Assessment of the immunogenicity and formulation of recombinant proteins from SARS-CoV-2 as vaccine antigens

Keser, Duygu
COVID-19 is an infectious disease caused by SARS-CoV-2. The virus was first detected in Wuhan, China in late 2019, and the outbreak was declared a pandemic in January 2020 by WHO, and continues to spread worldwide. As of July 2022, more than 575 million confirmed cases have been detected all over the world, and more than 6 million people died from the disease. One of the most important public health measures in combating the spread of infectious diseases is vaccination. Despite the existence of rapidly developed and administered vaccines in the later stages of the COVID-19 pandemic, there is still a need to develop more effective and safe vaccines against this pathogen. As a result, there is an urgent need to examine proteins with high immunogenicity that can be employed in novel vaccines. The genome of SARS-CoV-2 encodes four structural proteins and other accessory or nonstructural proteins. The present study is on the evaluation of the immunogenicity of the S1 and S2 region protein fragments in the Spike protein and the whole nucleocapsid protein for the formulation of a recombinant subunit vaccine against SARS-CoV-2. S1, S2 gene fragments and the N gene were amplified by PCR and cloned into the pGEM®-T Easy vector. The genes were then inserted into the pET-28a (+) vector and their gene expression was achieved in Escherichia coli BL21(DE3) cells. Recombinant proteins were purified by His-tag affinity chromatography. Western blot analyzes were performed with monoclonal antibodies and immunized mice sera. Serum-specific IgG levels were measured by the ELISA method, and the increased level of total antibodies to all three antigens showed that a robust humoral response was developed in the immunized group of mice. Moreover, in the analyzes depend on the ELISA results, a significant increase was observed in the antigen-specific IgG2a titers, which indicate the cellular response, in the immunized group compared to the control group. The increase in IFN-gamma levels observed as a result of the performed cytokine ELISAs indicated a strong cellular response to recombinant antigens and, in addition to the antibody response, indicates that these three antigens are suitable vaccine antigen candidates to combat the COVID-19 pandemic.


Characterization of leishmania RNA virus infected leishmania major exosomes and evaluation of the impact of cytosolic DNA sensing related pathways in L. Major infection
Yılmaz, İsmail Cem; Gürsel, Mayda; Department of Biology (2020)
Leishmaniasis is a neglected infectious disease caused by Leishmaniaparasite. The disease represents a significant health problem, necessitating urgent development of effective treatment strategies. New World Leishmaniaspecies harboringLeishmania RNA virus (LRV) causesevere,metastatic disease. In this thesis, we compared LRV2-1 deficient and proficient Old World L. majorstrains in terms of their exosomal content and infectivity. PCR and mass spectrometry based analysis revealed that virus infectedLeishm...
Assessment of immune protective capacities of the recombinant outer membrane protein q, iron superoxide dismutase and putative lipoprotein from bordetella pertussis
Yılmaz, Çiğdem; Özcengiz, Gülay; Department of Biology (2017)
Pertussis is a contagious disease which is commonly seen among infants and children and caused by a human pathogen known as Bordetella pertussis. There are currently two vaccine types available against the disease; whole-cell (wP) and acellular pertussis (aP) vaccines. Due to the side effects of wP vaccine, aP vaccines are commonly preferred for vaccination. Despite high vaccination coverage, high incidence rates among adolescents and adults have been reported causing the resurgence of pertussis to be the f...
A comprehensive analysis of Bordetella pertussis surface proteome and identification of new immunogenic proteins
Tefon, Burcu E.; Maass, Sandra; Ozcengiz, Erkan; Becher, Doerte; Hecker, Michael; Özcengiz, Gülay (2011-04-27)
Whooping cough, caused by the gram negative pathogen Bordetella pertussis, is a worldwide acute respiratory disease that predominantly involves infants. In the present study, surface proteins of B. pertussis Tohama I and Saadet strains were identified by using 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these approaches it was possible to identify 45 and 226 proteins, respectively. When surface proteins of the strains were separated by 2DE and analyzed by Western blotting for their re...
Determination of immunomodulatory effects of SARS-COV-2 structural, non-structural, and accessory proteins on macrophage-like cells in the context of type I IFN antagonism and inflammasome activation
Aydın, Yağmur; Gürsel, Mayda; Department of Biology (2022-2-02)
Infection with SARS-CoV-2 inhibits early type I interferon response but activates inflammasome signaling and pro-inflammatory cytokine production. The virus accomplishes these opposing effects by manipulating the host cell immunity through a multitude of encoded viral proteins of structural, non-structural, or accessory origin. In this thesis, to investigate the immunomodulatory effects of SARS-CoV-2 encoded proteins on type I IFN antagonism and inflammasome activation, five SARS-CoV-2 structural, nonstruct...
An integrative-omics analysis of an industrial clavulanic acid-overproducing Streptomyces clavuligerus
KURT KIZILDOĞAN, ASLIHAN; Celik, Gozde; unsaldi, Eser; ÖZCAN, SERVET; AYAZ GÜNER, ŞERİFE; Özcengiz, Gülay (2022-09-01)
Clavulanic acid (CA) is a clinically important secondary metabolite used to treat infectious diseases. We aimed to decipher complex regulatory mechanisms acting in CA biosynthesis by analyzing transcriptome- and proteome-wide alterations in an industrial CA overproducer Streptomyces clavuligerus strain, namely DEPA and its wild-type counterpart NRRL3585. A total of 924 differentially expressed genes (DEGs) and 271 differentially produced proteins (DPPs) were obtained by RNA-seq and nanoLC-MS/MS analyses, re...
Citation Formats
D. Keser, “Assessment of the immunogenicity and formulation of recombinant proteins from SARS-CoV-2 as vaccine antigens,” M.S. - Master of Science, Middle East Technical University, 2022.