on quality assessment / 中国生化药物杂志

Objective To quantitatively analyze the characteristics of a panel of murine anti-human papillomavirus(HPV)11 L1-derived virus-like particle( VLP ) monoclonal antibodies ( mAbs ) and establish the mAb-based methods for antigen quality analysis.Methods A panel of 22 murine anti-HPV11 mAbs were characterized in details with their isotype, and binding affinity, conformational sensitivity were examined quantitatively in the direct binding ELISA and Western blot.The hemagglutination inhibition activity of mAbs were identified using the hemagglutination inhibition assay and the pseudovirus ( PsV ) neutralization efficiency were examined quantitatively using the PsV-based neutralization assay.The type-specific, highly conformational sensitive and neutralizing mAbs were selected to be used in the sandwich ELISA assay.Results Based on the quantitative and semi-quantitative results, six type-specific, highly conformational sensitive and neutralizing mAbs (2A2, 4A1-3, 16G7, 14A6, 9C1 and 19C7) were identified.These mAbs, along with 10D6 were screened as the capture mAb or as the detection mAb in the sandwich ELISA.Conclusion The binding affinity, conformational sensitivity and neutralization efficiency of anti-HPV11 mAbs were characterized in details.A mAb-based sandwich ELISA assay (14A6:Ag:9C12-HRP) were developed, which could be used in the in vitro potency analysis of HPV11 VLP-based vaccine.

Chronic Hepatitis E Virus Infection and Treatment in Organ Transplant Recipients / 病毒学报

Hepatitis E, caused by hepatitis E virus (HEV) infection, usually leads to an acute clinical course, and is the most common diagnosis among cases of acute viral hepatitis. From 2008, there have been increasing reports of chronic HEV infection in immunocompromised patients such as organ transplant recipients. Without intervention with antiviral treatment, approximately 60% of HEV infections in organ transplant recipients evolve into chronic HEV infections. Of these chronic hepatitis E patients, 10% may develop liver fibrosis and progress to liver cirrhosis. This article reviews chronic HEV infection and treatment in organ transplant recipients.

The establishment of high-throughput neutralization titer evaluation model for hepatitis E virus (HEV) / 病毒学报

The lack of effective in vitro infection model for hepatitis E virus (HEV) has greatly hindered the quantitative analysis of neutralizing titers of anti-HEV antibodies and human sera, thus impeding further studies of HEV-stimulated antibody responses and the immunological mechanisms. In order to improve this situation, the infection of HepG2 cells that are inefficient for HEV replication was continuously monitored until the viral load reached the limit of detection on day 13, the results of which confirmed the feasibility of using this cell line to establish the infection model. Then, neutralization assays of five anti-HEV murine monoclonal antibodies and serum samples collected from four HEV vaccine recipients (collected before and after vaccination) were performed by 96 multi-channel parallel infections, nucleic acid extraction, and qPCR. The results showed that the cell model can be applied for quantitative evaluation of the neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients. In this study, we have successfully established a high-throughput in vitro HEV replication model, which will prove to be useful for the evaluation of HEV vaccines and studies of HEV epitopes.

Preparation of monoclonal antibodies against neutrophil gelatinase-associated lipocalin (NGAL) and development of an antibody-based chemiluminescence immune quantification assay / 中国生化药物杂志

Objective To obtain monoclonal antibodies ( mAbs ) against neutrophil gelatinase-associated lipocalin ( NGAL ) and a chemiluminescense immune quantification assay based one paired mAbs.Methods Six-to-eight weeks old female BALB/c mice were immunized with the purified recombinant human NGAL antigen( rhNGAL) that was produced by the Escherichia coil expression system.The spleen was fused with hybridoma for screening anti-NGAL monoclonal antibodies by indirect ELISA.Western blot was implemented to identify the reactivity with native NGAL. Results The rhNGAL antigen was found to form disulfide cross-linked dimers and present excellent immunogenicity.The reaction titer of the immune serum of NGAL immunized mice was about 106.Thirty mAbs were screened by indirect ELISA, hereinto;the EC50 values of mAb23C12 and 38D10 were 0.034 g/mL, 0.022 g/mL respectively.The antibodies pair, 38D10/23C12-SAE labeled with AcridiniumEster(AE), were shown to work well in chemiluminescense immune response quantitative detection which was screened by NGAL standardand clinical urine samples.This detection can resolve positive and negative samples with a statistically significant difference (P<0.0001).And the correlation coefficient R2between NGAL quantitative results and that of the Abbott's NGAL chemiluminescence immune assay kit was greater than 0.97.The detection linear range was 10-1500 ng/mL, analytical sensitivity of the method was 0.63 ng/mL.Conclusion Highly purified rhNGAL antigen and specific anti-NGAL monoclonal antibodies are generated in this study.The detection capability of method is comparable with that of the international commercial kit.

Establishment of human papillomavirus pseudovirion infection model in mouse for potency evaluation of HPV16 VLP Vaccine / 中国生化药物杂志

Objective To establish a mouse model of genital human papillomavirus (HPV) pseudovirion (PsV) transmission and evaluate the protective potency of HPV16 VLP vaccine.Methods HPV16 PsV with the encapsidated luciferase expressing plasmid Luc were generated from 293FT cells and purified by size-exclusion chromatography.The endpoint titers of HPV16 PsV-Luc were determined on 293FT cells, denoted as TRLU/mL.For in vivo genital challenge, mice were synchronized in a diestrus-like status by a subcutaneous injection with 0.1 μg β-estradiol and then with 3mg DepoProvera after 24 hours.Six hours prior to HPV16 PsV-Luc challenge, deeply anesthetized mice were intravaginally pretreated with 50 μL of 4%nonoxynol-9 ( N-9 ).HPV16 PsV-Luc was thoroughly mixed with 20 μL solution containing 4%carboxymethylcellulose ( CMC ) and intravaginally instilled using a positive-displacement pipette.Forty-eight hours after PsV-Luc challenge, mice were anesthetized and D luciferin was intravaginally instilled.After 3 minites, bioluminescence was measured with a cryogenically cooled Xenogen IVIS camera system.Then,the murine genital challenge model was used to determine the potency that HPV16 VLP vaccine is efficient at preventing HPV infection.Results HPV16 PsV-Luc was generated and purified from 293FT cells.HPV16 PsV-Luc was verified to containe L1 and L2 protein by Western blot.HPV 16 PsV-Luc successfully infected vaginal epithelial cells of mouse and the murine genital challenge model was established.To achieve consistent bioluminescence, the minimal dose of HPV16 PsV-Luc was 1.7 ×104 TRLU.The protective potency of HPV16 VLP vaccine was shown using this murine model.Our data showed that immune serum with over neutralizing antibody titer of 256-fold was sufficient to confer protection against HPV PsV genital infection .Conclusion The murine genital challenge model of HPV16 was successfully established, and the model is used to evaluate the potency of HPV16 VLP vaccine against in vivo genital HPV16 PsV challenge.Our model will benefit for the investigation of HPV neutralization and the potency evaluation of the HPV vaccine .

The laboratory diagnosis of viral hepatitis in the era of antiviral therapy / 中华检验医学杂志

In recent years, great achievements have been made in the therapy of viral hepatitis B and viral hepatitis C, as majority of hepatitis C patients can be clinically cured.Though current antiviral therapy is still unable to eradicate hepatitis B virus in infected hepatocyte and few patients could achieve HBsAg loss or seroconversion, end-stage liver disease like cirrhosis, liver failure and hepatocellular carcinoma have been dramatically prevented.The advances in treatments have prompted the progress in laboratory diagnosis of viral hepatitis.Here we review the progress in the field.

Construction and in vitro antiviral activity of highly effective artificial miRNA targeting to HIV-1 pol / 生物工程学报

RNA interference (RNAi) has exhibited huge potentials on anti-HIV-1 therapy research. The obtainment of RNAi element targeting to HIV-1 highly effectively and specifically was crucial for relevant research. Recent reports had described that microRNAs (miRNAs) posses more characteristics of inhibition and expression mechanisms than small interfering RNAs (siRNAs). In this study we explored the construction of artificial miRNA targeting to HIV-1 effectively and specifically. Sixteen siRNAs sequences were selected based on the conserved regions in the HIV-1 pol gene. ShRNA expression vectors were co-transfected with HIV-1 clone pNL4-3 to evaluate the abilities of siRNAs to inhibit HIV-1 expression. The pol1026 sequence was selected from candidates. The target sequence in the stem-loop structure of the well-characterized native miR-30a was replaced with pol1026 sequences, and the artificial miRNA expression vectors were co-transfected with the HIV-1 clone pNL4-3, results showed that HIV-1 can be effectively inhibited by miR-1026E. Target specificity of miR-1026E was confirmed by co-transfection assay with reporter plasmids containing different target sequences. The miR-1026E expression element was then inserted into Lentivirus which was used as a vector to transduce the MT-4 cells, MT-4-miR1026E expressing miR-1026E stably was cloned from transduced cells. The MT-4-miR1026E cell effectively inhibited HIV-1 replication in vitro. And the intracellular miR-181 and miR-16 expression levels and statl mRNA levels were not affected by the expression of miR-1026E in MT-4-miR1026E cells. miR-1026E is a promising candidate for future research.

The research of genotype 4 hepatitis E virus(HEV) capsid recombinant protein and the construction of cellular model for the investigation of viral infection early period / 中华微生物学和免疫学杂志

Objective To express the recombinant caspid of genotype 4 hepatitis E virus(HEV) ORF2. Methods HEV recombinant capsid protein D66 was expressed in E. coli, using the ORF2 fragment (aa368-606, obtained from swine bile) of genotype 4 HEV. Results The recombinant capsid proteins D66 self-assemble to be particle with a radius of 13 nm through dimeric form in neutral solution. Coated particles reacted well with sera obtained from patients during acute or recovered phase of HEV infection. Immunofluorescence and immnoblot assay suggested that D66 bound and penetrated HepG2 cell lines, and the process of attachment was blocked by sera collected from patients during acute or recovered phase of HEV infection.Conclusion Recombinant D66 particles simulate the structure at the surface of genotype 4 HEV well and specifically adhere and penetrate the host cells, which lays the foundation for the investigation of the molecular mechanism of genotype 4 HEV infection.