Mucosal IgA fused nanobody as a non-invasive, cost-effective prophylaxis and therapeutic option against major SARS-CoV-2 variants

In a recent study published in Frontiers in Immunology, a team of researchers from the United States developed an affordable, non-invasive, inhalable prophylactic treatment consisting of immunoglobulin (Ig) A fused nanobodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant.

Background

The remarkably rapid development of SARS-CoV-2 vaccines and monoclonal antibody therapies have drastically reduced the morbidity and mortality associated with the coronavirus disease 2019 (COVID-19) pandemic. However, emergent SARS-CoV-2 variants such as Omicron and its subvariants carry numerous novel mutations in the spike protein receptor binding domain (RBD) that allow immune escape and increase viral transmissibility. Furthermore, a majority of the monoclonal antibodies in clinical use are also becoming ineffective against these emergent variants.

Although neutralizing anti-RBD IgG antibodies have been effective against COVID-19, the manufacturing of monoclonal IgG antibodies is expensive, and the intravenous administration of IgG therapy is invasive. Moreover, the emergent variants are also decreasing the effectiveness of IgG therapy. In comparison, studies have shown that monoclonal IgA antibodies exhibit higher neutralizing capacity against SARS-CoV-2 than the natural mucosal forms. Additionally, intranasal administration of the SARS-CoV-2 vaccine in mice models has shown increased levels of mucosal IgA and protective effects against SARS-CoV-2 infections, indicating the potential for a nasally administered IgA prophylactic therapy.

About the study

In the present study, the affinity-matured clone VHH1.1 identified by the same team of researchers in an earlier study was used to develop the IgA fusion nanobody. Nanobodies are efficient at recognizing conserved regions in hypervariable pathogens, giving them an advantage over monoclonal antibodies. The complementarity-determining region 3 on nanobodies is longer, and the paratope diameters are smaller, enabling them to bind to epitopes that are conserved and found in spatially restricted recesses of viral glycoproteins. These advantages make nanobodies excellent candidates in mucosal therapy for respiratory diseases. Nanobody production using yeast or soybean platforms are also cost-effective.

The fragment crystallizable (Fc) region of  IgG1 and IgA1 were fused with the VHH1.1 monomer sequence, and the fusion protein was expressed in Expi293 cells. The cross-binding efficiency of the nanobody fusion proteins was then tested against the spike protein RBD of a wide range of SARS-CoV-2 variants, including WA1/2020, Alpha, Beta, Gamma, Kappa, Mu, Delta, and Omicron. The binding results were confirmed by testing the fusion nanobodies against soluble ectodomain spike protein trimers of all the SARS-CoV-2 variants.

Mutagenesis scanning was conducted using alanine, tryptophan, and lysine, which introduce a loss of interaction, steric challenge, and charge, respectively, to define the binding epitope on the VHH-IgA1.1 fusion nanobody. The binding residues of VHH-IgA1.1 were also compared with those on the human angiotensin-converting enzyme 2 (hACE2) interacting interface on the spike RBD.

Pseudovirus assays on 293T cells transfected with hACE2 were used to test the neutralizing efficacy of VHH-IgA1.1, with the efficacy expressed as half-maximal inhibitory concentration (IC50) of inhibited pseudoviral entry. The neutralization efficacy of VHH-IgA1.1 against live viral isolates of D614G, WA1/2020, Alpha, and Omicron variants was also evaluated using plaque reduction neutralization tests.

The in vivo neutralization of SARS-CoV-2 by VHH-IgA1.1 was tested by intranasally administering VHH-IgA1.1 to SARS-CoV-2 infected keratin 18 (K18) transgenic mice expressing hACE2. Additionally, VHH-IgA1.1 engineered into a PichiaPink high copy expression system was used to produce the fusion nanobodies in Pichia pastoris yeasts.

Results

The results reported that the VHH-IgA1.1 fusion nanobody recognized and bound to the conserved RBD epitopes and neutralized all the major SARS-CoV-2 variants, including the Omicron subvariants BA.1.1, BA.2, and BA.2.12.1. The IgA fusion nanobody was also found to have a higher potency against the Omicron subvariants than the IgG fusion nanobody, highlighting the efficacy of a mucosal IgA mediated treatment.

VHH-IgA1.1 also demonstrated significant protective effects in vivo against SARS-CoV-2 when administered intranasally to K18-ACE2 transgenic mice before or after viral challenge. The prophylactic and post-exposure treatment with VHH-IgA1.1 exhibited higher protection in the mucosal lining of the lungs and nasal passages than the monomeric nanobody VHH1.1.

Furthermore, when the potency of VHH-IgA1.1 produced in P. pastoris and mammalian cells was compared, the VHH-IgA1.1 produced in P. pastoris demonstrated comparable epitope binding activity and neutralization ability in vitro against the authentic and pseudotyped WA1/2020 strain of SARS-CoV-2.

Conclusions

Overall, the results indicated that the intranasal administration of the IgA-fused nanobody VHH-IgA1.1 granted effective mucosal protection against SARS-CoV-2 in vivo. VHH-IgA1.1 demonstrated effective binding activity and neutralization of major SARS-CoV-2 variants, including the Omicron variant and its subvariants carrying spike protein mutations with immune evading abilities.

The production of VHH-IgA1.1 in P. pastoris yeast with comparable potency as mammalian cell-produced VHH-IgA1.1 makes this fusion nanobody an affordable and effective therapeutic and prophylactic option against the emergent SARS-CoV-2 variants.