Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides (preprint)

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors can be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped VSV vectors and viral plaque assays. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed for the Spike proteins from Beta, Gamma and Omicron. The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days.

Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation (preprint)

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the proteins function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions.

Covid-19: Global Progress in Developing Vaccines and Mechanisms for Equitable Distribution

The serious unique infectious pneumonia (COVID-19), caused by the new coronavirus (SARS-coV-2) in Wuhan, China in late 2019, has rapidly spread and become a global pandemic. It resulted in crises menacing people's health, lives, international engagement and economic systems. Thus, a vaccine holds most potential for a rapid means of resolving the pandemic before the end of 2021. There are 23 different candidate vaccines worldwide that have entered into clinical trials. Among them, the two that have progressed the fastest are Sinovac Biotech's inactivated vaccine and the recombinant vaccine (ChAdOx1-S) developed by Oxford University, which are already in the third phase of clinical trials. In late April 2020, WHO, EU and the Bill and Melinda Gates Foundation launched the ACT Accelerator Plan to acquire more COVID-19 tools. Also, GAVI, CEPI and WHO are jointly promoting the COVAX Facility, responsible for coordinating and integrating resources among worldwide vaccine developers and manufacturers. In addition to assuming risks of vaccine development, they also provide early investment in candidate vaccine products. These efforts increase chances of successful vaccine development as they expedite safe, efficient development and mass manufacturing of COVID-19 vaccines. This will result in the common goal of equitable distribution of vaccines for every nation.