Human adenoviruses(HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3,-7,-4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respi...Human adenoviruses(HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3,-7,-4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population.Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies(NAb) against HAdV-3,-4,-7,-14,-55,and-11 in total 278 healthy populations between 0 months and 49 years of age(228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1–2, 3–5, and 6–17 years,respectively. The seroprevalence was very low(0% - 8.1%) for all other four types. In adults aged between 18 and49 years, HAdV-3,-4, and-7(> 50.00%) were the most common types, followed by HAdV-14(38.00%),-55(34.00%),and-11(24.00%). Adults tended to have high NAb titers against HAdV-4 and-55. HAdV-55-seropositive donors tended to be HAdV-11-and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14,-55,-11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.展开更多
Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome ...Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.展开更多
The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including in...The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.展开更多
Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous vir...Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.展开更多
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...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.展开更多
AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injecti...AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.·展开更多
The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were ...The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regar...Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.展开更多
Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therape...Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.展开更多
Human adenoviruses type 26(HAdV26)and type 35(HAdV35)have increasingly become the choice of adenovirus vectors for vaccine application.However,the population pre-existing immunity to these two adenoviruses in China,wh...Human adenoviruses type 26(HAdV26)and type 35(HAdV35)have increasingly become the choice of adenovirus vectors for vaccine application.However,the population pre-existing immunity to these two adenoviruses in China,which may reduce vaccine efficacy,remains largely unknown.Here,we established micro-neutralizing(MN)assays to investigate the seroprevalence of neutralizing antibodies(nAbs)against HAdV26 and HAdV35 in the general population of Guangdong and Shandong provinces,China.A total of 1184 serum samples were collected,47.0%and 15.8%of which showed HAdV26 and HAdV35 nAb activity,respectively.HAdV26-seropositive individuals tended to have more moderate nAbs titers(201-1000),while HAdV35-seropositive individuals appeared to have more low nAbs titers(72-200).The seropositive rates of HAdV26 and HAdV35 in individuals younger than 20 years old were very low.The seropositive rates of HAdV26 increased with age before 70 years old and decreased thereafter,while HAdV35 seropositive rates did not show similar characteristics.Notably,the seropositive rates and nAb levels of both HAdV26 and HAdV35 were higher in Guangdong Province than in Shandong Province,but did not exert significant differences between males and females.The seroprevalence between HAdV26 and HAdV35 showed little correlation,and no significant cross-neutralizing activity was detected.These results clarified the characteristics of the herd immunity against HAdV26 and HAdV35,and provided information for the rational development and application of HAdV26 and HAdV35 as vaccine vectors in China.展开更多
SARS-CoV-2 infection is a serious threat to human life and health all over the world,and COVID-19 is a global epidemic caused by SARS-CoV-2 infection.SARS-CoV-2 is highly infectious,strange and variable.Therefore,the ...SARS-CoV-2 infection is a serious threat to human life and health all over the world,and COVID-19 is a global epidemic caused by SARS-CoV-2 infection.SARS-CoV-2 is highly infectious,strange and variable.Therefore,the treatment of COVID-19 must be urgent and targeted.However,vaccines and currently used drugs generally do not have the above-mentioned characteristics.Although convalescent plasma of COVID-19 has shown a clinical application value in the emergency treatment of critical patients,it shows great limitations.All human recombinant multivalent neutralizing nano-antibodies may meet the deficiency of COVID-19 therapy.Gene engineering technologies have been used to develop specific neutralizing antibody(nAB)drugs for the treatment of COVID-19 worldwide.Some of the candidate nAB drugs have been entered the clinical trials and can be used for the therapy of COVID-19 shortly.In the present review,we studied and analyzed n ABs for the treatment of COVID-19 and the progress and prospect from the following five aspects:1)The biological and clinical characteristics of SARS-CoV-2 infection;2)The feasibility of plasma therapy for convalescents with COVID-19;3)The technical routes of developing n Ab drugs;4)The current status of developing global COVID-19 antibodies;5)The difficulties and clinical use.展开更多
Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious...Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.展开更多
New variants of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) appear rapidly every few months.They have showed powerful adaptive ability to circumvent the immune system. To further understand SARS-CoV-2...New variants of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) appear rapidly every few months.They have showed powerful adaptive ability to circumvent the immune system. To further understand SARS-CoV-2’s adaptability so as to seek for strategies to mitigate the emergence of new variants, herein we investigated the viral adaptation in the presence of broadly neutralizing antibodies and their combinations. First, we selected four broadly neutralizing antibodies, including pan-sarbecovirus and pan-betacoronavirus neutralizing antibodies that recognize distinct conserved regions on receptor-binding domain(RBD) or conserved stem-helix region on S2 subunit.Through binding competition analysis, we demonstrated that they were capable of simultaneously binding.Thereafter, a replication-competent vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein was employed to study the viral adaptation. Twenty consecutive passages of the virus under the selective pressure of individual antibodies or their combinations were performed. It was found that it was not hard for the virus to adapt to broadly neutralizing antibodies, even for pan-sarbecovirus and pan-betacoronavirus antibodies. The virus was more and more difficult to escape the combinations of two/three/four antibodies. In addition, mutations in the viral population revealed by high-throughput sequencing showed that under the selective pressure of three/four combinational antibodies, viral mutations were not prone to present in the highly conserved region across betacoronaviruses(stem-helix region), while this was not true under the selective pressure of single/two antibodies.Importantly, combining neutralizing antibodies targeting RBD conserved regions and stem helix synergistically prevented the emergence of escape mutations. These studies will guide future vaccine and therapeutic development efforts and provide a rationale for the design of RBD-stem helix tandem vaccine, which may help to impede the generation of novel variants.展开更多
From December 2022 to January 2023,the SARS-CoV-2 Omicron BA.5/BF.7 variant significantly impacted China’s mainland.While most COVID-19 patients experienced mild symptoms and were treated as outpatients or at home,so...From December 2022 to January 2023,the SARS-CoV-2 Omicron BA.5/BF.7 variant significantly impacted China’s mainland.While most COVID-19 patients experienced mild symptoms and were treated as outpatients or at home,some cases progressed to severe illness,necessitating hospitalization or even resulting in death.To better understand this outbreak and forecast future waves as SARS-CoV-2 continues to evolve,it is crucial to assess the titer of neutralizing antibodies(Nab)for evaluating the establishment of an immune barrier.In this study,we investigated the dynamic evolution of humoral immunity following the breakthrough infection wave driven by the SARS-CoV-2 Omicron BA.5/BF.7 variant in southwest China.展开更多
A study published in Nature'by Gonelli and colleagues reveals that potent,broadly neutralizing antibodies(bNAbs)delay viremic simian immunodeficiency virus(SIV)infection in rhesus macaques but do not fully prevent...A study published in Nature'by Gonelli and colleagues reveals that potent,broadly neutralizing antibodies(bNAbs)delay viremic simian immunodeficiency virus(SIV)infection in rhesus macaques but do not fully prevent subclinical infections.Despite bNAb concentrations being significantly higher than supposed protective thresholds,transient viral"blips"occurred,which suggests that bNAb prophylaxis can mask subclinical infections and has implications for the interpretation of HIV-1 prevention trials.展开更多
Snakebite envenoming has been designated by the World Health Organization(WHO)as a neglected tropical disease,responsible for over 100,000 deaths and more than 300,000 cases of permanent disability annually.The burden...Snakebite envenoming has been designated by the World Health Organization(WHO)as a neglected tropical disease,responsible for over 100,000 deaths and more than 300,000 cases of permanent disability annually.The burden is especially pronounced in Southeast Asia,sub-Saharan Africa,India,and Latin America1.Despite this,current clinical management still relies on polyclonal antivenoms derived from hyperimmunized animals—a century-old technology first introduced in the 1890s.These sera suffer from major limitations,including species specificity,high immunogenicity,protein impurity,short half-life,and cold-chain dependence,all of which hinder their global accessibility and therapeutic efficacy^(2,3).展开更多
Japanese encephalitis(JE),a vector-borne disease caused by the Japanese encephalitis virus(JEV),remains a major public health concern in South and Southeast Asia[1].JEV,a Flaviviridae family virus,is primarily transmi...Japanese encephalitis(JE),a vector-borne disease caused by the Japanese encephalitis virus(JEV),remains a major public health concern in South and Southeast Asia[1].JEV,a Flaviviridae family virus,is primarily transmitted by Culex mosquitoes,especially the Culex vishnui subgroup,which breeds extensively in rice fields[1].Birds(Ardeidae family)act as natural reservoirs,while pigs serve as amplifying hosts,and humans are incidental hosts[1].展开更多
Nipah virus(NiV)and Hendra virus(HeV)are highly pathogenic henipaviruses within the Paramyxoviridae family,causing severe respiratory and neurological diseases in humans and animals with fatality rates up to 75%,and n...Nipah virus(NiV)and Hendra virus(HeV)are highly pathogenic henipaviruses within the Paramyxoviridae family,causing severe respiratory and neurological diseases in humans and animals with fatality rates up to 75%,and no licensed human vaccines or therapeutics.In this study,we identified a unique vulnerable epitope on the NiV attachment glycoprotein(G)recognized by the potent neutralizing antibody 14F8,which targets a receptor-binding site and neutralizes NiV effectively.Using the 2.8Åcrystal structure of the 14F8 Fab–NiV-G complex as a guide,we reconstructed this epitope on HeV-G via a single amino acid substitution(S586N),creating the HeV-G_(S586N) mutant.Immunization with HeV-G_(S586N) in BALB/c mice and cynomolgus monkeys elicited robust,broadly neutralizing antibody responses against both NiV and HeV,achieving higher NiV-neutralizing titers post-prime compared to wild-type HeV-G,as confirmed by pseudovirus and live-virus assays.Crystal structures of HeV-G_(S586N)(3.3Å)and its 14F8 complex(3.2Å)showed the S586N substitution induced a 9Åconformational rearrangement inβ-propeller blade 6,reshaping the molecular skeleton and solvent-accessible surface without direct N586–14F8 interaction,thus mimicking the NiV epitope.These findings position HeV-G_(S586N) as a promising broad-spectrum antigen for henipavirus prevention and demonstrate the value of structure-guided epitope reconstruction in universal vaccine design for emerging viral threats.展开更多
Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),can induce 80–100%mortality in newborn piglets;therefore,specific and rapid detection methods are important for the prevention of this vi...Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),can induce 80–100%mortality in newborn piglets;therefore,specific and rapid detection methods are important for the prevention of this viral infection.In particular,methods for detecting neutralizing antibodies(nAbs)can be used to evaluate the immunization effect of PEDV vaccines.The spike protein of PEDV(PEDV-S)has been universally used as an antigen to develop immunoassays to detect nAbs.Nanobodies(Nbs)offer advantages such as ease of genetic engineering and low production costs,making them promising for diagnostic applications.In this study,PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels.A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase(HRP)in HEK293T cells.A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay(cELISA)to detect anti-PEDV nAbs.Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration.The optimal dilution of PEDV-S-Nb13-HRP was 1:200,and the optimal serum dilution was 1:10.The cutoff value of cELISA was determined as 28.1%,demonstrating high specificity,repeatability,stability,and good agreement rates with two commercial ELISA kits(93.6%)and a serum neutralization test(96.34%).Additionally,the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit.These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.展开更多
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neu...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2,we constructed a codon-optimized,full-length C-terminal mutant spike(S)gene of SARS-CoV-2.We generated a luciferase(Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone.The key parameters for this pseudovirus-based assay,including the S mutants and virus incubation time,were optimized.This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2(ACE2)-expressing 293T cells.Cathepsin(Cat)B/L inhibitor E64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells.Furthermore,the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1.Thus,we developed a pseudovirus-based assay for SARS-CoV-2,which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.展开更多
基金supported by the National Key Research and Development Program of China (2018YFC1200100)the Guangzhou Science and Technology Program Key Project, China (201803040004)+2 种基金the National Science and Technology Major Project of China (2017ZX10103011003, 2018ZX10102001)the National Natural Science Foundation of China, China (31570163)the Youth Project of State Key Laboratory of Respiratory Disease, China (SKLRD-QN-201713)。
文摘Human adenoviruses(HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3,-7,-4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population.Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies(NAb) against HAdV-3,-4,-7,-14,-55,and-11 in total 278 healthy populations between 0 months and 49 years of age(228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1–2, 3–5, and 6–17 years,respectively. The seroprevalence was very low(0% - 8.1%) for all other four types. In adults aged between 18 and49 years, HAdV-3,-4, and-7(> 50.00%) were the most common types, followed by HAdV-14(38.00%),-55(34.00%),and-11(24.00%). Adults tended to have high NAb titers against HAdV-4 and-55. HAdV-55-seropositive donors tended to be HAdV-11-and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14,-55,-11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.
基金support by National Key Technology R&D Program of China (2013BAD12B05)
文摘Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.
基金supported by the Science and Technology Basic Work Program (2013FY113500) from the Ministry of Science and Technology of Chinathe strategic priority research program of the Chinese Academy of Sciences (ZDRW-ZS2016-4)
文摘The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.
基金Inserm, France Université Louis Pasteur, France+3 种基金the European Union (Virgil Network of Excellence)the DeutscheForschungsgemeinschaft (Ba1417/11-1), Germanythe ANRchair of excellence program and ANRS, FranceInserm "PosteVert" research fellowship in the framework of Inserm EuropeanAssociated Laboratory Inserm U748-Department of Medicine Ⅱ,University of Freiburg, Germany
文摘Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.
文摘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.
文摘AIM:To determine the involvement of the transforming growth factor(TGF)-β with the development of experimental subretinal fibrosis in a mouse model.· METHODS:Subretinal fibrosis was induced by subretinal injection of macrophage-rich peritoneal exudate cells(PECs) and the local expression of TGF-β isoforms was assessed by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA) at various time points.In addition,we investigated the effect of TFG-β-neutralizing antibodies(TGF-β NAb) on subretinal fibrosis development.· RESULTS:TGF-β1 and TGF-β2 mRNA level was significantly elevated at day 2 after subretinal fibrosis induction and increased further to 5 and 6.5-fold respectively at day 5,reaching the peak.TGF-β3 mRNA was not detected in the present study.The result of ELSIA showed that active TGF-β1 and TGF-β2 levels were upregulated to 10-fold approximately,while total TGF-β1 and TGF-β2 levels were even upregulated more than 10-fold and more than 20-fold respectively in subretinal fibrosis mice in comparison with na?觙ve mice at day 5.TGF-β NAb resulted in a reduced subretinal fibrosis areas by 65% compared to animals from control group at day 7.· CONCLUSION:Our results indicate that TGF-β signaling may contribute to the pathogenesis of subretinal fibrogenesis and TGF-β inhibition may provide an effective,novel treatment of advanced and late-stage neovascular age-related macular degeneration.·
文摘The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.
基金supported by the Major Program of National Natural Science Foundation of China (31490603, 31572549)the National Key Technology R & D Program of China (2015BAD12B01-2)
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)is characterized by its genetic variation and limited cross protection among heterologous strains.Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies(NAs)against PRRSV,the mechanism underlying limited cross-neutralization among heterologous strains is still controversial.In the present study,examinations of NA cross reaction between a highly pathogenic PRRSV(HP-PRRSV)strain,JXwn06,and a low pathogenic PRRSV(LP-PRRSV)strain,HB-1/3.9,were conducted with viral neutralization assays in MARC-145 cells.None of the JXwn06-hyperimmuned pigs’sera could neutralize HB-1/3.9 in vitro and vice versa.To address the genetic variation between these two viruses that are associated with limited crossneutralization,chimeric viruses with coding regions swapped between these two strains were constructed.Viral neutralization assays indicated that variations in nonstructural protein 2(nsp2)and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9.Furthermore,we substituted the nsp2-,glycoprotein2(GP2)-,GP3-,and GP4-coding regions together,or nsp2-,GP5-,and membrane(M)protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses.The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells.Taken together,these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.
基金supported by the National Natural Science Foundation of China(Nos.31970130,31600672,31670831,and 31370813).
文摘Coronavirus disease 2019 is threatening thousands of millions of people around the world.In the absence of specific and highly effective medicines,the treatment of infected persons is still very challenging.As therapeutics,neutralizing antibodies(NAbs)have great potential.Many NAbs have been reported,and most target various regions on the receptor-binding domain of the spike(S)protein,or the N-terminal domain.Several NAbs and NAb cocktails have been authorized for emergency use,and more arc in clinical trials or are under development.In this review,considering the angle of binding epitopes on the S protein,we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies.In addition,we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29050701)the Emergency Key Program of Guangzhou (EKPG21-20)+2 种基金the China Evergrande Group funding for SARS-Co V-2 (2020GIRHHMS22)the Zhongnanshan Medical Foundation of Guangdong Province (ZNSA-2022009)the China Postdoctoral Science Foundation (2020M682942)
文摘Human adenoviruses type 26(HAdV26)and type 35(HAdV35)have increasingly become the choice of adenovirus vectors for vaccine application.However,the population pre-existing immunity to these two adenoviruses in China,which may reduce vaccine efficacy,remains largely unknown.Here,we established micro-neutralizing(MN)assays to investigate the seroprevalence of neutralizing antibodies(nAbs)against HAdV26 and HAdV35 in the general population of Guangdong and Shandong provinces,China.A total of 1184 serum samples were collected,47.0%and 15.8%of which showed HAdV26 and HAdV35 nAb activity,respectively.HAdV26-seropositive individuals tended to have more moderate nAbs titers(201-1000),while HAdV35-seropositive individuals appeared to have more low nAbs titers(72-200).The seropositive rates of HAdV26 and HAdV35 in individuals younger than 20 years old were very low.The seropositive rates of HAdV26 increased with age before 70 years old and decreased thereafter,while HAdV35 seropositive rates did not show similar characteristics.Notably,the seropositive rates and nAb levels of both HAdV26 and HAdV35 were higher in Guangdong Province than in Shandong Province,but did not exert significant differences between males and females.The seroprevalence between HAdV26 and HAdV35 showed little correlation,and no significant cross-neutralizing activity was detected.These results clarified the characteristics of the herd immunity against HAdV26 and HAdV35,and provided information for the rational development and application of HAdV26 and HAdV35 as vaccine vectors in China.
基金China Postdoctoral Science Foundation(Grant No.2020T1300011ZX)National Science and Technology Major Project(Grant No.2018ZX10712001)Key Research and Development Projects of Tianjin Science and Technology Committee(Grant No.17YFZCSY00660)。
文摘SARS-CoV-2 infection is a serious threat to human life and health all over the world,and COVID-19 is a global epidemic caused by SARS-CoV-2 infection.SARS-CoV-2 is highly infectious,strange and variable.Therefore,the treatment of COVID-19 must be urgent and targeted.However,vaccines and currently used drugs generally do not have the above-mentioned characteristics.Although convalescent plasma of COVID-19 has shown a clinical application value in the emergency treatment of critical patients,it shows great limitations.All human recombinant multivalent neutralizing nano-antibodies may meet the deficiency of COVID-19 therapy.Gene engineering technologies have been used to develop specific neutralizing antibody(nAB)drugs for the treatment of COVID-19 worldwide.Some of the candidate nAB drugs have been entered the clinical trials and can be used for the therapy of COVID-19 shortly.In the present review,we studied and analyzed n ABs for the treatment of COVID-19 and the progress and prospect from the following five aspects:1)The biological and clinical characteristics of SARS-CoV-2 infection;2)The feasibility of plasma therapy for convalescents with COVID-19;3)The technical routes of developing n Ab drugs;4)The current status of developing global COVID-19 antibodies;5)The difficulties and clinical use.
基金Supported by the National Natural Science Foundation of China(No.20872048)
文摘Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.
基金funded by the National Natural Science Foundation of China(81773621,82073751 to J.Z.)the National Science and Technology Major Project“Key New Drug Creation and Manufacturing Program”of China(No.2019ZX09732001-019 to J.Z.)+1 种基金the Key R&D Supporting Program(Special Support for Developing Medicine for Infectious Diseases)from the Administration of Chinese and Singapore Tianjin Eco-city to Jecho Biopharmaceuticals Ltd.Co.the Shanghai Jiao Tong University“Crossing Medical and Engineering”grant(20X190020003 to J.Z.)
文摘New variants of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) appear rapidly every few months.They have showed powerful adaptive ability to circumvent the immune system. To further understand SARS-CoV-2’s adaptability so as to seek for strategies to mitigate the emergence of new variants, herein we investigated the viral adaptation in the presence of broadly neutralizing antibodies and their combinations. First, we selected four broadly neutralizing antibodies, including pan-sarbecovirus and pan-betacoronavirus neutralizing antibodies that recognize distinct conserved regions on receptor-binding domain(RBD) or conserved stem-helix region on S2 subunit.Through binding competition analysis, we demonstrated that they were capable of simultaneously binding.Thereafter, a replication-competent vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein was employed to study the viral adaptation. Twenty consecutive passages of the virus under the selective pressure of individual antibodies or their combinations were performed. It was found that it was not hard for the virus to adapt to broadly neutralizing antibodies, even for pan-sarbecovirus and pan-betacoronavirus antibodies. The virus was more and more difficult to escape the combinations of two/three/four antibodies. In addition, mutations in the viral population revealed by high-throughput sequencing showed that under the selective pressure of three/four combinational antibodies, viral mutations were not prone to present in the highly conserved region across betacoronaviruses(stem-helix region), while this was not true under the selective pressure of single/two antibodies.Importantly, combining neutralizing antibodies targeting RBD conserved regions and stem helix synergistically prevented the emergence of escape mutations. These studies will guide future vaccine and therapeutic development efforts and provide a rationale for the design of RBD-stem helix tandem vaccine, which may help to impede the generation of novel variants.
基金supported by National Science and Technology Major Project of the Ministry of Science and Technology of China(2024ZD0523902)the National Natural Science Foundation of China(81790643,82121003,82103986)+5 种基金the CAMS Innovation Fund for Medical Sciences(2019-I2M-5-032)the Sichuan Science and Technology Program(2025ZNSFSC0793,2022YFS0002)Sichuan Medical Association(Q2024079)the Health Department of Sichuan Province(2022-214,21PJ081,S21010)Chengdu Science and Technology Bureau(2022-YF05-01644-SN)the Sichuan Provincial People’s Hospital(2022QN41).
文摘From December 2022 to January 2023,the SARS-CoV-2 Omicron BA.5/BF.7 variant significantly impacted China’s mainland.While most COVID-19 patients experienced mild symptoms and were treated as outpatients or at home,some cases progressed to severe illness,necessitating hospitalization or even resulting in death.To better understand this outbreak and forecast future waves as SARS-CoV-2 continues to evolve,it is crucial to assess the titer of neutralizing antibodies(Nab)for evaluating the establishment of an immune barrier.In this study,we investigated the dynamic evolution of humoral immunity following the breakthrough infection wave driven by the SARS-CoV-2 Omicron BA.5/BF.7 variant in southwest China.
文摘A study published in Nature'by Gonelli and colleagues reveals that potent,broadly neutralizing antibodies(bNAbs)delay viremic simian immunodeficiency virus(SIV)infection in rhesus macaques but do not fully prevent subclinical infections.Despite bNAb concentrations being significantly higher than supposed protective thresholds,transient viral"blips"occurred,which suggests that bNAb prophylaxis can mask subclinical infections and has implications for the interpretation of HIV-1 prevention trials.
文摘Snakebite envenoming has been designated by the World Health Organization(WHO)as a neglected tropical disease,responsible for over 100,000 deaths and more than 300,000 cases of permanent disability annually.The burden is especially pronounced in Southeast Asia,sub-Saharan Africa,India,and Latin America1.Despite this,current clinical management still relies on polyclonal antivenoms derived from hyperimmunized animals—a century-old technology first introduced in the 1890s.These sera suffer from major limitations,including species specificity,high immunogenicity,protein impurity,short half-life,and cold-chain dependence,all of which hinder their global accessibility and therapeutic efficacy^(2,3).
文摘Japanese encephalitis(JE),a vector-borne disease caused by the Japanese encephalitis virus(JEV),remains a major public health concern in South and Southeast Asia[1].JEV,a Flaviviridae family virus,is primarily transmitted by Culex mosquitoes,especially the Culex vishnui subgroup,which breeds extensively in rice fields[1].Birds(Ardeidae family)act as natural reservoirs,while pigs serve as amplifying hosts,and humans are incidental hosts[1].
基金funding support from the National Natural Science Foundation of China(32200762)the Defense Industrial Technology Development Program(JCKY2020802B001).
文摘Nipah virus(NiV)and Hendra virus(HeV)are highly pathogenic henipaviruses within the Paramyxoviridae family,causing severe respiratory and neurological diseases in humans and animals with fatality rates up to 75%,and no licensed human vaccines or therapeutics.In this study,we identified a unique vulnerable epitope on the NiV attachment glycoprotein(G)recognized by the potent neutralizing antibody 14F8,which targets a receptor-binding site and neutralizes NiV effectively.Using the 2.8Åcrystal structure of the 14F8 Fab–NiV-G complex as a guide,we reconstructed this epitope on HeV-G via a single amino acid substitution(S586N),creating the HeV-G_(S586N) mutant.Immunization with HeV-G_(S586N) in BALB/c mice and cynomolgus monkeys elicited robust,broadly neutralizing antibody responses against both NiV and HeV,achieving higher NiV-neutralizing titers post-prime compared to wild-type HeV-G,as confirmed by pseudovirus and live-virus assays.Crystal structures of HeV-G_(S586N)(3.3Å)and its 14F8 complex(3.2Å)showed the S586N substitution induced a 9Åconformational rearrangement inβ-propeller blade 6,reshaping the molecular skeleton and solvent-accessible surface without direct N586–14F8 interaction,thus mimicking the NiV epitope.These findings position HeV-G_(S586N) as a promising broad-spectrum antigen for henipavirus prevention and demonstrate the value of structure-guided epitope reconstruction in universal vaccine design for emerging viral threats.
基金funded by grants from the National Key R&D Program of China(2023YFD1800304)the National Natural Science Foundation of China to QZ(32273041)+1 种基金the Natural Science Foundation of Shaanxi Province of China(2022JC-12)the Central Public interest Scientific Institution Basal Research Fund,National Data Center of Animal Health.
文摘Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),can induce 80–100%mortality in newborn piglets;therefore,specific and rapid detection methods are important for the prevention of this viral infection.In particular,methods for detecting neutralizing antibodies(nAbs)can be used to evaluate the immunization effect of PEDV vaccines.The spike protein of PEDV(PEDV-S)has been universally used as an antigen to develop immunoassays to detect nAbs.Nanobodies(Nbs)offer advantages such as ease of genetic engineering and low production costs,making them promising for diagnostic applications.In this study,PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels.A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase(HRP)in HEK293T cells.A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay(cELISA)to detect anti-PEDV nAbs.Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration.The optimal dilution of PEDV-S-Nb13-HRP was 1:200,and the optimal serum dilution was 1:10.The cutoff value of cELISA was determined as 28.1%,demonstrating high specificity,repeatability,stability,and good agreement rates with two commercial ELISA kits(93.6%)and a serum neutralization test(96.34%).Additionally,the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit.These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.
基金This work was supported by the Emergency Project from the Science&Technology Commission of Chongqing(cstc2020jscx-fyzx0053)a Major National Science&Technology Program grant(2017ZX10202203)from the Science&Technology Commission of China,the Leading Talent Program of CQ CSTC(CSTCCXLJRC201719)+1 种基金the Scientific Research Innovation Project for Postgraduate in Chongqing(CYB19168)the Emergency Project for Novel Coronavirus Pneumonia from the Chongqing Medical University(CQMUNCP0302).
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2,we constructed a codon-optimized,full-length C-terminal mutant spike(S)gene of SARS-CoV-2.We generated a luciferase(Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone.The key parameters for this pseudovirus-based assay,including the S mutants and virus incubation time,were optimized.This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2(ACE2)-expressing 293T cells.Cathepsin(Cat)B/L inhibitor E64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells.Furthermore,the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1.Thus,we developed a pseudovirus-based assay for SARS-CoV-2,which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.
