The hunt for an effective treatment for COVID-19 has led one team of researchers to find an improbable ally for their work: a llama named Winter.
The team — from The University of Texas at Austin, the National Institutes of Health and Ghent University in Belgium — reports their findings about a potential avenue for a coronavirus treatment involving llamas on May 5 in the journal Cell.
The paper is currently available online as a “pre-proof,” meaning it is peer-reviewed but undergoing final formatting.
The researchers linked two copies of a special kind of antibody produced by llamas to create a new antibody that binds tightly to a key protein on the coronavirus that causes COVID-19.
This protein, called the spike protein, allows the virus to break into host cells. Initial tests indicate that the antibody blocks viruses that display this spike protein from infecting cells in culture.
“This is one of the first antibodies known to neutralize SARS-CoV-2,” said Jason McLellan, associate professor of molecular biosciences at UT Austin and co-senior author, referring to the virus that causes COVID-19.
The team is now preparing to conduct preclinical studies in animals such as hamsters or nonhuman primates, with the hopes of next testing in humans.
The goal is to develop a treatment that would help people soon after infection with the virus.
“Vaccines have to be given a month or two before infection to provide protection,” McLellan said. “With antibody therapies, you’re directly giving somebody the protective antibodies and so, immediately after treatment, they should be protected.
The antibodies could also be used to treat somebody who is already sick to lessen the severity of the disease.”
This would be especially helpful for vulnerable groups such as elderly people, who mount a modest response to vaccines, which means that their protection may be incomplete.
Health care workers and other people at increased risk of exposure to the virus can also benefit from immediate protection.
When llamas’ immune systems detect foreign invaders such as bacteria and viruses, these animals (and other camelids such as alpacas) produce two types of antibodies: one that is similar to human antibodies and another that’s only about a quarter of the size.
These smaller ones, called single-domain antibodies or nanobodies, can be nebulized and used in an inhaler.
“That makes them potentially really interesting as a drug for a respiratory pathogen because you’re delivering it right to the site of infection,” said Daniel Wrapp, a graduate student in McLellan’s lab and co-first author of the paper.
Winter, the llama, is 4 years old and still living on a farm in the Belgian countryside along with approximately 130 other llamas and alpacas. Her part in the experiment happened in 2016 when she was about 9 months old and the researchers were studying two earlier coronaviruses: SARS-CoV-1 and MERS-CoV.
In a process similar to humans getting shots to immunize them against a virus, she was injected with stabilized spike proteins from those viruses over the course of about six weeks.
Next, researchers collected a blood sample and isolated antibodies that bound to each version of the spike protein. One showed real promise in stopping a virus that displays spike proteins from SARS-CoV-1 from infecting cells in culture.
“That was exciting to me because I’d been working on this for years,” Wrapp said. “But there wasn’t a big need for a coronavirus treatment then. This was just basic research. Now, this can potentially have some translational implications, too.”
The team engineered the new antibody that shows promise for treating the current SARS-CoV-2 by linking two copies of the llama antibody that worked against the earlier SARS virus.
They demonstrated that the new antibody neutralizes viruses displaying spike proteins from SARS-CoV-2 in cell cultures.
The scientists were able to complete this research and publish it in a top journal in a matter of weeks thanks to the years of work they’d already done on related coronaviruses.
McLellan also led the team that first mapped the spike protein of SARS-CoV-2, a critical step toward a vaccine. (Wrapp also co-authored that paper along with other authors on the current Cell paper, including UT Austin’s Nianshuang Wang, and Kizzmekia S. Corbett and Barney Graham of the National Institute of Allergy and Infectious Diseases’ Vaccine Research Center.)
Besides Wrapp, the paper’s other co-first author is Dorien De Vlieger, a postdoctoral scientist at Ghent University’s Vlaams Institute for Biotechnology (VIB), and the other senior authors besides McLellan are Bert Schepens and Xavier Saelens, both at VIB.
The first antibodies the team identified in the initial SARS-CoV-1 and MERS-CoV tests included one called VHH-72, which bound tightly to spike proteins on SARS-CoV-1.
In so doing, it prevented a pseudotyped virus — a virus that can’t make people sick and has been genetically engineered to display copies of the SARS-CoV-1 spike protein on its surface — from infecting cells.
When SARS-CoV-2 emerged and triggered the COVID-19 pandemic, the team wondered whether the antibody they discovered for SARS-CoV-1 would also be effective against its viral cousin.
They discovered that it did bind to SARS-CoV-2’s spike protein too, albeit weakly. The engineering they did to make it bind more effectively involved linking two copies of VHH-72, which they then showed neutralizes a pseudotyped virus sporting spike proteins from SARS-CoV-2. This is the first known antibody that neutralizes both SARS-CoV-1 and SARS-CoV-2.
Four years ago, De Vlieger was developing antivirals against influenza A when Bert Schepens and Xavier Saelens asked whether she would be interested in helping to isolate antibodies against coronaviruses from llamas.
“I thought this would be a small side project,” she said. “Now the scientific impact of this project became bigger than I could ever expect. It’s amazing how unpredictable viruses can be.”
The paper’s other authors are Gretel M. Torres, Wander Van Breedam, Kenny Roose, Loes van Schie, Markus Hoffmann, Stefan Pöhlmann, Barney S. Graham and Nico Callewaert.
This work was supported by the National Institute of Allergy and Infectious Diseases (U.S.), VIB, The Research Foundation–Flanders (Belgium), Flanders Innovation and Entrepreneurship (Belgium) and the Federal Ministry of Education and Research (Germany).
Numerous anti-SARS-CoV-1 RBD and anti-MERS-CoV RBD antibodies have been reported and their mechanisms of neutralization can be attributed to the occlusion of the receptor-binding site and to trapping the RBD in the unstable up conformation, effectively acting as a receptor mimic that triggers a premature transition from the prefusion-to-postfusion conformation (Hwang et al., 2006; Walls et al., 2019; Wang et al., 2018; Wang et al., 2015).
Heavy chain-only antibodies (HCAbs), present in camelids, contain a single variable domain (VHH) instead of two variable domains (VH and VL) that make up the equivalent antigen-binding fragment (Fab) of conventional IgG antibodies (Hamers-Casterman et al., 1993).
This single variable domain, in the absence of an effector domain, is referred to as a single-domain antibody, VHH or Nanobody® and typically can acquire affinities and specificities for antigens comparable to conventional antibodies.
VHHs can easily be constructed into multivalent formats and are known to have enhanced thermal stability and chemostability compared to most antibodies (De Vlieger et al., 2018; Dumoulin et al., 2002; Govaert et al., 2012; Laursen et al., 2018; van der Linden et al., 1999). Their advantageous biophysical properties have led to the evaluation of several VHHs as therapeutics against common respiratory pathogens, such as respiratory syncytial virus (RSV) (Detalle et al., 2016; Rossey et al., 2017).
The use of VHHs as biologics in the context of a respiratory infection is a particularly attractive application, since the highly stable VHHs can be nebulized and administered via an inhaler directly to the site of infection (Respaud et al., 2015).
Moreover, due to their stability after prolonged storage, VHHs could be stockpiled as therapeutic treatment options in case of an epidemic. Although therapeutics against MERS-CoV and SARS-CoV-2 are sorely needed, the feasibility of using VHHs for this purpose has not yet been adequately explored. Several MERS-CoV S-directed VHHs have been reported, but their epitopes remain largely undefined, other than being classified as RBD-directed (Stalin Raj et al., 2018; Zhao et al., 2018).
Here we report the isolation of two potently neutralizing VHHs directed against the SARS-CoV-1 and MERS-CoV RBDs. These VHHs were elicited in response to immunization of a llama with prefusion-stabilized SARS-CoV-1 and MERS-CoV S proteins. We solved the crystal structures of these two VHHs in complex with their respective viral epitopes and determined that their mechanisms of neutralization were occlusion of the receptor binding interface and trapping of the RBDs in the up conformation.
We also show that the SARS-CoV-1 RBD-directed VHH exhibits cross-reactivity against the SARS-CoV-2 RBD and is capable of blocking the receptor-binding interface. After engineering this VHH into a bivalent Fc-fusion, we show that this cross-reactive VHH is also capable of potently neutralizing SARS-CoV-2 S pseudoviruses.
In addition, we demonstrate that the VHH-Fc fusion can be produced at high yields in an industry-standard CHO cell system, suggesting that it merits further investigation as a potential therapeutic for the ongoing COVID-19 pandemic.
Isolation of betacoronavirus S-directed VHHs
Our initial aim was to isolate VHHs that could potently neutralize MERS-CoV and SARS-CoV-1.
Therefore, a llama was sequentially immunized subcutaneously twice with SARS-CoV-1 S protein, twice with MERS-CoV S protein, once again with SARS-CoV-1 S and finally with both SARS-CoV-1 and MERS-CoV S protein (S. Figure 1A).
To obtain VHHs directed against these spike proteins, two consecutive rounds of panning were performed using either SARS-CoV-1 S or MERS-CoV S protein. Positive clones were sequenced and multiple sequence alignment and phylogenetic analysis using the neighbor-joining method revealed that seven unique MERS-CoV S and five unique SARS-CoV-1 S VHHs were isolated (S. Figure 1B).
These VHHs and an irrelevant control (RSV F-VHH, directed against the F protein of human respiratory syncytial virus) were subsequently expressed in Pichia pastoris and purified from the yeast medium (Rossey et al., 2017).
The binding of the purified VHHs to prefusion-stabilized MERS-CoV S and SARS-CoV-1 S was confirmed by ELISA (S. Figure 1C). Four clones (MERS VHH-55, −12, −34 and −40), obtained after panning on MERS-CoV S protein, bound with high affinity to prefusion stabilized MERS-CoV S, whereas the affinity of VHH-2, −20 and −15 was 100- to 1000-fold lower. Of the five clones isolated after panning on SARS-CoV-1 S protein, three VHH clones (SARS VHH-72, −1 and −6) interacted strongly with prefusion stabilized SARS-CoV-1 S protein.
VHHs neutralize coronavirus S pseudotyped viruses
To assess the antiviral activity of the MERS-CoV and SARS-CoV S-directed VHHs, in vitro neutralization assays using MERS-CoV England1 S and SARS-CoV-1 Urbani S pseudotyped lentiviruses were performed.
The high affinity MERS VHH-55, −12, −34 and −40 neutralized MERS-CoV S pseudotyped virus with IC50 values ranging from 0.014 to 2.9 µg/mL (0.9 nM to 193.3 nM), while no inhibition was observed for the lower affinity MERS-CoV or SARS-CoV-1 specific VHHs (Table 1).
SARS VHH-72 and −44 were able to neutralize lentiviruses pseudotyped with SARS-CoV-1 S with an IC50 value of 0.14 (9 nM) and 5.5 µg/mL (355 nM), respectively. No binding was observed for SARS VHH-44 to prefusion stabilized SARS-CoV-1 S protein in the ELISA assay. Sequence analysis revealed that the neutralizing MERS-CoV specific VHHs −12, −40 and −55 have highly similar complementarity-determining regions (CDRs), indicating that they likely bind to the same epitope (S. Figure 2). In contrast, the CDRs from the SARS-CoV S-specific VHHs −44 and −72 are highly divergent.
SARS VHH-72 is cross-reactive against WIV1-CoV and SARS-CoV-2
Analysis of 10 available SARS-CoV-1 strain sequences revealed a high degree of conservation in the residues that make up the SARS VHH-72 epitope, prompting us to explore the breadth of SARS VHH-72 binding (S. Figure 5A).
WIV1-CoV is a betacoronavirus found in bats that is closely related to SARS-CoV-1 and also utilizes ACE2 as a host-cell receptor (Ge et al., 2013). Due to the relatively high degree of sequence conservation between SARS-CoV and WIV1-CoV, we expressed the WIV1-CoV RBD and measured binding to SARS VHH-72 by SPR (S. Figure 5B).
SARS VHH-72 also exhibits high-affinity binding to the WIV1-CoV RBD (7.4 nM), demonstrating that it is cross-reactive between these two closely related coronaviruses (S. Figure 5C).
ased on the high degree of structural homology that has been reported between SARS-CoV-1 S and SARS-CoV-2 S (Walls, 2020; Wrapp et al., 2020), we also tested SARS VHH-72 for cross-reactivity against the SARS-CoV-2 RBD-SD1 by SPR (Figure 4). The binding affinity of SARS VHH-72 for the SARS-CoV-2 RBD-SD1 was ∼39 nM.
This diminished binding affinity, compared to the binding of SARS VHH-72 to SARS-CoV-1 RBD, can primarily be attributed to an increase in the dissociation rate constant of this interaction (Figure 4A). The only variant residue on the SARS-CoV-1 RBD that makes direct contact with SARS VHH-72 is Arg426, which is Asn439 in the SARS-CoV-2 RBD (Figure 3C).
VHHs disrupt RBD dynamics and receptor-binding
As stated previously, the RBDs of MERS-CoV S, SARS-CoV-1 S and SARS-CoV-2 S undergo dynamic conformational rearrangements that alternately mask and present their receptor-binding interfaces and potential neutralizing epitopes to host molecules. By aligning the crystal structures of MERS VHH-55 and SARS VHH-72 bound to their respective targets to the full-length, cryo-EM structures of the MERS-CoV, SARS-CoV-1 and SARS-CoV-2 spike proteins, we can begin to understand how these molecules might function in the context of these dynamic rearrangements.
When the MERS-CoV RBDs are all in the down conformation or all in the up conformation, MERS VHH-55 would be able to bind all three of the protomers making up the functional spike trimer without forming any clashes. However, if a down protomer was bound by MERS VHH-55 and the neighboring protomer sampled the up conformation, this RBD would then be trapped in this state by the presence of the neighboring MERS VHH-55 molecule (Figure 5A). This conformational trapping would be even more pronounced upon SARS VHH-72 binding to the full-length SARS-CoV-1 S protein or the full-length SARS-CoV-2 S protein.
Due to the binding angle of SARS VHH-72, when a bound SARS-CoV-1 or SARS-CoV-2 RBD samples the down conformation, it would form dramatic clashes with the S2 fusion subunit, regardless of the conformations of the neighboring RBDs (Figure 5B-C).
Therefore, once a single SARS VHH-72 binding event took place, the bound protomer would be trapped in the up conformation until either SARS VHH-72 was released or until the S protein was triggered to undergo the prefusion-to-postfusion transition. Based on the binding angles of MERS VHH-55 and SARS VHH-72, we can conclude that these molecules would likely disrupt the RBD dynamics in the context of a full-length S protein by trapping the up conformation.
Because this up conformation is unstable and leads to S protein triggering, it is possible that this conformational trapping may at least partially contribute to the neutralization mechanisms of these VHHs.
Based on our structural analysis, we hypothesized that another mechanism by which both MERS VHH-55 and SARS VHH-72 neutralize their respective viral targets is by blocking the interaction between the RBDs and their host-cell receptors.
To test this hypothesis, we performed a BLI-based assay in which the SARS-CoV-1, SARS-CoV-2 and MERS-CoV RBDs were immobilized to biosensor tips, dipped into VHHs and then dipped into wells containing the recombinant, soluble host cell receptors. We found that when tips coated in the MERS-CoV RBD were dipped into MERS VHH55 before being dipped into DPP4, there was no increase in response that could be attributed to receptor binding.
When tips coated with the MERS-CoV RBD were dipped into SARS VHH-72 and then DPP4, a robust response signal was observed, as expected. Similar results were observed when the analogous experiments were performed using the SARS-CoV-1 or SARS-CoV-2 RBDs, SARS VHH-72 and ACE2 (Figure 5D). These results support our structural analysis that both MERS VHH-55 and SARS VHH-72 are capable of neutralizing their respective viral targets by directly preventing host-cell receptor binding.
Bivalent SARS VHH-72 is capable of potently neutralizing SARS-CoV-2 S pseudoviruses
Despite the relatively high-affinity binding that was observed by SPR between SARS VHH-72 and the SARS-CoV-2 RBD, this binding could not be detected by ELISA nor was SARS VHH-72 capable of neutralizing SARS-CoV-2 S VSV pseudoviruses, possibly due to the high off-rate constant, whereas SARS-CoV-1 pseudotypes were readily neutralized (Figure 6A-D).
In an attempt to overcome this rapid dissociation, we engineered two bivalent variants of SARS VHH-72. These included a tail-to-head fusion of two SARS VHH-72 molecules connected by a (GGGGS)3 linker (VHH-72-VHH-72) and a genetic fusion of SARS VHH-72 to the Fc domain of human IgG1 (VHH-72-Fc) (S. Figure 6A-C).
These bivalent SARS VHH-72 constructs were able to bind to both prefusion SARS-CoV-1 S and SARS-CoV-2 RBD-SD1 as demonstrated by ELISA and by a dose-dependent reduction in the binding of SARS-CoV-2 RBD-SD1 to Vero E6 cells (Figure 6C-D and S. Figure 6B-C). We could also detect binding of both of these constructs to full length SARS-CoV-1 S and SARS-CoV-2 S expressed on the surface of mammalian cells (S. Figure 6D-E).
Supernatants of HEK 293S cells transiently transfected with VHH-72-Fc exhibited neutralizing activity against both SARS-CoV-1 and SARS-CoV-2 S VSV pseudoviruses in the same assay which showed no such cross-reactive neutralization for monovalent SARS VHH-72 (Figure 6E-F).
A BLI experiment measuring binding of VHH-72-Fc to immobilized SARS-CoV-2 RBD-SD1 further confirmed that bivalency was able to compensate for the high off-rate constant of the monomer (Figure 7A). Furthermore, cross-neutralizing VHH-72-Fc construct reached expression levels of ∼300 mg/L in ExpiCHO cells (Figure 7B). Using VHH-72-Fc purified from ExpiCHO cells and a SARS-CoV-2 S pseudotyped VSV with a luciferase reporter, we evaluated the neutralization capacity of VHH-72-Fc and found that it was able to neutralize pseudovirus with an IC50 of approximately 0.2 μg/mL (Figure 7C).
Here we report the isolation and characterization of two potently neutralizing single-domain antibodies from a llama immunized with prefusion-stabilized MERS-CoV and SARS-CoV-1 spikes. These VHHs bind to the spike RBDs with high affinity and are capable of neutralizing S pseudotyped viruses in vitro.
To our knowledge, the isolation and characterization of SARS-CoV-1 S-directed VHHs have not been described before. Several MERS-CoV S-specific VHHs have been described, all of which have been directed against the RBD. Several of these VHHs have also been reported to block DPP4 binding, much like MERS VHH-55 (Stalin Raj et al., 2018; Zhao et al., 2018).
By solving the crystal structures of these newly isolated VHHs in complex with their respective viral targets, we provide detailed insights into epitope binding and their mechanisms of neutralization.
A number of RBD-directed conventional antibodies have been described that are capable of neutralizing SARS-CoV-1 or MERS-CoV. The epitope of MERS VHH-55 overlaps with the epitopes of several of these MERS-CoV RBD-directed antibodies including C2, MCA1, m336, JC57-14, D12, 4C2 and MERS-27 (Chen et al., 2017; Li et al., 2015; Wang et al., 2018; Wang et al., 2015; Ying et al., 2015; Yu et al., 2015) (S. Figure 7A).
The epitope of SARS VHH-72 does not significantly overlap with the epitopes of any previously described antibodies other than that of the recently described CR3022, which is also capable of binding to the RBDs of both SARS-CoV-1 and SARS-CoV-2 S (Hwang et al., 2006; Pak et al., 2009; Prabakaran et al., 2006; Walls et al., 2019; Yuan M, 2020) (S. Figure 7B).
However, unlike SARS VHH-72, CR3022 does not prevent the binding of ACE2 and it lacks neutralizing activity against SARS-CoV-2 (Tian et al., 2020; Yuan M, 2020). Because SARS VHH-72 binds with nanomolar affinity to a portion of the SARS-CoV-1 S RBD that exhibits low sequence variation, as demonstrated by its cross-reactivity toward the WIV1-CoV and SARS-CoV-2 RBDs, it may broadly bind all S proteins from SARS-CoV-like viruses.
We show that by engineering a bivalent VHH-72-Fc construct, we are able to compensate for the relatively high off-rate constant of the monovalent SARS VHH-72. This bivalent molecule expresses well in transiently transfected ExpiCHO cells (∼300 mg/L) and is capable of potently neutralizing SARS-CoV-2 S pseudoviruses in vitro.
Due to the inherent thermostability and chemostability of VHHs, they have been investigated as potential therapeutics against a number of diseases. Several HIV- and influenza-directed VHHs have been reported previously, and there are multiple RSV-directed VHHs that have been evaluated (Detalle et al., 2016; Ibanez et al., 2011; Koch et al., 2017; Rossey et al., 2017).
The possibility of administering these molecules via a nebulized spray is particularly attractive in the case of respiratory pathogens because the VHHs could theoretically be inhaled directly to the site of infection in an effort to maximize bioavailability and function (Larios Mora et al., 2018).
Due to the current lack of treatments for MERS, SARS and COVID-19 and the devastating effects associated with pandemic coronavirus outbreaks, both prophylactic and therapeutic interventions are sorely needed. It is our hope that due to their favorable biophysical properties and their potent neutralization capacity, MERS VHH-55, SARS VHH-72 and VHH-72-Fc may serve as both useful reagents for researchers and as potential therapeutic candidates.
University of Texas