University of Pittsburgh School of Medicine scientists have isolated the smallest biological molecule to date that completely and specifically neutralizes the SARS-CoV-2 virus, which is the cause of COVID-19.
This antibody component, which is 10 times smaller than a full-sized antibody, has been used to construct a drug – known as Ab8 – for potential use as a therapeutic and prophylactic against SARS-CoV-2.
The researchers report today in the journal Cell that Ab8 is highly effective in preventing and treating SARS-CoV-2 infection in mice and hamsters.
Its tiny size not only increases its potential for diffusion in tissues to better neutralize the virus, but also makes it possible to administer the drug by alternative routes, including inhalation.
Importantly, it does not bind to human cells – a good sign that it won’t have negative side-effects in people.
Ab8 was evaluated in conjunction with scientists from the University of North Carolina at Chapel Hill (UNC) and University of Texas Medical Branch (UTMB) at Galveston, as well as the University of British Columbia and University of Saskatchewan.
“Ab8 not only has potential as therapy for COVID-19, but it also could be used to keep people from getting SARS-CoV-2 infections,” said co-author John Mellors, M.D., chief of the Division of Infectious Diseases at UPMC and Pitt.
“Antibodies of larger size have worked against other infectious diseases and have been well tolerated, giving us hope that it could be an effective treatment for patients with COVID-19 and for protection of those who have never had the infection and are not immune.”
The tiny antibody component is the variable, heavy chain (VH) domain of an immunoglobulin, which is a type of antibody found in the blood.
It was found by “fishing” in a pool of more than 100 billion potential candidates using the SARS-CoV-2 spike protein as bait.
Ab8 is created when the VH domain is fused to part of the immunoglobulin tail region, adding the immune functions of a full-size antibody without the bulk.
John Mellors, M.D., chief of infectious diseases, UPMC and the University of Pittsburgh, discusses a scientific breakthrough that is a major step toward a potential drug to treat and prevent COVID-19. Credit: UPMC
Abound Bio, a newly formed UPMC-backed company, has licensed Ab8 for worldwide development.
Dimiter Dimitrov, Ph.D., senior author of the Cell publication and director of Pitt’s Center for Antibody Therapeutics, was one of the first to discover neutralizing antibodies for the original SARS coronavirus in 2003.
In the ensuing years, his team discovered potent antibodies against many other infectious diseases, including those caused by MERS-CoV, dengue, Hendra and Nipah viruses.
The antibody against Hendra and Nipah viruses has been evaluated in humans and approved for clinical use on a compassionate basis in Australia.
Clinical trials are testing convalescent plasma – which contains antibodies from people who already had COVID-19 – as a treatment for those battling the infection, but there isn’t enough plasma for those who might need it, and it isn’t proven to work.
That’s why Dimitrov and his team set out to isolate the gene for one or more antibodies that block the SARS-CoV-2 virus, which would allow for mass production. In February, Wei Li, Ph.D., assistant director of Pitt’s Center for Therapeutic Antibodies and co-lead author of the research, began sifting through large libraries of antibody components made using human blood samples and found multiple therapeutic antibody candidates, including Ab8, in record time.
Then a team at UTMB’s Center for Biodefense and Emerging Diseases and Galveston National Laboratory, led by Chien-Te Kent Tseng, Ph.D., tested Ab8 using live SARS-CoV-2 virus. At very low concentrations, Ab8 completely blocked the virus from entering cells.
With those results in hand, Ralph Baric, Ph.D., and his UNC colleagues tested Ab8 at varying concentrations in mice using a modified version of SARS-CoV-2 . Even at the lowest dose, Ab8 decreased by 10-fold the amount of infectious virus in those mice compared to their untreated counterparts.
Ab8 also was effective in treating and preventing SARS-CoV-2 infection in hamsters, as evaluated by Darryl Falzarano, Ph.D., and colleagues at the University of Saskatchewan. Sriram Subramaniam, Ph.D., and his colleagues at the University of British Columbia uncovered the unique way Ab8 neutralizes the virus so effectively by using sophisticated electron microscopic techniques.
“The COVID-19 pandemic is a global challenge facing humanity, but biomedical science and human ingenuity are likely to overcome it,” said Mellors, also Distinguished Professor of Medicine, who holds the Endowed Chair for Global Elimination of HIV and AIDS at Pitt. “We hope that the antibodies we have discovered will contribute to that triumph.”
Potent neutralization of SARS-CoV-2 by VH-Fc ab8 in vitro
We used four different assays to evaluate VH-Fc ab8 mediated inhibition of SARS-CoV-2 infection in vitro: a β- galactosidase (β-Gal) reporter gene-based quantitative cell-cell fusion assay (Xiao et al., 2003); an HIV-1 backbone- based SARS-CoV-2 pseudovirus assay (Zhao et al., 2013); and two different replication-competent virus neutralization assays (a luciferase reporter gene assay and a microneutralization (MN)-based assay) (Scobey et al., 2013; Yount et al., 2003). VH-Fc ab8 inhibited cell-cell fusion much more potently than VH ab8 (Figure 5A).
The inhibitory activity of VH-Fc ab8 was also higher than that of ACE2-Fc. The control anti MERS-CoV antibody IgG1 m336 did not show any inhibitory activity. VH-Fc ab8 neutralized pseudotyped SARS-CoV-2 virus (IC50 = 0.03 µg/ml) more potently than ACE2-Fc (IC50 = 0.40 µg/ml) and VH ab8 (IC50 = 0.65 µg/ml) (Figure 5B).
The pseudovirus neutralization IC50 for ACE2-Fc in our assay is comparable to the one reported by Changhai Lei et al. (0.03-0.1 µg/ml) (Lei et al., 2020). Interestingly, the maximum neutralization by VH ab8 was only 50% compared to the 100% by VH-Fc ab8 and ACE2-Fc, which was also observed for another antibody S309 (Pinto et al., 2020). The complete neutralization by VH-Fc ab8/ACE2-Fc emphasizes the role of bivalency and related avidity in neutralization (Klasse and Sattentau, 2002). Furthermore, in the reporter gene assay VH-Fc ab8 neutralized live SARS-CoV-2 with an IC50 of 0.04 µg/ml (Figure 5C), which is much lower than that for ACE2-Fc (IC50 of 6.1 µg/ml) and VH ab8 (IC50 = 29 µg/ml).
ACE2-Fc seemed to be much less potent against the live virus compared to the pseudovirus, which is also observed by others (IC50 = 12.6 µg/ml) (Case et al., 2020) and may relate to the S expression levels and RBD/S conformation on the virus surface. We also confirmed the high VH-Fc ab8 live virus neutralization potency by a microneutralization (MN) assay-100% neutralization (NT100) at 0.1 µg/ml (Figure 5D).
The NT100 from the MN assay (0.1 µg/ml) was close to the IC100 (0.2 µg/ml) from the reporter gene assay suggesting consistency in the live virus neutralizing activity of VH-Fc ab8 obtained with two independent assays at two different laboratories. These results suggest that VH-Fc ab8 is a potent neutralizer of SARS-CoV-2, which correlates with its strong competition with ACE2 for binding to RBD.
High prophylactic efficacy of VH-Fc ab8 in a mouse ACE2 adapted SARS-CoV-2 infection model
To evaluate the prophylactic efficacy of VH-Fc ab8 in vivo, we used a recently developed mouse ACE2 adapted SARS-CoV-2 infection model, in which wild type BALB/c mice are challenged with SARS-CoV-2 carrying two mutations Q498T/P499Y at the ACE2 binding interface in the RBD (Dinnon et al., 2020). It was shown that in this model, the aged BALB/c mice exhibited more clinically relevant phenotypes than those seen in hACE2 transgenic mice (Dinnon et al., 2020).
Groups of 5 mice each were administered 36, 8, 2 mg/kg VH-Fc ab8 prior to high titer (105 pfu) SARS-CoV-2 challenge followed by measurement of virus titer in lung tissue 2 days post infection. VH-Fc ab8 effectively inhibited SARS-CoV-2 in the mouse lung tissue in a dose dependent manner (Figure 6A).
There was complete neutralization of infectious virus at the highest dose of 36 mg/kg, and statistically significant reduction by 1000-fold at 8 mg/kg. Remarkably, even at the lowest dose of 2 mg/kg it significantly decreased virus titer by 10- fold (two tailed, unpaired t test, p = 0.0075). To exclude possible effects of residual ab8 on viral titration, we performed another experiment in which mouse lungs were perfused with 10 ml of PBS before harvesting for titration. The perfusion did not affect to any significant degree the infectious virus in the lungs (Figure 6B).
The VH-Fc ab8 completely neutralized the virus in the lungs at 36 mg/kg and significantly reduced infectious virus at 8 mg/kg. VH– Fc ab8 also reduced viral RNA in the lungs (Figure 6C). These results demonstrate the neutralization potency of VH-Fc ab8 in vivo. They also suggest that the double mutations Q498T/P499Y on RBD did not influence VH-Fc ab8 binding and contribute to the validation of the mouse adapted SARS-CoV-2 model for evaluation of neutralizing antibody efficacy.
VH-Fc ab8 exhibited both prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection
Recently hamsters were demonstrated to recapitulate clinical features of SARS-CoV-2 infection (Chan et al., 2020) (Imai et al., 2020). To evaluate the VH-Fc ab8 efficacy in hamsters, it was intraperitoneally administered either 24 hours before (prophylaxis) or 6 hours after (therapy) intranasal 105 TCID50 virus challenge.
In the therapeutic group, the rationale for administration of the antibody six hours post viral infection is based on the replication cycle length of 5-6 hours after initial infection for SARS-CoV in VeroE6 cells (Keyaerts et al., 2005). Six hours after challenge with a high dose of 105 TCID50, approximately the same number of susceptible cells could become infected and likely produce much more infectious virus, which would need to be neutralized by the antibody to prevent subsequent cycles of infection. Nasal washes and oral swab at 1, 3, 5 days post infection (dpi) and different lung lobes at 5 dpi were collected. VH-Fc ab8 decreased viral RNA by 1.7 log in the lung when administered prophylactically.
The lung viral RNA decrease in the therapeutic groups was slightly lower (by 1.2 log) (Figure 6D). Interestingly, the viral RNA load in the therapeutic groups was to some extent tissue location dependent (Figure 6F). The variation of the viral load in different lung lobes may relate to nonuniform antibody transport and viral spread inside the lung. Remarkably, VH-Fc ab8 alleviated hamster pneumonia and reduced the viral antigen in the lung (H&E staining, Figure 7A and C and immunohistochemistry Figure 7B and D).
The lung viral RNA decrease in the therapeutic groups was slightly lower (by 1.2 log) (Figure 6D). Interestingly, the viral RNA load in the therapeutic groups was to some extent tissue location dependent (Figure 6F). The variation of the viral load in different lung lobes may relate to nonuniform antibody transport and viral spread inside the lung. Remarkably, VH-Fc ab8 alleviated hamster pneumonia and reduced the viral antigen in the lung (H&E staining, Figure 7A and C and immunohistochemistry Figure 7B and D).
The control hamsters exhibited severe interstitial pneumonia characterized by extensive inflammatory cell infiltration, presence of type II pneumocytes, alveolar septal thickening and alveolar hemorrhage. Both prophylactic and therapeutic treatment of VH-Fc ab8 reduced the lesions of alveolar epithelial cells, focal hemorrhage and inflammatory cells infiltration. VH-Fc ab8 also reduced the shedding from mucosal membranes including in nasal washes and oral swabs (Figure S4).
The decrease in viral RNA in nasal washes and oral swabs were not as large as the decrease observed in the lung tissue, similar to a recent finding in hamsters (Imai et al., 2020). Overall, the
prophylactic treatment was more effective than the therapeutic treatment in decreasing viral load in nasal washes and oral swabs. Notably, prophylactic administration of VH-Fc ab8 effectively reduced the infectious virus in the oral swab at 1 dpi, while the post-exposure treatment did not (Figure S4C and G).
Interestingly, viral reduction (except the viral titer in the oral swab at 1 dpi) was more effective at 3 and 5 dpi compared to that at 1 dpi, likely due to the infection peak occurring before day 3 as reported in hamsters (Sia et al., 2020). A striking finding is that VH-Fc ab8 given therapeutically at as low dose as 3 mg/kg can still decrease viral loads in the lung, nasal washes and oral swabs (Figure S5).
We measured the VH-Fc ab8 concentrations at both doses (10 and 3 mg/kg) in the sera at 1 dpi and 5 dpi in the post-exposure treatment groups (Figure S5C). The higher dose (10 mg/kg) resulted in higher antibody concentration and better inhibitory activity than the lower dose (3 mg/kg).
The relatively high concentration of VH-Fc ab8 five days after administration also indicates good pharmacokinetics. Furthermore, we also compared the VH-Fc ab8 concentration in both the sera and lung with that of IgG1 ab1, which has a similar affinity to SARS-CoV-2 and similar degree of competition with the receptor ACE2 as VH-Fc ab8 (Li et al., 2020a).
We found that the concentration of VH-Fc ab8 in hamster sera is significantly higher than that of IgG1 ab1 at 1 and 5 dpi after post- exposure administration of the same dose of 10 mg/kg (Figure 7E), possibly indicating more effective delivery of VH-Fc ab8 from the peritoneal cavity to the blood than that of IgG1 ab1.
We also found that the VH-Fc ab8 concentration in all hamster lung lobes was higher than that of the IgG1 ab1 (Figure 7F), suggesting that VH-Fc ab8 appears to penetrate the lung tissue more effectively than IgG1 ab1. These results indicate that the in vivo delivery of VH-Fc ab8 may be more effective than that of full-size antibodies in an IgG1 format.
More information: Wei Li et al, High potency of a bivalent human VH domain in SARS-CoV-2 animal models, Cell (2020). DOI: 10.1016/j.cell.2020.09.007
