Neutralizing Antibody And Cellular Responses To SARS-CoV-2 Natural Infection And Inactivated SARS-CoV-2 Vaccines

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)is the third highly pathogenicβcoronavirus in the 21st century,causing Coronavirus disease 2019(COVID-19).Vaccination is regarded as the most promsing solution to achieve herd immunity.However,the continuously emerging variants showed higher infectivity than the wild-type strains and may escape immunity obtained from previous infection and vaccination.SARS-CoV-2 can only be operated in the Biosafety level 3(BSL-3)laboratories,which seriously hinders the research of SARS-CoV-2 and the development of vaccines and treatments,and there is an urgent need for a safe,effective,and widely applicable alternative research model.The SARS-CoV-2 infection causes strong neutralizing antibodies and cellular immune responses in the early stage of the disease.However,the persistence of the immune response caused by SARS-CoV-2 is unclear.In the real world,the immune response induced by SARS-CoV-2 inactivated vaccines in SARS-CoV-2 na(?)ve persons and COVID-19 convalescents remains unclear,especially the cellular immune response.SARS-CoV-2 variants may escape immune protection induced by SARS-CoV-2infection and vaccines.Therefore,it is important to elucidate the immune response elicited by SARS-CoV-2 and inactivated vaccine,so as to lay a foundation for the prevention and control of COVID-19 pandemic.Objective1.Construction of the SARS-CoV-2 pseudovirus,including wild-type and variants of concerns(VOCs)and variants of interests(VOIs).2.To explore the specific immune response in COVID-19 hospitalized patients and recovered patients at different times,including IgG antibody,neutralizing antibodies(NAbs),and cellular immune response.To comprehensively understand the immune response of SARS-CoV-2 infection and the inactivated vaccine in recovered COVID-19 patients.3.To explore the immune response of inactivated SARS-CoV-2 vaccine in healthy people who had no history of SARS-CoV-2 infection in the real world.4.To explore the escape of SARS-CoV-2 VOCs and VOIs to the immunity of SARS-CoV-2 natural infection and the inactivated vaccine.Methods1.Establishment of SARS-CoV-2 pseudovirus system and NAbs method.Molecular biology methods,including gene cloning and lentivirus infection,were used to conduct the SARS-CoV-2 pseudovirus system.The SARS-CoV-2 pseudovirus was neutralized with COVID-19 patient’s serum and the specificity of the pseudovirus-based neutralizing antibody assay were verified.2.Study on the immune response of SARS-CoV-2 natural infection.COVID-19patients with mild to severe disease in the early stage of infection,as well as individuals who recovered 10–11 and 17–18 months after illness onset were recruited(N=170).Enzyme-linked immunosorbent assay(ELISA)and SARS-CoV-2 pseudovirus neutralizing antibody assay were used to measure the virus-specific IgG antibody and NAbs.Peripheral blood mononuclear cells(PBMCs)from COVID-19 convalescents were isolated.Enzyme-linked immunospot(ELISpot)assay was used to evaluate SARS-CoV-2 S-specific IFN-γ-and IL-2-secreting T cell response.3.Study on the immune response of the inactivated SARS-CoV-2 vaccine in COVID-19 recovered patients.We investigated the immune level changes in 15–17 months recovered patients after receiving the inactivated vaccines(N=181).4.Study on the immune response of the SARS-CoV-2 inactivated vaccine in healthy people and breakthough infection persons.240 healthy SARS-CoV-2 na(?)ve persons who received the inactivated vaccine were recruited to explored the NAbs and T cell immune response dynamic from 1 to 6 months.Results1.Establishment of SARS-CoV-2 pseudovirus system and neutralizing antibody detection method(1)Construction of SARS-CoV-2 wild-type and variants spike plasmids:the SARS-CoV-2 S genes were obtained by sequence optimization synthesis,including wild-type,D614G strain,VOCs such as Alpha(B.1.1.7),Beta(B.1.351),Delta(B.1.617.1)and Omicron(B.1.1.529),and Iota and Kappa VOIs.S segments were lingated to the eukaryotic expression vector p CMV to obtain the spike plasmids.(2)Establish SARS-CoV-2 pseudovirus neutralizing antibody detection method:construct different cells stably expressing human angiotensin-converting enzyme 2(ACE2),and obtain SARS-CoV-2 wild-type and variants pseudoviruses by lentiviral double-plasmid transfection.After screening,Vero-E6-ACE2 and HEK293T-ACE2were found as the most sensitive cell lines to SARS-CoV-2 pseudoviruses infection.S plasmid and lentiviral backbone plasmid p NL4.3-luc.R-E-were transfected into HEK293T cells at a ratio of 1:3,and the supernatant was collected in 24 hours and 48hours to obtain high-titer SARS-CoV-2 pseudoviruses.Cell density 2×10~4/well and infection for 72 hours were the optimal cell density and measurement time for SARS-CoV-2 pseudoviruses infection.The serum of COVID-19 patients can specifically neutralize SARS-CoV-2 pseudoviruses,and the serum of healthy control cannot neutralize the pseudovirus,indicating that the SARS-CoV-2 pseudovirus neutralization method has high specificity and sensitivity which can replace live SARS-CoV-2 virus to test neutralizing antibodies.2.Characteristics of the humoral and cellular immune responses after SARS-CoV-2natural infection(1)COVID-19 patients had a strong humoral immune response in the early stage of viral infection:After viral infection,the S and N protein IgG positive rates were 95.3%(41/43)and 97.6%(42/43)at 1-to 2.5-month sera of the patients.And 97.7%(42/43)individuals developed NAbs with median of 1:237.In the early stage of the disease,the levels of IgG antibodies and NAbs in severe patients were significantly higher than those in mild patients.(2)SARS-CoV-2 infection induced persistent humoral and cellular immune responses:10–11 months after SARS-CoV-2 infection,91.9%(57/62)individuals developed NAbs with median of 1:99.The S and N protein IgG positive rates were 85.5%(53/62)and 82.3%(51/62)at 10-to 11-month sera of the recovered patients.17–18months after SARS-CoV-2 infection,88.9%(40/45)individuals developed NAbs with median of 1:80.The S and N protein IgG positive rates were 80.0%(36/45)and 86.7%(39/45)at 17-to 18-month sera of the recovered patients.The humoral immunity responses decreased significantly from 1–10 months after virus infection,but remained relatively stable from 10–18 months.95.6%(43/45)and 93.8%(15/16)of recovered COVID-19 patients developed strong virus-specific IFN-γ-and IL-2-secreting T-cell immune responses 17–18 months after virus infection.There was no significant difference of T cell immune response between severe and mild patients.After SARS-CoV-2 infection,NAb titers were significantly correlated with S/N protein IgG levels.Virus-specific T cell immune responses were significantly correlated with NAbs.3.Characteristics of the humoral and cellular immune responses of inactivated SARS-CoV-2 vaccine in recovered COVID-19 patientsInactivated SARS-CoV-2 vaccines can enhance humoral and cellular immune responses in recovered COVID-19 patients:All recovered COVID-19 patients(15–17months after illness onset)developed NAbs after one and two doses of the inactivated vaccine with positive rates of 100%(53/53)and 100%(83/83),and NAbs(geometric mean titer,GMT were 1:178 and 1:199)were significantly higher than those of unvaccinated convalescents.Vaccinated convalescents generated significantly higher virus-specific T-cell immune responses than unvaccinated individuals.Patients with higher virus-specific IFN-γsecreting T-cell response also had higher IL-2 secreting T-cell response.In COVID-19 convalescents,humoral and cellular immune responses generated by one dose of the vaccine was comparable to that of two doses of the vaccine.4.Characteristics of the immune response in inactivated SARS-CoV-2 vaccine-received healthy people and the cellular immune response of breakthrough infection(1)Neutralizing antibody response:41.2%(7/17)of healthy people had NAbs after receiving first dose of inactivated vaccine,with a GMT of 1:12.4,and 94.1%(16/17)of those people had NAbs after receiving the second dose with a GMT of 1:56.7,indicating the second dose of inactivated SARS-CoV-2 vaccine was essential to enhance NAbs in healthy individuals.During the observation from 14 to 182 days after the second dose of vaccine,the NAbs waned as time post vaccination increased.The positive rates of NAbs remained relatively high within two months after the second dose(2–4 weeks,94.7%,GMT 1:54 and 5–8 weeks,90.0%,1:41.5).We founded that13–16 weeks after the second dose,the positive of NAbs declined quickly.Only 37.0%of immunized persons maintained NAbs 21–24 weeks after the second dose.(2)Cellular immune response:after the second dose of vaccine,the T-cell immune responses remained relatively high within two months,and all individuals had SARS-CoV-2-specific IFN-γ-secreting T-cell response within 2–4 weeks.During the observation period of 5–8 weeks,92.3%(24/26)of the individuals had T-cell immune response.The level of T-cell immune response decreased rapidly from the third month.At 17–20 and 21–24 weeks after the second dose,only 41.7%(5/12)and 44%(11/25)of immunized persons developed virus-specific T-cell immune response.(3)SARS-CoV-2 VOCs and VOIs escape NAbs from vaccine:the NAbs against Delta and Omicron pseudoviruses were 1:42.4 and 1:9.1,respectively,which represented about 3.1(95%CI:1.5–4.8)and 14.7(8.3–21.1)fold decrease of neutralization compared with the wild-type pseudovirus(GMT:133.1).Compared with the wild-type virus,sera obtained from vaccine were equally effective in neutralizing D614G,Alpha,and Iota variants,with a decrease of 1.1(1.0–1.3),1.5(0.7–2.4),and1.2(0.9–1.6)fold,respectively.As for Beta and Kappa variants,the neutralization activities were decreased about 4.0(1.3–6.7)and 2.2(0.3–4.1)fold,respectively(4)The Omicron breakthrough infection rate(28/28)was high in vaccine received individuals.Individuals who recovered from Omicron infection developed IFN-γ-secreting T-cell immune response which showed extensive cross-reactivity against both wild-type SARS-CoV-2 and Omicron variants(P=0.155).Conclusions1.Established pseudoviruses of SARS-CoV-2 wild-type strain,D614G,VOCs(Alpha,Beta,Delta,and Omicron)and VOIs(Iota,Kappa),which were safe replacement models of live SARS-CoV-2.2.SARS-CoV-2 infection elicited a robust and persistent neutralizing antibody and memory T-cell response in COVID-19 patients,indicating that these sustained immune responses,among most SARS-CoV-2-infected people,may play a crucial role in protection against reinfection.3.Inactivated SARS-CoV-2 vaccine induced robust NAbs and T-cell responses to SARS-CoV-2 in COVID-19 convalescent patients and immune responses after one dose were equal to that after receiving two doses,which highlighted that robust humoral and cellular immune response can be reactivated by the inactivated vaccine in SARS-CoV-2 convalescent patients.4.The inactivated SARS-CoV-2 vaccine stimulated robust NAbs and T-cell immune responses in the first two months after the second dose but the immune effect drops rapidly,which highlights that a third or more dose boost shot may be required to boost immunity against SARS-CoV-2.5.Immune serum retained most of neutralizing potency against Alpha and Iota variants,but significantly lost neutralizing potency against Beta,Kappa,and Delta variants.Omicron variant dramatically evated neutralizing antibody response to inactivated SARS-CoV-2 vaccine,however,the T cell immune responses to wild-type strain and Omicron variant were highly cross-reactive,which provide immunological context for the observation that inactivated vaccines showed robust protection against severe disease with Omicron infection despite greatly reduced neutralizing antibody response.