Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative

Abstract

The Receptor Binding Domain of SARS-CoV-2 is the primary target of neutralizing antibodies. We fused our previously described, highly thermotolerant glycan engineered monomeric RBD to a heterologous non-immunogenic trimerization domain derived from cartilage matrix protein. The protein was expressed at a good yield of ∼80-100 mg/liter in Expi293 cells, as well as in both CHO and HEK293 stable cell lines. The designed trimeric RBD was observed to form homogeneous disulfide-linked trimers. When lyophilized, the trimer possessed remarkable functional stability to transient thermal stress of upto 100 °C and was stable to long term storage of over 4 weeks at 37 °C. Two immunizations with an AddaVax adjuvanted formulation elicited antibodies with high endpoint neutralizing titers against replicative virus with geometric mean titers of ∼1114 and 1940 in guinea pigs and mice respectively. In pseudoviral assays, corresponding titers were ∼3600 and ∼16050, while the corresponding value for human convalescent sera was 137. Similar results were obtained with an Alhydrogel, CpG combination adjuvant. The same immunogen was expressed in Pichia pastoris, but this formed high molecular weight aggregates and elicited much lower ACE2 competing antibodies than mammalian cell expressed protein. The excellent thermotolerance, high yield, and robust immunogenicity of such trimeric RBD immunogens suggest that they are a promising modality to combat COVID-19.ImportanceSARS-CoV-2 is the causative agent of the ongoing COVID-19 pandemic. The viral surface exposed Spike glycoprotein is the target of neutralizing antibodies of which a major fraction targets the receptor binding domain (RBD). Thus RBD derived immunogens are attractive vaccine candidates. Monomeric, mammalian cell expressed RBD protein elicits low to moderate titers of neutralizing antibodies. We designed a highly expressed, trimeric RBD derivative with a non-immunogenic trimerization domain. In guinea pigs and mice respectively, this derivative induces 20-300 fold higher neutralizing antibody titers relative to convalescent human sera, while remaining conformationally intact after incubation for over four weeks at 37 °C and for ninety minutes at 100 °C when lyophilized. Such trimeric RBD formulations should not require a cold chain. Additionally, the high titers of neutralizing antibodies should buffer against viral sequence variation. These are both highly desirable attributes for a COVID-19 vaccine, especially in resource limited settings.

Periodic Mesoporous Organosilica as a Nanoadjuvant for Subunit Vaccines Elicits Potent Antigen-Specific Germinal Center Responses by Activating Naive B Cells

“…Unlike the HIV-1 vaccine, the PMO-RBD and alum adjuvant groups induced relatively low and similar levels of IgG titers after the third immunization, and this may be due to the weakened immunogenicity of monomeric RBD compared to ZCM. 37 The antibody isotype revealed that both PMO-RBD and alum adjuvant primarily induced IgG1 that is the primary antibody in serum that protects patients 38 (Supporting Information Figure 15B). Antibody affinity analysis demonstrated that the antibodies induced by all three vaccine groups possessed similar affinities (Supporting Information Figure 15C), and this was consistent with the HIV-1 vaccine results.…”