Preparation And In Vitro Neutralizing Efficacy Assessment Of Antigen-specific F?ab'?₂ Against Middle East Respiratory Syndrome Coronavirus

Middle East Respiratory Syndrome(MERS)is another respiratory disease infected by human coronavirus since the SARS 2003.The pathogen of MERS,the Middle East Respiratory Syndrome Coronavirus(MERS-CoV)was first discovered in Saudi Arabia in 2012.Since 2012,there are no vaccines for MERS or any other specific therapeutic drugs available on the market.In order to cope with any possible outbreak of MERS,it is necessary to have effective emergency treatment in store.Equine antiserum products have developed for more than a 100 years.For a long time,when dealing with some of the severe disease or toxins poisoning which there are no proper treatments,such as rabies,tetanus,diphtheria or a variety of snake or scorpion venom poisoning,administration of antiserum or immunoglobulin derived from animals(horses,sheep,rabbits,etc.)always work well.With a lot of advantages such as a high yield,well eatablished manufacturing,affordable price and so on,equine antiserum product has been proven to be safe and effective antibody drugs,especially for emergency prevention and treatment.The IgG was extracted from equine hyperimmuned serum obtained by immunization of MERS-CoV virus-like particles(VLPs)and then submitted to the pepsin digestion to make F(ab')2.Next,the MERS-CoV specific immunoglobulin F(ab')2 was extracted by immunoaffinity chromatography.MERS-CoV pseudovirus was used to evaluate the neutralizing potency.For the preparation of immunoaffinity columns,this we use the receptor binding domain(RBD)derieved from prokaryotic expression as antigen,and couples with Sepharose 4B gel.Chapter one: instead of traditional ammonium sulfate precipitation or octanoic acid precipitation method,and the preparation and purification process of equine immunoglobulin F(ab')2 was produced by chromatographic methods.Protein A column and Protein G column were used to extract IgG from the serum.The extracted IgG was digested using pepsin.The highly puritied F(ab')2 was obtained by gel filtration chromatography.The neutralizing test using pseudovirus showed a neutralizing activity loss in the final F(ab')2 product.But at the same concentration,due to the smaller molecular weight,the F(ab')2 shared a higher neutralizing activity compared with whole IgG,which was consistent with the theoretical expectations.In this part,what we did on the one hand laid foundation for the follow-up study,on the other hand,also laid foundation for establishment of a modern manufacturing process for human use of the equine immunoglobulin F(ab')2 against MERS-CoV.Chapter 2: Preparation of MERS-CoV specific F(ab')2.The preparation of total F(ab')2 from horse serum was conducted using methods described in chapter 1.The MERS-CoV specific F(ab')2 was then extracted using S-RBD immunoaffinity column.The neutralizing test using pseudovirus showd a higher neutralizing activity of the MERS-CoV specific F(ab')2 extracted by S-RBD immunoaffinity columns compared with total F(ab')2.We also had the same result on the MERS-CoV specific IgG against total IgG.At the same concentration,the neutralizing activity of MERS-CoV specific F(ab')2 was higher than that of the MERS-CoV specific IgG.It was noted that,the yield of the MERS-CoV specific antibodies extracted using a prokaryoticly expressed S-RBD column is low,though high in the neutralizing activity.And the flowthrough of the S-RBD column was still able to neutralize the MERS pseudovirus.This suggested that only some parts of the MERS-CoV specific antibodies can be extracted using a prokaryoticly expressed S-RBD column.Therefore,in order to extract MERS-CoV specific antibodis in a complete way,a competent antigen used for building a immunoaffinity column is to be taken into consideration.In summary,this studiy showed the removal of non-antigen-specific antibodies from equine antiserum and only the antigen specific antibodies against MERS-CoV remained,thus presented highly purified MERS-CoV specific immunoglobulin F(ab')2.The principles and methods of immunoaffinity chromatography can be extended to any manufacturing process of equine antiserum products,thus laying the foundation for safer use of these antiserum products.