A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.

Smaller, Stronger, More Stable: Peptide Variants of a SARS-CoV-2 Neutralizing Miniprotein.

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.

Characterization of SARS-CoV-2 Escape Mutants to a Pair of Neutralizing Antibodies Targeting the RBD and the NTD.

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.

Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Background: Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. Methods: To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Results: Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Conclusion: Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

HIV-1 neutralizing antibodies provide sterilizing immunity by blocking infection of the first cells.

Neutralizing antibodies targeting HIV-1 Env have been shown to protect from systemic infection. To explore whether these antibodies can inhibit infection of the first cells, challenge viruses based on simian immunodeficiency virus (SIV) were developed that use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env usage. Antibodies binding to Env of HIV-1, but not SIV, block HIV-1 Env-mediated infection events after rectal exposure of non-human primates to the switching challenge virus. After natural exposure to HIV-1, such a reduction of the number of first infection events should be sufficient to provide sterilizing immunity in the strictest sense in most of the exposed individuals. Since blocking infection of the first cells avoids the formation of latently infected cells and reduces the risk of emergence of antibody-resistant mutants, it may be the best mode of protection.