目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫...目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫PBS作为对照,每组10只。初次免疫后第7、14、21、28、35和42天采集血清,间接ELISA法检测血清中针对Delta株病毒S和N蛋白的结合抗体效价,微量中和试验检测针对Delta株病毒的中和抗体效价。初次免疫后第42天,取小鼠脾脏,进行Elispot检测,评价细胞免疫水平。结果初次免疫后第7天即可检测到S蛋白结合抗体,加强免疫后抗体效价进一步升高,至第21天抗体几何平均滴度(geometric mean titer,GMT)为89144;而初次免疫后N蛋白结合抗体水平较低,加强免疫后迅速升高,与S蛋白抗体水平相当。初次免疫后第7、14天小鼠中和抗体阳转数为4/10和8/10,加强免疫后全部小鼠抗体阳转,中和抗体GMT达391。初次免疫后第42天,疫苗组IFNγ和IL-2平均斑点数均显著高于对照组(t分别为8.094和13.08,P均<0.0001)。结论SARS-CoV-2灭活疫苗2次免疫能够有效刺激小鼠产生针对Delta株病毒的体液免疫和细胞免疫。展开更多
Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role i...Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016μM.The mechanism was related to binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quantum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)pathways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.展开更多
Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines....Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.展开更多
The emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants capable of evading both convalescent and vaccine-triggered antibody responses has underscored the pivotal role of T-cell immunity in...The emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants capable of evading both convalescent and vaccine-triggered antibody responses has underscored the pivotal role of T-cell immunity in antiviral defense.Here,we develop the ConFormer network for epitope prediction,which couples convolutional neural network(CNN)local features with Transformer global representations to enhance binding prediction performance,and employ the deep learning algorithm and bioinformatics workflows to identify conserved T-cell epitopes within the SARS-CoV-2 proteome.Five epitopes are identified as potential inducers of T-cell immune responses.Notably,the multi-valent vaccine composed of these five peptides significantly activates cluster of differentiation(CD)8^(+)and CD4^(+)T cells both in vitro and in vivo.The serum of mice immunized with this vaccine is able to neutralize the five major SARS-CoV-2 variants of concern.This study provides a candidate peptide vaccine with the potential to trigger antiviral T-cell responses,thereby offering the prospect of immune protection against SARS-CoV-2 variants.展开更多
Sustained antigen release from delivery systems is a pivotal strategy to enhance vaccine-induced immune responses,primarily by mimicking the antigen exposure kinetics of natural infections to synchronously boost humor...Sustained antigen release from delivery systems is a pivotal strategy to enhance vaccine-induced immune responses,primarily by mimicking the antigen exposure kinetics of natural infections to synchronously boost humoral and cellular immunity.However,the absence of an"antigen boost"effect in current approaches stands as a critical bottleneck,limiting the intensity and durability of immune responses.To address the critical gap of insufficient antigen boosting in sustained-release vaccine platforms,we engineered an ultrasound-responsive hydrogel(URH)with diselenide-functionalized 4-arm PEG-ONH_(2)(4-arm PEG-Se-Se-ONH_(2)),4-arm PEG-ONH_(2)and ODEX.Leveraging its exceptional ultrasonic sen-sitivity,the URH enables timely controlled,multiple-boost antigen release both in vitro and in vivo,overcoming the limitations of conventional sustained-release systems.With the multiple boost release mode triggered by ultrasound,the immune response in lymph nodes was significant-ly stronger than that in sustained release group without ultrasonic trigger.At the same time,it also greatly improved the humoral immunity level,URH+US-OVA elicited 7.5×10^(4)-fold higher anti-OVA IgG titers over commercial AI-OVA vaccines and 440-fold higher than URH-OVA vaccines at day 40 post-vaccination,while the levels of blood routine and inflammatory factors were within the normal range,which proved that the safety of URH vaccines.The results support that the antigen release mode is a key factor affecting the immunological efficacy of vaccines,and URH can be modularized to regulate the multiple boost antigen releasemode.展开更多
Since the outbreak of COVID-19 in late 2019,the cumulative number of confirmed cases worldwide has surpassed 778 million,and the number of deaths has exceeded 7 million,posing a significant threat to human life and he...Since the outbreak of COVID-19 in late 2019,the cumulative number of confirmed cases worldwide has surpassed 778 million,and the number of deaths has exceeded 7 million,posing a significant threat to human life and health while inflicting enormous losses on the global economy.At the stage where sequential immunization is recommended,there is a pressing demand for mRNA vaccines that can be rapidly adapted to new sequences,are easy to industrialize,and exhibit high safety and effectiveness.We developed a lipid nanoparticle(LNP)system,designated as WNP,which facilitates essentially in situ expression at the injection site and results in lower levels of pro-inflammatory factors in the liver,thus enhancing its safety compared to liver-targeted alternatives.Furthermore,in light of the swiftly mutating characteristic of SARS-CoV-2,a study has used cross-lineage chimeras and mutation patch strategies to design an antigen that is highly immunogenic and can stimulate the production of a broad range of effective antibodies.Therefore,we used the same antigenic configuration of RBD including five key mutation sites(K417T,L452R,T478K,E484K,and N501Y)to achieve optimal broad-spectrum efficacy.Our results indicate that WNP can elicit a humoral immunity response that is as robust as that of SM-102,a stronger cellular immune response,and provide a certain protective effect.On top of that,WNP can be applied to the development of vaccines targeting other pathogens and will contribute to a quicker response to the spillovers of unknown mammalian viruses.展开更多
文摘目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫PBS作为对照,每组10只。初次免疫后第7、14、21、28、35和42天采集血清,间接ELISA法检测血清中针对Delta株病毒S和N蛋白的结合抗体效价,微量中和试验检测针对Delta株病毒的中和抗体效价。初次免疫后第42天,取小鼠脾脏,进行Elispot检测,评价细胞免疫水平。结果初次免疫后第7天即可检测到S蛋白结合抗体,加强免疫后抗体效价进一步升高,至第21天抗体几何平均滴度(geometric mean titer,GMT)为89144;而初次免疫后N蛋白结合抗体水平较低,加强免疫后迅速升高,与S蛋白抗体水平相当。初次免疫后第7、14天小鼠中和抗体阳转数为4/10和8/10,加强免疫后全部小鼠抗体阳转,中和抗体GMT达391。初次免疫后第42天,疫苗组IFNγ和IL-2平均斑点数均显著高于对照组(t分别为8.094和13.08,P均<0.0001)。结论SARS-CoV-2灭活疫苗2次免疫能够有效刺激小鼠产生针对Delta株病毒的体液免疫和细胞免疫。
基金supported by National Natural Science Foundation of China(Grant Nos.:81891010/81891011,81725023,82003614,82173950,31770192,32070187,32161133003 and 82003681)China Postdoctoral Science Foundation(Grant No:2022T150029).
文摘Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016μM.The mechanism was related to binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quantum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)pathways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.
基金supported by funds from the scientific and technical program BR218004/0223“Improving measures to ensure biological safety in Kazakhstan:counteracting dangerous and especially dangerous infections”.Part of this work carried out in Russian Federation was supported by internal grant funding from the Scientific Centre for Family Health and Human Reproduction Problems:grant number 121022500179-0.Title:“Molecular,Organismal,and Population Patterns of the Epidemic Process of Anthropozoonotic and Transmissible Infections in the Territory of Northern Asia and Adjacent Areas".
文摘Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.
基金supported in part by the National Natural Science Foundation of China(82150208 and 82425104)the National Key Research and Development Program of China(2022YFC3400501)the Shanghai Rising-Star Program(23QA1402800).
文摘The emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants capable of evading both convalescent and vaccine-triggered antibody responses has underscored the pivotal role of T-cell immunity in antiviral defense.Here,we develop the ConFormer network for epitope prediction,which couples convolutional neural network(CNN)local features with Transformer global representations to enhance binding prediction performance,and employ the deep learning algorithm and bioinformatics workflows to identify conserved T-cell epitopes within the SARS-CoV-2 proteome.Five epitopes are identified as potential inducers of T-cell immune responses.Notably,the multi-valent vaccine composed of these five peptides significantly activates cluster of differentiation(CD)8^(+)and CD4^(+)T cells both in vitro and in vivo.The serum of mice immunized with this vaccine is able to neutralize the five major SARS-CoV-2 variants of concern.This study provides a candidate peptide vaccine with the potential to trigger antiviral T-cell responses,thereby offering the prospect of immune protection against SARS-CoV-2 variants.
基金supported by the National Natural Science Foundation of China(Nos.W2412020,22222509)China Science and Technology Exchange Center Joint Project on Vaccine Research and Development(No.2024VTJP1004)Jilin Provincial International Cooperation Key Laboratory of Biomedical Polymers(No.YDZJ202402077CXJD).
文摘Sustained antigen release from delivery systems is a pivotal strategy to enhance vaccine-induced immune responses,primarily by mimicking the antigen exposure kinetics of natural infections to synchronously boost humoral and cellular immunity.However,the absence of an"antigen boost"effect in current approaches stands as a critical bottleneck,limiting the intensity and durability of immune responses.To address the critical gap of insufficient antigen boosting in sustained-release vaccine platforms,we engineered an ultrasound-responsive hydrogel(URH)with diselenide-functionalized 4-arm PEG-ONH_(2)(4-arm PEG-Se-Se-ONH_(2)),4-arm PEG-ONH_(2)and ODEX.Leveraging its exceptional ultrasonic sen-sitivity,the URH enables timely controlled,multiple-boost antigen release both in vitro and in vivo,overcoming the limitations of conventional sustained-release systems.With the multiple boost release mode triggered by ultrasound,the immune response in lymph nodes was significant-ly stronger than that in sustained release group without ultrasonic trigger.At the same time,it also greatly improved the humoral immunity level,URH+US-OVA elicited 7.5×10^(4)-fold higher anti-OVA IgG titers over commercial AI-OVA vaccines and 440-fold higher than URH-OVA vaccines at day 40 post-vaccination,while the levels of blood routine and inflammatory factors were within the normal range,which proved that the safety of URH vaccines.The results support that the antigen release mode is a key factor affecting the immunological efficacy of vaccines,and URH can be modularized to regulate the multiple boost antigen releasemode.
基金supported by the National Key R&D Program of China(2021YFF0702000 to Y.Chen,2021YFC2300700 to L.Zhou)the National Natural Science Foundation of China(grants 82341061,82172243 and 82372223 to Y.Chen)+2 种基金the Fundamental Research Funds for the Central Universities(2042022dx0003 to Y.Chen)the Hubei Provincial Natural Science Foundation(2023AFB201 to Z.Zhang,and 2024AFB906 to L.Zhou.)the China Postdoctoral Science Foundation(2023M732705 to Q.Liu).
文摘Since the outbreak of COVID-19 in late 2019,the cumulative number of confirmed cases worldwide has surpassed 778 million,and the number of deaths has exceeded 7 million,posing a significant threat to human life and health while inflicting enormous losses on the global economy.At the stage where sequential immunization is recommended,there is a pressing demand for mRNA vaccines that can be rapidly adapted to new sequences,are easy to industrialize,and exhibit high safety and effectiveness.We developed a lipid nanoparticle(LNP)system,designated as WNP,which facilitates essentially in situ expression at the injection site and results in lower levels of pro-inflammatory factors in the liver,thus enhancing its safety compared to liver-targeted alternatives.Furthermore,in light of the swiftly mutating characteristic of SARS-CoV-2,a study has used cross-lineage chimeras and mutation patch strategies to design an antigen that is highly immunogenic and can stimulate the production of a broad range of effective antibodies.Therefore,we used the same antigenic configuration of RBD including five key mutation sites(K417T,L452R,T478K,E484K,and N501Y)to achieve optimal broad-spectrum efficacy.Our results indicate that WNP can elicit a humoral immunity response that is as robust as that of SM-102,a stronger cellular immune response,and provide a certain protective effect.On top of that,WNP can be applied to the development of vaccines targeting other pathogens and will contribute to a quicker response to the spillovers of unknown mammalian viruses.
