Author response: Multimeric single-domain antibody complexes protect against bunyavirus infections (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies. Introduction Facilitated by globalization and climate change, arthropod-borne viruses (arboviruses) increasingly pose a threat to human and animal health (Gould et al., 2017). Although for some arboviruses vaccines are available that can be used to prevent or control outbreaks, for the vast majority of emerging arboviruses no countermeasures are available. Rift Valley fever virus (RVFV), a phlebovirus within the order Bunyavirales, is prioritized by the World Health Organization (WHO) as being likely to cause major epidemics for which no, or insufficient countermeasures exist. RVFV is currently confined to the African continent, the Arabian Peninsula and several islands off the coast of Southern Africa, where it causes recurrent outbreaks (Clark et al., 2018). The world-wide distribution of competent mosquito vectors and susceptible animals underscores the risk for emergence in currently unaffected areas. In endemic areas, RVFV causes major epizootics among livestock, characterized by abortion storms and large-scale mortality among newborn ruminants. Importantly, the virus also infects humans, either through direct contact with infected animal tissues or via the bites of infected mosquitoes (Hartman, 2017). Infected individuals generally present with mild to severe flu-like symptoms, however a minority of patients may develop encephalitis or hemorrhagic fever. In addition to members of the family Phenuiviridae, like RVFV, members of the family Peribunyaviridae, genus Orthobunyavirus, may also cause severe disease in humans and animals. In 2011, the incursion of Schmallenberg virus (SBV) in Europe demonstrated that orthobunyaviruses are capable of spreading very efficiently across new territories. SBV infections are associated with fever and reduced milk production in cows and severe malformations in offspring of both large and small ruminants (Hoffmann et al., 2012; van den Brom et al., 2012). SBV is categorized as a biosafety level-2 pathogen and is not pathogenic to humans, facilitating its use as a model of zoonotic orthobunyaviruses that require a higher level of containment (Golender et al., 2015; van Eeden et al., 2014; van Eeden et al., 2012). At the moment, no effective antiviral therapy is available to treat bunyavirus infections in humans. Patients essentially rely on supportive care. Some promising pre-clinical and clinical data have been obtained with small molecules, mainly nucleoside analogues, that interfere with viral replication (Gowen and Hickerson, 2017). Recent X-ray crystallography and cryo-electron microscopy/tomography data have provided novel insights into the bunyavirus glycoprotein architecture that may facilitate the development of bunyavirus antibody therapies in the future (Wu et al., 2017b; Hellert et al., 2019; Halldorsson et al., 2018). After vaccines, antibody therapies are considered the most effective tools to fight (re)emerging life-threatening viral infections (Jin et al., 2017). A very wide range of approaches are currently used to isolate natural antibodies and to design synthetic constructs. Most of these efforts are based on neutralizing antibodies or antibody fragments of murine or human origin. In general, these molecules consist of heavy and light polypeptide chains linked by disulphide bonds. Interestingly, in only a few species such as camelids and nurse sharks, heavy chain-only antibodies (HCAbs) are found, of which the antigen binding domain can be expressed as a single-domain antibody (sdAb) (Arbabi-Ghahroudi, 2017). Camelid derived sdAbs, known as VHHs, are increasingly used as tools in medicine, including virus neutralization (Wu et al., 2017a; De Vlieger et al., 2018). VHHs are intrinsically highly soluble molecules and due to their distinctive structure with extended antigen-binding CDR3 region and overall small size, some are able to target unique (cryptic) antigenic sites not accessible to conventional antibodies (Muyldermans, 2013). VHHs with nanomolar or even picomolar affinity targeting a broad spectrum of antigens have been described (Jin et al., 2017; Arbabi-Ghahroudi, 2017). The single-domain nature additionally allows easy genetic manipulation and favors efficient expression in various heterologous systems including microorganisms (Harmsen and De Haard, 2007a; Liu and Huang, 2018). So far, VHHs have been explored as tools for a wide range of applications including their use as therapeutic agents (Wu et al., 2017a; Harmsen and De Haard, 2007a; Bannas et al., 2017; Traenkle and Rothbauer, 2017; Gonzalez-Sapienza et al., 2017). With respect to virus neutralization, a multimeric VHH has shown great promise in counteracting severe pulmonary disease in infants caused by respiratory syncytial virus (RSV) (Detalle et al., 2016). The therapeutic potency of complexes consisting of multimers of the same VHH (multivalent) or combinations of VHHs targeting different antigenic sites (multispecific) is explained by their improved avidity, selectivity and kinetics compared to individual VHHs (Hultberg et al., 2011). Although techniques to use VHHs as building blocks to generate multifunctional molecules are well established, selection of the optimal VHH combination and optimal VHH formats is still challenging (Iezzi et al., 2018). Recently, the discovery of 'bacterial superglues' has enabled the creation of unique protein architectures. The superglues are comprised of a bacterially-derived peptide and a small protein able to form unbreakable isopeptide bonds (Veggiani et al., 2014). The first bacterial superglue was developed by splitting the immunoglobulin-like collagen adhesion domain (CnaB2) of the fibronectin binding protein (FbaB) of Streptococcus pyogenes (Zakeri et al., 2012) into a peptide and a protein fragment referred to as SpyTag and SpyCatcher. When the two peptides meet, an amide bond is formed which is highly specific and irreversible. Covalent bond formation occurs within minutes upon mixing and is highly robust under various conditions (Zakeri et al., 2012). The potency of SpyTag:SpyCatcher has already found applications in various disciplines and triggered the search for various additional protein and peptide partners (Veggiani et al., 2016; Tan et al., 2016). Here, we selected and characterized RVFV and SBV-specific VHHs targeting receptor binding glycoprotein domains, and used bacterial superglues to create virus neutralizing VHH complexes that reduced and prevented morbidity and mortality in mouse models. Selected RVFV VHHs were subsequently reformatted into human Fc-based bispecific chimeric HCAbs that showed protection in pre- and post-exposure treatments. Results Selection of RVFV and SBV-specific VHHs To obtain a source of RVFV and SBV-specific VHHs with potential neutralizing activity for the respective viruses, llamas were inoculated with the live-attenuated RVFV vaccine strain Clone 13, RVFV virus-like particles (VLPs) or SBV NL-F6 (Figure 1A). All inoculated animals responded well with virus-specific neutralizing antibodies in serum above a 50% neutralizing titer of 500 (Figure 1B; Wichgers Schreur et al., 2017; Loeffen et al., 2012). The B-cell-derived VHH phagemid libraries contained >107 unique clones per animal. Figure 1 with 2 supplements see all Download asset Open asset Selection and expression of RVFV and SBV-specific VHHs. (A) Illustration of the llama immunization strategy to obtain a source for RVFV and SBV-specific VHHs. (B) RVFV and SBV-specific virus neutralizing responses in llama sera determined by virus neutralization test (VNT) before (pre) and post immunization. Neutralizing antibody titers were measured by endpoint titration and calculated as 50% neutralization. (C) Schematic presentation of the VHH library construction and phage display selections. Blood lymphocyte RNA was used as a template for VHH-specific PCRs and purified fragments were cloned into phagemids generating a phagemid library. Following phagemid transduction, the purified phage library was screened for antigen-specific phages. (D) Summarizing table of the VHH clone selection showing the number (#) of unique clones identified and the number of CDR groups per antigen. (E) SDS-PAGE of yeast-expressed RVFV and SBV-specific VHHs. SB10 was shown to be partly N-glycosylated as confirmed by PNGase F based deglycosylation (lane SB10*). (F) RVFV-Gnecto and (G) SBV-Gchead-specific indirect ELISAs. The color coding of the individual VHHs is based on the outcome of the competition ELISA result as presented in Figure 2A and B. RVFV and SBV-specific VHHs were isolated from VHH phage display libraries by pannings with the RVFV Gn ectodomain (RVFV-Gnecto) (de Boer et al., 2010) or the SBV Gc head domain (SBV-Gchead) (Figure 1C; Hellert et al., 2019), previously described as the Gc N-terminal subdomain of 234 amino acids (Gc Amino) (Wernike et al., 2017). Both RVFV-Gnecto and SBV-Gchead are considered to be the main targets of neutralizing antibodies against the respective viruses (Hellert et al., 2019; Wernike et al., 2017; Roman-Sosa et al., 2017; Kortekaas et al., 2010; Kortekaas et al., 2012). Supernatants of induced clones were screened for RVFV-Gnecto and SBV-Gchead specificity by indirect ELISA and immunoperoxidase monolayer assay (IPMA). In total 62 unique RVFV-Gnecto-specific clones that grouped into 16 CDR groups (15 CDR3 groups) and 15 unique SBV-Gchead-specific clones that grouped into 6 CDR3 groups were identified (Figure 1D; Figure 1—figure supplement 1). One representative VHH per CDR group was subsequently produced in yeast (Figure 1E). All yeast expressed VHHs were confirmed to recognize soluble RVFV-Gnecto, or SBV-Gchead by ELISA (Figure 1F,G) and/or were found to bind to fixed viral antigen in infected cells (Figure 1—figure supplement 2). RVFV and SBV VHHs recognize distinct antigenic sites To determine the number of antigenic sites targeted by the different VHHs, RVFV-Gnecto and SBV-Gchead-specific competition ELISAs were performed. The RVFV VHH panel (N = 16) was shown to target three independent antigenic sites within RVFV-Gnecto, referred to as the red, blue and yellow antigenic sites (Figure 2A). When compared to the VHHs of the blue and the yellow antigenic sites, all of the VHHs of the red antigenic site did not show typical sigmoidal curves, suggesting markedly reduced avidity for monomeric Gnecto (Figure 1F). The SBV-specific VHHs showed a more complex pattern of competition (Figure 2B). SB10 (beige) clearly targets an antigenic site that does not overlap with the sites targeted by the other five VHHs. SB12 and SB13 (orange) recognize the same antigenic site that does not overlap with the site targeted by SB11 (green). However, SB9 and SB14 (violet) show reciprocal competition with each other and SB9 competes (reciprocally) with SB11. SB14 shows non-reciprocal competition with SB12 and SB13, which strongly suggests that the epitopes of SB9, SB11, SB12, SB13 and SB14 are all in very close proximity (Figure 2B). Figure 2 Download asset Open asset Characterization of VHH binding sites. (A) RVFV and (B) SBV-specific VHHs and antibodies were tested in competition ELISA using RVFV-Gnecto and SBV-Gchead as antigens. Competition is expressed as percentage of blocking. A percentage above 50% is considered efficient blocking and strongly suggests that the test VHH binds at an overlapping site in the antigen as the blocking VHH. For SBV, competition was additionally assessed for the 1C11 and 4B6 mouse mAbs (Hellert et al., 2019). The RVFV-specific VHHs clustered into three groups each targeting a different antigenic site in RVFV-Gnecto and the SBV VHH panel was shown to target 4 different antigenic sites in SBV-Gchead (of which the green, violet and orange sites are shown to be partly overlapping). Since SB11 and SB14 showed efficient competition (although non-reciprocal) with 1C11, the green, violet and orange antigenic sites are expected to be located at the apical end of the SBV-Gchead domain. (C) SDS-PAGE of the baculovirus produced head, A, β-ribbon and B subdomains of RVFV-Gnecto. The lowest band in the subdomain A lane is expected to be the result of proteolytic cleavage. (D) RVFV-Gnecto subdomain-based VHH ELISAs. Wells were coated with the indicated subdomains and VHH binding was assessed with a HRP-conjugated goat anti-llama IgG. (E) SB10 escape mutant selection. Briefly, wildtype SBV was sequentially passaged in the absence or presence of SB10. Following passage 6, the Gchead coding region was sequenced and SB10 neutralization was quantitatively assessed. (F) Model of a pentameric RVFV spike. The RVFV Gn/Gc heterodimer within a penton (PDB: 6F9F, Halldorsson et al., 2018) is color-coded by domains. The Gn head domain is outlined in black. Arrows indicate approximate positions of the two mapped antigenic sites. (G) Model of a trimeric SBV spike based on the monomeric SBV crystal structure (PDB: 6H3S, Hellert et al., 2019) and the trimeric BUNV cryo-ET map (EMDB: EMD-2352, Bowden et al., 2013). A protomer is color-coded by domains and the Gc head domain is outlined in black. The two N-glycans are labelled with the positions of their respective asparagine residues and the position of the SB10 escape mutation is indicated with a star. Arrows indicate approximate positions of the antigenic sites. Antigenic sites for which evidence is inconclusive are indicated with question marks (green and beige sites). Mapping of the RVFV VHHs to the RVFV-Gn head domain The N-terminal 154–469 amino acids of RVFV-Gnecto, now known as the RVFV-Gn head domain, was recently crystallized and shown to comprise three subdomains; subdomain A (or I), β ribbon domain (II) and domain B (III) (Wu et al., 2017b; Halldorsson et al., 2018). To assess whether the VHHs target a specific subdomain, the head domain as well as the individual subdomains were expressed using the baculovirus expression system. The subdomains were expressed successfully (Figure 2C) and binding to the RVFV VHHs was assessed by indirect ELISA (Figure 2D). The results show that the yellow antigenic site, which is targeted by RV104, RV136 and RV148, is located on subdomain B and that the blue antigenic site, recognized by RV107 and RV115, is primarily located on the β-ribbon domain. The red antigenic site could not be mapped to a specific subdomain, suggesting that this site is present at a subdomain interface. Gn, as part of the Gn/Gc heterodimer, is organized similarly in both hexamers and pentamers on the RVFV particle, of which Gn is most exposed to the immune system. We mapped the antigenic sites to the previously proposed Gn structure within the pentameric capsomer density (Figure 2F). Mapping of the SBV VHHs on the SBV-Gc head domain The SBV-Gc head domain is the most exposed feature on the virus envelope, and based on homology with other orthobunyaviruses, it is believed to reversibly form homotrimers among neighboring Gc molecules (Hellert et al., 2019). Crystal structures of the head domain in complex with two neutralizing murine mAbs, referred to as 1C11 and 4B6, have recently shown that 1C11 binds to the apical end of the head domain, near the N686 glycosylation site, and that 4B6 binds to the opposite, basal end of the head domain, near its connection to the stalk (Hellert et al., 2019). Binding of either antibody is sterically incompatible with head domain trimerization, suggesting that neutralization may at least be partially mediated by destabilization of the spike’s quaternary structure (Hellert et al., 2019). Competition ELISAs with 1C11 and 4B6 and our SBV VHH panel suggests that SB11 and SB14 target the same antigenic site as 1C11 at the apical end of the domain, whereas no competition was observed with 4B6 (Figure 2B). For SB10, of which the antigenic site is independent from the others, we generated an escape mutant carrying the single point mutation Y541C located directly at the head domain’s contact area with the stalk (Figure 2E,G). This site likely acts as a hinge allowing the head domain to adopt different orientations relative to the virion surface (Hellert et al., 2019), and antibody binding is thus expected to restrain the local mobility of the spike. Multimeric VHH complexes show superior neutralization activity The virus neutralizing activity of selected VHHs, representative for each antigenic site, was evaluated with RVFV and SBV-specific virus neutralization tests (VNTs). At nM range concentrations, no neutralization was observed for any of the monovalent VHHs (Figure 3; left panel of the figure and diagonal set of bars). Since synergistic effects have been reported for combinations of monoclonal antibodies in bunyavirus neutralization (Besselaar and Blackburn, 1992), we subsequently tested mixtures of VHHs. Combining two VHHs in the VNT assays resulted in moderate neutralization for some SBV VHH combinations (ND50 around 200 nM) whereas still no neutralization was observed for RVFV VHH mixtures (Figure 3; left part of the figure with double colored bars). We therefore assessed whether multimerization supports efficient neutralization. In order to facilitate the screening of a large set of bivalent and bispecific complexes including various linker lengths and without the need to construct a lot of expression plasmids, we used the advantageous properties of bacterial superglue. VHHs were expressed with Spy- and SnoopTags and flexible scaffolds consisting of one, two or three elastin-like protein (ELP) domains were expressed with Spy- and SnoopCatcher domains. By simply mixing the tagged VHH versions with the Catcher containing ELP scaffolds, bivalent and bispecific complexes were constructed (Figure 4). The VNT assays subsequently revealed that several multimeric complexes very efficiently neutralized with nM (Figure 3; and For RVFV, targeting of two distinct antigenic sites a to efficient neutralization (Figure whereas for SBV both bivalent and bispecific complexes were capable of neutralization (Figure Interestingly, the of the scaffolds did not have a major on neutralization of the RVFV VHH whereas the did of the SBV based on the showed markedly higher neutralization Figure Download asset Open asset RVFV and SBV-specific neutralization by monovalent VHHs and bacterial VHH (A) and (B) SBV-specific monovalent VHHs, VHH combinations and bivalent and bispecific VHH complexes were tested in a RVFV or SBV-specific VNT were formed by mixing the tagged versions of the VHHs with the indicated ELP scaffolds of different lengths (Figure 4). were subsequently with virus before to susceptible The to total VHH per as neutralization be the result of of VHH complexes and VHHs. of two are VHHs are color-coded based on the antigenic site recognize as determined by competition ELISA (Figure 2A and most efficient neutralization was observed with multimerization of VHHs that each target a different antigenic Figure 4 Download asset Open asset VHH (A) SDS-PAGE of scaffolds with an N-terminal SnoopCatcher and a domain. (B) SDS-PAGE of RVFV and (C) SBV-specific VHHs expressed with a or The were expressed with the llama antibody hinge (D) Illustration of the bacterial formation of a bispecific or bivalent VHH (E) SDS-PAGE of the bacterial formation of bivalent and bispecific VHH complexes using of various VHH and VHH were used as an for and of VHH complexes morbidity and mortality in mice Following the in neutralization selected bivalent and bispecific complexes were evaluated in mouse To the in the complexes were expressed with an binding domain (Figure Binding of a VHH complex to is expected to the not only due to an of the also as a result of through the Fc receptor et al., et al., RVFV VHH complex was constructed and evaluated to the bacterial superglue to the level and to a construct targeting the red, blue and yellow antigenic sites within the domain To the construction of this complex we use of a bacterial and (Veggiani et al., 2014). The specificity of all multimeric complexes to bind to RVFV-Gnecto or SBV-Gchead was confirmed by indirect ELISA (Figure and efficient neutralization at nanomolar was confirmed by VNT (Figure Figure with 1 supplement see all Download asset Open asset of RVFV and SBV-specific VHH complexes in (A) Illustration of VHH complexes an binding domain (B) SDS-PAGE of purified RVFV and SBV-specific VHHs were by a based the The indicate the with higher (C) ELISA of the containing ELP mouse or was coated at a and the ELP containing an domain was in a Binding was with a (D) RVFV-Gnecto and (E) SBV-Gchead ELISA of purified RVFV and SBV-specific VHH (F) Neutralizing activity of the purified VHH complexes as determined by RVFV and SBV-specific The to the of (G) Illustration of the RVFV and SBV mouse models. of RVFV infected mice with of the RVFV-specific VHH of mice with either 200 of or were inoculated with the by a of either RVFV or RVFV-specific complexes were evaluated in a mouse model and SBV-specific complexes in mice (Figure Wernike et al., 2012). The results of the RVFV mouse showed that in the absence of the domain morbidity and mortality could not be prevented or whereas with both and resulted in a in of which the was most for (Figure Interestingly, the complex the mice most suggesting that targeting of to three antigenic sites is very For SBV, the complex prevented mortality in of 6 mice whereas the complex all of the inoculated mice (Figure In both of the animals showed any clinical of disease or In for all the antibody mice viral RNA in and were markedly reduced in to the control animals (Figure supplement 1). and in potency of bispecific chimeric antibodies To the potency of the RVFV VHHs, we and constructed a chimeric bispecific that as conventional we to generate HCAbs that are for therapeutic in humans and with We with a human and which binding to and Fc of immune cells their The the domains also the binding site for the for the of and of through et al., 2014). bivalent and/or bispecific were constructed (Figure and produced at small in cells et al., 2011). In with the ELP efficient neutralization at nM was mainly observed with the bispecific (Figure binding to Gnecto that were highly (Figure The two most promising bispecific Fc and were subsequently produced at using a expression in et al., 2014). The purified was subsequently tested in the RVFV mouse In addition to prophylactic therapeutic was also evaluated (Figure The results show that protection was with prophylactic protection was therapeutic (Figure these results that VHH-based great promise for the of bunyavirus infections either or Figure 6 Download asset Open asset chimeric antibodies mice from RVFV (A) Illustration of the VHH and chimeric (B) SDS-PAGE of the purified chimeric antibodies under and (C) Neutralizing activity of the purified chimeric antibodies expressed as (D) ELISA of the chimeric (E) Illustration of the mouse (F) of mice with 200 (G) of mice with 200 chimeric antibodies were produced at in Discussion pose a and present to both animal and human The recognized the threat for human health by including three RVFV, hemorrhagic fever virus and severe fever with virus on the Blueprint list of viruses likely to cause future epidemics and for which no countermeasures are currently available Although for some bunyaviruses vaccines are available or in for humans vaccines effective therapies exist. The of bunyavirus glycoprotein structures and domains with on of neutralization, have the development of bunyavirus therapies. However, novel data on the obtained from X-ray and cryo-electron microscopy/tomography of for some members of the now new (Wu et al., 2017b; Hellert et al., 2019; Halldorsson et al., et al., 2018). For RVFV and SBV, a number of conventional mouse and mAbs as well as human antibodies have been described with neutralizing activity (Wu et al., 2017b; Hellert et al., 2019; and Blackburn, et al., et al., 2019; Wernike et al., With this we a panel of VHHs targeting RVFV or SBV, show neutralizing activity of VHH-based complexes in and in a broadly applicable method for the development of VHH-based biotherapeutics. We the advantageous of VHHs in combination with bacterial superglues to develop highly potent virus neutralizing A major of the presented more conventional approaches is the and and in complex selection of the most potent complexes a easy