Comparative Immunogenicity of the Recombinant Receptor-Binding Domain of Protein S SARS-CoV-2 Obtained in Prokaryotic and Mammalian Expression Systems

The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

A novel bacterial protease inhibitor adjuvant in RBD-based COVID-19 vaccine formulations increases neutralizing antibodies, specific germinal center B cells and confers protection against SARS-CoV-2 infection.

In this work we evaluated recombinant receptor binding domain (RBD) based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus Alum, AddaS03, AddaVax or the combination of Alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and Alum as adjuvants have a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus Alum, AddaVax or AddaS03. Antibodies induced with the formulation containing U-Omp19 not only increased their neutralization capacity against the wild-type virus but also cross neutralized alpha, lambda and gamma variants with similar potency. Also, addition of U-Omp19 to vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+Alum formulation induced RBD-specific Th1 and CD8+ T cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal SARS-CoV-2 challenge of K18-hACE2 mice.

Generation and Characterization of a Nanobody Against SARS-CoV

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

Longitudinal analysis to characterize classes and subclasses of antibody responses to recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients in Thailand

Serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might contribute to confirming the suspected coronavirus disease 2019 (COVID-19) in patients not detected with molecular assays. Human antibodies that target the host angiotensin-converting enzyme 2-binding domain of the viral spike protein are a target for serodiagnosis and therapeutics. This study aimed to characterize the classes and subclasses of antibody responses to a recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients and investigated the reactivity of these antibodies in patients with other tropical infections and healthy individuals in Thailand. ELISAs for IgM, IgA, IgG and IgG subclasses based on RBD antigen were developed and tested with time series of 27 serum samples from 15 patients with COVID-19 and 60 samples from pre-COVID-19 outbreaks including acute dengue fever, murine typhus, influenza, leptospirosis and healthy individuals. Both RBD-specific IgA and IgG were detected in only 21% of the COVID-19 patients in the acute phase. The median IgA and IgG levels were significantly higher in the convalescent serum sample compared to the acute serum sample (P < 0.05). We observed the highest correlation between levels of IgG and IgA (rho = 0. 92). IgG1 and IgG3 were the major IgG subclasses detected in SARS-CoV-2 infection. Only acute IgG3 level was negatively associated with viral detection based on RT-PCR of ORF1ab gene (rho = -0.57). The median IgA and IgG levels in convalescence sera of COVID-19 patients were significantly higher than healthy individuals and convalescent sera of other febrile infectious patients. The analyses of antibody classes and subclasses provide insights into human immune responses against SARS-CoV-2 during natural infection and interpretation of antibody assays.

The SARS-CoV-2 spike L452R-E484Q co-variant in the Indian B.1.617 strain Showed Significant Reduction in the Neutralization Activity of Immune Sera

To assess the impact of the key non-synonymous amino acid substitutions in the RBD of the spike protein of SARS-CoV-2 variant B.1.617.1 (dominant variant identified in the current India outbreak) on the infectivity and neutralization activities of the immune sera, L452R and E484Q (L452R-E484Q, “co-variant”) pseudotyped virus was constructed (with the D614G background). The impact on the binding with the neutralizing antibodies was also assessed by an ELISA assay. Pseudotyped virus carrying a L452R-E484Q co-variant showed a comparable infectivity compared with D614G. However, there was a significant reduction in the neutralization activity of the immune sera from non-human primates vaccinated with a recombinant Receptor binding domain (RBD) protein, convalescent patients, and healthy vaccinees vaccinated with an mRNA vaccine. In addition, there was a reduction in the binding of L452R-E484Q-D614G protein to the antibodies of the immune sera from vaccinated non-human primates. These results highlight the interplay between infectivity and other biologic factors involved in the natural evolution of SARS-CoV-2. Reduced neutralization activities against the L452R-E484Q co-variant will have impact to the health authorities planning and implications to the vaccination strategy/new vaccine development.

SARS-COV-2 Recombinant Receptor-Binding-Domain (RBD) Induces Neutralising Antibodies Against Variant Strains of SARS-CoV-2 and SARS-CoV-1

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them are targeting the spike protein (or virion) in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested the utility of SARS-CoV-2 spike RBD as a vaccine antigen and explore the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results indicate RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralisation of variants B1.1.7 and B.1.351 and to a lesser extent, SARS-CoV- 1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.