COVID-19: SARS2-38 antibody easily neutralized all the variants


The virus that causes COVID-19 today is not the same as the one that first sickened people way back in December 2019. Many of the variants circulating now are partially resistant to some of the antibody-based therapeutics that were developed based on the original virus.

As the pandemic continues, more variants inevitably will arise, and the problem of resistance will only grow.

Researchers at Washington University School of Medicine in St. Louis have identified an antibody that is highly protective at low doses against a wide range of viral variants.

Moreover, the antibody attaches to a part of the virus that differs little across the variants, meaning that it is unlikely for resistance to arise at this spot.

The findings, available online in the journal Immunity, could be a step toward developing new antibody-based therapies that are less likely to lose their potency as the virus mutates.

“Current antibodies may work against some but not all variants,” said senior author Michael S. Diamond, MD, Ph.D., the Herbert S. Gasser Professor of Medicine. “The virus will likely continue to evolve over time and space. Having broadly neutralizing, effective antibodies that work individually and can be paired to make new combinations will likely prevent resistance.”

SARS-CoV-2, the virus that causes COVID-19, uses a protein called spike to attach to and invade cells in the body’s respiratory tract. Antibodies that prevent spike from attaching to cells neutralize the virus and prevent disease.

Many variants have acquired mutations in their spike genes that allow them to evade some antibodies generated against the original strain, undermining the effectiveness of antibody-based therapeutics.

To find neutralizing antibodies that work against a wide range of variants, the researchers began by immunizing mice with a key part of the spike protein known as the receptor-binding domain. Then, they extracted antibody-producing cells and obtained 43 antibodies from them that recognize the receptor-binding domain.

Along with Diamond, the research team included co-first authors Laura VanBlargan, Ph.D., a staff scientist; Lucas J. Adams, an MD/Ph.D. student; and Zhuoming Liu, Ph.D., a staff scientist; as well as co-author Daved Fremont, Ph.D., a professor of pathology & immunology, of biochemistry & molecular biophysics and of molecular microbiology.

The researchers screened the 43 antibodies by measuring how well they prevented the original variant of SARS-CoV-2 from infecting cells in a dish.

Nine of the most potent neutralizing antibodies were then tested in mice to see whether they could protect animals infected with the original SARS-CoV-2 from disease. Multiple antibodies passed both tests, with varying degrees of potency.

The researchers selected the two antibodies that were most effective at protecting mice from disease and tested them against a panel of viral variants. The panel comprised viruses with spike proteins representing all four variants of concern (alpha, beta, gamma and delta), two variants of interest (kappa and iota), and several unnamed variants that are being monitored as potential threats.

One antibody, SARS2-38, easily neutralized all the variants. Moreover, a humanized version of SARS2-38 protected mice against disease caused by two variants: kappa and a virus containing the spike protein from the beta variant.

The beta variant is notoriously resistant to antibodies, so its inability to resist SARS2-38 is particularly remarkable, the researchers noted.

Further experiments pinpointed the precise spot on the spike protein recognized by the antibody, and identified two mutations at that spot that could, in principle, prevent the antibody from working.

These mutations are vanishingly rare in the real world, however. The researchers searched a database of nearly 800,000 SARS-CoV-2 sequences and found escape mutations in only 0.04% of them.

“This antibody is both highly neutralizing (meaning it works very well at low concentrations) and broadly neutralizing (meaning it works against all variants),” said Diamond, who is also a professor of molecular microbiology and of pathology & immunology. “That’s an unusual and very desirable combination for an antibody.

Also, it binds to a unique spot on the spike protein that isn’t targeted by other antibodies under development. That’s great for combination therapy. We could start thinking about combining this antibody with another one that binds somewhere else to create a combination therapy that would be very difficult for the virus to resist.”

n this study, we describe a panel of potently neutralizing murine mAbs against the RBD of SARS-CoV-2 that bind several epitopes proximal to the receptor binding motif (RBM) of the RBD or at the base of the RBD. Although some neutralizing mAbs demonstrated limited ability to protect against infection by the historical SARS-CoV-2 WA1/2020 strain in a mouse disease model and selected for rapid escape in vivo, others protected completely in the context of prophylactic or therapeutic administration.

Two protective mAbs, SARS2-02 and SARS2-38, showed variable capacity to neutralize variants of concern (VOCs): SARS2-02 binds an epitope that includes residues E484 and L452 and has reduced potency against strains (B.1.429, B.1.351, and B.1.1.28) encoding these mutations. In contrast, SARS2-38 binds an epitope centered on residues K444 and G446 and potently neutralized all tested VOCs.

Analysis of a cryo-electron microscopy (cryo-EM) structure of SARS2-38 bound to spike reveals that this mAb binds a conserved epitope on the RBD that is also engaged, albeit through distinct geometries, by other neutralizing and protective human mAbs. Thus, treatment with mAbs or induction of pAbs targeting this conserved region of the RBD may confer protection against many emerging SARS-CoV-2 variants.

In this study, we describe and characterize extensively a panel of mAbs that bind the RBD of the SARS-CoV-2 spike protein. Several anti-RBD mAbs protected in vivo against SARS-CoV-2 infection in K18-hACE2 transgenic mice. While the less potently neutralizing mAbs directed against epitopes on the base of RBD (SARS2-10, SARS2-31, and SARS2-03) exhibited diminished protection against weight loss, induction of inflammatory cytokines and chemokines in the lung, and viral infection in the lung and nasal wash than mAbs recognizing the RBM, neutralization potency was not the only predictor of in vivo efficacy.

Indeed, SARS2-71 neutralized SARS-CoV-2 with a potency similar to that of protective mAbs SARS2-02 and SARS2-38, yet failed to confer protection in mice. Notwithstanding this result, antibodies targeting proximal competing epitopes as SARS2-71, such as COV2-2196, have been shown to confer protection in vivo (Zost et al., 2020). The failure of SARS2-71 to protect in particular is likely due to the emergence of the escape variant S477N in vivo.

This finding demonstrates that SARS-CoV-2 can rapidly escape from mAb inhibition in vivo, and that mAb or mAb cocktails that prevent or limit rapid escape mutant generation likely will have greater therapeutic utility. While currently authorized mAb treatments include cocktails, the emergence of VOCs that are resistant to one or both component mAbs could compromise drug efficacy.

The most potently inhibitory mAbs in our panel bind epitopes within or proximal to the RBM and inhibit spike interaction with human ACE2 by ELISA, as observed for other anti-SARS-CoV-2 mAbs (Zost et al., 2020). Several of these mAbs inhibited viral attachment to Calu-3 and Vero-TMPRSS2-ACE2 cells, but not to Vero E6 cells or Vero-TMPRSS2 cells. Infection of Vero E6 cells by SARS-CoV-2 is dependent on endogenous levels of monkey ACE2 expression, as pretreatment with anti-ACE2 mAbs inhibits infection (Hoffmann et al., 2020).

However, other host factors such as heparan sulfate also can mediate virus attachment to cells (Chu et al., 2021; Clausen et al., 2020). If binding to other cell surface ligands occurs prior to the RBD-ACE2 interaction, mAbs that block ACE2 binding may not efficiently inhibit SARS-CoV-2 attachment, but instead block a downstream ACE2-dependent entry step. This idea is supported by our data showing that several neutralizing mAbs block viral internalization in Vero E6 cells. Moreover, anti-RBD mAbs have only moderate decreases in neutralization potency when added after virus absorption to Vero E6 cells.

In contrast, when SARS-CoV-2 attaches to the cell surface via human ACE2 interaction, such as in Vero-TMPRSS2-ACE2 cells, the addition of anti-RBD mAbs after attachment failed to neutralize virus infection. A higher density of ACE2 or higher affinity of spike protein for human ACE2 (relative to monkey ACE2) on the Vero-TMPRSS2-ACE2 cells may drive initial virus attachment through the RBD-ACE2 interaction and explain why mAbs can block this step in these cells.

Together, these data suggest that the ability of anti-RBD mAbs to inhibit SARS-CoV-2 attachment depends on cellular ACE2 expression levels and thus can be cell-type dependent. As these mechanistic differences did not markedly affect mAb potency on the different cellular substrates, we conclude that in the cells we tested there is a required entry interaction with ACE2 either at attachment, post-attachment, or internalization steps.

Several mutations and deletions in emerging VOCs occur in the NTD and RBD that allow them to avoid antibody recognition, including RBD mutations K417N/T (B.1.351 and B.1.1.28), N439K (B.1.222), L452R (B.1.429), Y453R (B.1.1.298), E484K (B.1.351 and B.1.1.28), and N501Y (B.1.1.7, B.1.351, and B.1.1.28) (reviewed by (Plante et al., 2021)), highlighting the importance of developing mAbs against a variety of spatially distinct epitopes.

In our panel, SARS2-38 potently neutralized viruses encoding any of the above mutations, did not readily select for escape mutations with authentic SARS-CoV-2 strains, and retained therapeutic activity in vivo against a virus containing substitutions of one of the key VOCs (B.1.351). Moreover, functional mapping and structural analysis of the binding footprint of SARS-CoV-2 defined a conserved RBD epitope that could be recognized by other potently neutralizing and protective human mAbs.

Relatively few antibodies targeting similar epitopes to SARS2-38 have been described, and those characterized bind the RBD in distinct orientations with heavy chain predominance (Fig 7A). These include murine mAb 2H04, as well as human mAbs REGN10987, COV2-2130, and, though less similar, S309 (Dong et al., 2021; Hansen et al., 2020; Liu et al., 2021; Pinto et al., 2020). SARS2-38 differs in two respects: (a) the baseline neutralizing activity of SARS2-38 against WA1/2020 in Vero cells (EC50, ∼5 ng/mL) is 30-fold, 20-fold, and 16-fold more potent than that of 2H04, COV2-2130, and S309, respectively (Alsoussi et al., 2020; Pinto et al., 2020; Zost et al., 2020); and (b) SARS2-38 retains strong neutralization potency against all VOCs evaluated in this study, whereas the inhibitory activity 2H04, COV2-2130, and S309 is reduced somewhat against B.1.1.7, B.1.429, and B.1.351, respectively ((Chen et al., 2021c; Chen et al., 2021d) and R.E.C. and M.S.D. unpublished results). Similarly, REGN10987 exhibited a 10-fold reduction in neutralizing activity against B.1.429 compared to WA1/2020 (Chen et al., 2021c; Hansen et al., 2020; Wang et al., 2021).

A structural examination of these other antibody footprints within the context of VOC mutations does not provide a direct explanation for some of the resistance (Fig 7B). Instead, allostery may play a role. Whereas other broadly and potently neutralizing mAbs (including mAbs 2C08, COV2-2196, 58G6, 510A5, and S2X259) have been reported that bind RBD epitopes at residues G476, F486, and N487, or loops near residues 369-386, 404-411, 450-458, and 499-508 (Dong et al., 2021; Li et al., 2021; Schmitz et al., 2021; Tortorici et al., 2021), SARS2-38 targets a distinct epitope proximal to the RBM and has been evaluated functionally against a larger panel of authentic viruses containing sequences corresponding to emerging SARS-CoV-2 variants.

Figure 7.
Figure 7.
Similarity of SARS2-38 epitope to other mAbs.
(A) Structural comparison of SARS2-38 to mAbs targeting a similar region of the RBD. (B) Multiple sequence alignment of the SARS-CoV-2 RBD (residues 333-518) with mAb binding footprints as determined by qtPISA analysis. For SARS2-38, heavy chain, light chain, and shared contacts are shown in blue, cyan, and dark blue, respectively. For 2H04, heavy chain, light chain, and shared contacts are shown in orange, pale orange, and dark orange, respectively. For REGN10987, heavy chain, light chain, and shared contacts are shown in green, pale green, and dark green, respectively. For S309, heavy chain, light chain, and shared contacts are shown in magenta, pale purple, and purple, respectively. For COV2-2130, heavy chain, light chain, and shared contacts are shown in red, pale red, and brick red, respectively. Secondary structure annotation is displayed above the alignment in yellow, with ACE2 contacts designated by green triangles (Lan et al., 2020). VOC substitutions are designated below the alignment by red triangles.

In summary, we have characterized a panel of anti-SARS-CoV-2 mAbs, defined their cellular mechanism of action in different cells, tested in vitro neutralizing and in vivo protection capacity against historical and circulating variants, and determined the structure of the viral spike protein bound to SARS2-38, a potently and broadly neutralizing mAb that recognizes emerging VOCs.

A humanized version of SARS2-38 confers therapeutic protection against the WA1/2020 isolate and a SARS-CoV-2 strain expressing the spike protein of B.1.351. The conserved epitope bound by SARS2-38 thus may be a potential target for antibodies with therapeutic potential or that are induced by effective vaccines with more limited potential for resistance against VOCs.

reference link :

More information: Laura A. VanBlargan et al, A potently neutralizing SARS-CoV-2 antibody inhibits variants of concern by utilizing unique binding residues in a highly conserved epitope, Immunity (2021). DOI: 10.1016/j.immuni.2021.08.016


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