Production and Characterization of Recombinant Antibody Variants Neutralizing SARS-CoV-2

Coronaviruses are ssRNA viruses, some of which can infect human lung epithelium via binding the ACE2 receptor. The outbreak of the severe acute respiratory syndrome corona-virus-2 (SARS-CoV-2) reported at the end of 2019 has forced the biomedical field to shift the focus to intensive studying of the novel virus, its diagnostics and treatment strategies. This thesis work aimed to produce recombinant IgG, Fab, scFv and scFv-Fc antibodies against receptor binding site (RBD) of SARS-CoV-2 and to perform their characterization regarding their specificity and overall functionality. The antibody constructs are based on two antibodies (REGN10933 and REGN10987) developed by Regeneron Pharmaceuticals, which have demonstrated high efficiency in neutralizing SARS-CoV-2 in animal testing and humans. The approach for recombinant antibody production includes the expression of antibodies and antibody fragments using mammalian expression vectors comprising single chain frag-ment variable (scFv) and IRES-mediated tricistronic genes as well as fusions with mFc and hFc regions of IgG. In particular, this approach allows beneficial outcomes regarding protein yields and stability. The DNA for the antibody constructs was cloned in E.coli and expressed in HEK293 cells. Proteins were purified with affinity chromatography techniques. The quality of the product was assessed with SDS-PAGE gel staining and Western blotting. Obtained results have demonstrated satisfactory purity and yields for most proteins as well as the expected formation of covalent bonds between antibody chains (di-, tetramerization). However, some of the antibody variants’ expression and functionality failed presumably due to the possible flaw in the sequence alignment. Analysis of binding properties of the antibody constructs was performed by pseudovirus neutralization assay against Wuhan, Beta, Delta and Omicron SARS-CoV-2 variants. The assay was carried out on ACE2 overexpressing HEK cells, which allowed to assess specific inhibition of viral entry by neutralizing anti-RBD antibodies and antibody fragments. The results demonstrated that SCF1-mFc and LIH2-mFc appear to be the most efficient and specific recombinant antibody constructs against RBD of certain SARS-CoV-2 variants, able to neutralize pseudovirus in a dose-dependent manner. However, Omicron variant fully resisted the neutralization. The results have also demonstrated that the presence of Fc region significantly improves the stability and neutralizing ability of recombinant antibodies. The production system of tricistronic, scFv and scFv-Fc constructs as well as their func-tionality was mainly successful. Most efficient recombinant antibody constructs will be poten-tially utilized for research and diagnostic purposes, e.g. for immunological assays detecting SARS-CoV-2

Ohje MININEPHPLUS™ -nefelometrin käyttöön : opetusvideo bioanalytiikan opiskelijoille

Nefelometria on menetelmä, jota kliinisessä kemiassa käytetään kliinisesti merkittävien spesifisten proteiinien mittauksissa. Menetelmä kuuluu immunologisiin määritysmenetelmiin, joista suomenkielellä saatavaa materiaalia löytyy rajoitetusti. Nefelometria on yksi bioanalyytikkokoulutuksessa opetettavista perusmenetelmistä. Tampereen ammattikorkeakoulun bioanalyytikkokoulutuksen harjoitustunneille tarvitaan selkeä suomenkielinen video MININEPHPLUS™ -nefelometrin käytöstä. Videollinen ohje toimii oppimista tukevana oppimateriaalina, jonka kautta opiskelijat voivat perehtyä analysaattorin toimintaan. Opinnäytetyön toimeksiantajana on Tampereen ammattikorkeakoulun bioanalyytikkokoulutus. Opinnäytetyön tavoitteena oli parantaa opiskelijoiden tietoa nefelometriasta sekä kehittää taitoja käyttää mittausmenetelmää lähes itsenäisesti. Tuotoksena tehtiin mahdollisimman selkeä videollinen analysaattorin käyttöohje. Videon tarkoituksena on toimia opiskelua tukevana, mutta ei korvaavana oppimateriaalina. Tehtävänä oli tuottaa videollinen oppimateriaali, jonka avulla opiskelijat perehtyvät nefelometriseen mittausmenetelmään sekä MININEPHPLUSTM -analysaattorin käyttöön. Tämä mahdollistaa oppimisprosessin tehostamisen sekä opiskelijoiden itsevarmuuden lisäämisen itsenäisempään työskentelyyn. Opinnäytetyössä käytettiin toiminnallista menetelmää, joka sisältää tuotoksen eli opetusvideon sekä kirjallisen raportin. Videossa esiteltiin MININEPHPLUS™ -analysaattorin periaatteen sekä työskentelyprosessin ja raportissa keskitytettiin teoreettisiin lähtökohtiin sekä työn toteutusprosessin pohdintaan.Nephelometry is a method used in clinical chemistry for the measurement of clinically relevant specific proteins. The method is one of the immunological assay methods and a part of study programme in Biomedical Laboratory Science. Information on the topic available in Finnish is somewhat limited. The thesis was requested by Tampere University of Applied Sciences as there was a need for an educational video on nephelometry with the aim of supporting students’ learning process. The objective of this study was to improve students’ knowledge about nephelometry in order to be able to use the measurement method almost independently. Therefore, the video guide was made as user-friendly as possible. The purpose of this study was to create the video material that can be used as a teaching tool to familiarise students with the nephelometric measurement method and to improve their self-confidence in more independent work. As a result, students would be able to work independently using the video in the university’s laboratory of clinical chemistry. The purpose of this study is to act as a supportive, but not a substitute learning material. The study has a functional approach. It is a practice-based study with an output, the educational video, and a written report supplementing the theoretical content. The video introduces the principle of the MININEPHPLUS™ analyser and its working process. In the report, we deepened the theory presented in the video and analysed the implementation process of the study, along with its outcomes.Tuotos eli opetusvideo on julkaistu Youtube-kanavalla ja on saatavilla verkko-osoitteen kautta saatavaksi rajoitetusti Tampereen ammattikorkeakoulun käyttöön

Coronavirus spike protein-specific antibodies indicate frequent infections and reinfections in infancy and among BNT162b2-vaccinated healthcare workers

The prevalence of seasonal human coronavirus (HCoV) infections in early childhood and adults has not been well analyzed in longitudinal serological studies. Here we analyzed the changes in HCoV (229E, HKU1, NL63, OC43, MERS, and SARS-CoV-2) spike-specific antibody levels in follow-up serum specimens of 140 children at the age of 1, 2, and 3 years, and of 113 healthcare workers vaccinated for Covid-19 with BNT162b2-vaccine. IgG antibody levels against six recombinant HCoV spike subunit 1 (S1) proteins were measured by enzyme immunoassay. We show that by the age of three years the cumulative seropositivity for seasonal HCoVs increased to 38-81% depending on virus type. BNT162b2 vaccinations increased anti-SARS-CoV-2 S1 antibodies, but no increase in seasonal coronavirus antibodies associated with vaccinations. In healthcare workers (HCWs), during a 1-year follow-up, diagnostic antibody rises were seen in 5, 4 and 14% of the cases against 229E, NL63 and OC43 viruses, respectively, correlating well with the circulating HCoVs. In 6% of the HCWs, a diagnostic antibody rise was seen against S1 of HKU1, however, these rises coincided with anti-OC43 S1 antibody rises. Rabbit and guinea pig immune sera against HCoV S1 proteins indicated immunological cross-reactivity within alpha-CoV (229E and NL63) and beta-CoV (HKU1 and OC43) genera.Peer reviewe

Neutralizing antibodies after the third COVID-19 vaccination in healthcare workers with or without breakthrough infection

Abstract Background Vaccinations against the SARS-CoV-2 are still crucial in combating the ongoing pandemic that has caused more than 700 million infections and claimed almost 7 million lives in the past four years. Omicron (B.1.1.529) variants have incurred mutations that challenge the protection against infection and severe disease by the current vaccines, potentially compromising vaccination efforts. Methods We analyzed serum samples taken up to 9 months post third dose from 432 healthcare workers. Enzyme-linked immunosorbent assays (ELISA) and microneutralization tests (MNT) were used to assess the prevalence of vaccine-induced neutralizing antibodies against various SARS-CoV-2 Omicron variants. Results In this serological analysis we show that SARS-CoV-2 vaccine combinations of BNT162b2, mRNA-1273, and ChAdOx1 mount SARS-CoV-2 binding and neutralizing antibodies with similar kinetics, but with differing neutralization capabilities. The most recent Omicron variants, BQ.1.1 and XBB.1.5, show a significant increase in the ability to escape vaccine and infection-induced antibody responses. Breakthrough infections in thrice vaccinated adults were seen in over 50% of the vaccinees, resulting in a stronger antibody response than without infection. Conclusions Different three-dose vaccine combinations seem to induce considerable levels of neutralizing antibodies against most SARS-CoV-2 variants. However, the ability of the newer variants BQ1.1 and XBB 1.5 to escape vaccine-induced neutralizing antibody responses underlines the importance of updating vaccines as new variants emerge