Uncovering the structural flexibility of SARS-CoV-2 glycoprotein spike variants

The severe acute respiratory syndrome CoV-2 rapidly spread worldwide, causing a pandemic. After a period of evolutionary stasis, a set of SARS-CoV-2 mutations has arisen in the spike, the leading glycoprotein at the viral envelope and the primary antigenic candidate for vaccines against the 2019 CoV disease (COVID-19). Here, we present comparative biochemical data of the glycosylated full-length ancestral and D614G spike together with three other highly transmissible strains classified by the World Health Organization as variants of concern (VOC): beta, gamma, and delta. By showing that only D614G early variant has less hydrophobic surface exposure and trimer persistence at mid-temperatures, we place D614G with features that support a model of temporary fitness advantage for virus spillover worldwide. Further, during the SARS-CoV-2 adaptation, the spike accumulates alterations leading to less structural rigidity. The decreased trimer stability observed for the ancestral and the gamma strain and the presence of D614G uncoupled conformations mean higher ACE-2 affinities when compared to the beta and delta strains. Mapping the energetic landscape and flexibility of spike variants is necessary to improve vaccine development.

Equine hyperimmune globulin raised against the SARS-CoV-2 spike glycoprotein has extremely high neutralizing titers

We used the trimeric spike (S) glycoprotein (residues 1-1208) in the prefusion conformation to immunize horses for production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by anti-spike ELISA were above 1:1,000,000, and neutralizing antibody titer was 1:14,604 (average PRNT90), which is 140-fold higher than the average neutralizing titer of plasma from three convalescent COVID-19 patients analyzed for comparison. Using the same technology routinely used for industrial production of other horse hyperimmune products, plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding a F(ab)2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Repeating the hyperimmunization in a second group of horses confirmed the very high neutralizing titers in serum and in a GMP clinical F(ab)2 lot. Virus-neutralizing activity in samples from mice that received the F(ab)2 preparation was detected even three days after injection, indicating an appropriate half-life for therapeutic intervention. These results supported the design of a clinical trial (identifier NCT04573855) to evaluate safety and efficacy of this horse F(ab)2 preparation.

AN AFFORDABLE ANTI-SARS-COV-2 SPIKE ELISA TEST FOR EARLY DETECTION OF IgG SEROCONVERSION SUITED FOR LARGE-SCALE SURVEILLANCE STUDIES IN LOW-INCOME COUNTRIES

Accurate serological tests are essential tools to allow adequate monitoring and control of COVID-19 spread. Production of a low-cost and high-quality recombinant viral antigen can enable the development of reliable and affordable serological assays, which are urgently needed to facilitate epidemiological surveillance studies in low-income economies. Trimeric SARS-COV-2 spike (S) protein was produced in serum-free, suspension-adapted HEK293 cells. Highly purified S protein was used to develop an ELISA, named S-UFRJ test. It was standardized to work with different types of samples: (i) plasma or serum from venous blood samples; (ii) eluates from dried blood spots (DBS) obtained by collecting blood drops from a finger prick. We developed a cost-effective, scalable technology to produce S protein based on its stable expression in HEK293 cells. Using this recombinant antigen, we presented a workflow for test development in the setting of a pandemic, starting from limited amounts of samples up to reaching final validation with hundreds of samples. Test specificity was determined to be 98.6%, whereas sensitivity was 95% for samples collected 11 or more days after symptoms onset. A ROC analysis allowed optimizing the cut-off and confirming the high accuracy of the test. Endpoint titers were shown to correlate with virus neutralization assessed as PRNT90. There was excellent agreement between plasma and DBS samples, significantly simplifying sample collection, storing, and shipping. An overall cost estimate revealed that the final retail price could be in the range of one US dollar. The S-UFRJ assay developed herein meets the quality requirements of high sensitivity and specificity. The low cost and the use of mailable DBS samples allow for serological surveillance and follow-up of SARS-CoV-2 vaccination of populations regardless of geographical and socio-economic aspects. We hope the detailed guidelines for the development of an affordable and accurate anti-spike SARS-COV-2 ELISA, such as S-UFRJ described here, will stimulate governmental and non-governmental health agencies in other countries to engage in much-needed large-scale studies monitoring the spread and immunity to SARS-COV-2 infection.