| Respiratory syncytial virus(RSV)is the main viral pathogen causing acute lower respiratory tract infections among children under 5 years old,the primary cause of hospitalization under infants less than 6 months,and the third leading cause of mortality among children with lower respiratory tract infections.RSV has brought serious health and life threats to children and the elderly worldwide,and has caused a heavy burden of disease and health care to governments,especially in developing countries.At present,there is no safe and effective RSV vaccine on the market,and there is a lack of specific anti-RSV therapeutic drugs worldwide.Only one prophylactic antibody against RSV F protein,Palivizumab,has been approved by US FDA to prevent high-risk children from severe RSV infection,but this antibody shares a low neutralization titer and high cost,requiring multiple injections.Multi-dose administration greatly reduces the compliance of infants and parents,and the high cost significantly restricts the promotion and application of this mAb in developing countries.Infants aged<6 months are the main population of RSV infection among children younger than 5 years old,and a high-risk group of severe RSV pneumonia.Except premature infants and children with congenital diseases,other healthy infants aged 0-6 months should also receive the same protection.RSV vaccines currently under development are divided into non-replicating and replicating vaccines.Among them,non-replicating vaccines are presented exogenously,induce a Th2-biased immune response and could cause ERD(enhanced respiratory disease).Although the novel adjuvants may improve this problem,it is difficult to be approved for use in neonates.In addition,although replicating vaccines can induce a Th1/Th2 balanced immune response through endogenous presentation,they will induce adverse immune responses.At the same time,the immune effect is unstable,and repeated immunizations cannot be performed.Therefore,none of these vaccines are currently available for RSV-na(?)ve populations with immature immune systems.For this part of infants and young children,two passive immunization strategies are mainly adopted at present.One is to provide passive protection for neonates through maternally-transmitted antibodies produced by maternal immunization.However,it can only provide 3 months of protection.Whether it is effective to increase maternal antibody titers is still unknown.Furthermore,in February 2022,GSK pre-F subunit vaccine for pregnant women in the clinical trial phase 3 was stopped due to safety.Therefore,whether the maternal immunization is effective is currently inconclusive.Another immunization strategy is to provide protection through RSV F antibodies.It is also a safer and more effective immunization strategy recommended by WHO.Our study group obtained a high-potent neutralizing monoclonal antibody,5C4,previously.Unfortunately,5C4 is not a broad-spectrum antibody,only effectively neutralizes RSV type A and a small part of RSV type B strains.Therefore,we abandoned the clinical translation of this antibody and turned to seek more broad-spectrum,high-potent neutralizing antibodies.This study is based on this part.In this study,Balb/C mice were immunized with nucleic acid(DNA-F)and adenovirus vector(rAd-F)combination immunization by high pressure of injection via tail vein.8 RSV pre-F-specific high-potent neutralizing antibody candidates were obtained by high-throughput RSV neutralization model and cell-based ELISA.The detection of binding activity with purified F protein showed that all 8 antibodies were RSV pre-F specific antibodies.The neutralization results showed that all 8 antibodies could neutralize RSV A/B strains.The neutralization titers were all higher than or equal to that of 1129.Sequence alignment implied that the light and heavy chain CDR regions sequences of 6 antibodies including 6B2 were highly consistent.It was speculated that they might be from the same clone.Finally,6B2 with the best neutralizing activity,5B11 and 7G5 were selected as the three candidate antibodies for further in vitro evalution.The neutralization assay showed that all three antibodies could neutralize RSV A/B strains with broad-spectrum neutralizing activity.Among them,5B11 had the strongest neutralizing ability,and its neutralizing titer was about 30-fold more than that of 1129,comparable to D25 and MEDI8897.The affinity test by Biacore showed that the affinity constants of 5B11 with RSV A DS-Cav1(pre-F)and RSV B DSCav1 were 1.44 pM and 4.58 nM respectively.Prophylactic evaluation in Balb/C mice showed that a low dose of three candidates antibody protected against weight loss induced by RSV infection in mice,but 5B11 presented better protection against weight loss in mice.Nose and lung plaque assays showed that three antibodies could completely clear the virus in lungs and reduce the virus titer in noses to a certain extent.Lung histopathology showed that 5B11-treated mice shared the lowest pathology score of lungs,indicating that 5B11 could better reduced RSV-related inflammation,compared with 6B2 and 7G5.In addition,the prophylactic efficacy of 5B11 with 5C4 and D25 in Balb/C illustrated that 5B11 exhibited the same ability to prevent RSV infection as 5C4 and D25.In summary,5B11 was identified as the optimal candidate antibody in this study.The high-resolution complex structure of 5B11 and DS-CAV1 was obtained by cryoelectron microscopy(cryo-EM).The results showed that 5B11 recognized a novel binding site between 5C4,AM 14 and hRSV90 on the RSV pre-F protein,which was different from the binding sites of all antibodies previously reported.The 5B11 binding site is highly conserved(94.7%),which is more conserved than site (?)(90.0%).Next,in vivo antiviral evaluation of 5B11 were conducted in Balb/C and cotton rat models to evaluate the prophylactic and therapeutic effects of 5B11 on RSV A and B strains.Both animal models showed that high and low doses of 5B11 can effectively protect animals from RSV infection,and low dose of 5B11 was comparable to high dose of 1129 for preventing RSV infection.Next,the murine antibody 5B11 was humanized.The neutralization and binding activity of the humanized antibody N5B11 were consistent with those of murine antibody 5B11.To obtain a long-acting neutralizing antibody,the Fc domain of N5B11 was mutated in this study to prolong the half life of the antibody.In vitro and in vivo evaluation experiments showed that N5B11-YTE had the longest half life.In low-dose prophylactic evaluation in cotton rat model,N5B11-YTE was comparable to MEDI8897 for preventing RSV A2 at the same dose.Nevertheless,N5B11-YTE was better than MEDI8897 for preventing RSV B 18537 at the same dose.In summary,this study finally obtained the humanized,long-acting antibody N5B11-YTE,which is expected to become a new generation of long-acting broad-spectrum antibody against RSV. |
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