Single-domain antibodies inhibiting viral RNA polymerase activity

Abstract

The present invention relates notably to specific single-domain antibodies (sdAbs) targeting RNA-dependent RNA polymerase (RdRp) activity and their use in the prevention and / or treatment of a virus infection from Coronaviruses, and more particularly of SARS-CoV-2.

Description

[0001] SINGLE-DOMAIN ANTIBODIES INHIBITING VIRAL RNA POLYMERASE ACTIVITY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to single-domain antibodies that specifically target RNA-dependent RNA polymerase (RdRp) and block RdRp activity, in particular of Coronaviruses. The antibodies contain a single variable domain which comprises three complementarity-determining regions (CDRs), / .e. any CDR1 , CDR2, and CDR3 as disclosed herein, and may have residue changes in the non-CDR segments. Representative antibody sequences are SEQ ID NO: 25-32. The invention relates notably to the described single-domain antibodies, complexes of said antibodies, compositions comprising said antibodies, and its use in the prevention and / or treatment of a virus infection from Coronaviruses.

[0004] BACKGROUND

[0005] According to the World Organization for Animal Health, 60% of the 1 ,400 human pathogens are of animal origin, and three-quarters of emerging animal diseases can be transmitted to humans.

[0006] This threat of infections transmitted from vertebrate animals to humans is not new, and dates back thousands of years. Over the last 50 years alone, the frequency of viral zoonoses has surged with the emergence of the human immunodeficiency virus, SARS- CoV, Ebola virus, Zika virus, Chikungunya virus, MERS-CoV, SARS-CoV-2, for examples. Notably, as a result of modern activities and climate disruption, the incidence of epidemics and pandemics caused by viral zoonoses will only increase, and will therefore be associated with deleterious effects on human health and the global economy.

[0007] The Covid-19 pandemic was just another warning to remind us of the need to be prepared. This preparation / anticipation requires research upstream of health crises, enabling the development of diagnostic tools, vaccines and specific therapeutic treatments.

[0008] Coronavirus (CoV) family includes important zoonotic veterinary and human pathogens.

[0009] In the 21thcentury, CoVs became a real threat to mankind, with the emergence in 2003 of the Severe Acute Respiratory Syndrome-CoV (SARS-CoV), in 2012 of the Middle East Respiratory Syndrome-CoV (MERS-CoV) and now with the SARS-CoV-2.

[0010] More particularly, the Covid-1 pandemic, caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a profound global impact, the likes of which has not been seen in more than a century. The remarkably rapid development and distribution of vaccines undoubtedly saved many millions of lives; nevertheless, at the time of writing, mortality estimates about 7 million (World Health Organization data), with additional profound long-lasting health impacts for many survivors (e.g. Long COVID). The disease appears to be transitioning to an endemic phase, thus presenting a serious worldwide health problem for the foreseeable future and demanding a large- scale ongoing implementation of new prophylactics and therapeutics.

[0011] Major therapeutic strategies have utilized antibodies directed against the major Spike (S) surface envelope glycoprotein of the SARS-CoV-2 virion, various fragments of which are also the immunogens for most vaccines. Spike is a homotrimer of an extensively glycosylated -200 kDa protein composed of two major domains: SI , which contains the host receptor binding domain (RBD) that targets the angiotensin-converting enzyme 2 (ACE2) surface receptor on host cells; and S2, which upon host cell binding undergoes major conformational changes to enable viral - host membrane fusion, resulting in virus entry into the cytoplasm. Thus, antibodies that target Spike, and particularly RBD have the potential to block viral binding and entry into the cells of the host.

[0012] Unfortunately, the continuing emergence of new SARS-CoV-2 Variants of Concern (VoCs; Alpha, Beta, Gamma, Delta, Omicron and subvariants) presents a significant barrier to attaining complete control of Covid-19. These VoCs usually have many Spike mutations (especially in the RBD), and thus are relatively poorly neutralized by current vaccines and antibody therapies. For instance, monoclonal antibodies (mAbs) have proven to be an effective therapeutic strategy, though sensitive to emerging variants. Moreover, mAbs are limited by challenges in the ease and cost of their large-scale manufacturing, distribution, and parenteral administration.

[0013] A particular class of single domain antibodies termed VHH-antibody. VHH- antibodies are “mini-antibodies”, some 1 / 10ththe size of regular IgGs, derived from the variable domain (VHH) of variant heavy chain-only IgGs (HCAbs) found in camelids (e.g. llamas). Each VHH-antibody molecule consists of four framework regions (FRs) that intersperse and orient three complementarity determining regions (CDRs) that form the VHH-antibody paratope. These regions are comparable to FRs and CDRs of conventional antibodies. As with conventional antibodies, CDR3 is formed by VDJ recombination of germline DNA; CDR1 and CDR2 come from the germline V region, and all three CDRs are then subject to somatic hypermutation, with selection for improved binding affinity to antigens.

[0014] VHH-antibodies have several attractive advantages over mAbs, including: fast on- rates leading to high overall affinities; characteristics of small molecules in terms of higher tissue penetration and accessibility to regions not accessible to the larger mAbs or occluded by glycosylation, greatly enhancing their potential to synergize in combination, a profound advantage they have over often poorly-synergizing conventional antibodies. They can be readily engineered, including humanization to minimize immunogenicity; they are highly denaturation-resistant, giving them long shelf lives and making them suitable for a broader range of delivery methods (e.g., via nebulization directly into lungs). In view of limitations of mAbs / VHH-antibodies targeting the viral spike surface protein subject to a high mutation rate (VoCs), the invention through VHH-antibody targets the RNA-dependent RNA polymerase (RdRp) activity as a more effective therapy against the continuing emergence of new SARS-CoV-2 VoCs (Figure 1 ). The present disclosure addresses these and related needs.

[0015] CoV genomes consist of a positive-sense single-stranded RNA genome, and are among the largest viral RNAs known to date (~30-kb). To replicate their exceptionally large genome, they employ a unique and sophisticated RNA-synthesis machinery, comprising a variety of nonstructural proteins (nsps).

[0016] The coronavirus replication complex is a multi-protein machinery essential for the replication and transcription of the viral RNA genome. At its core is the RNA-dependent RNA polymerase (RdRp), also referred to as nsp! 2 (non-structural protein 12). This enzyme catalyzes the synthesis of RNA using the viral RNA as a template.

[0017] The functionality of nspl2 is critically dependent on two accessory proteins, nsp7 and nsp8, which act as cofactors. Structural studies have demonstrated that nsp7 and nsp8 form a stable complex with nspl2, significantly enhancing its processivity and fidelity during RNA synthesis (Gao ef al., “Structure of the RNA-dependent RNA polymerase from COVID-19 virus”, Science, 2020, Vol 368, Issue 6492, pp. 779-782). Nsp8 also serves as a primase, capable of synthesizing short RNA primers necessary for initiating RNA polymerization (Imbert ef al., “A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus”, EMBO J, 2006, 4933-4942). Together, the nspl2-nsp7- nsp8 complex ensures efficient replication of the viral RNA, even on long templates.

[0018] Additionally, this replication complex operates within a larger framework of viral and host proteins, including nspl 3, nspl4, and nspl 6, and forms part of the replicationtranscription organelles (DMVs) that shield the viral genome from host defenses (Snijder ef al., “A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis”, PLoS Biology, 2020). The coordinated activity of the complex is central to the coronavirus life cycle and represents a critical target for antiviral therapies, including remdesivir, which binds to the nspl2 active site and inhibits RNA synthesis (Yin ef al., “Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir”, Science, 2020, Vol 368, Issue 6498 p. 1499- 1504).

[0019] Among these viral replicases, the key enzyme for RNA viruses, the RNA-dependent RNA polymerase (RdRp) resides in the C-terminal domain of nspl 2. Remarkably, CoV RNA polymerase activity requires a processivity factor, made up of nsp7 (10 kDa) and nsp8 (22 kDa) (Subissi ef al., 2014, “One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities”, Proc. Natl. Acad. Sci. USA 1 1 1 , E3900-3909). Indeed, these two viral proteins are essential for nspl2- RNA polymerase activity, acting in a similar fashion to a clamp. Indeed, nsp7 / nsp8 prevent the nspl2-RNA polymerase falling off the RNA pattern during RNA polymerization, which is critical for efficient and fast viral replication. Importantly, in vivo study (by reverse genetics approach) established that nsp8 / nsp!2-RNA polymerase interaction is critical for viral replication and therefore the production of new viruses.

[0020] Moreover, since 2019 the molecular details of the cooperation between nspl2- RdRp and nsp7 / nsp8 have been obtained by cryo-electron microscopy (cryo-EM) 3D structures (Hillen ef al., 2020, “Structure of replicating SARS-CoV-2 polymerase”, Nature 584, 154-156; Peng ef al., 2020, “Structural and Biochemical Characterization of the nspl 2-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2”, Cell Rep. 31 , 107774; Yin ef al., 2020, “Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir”, Science 368, 1499-1504). In all the available structures of the nspl 2 / nsp8 / nsp7 complex, one nspl 2 molecule interacts with one nsp7 and two molecules of nsp8.

[0021] In light of these structural data, the nsp7-nsp8 heterodimer plays a critical role in stabilizing the polymerase domain, thereby enabling pattern recognition and binding. Moreover, in the presence of an RNA duplex (corresponding to a pattern product), the long a-helical amino-terminal extensions of the two nsp8 are stabilised and bound at the opposite sides of the polymerase active site cleft, forming “sliding poles” in which positively charged residues interact with and guide the exiting RNA. Thus, these highly conserved helical extensions act as an electrostatic guide through which the product / pattern RNA duplex is extruded. This “sliding clamp” helps the polymerase to grip the RNA and prevent the premature dissociation of the RdRp from RNA during replication, thereby promoting polymerisation processivity.

[0022] Nsp8 is unique and well-conserved protein, like nsp7, among viruses of the Coronavirus genus. Within all SARS-CoV-2 variants isolated so far, sequence conservation of 100% is observed and 97% sequence identity with SARS-CoV (Figure 1 ). In addition to its essential role in viral RNA synthesis, more and more evidences also point out to an involvement of nsp8 in host immune and inflammatory responses suppression. Indeed, it has been shown that SARS-CoV-2 nsp8 disrupts host protein trafficking to the cell membrane upon infection, contributing to host defense suppression via the shutdown of the IFN response (Banerjee ef al., 2020, “SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses”, Cell 183, 1325-1339). A translational study analysed a large cohort of sera from COVID-19-infected patients and defined their antibody response profiles against the SARS-CoV-2 proteome. They revealed that in addition to S and N proteins, some nonstructural and accessory proteins also elicit prevalent antibody responses (Li ef al., 2021 , “Antibody landscape against SARS-CoV-2 reveals significant differences between non-structural / accessory and structural proteins”, Cell Rep. 36, 109391 ). In addition, they showed that the intensity of the humoral response against nonstructural and accessory proteins is correlated with disease severity. Interestingly, among viral replicases, strong IgG responses are detected against nsp8, nspl 2, and nsp7. Very recently, SARS-CoV-2 nsp8 was reported to down-regulate the expression of type I interferon, IFN-stimuloted genes and proinflammatory cytokines (Zhang ef al., 2023, “SARS-CoV-2 Nsp8 suppresses MDA5 antiviral immune responses by impairing TRIM4-mediated K63-linked polyubiquitination”, PLoS Pathog. 19).

[0023] Undeniably, this CoV nonstructural protein nsp8 of 22 kDa represents a very attractive antiviral target with its multiple essential roles for the virus.

[0024] With no human homolog and a conserved-3D structure, viral RNA-dependent RNA polymerases are considered as prime targets for broad spectrum direct-acting antivirals (DAAs).

[0025] Currently, it exists two types of RdRp inhibitors: nucleoside and non-nucleoside analogs, with the first class that are incorporated into replicating viral RNA chain and blocked its extension while the second type induces conformational change preventing the enzyme from working properly. DAAs should have high potency, high therapeutic index and a high barrier to viral resistance. Their discovery is a very long road time and there are a number of cases where such molecules are not (yet) identified (e.g., SARS- CoV-2, Ebola virus, Dengue virus...).

[0026] For example, we know the publication Gharui ef al. (“Characterization of the Conformational Hotspots of the RNA-Dependent RNA Polymerase Complex Identifies a Unique Structural Malleability of nsp8”, 2024, The Journal of Physical Chemistry B, Vol 128 Issue 41 ) describing a mechanistic insight into how nsp7 and nsp8 cofactors regulate the polymerase activity of nspl 2 and suggest a potential new intervention interface, in addition to the canonical polymerase active center, in RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 for antiviral design. Nevertheless, no VHH is explicitly disclosed.

[0027] Furthermore, the publications Espositio ef al. (“NMR-based analysis of VHH- antibodies to SARS-CoV-2 Nsp9 reveals a possible antiviral strategy against COVID-1 ”, Adv Biol (Weinh), 2021 Dec;5(12):e2101 1 13) and Venit et al. (“Nanobody against SARS- CoV-2 non-structural protein Nsp9 inhibits viral replication in human airway epithelia”, Molecular Therapy: Nucleic Acids Vol. 35, September 2024) disclose VHH-antibodies with alternative targets to the SARS-CoV-2 Spike protein such as nsp9, a non-structural protein essential in the SARS-CoV-2 replication complex. Nsp9 interacts with nspl2, described as the core subunit of the SARS-CoV-2 replication complex responsible for RNA-dependent RNA polymerase activity. Specifically, the replication complex is composed of nsp7, nsp8, nspl2, nspl 3 and nsp9, which is required for the function of this complex.

[0028] As same, the publication Wang ef al. (“Applications of VHH-antibodies in the prevention, detection, and treatment of the evolving SARS-CoV-2”, Biochem Pharmacol, 2023 Feb:208:l 15401 ) summarizes the progress in the prevention, detection, and treatment of SARS-CoV-2 using VHH-antibodies as well as strategies to combat the evolving SARS-CoV-2 variants. However, it has not been shown any results proving that VHH-antibodies against either nsp3, nsp7, nsp8, or RdRp inhibit the viral replication of SARS-Cov-2. Patent application WG2023041985 refers to VHHs directed against SARS-CoV-2 nsp7, nsp8, nsp9, nsp!2 and nsp!3. However, in practice, only anti-nsp9 VHHs are actually generated and characterized, and only one of them is presented as having an inhibitory effect on viral replication, and at a very low multiplicity of infection (MOI).

[0029] In the publication Tanaka et al. (“Establishment of a stable SARS-COV-2 replicon system for application in high-throughput screening”, Antiviral Search, 2022), a SARS- CoV-2 replicon system has been described as a screening platform, and an anti-nsp8 IgG antibody is used. However, said IgG is used therein solely for analytical purposes in Western blotting, without any demonstrated effect on replication, or even convincing detection of nsp8. No antiviral activity of an antibody directed against nsp8 is reported in this context.

[0030] Finally, we know from the publication Chouchane ef al. (“Dromedaray camels as a natural source of neutralizing VHH-antibodies against SARS-CoV-2”, JCI Insight, 2021 Mar 8;6(5):el 45785) that cross-neutralizing antibodies against SARS-CoV-2 were found in dromedary camels that were Middle East respiratory syndrome coronavirus (MERS-CoV) seropositive but MERS-CoV free. The latter had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins. Analysis of these antibodies from camel sera revealed a remarkable cross-reactivity between camel IgG and VHH antibodies and SARS-CoV-2 proteins. Several non-structural proteins of SARS- CoV-2 such as nsp7 and nsp8 and RdRp elicited strong reactivities of IgGl and VHH antibodies. These analyses demonstrate that the development of hyperimmune camels against SARS-CoV-2 could be interesting for the development of therapeutic agents for the prevention and / or treatment of Covid-19. However, these cross-reactivities were only assessed qualitatively on camel sera, with no negative controls, and the antibodies against nsp7, nsp8, and nspl2 were never tested for actual viral replication inhibition. The observed neutralization of SARS-CoV-2 was mediated by anti-Spike antibodies, and the sequences of anti-nsp VHHs were not determined.

[0031] PROBLEM TO BE SOLVED

[0032] The technical problem underlying the present invention is the provision of a first-in- class product targeting and blocking the catalytic core of a virus replication, notably SARS-CoV-2 replication, which is relatively fast and costless to implement and offers a pan-viral protection against SARS-CoV-2 variants and also more broadly against Coronaviruses, with a risk of selecting treatment-resistant viruses which is extremely low.

[0033] The specific aim of the invention is to target the RNA-dependent RNA polymerase (RdRp), in particular SARS-CoV-2 RdRp, with a different angle of attack in view of the prior art, in preventing its interaction with cofactor like nsp8 which is essential to its activity.

[0034] To this end, single-domain antibodies have been specifically generated (also named hereafter VHHs) against SARS-CoV-2 nsp8. Although nsp8 has been identified in light of the state of the art as essential subunit of the SARS-CoV-2 viral replication / transcription complex, no VHHs has been explicitly described against the latter.

[0035] In comparison with the typical IgG antibody of 150 kDa, VHHs are smaller ( 15 kDa) and therefore, can recognize more epitopes, particularly some hidden in pockets or clefts or even cryptic epitopes, with high affinity. Furthermore, the affinity of the VHHs according to the invention for notably nsp8 is much higher than the affinity of this cofactor with nsp! 2.

[0036] SUMMARY OF THE INVENTION

[0037] A first object of the invention is to provide single-domain antibodies (sdAbs) that specifically bind to a RNA-dependent RNA polymerase (RdRp) complex, said singledomain antibodies comprising an amino acid sequence consisting of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3) according to the formula FR1 -CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences have at least 85% sequence identity to the SEQ ID below : CDR1 is SEQ ID NO:1 , CDR2 is SEQ ID:2 and CDR3 is SEQ ID NO: 3; or CDR1 is SEQ ID NO:4, CDR2 is SEQ ID:5 and CDR3 is SEQ ID NO: 6; or CDR1 is SEQ ID NO:7, CDR2 is SEQ ID:8 and CDR3 is SEQ ID NO: 9; or CDR1 is SEQ ID NQ:10, CDR2 is SEQ ID:1 1 and CDR3 is SEQ ID NO: 12; or CDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO: 15; or CDR1 is SEQ ID NO:16, CDR2 is SEQ ID:17 and CDR3 is SEQ ID NO: 18; or CDR1 is SEQ ID NO:19, CDR2 is SEQ ID:20 and CDR3 is SEQ ID NO: 21 ; or CDR1 is SEQ ID NO:22, CDR2 is SEQ ID:23 and CDR3 is SEQ ID NO: 24.

[0038] A second object is to provide a complex comprising an homodimer or an heterodimer of sdAbs according to the invention linked by a linker.

[0039] A further object of the invention is to provide a complex comprising a sdAb according to the invention and a RdRp SARS-CoV-2 cofactor chosen from nsp8 alone, nsp8-nsp7 complex and nsp8-nsp7-nspl 2 complex.

[0040] Another object is a nucleic acid molecule encoding sdAbs according to the invention.

[0041] Another object is a lipid nanoparticle carrying a nucleic acid molecule according to the invention.

[0042] Finally, another object is a composition comprising sdAbs according to the invention and optionally a pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant.

[0043] Further aspects and advantages of the present invention are described in the following description (with reference to Figures), which should be regarded as illustrative and not limiting the scope of the present application.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a comparison of the nsp8 protein sequence of SARS-CoV and of 12 SARS-CoV-2 variants (respectively corresponding to SEQ ID NO: 33-45).

[0045] Figure 2 shows in vitro SARS-CoV-2 RNA polymerase activity in presence of anti- nsp8 VHHs.

[0046] Figure 3 shows real-time association and dissociation between 3 of the 8 VHHs and the SARS-CoV-2 polymerase complex (formed by nsp! 2 / nsp8 / nsp7) by BioLayer interferometry (BLI). Ctrl- corresponds to a VHH against the SARS-CoV-2 Spike protein incubated with the SARS-CoV-2 polymerase complex.

[0047] Figure 4 shows ex vivo analysis of VHHs anti-nsp8 on SARS-CoV-2 replication in Vero cells.

[0048] Figure 5 shows real-time association and dissociation between 3 of the 8 VHHs and the nsp8 SARS-CoV-2 polymerase subunit by BioLayer interferometry (BLI).

[0049] Figure 6 shows in vitro SARS-CoV-2 RNA polymerase activity in presence of anti- nsp8 VHHs for the determination of ICso.

[0050] Figure 7 shows analysis of the protective effect of VHHs anti-nsp8 in Vero cells infected with SARS-CoV-2 (BA.5 strain, at an MOI of 1 ).

[0051] Figure 8 shows analysis of the protective effect of VHHs anti-nsp8 in Vero cells infected with SARS-CoV-2 ( KP3 strain, at an MOI of 1 ).

[0052] Figure 9 shows in vitro SARS-CoV-2 RNA polymerase activity in presence of anti- nsp8 VHHs and bi-VHHs.

[0053] Figure 10 shows in vitro SARS-CoV-2 RNA polymerase activity in presence of anti- nsp8 bi-VHH F5-F5 for the determination of ICso.

[0054] The sequences used in the present invention are detailed in the following table:

[0055] DETAILED DESCRIPTION

[0056] According to a first aspect of the invention, the present inventors have identified single-domain antibodies (sdAbs) that specifically bind to a RNA-dependent RNA polymerase (RdRp) complex, said single-domain antibodies comprising an amino acid sequence consisting of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3) according to the formula FR1 -CDR1 -FR2-CDR2-FR3- CDR3-FR4, wherein the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below : CDR1 is SEQ ID NO:1 , CDR2 is SEQ ID:2 and CDR3 is SEQ ID NO: 3; or CDR1 is SEQ ID NO:4, CDR2 is SEQ ID:5 and CDR3 is SEQ ID NO: 6; or CDR1 is SEQ ID NO:7, CDR2 is SEQ ID:8 and CDR3 is SEQ ID NO: 9; or CDR1 is SEQ ID NQ:10, CDR2 is SEQ ID:1 1 and CDR3 is SEQ ID NO: 12; or CDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO: 15; or CDR1 is SEQ ID NO:16, CDR2 is SEQ ID:17 and CDR3 is SEQ ID NO: 18; or CDR1 is SEQ ID NO:19, CDR2 is SEQ ID:20 and CDR3 is SEQ ID NO: 21 ; or CDR1 is SEQ ID NO:22, CDR2 is SEQ ID:23 and CDR3 is SEQ ID NO: 24.

[0057] According to a preferred embodiment, the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below:

[0058] CDR1 is SEQ ID NO:13, CDR2 is SEQ ID NO:14, and CDR3 is SEQ ID NO:15.

[0059] Even more preferably, the CDR sequences consist in with the following sequences: CDR1 is SEQ ID NO:13, CDR2 is SEQ ID NO:14, and CDR3 is SEQ ID NO:15.

[0060] As used herein, the terms “single-domain antibody” (sdAb), “nanobody” (Nb), “Variable domain of Heavy-chain-only antibody (VHH)", “VHH-antibody”, “VHH antibody fragment” and “VHH chain” can be used interchangeably herein and have the same meaning referring to a variable region of a single heavy chain of an antibody, and construct a single-variable domain antibody consisting of only one heavy chain variable region, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding-domain. A single-domain antibody may derive from, or consist in, a VHH that refers to a single-variable domain found in HCAb of Camelidae, which are naturally devoid of light chains. It is the smallest antigen-binding fragment with complete function.

[0061] As used herein, the terms “antigen-binding fragment”, “antigen-binding domain”, “antigen-binding region”, and similar terms refer to that portion of a binding molecule, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g, the CDRs). “Antigenbinding fragment” as used herein includes “antibody fragment,” which comprises a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab’, F(ab’)2, and Fv fragments; diabodies and di-diabodies; single-chain antibody molecules; dual variable domain antibodies; single variable domain antibodies (sdAbs); and multispecific antibodies formed from antibody fragments.

[0062] As used herein, the term “variable” means that certain portions of the variable region in the VHH-antibody vary in sequences, which forms the binding and specificity of various specific antibodies to their particular antigen. However, variability is not uniformly distributed throughout the VHH-antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of heavy chain. The more conserved part is called the framework region (FR). The variable regions of the natural heavy chains contain four FR regions, which are present in a pi-folded configuration, joined by three CDRs which form a linking loop, and in some cases can form a partially pi-folded structure.

[0063] The CDR sequences of the single-domain antibodies (sdAbs) according to the invention have at least 85%, e.g. 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below: CDR1 is SEQ ID NO:1 , CDR2 is SEQ ID:2 and CDR3 is SEQ ID NO: 3; or CDR1 is SEQ ID NO:4, CDR2 is SEQ ID:5 and CDR3 is SEQ ID NO: 6; or CDR1 is SEQ ID NO:7, CDR2 is SEQ ID:8 and CDR3 is SEQ ID NO: 9; or CDR1 is SEQ ID NQ:10, CDR2 is SEQ ID:1 1 and CDR3 is SEQ ID NO: 12; or CDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO: 15; or CDR1 is SEQ ID NO:16, CDR2 is SEQ ID:17 and CDR3 is SEQ ID NO: 18; or CDR1 is SEQ ID NO:19, CDR2 is SEQ ID:20 and CDR3 is SEQ ID NO: 21 ; or CDR1 is SEQ ID NO:22, CDR2 is SEQ ID:23 and CDR3 is SEQ ID NO: 24.

[0064] “Identity”, as used herein, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in the following references (Computational Molecular Biology, Lesk A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and genome Projects, Smith D.W., ed., Academic Press, New York. 1993; Computer Analysis of sequence Data, Part I, Griffin A.M., and Griffin H.G., eds., Humana Press. New jersey, 1994; sequence Analysis in Molecular Biology, von Heinje G., Academic Press, 1987; and sequence Analysis Primer, Gribskov M. and Devereux J., eds., M Stockton Press, New York, 1991 ; and Carillo H., and Lipman D., SIAM J. Applied Math., 48:1073 (1998)). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux J. et al., Nucleic Acids Research 12(1 ): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul S.F. et al., J. Molec. Biol. 215: 403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul S. etal., NCBI NLM NUH Bethesda, MD 20894; Altschul S. et al., J. Mol Biol. 215: 403-410 (1990)).

[0065] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense of “consisting of” or “consisting essentially of”; that is to indicate, in the sense of “including, but not limited to”. Preferably, the sdAbs according to the invention, comprise an amino acid sequence having at least 85%, e.g. 86%, 88%, 90%, 92%, 94%, 96%, 98%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID NO: 25-32, preferably SEQ ID NO: 29.

[0066] As an illustrated example, comprising an amino acid sequence having at least 85% identity to the e.g. SEQ ID NO:1 (for CDR1 ) means some variant amino acid sequences having at most one mutation compared to SEQ ID NO:1 , i.e. one insertion, one deletion or one substitution compared to SEQ ID NO:1 .

[0067] An insertion is a mutation in which an extra amino acid is inserted into a new place in the peptide or protein.

[0068] Deletion is a mutation in which an amino acid is lost or deleted.

[0069] A substitution is a mutation that exchanges one amino acid for another.

[0070] More preferably, the sdAbs according to the invention, consist of an amino acid sequence chosen from SEQ ID NO: 25-32, preferably SEQ ID NO: 29.The RNA-dependent RNA polymerase (RdRp) complex targeted by the sdAbs according to the invention is preferably of a virus from Coronaviruses.

[0071] For the present disclosure, it is envisaged that the term “coronavirus” comprises any coronavirus, irrespective of strain or origin. Suitably, the term “coronavirus” encompasses, at least, those coronavirus strains of SARS-CoV-2 Wuhan, alpha, beta, gamma, delta and omicron lineages.

[0072] As illustrated examples, the coronavirus provided herein comprises a coronavirus strain selected from the group consisting of: omicron BA.5.

[0073] The RNA-dependent RNA polymerase (RdRp) activity targeted by the sdAbs according to the invention is more preferably of SARS-CoV-2.

[0074] Preferably, the single-domain antibodies according to the invention specifically bind to or interact with one or more residues of a RdRp SARS-CoV-2 cofactor chosen from nsp8 alone, nsp8-nsp7 complex and nsp8-nsp7-nspl2 complex.

[0075] The terms "that specifically binds to RNA-dependent RNA polymerase (RdRp) complex” and analogous terms, as used herein, refer to constructs that specifically recognize nsp7, nsp8 and nspl 2 of RdRp and do not or weakly recognize other antigen(s).

[0076] A further aspect of the invention provides a complex comprising an homodimer or an heterodimer of a sdAb (=bi-sdAb) according to the invention linked by a linker.

[0077] In other words, the bi-sdAb comprises two sdAbs according to the invention, identical (i.e. homodimer) or different (i.e. heterodimer), each sdAb comprising an amino acid sequence consisting of four framework regions (FR1 to FR4) and three complementary determining regions (CDR1 to CDR3) according to the formula FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein for each sdAb the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below:

[0078] CDR1 is SEQ ID NO:1 , CDR2 is SEQ ID:2 and CDR3 is SEQ ID NO: 3; or

[0079] CDR1 is SEQ ID NO:4, CDR2 is SEQ ID:5 and CDR3 is SEQ ID NO: 6; or

[0080] CDR1 is SEQ ID NO:7, CDR2 is SEQ ID:8 and CDR3 is SEQ ID NO: 9; or

[0081] CDR1 is SEQ ID NQ:10, CDR2 is SEQ ID: 1 1 and CDR3 is SEQ ID NO:12; or

[0082] CDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO:15; or CDR1 is SEQ ID NO:16, CDR2 is SEQ ID:17 and CDR3 is SEQ ID NO:18; or CDR1 is SEQ ID NO:19, CDR2 is SEQ ID:20 and CDR3 is SEQ ID NO:21 ; or CDR1 is SEQ ID NO:22, CDR2 is SEQ ID:23 and CDR3 is SEQ ID NO:24.

[0083] Preferably, for at least one sdAb of the bi-sdAb, the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below:

[0084] CDR1 is SEQ ID NO:13, CDR2 is SEQ ID NO:14 and CDR3 is SEQ ID NO:15.

[0085] More preferably, for both sdAbs of the bi-sdAb, the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below:

[0086] CDR1 is SEQ ID NO:13, CDR2 is SEQ ID NO:14 and CDR3 is SEQ ID NO:15.

[0087] As illustrated non limitative examples, the linker is a type of flexible, unstructured synthetic peptide linker sequence often leveraged to connect two VHH-antibodies chosen from G4S linker proteins, which is a poly-Glycine-Serine (G4S) linker consisting of a core pentapeptide sequence, Gly-Gly-Gly-Gly-Ser, that is repeated and commonly found as either a 15-mer (G4S)s or 20-mer (G4S)4. The length of the linker will be advantageously determined rationally, based on epitope-mapping data between VHHs and the polymerase complex by electron microscopy.

[0088] Another aspect of the invention provides a complex comprising a sdAb according to the invention and a RdRp SARS-CoV-2 cofactor chosen from nsp8 alone, nsp8-nsp7 complex and nsp8-nsp7-nspl 2 complex.

[0089] A further aspect of the invention provides a nucleic acid molecule encoding sdAbs according to the invention.

[0090] Another object is a lipid nanoparticle carrying a nucleic acid molecule according to the invention.

[0091] Lipid nanoparticles mean hollow spheres made of different types of lipids such as ionizable lipids, phospholipids, cholesterol and lipids modified by polyethylene glycol (PEG). Lipid nanoparticles are able to merge with the cytoplasmic membrane of the cells by incorporating their lipids into the lipids of said cytoplasmic membrane. In this way, lipid nanoparticles release their content inside said cells. For production of the sdAbs according to the invention, one method is to produce by in vitro transcription (RNA polymerase of the T7 phage) the mRNAs encoding the VHHs and then to deliver them by lipid nanoparticle.

[0092] Finally, an object of the invention is to provide a composition comprising the sdAbs according to the invention and optionally a pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant.

[0093] A pharmaceutically acceptable carrier includes any, and all solvents, lipids, polymers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like suitable for administration to a mammalian host. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition of the present invention.

[0094] Preferably, the composition according to the invention is used in the prevention and / or treatment of a virus infection from Coronaviruses, and / or disease, disorder or condition associated therewith in a subject in need thereof, in particular a virus infection of SARS-CoV-2.

[0095] The term "treat” or "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and / or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and / or eliminating, reducing and / or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and / or delaying the progression and / or the incidence of a disease or the risk of its occurrence. In certain embodiments, such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.

[0096] As used herein, the term "disorder” or "disease” refers to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g. an illness that disrupts normal physical or mental functions.

[0097] As used herein, the term "subject” or "patient” refers to human and veterinary subjects particularly to an animal, preferably to a mammal, even more preferably to a human, including adult and child. However, the term "subject" also encompasses non- human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep and non- human primates, among others.

[0098] Embodiments of the present invention will now be described by way of the following examples.

[0099] EXAMPLES

[0100] EXAMPLE 1 : In vitro RNA polymerase activity of SARS-CoV-2 RNA polymerase complex (formed of nspl2 / nsp8 / nsp7) in presence of 8 VHHs generated against SARS- CoV-2

[0101] Primer extension polymerase assays were performed using an RNA hairpin as substrate, purified nspl 2, nsp8 and nsp7, and in presence of the eight VHHs (Fl to F8 of respective sequences SEQ ID NO: 25-32) generated against nsp8 at 30 M each.

[0102] Specific single domain antibodies were selected after 3 rounds of panning using SARS-CoV-2 nsp8 as bait. Sequencing analysis of positive clones from the third round of panning revealed eight different nsp8-specific VHHs (named Fl to F8). The eight selected VHHs show sequence differences in the three variable complementary determining regions (CDRs), suggesting that they bind to distinct regions on nsp8 protein.

[0103] The eight VHH-antibodies were expressed in bacteria system and purified by affinity and size exclusion chromatography. Their potential SARS-CoV-2 replication inhibition activity were analysed in vitro, as described in (Subissi et al., 2014). This in vitro analysis revealed that all 8 VHHs negatively affect SARS-CoV-2 nspl 2-RNA polymerase activity and with VHH F5 and F7 (at 30 M) that totally prevent the latter, as shown in Figure 2. To characterize more deeply these 8 VHHs, a real-time association and dissociation were monitored between at least 3 VHHs (F4, F5 and F7) and the polymerase complex (formed of nspl 2 / nsp8 / nsp7) using BioLayer interferometry (BLI) (Figure 3).

[0104] As expected, the 3 VHHs bind to the polymerase complex, albeit with differences in the binding curves. Importantly, none of the 3 VHHs dissociate over the time range tested, indicating a very high affinity.

[0105] Two parameters may be of interest. The slope at origin is the kinetics factor that characterizes the association speed between a VHH and the polymerase complex. The terminal value characterizes the quantity of VHH bound to the polymerase complex.

[0106] As a result, VHH F5 and VHH F7 bind rapidly to the polymerase complex, whereas the VHH F4-polymerase complex association is slower. This behavior could explain the high potent of VHH F5 and F7 revealed in the in vitro assay.

[0107] There is no obvious dissociation for VHH F4, F5 and F7.

[0108] As negative control, VHH72 was used as it recognizes the spike protein of SARS- CoV-2 (Ctrl-). EXAMPLE 2: Ex vivo activity test of VHH F2, F4, F5 and F6 in SARS-CoV-2 infected Vero cells

[0109] The inhibition potential of VHH F2, F4, F5 and F6 in cellular context were analysed. Vero cells stably expressing these VHHs were established after lentivirus transduction. Then, these cells were infected with SARS-CoV-2 (Omicron variant, BA.5 strain) at a MOI of 1.

[0110] As SARS-CoV-2 is a lytic virus, cellular viability was assessed 5 days after infection.

[0111] As shown in Figure 4, and in comparison with the NT+ control, corresponding to SARS-CoV-2 infected cells without VHH, a protector effect is observed for cells expressing VHH F2 and F5.

[0112] These first results indicate VHH specificity of action in vitro and ex vivo.

[0113] EXAMPLE 3: Characterization of interactions and in vitro inhibitory potential of VHH F4, VHH F5 and VHH F7 targeting SARS-CoV-2 nsp8

[0114] Real-time binding interactions between SARS-CoV-2 nsp8 and at least three VHHs F4, F5, and F7 were analyzed using BioLayer Interferometry (BLI).

[0115] For this purpose, nsp8 was biotinylated and immobilized on streptavidin-coated biosensors. The sensors were then incubated with serial dilutions of each VHH, and association and dissociation phases were monitored over several hundred seconds. All binding curves were fitted using a 1 :1 binding model with Sartorius Octet-R2 analysis software, yielding dissociation constants (KD) of 0.22 pM for VHH F4, 3.8 nM for VHH F5, and 0.4 nM for VHH F7 (Figure 5).

[0116] These KD values indicate that VHH F5 and F7 bind nsp8 with high affinity in the low nanomolar range, whereas VHH F4 exhibits a weaker, micromolar interaction. The strong binding of VHH F5 and F7 suggests a higher potential for effective inhibition of nsp8- mediated polymerase activity, highlighting their promise as candidate therapeutic agents targeting viral replication.

[0117] In addition to binding affinity measurements, the inhibitory potency of the VHHs against nsp8 activity was evaluated by determining their IC50values. The IC50(half- maximal inhibitory concentration) is defined as the concentration of an inhibitor required to reduce a specific biological or enzymatic activity by 50%.

[0118] A labelled-RNA hairpin was incubated with the purified nspl 2 / nsp8 / nsp7 complex allowing primer extension of a 4-mer product and in presence of increasing concentrations of VHH. The RNA products were analysed after 30 min of reaction incubation by denaturing PAGE and quantified using Typhoon Imager software.

[0119] The ICso values were 1 .2 pM for VHH F4, 4 pM for VHH F5, and 2 pM for VHH F7 (Figure

[0120] 6). The KD values (VHH F4: 0.22 M; VHH F5: 3.8 nM; VHH F7: 0.4 nM) together with the IC50measurements (VHH F4: 1 .2 pM; VHH F5: 4 pM; VHH F7: 2 pM) indicate that VHH F4, F5 and F7 each bind to nsp8 and exert a measurable inhibition of its activity in vitro.

[0121] Taken together, these quantitative data support all three VHHs as comparable candidates with demonstrated interaction with nsp8 and inhibitory potential on the polymerase complex.

[0122] EXAMPLE 4: Evaluation of the protective effect of anti-nsp8 VHHs in SARS-CoV-2- infected Vero cells

[0123] Analysis of the protective effect of anti-nsp8 VHHs in Vero cells infected with SARS- CoV-2 was performed using two viral strains, BA.5 and KP3.

[0124] Vero cells were stably transduced with lentiviral vectors expressing anti-nsp8 VHHs Fl to F8, followed by selection using the pLVX-IRES-ZsGreen plasmid. Cells expressing an irrelevant VHH (7bnw) or only the ZsGreen reporter (0) were included as controls, along with infected (NT inf.) and uninfected (NT non inf.) Vero cells. The transduced cells were infected with the respective SARS-CoV-2 strains (BA.5 and KP3) at an MOI of 1, and cell viability was assessed five days post-infection.

[0125] For the BA.5 strain, VHHs Fl , F2, F3, F5, and F7 demonstrated significantly improved cell survival compared to controls (Figure 7). A similar protective pattern was observed with the KP3 strain (Figure 8).

[0126] In the cellular context, the VHHs were delivered either as DNA via lentiviral transduction or as mRNA, allowing intracellular expression of the anti-nsp8 antibodies. Notably, VHH F4 exhibited poor expression in Vero cells regardless of the delivery format, which likely accounts for its lack of protective effect in these assays. This contrasts with its strong in vitro performance observed in Examples 1 and 3, highlighting that efficient cellular expression is a critical factor for translating in vitro activity into functional antiviral effects in vivo.

[0127] These results indicate that specific anti-nsp8 VHHs can confer measurable protection against SARS-CoV-2-induced cytopathic effects. The observed protection is consistent with the VHHs’ ability to interfere with viral replication, suggesting that these candidates, particularly Fl , F2, F3, F5, and F7, represent promising tools for further development as antiviral agents targeting nsp8-mediated replication processes.

[0128] EXAMPLE 5: In vitro inhibition of SARS-CoV-2 RNA polymerase activity by monovalent and bi-valent anti-nsp8 VHHs

[0129] The potential inhibition of SARS-CoV-2 replication by anti-nsp8 VHHs was evaluated in vitro using monovalent VHHs F4 and F5, as well as bi-valent constructs combining F4 and F5 in different orientations (F4-F5, F5-F4, F5-F5) with a [G4S]4linker, following the primer-dependent polymerase assay protocol described by Subissi et al., 2014. RNA products generated by the SARS-CoV-2 nspl 2-RNA polymerase complexwere analysed by denaturing PAGE and visualized using a Typhoon Imager.

[0130] All VHHs and bi-VHHs tested inhibited polymerase activity to varying degrees (Figure 9). Notably, the bi-VHH F5-F5 exhibited an IC50of 0.58 M (Figure 10), corresponding to a six-fold improvement in inhibitory potency compared to the monovalent VHH F5 (IC50= 4 M).

[0131] This enhancement demonstrates the benefit of bivalency in increasing effective inhibition, likely due to cooperative binding and prolonged engagement with nsp8, as well as by the fact that two nsp8 monomers are required for an RNA polymerase nspl 2 molecule. Indeed, in all the available structures of the nspl 2 / nsp8 / nsp7 complex, one nspl 2 molecule interacts with one nsp7 and two molecules of nsp8.

[0132] The data indicate that combining VHHs into bi-valent formats can significantly enhance functional inhibition of the SARS-CoV-2 polymerase complex, providing a promising strategy for the development of potent antiviral molecules targeting viral replication.

[0133] Overall, these results identify both monovalent and bi-valent anti-nsp8 VHHs as effective inhibitors of SARS-CoV-2 polymerase activity, with bi-VHH formats showing superior efficacy, supporting their prioritization for further preclinical evaluation.

Claims

CLAIMS1. A single-domain antibody (sdAb) that specifically binds to a RNA-dependent RNA polymerase (RdRp) complex, said single-domain antibody comprising an amino acid sequence consisting of 4 framework regions (FR1 to FR4) and 3 complementary determining regions (CDR1 to CDR3) according to the formula FR1 -CDR1 -FR2-CDR2-FR3- CDR3-FR4, wherein the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ ID below:CDR1 is SEQ ID NO:1 , CDR2 is SEQ ID:2 and CDR3 is SEQ ID NO: 3; orCDR1 is SEQ ID NO:4, CDR2 is SEQ ID:5 and CDR3 is SEQ ID NO: 6; orCDR1 is SEQ ID NO:7, CDR2 is SEQ ID:8 and CDR3 is SEQ ID NO: 9; orCDR1 is SEQ ID NQ:10, CDR2 is SEQ ID:1 1 and CDR3 is SEQ ID NO: 12; orCDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO: 15; orCDR1 is SEQ ID NO:16, CDR2 is SEQ ID:17 and CDR3 is SEQ ID NO: 18; orCDR1 is SEQ ID NO:19, CDR2 is SEQ ID:20 and CDR3 is SEQ ID NO: 21 ; orCDR1 is SEQ ID NO:22, CDR2 is SEQ ID:23 and CDR3 is SEQ ID NO: 24.

2. The sdAb according to claim 1 , comprising an amino acid sequence having at least 85% sequence identity to the SEQ ID NO: 25-32, preferably SEQ ID NO: 29.

3. The sdAb according to claim 1 or 2, consisting of an amino acid sequence chosen from SEQ ID NO: 25-32, preferably SEQ ID NO: 29.

4. The sdAb according to claim 1 , wherein the CDR sequences have at least 85% sequence identity to the SEQ ID below:CDR1 is SEQ ID NO:13, CDR2 is SEQ ID:14 and CDR3 is SEQ ID NO:15.

5. The sdAb according to any of the preceding claims, wherein the RNA- dependent RNA polymerase (RdRp) complex is of a virus from Coronaviruses.

6. The sdAb according to claim 5, wherein the RNA-dependent RNA polymerase (RdRp) complex is of SARS-CoV-2.

7. The sdAb according to claim 6, wherein the single-domain antibody specifically binds to or interacts with one or more residues of a RdRp SARS-CoV-2 cofactor chosen from nsp8 alone, nsp8-nsp7 complex and nsp8-nsp7-nspl 2 complex.

8. A complex comprising an homodimer or an heterodimer of a sdAb (=bi-sdAb) according to any of claims 1 to 7 linked by a linker.

9. The complex according to claim 8, wherein for at least one sdAb of the bi-sdAb, preferably for both sdAbs of the bi-sdAb, the CDR sequences have at least 85%, preferably at least 95%, more preferably at least 99%, sequence identity to the SEQ IS below:CDR1 is SEQ ID NO:13, CDR2 is SEQ ID NO:14 and CDR3 is SEQ ID NO:1510. A complex comprising a sdAb according to any of claims 1 to 7 and a RdRp SARS-CoV-2 cofactor chosen from nsp8 alone, nsp8-nsp7 complex and nsp8-nsp7-nspl2 complex.1 1 . A nucleic acid molecule encoding a sdAb according to any of claims 1 to 7.

12. A lipid nanoparticle carrying a nucleic acid molecule according to claim 1 1 .

13. A composition comprising the sdAb according to any of claims 1 to 7 and optionally a pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant.

14. The composition according to claim 13 for use in the prevention and / or treatment of a virus infection from Coronaviruses, and / or disease, disorder or condition associated therewith in a subject in need thereof.

15. The composition for use according to claim 14, wherein the virus infection is of SARS-CoV-2.

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