Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody

Nieto, Guillermo Valenzuela; Jara, Ronald; Watterson, Daniel; Modhiran, Naphak; Amarilla, Alberto A.; Himelreichs, Johanna; Khromykh, Alexander A.; Salinas, Constanza; Pinto, Teresa; Cheuquemilla, Yorka; Margolles, Yago; Rey, Natalia López González del; Miranda-Chacón, Zaray; Cuevas, Alexei; Berking, Anne; Deride, Camila; González-Moraga, Sebastián; Mancilla, Héctor; Maturana, Daniel; Langer, Andreas; Toledo, Juan Pablo; Müller, Ananda; Uberti, Benjamín; Krall, Paola; Ehrenfeld, Pamela; Blesa, Javier; Chaná-Cuevas, Pedro; Rehren, Germán; Schwefel, David; Fernández, Luis A.; Rojas-Fernández, Alejandro

doi:10.1101/2020.06.09.137935

Cited by 11 publications

(9 citation statements)

References 71 publications

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“…Nbs can be efficiently selected against different epitopes on the same antigen and can be easily converted into multivalent formats 9 . The potential of Nbs to address SARS-CoV-2 has been impressively demonstrated by the recent identification of several RBD specific Nbs from naïve/ synthetic libraries [10][11][12] or immunized animals [12][13][14][15][16] .…”

Section: Introductionmentioning

confidence: 99%

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

Wagner

Kaiser

Gramlich

et al. 2020

Preprint

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Facing the worldwide disease progression of COVID-19 caused by the SARS-CoV-2 virus, the situation is highly critical and there is an unmet need for effective vaccination, reliable diagnosis and therapeutic intervention. Neutralizing binding molecules such as antibodies or derivatives thereof have become important tools for acute treatment of COVID-19. Additionally, such binders provide the unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we describe a set of 11 unique nanobodies (Nbs), originated from an immunized alpaca which bind with high affinities to the glycosylated SARS-CoV-2 Spike receptor domain (RBD). Using a multiplex in vitro binding assay we showed that eight of the selected Nbs effectively block the interaction between RBD, S1-domain and homotrimeric Spike protein with the angiotensin converting enzyme 2 (ACE2) as the viral docking site on human cells. According to competitive binding analysis and detailed epitope mapping, we grouped all Nbs blocking the RBD:ACE2 interaction in three distinct Nb-Sets and demonstrated their neutralizing effect with IC50 values in the low nanomolar range in a cell-based SARS-CoV-2 neutralization assay. Tested Nb combinations from different sets showed substantially lower IC50 values in both functional assays indicating a profound synergistic effect of Nbs simultaneously targeting different epitopes within the RBD. Finally, we applied the most potent Nb combinations in a competitive multiplex binding assay which we termed NeutrobodyPlex and detected a neutralizing immune response in plasma samples of infected individuals. We envisage that our Nbs have a high potential for prophylactic as well as therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals thus helping to monitor the immune status or to guide vaccine design.

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Section: Introductionmentioning

confidence: 99%

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

Wagner

Kaiser

Gramlich

et al. 2020

Preprint

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“…Blue highlights indicate sequence diversity with NIH-CoVnb-112, highlighted in gray, set as the reference sequence for comparison. For comparison, seven previously reported nanobody sequences have clearly distinct sequences: Ty1 23 , VHH72 24 , H11-D4 19 , MR3 20 , Sb#14 21 , Sb23 22 , and W25UACh 25 and possess shorter CDR3 domains (represented in NIH-CoVnb-112 by amino acids 99-120). Affinity binding curves of isolated anti-SARS-CoV-2 RBD nanobodies.…”

mentioning

confidence: 99%

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

Esparza

Martin

Anderson

et al. 2020

Sci Rep

104

118

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There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12–15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

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“…HeLa cells (ATCC ® CCL-2) were transient transfected with a mammalian expression vector expressing a Spike-GFPSpark tag codon optimized fusion SinoBiological VG40589-ACGLN in 10cm plates, 24 h after transfection ~ 8000 cells per well were deposited into 96 well optical plate (Themofisher), after 24 h incubation cells were washed with PBS (phosphate buffered saline solution) 1X 3 times and fixed with 4% paraformaldehyde at room temperature for 30 min. After fixation, cells were washed with PBS 1X and permeabilized in PBS 1X 0.2% Triton X-100 and further washed 3 times in PBS 1X and store at 4°C (36). Random serum samples from COVID-19 recovered patients were assessed for staining Spike protein by immunofluorescence.…”

Section: Methodsmentioning

confidence: 99%

Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients.

Cereceda

González-Stegmaier

Briones

et al. 2021

Preprint

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Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has been a pandemic since March 2020. Currently, the virus has infected more than 50 million people worldwide and more than half a million in Chile. For many coronaviruses, Spike (S) and Nucleocapsid (N) proteins are described as major antigenic molecules, inducing seroconversion and production of neutralizing antibodies. In this work, we evaluated the presence in serum of IgM, IgA and IgG antibodies against N and S proteins of SARS-CoV-2 using western blot, and developed an ELISA test for the qualitative characterization of COVID-19 patients. Patients with an active infection or who have recovered from COVID-19 showed specific immunoblotting patterns for the recombinants S protein and its domains S1 and S2, as well as for the N protein of SARS-CoV-2. Anti-N antibodies were more frequently detected than anti-S or anti-S1-RBD antibodies. People who were never exposed to SARS-CoV-2 did not show reactivity. Finally, indirect ELISA assays using N and S1-RBD proteins, alone or in combination, were established with variable sensitivity and specificity depending on the antigen bound to the solid phase. Overall, Spike showed higher specificity than the nucleocapsid, and comparable sensitivity for both antigens. Both approaches confirmed the seroconversion after infection and allowed us to implement the analysis of antibodies in blood for research purposes in a local facility.

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Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody

Cited by 11 publications

References 71 publications

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

Serological Profile Of Specific Antibodies Against Dominant Antigens Of SARS-CoV-2 In Chilean COVID-19 Patients.

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