A Recombinant Vaccine Targeting The RBD Of The S Protein Of SARS-CoV-2 Induces Protective Immunity

COrona VIrus Disease 2019（COVID-19）is an infectious disease caused by the infection of New Coronavirus Acute Respiratory Syndrome Coronavirus-2（SARS-CoV-2）.At the end of December 2019,Wuhan,China first reported cases of pneumonia of unknown cause.The first case appeared symptoms on December 1st,2019,and then the rapid spread began among people.At the end of February 2020,it began to erupt in Europe,North America,and other places.The World Health Organization declared it a pandemic on March 11,2020.Since then,in 235 countries around the world,as of 11 April 2021,135,646,617 confirmed cases of covid-19 have been reported to who,including 2,930,732 deaths（https://covid19.who.int/）.Over the past two decades,coronavirus has caused three major outbreaks:severe acute respiratory syndrome（SARS）,Middle Eastern respiratory syndrome（MERS）and current SARS-CoV-2.Although the mortality of SARS-CoV-2 is not as high as that of SARS CoV or MERS CoV,its transmission ability is much higher than that of the latter.The popularity of COVID-19 has damaged human health and seriously affected human society,economy,environment,and other fields.Therefore,an effective vaccine is urgently needed to prevent SARS-CoV-2.Vaccine is one of the greatest successes of modern medicine.The emergence of vaccines has eliminated smallpox,almost eliminated poliomyelitis,and greatly reduced the incidence rate and mortality of large populations caused by infectious diseases.Since the outbreak of SARS in 2003,many countries have carried out vaccine research.The nucleotide sequence similarity between SARS-CoV-2 and SARS CoV is79%,and both mediate virus invasion and infection through the interaction between spike（s）and host receptor protein ACE2.Complete spike protein S and its S1 subunit can induce neutralizing antibody reaction in immunized animals.However,they may also produce antibodies that enhance the intensity of virus infection,or lead to liver injury in animals infected with SARS coronavirus.The Receptor Binding Domain（RBD）of spike protein S is the key part of antibody neutralization reaction,so it is more necessary to design s protein precisely.Some studies have confirmed that RBD is an ideal protein subunit vaccine in the process of developing SARS vaccine,which provides ideas for the development of new coronal vaccine.Vaccine types include inactivated vaccine,live attenuated vaccine,subunit vaccine,recombinant protein vaccine,m RNA vaccine,DNA vaccine,viral vector vaccine,etc.Among them,the recombinant protein vaccine is made of effective antigen that can stimulate the body to produce protective immunity,which is safe,efficient and can be produced on a large scale.Therefore,we selected the RBD domain of SARS-CoV-2 S protein as the antigen to precisely design the recombinant vaccine;EK digestion site to construct the tag less antigen protein;used insect thioredoxin（TRX）to help the protein fold correctly to improve the stability of the antigen structure;used mature recombinant protein expression technology insect cells and BAC to BAC baculovirus expression system to improve the efficiency Protein content and yield.The recombinant RBD of the spike protein of SARS-CoV-2 was prepared using insect cells and the Bac-to-Bac baculovirus expression system.A GP67 signal peptide sequence was added to ensure protein secretion.Recombinant RBD was successfully obtained from the cell culture supernatant and purified to high homogeneity.The purity of the final protein preparation was more than 98%.The apparent molecular mass of the purified RBD protein was determined to be around 34 k Da,which was approximately a quarter larger than the molecular mass calculated using the RBD amino acid sequence alone（about 27 k Da）,suggesting that the RBD is densely glycosylated.Using mass spectrometry（MS）,the intact N-glycopeptides and glycans were analyzed with the GPSeeker software.Three N-glycosylation sites on asparagine were identified.O-Glycosylation sites were also evaluated by analyzing the MS results using SEQUEST in Proteome Discoverer and ten O-glycosylation sites were identified.To determine the abundance of glycosylation,the number of MS/MS spectra of glycosylated peptides and their corresponding unmodified peptides were determined.A much higher degree of N-glycosylation than O-glycosylation was observed.These identified glycosylation sites were further mapped on the complex structure of the RBD of SARS-CoV-2 bound to ACE2.Most of the sites were located in the RBD core subdomain.In addition,all the sites were found to be distant from the bound ACE2,indicating that decorated glycans may not interfere with receptor recognition and/or binding.The binding of our RBD protein with ACE2 was then confirmed using surface plasmon resonance（Biacore）.In agreement with a previous study,potent interactions were observed.The binding affinity was calculated to be about Kd=1.54 n M（dissociation constant）,with an association rate constant（ka）=1.33×10⁷ M^-1 s^-1 and a dissociation rate constant（kd）of 2.05×10^-2s^-1.This finding shows that our RBD binds to ACE2 with a high affinity,which is a good reflection of the native conformation of our RBD protein.In order to prove the antigenicity of recombinant RBD,we measured the antibody response to the recombinant RBD protein in the serum of patients with COVID-19 by ELISA.Compared with 20 healthy blood donors,the serum Ig G and Ig M of all 16patients were significantly increased（P<0.0001）.The results show that the RBD of SARS-CoV-2 S protein can be specifically recognized and trigger antibody reaction,and has good antigenicity,which provides a clinical basis for the development of vaccine based on recombinant RBD.Aluminum salt adjuvant has been widely used in human and animal vaccines for nearly 90 years.The aluminum salt adjuvant assisted mechanism is relatively clear,and it is also safe,effective,and well tolerated.It is the most widely used adjuvant and the only approved adjuvant in China.In order to rapidly develop a safe and efficient vaccine,we chose aluminum hydroxide as the adjuvant of recombinant RBD vaccine.The results of adsorption test and stability test showed that the recombinant RBD could be combined with aluminum hydroxide to prepare vaccine.We explored the immunogenicity of the recombinant RBD vaccine in mice,New Zealand rabbits and Rhesus Macacas,and found that the recombinant RBD vaccine could produce good RBD specific antibody in different species of animal immune models.We also characterized the effect of recombinant RBD vaccine immune serum by binding inhibition test of RBD and ACE2 receptor,neutralization test of pseudovirus,neutralization test of SARS-CoV-2 virus and adoptive protection test of immune serum in mice challenged with ACE2.Effective functional antibody responses could be induced in immunized mice,rabbits,and Rhesus Macacas as early as 7 or 14 days after injection.A dose of vaccine.Serum from immunized animals blocked the binding of RBD to ACE2,which was expressed on the cell surface and neutralized the infection of SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro.It is worth noting that the immune serum after a single immunization can prevent mice from being infected with SARS-CoV-2 live virus.In novel coronavirus pneumonia,we have optimized the immunization and immunization procedures.We also consider the rapid deterioration of the epidemic trend of new crown pneumonia.We urgently need vaccines that can produce effective protection in a short time.Therefore,we have chosen the immunity program of muscle immunity and zeroth,seventh days of immunity 2 times.In the subsequent experiments,we also tested whether the RBD vaccine could block the infection in non-human primates inoculated with live SARS-CoV-2.We immunized non-human primates with two intramuscular injections on days 0 and 7 of20μg or 20μg per dose and then challenged the monkeys with live SARS-CoV-2viruses 28 days after the first vaccination.The control groups included treatment with PBS or aluminum hydroxide gel alone.Neutralizing antibodies against live SARS-CoV-2 were detected in all vaccinated non-human primates,whereas no neutralizing antibodies were detected in the two control groups.A quantitative real-time reverse-transcription PCR（RT-q PCR）assay was used to measure viral genomic RNA（g RNA）and viral sub genomic RNA（sg RNA）.The expression of sg RNA is indicative of viral replication.Lung tissues of non-human primates were collected on day 7 after challenge to determine the viral replication status.Lung tissues from the control groups showed high levels of viral g RNA and sg RNA copies.By contrast,no detectable viral g RNA or sg RNA was present in the vaccinated groups that received 20μg and 40μg of the vaccine with adjuvant.In addition,peak loads of viral g RNA were observed in throat swabs from the control monkeys 3 days after inoculation.The peak loads were reduced after the vaccination;only around 1.6 and 3.8 ppm of the viral loads were found in the monkeys vaccinated with 20μg and 40μg of the vaccine,respectively,compared with the viral loads of the control group.Notably,no sg RNA was detected in the throat swabs of monkeys vaccinated with either the 20-μg or 40-μg dose after challenge with SARS-CoV-2,whereas high levels of sg RNA were observed in the control monkeys.Peak levels of viral g RNA and sg RNA in anal swabs were observed in the control groups 5 and 6 days after inoculation,whereas only very low levels of g RNA were detected in the vaccinated monkeys and no sg RNA was detected in the anal swabs of non-human primates vaccinated with either the 20μg or 40μg dose.These data indicated that the low level of g RNA that was detected was due to the high-inoculation dose—which may have been neutralized already—and there is no evidence of viral replication,no sg RNA was detected.The lung tissues of monkeys from the two control groups（PBS or aluminum hydroxide alone）showed typical histopathological changes of viral interstitial pneumonia,a key feature of COVID-19.As shown in Fig.3g,the microscopy findings included apparent thickened alveolar walls and heavy interstitial infiltrates of mononuclear inflammatory cells.The alveolar space also showed heavy infiltration of mononuclear inflammatory cells and exudates with the disappearance of recognizable architecture.In addition,diffuse hemorrhage,and type-II pneumocyte hyperplasia was also observed.By contrast,lung tissues from non-human primates vaccinated with the RBD vaccine（40μg or 20μg）showed no significant histopathological changes and appeared to be normal.These results suggest that the recombinant RBD vaccine had a good protective effect on Rhesus Macacas.Next,we studied the potential pathway of humoral immune response induced by recombinant RBD vaccine.Compared with wild-type mice,reduced Ig G levels induced by RBD protein vaccine were found in CD4^-/-,STING^-/-,Casp1^-/-,NLRP3^-/-,IL1β^-/-,TLR2^-/-and TLR4^-/-mice,while no effect on Ig G levels was found in other knockout mice(CD8a^-/-mice).Cellular immune response may also be involved in the clearance of SARS CoV infection,including CD4 and CD8 T cells.We studied the cellular immunity of recombinant RBD vaccine.When stimulated by recombinant RBD vaccine,the lymphocytes isolated from mice immunized with the vaccine induced increased levels of IFN-γand IL-4.In addition,the percentages of memory lymphocytes(including CD4⁺CD44^highIL-4⁺,CD4⁺CD44^highIFN-γ⁺and CD8⁺CD44^high IFN-γ⁺cells)increased in mice immunized with recombinant RBD vaccine by flow cytometry.These results suggest that vaccination with recombinant RBD protein could trigger T cell response and induce the increase of memory T cells.This study demonstrated that the combination of B cells and T cells coordinated the good antibody response of mice to the recombinant RBD candidate vaccine.In order to investigate the protective effect of immune serum or spleen lymphocytes on SARS-CoV-2 challenged mice,adoptive therapy was carried out with immune serum or spleen lymphocytes of the vaccine immunized mice.Compared with PBS control,adoptive treatment with 0.1m L mixed serum of the vaccine immunized mice did not show detectable viral replication,significant histopathological changes and weight loss in the recipient mice.Even the serum of mice on day 7 after single dose vaccination could completely protect mice from SARS-CoV-2 infection.In addition,no evidence of antibody dependent enhancement or accelerated pneumonia was observed,as there was no evidence of pneumonia in mice receiving recombinant RBD immunized serum.Although the levels of IFN-γand IL-4 produced by lymphocytes stimulated by RBD in vitro were increased,there was no significant increase of these two cytokines and other inflammatory cytokines in the plasma of mice immunized with RBD vaccine,suggesting that the recombinant RBD vaccine did not cause systemic inflammatory response.In the vaccine safety evaluation experiment,the results showed that there was no abnormal change in weight,food intake and action of the mice.After the administration,the animals in each group were dissected to observe the brain,heart,liver,spleen,lung,kidney,gastrointestinal tract,reproductive system and other organs or tissues with naked eyes.No abnormal changes were found in the biochemical indexes of each group of mice,and no abnormal histopathological changes were found in the organs or tissues of heart,liver,spleen,lung,kidney and so on.In order to further explain the safety of the recombinant RBD candidate vaccine,the potential toxicity of the vaccine in non-human primates（n=50）was evaluated according to good laboratory practice.No adverse events were observed in terms of weight,fur wrinkles,behavioral changes or appetite.No abnormal changes were found in size,shape,color and texture of the organs or tissues related to the drug,and no redness,sclerotia,scab and other reactions were found in the injection site.The histology of liver,lung,kidney,spleen,brain and heart of primates were observed under microscope,the result of which turned out to be pleasant.No changes in peripheral blood count and difference were found.In conclusion,we produced recombinant RBD protein in insect cell expression system,and found that the RBD protein has abundant glycosylation sites and high affinity with ACE2.The recombinant RBD vaccine was prepared by adding aluminum hydroxide adjuvant.Our recombinant RBD vaccine could stimulate specific antibodies in mice,rabbits and Rhesus Macacas,which is dose-dependent and time-dependent.The results of the virus challenge test in Rhesus Macacas showed that the viral loads of the animals immunized with the recombinant RBD vaccine were significantly reduced comparing with the untreated and AL group,the RBD positive binding antibody and neutralization antibody increased significantly,and no pathological damage was found in lung tissue,which indicating that the recombinant RBD vaccine had obvious effect of preventing SARS-CoV-2 infection.The study of immune pathway of the RBD vaccine was formulated as muscle injection,and the immune procedure was 2 injections on day 0,7.The results of the experiments on the protective effects of recombinant RBD vaccine in mice with h ACE2 transgenic background showed that passive immunotherapy had potential in the prevention of SARS-CoV-2infection.The mechanism studies showed that the inflammatory corpuscles pathway,Sting,TLR-4 and TLR-2 pathway and CD4 positive T-lymphocytes may be involved in the induction of specific Ig G by recombinant RBD protein candidate vaccine.In conclusion,our results emphasize the importance of RBD domain of spike protein in the design of sars-cov-2 vaccine,and provide a theoretical basis for the development of protective vaccines by inducing antibodies against RBD.The recombinant RBD protein we used is technically and commercially feasible and could be produced on large scale.All these characteristics support the further development of the candidate vaccine.