ABSTRACT
As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 [A] and 2.42 [A], respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 [A], 7.76 [A], 6.47 [A], 3.45 [A], and 7.32 [A]) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.
ABSTRACT
Summary ParagraphWe identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone1, a compound in clinical trials for anti-fibrotic and anti-inflammatory applications2, as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry3. We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir4. Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginones translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
ABSTRACT
The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
