| Objective:To investigate the correlation between serum soluble angiotensin converting enzyme 2（s ACE2）concentration or ACE activity and disease severity in patients with acute lung injury/acute respiratory distress syndrome（ALI/ARDS）.The recombinant Fc-fusion protein ACE2-Ig and enzyme inactivation mutant ACE2-Ig（m ACE2-Ig）were constructed by the extracellular domain of human ACE2 and the Fc fragment of human Ig G1.The pharmacokinetic parameters and protective effect of recombinant Fc-fusion protein in Lipopolysaccharide（LPS）induced acute lung injury in mice were investigated.The neutralization effect of Fc fusion protein on SARS-Co V-2 or SARS-Co V pseudovirus in vitro and the protective effect on SARS-Co V-2 spike protein（S protein）induced acute lung injury model in mice were investigated.Methods:Clinical data of ALI/ARDS patients in Shanghai Changhai Hospital from2019 to 2022 were collected,and serum samples were collected to analyze serum ACE2concentration and ACE activity.The difference of serum ACE2 concentration and ACE activity in ALI/ARDS patients and the correlation between serum ACE2 concentration or ACE activity and oxygenation index（Pa O2/Fi O2）were analyzed.The wild type or enzyme inactivated human ACE2extracellular domain was linked with human Ig G1 Fc gene and insert into pc DNA3.4 vector to construct recombinant plasmids ACE2-Ig and m ACE2-Ig（mutant ACE2-Ig）.The protein was expressed using Freestyle293F mammalian expression system and purified by SPA affinity chromatography.The pharmacokinetic parameters of ACE2-Ig were determined.The lung injury model of BALB/C mice induced by LPS was constructed.The mice were divided into three groups,with 18 mice in each group.After anesthetized,the mice were intubated under direct vision and injected LPS（5mg/kg）.The control group（LPS group）was intraperitoneally injected with normal saline,and the low dose or high dose ACE2-Ig treatment group was intraperitoneally injected with ACE2-Ig（50 mg/kg or 100 mg/kg）.The changes of body weight of mice were recorded.After surgery,6 mice in each group were sacrificed 24h,72h,and 168h respectively.Bronchoalveolar lavage fluid（BALF）was collected and the pathological changes of lung in each group were compared.Surface Plasmon Resonance（SPR）technique was used to evaluate the binding ability of ACE2-Ig to SARS-Co V-2 and SARS-Co V spike protein receptor binding regions.SARS-Co V-2 and SARS-Co V pseudoviruses were constructed using HIV-1 defective virus as vectors to evaluate the neutralization activity of ACE2-Ig.The inhibitory effect of ACE2-Ig on SARS-Co V-2 and SARS-Co V spike protein induced cell fusion was evaluated byβ-galactosidase reporter gene system.SARS-COV-2 spike protein induced acute lung injury model of human ACE2（h ACE2）transgenic mice by was established.The mice were divided into three groups,with 6 mice in each group.The mice in the sham operation group（h ACE2+Vehicle）were exposed to the trachea and sutured.The control group（h ACE2+S1）was treated with airway dripping SARS-Co V-2 S protein and mechanical ventilation for 4h.In the treatment group（h ACE2+S1+ACE2-Ig）,SARS-Co V-2 S protein was intravenously injected into the airway and ACE2-Ig was intraperitoneally injected.Mice in the three groups were mechanically ventilated and inhaled oxygen for 4h after intubation,while mice in the sham operation group and the control group were intraperitoneally injected with normal saline.The body weight of mice was recorded every 24 hours,and the mice were sacrificed72 hours after surgery.BALF was collected,and the total protein content and cytological classification were determined.The lung tissues were fixed,embedded in paraffin,and sectioning were performed for HE staining.The pathological changes of lung in each group were comparedTo explore other antiviral treatments for COVID-19,c DNA sequence of chimeric antigen receptor（CAR）that specifically recognizes SARS-Co V-2 S protein was cloned into lentiviral vector,and THP-1 cell line or primary human macrophages were engineered by lentiviral transfection method.The cell killing ability and phagocytosis ability of engineered cells against SARS-Co V-2 pseudovirus were detected.The susceptibility of engineered cells to pseudovirus and cytokine release after phagocytic pseudovirus were detected.Results:Statistics and analysis of clinical data of 46 ALI/ARDS patients has been processed.The concentration of s ACE2 in ARDS patients was significantly higher than that in ALI patients（P<0.001）.There was no significant difference in ACE activity（P=0.2113）but a significant negative correlation between oxygenation index and s ACE2 level（r=-0.5537,P<0.0001）was observed.Recombinant plasmids pc DNA3.4-ACE2-Ig and pc DNA3.4-m ACE2-Ig were constructed,and the recombinant Fc fusion proteins ACE2-Ig and m ACE2-Ig were expressed and purified by mammalian cell expression system and SPA affinity chromatography.It was confirmed that recombinant Fc fusion proteins have high stability in vivo and in vitro.In the LPS-induced acute lung injury model in mouse,compared with the control group（LPS group）,mice in the low-dose ACE2-Ig treatment group and the high-dose ACE2-Ig treatment group had less weight loss and lower Smith score（P<0.05）.The TNF-αconcentration in BALF of mice in low-dose treatment group significant decreased24h after operation（P<0.05）.There was no significant difference between the low-dose treatment group and the high-dose treatment group.Lung pathological sections of mice in each group were observed at different time.ACE2-Ig treatment significantly improved pulmonary inflammatory cell infiltration and fluid exudation,reduced pulmonary edema and lesion scope,and helped mice recovered from inflammation in an earlier period.ACE2-Ig and m ACE2-Ig have potent cross neutralizing activity against SARS-Co V and SARS-Co V-2 spike proteins.The binding constants KD of ACE2-IG to SARS-Co V and SARS-Co V-2 were 177.1n M and 11.2n M respectively.Furthermore,ACE2-Ig and m ACE2-Ig could inhibit cell infection by pseudovirus or cell fusion induced by spike proteins.ACE2-Ig inhibited SARS-Co V and SARS-Co V-2 spike protein mediated cell fusion with IC50values of 0.85μg/m L and 0.65μg/m L,respectively.m ACE2-Ig inhibited SARS-Co V and SARS-Co V-2 spike protein mediated cell fusion with IC50 values of 0.76μg/m L and 0.48μg/m L,respectively.The effect or pharmacokinetic characteristics of ACE2-Ig and m ACE2-Ig have no significant difference.In the model of acute lung injury induced by SARS-Co V-2spike protein in mouse,compared with sham operation group（h ACE2+Vehicle）,total protein contention and white blood cell count in bronchoalveolar perfusion fluid（BALF）of mice in control group（h ACE2+S1）and treatment group（h ACE2+S1+ACE2-Ig）were significantly increased（P<0.05）,there was no significant difference between the modeling group and the treatment group.Engineered THP-1 or primary human macrophages cells expressing different CAR have been construct successfully.All the engineered cells can be combined with the SARS-Co V-2 spike protein remove the pseudovirus by phagocytosis without be infected by the SARS-Co V-2 pseudovirus.THP-1 or primary human macrophage with CARMERTK does not have the ability to phagocytose and kill the cells express SARS-Co V-2 S protein,but they are able to deplete the SARS-Co V-2 pseudovirus particles while not increasing the release of cytokines.Conclusions:1.Serum s ACE2 concentration increased in patients with ALI and ARDS,and was significantly negatively correlated with Pa O2/Fi O2,suggesting that serum s ACE2concentration is one of the predictors of poor prognosis in ALI/ARDS patients.2.Recombinant fusion proteins ACE2-Ig and m ACE2-Ig were expressed and purified.The fusion protein has relatively stable pharmacokinetic parameters in vivo,and can effectively neutralize SARS-Co V-2 and SARS-Co V pseudoviruses by binding to the receptor binding domain of the spike protein,and significantly inhibit the cell fusion induced by the spike protein.3.The protective effect of ACE2-Ig on spike protein induced acute lung injury was not obvious,but in LPS-induced mouse acute lung injury model,ACE2-Ig could alleviate weight loss,improve lung pathological injury,and down-regulate the concentration of TNF-αand other inflammatory factors in BALF.Engineered primary human macrophages CARMERTK-macrophages may be a novel antiviral cell therapy that inhibits viral replication without triggering an inflammatory response and may be one of the potential therapeutic options for patients with severe COVID-19. |
PDF Full Text Request