Targeting SARS-CoV-2 with Nanobodies and Antibodies: Neutralization and Escape Variants
by Priya Kumar
Date of Examination:2025-03-26
Date of issue:2025-05-15
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Stefan Pöhlmann
Referee:Dr. Anne PD. Balkema-Buschmann
Persistent Address:
http://dx.doi.org/10.53846/goediss-11258
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Abstract
English
The SARS-CoV-2 pandemic revealed significant limitations in existing preventive measures against coronavirus infections. Before vaccines were approved, over 64 million infections and 1.6 million deaths were reported globally (World Health Organization, 2024), underscoring the need for rapid therapeutic interventions. We hypothesized that blocking the initial step of SARS-CoV-2 infection, i.e. the binding of the viral Spike protein to the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, could neutralize infection. To disrupt this critical interaction, we made use of VHH antibodies (also called “nanobodies”), which are variable domains of heavy-chain-only antibodies. Nanobodies targeting the SARS-CoV-2 Spike protein are well suited for the viral neutralization due to their specificity and high-affinity. We demonstrated that nanobodies could be produced rapidly against multiple SARS-CoV-2 variants of concern, and that they were capable of potently neutralizing both authentic virus variants and vesicular stomatitis virus (VSV)-based pseudotyped viruses. Importantly, one representative nanobody exhibited potent neutralization of SARS-CoV-2 even in an aerosolized form and its inhalation provided near-complete protection to Syrian golden hamsters, either as a prophylactic treatment prior to infection or as a therapeutic agent administered 24 hours after infection. In summary, these findings highlight the potential of aerosolized nanobodies as a promising therapeutic or preventive strategy against SARS-CoV-2. While no nanobody-based therapeutic solutions for SARS-CoV-2 are currently clinically available, therapeutic monoclonal antibodies also targeting the viral Spike protein have played a critical role in mitigating disease severity, particularly in patients at high risk of hospitalization. However, as the pandemic progressed, the emergence of SARS-CoV-2 variants with Spike protein mutations posed significant challenges, most notably the reduced efficacy of vaccines and therapeutic antibodies. To make the occurrence of such mutations more predictable, we developed a method to efficiently identify antibody-resistant SARS-CoV-2 mutants by selecting them from mutagenized virus pools. Mutations were induced using N4-hydroxycytidine (NHC), the active compound of the antiviral drug molnupiravir, followed by virus passaging in the presence of antibodies. This approach enabled the identification of specific Spike mutations potentially associated with resistance. These mutations were subsequently validated through VSV-pseudotype assays and immunofluorescence analyses. Some of them, such as F490S, E484K, and K444R, were also detected in circulating variants, while others, e.g. D428G and K462E, were novel. Crucially, all identified mutations retained the ability of the virus to bind ACE2 and remain infectious, highlighting SARS-CoV-2’s remarkable adaptability to immune pressures. Thus, in the second study, we developed a strategy for predicting the therapeutic efficacy of antibodies against emerging SARS-CoV-2 variants.
Keywords: SARS-CoV-2; Nanobodies; N4-Hydroxycytidine; Mutagenesis; Selection Pressure; Spike Evolution; COVID-19; Coronavirus; Therapeutic antibodies; Vesicular stomatitis virus; Pseudotype; Variants of concern; Receptor binding domain
