California [US], April 24 (ANI): A cocktail of high-affinity nanobodies, which are small antigen recognition domains, from alpacas were able to neutralize both wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the N501Y D614G variant, said researchers in a new study published in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).
According to the authors, neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19.
“We identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2,” the researchers said.
Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.
Coronaviruses are enveloped RNA viruses that infect many mammalian and avian species. The current devastating COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has resulted in over 90 million infections and over 2 million deaths worldwide.
“To identify nanobodies that are effective at neutralizing SARS-CoV-2, we immunized two alpacas with combinations of recombinant spike and RBD proteins using a 42 d immunization schedule. Postimmunization sera showed higher reactivity to the respective antigens relative to preimmunization sera,” the researcher said.
They further said that two nanobodies, WNb 21 and WNb 23, which displayed no or low-level detection to SARS-CoV-2 RBD, did bind to SARS-CoV RBD.
As expected, an irrelevant nanobody control B12 (directed to a Plasmodium falciparum antigen) had no detectable binding to both SARS-CoV-2 and SARS-CoV RBD.
“We tested the potential of the 50 nanobodies to neutralize WT SARS-CoV-2 virus (hCoV-19/Australia/VIC01/2020) using a microneutralization assay (MNV) with viral cytopathic effect as a measurement of infectivity. Neutralizing activity ranged from 3 to 36,108 nM, with 10 nanobodies (WNb 67, 41, 36, 2, 53, 7, 50, 15, 10, and 19) neutralizing virus at <=80 nM. These 10 nanobodies also potently abolished RBD-ACE2 complex formation in the FRET assay,” they added.
The results show that the WNbFc fusions bound to the N501Y RBD variant at similar antibody affinities compared to WT RBD and neutralized SARS-CoV-2 N501Y D614G virus at comparable potencies to WT SARS-CoV-2, with concentrations between 0.1 to 5.1 nM.
Collectively, these results suggest that the mixture combination of WNb 2 + 7 or WNb 36 + 7 are promising candidates for the prevention of COVID-19.
Neutralizing antibodies against SARS-CoV-2 are an important therapeutic option against COVID-19. Nanobody and antibody mixtures targeting nonoverlapping epitopes on the RBD have shown promise in preventing occurrence of resistance mutations.
The use of nanobody mixtures may be advantageous for controlling more highly infectious variants and reducing the potential for virus escape mutations to develop.