| dc.description.abstract | Coronaviruses (CoVs) are a global health threat due to their potential for widespread transmission including in humans. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent responsible for the COVID-19 pandemic, is the ninth known human-infecting coronavirus and the seventh to be identified within the past two decades. The majority of coronaviruses infecting humans are of zoonotic origin, including all previously known human CoV strains. Animal coronaviruses such as bovine coronavirus (BCoV), porcine transmissible gastroenteritis virus (TGEV), infectious bronchitis virus (IBV), and feline infectious peritonitis virus (FIPV) have been recognized since the late 1930s. Bats, in particular, serve as a significant natural reservoir for CoVs, a fact underscored by the identification of approximately 400 novel coronavirus strains through extensive surveillance efforts. Multiple zoonotic alpha-CoV and beta-CoV strains have also been detected in bat populations across Western Europe. Sarbecoviruses are a subgenus of the Betacoronavirus genus within the Coronaviridae family. This group includes several zoonotic and human-infecting coronaviruses, most notably SARS-CoV-1 and SARS-CoV-2. Sarbecoviruses primarily originate from bat reservoirs, particularly those of the Rhinolophus (horseshoe bat) genus, and exhibit significant genetic diversity. Due to their zoonotic potential and history of causing severe human disease, sarbecoviruses are a major focus of surveillance and vaccine development efforts aimed at preventing future outbreaks.
The first emergence of SARS-CoV-1 was reported in China in November 2002, subsequently escalating into a global epidemic that affected 28 countries, resulting in numerous infections and fatalities. Later investigations identified Chinese horseshoe bats (Rhinolophus spp.) as hosts for genetically diverse CoVs closely related to both SARS-CoV-1 and SARS-CoV-2. Similarly, in 2012, the initial case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in humans was documented in Saudi Arabia. To date, over 2,000 cases of MERS-CoV have been reported across 27 countries, with a high associated mortality rate. The entry of CoVs into host cells is mediated by the spike (S) glycoprotein, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The S protein comprises two subunits: S1 and S2. The S1 subunit, located at the N-terminal region, contains the receptor-binding domain (RBD), which is responsible for recognizing and binding to the host cell receptor. The RBDs of SARS-CoV-2, SARS-CoV-1, and MERS-CoV have been identified as highly immunogenic, making them promising targets for vaccine development across various platforms due to their immunodominant nature, robust antigenic properties and capacity to elicit strong immune responses. Building upon these insights, our objective was to utilize stabilized RBDs to develop a broad-spectrum pan-sarbecovirus vaccine formulation.
Chapter 1 provides a brief overview of coronaviruses with an emphasis on SARS-CoV-2 and SARS-related sarbecoviruses, structural organization and function of the spike (S) surface glycoprotein. It summarizes the different vaccine strategies used to combat coronavirus mediated respiratory infections and mentions the approaches used to develop effective and broadly protective SARS-CoV-2 and pan-sarbecovirus vaccines.
In Chapter 2, we have compared the immunogenicity of stabilized RBD based immunogens derived from mammalian (Expi293F) and Pichia (Komagatella phaffi) cells. The Pichia expression system provides a cost-effective alternative for large scale immunogen production. Even though Pichia expressed protein (pRBD) is hyperglycosylated, in comparison to mammalian derived RBD (mRBD), we observed elicitation of comparable RBD specific antibody titres in pRBD immunized animals, however the breadth of neutralization and protection against morbidity was better for the mRBD immunized group in a high dose immunization study. To understand the differences in epitope targeting between these groups because of differences in glycan shielding, we performed epitope mapping experiment using yeast surface display of SARS-CoV-2 mutant library coupled with FACS and NGS.
Chapter 3 discusses a stabilization approach for SARS-CoV-2 RBD through incorporation of cross strand disulfides. Previous work from the lab has demonstrated that engineered disulfides at exposed non-hydrogen bonded (NHB) registered pairs in antiparallel strands significantly enhance protein stability. An automated procedure for identification of NHB pairs was developed in-house. In this study, we have experimentally validated the effect of disulfide incorporation at NHB pairs in the antiparallel -strands of SARS-CoV-2 RBD. We observed an increased thermal and proteolytic stability of the SARS-CoV-2 RBD derivatives upon introduction of the predicted cross-strand disulfide. However, this did not translate to increased immunogenicity and the stabilized RBD derivatives with or without the introduced disulfide showed higher antibody titers in mice as compared to the wild type (WT) RBD. Neutralization assays are underway.
In Chapter 4, we describe the use of an RBD based cocktail to generate pan-sarbecovirus antibody responses in mice. We expressed and purified stabilized, trimeric RBDs derived from diverse sarbecoviruses from all clades and used them in an adjuvanted cocktail format to immunize animals. We observed increased yields and thermal stability upon incorporating stabilizing mutations without any effect on the conformational integrity. Broad pseudoviral neutralization response was elicited in naïve as well as SARS-CoV-2 pre immunized mice. The stabilized RBD cocktail also showed good long-term thermal stability upon lyophilization.
Within the spike protein, the S2 subunit is highly conserved across sarbecoviruses. In a recent study from the lab we showed that fusion of SARS-CoV-2 RBD and S2 subunits resulted in enhanced purification yield and immunogenicity. In Chapter 5, we attempted to utilize this approach for our pan-sarbecovirus vaccine formulation. We made genetic fusions of stabilized sarbecovirus RBDs with the S2 subunit derived from SARS-CoV-2 and immunized mice with stabilized RBD-S2 monomer cocktail. Our stabilized RBD-S2 cocktail elicited anitbodies that neutralized strains across sarbecovirus clades in naïve and pre-immunized mouse models. The cocktail also exhibited good thermal stability. We are currently comparing protection against sarbecovirus challenge in mice immunized with either stabilized RBD trimer or stabilized RBD-S2 cocktails.
In Chapter 6, we explored the use of chimeric RBDs for elicitation of a broad neutralization response. We designed and expressed second-generation chimeric RBDs comprising of receptor binding motif (RBM) from a highly divergent strain of SARS-CoV-2 (XBB1.5) graft on to a scaffold derived from distant sarbecoviruses. These chimeric RBDs were first screened for proper folding and expression by yeast surface display (YSD) and then expressed in mammalian cells via transient transfection. The purified proteins elicited neutralizing responses in a SARS CoV-2 pre-immunized mouse model. The neutralization breadth is currently being assessed.
Appendix 1 describes the design and biophysical characterization of MERS-CoV immunogens. These immunogens were RBD and RBD-S2 derivatives of MERS spike protein expressed in mammalian cells and purified for characterization.
In Appendix 2, we discuss the design and characterization of Hemagglutinin (HA) stem based immunogens for generating a broad response against group-1 Influenza viruses. | en_US |
