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A variable fragment of a heavy chain antibody (VHH) directed against rotavirus, also referred to as anti-rotavirus protein 1 (ARP1), was shown to confer protection against rotavirus induced diarrhea in infant mouse model of rotavirus induced diarrhea. In this study, we have fused the mouse IgG1 Fc to ARP1 to improve the protective capacity of ARP1 by inducing an Fc-mediated effector function.
We have shown that the Fc-ARP1 fusion protein confers significantly increased protection against rotavirus in a neonatal mouse model of rotavirus-induced diarrhea by reducing the prevalence, duration and severity of diarrhea and the viral load in the small intestines, suggesting that the Fc part of immunoglobulins may be engaged in Fc-mediated neutralization of rotavirus. Engineered conventional-like antibodies, by fusion of the Fc part of immunoglobulins to antigen-specific heavy-chain only VHH fragments, might be applied to novel antibody-based therapeutic approaches to enhance elimination of pathogens by activation of distinct effector signaling pathways. Rotavirus is a non-enveloped double stranded RNA virus that is associated with a severe dehydrating diarrhea, infecting infants and children less than 5 years of age worldwide. The rotavirus recognition involves the cell-surface Lewis b blood group antigen and several intracellular receptors, and its replication is limited to mature enterocytes of the small intestinal villi.
Protection against rotavirus involves blocking of enterocyte infection by neutralizing antibodies against outer capsid proteins VP4 and VP7. However, the vast majority of antibodies is directed against the most abundant and highly conserved rotavirus inner capsid protein VP6, and has been shown to mediate intracellular neutralization.IgG-based therapeutics have gained increasing importance for the treatment of a wide range of infectious diseases including rotavirus infection.
In addition to the receptor or ligand blocking capacity of antibodies, they can also trigger potent biological responses such as regulation of immune responses in cells through Fc/Fc receptor interactions. The receptors for IgG can be classified into the well-known Fc gamma receptor (FcγR) family, consisting of different proteins expressed on the surface of myeloid cells, and the neonatal Fc receptor (FcRn), expressed at various levels in different cell types. FcRn is the only receptor known to be engaged in bidirectional transcytosis of IgG across the mucosal epithelium in individuals at any age. It protects the captured antibody from lysosomal degradation and thus prolongs its half-life.
Another more recent and less-characterized receptor is the tripartite-motif containing protein 21 (TRIM21), a cytosolic Fc receptor found in all cells, but with high expression levels in immune and endothelial cells. TRIM21 is involved in intracellular antibody-mediated adenovirus recognition and destruction of virus-antibody complexes using the proteasome degradation machinery. According to the site and level of infection, either one or more Fc receptor(s) might be activated in concert to drive some well-defined effector functions, including virus degradation or cell phagocytosis.Single domain variable fragments of camelid heavy chain-only antibodies (referred to as Nanobodies® or VHHs) show high solubility and stability under different extreme conditions and exhibit similar affinities as compared to full-sized antibodies. The VHH molecules have been used in various prophylactic and therapeutic applications, including treatment for numerous viruses. Even though mono- or multivalent VHHs are highly efficient in anti-viral protection at mucosal surfaces, the viral neutralization through VHHs potentially enroll distinct mechanisms as compared to conventional antibodies with Fc effector functions.
An anti-rotavirus VHH (ARP1), capable of protecting mouse pups against rotavirus-induced diarrhea when produced in yeast, rice and lactobacilli, has previously been described. Orally administered yeast produced ARP1 was found to be safe and effective in reducing the severity of diarrhea in children in a recent clinical trial conducted in Bangladesh. ARP1 binds to abundant VP6 protein, containing the group and subgroup epitope specificities, and neutralize a broad range of mammalian rotavirus serotypes/genotypes in vitro. For evaluation of the role of Fc-mediated effector functions in protection against rotavirus, we developed a Fc fusion protein containing a mouse IgG1 Fc fragment and ARP1 (Fc-ARP1). We further addressed the involvement of FcRn and TRIM21 in rotavirus protection by generating a single point mutation within the TRIM21 and FcRn binding “HNH” motif of Fc (N434D). Substitutions at position N434 (N434D, N434H or N434A) were previously shown to be crucial for TRIM21 mediated intracellular antibody-bound pathogen neutralization in primary cells, and for FcRn-mouse IgG1 interaction, respectively.
We thus evaluated the protection by Fc-ARP1 and mutant Fc N434D-ARP1 fusion protein in a neonatal mouse model of rotavirus induced diarrhea as compared to ARP1 and bivalent (ARP1′) 2. Analysis of antibody fragmentationSamples including ARP1, Fc-ARP1, mutant Fc N434D-ARP1, bivalent (ARP1′) 2 and conventional irrelevant mouse IgG1 antibodies were detected in both non-reducing and reducing conditions in Coomassie blue-stained SDS-PAGE gels. Under reducing conditions, the chimeric heavy chain of Fc-ARP1 and mutant Fc N434D-ARP1 were detected with an estimated molecular weight of 40 kDa, while the light and heavy chains of the IgG1 migrated at around 25 and 50 kDa, respectively. The Fc-ARP1 cleaved with pre-activated papain, contained a mixture of Fc (25.5 kDa for each chain) and ARP1′ (ARP1 plus a part of the Fc hinge region) (14.5 kDa). The other bands observed between 16 and 30 kDa are probably due to additional Fc digestion sites. Furthermore, under non-reducing conditions, bands detected for Fc-ARP1 and mutant Fc N434D-ARP1 (100 kDa), as well as mouse IgG1 antibody (250 kDa) were higher than the theoretical molecular weights (80 kDa and 150 kDa, respectively), most likely due to dimerization by disulfide bridges.
Furthermore, bands corresponding to bivalent (ARP1′) 2 (29 kDa) and each chain of the Fc part (25.5 kDa) were detected in the cleaved Fc-ARP1 preparation. Detection of proteins in non-reducing ( A) and reducing ( B) conditions in Coomassie blue-stained SDS-PAGE gels.
( C) Detection of ARP1 by an anti-rabbit anti-VHH (K212) antibody, and ( D) detection of Fc fragments by a rabbit anti-mouse IgG antibody, both in reducing and non-reducing conditions in Western blot. Under denaturing and non-reducing conditions, the theoritical MW of the various antibody forms are for Fc-ARP1: 80 kDa, Fc N434D-ARP1: 80 kDa, cleaved preparation: 29 kDa for the bivalent (ARP1′) 2 when detected with an anti-VHH antibody and 25.5 kDa for each chain of the Fc part when detected with an anti-mouse IgG antibody, irrelevant mouse IgG1: 150 kDa, and ARP1: 13 kDa. Under denaturing and reducing conditions, the theoretical MW are for Fc-ARP1: 40 kDa for each chain, Fc N434D-ARP1: 40 kDa for each chain; cleaved preparation: 14.5 kDa for each chain of the bivalent (ARP1′) 2 detected with an anti-VHH antibody and 25.5 kDa for each chain of the Fc part when detected with an anti-mouse IgG antibody; mouse irrelevant IgG1: 50 kDa for the heavy chain and 25 kDa for the light chain, and ARP1: 13 kDa. The same samples were analyzed in both reducing and non-reducing conditions in Western blot, using an anti-ARP1 antibody and an anti-mouse IgG to detect Fc , confirming the nature of the bands observed with Coomassie staining (,B).
The preparation of Fc-ARP1 cleaved with pre-activated papain and detected with an anti-ARP1 antibody showed a 14.5 kDa band corresponding to ARP1′ under reducing conditions , and a 29 kDa band corresponding to (ARP1′) 2 under non-reducing conditions. The cleaved Fc-ARP1 detected with an anti-mouse IgG antibody results in a band of 25.5 kDa corresponding to each chain of the Fc part in reducing and non-reducing conditions.
Assessment of binding to a rhesus rotavirus strain (RRV) in vitroARP1, Fc-ARP1, mutant Fc N434D-ARP1 and bivalent (ARP1′) 2 were all shown to bind to rotavirus in vitro when detected by anti-ARP1 antibody (K212) in ELISA. At equivalent amount of ARP1 molecules added (from 3.23 nM to 0.41 nM), the binding of the bivalent (ARP1′) 2, Fc-ARP1 and mutant Fc N434D-ARP1 to rhesus rotavirus (RRV) was similar.
The binding of ARP1 to RRV was not as high as compared to the aforementioned ones, which may be due to its monovalency. When detecting the complex with an anti-mouse IgG instead of anti-ARP1, no signal was observed with bivalent (ARP1′) 2, confirming the complete removal of the Fc part.
The commercial mouse IgG1 antibodies did not bind to rotavirus in vitro , which made it possible to use it as a control for Fc effector functions that were independent of antigen specificity in animal model. Affinity of interaction between recombinant antibodies and Fc receptorsThe interactions of Fc-ARP1 and Fc N434D-ARP1 with TRIM21 (the PRYSPRY-domain) and FcRn (the extracellular domain) were investigated by surface plasmon resonance analysis (SPR). When injected at 100 nM concentration, Fc-ARP1 could interact with TRIM21 and FcRn whereas Fc N434D-ARP1 could not. Even injection of higher concentrations of Fc N434D-ARP1 (up to 2000 nM) did not result in any interaction with TRIM21 or FcRn. The equilibrium dissociation constant (K D) between Fc-ARP1 and TRIM21 was determined by injecting a dilution series of Fc-ARP1 over immobilized TRIM21. Similarly, the K D of the interaction between Fc-ARP1 and FcRn was determined by injecting a dilution series of Fc-ARP1 over immobilized FcRn. Since there are two TRIM21 and FcRn interaction sites on Fc-ARP1, the resulting curves were evaluated with a bivalent analyte model.
From the analysis, the K D (K D1) for the interaction of Fc-ARP1 with TRIM21 and FcRn (at pH 6.0) was determined to be 28 nM and 0.76 μM , respectively. The interaction between Fc and FcRn is pH dependent and injection of Fc-ARP1 (100 nM) at pH 7.4 gave no interaction, as expected (data not shown). Panel A and C shows an overlay of sensorgrams after injection of 100 nM of Fc-ARP1 and Fc N434D-ARP1 over surfaces with immobilized TRIM21 or FcRn, respectively. Panel B and D shows an overlay of sensorgrams after injection of dilution series of Fc-ARP1 over surfaces with immobilized TRIM21 or FcRn, respectively. The numbers next to the arrows indicate the span of the dilution series (0.25, 0.5, 1, 2, 4, 8 nM) or (31.2, 62.5, 125, 250, 500, 1000, 2000 nM).
All experiments were performed twice and the duplicate sensorgrams are displayed in the panels. In vivo protection against rotavirus-induced diarrheaThe infant mice were randomly distributed in groups, receiving one of the following oral prophylactic treatments: ARP1, Fc-ARP1, mutant Fc N434D-ARP1, bivalent (ARP1′) 2, generated by enzymatic fragmentation of Fc-ARP1 (comprised of two ARP1 fragments joined by a disulfide bridge at the hinge region and free Fc fragments), an irrelevant mouse IgG1 or PBS. The monovalent ARP1 was given at a suboptimal dose known to be non-protective in the mouse model in order to evaluate the effect of the fused Fc part. At equivalent amount of administered ARP1 molecules, the Fc-ARP1 treatment markedly reduced the prevalence of diarrhea (3.23%) at the peak of infection as compared to all other groups, including Fc N434D-ARP1 (20%). Furthermore, Fc-ARP1 treatment causes a significant reduction in duration and severity, as compared to bivalent antibodies and negative control groups. Pups treated with Fc N434D-ARP1 resulted in more prolonged disease duration and higher severity, as compared to Fc-ARP1, even though it is not significant.
Bivalent (ARP1′) 2 did not confer any protection at the given dose. Quantification of infection markers (VP7, IFN-β) and TRIM21 from the infant mice intestinal tissue sectionsThe rotavirus load in the small intestinal sections, assessed by VP7 transcript copies, was significantly decreased in mice treated with Fc-ARP1 as compared to groups receiving bivalent (ARP1′) 2, IgG1 and PBS.
The viral load in mice receiving Fc N434D-ARP1 was also reduced but not significantly. The IFN-β gene expression level was significantly decreased in mice given Fc-ARP1 as compared to groups receiving bivalent (ARP1′) 2, IgG1 and PBS. In addition, TRIM21 gene expression was significantly reduced in mice treated with Fc-ARP1 than those receiving IgG1 treatment.