Nanoscale warriors against viral invaders: a comprehensive review of Nanobodies as potential antiviral therapeutics

ABSTRACT

Viral infections remain a significant global health threat, with emerging and reemerging viruses causing epidemics and pandemics. Despite advancements in antiviral therapies, the development of effective treatments is often hindered by challenges, such as viral resistance and the emergence of new strains. In this context, the development of novel therapeutic modalities is essential to combat notorious viruses. While traditional monoclonal antibodies are widely used for the treatment of several diseases, nanobodies derived from heavy chain-only antibodies have emerged as promising “nanoscale warriors” against viral infections. Nanobodies possess unique structural properties that enhance their ability to recognize diverse epitopes. Their small size also imparts properties, such as improved bioavailability, solubility, stability, and proteolytic resistance, making them an ideal class of therapeutics for viral infections. In this review, we discuss the role of nanobodies as antivirals against various viruses. Techniques used for developing nanobodies, delivery strategies are covered, and the challenges and opportunities associated with their use as antiviral therapies are discussed. We also offer insights into the future of nanobody-based antiviral research to support the development of new strategies for managing viral infections.

Introduction

Viral infections present a persistent and formidable challenge to global health, underscored by the emergence and reemergence of various viral pathogens that can lead to epidemics and pandemics. Despite advancements in antiviral therapies, the development of effective treatments is frequently affected by issues such as immune evasion,¹ continuous emergence of new strains causing drug resistance, and costs associated with drug development.² This necessitates the development of new therapeutic strategies to overcome the existing challenges. Monoclonal antibodies (mAbs) have been recognized as an important class of targeted therapeutic agents for the treatment of a wide range of disease conditions.^3,4 Typically, they exhibit high specificity and affinity for their target with relatively low toxicity.⁵ In addition to their use in the treatment of cancer, autoimmunity, and inflammation, they have also been recognized as potent antiviral molecules. MAbs have been developed to treat various viral infections, including infections caused by human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), and SARS-CoV-2.^6–8 Primarily, mAbs neutralize viruses by disrupting binding between the host and viral proteins. This is achieved by targeting the viral⁷ or host proteins.⁵ They also promote clearance of infected cells through Fc-mediated effector functions.⁷ However, their large size (~150 kDa) and complex multimeric structure may pose limitations, including reduced tissue penetration and potential immunogenicity.⁹ Furthermore, mAbs are challenging to produce, with high costs and long development timelines.¹⁰

In recent years, nanobodies derived from the antigen-binding domain of heavy chain-only antibodies (HCAbs) expressed in camelids, have garnered attention as promising candidates for the fight against viral infections.¹¹ Discovered serendipitously by the research group of Raymond Hammers, these molecules have been extensively studied for diagnostic and therapeutic applications. Their unique structural properties enable the recognition of a broader range of epitopes, enhancing their versatility for targeting diverse viruses.¹² Their small size offers several advantages, including improved bioavailability, stability, solubility, and ease of genetic engineering for tailoring properties. Nanobodies are also economically produced through recombinant expression and purification, positioning them as cost-effective antiviral candidates.¹³ In addition, their favorable biophysical characteristics allow for the exploration of alternative delivery routes for respiratory and intestinal viral infections.^14,15 Target-specific nanobodies are typically identified using phage-displayed libraries derived from immunized camelids.¹¹ Recently, phage-displayed semi-synthetic and synthetic libraries have become popular, eliminating the need for animal immunization.¹⁶ In addition to phage display, other surface display technologies, including yeast display and ribosome display have also been used to discover nanobodies.

Nanobody-based research gained traction in 2001 with the establishment of Ablynx, a company focused on developing nanobody therapeutics.¹¹ These efforts eventually led to the approval of the first nanobody-based therapeutic, caplacizumab, in 2019 for the treatment of thrombotic thrombocytopenic purpura (TPP), a blood-clotting disorder.¹⁷ Since then, the development of nanobody therapies has accelerated, particularly after intellectual property restrictions were relaxed.^11,16 In this review, we discuss the potential of nanobodies as antiviral agents against a broad range of viral pathogens, including SARS-CoV-2, MERS-CoV, HIV, and influenza. We provide an overview of the structural and functional properties of nanobodies, discuss the technologies used for nanobody discovery and development, examines strategies for their delivery, and highlight the challenges and opportunities associated with their use as antivirals. Finally, we also provide insights into the future of nanobody-based antiviral research.

Structural and functional properties of Nanobodies

Nanobodies, the antigen-binding domains of heavy-chain-only antibodies (HCAbs), were first discovered in 1989.¹³ In contrast to traditional antibodies, which consist of two heavy chains and two light chains, HCAbs lack the light chain and CH1 domain due to a point mutation in the CH1-Hinge intron that disrupts the consensus splice site (Figure 1).^13,18,19 This mutation results in the expression of antibodies without a light chain, with the variable heavy-chain domain (~110 amino acids) being the primary antigen-binding structure, known as the VHH or Nanobody, a term coined by Ablynx (Figure 1). Later, similar antibodies called Ig New Antigen Receptors (IgNARs) encoded by one variable domain and five constant domains were discovered in cartilaginous fish, including sharks.²⁰ The variable domain of these antibodies, called VNAR, is one of the smallest antigen-binding structures (~10–12 kDa), resembling traditional antibodies.²⁰

Figure 1.

Comparison of conventional and camelid-derived antibody structures. (a) Conventional IgG antibodies consist of two heavy chains and two light chains. The antigen-binding site is formed by the variable domains of the heavy (VH) and light (VL) chains. (b) Camelid heavy chain-only antibodies (HCAbs) lack the light chain and have a single variable heavy domain (VHH) responsible for antigen binding. (c) The VHH domain of HCAbs is called a nanobody. Sequence differences between VH and VHH at key positions (amino acid 37, 44, 45, 47 as per Kabat numbering system) are shown. (the figure has been made using Microsoft PowerPoint).

Nanobodies with a single-domain structure are significantly smaller (~15 kDa) than the antigen-binding domains of traditional antibodies (scFv ~25–30 kDa and Fab ~ 50 kDa). The VHH domain is composed of a typical immunoglobulin (Ig) fold composed of 4-stranded beta-sheet and a 5-stranded beta-sheet stabilized by a disulfide bond.¹⁸ The structure includes three complementarity-determining regions (CDRs) and four framework regions (FRs), with specific structural differences to compensate for the lack of a light chain. In traditional antibodies, four hydrophobic residues in FR2 of the VH domain (V-37, G-44, L-45, and F-47/W-47; Kabat numbering system; Figure 1) form a part of the interface that interacts with the VL domain. However, in VHH, these residues are replaced by smaller, more hydrophilic residues, (Y-37/F-37, E-44/Q-44, R-45/C-45, and G-47/R-47/L-47/S-47; Kabat numbering system²¹ reducing the area of the exposed hydrophobic patch.^22,23 In addition, CDR3 of VHH folds over to cover the region previously covered by the VL domain of the light chain enhancing the hydrophilicity and solubility of the nanobody molecules compared to the VH domains or scFv(s).⁹ The elongated CDRs of VHH, especially CDR1 and CDR3 compensate for the loss of antigen-binding diversity caused by the lack of a light chain. The extended length of CDR3 in naturally occurring nanobodies provides a large area of 600–800 angstroms, thereby contributing to the large antigen-binding diversity in the VHH paratope, similar to that provided by the six CDRs (3 each in VL and VH) in traditional antibodies.^24,25 The long CDR3 forms convex-shaped or finger-like extensions that can extend into the inner regions of antigens, targeting epitopes that are often unreachable by traditional mAbs. This property is particularly beneficial in cases of viral infections that require targeting of recessed epitopes on viral glycoproteins or enzyme active sites.²⁶ To compensate for the increased entropy due to the higher flexibility of the long CDR3 loop during antigen binding, the VHH domain has also evolved to possess an extra non-conserved disulfide bond between CDR1 and CDR3, or CDR2 and CDR3.²² Due to their unique structure, despite the small size, the affinity of nanobodies for target antigens is comparable to that of the traditional antibodies.¹³ VNARs form a beta-sandwich structure with only two CDRs—CDR1 and CDR3. The lack of CDR2 is compensated for by the long, protruding and highly diverse CDR3, and the additional disulfide bond in CDR3, which provides extra stability to the VNAR structure.

The presence of more hydrophilic residues and an extra disulfide bond contributes to an overall increase in the stability of nanobodies compared to that of scFv(s). The extra disulfide bonds also make them less prone to thermal denaturation and aggregation. Nanobodies exhibit better refolding efficiency and temperature changes generally do not affect their conformation.²³ Their high solubility, stability, rigidity and low aggregation tendency make them promising candidates for therapeutic applications, minimizing immunogenicity and the chances of formation of anti-drug antibody responses.^23,27 Furthermore, the compact structure makes them more stable in the presence of proteases, organic solvents, pH changes, with increased resistance to high pressure and chemical denaturants without compromising their antigen recognition ability.^12,28 These properties also facilitate the formulation of nanobodies as therapeutic agents and allow for the exploration of alternative drug delivery routes including oral delivery and nasal inhalation.^29,30

Strategies for production of nanobodies

Efficient strategies for the discovery and production of nanobody-based therapeutics are important for their development. Typically, animals from the Camelidae family (llamas, alpacas, camels) are immunized with the target antigen leading to the production of target-specific HCAbs (encoding VHH). These antibodies can be isolated using various strategies (Figure 2).¹² One of the most commonly used technologies is phage display, which involves the construction of a phage-displayed nanobody library by amplification of VHH sequences from the B cells of immunized animals and their cloning into phage display vectors.^11,19 In the phage-displayed nanobody library, each phage displays only one unique nanobody sequence. This library is then subjected to selection against the target antigen, enriching phages that display nanobodies specific to the target antigen.^31–33 Individual nanobody clones can be sequenced from the enriched library. The specific protocols used during the selection process vary considerably based on the nature of the target antigen, desired affinity, and specificity of the nanobodies.³⁴ For example, the selection process can be integrated with octet biolayer interferometry sensors to achieve high-throughput selection, maintain precise control over the selection process, and reduce overall selection time.³⁵

Figure 2.

Strategies for production of nanobodies. Target-specific nanobodies can be discovered using (a) phage display-based selection from immunized, naïve, semi-synthetic or synthetic camelid libraries (b) immunization of transgenic mice encoding HCAbs (c) ai/ml-based prediction (d) next-generation sequencing (NGS) of selected phage libraries (e) after selection, VHHs can be produced in prokaryotic or eukaryotic systems for downstream applications (the figure has been made using Microsoft PowerPoint).

Alternatively, nanobodies can be isolated from naïve libraries that encode nanobodies from unimmunized animals or from semi-synthetic or fully synthetic libraries made with synthetic DNA.^36,37 These libraries hold the potential to yield nanobody binders even for targets that do not provoke an immune response and toxic antigens that could be lethal to animals.¹² Such libraries also reduce the dependence on large animals such as camelids, which are expensive to maintain in animal husbandry facilities.³⁸ Other surface display technologies, including yeast display,³⁹ ribosome display,⁴⁰ E. coli surface display,²² and mammalian cell display,⁴¹ have also been explored for the discovery of nanobodies. Transgenic mouse platforms expressing only llama IgH molecules have also been developed to facilitate the production of nanobodies without the need for camelids, which is expensive and time-consuming.³⁸ Next-generation sequencing (NGS) has enabled researchers to rapidly analyze vast libraries of nanobodies and identify rare clones that may be missed by conventional screening processes.^42,43 By sequencing nanobody genes after selection, researchers can identify promising candidates for further development.^43,44 The integration of experimental methods with computational approaches, including artificial intelligence (AI) and machine learning (ML), also holds promise for improving the selection of specific nanobody molecules against the desired targets.^45,46

Once the target-specific nanobodies are identified, selected clones are expressed, purified, and characterized for their affinity toward the antigen using techniques such as biolayer interferometry or surface plasmon resonance.⁴⁷ The selection of an appropriate expression system is crucial to obtain high yields of functional nanobodies. This choice is often dictated by multiple factors, including the scalability of the process, requirement of post-translational modifications, the complexity of the nanobody structure, and production costs. E. coli remains one of the most commonly used systems for expression of nanobodies due to its ease of manipulation, rapid growth rate, and the low cost of required reagents. Several studies have reported the use of E. coli for the production of nanobodies.^48–51 Although cytoplasmic expression in E. coli results in high yields, it may lead to the formation of inclusion bodies, thereby necessitating the additional steps of denaturation and refolding that can increase production costs.⁴⁸ Periplasmic expression allows proper folding of nanobodies containing disulfide bonds, but has low-expression yields.⁵² The choice of fusion partners, induction strategies, strains, and signal peptides also affect the solubility, expression levels and purity of the nanobodies.^50,51

Yeast expression systems using hosts such as Pichia pastoris and Saccharomyces cerevisiae have also been explored for nanobody expression, especially when post-translational modifications (PTMs) are important for functional expression.^53–55 These systems provide a cost-effective alternative for large-scale production and under optimal conditions, the yield of nanobodies can be as high as 20 g/L.⁵⁶ Baculovirus^57,58 and mammalian cell-based expression systems allow complex PTMs, which are often required for the activity and stability of nanobodies. For example, the expression of GFP-targeting nanobodies in HEK293T cells allows for the identification of PTMs that affect binding ability, which is not possible in prokaryotic systems.⁵⁹ Mammalian systems are especially useful for producing complex molecules such as multimeric nanobodies or nanobody-fusion molecules.⁶⁰ However, these systems can be expensive and require establishment of cell lines and selection of appropriate cell culture medium for optimal protein production.⁶¹ Plant-based systems offer a more cost-effective and scalable platform for producing functional nanobodies compared to mammalian expression systems.^3,58,60,62 Furthermore, unlike mammalian or bacterial expression systems, plant-based expression platforms neither support the replication of human pathogens nor produce endotoxins, thereby significantly minimizing the risk of contamination by endotoxins, viruses, or prions.⁶³ Recent advancements in AI-driven prediction tools hold promise to streamline nanobody production.⁶⁴ AI-guided analysis of physicochemical properties, such as melting temperature using the TEMPRO tool, enables the identification of thermally stable nanobody candidates, facilitating solubility optimization and expediting the development process.⁶⁴

Nanobodies as an emerging class of antivirals

Nanobodies against coronaviruses

Coronaviruses, such as SARS-CoV, SARS-CoV-2, and MERS-CoV, are enveloped RNA viruses responsible for severe respiratory diseases in humans. SARS-CoV-2 employs the receptor-binding domain (RBD) on the spike (S) protein to bind to the human angiotensin-converting enzyme 2 (ACE2) receptor, leading to viral entry into host cells. Consequently, the RBD serves as an important target for neutralizing antibodies and nanobodies, with several nanobodies specifically designed to target its ACE2-binding sites (Table 1).¹⁰³ Nanobodies, such as aRBD-2, aRBD-5, aRBD-7, aRBD42, NB1B5, NB1C6, Nb-015, Nb-021, and NIH-CoVnb-112, have shown promising antiviral effects by blocking the ACE2-RBD interaction and effectively preventing viral entry.^73,90,91,104 These nanobodies exhibit high binding affinity, with KD values in the nanomolar range. Despite their high binding affinity, some nanobodies demonstrate weak ACE2-RBD blocking activity, suggesting that they occupy different epitopes on the RBD.

Table 1.

Summary of nanobodies isolated against coronaviruses.

Virus	Target protein/Immunogen	Nanobody	Technology used for Nanobody discovery	Potential mechanism of action/Binding site	Reference
SARS-CoV-2	RBD	Multiple nanobodies	Naïve and synthetic humanized llama libraries	Blockage of RBD and Human ACE2 receptor interaction	65
SARS-CoV-2	RBD	H11-D4 and H11-H4	Naive Llama library and PCR-based maturation	Blockage of RBD and Human ACE2 receptor interaction	66
SARS-CoV-2	RBD	n3088 and n3130	Human single-domain antibody library	Blockage of RBD and Human ACE2 receptor interaction	67
SARS-CoV/SARS-CoV-2	RBD	Ty1, S14, NM1267, NM1268, aSA3, aRBD-2	Immunized Alpaca library	Blockage of RBD and Human ACE2 receptor interaction	68–71
SARS-CoV-2	RBD	Ty1 (modified)	Immunized Alpaca library followed by Azide-alkyne click chemistry for multimeric constructs	Blockage of RBD and Human ACE2 receptor interaction	72
SARS-CoV-2	S or RBD	VHH72, VHH6 and NIH-CoVnb-112	Immunized Llama library	Blockage of RBD and Human ACE2 receptor interaction	73,74
SARS-CoV-2	RBD	4A8, Sb23	Synthetic nanobody library	Blockage of RBD and Human ACE2 receptor interaction	75,76
SARS-CoV-2	NTD of S protein	Multiple nanobodies	Direct isolation from B cells of infected patients	Restraining the conformational changes of the S protein and used as cocktail therapeutics	76,77
SARS-CoV-2	RBD	n3113.1-Fc	Human single-domain antibody library	Inhibition of viral-cell fusion with no competition with ACE2 for RBD binding	78
SARS-CoV-2	RBD	PiN-21	Immunized Llama library followed by production of homotrimeric construct	Blockage of RBD and Human ACE2 receptor interaction	79
SARS-CoV-2	S1 protein	K-874A	VHH-cDNA display library	Conformational change of S protein	80
SARS-CoV-2	RBD	SR31	Ribosome and phage-displayed synthetic libraries	Blockage of RBD and Human ACE2 receptor interaction	81
SARS-CoV-2	RBD	SR4, MR17, and MR3	Ribosome and phage-displayed synthetic libraries	Blockage of RBD and Human ACE2 receptor interaction	82
SARS-CoV-2	RBD	3-2A2-4	Immunized Alpaca library	Blockage of RBD and Human ACE2 receptor interaction	83
SARS-CoV-2	RBD	H7-Fc and G12×3-Fc	Synthetic nanobody library	Blockage of RBD and Human ACE2 receptor interaction	84
SARS-CoV-2	NTD of S protein	Multiple nanobodies	Immunized Llama library	Restraining the conformational changes of the S protein	85,86
SARS-CoV/SARS-CoV-2	RBD	saRBD-1, Nanosota-2, -3, and 4	Immunized Alpaca library	Blockage of RBD and Human ACE2 receptor interaction	87,88
SARS-CoV-2	RBD	S2-3-IgA2m2	Engineered nanobody	Blockage of RBD and Human ACE2 receptor interaction	89
SARS-CoV-2	RBD	NB1B5, NB1C6	Immunized camel library	Blockage of RBD and Human ACE2 receptor interaction	90
SARS-CoV-2	RBD	Nb-015 and Nb-021	Immunized Alpaca library	Blockage of RBD and Human ACE2 receptor interaction	91
SARS-CoV-2	PLpro	NB1A1 and NB1F7	Immunized camel library	Inhibition of PLpro activity	92
SARS-CoV-2	RBD	VHH60	Synthetic nanobody library	Competition with human ACE2 to bind the RBD of the Spike protein at S351, S470-471and S493–494	93
SARS-CoV-2	RBD	B11‐E8‐F3 (trivalent)	Immunized and synthetic nanobody library	Blockage of RBD and Human ACE2 receptor interaction	94
SARS-CoV-2	RBD	A8, H6 and B5-5	Immunized Llama library	Blockage of RBD and Human ACE2 receptor interaction	95
SARS-CoV-2	NTD of S protein	N235	Immunized Alpaca library	Induce S1 shedding from trimeric S protein	96
SARS-CoV-2	Nsp9	2NSP23 and 2NSP90	Immunized Llama library	Block SARS-CoV-2 genome replication	97,98
SARS-CoV-2	RBD	C11 and K9	Error-prone PCR and FLI-TRAP technique	Blocking RBD and Human ACE2 receptor interaction	99
MERS-CoV	Modified vaccinia virus Ankara expressing the MERS-CoV-S protein	Multiple nanobodies	Immunized Llama library	Blockage of RBD binding to DPP4	100
MERS-CoV	Recombinant RBD-Fc	NbMS10	Immunized Alpaca library	Blockage of RBD binding to DPP4	101
MERS-CoV	Extracellular domain of MERS-CoV S protein	Multiple nanobodies	Immunized Alpaca library	Disruption of RBD interaction with the carbohydrate moiety on DPP4	102

Abbreviations: SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; MERS-CoV, Middle East Respiratory Syndrome Coronavirus; RBD, Receptor Binding Domain; S protein, Spike protein; NTD, N-Terminal Domain; ACE2, Angiotensin-Converting Enzyme 2; DPP4, Dipeptidyl Peptidase-4; PLpro, Papain-like Protease.

Most isolated nanobodies binding to SARS-CoV-2 spike protein are either derived after immunization with SARS-CoV protein or from naïve, semi-synthetic or synthetic nanobody libraries. Nanobody Ty1 is a single-domain antibody isolated from an animal immunized with a SARS-CoV-2 protein with high affinity to RBD with a KD of 9 nM.⁶⁸ Humanized single-domain antibodies (sdAbs) derived from a synthetic library, such as 1E2, 2F2, 3F11, 4D8, and 5F8, exhibit nanomolar affinities (KD as low as 0.996 nM) with potent neutralization of SARS-CoV-2 S-pseudotyped particles.¹⁰⁵ Sybodies Sb23 and SR31 isolated from synthetic libraries, also exhibit high affinity and potent pseudovirus neutralization.^75,81 Nanobodies H11-D4 and H11-H4 isolated from a naive llama library and matured via PCR recognize all three RBDs in the S protein trimer with high affinity, effectively blocking its interaction with ACE2.⁶⁶ When used in combination with the CR3022 antibody, these nanobodies exhibit enhanced neutralization. Such therapeutic antibody cocktails, which target multiple viral epitopes, also reduce the likelihood of escape mutants. Nanobody VHH60 derived from an engineered library based on caplacizumab, a commercially approved nanobody competes with ACE2 for RBD binding resulting in inhibition of infection with both wild-type SARS-CoV-2 and pseudotyped viruses at the nanomolar levels.⁹³

One of the advantages of nanobodies over conventional mAbs is their ability to target cryptic epitopes. Nanobodies that bind to conserved or cryptic epitopes outside the ACE2-binding interface have the potential to neutralize viruses through mechanisms that reduce the risk of immune escape. Three such nanobodies, namely, 1-2C7, Nb70, and 3-2A2-4, have been reported to exhibit distinct mechanisms of action for the neutralization of SARS-CoV-2.⁸³ Nanobody 1-2C7 competes directly for binding to RBD, while Nb70 acts indirectly by inducing premature shedding of S1 and triggering premature formation of the core before interaction of virus with ACE2 receptor on the target cells. Nanobody 3-2A2-4 suppresses RBD in the “down” conformation, blocking its transition to the “up” conformation required for ACE2 binding and viral entry.⁸³ Nanobodies n3088 and n3130 neutralize SARS-CoV-2 by targeting a hidden epitope on the interface of the S trimer, which is not accessible to the mAb CR3022, underscoring the advantage of nanobodies in targeting cryptic epitopes.⁶⁷ High-affinity nanobody VHH-72 targets the RBD and prevents ACE2 binding by stabilizing the RBD in a “locked” conformation.⁷⁴ Inhalable nanobody IBT-CoV144 has a broad neutralization spectrum because it binds to the lateral side of the RBD, facilitating a unique trimer-dimer conformation that prevents RBDs from attaching to ACE2 receptors on host cells and causes aggregation of S proteins.¹⁰⁶ Nanobodies targeting conserved epitopes have also shown efficacy across various SARS-CoV-2 variants, including Omicron and its sub variants. Nanobodies such as Nanosota-2, Nanosota-3, and Nanosota-4 initially developed for the Omicron BA.1 subvariant, have also been engineered through in vitro affinity maturation to inhibit the XBB.1.5 subvariant and other coronaviruses, including bat SARS-2 and SARS-CoV-1.⁸⁷

Multivalent nanobodies, which combine two or more nanobodies targeting distinct epitopes on the RBD enhance the neutralization potency and offer broader coverage against viral strains.^99,107,108 XVR011, a bivalent Fc-tagged version of the nanobody VHH-72 with affinity enhancement, demonstrated increased potency compared to its parental nanobody.^74,109 It was evaluated in one Phase 1/2 study (NCT04884295) that was terminated following the completion of Phase 1 due to a change in company strategy for the Phase 2 design. Hetero-bivalent fused nanobodies, such as aRBD-2-5 and aRBD-2-7, exhibit more than 10-fold increase in RBD binding affinity. They target non-overlapping epitopes and demonstrate strong neutralization across variants.¹¹⁰ Biparatopic nanobodies, such as NM1267, NM1268, and MR3, which recognize both conserved and variant-specific epitopes, have shown excellent efficacy against various SARS-CoV-2 variants.^69,82 Bispecific VHH-Fc antibodies are more effective in binding to SARS-CoV-2 S1 RBD and blocking S/ACE2 compared to monospecific counterparts due to their ability to simultaneously target two distinct epitopes, improving avidity and reducing viral escape potential. Similarly, tri-specific VHH-Fcs 3F-1B-2A, 1B-3F-2A, in which the third VHH was added to increase the synergistic potency of antibodies showed enhanced binding, blocking, and pseudovirus neutralization, highlighting their therapeutic potential and scalability.⁶⁵ Multivalent nanobodies such as NB1C6, NB1B5, Nb15 and Nb56 exhibit enhanced RBD affinity and prevent viral escape.^90,111 SaRBD-1, an alpaca-derived nanobody exhibits high potency in multivalent forms, and is tolerant to elevated temperatures, freeze-drying, and nebulization, making it a promising therapeutic candidate.⁸⁸ A trivalent broad-spectrum nanobody B11-E8-F3 exhibits efficient neutralization activity against both SARS-CoV-1 and SARS-CoV-2 variants.⁹⁴ Tetrameric Ty1 construct exhibits picomolar neutralization efficacy, outperforming its monomeric and dimeric counterparts.⁷² Similarly, homotrimeric nanobodies like PiN-21 display remarkable efficacy in reducing lung viral titers, and mitigating lung pathology when administered intranasally at ultra-low doses.^79,112 Other trimeric nanobodies, such as H6 and B5–5, exhibit neutralization activity against the original Wuhan strain and the SARS-CoV-2 variants Beta (B.1.351) and Alpha (B.1.1.7).⁹⁵ Nanobody cocktails targeting both RBD and non-RBD regions of the S protein further enhance neutralization and confer resistance against escape variants, including Alpha, Beta, and N501Y D614G, by exploiting synergistic binding to diverse epitopes.^113,114 While the multimeric nanobodies exhibit enhanced neutralization of virus due to increased RBD binding affinity and the additional steric hindrance posed by the increased size, further investigations are required to understand the underlying mechanisms.

To further improve the binding affinity and neutralization potency of nanobodies, fusion tags have also been used. Fc-tagged nanobodies, where nanobodies are fused with the Fc domain of immunoglobulin G (IgG), IgA, or other immunoglobulin tags, have shown improved neutralizing activity across different variants. For example, the G12 × 3-Fc nanobody exhibits high affinity for the original RBD and effectively neutralizes the Delta and Beta variants.⁸⁴ Similarly, hetero-bivalent nanobodies when Fc-tagged also exhibit increased neutralization potency across multiple variants. An Fc-based tetravalent nanobody conjugate of Nb-015 and Nb-021 or bivalent n3113.1-Fc, both exhibit potency against SARS-CoV-2 variants, including Omicron sub-lineages.^78,91 Furthermore, S2-3IgA2m2, a nanobody fused to secretory IgA exhibits superior neutralization capability, particularly against emerging variants, such as XBB and BQ.1.1, when compared to its IgG-fused counterpart.⁸⁹ Dimeric Fc-tagged nanobodies, Nanosota-2A-Fc, Nanosota-3A-Fc, and Nanosota-4A-Fc, exhibit higher affinity for the prototypic RBD than ACE2. A lead drug candidate, Nanosota-1C-Fc, binds to the SARS-CoV-2 RBD approximately 3000-fold more tightly than ACE2, inhibiting the SARS-CoV-2 pseudovirus with ~ 160 times greater efficiency than ACE2.⁸⁷

While most SARS-CoV-2 neutralizing antibodies target the viral RBD, nanobodies that target the N-terminal domain (NTD) of the S1 subunit and S2 subunit, which mediate fusion between virus and host cells, have also been developed.¹¹⁵ VNAR nanobodies from sharks, such as S2A9, bind to conserved regions in the S2 subunit, which is involved in viral fusion and entry into host cells. These nanobodies may be particularly useful against variants with heavily mutated RBDs, offering an alternative strategy for viral neutralization.^116,117 Nanobodies targeting other structural and non-structural proteins of SARS-CoV-2 have also shown promise. Nanobodies engineered against the S1 site of the SARS-CoV-2 Nsp3 protease inhibit its ability to remove ubiquitin (deubiquitination) and ISG15 (deISGylation) from host proteins.¹¹⁸ Since these modifications play an important role in antiviral immune signaling, their inhibition may contribute to restoring the host immune response and inhibiting viral replication. Nanobodies such as NB1A1 and NB1F7, targeting Papain-like Protease (PLpro), have also shown the ability to inhibit the enzymatic activity of PLpro in experimental assays.⁹² Nanobody 2NSP23, targeting SARS-CoV-2 Nsp9, blocks the viral genome replication and exhibits broad efficacy against coronaviruses when encapsulated into lipid nanoparticles as mRNA.⁹⁷

The small size and stability of nanobodies allow for administration through inhalation, which is advantageous for treating respiratory infections like COVID-19. Nasal delivery of the camelid single-domain nanobody K-874A significantly reduced the viral load in the lungs of Syrian hamsters without adverse effects such as weight loss or cytokine induction.⁸⁰

MERS-CoV, another coronavirus that causes respiratory disease outbreaks, also employs an RBD on the S protein to interact with its entry receptor, dipeptidyl peptidase-4 (DPP4). Nanobody VHH-83 neutralizes MERS-CoV by blocking the interaction of RBD with DPP4 at concentrations as low as 30 pM.¹⁰⁰ Nanobody NbMS10 and its Fc-fusion counterpart NbMS10-Fc recognize a conserved region on the RBD interface and neutralize multiple MERS-CoV strains isolated from camels and humans.¹⁰¹ Furthermore, the dimeric and trimeric versions of NbMS10 exhibit a stronger potential to neutralize various MERS-CoV strains, which can be advantageous in preventing camel-to-human and human-to-human transmission of MERS-CoV.¹¹⁹ Nanobody Nb14 neutralizes MERS-CoV by disrupting the interaction of RBD with the carbohydrate moiety on DPP4, showing potent neutralization activity.¹⁰²

Nanobodies against human immunodeficiency virus

Human Immunodeficiency Virus (HIV) is a member of the Retroviridae family, with a positive-sense single-stranded RNA genome. HIV primarily targets the CD4+ T lymphocytes attaching to the CD4 receptor via envelope protein gp120, followed by interaction with coreceptors CCR5 or CXCR4.¹²⁰ These interactions induce a conformational change in the virus, enabling fusion with the host cell membrane via glycoprotein gp41 and the release of its RNA genome in the cytoplasm. Inside the host cell, the RNA genome is reverse transcribed using viral reverse transcriptase enzyme and the resulting viral DNA integrates into the genome of host cells using viral integrase. Regulatory proteins Tat and Rev regulate viral transcription and RNA transport from nucleus to cytoplasm. Other HIV proteins including Nef, Vif, Env, and Vpr play key roles in counteracting host restriction proteins and enhancing HIV infectivity.¹²¹ Current antiretroviral therapy (ART) aims to control HIV replication, but this approach does not affect the latent virus reservoir or cure the infection. In contrast, broadly neutralizing antibodies show promise for the prevention, treatment, and potential cure of HIV.¹²² Several groups have developed nanobodies as alternative therapeutics targeting different classes of HIV proteins, ranging from proteins involved in the attachment and infection of HIV (gp120, gp41), accessory proteins (Vif, Nef), and regulatory proteins (Rev, Vpr) (Table 2).

Table 2.

Summary of nanobodies isolated against viruses other than coronaviruses.

Virus	Target protein/Immunogen	Nanobody	Technology used for Nanobody discovery	Potential mechanism of action/Binding site	Reference
HIV	gp120	Multiple nanobodies	Immunized Llama library	Blockage of CD4 binding site on gp120	123
HIV	Mixture of gp140 Subtype A and B/C	Multiple nanobodies	Immunized Llama library	Various epitopes on gp120; exact location not determined	124
HIV	Mixture of gp140 trimers from HIV strains	J3	Immunized Llama library	Blockage of binding of CD4 to a prefusion-closed Env trimer	125
HIV	Free gp140 or cross-linked gp140-CD4 mimic (M64U1) complex	Multiple nanobodies	Immunized Llama library	Recognition of CD4 binding sites or CD4-induced binding sites	126
HIV	gp41CHRTM proteoliposomes (encodes CHR, MPER and TM region of gp41 from clade B)	2H10	Immunized Llama library	Binding to a linear helical epitope in the late fusion intermediate of gp41	127
HIV	gp140 SOSIPs from three HIV-1 subtype C strains	Multiple nanobodies	Immunized dromedary library	Blockage of CD4 binding site	128
HIV	gp41 NHR-trimer mimic	Nb-172	Immunized Alpaca library	Inhibition of binding between CHR and NHR, preventing formation of 6-helix bundle for fusion of viral envelope to host cell membrane	129
HIV	Vif	Multiple nanobodies	Camelized rabbit VH domain	Neutralization of HIV Vif resulting in increased expression of Apobec3G	130
HIV	Rev	Nb190	Immunized Llama library	Nanobody Nb190 inhibits the multimerization of Rev resulting in reduced expression of late viral RNA molecules during infection	131
HIV	Nef	sdAb19	Immunized Llama library	Recognition of a conformational epitope on Nef leading to inhibition of its interaction with other partners	132
HIV	Vpr and Capsid	Multiple nanobodies	Immunized Llama library; modified Yeast two-hybrid	Not known	133
HIV	CXCR4	Multiple nanobodies	Immunized Llama library	Interaction with ECL2 on CXCR4	134
HIV	Human CD4	Nb457	Immunized Alpaca library	Conformational change in the CD4, which impairs binding to gp120	135
HIV	CD9 large ECL	Multiple nanobodies	Immunized Llama library	Not known	136
Rotavirus	Rhesus-monkey rotavirus serotype G3, strain RRV	Multiple nanobodies	Immunized Llama library	Blockage of crucial epitopes on RV	137
Rotavirus	VP6 protein	Multiple nanobodies	Immunized Llama library	Blockage of binding between VP6 and cellular receptor or conformational change in virus affecting its attachment to the host cells	138
Hepatitis B virus	Non-infectious Plasma-purified HbsAg particles	Multiple nanobodies	Immunized Llama library	Likely the disruption of interaction between S- protein in the ER and the capsid in the cytoplasm or inhibition of interaction between individual S-protein molecules in the ER	139
Hepatitis B virus	Core antigen of HBV (HbcAg)	Multiple nanobodies	Immunized Llama library	Not known; likely affect the HBV life cycle	140
Hepatitis B virus	HbsAg	125s	Immunized Alpaca library	Binds to conserved C-terminal motif of HbsAg	141
Hepatitis C virus	RNA dependent RNA polymerase (RdRp NS5B) lacking 55 amino acid residues from C-terminus	Multiple nanobodies	Humanized-camel VH/VHH Naïve Library	Inhibition of RdRp polymerase activity by blockage of active site	142
Hepatitis C virus	HCV Helicase	Multiple nanobodies	Humanized-camel VH/VHH Naïve Library	Inhibition of Helicase activity	143
Hepatitis C virus	E2 glycoprotein	D03	Immunized Alpaca library	Disruption of binding between E2 and CD81	144
Hepatitis C virus	HCV Serine Protease	Multiple nanobodies	Humanized-camel VH/VHH Naïve Library	Inhibition/alteration of protease activity	145
Hepatitis C virus	NS4B	Multiple nanobodies	Humanized-camel VH/VHH Naïve Library	Interactions of VHHs with NS4B residues (VHH7-R207 and E226; VHH9-RNA binding motif) resulting in inhibition of HCV replication	146
Hepatitis E virus	Capsid proteins of different HEV genotypes (p239 region)	Nb55	Immunized Bactrian camel	Blockage of binding between capsid protein and target cells	147
Respiratory Syncytial Virus	Fusion protein (F) trimer	RSV-D3	Immunized Llama library	Binding to antigenic site II on F protein and blockage of fusion with host cell membrane	148,149
Respiratory Syncytial Virus	Recombinant F protein and inactivated RSV-A strain or a combination of both	Nb017 (trimer ALX-0171)	Immunized Llama library	Binding to antigenic site II on F protein	150
Respiratory Syncytial Virus	DS-Cav1 (recombinant F protein stabilized in pre-fusion state)	F-VHH-4 and F-VHH-L66	Immunized Llama library	Binding to cavity formed by two F protomers in pre-fusion state	151
HPV	L1 capsid protein	sm5 and sm8	Immunized dromedary library	Not known	152
HPV	HPV16-E6	Multiple nanobodies	Immunized Llama library	Not known	153
HPV	HPV16-E6	Nb9	Immunized Camel library	Not known	154
HPV	HPV16-E7	Nb2	Immunized Bactrian camel	Not known	155
Influenza virus H5N1	H5N1 Hemagglutinin	Multiple nanobodies	Immunized Llama library	Binding to sialic acid binding site on HA and prevents virus attachment	148
Influenza virus H5N2	Inactivated influenza virus (H5N2) A/Mallard duck/Pennsylvania/10218/84	Multiple nanobodies	Immunized two-hump camel library	Not known	156
Influenza virus H1N1	H1N1 Hemagglutinin	Multiple nanobodies	Immunized Alpaca library	Four nanobodies affect post virus attachment events and one recognize HA1 region	157
Influenza virus H1N1	Monovalent influenza H1N1 vaccine (Novartis) and trivalent vaccine Fluvirin	E13	Immunized Llama library	E13 recognizes a highly conserved region on HA1 of H1 subtype	158
Influenza virus A and B	Influenza vaccine and recombinant HA (H7 and H2)	Multiple nanobodies	Immunized Llama library	SD36, SD38, SD83 bind to HA stem and SD84 binds to HA head region	159
Influenza virus B	Recombinant HA from B/Yamagata and B/Victoria lineage	Vic1b-10	Immunized Alpaca library	Cross neutralizing Vic1b-10 recognizes epitope in HA stem region	43
Influenza virus H7N9	Recombinant HA (H7)	NB7–14	Immunized Alpaca library (Phage and Yeast display)	NB7–14 recognizes head domain	160
Influenza virus H7N9	Inactivated influenza virus H7N9	E10	Immunized Alpaca library	E10 recognizes conserved lateral patch on HA	161
Influenza virus A	M2 ion channel	M2-7A	Synthetic camelid library	Conformation changes in M2 channel leading to inhibition of proton influx	162
Influenza virus A	Neuraminidase; Mixture of influenza viruses covering all H and N subtypes	Multiple nanobodies	Immunized Llama library	IV512F nanobody recognizes a conserved epitope on Neuraminidase	163
Influenza virus A	Nucleoprotein; PR8 (mouse-adapted H1N1 strain)	Multiple nanobodies	Immunized Alpaca library	Inhibition of genome replication or blockage of a step before replication	164
Influenza virus A	Nucleoprotein; PR8 (mouse-adapted H1N1 strain)	Multiple nanobodies	Lentiviral plasmid library	Majority lead to blockage of nuclear import of viral ribonucleoproteins and some lead to blockage of nucleoprotein-dependent viral RNA polymerase	165
Influenza virus A	Core of H3N2 RNA-dependent RNA-polymerase	VHH16	Immunized Llama library	Blockage of transport of PA-PB1 dimer of viral RNA-dependent RNA-polymerase to the nucleus	166
Rabies Virus	Rabies genotype 1, PV glycoprotein	Multiple nanobodies	Immunized Llama library	Binding to antigenic site IIa on glycoprotein for most VHHs	148
Rabies Virus	Glycoprotein G	26424 and 26,434	Naïve Llama library	Blockage of binding between G protein and host receptor	167
Human norovirus	GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs	Multiple nanobodies	Immunized Llama library	Blockage of binding with HBGAs	168
Human Norovirus	VP1 on GII.10 VLP	Multiple nanobodies	Immunized Alpaca library	Disassembly of Norovirus VLPs and inhibition of VLP binding to HBGAs	169,170
Human Norovirus	GI.1 VLP	Multiple nanobodies	Immunized Alpaca library	Inhibition of VLP binding to HBGAs	171
Human Norovirus	VLPs of GII.4 Sakai08-403_2006b	7C6 and 1E4	Immunized Llama library	Direct competition for HBGA binding site or steric hindrance to prevent virus binding	172

Abbreviations: NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; MPER, Membrane-proximal external region; TM, Transmembrane; SOSIP, Stabilized native-like soluble HIV-1 envelope glycoprotein trimer; ECL, Extracellular loop; HbsAg, Hepatitis B surface antigen; HBV, Hepatitis B virus; HCV, Hepatitis C virus; NS5B, RNA-dependent RNA polymerase of HCV; NS4B, Nonstructural protein 4B of HCV; HPV, Human papillomavirus; HA, Hemagglutinin; NA, Neuraminidase; M2, Matrix protein 2 ion channel; PA-PB1, Polymerase acidic and polymerase basic subunits of Influenza virus RNA polymerase; RSV, Respiratory syncytial virus; DS-Cav1, RSV F protein stabilized in the pre-fusion state; VP, Viral protein; VLP, Virus-like particle; HBGA, Histo-blood group antigens.

Three cross-subtype neutralizing nanobodies, A12, D7, and C8, targeting gp120 have demonstrated the ability to neutralize multiple HIV strains with A12 exhibiting the highest neutralization potency, by neutralizing 42% of the tested viruses (27 of 65).¹²³ These nanobodies preferentially bind to HIV-1 strains with more flexible and open Env trimers, which limits their neutralization breadth.¹⁷³ To address this, Koh et al. developed an A12/D7 family-specific phage library using RNA from the same immunized llama, and identified several A12/D7 sequence-homologous nanobodies with varied affinity and neutralization properties.¹⁷⁴ In a different study, Strokappe et al. used a mixture of trimeric gp140 proteins from HIV subtypes A and B/C to immunize llamas, leading to the development of broadly neutralizing nanobodies.¹²⁴ To identify nanobodies with superior neutralizing properties, McCoy et al. focused on evaluation of neutralization ability over assessment of binding strength to target protein and identified nanobody J3, which had a neutralization breadth equivalent to antibodies isolated in natural infections and neutralized 96% of the viruses tested.¹²⁵ J3 mimics the binding of CD4 to a prefusion-closed Env trimer, effectively neutralize a diverse range of HIV-1 strains and inhibits cell-to-cell transmission of virus in HIV-1 infected Jurkat cells.^173,175 Furthermore, combining J3 with nanobody 3E3, and other gp120 targeting nanobodies resulted in highly potent neutralization across a wide range of viral clades.¹⁷⁶ Similarly, Nanobody VHH-A6 developed using soluble SOSIPs exhibits broad neutralization potency with neutralization of subtypes B, C, AC/AE and G with varying potency.¹²⁸ A combination of VHH-A6 and VHH-28 exhibited complementarity in their neutralization potential and covered 19/21 strains from the panel. Matz et al. developed multiple nanobodies (JM1-JM5) out of which nanobody JM3 targets an epitope exposed after gp120-CD4 binding and neutralizes not only HIV-1 subtype B (used for immunization), but also strains from subtypes A, C, G, and CRF01_AE.¹²⁶ In addition, nanobody JM4, engineered with a glycosyl-phosphatidylinositol (GPI) anchor, demonstrated the ability to neutralize both cell-free and T-cell-to-T-cell viral transmission.¹⁷⁷ Like SARS-CoV-2, bispecific and multivalent nanobodies exhibit enhanced neutralization potency against HIV-1 as well. Bispecific nanobodies constructed from anti-HIV-1 nanobodies targeting independent epitopes enhance the neutralization potency up to 1400-fold compared to their monovalent counterparts.¹⁷⁸

Nanobodies targeting gp41 have also shown promise. Various regions of gp41 have been targeted using nanobodies. Lutje-Hulsik et al. developed nanobodies targeting the membrane proximal external region (MPER) of gp41, which plays a critical role during the fusion process and encodes highly conserved regions.^127,179 Anti-MPER nanobody 2H110 exhibited 20-fold higher affinity in its bivalent form, neutralizing multiple HIV-1 strains using its hydrophobic CDR3, which interacts with MPER regions potentially embedded in the membrane. Similarly, Nb-172, targeting the N-terminal heptad repeat, displayed broad neutralization potency against diverse HIV-1 pseudoviruses, primary isolates and T20-resistant strains.¹²⁹ A combination of Nb-172 with MD1.22, a soluble CD4 subunit D1 analog, further improved neutralization potency. Beyond Env protein, HIV-1 capsid protein required for the viral genome replication and assembly has also been targeted. Anti-capsid nanobodies, such as 59H10 and VHH9 reduced Gag processing leading to a reduction in HIV-1 infection and virus release.¹⁸⁰

Nanobodies targeting HIV accessory and regulatory proteins have demonstrated notable potential for inhibiting viral replication and immune evasion. A camelized rabbit nanobody targeting the HIV accessory protein Vif, an HIV accessory protein that suppresses Apobec3G, a cytidine deaminase inducing hypermutations in viral RNA demonstrated increased Apobec3G expression and reduced proviral DNA integration, effectively reducing HIV replication.¹³⁰ Nanobodies such as sdAb19, which bind specifically to Nef, a key protein aiding immune evasion by downregulating CD4 and MHC-I receptors, were able to selectively inhibit CD4 receptor downregulation.¹³² Engineered constructs such as neffins, developed by fusing sdAb19 with Nef-specific SH3 polypeptides, further inhibited all major Nef functions, including the downregulation of CD4 and MHC-1 resulting in reduced HIV-1 infectivity.¹⁸¹ Similarly, nanobodies targeting Rev, a regulatory protein critical for transporting viral RNA from the nucleus to the cytoplasm during the late stages of the viral lifecycle, showed strong antiviral activity.¹³¹ By inhibiting the oligomerization of Rev protein, intrabody Nb190 reduced the expression of late viral RNAs and its stable expression in different cell lines made cells resistant to HIV replication without any cytotoxicity.¹⁸² Moreover, intracellular nanobodies developed against Vpr, a protein that enhances HIV replication in macrophages, and against the HIV-1 capsid protein involved in viral particle assembly resulted in viral inhibition.¹³³

By focusing on conserved epitopes, host-targeting therapies such as the approved anti-CD4 mAb ibalizumab provide an alternative strategy to address challenges related to viral diversity, escape mutations and drug-resistance.¹³⁶ Nanobody Nb457 targets a unique conformational epitope on the CD4 receptor, inducing structural changes in its CD4-D1 domain, which impairs gp120 binding and prevents viral entry.¹³⁵ In its Fc-fusion form, Nb457-Fc demonstrated superior neutralization efficacy compared to ibalizumab, with a 3-fold lower IC50 and the ability to achieve 90% inhibition of infection in 91.4% of the tested pseudoviruses, compared to 54.2% for ibalizumab. In a trimeric format, Nb457-NbHSA-Nb457 (Nb457 fused to NbHSA for targeting human serum albumin to improve half-life) results in high neutralization potency, achieving 100% inhibition in a humanized mouse model challenged with the HIV-1CH058 strain, which is known to be resistant to neutralization by antibodies. Other host-targeting strategy includes nanobodies T2C001 and T2C002 targeting CD9, a tetraspanin involved in membrane fusion and viral exit, which effectively reduces viral release, syncytia formation, and cell-to-cell transmission.¹³⁶ Nanobodies 238D2 and 238D4 directed against the coreceptor CXCR4 effectively inhibit the replication of HIV-1 strains in cells by targeting extracellular loop 2 (ECL2) on CXCR4, which contacts gp120 during HIV infection.^134,183 Similarly, nanobody VUN402 targets CXCR4 and inhibits HIV infection.¹⁸⁴

Nanobodies against rotavirus

Nanobody-based antivirals have also been explored for the treatment of rotavirus (RV), a double-stranded RNA virus belonging to Reoviridae family. RV causes acute gastrointestinal infections in young children and is characterized by diarrhea and vomiting. It is a triple-layered virus, composed of a core protein (VP2), inner capsid (VP6), and outer capsid proteins (VP7 and VP4).¹³⁸ RV is classified into seven antigenic groups (A to G) based on variations in VP6. Group A RVs are further categorized into G and P types based on their capsid proteins. The outer capsid proteins are important for inducing neutralizing antibodies and conferring protection against infection.

Several studies have shown that nanobodies serve as potent antivirals against RV (Table 2). Vandervaart et al. demonstrated that a nanobody 2B10 maintained its functionality in the acidic environment of the stomach and could reduce RV-induced diarrhea in mice.¹³⁷ While complete inhibition of infection was not achieved, 2B10 exhibited dose-dependent neutralization in RV-infected mice. These nanobodies have an advantage over the orally administered IgG preparations from bovine and human origin that have limited use in developing countries due to high costs. Nanobodies such as ARP1 and ARP3 targeting the G3P strain of RV have shown cross-strain neutralization through the recognition of a linear epitope on VP6 protein.¹⁸⁵ Nanobody ARP1 was also evaluated in a randomized placebo-controlled, Phase 2 trial (NCT01259765) where it reduced total stool output, but did not significantly decrease diarrhea duration or improve outcomes in children with concomitant infections.¹⁸⁶ When expressed as an anchored dimer on Lactobacillus, ARP3-ARP1 increased the efficacy of reducing diarrhea in a mouse model compared to its monovalent counterparts.¹⁵ Furthermore, Günaydın et al. developed a Lactobacillus-based co-expression system where one nanobody was anchored on the surface of the bacterium, while the another nanobody targeting a different RV epitope was secreted from the same bacterium.¹⁸⁷ This dual-targeting strategy not only increased the treatment efficacy, but also minimized the chances of emergence of escape mutants. Garaicoechea et al. isolated three nanobodies (2KD1, 3A6, and 3B2) against VP6, two of which (3B2 and 2KD1) protected 60% of mice from RV-induced diarrhea.¹³⁸ Nanobody 3B2 demonstrated high efficacy and significantly reduced viral shedding in all animals when administered as a milk supplement over nine consecutive days.¹⁸⁸ These studies highlight that nanobodies can effectively neutralize RV and can be delivered as oral therapeutics owing to their stability in the acidic environment in the stomach to mitigate the impact of this prevalent viral pathogen.

Nanobodies against hepatitis virus

For Hepatitis B virus (HBV), which causes acute as well as chronic infections in the liver, nanobodies targeting the envelope (HBsAg) and core (HBcAg) antigens have shown promising antiviral effects (Table 2). Serruys et al. developed nanobodies targeting HBsAg, which when expressed in HepG2 cells as intrabodies reduced the secretion level of antigen and led to up to two log reduction in the levels of secreted HB virions in mouse model.¹³⁹ Wang et al. identified a nanobody 125s targeting a highly conserved epitope in the C-terminal motif of HBsAg, which exhibited broad cross-reactivity against HBV genotypes A, B, and D when expressed in fusion with Fc.¹⁴¹ Similarly, six nanobodies targeting the HBcAg have also expanded the arsenal of antivirals for HBV.¹⁴⁰

Different proteins of the Hepatitis C virus (HCV) have also been targeted using nanobodies. Thueng-in et al. developed nanobodies V(H)H6 and V(H)H24 against RNA-dependent RNA polymerase (RdRp), which reduced RdRp activity in ELISA and reduced HCV replication in HCV infected Huh7 cells when administered as VHH-penetratin fusion proteins.¹⁴² The same group also identified nanobodies against HCV helicase,¹⁴³ serine protease,¹⁴⁵ and NS4B protein¹⁴⁶ similarly inhibited HCV replication and reduced viral egress. Tarr et al. developed a broadly neutralizing nanobody D03 targeting E2 glycoprotein, which disrupted the binding between E2 and CD81, resulting in neutralization of HCV pseudo particles from multiple genotypes, as well as infectious HCV particles.¹⁴⁴ Wang et al. developed monovalent and polyvalent nanobodies targeting the p239 region of the Hepatitis E virus (HEV) capsid protein, which neutralized non-enveloped HEV both in vivo and in vitro.¹⁴¹

Nanobodies against respiratory syncytial virus

RSV is a known cause of acute lower respiratory tract infections in children, especially infants and younger children. Nanobody research for RSV has primarily focused on the Fusion (F) protein involved in viral fusion (Table 2). Hultberg et al. identified neutralizing nanobodies RSV-D3 and RSV-C4, with the bivalent RSV-D3 (F-VHHb) exhibiting 4000-fold higher neutralization potency than its monovalent counterpart and 2.6-fold higher than the Fab fragment derived from Synagis®, an approved mAb for the treatment of RSV.¹⁴⁸ Furthermore, intranasal administration of F-VHHb significantly reduces RSV replication in mouse models.¹⁴⁹ ALX-0171, a trivalent nanobody targeting antigenic site II on F protein has 160 times higher affinity compared to its monovalent counterpart Nanobody Nb017 and effectively neutralizes RSV A and B strains.¹⁵⁰ Its robust structure allows intranasal delivery using nebulization, resulting in a dose-dependent reduction in RSV titers in the nose and lungs of cotton rats. Furthermore, ALX-0171 delayed the emergence of escape mutants¹⁸⁹ and reduced the severity of symptoms in an RSV-infected newborn lamb model.³⁰ Despite these successes in preclinical models, the clinical trial program for ALX-0171, which included two Phase 2 studies (NCT02979431, NCT03418571), were ultimately discontinued due to a lack of improvement in patients with established RSV infections.^190,191 Other nanobodies, such as F-VHH-4 and -L66, targeting the pre-fusion state F protein, exhibited high affinities (KD of 18 pM and 154 pM) and inhibited RSV replication in mice when administered intranasally before RSV challenge.¹⁵¹

Nanobodies against human papilloma virus

Human papilloma virus (HPV) belongs to the Papillomaviridae family and several HPV types are associated with different types of cancers in humans, among which HPV16 is the most prevalent.¹⁹² The development of nanobody-based antivirals for HPV is focused on viral oncoproteins E6 and E7, which are major oncoproteins that interact with p53 and retinoblastoma proteins, respectively, resulting in a malignant phenotype in HPV-associated cancers (Table 2).¹⁵⁵

Nanobodies A05, 2A12, and 2A17 targeting E6 protein exhibit binding to the recombinant protein with affinity in the nanomolar range.¹⁵³ Another anti-E6 nanobody, Nb9 effectively inhibits E6-mediated p53 degradation, restoring apoptosis in HPV16-positive cells, and delays tumor development in mouse models.¹⁵⁴ Similarly, when expressed intracellularly, nanobody Nb2 targeting E7 protein suppresses the proliferation of HPV16-positive cells.¹⁵⁵ Nanobodies sm5 and sm8 targeting HPV16- L1 capsid protein lead to 75% and 60% neutralization of HPV-16 particles, respectively.¹⁵² These findings suggest that nanobodies targeting both oncogenic and structural HPV proteins may serve as promising therapeutic candidates for HPV.

Nanobodies against influenza virus

The influenza virus belongs to the Orthomyxoviridae family and is a significant pathogen responsible for seasonal epidemics and occasional pandemics. Influenza A and B are the primary causes of human illness, with influenza A further categorized into subtypes based on the surface proteins hemagglutinin (HA) and neuraminidase (NA), which participate in infection and viral spread. The HA protein facilitates influenza virus binding to sialic acid receptors present on respiratory epithelial cells, leading to internalization and viral RNA replication. Following this, the viral particles are assembled and released through the action of the NA protein, which facilitates infection spread.

Several groups have targeted HA proteins to develop neutralizing nanobodies for different influenza strains (Table 2). Nanobodies Infl-C8 and Infl-B12 targeting the sialic acid-binding site on HA inhibited the virus from attaching to the host cells and neutralized strains A/Vietnam/1194/04 and A/Vietnam/1203/04 with bivalent and trivalent formats showing enhanced potency.¹⁴⁸ Similarly, the bivalent H5-VHHb nanobody, when administered intranasally, reduced viral replication in mice with a 60-fold increase in potency compared to its monovalent form.¹⁹³ Tillib et al. developed trimerized nanobodies against H5N2 HA using an isoleucine zipper sequence, with aHA-7 exhibiting 50% protection in mice intraperitoneally and offered stronger protection when delivered intranasally.¹⁵⁶ Nanobodies developed against H1N1 by Hufton et al. exhibited cross-neutralization of H1N1 and H5N1, with the Fc-fusion version of R1a-B6 nanobody exhibiting complete protection against infection when challenged 42 days after intramuscular administration via an adeno-associated viral (AAV) vector.^157,194 Barbieri et al. further demonstrated the potential of nanobodies E13 and G41, with E13 providing sterilizing immunity against H1N1 strains at a low intranasal dose of 0.05 mg/kg body weight.¹⁵⁸

To achieve a wider neutralization breadth and potency against influenza viruses, Laursen et al. developed multidomain nanobodies from nanobodies targeting both Influenza A (nanobody SD36 and 38) and B (nanobody 83 and 84).¹⁵⁹ The multidomain construct MD2407 encoding all four nanobodies or MD3606 (MD2407 fused with Fc domain) neutralized all A and B strains except one exhibiting much wider neutralization breadth compared to individual nanobodies. MD3606 also led to complete protection of mice from the H1N1 strain when administered intravenously at 1.7 mg/kg body weight and from H3N2 at 5 mg/kg body weight. When administered seven days before challenge as a prophylactic using a recombinant AAV9 vector via intranasal route, humanized MD3606 also protected mice against H1N1, H3N2 and B strains, making MD3606 one of the most broadly acting anti-influenza intervention.¹⁹⁵ Such multidomain nanobodies may provide passive protection against different strains of Influenza virus particularly to the elderly and other high-risk groups for the entire influenza season.

Ramage et al. developed nanobody Vic1b-10 exhibiting high potency against influenza B strains from two lineages B/Yamagata and B/Victoria.⁴³ Gaiotto et al. and Huang et al. targeted H7N9, developing neutralizing nanobodies NB7–14 and Nb-Z77, while Chen et al. described nanobody E10 targeting a conserved lateral patch on HA from H7 subtype, which exhibited broad neutralization across different subtypes.^160,161,196

In addition to HA, nanobodies have also been developed against other influenza virus proteins (Table 2), including the M2 ion channel,^162,197 neuraminidase,^163,198 nucleoprotein^164,165,199 and viral RNA-dependent RNA polymerase.¹⁶⁶

Nanobodies against rabies virus

Rabies virus (RABV), a neurotropic negative-sense RNA virus of the family Rhabdoviridae, can cause lethal brain infections if left untreated. The viral glycoprotein (G protein) is an attractive drug target as it mediates RABV entry into host cells via endocytosis and fusion with the endosomal membrane, leading to viral replication in the cytoplasm. Boruah et al. reported the development of pentavalent nanobodies 26,424 and 26,434, which exhibited the neutralization of 85-fold higher amounts of RABV pseudotypes compared to their monovalent counterparts.¹⁶⁷ Similarly, Rab-E8/H7, a biparatopic nanobody exhibited about 1500-fold higher potency with IC50 of 0.14 nM compared to its monovalent counterparts.¹⁴⁸ In vivo studies have further highlighted the therapeutic potential of these nanobodies. Intracerebral administration of 33 μg Rab-E8/H7 in mice provided protection on the first day of infection, whereas systemic delivery required a significantly higher dose of 10 mg.²⁰⁰

The World Health Organization (WHO) recommends the administration of anti-rabies antibodies along with a vaccine after potential exposure to the virus. In line with this, a combination of Rab-E8/H7-ALB11 (a biparatopic nanobody linked to an anti-albumin antibody to enhance half-life) with a rabies vaccine significantly improved the survival rates in a mouse model of intranasal rabies challenge.²⁰¹ While only 19% of mice survived with nanobody treatment alone and none with the vaccine alone, the combination achieved a survival rate of 60%. Given the short incubation period of RABV in humans, this synergistic effect could close the gap between virus exposure and immune activation elicited by vaccination.²⁰¹

Nanobodies against noroviruses

Noroviruses are highly contagious positive-sense RNA viruses from the Caliciviridae family and are a leading cause of acute gastroenteritis. The capsid protein VP1, particularly its P-domain, interacts with histo-blood group antigens (HBGAs) on host cells, facilitating infection.²⁰² Koromyslova et al. identified Nano-85, a nanobody derived from immunized alpaca libraries, which disrupts norovirus virus-like particles (VLPs) by inserting its extended CDR3 loop between the S and P domains of VP1.¹⁶⁹ Four additional nanobodies (Nano-4, −26, −27, and −42) exhibited various degrees of VLP destabilization.¹⁷⁰ Nano-4 and −27 induced transitions to smaller VLPs, Nano-26 caused partial disassembly, and Nano-42 produced smaller, disassembled particles, though less effectively than Nano-85. Nano-26 and Nano-85 also prevented VLP attachment to HBGAs, highlighting their therapeutic potential against noroviruses. Fc-fusion of Nano-26 (Fc-NB26) enhanced the binding affinity to the norovirus P domain by 100-fold compared to the native nanobody, effectively neutralizing norovirus replication and causing particle disassembly and aggregation.²⁰³ Nano-7 and Nano-94 inhibited GI.1 norovirus VLP binding to HBGAs, with efficacy further enhanced by combining them with 2-fucosyllactose.¹⁷¹ A heterodimeric nanobody construct combining VHH 7C6 and 1E4 neutralized both GII.4 and GII.17 noroviruses.¹⁷²

Noroviruses frequently mutate, altering their binding specificity for HBGAs and complicating therapeutic development.²⁰⁴ To address this, nanobodies targeting conserved norovirus epitopes have been explored. Garaicoechea et al. identified M4, a nanobody binding multiple GII norovirus strains.¹⁶⁸ Structural analysis has revealed its interaction with a conserved epitope in the VP1 P-domain, reducing its conformational flexibility, inducing stress, and promoting VLP disassembly.²⁰⁴ Such strategies can facilitate the development of broadly neutralizing nanobodies to address the emerging norovirus variants.

Nanobodies against other human viruses

Nanobody-based therapies have also been developed for other human viral pathogens, including chikungunya virus,^205,206 poliovirus,²⁰⁷ Ebola virus,^208–210 Herpes Simplex virus,²¹¹ vaccinia virus,²¹² Marburg virus,²¹³ Vesicular stomatitis virus,¹⁶⁵ western equine encephalitis virus,²¹⁴ enterovirus,²¹⁵ bunyavirus^216,217 and porcine retroviruses.²¹⁸ Together, these studies underscore the versatility and potential of nanobody-based therapeutics across a broad spectrum of viral pathogens.

Strategies for delivering nanobody-based therapeutics

Owing to their robust structure and stability under extreme conditions, nanobody-based therapeutics offer flexibility in delivery through various drug administration routes tailored for specific viral infections. Amongst approved nanobody-based drugs, caplacizumab (used for TPP) is administered intravenously, which is the most common route, whereas ozoralizumab (used for rheumatoid arthritis) and envafolimab (used for various cancers) are administered through subcutaneous route.¹⁶ Alternative routes, such as oral administration are being investigated to reduce production costs and enhance patient comfort by providing pain-free noninvasive options.¹⁶ However, oral administration can present challenges for protein-based drugs like nanobodies due to variable pH levels in the gastrointestinal tract and enzymatic degradation, resulting in low bioavailability.^29,219

Nanobody stability can be enhanced during the discovery phase by selecting candidates with a higher resistance to harsh conditions.²⁹ Formulation strategies, such as encapsulation in polysaccharide-based nanoparticles (e.g., chitosan or dextran), can improve the stability of oral administration.²¹⁹ Examples of successful oral applications include supplementation of a daily milk diet with a monovalent nanobody targeting the VP6 protein of rotavirus, which provides protection against diarrhea in piglets.¹⁸⁸ Similarly, combining anti-VP6 nanobodies with oral rehydration solutions enhances nanobody resistance to stomach degradation and improves treatment efficacy.²²⁰ Another promising delivery strategy, MucoRice-ARP1, a transgenic rice expressing anti-rotavirus nanobodies, demonstrates the inherent stability of nanobodies and their suitability for oral administration.⁶⁰ This system has been shown to reduce diarrhea severity in rotavirus-inoculated mice. Furthermore, the nanobody retains its neutralizing activity after long-term storage and maintains its binding capacity even under extreme conditions, such as boiling.⁶⁰ A similar system has also been developed for norovirus.²²¹

Bacteria-based delivery systems have also emerged as a viable option. These co-expression systems use the host microbiota to provide sustained therapeutic benefits and expand the application of nanobody-based therapies. For example, Lactobacillus paracasei BL23 was engineered to express nanobodies targeting rotavirus and norovirus, offering protection against infection.^187,222 Similarly, the Lactobacillus rhamnosus DSM 14,870-based system was explored to deliver HIV-neutralizing nanobodies in the vaginal environment, maintaining activity in acidic pH conditions and providing a potential preventive strategy for women at high risk of HIV infection.²²³

Adeno-associated viral (AAV) vectors have been explored as efficient delivery systems for sustained nanobody expression.²²⁴ Laursen et al. demonstrated protection from influenza virus with intranasal delivery of recombinant AAV9 encoding multidomain nanobody MD3606.¹⁵⁹ Similarly, Del Rosario et al. used AAV8 to deliver an Fc-fused nanobody (R1a-B6) capable of neutralizing multiple influenza virus subtypes, achieving strong nanobody expression and protection against the virus with a single intramuscular injection.¹⁹⁴

Inhalation-based intranasal delivery has emerged as an effective route for treating respiratory viral infections due to its noninvasive nature, enhanced bioavailability, and direct targeting of the respiratory system.^225,226 The large surface area of lungs and unique pharmacokinetics facilitate rapid absorption of therapeutic proteins, while factors such as reduced mucociliary clearance and the presence of antiproteases further improve drug bioavailability.²²⁶ Structural and biochemical features of nanobodies including their compact size, high solubility, and stability make them well-suited for aerosolization and intranasal administration.^56,227–229

Intranasal delivery has been explored for the treatment of various respiratory viruses, including RSV,¹⁵⁰ influenza virus,^193,230,231 and SARS CoV-2.^14,232 The delivery of trimeric nanobody ALX-0171 via nebulization completely blocked RSV replication in the lungs and significantly reduced nasal viral titers.¹⁵⁰ Despite promising outcomes in preclinical studies, clinical trials for ALX-0171 were discontinued as it did not exhibit improvement in patients with established RSV infections.^190,191 Similarly, nanobodies such as F-VHH-4 and L66 reduced RSV replication and inflammation when administered intranasally before virus challenge.¹⁵¹ In the context of SARS-CoV-2, aerosolized nanobody PiN-21 reduced viral loads and prevented lung damage in Syrian hamsters.⁷⁹ Similarly, nanobody NIH-CoVnb-112 achieved a six-order magnitude reduction in viral load when delivered via nebulization.²³³ Bispecific nanobody dimer 2–3-Fc completely eliminated Omicron BA.1 virus particles after intranasal administration.⁷¹ Intranasal administration of VHH-IgA1.1, an anti-RBD nanobody fused to IgA-Fc, led to significant protection from severe disease due to SARS-CoV-2 variants both before and after exposure in K18-ACE2 transgenic mice.²³⁴ Fc-tagged Nanosota-3A-Fc also effectively neutralized the SARS-CoV-2 Omicron BA.1 variant.⁸⁷ Inhalation-based delivery of bn03, a bispecific nanobody targeting SARS-CoV-2 led to a higher effective concentration and effective neutralization of virus compared to intravenous administration.^235,236 Nanobody-based therapies delivered intranasally have also shown potential in treating viral infections in locations beyond the respiratory system. Nanobodies Nb-39, Nb-104, and Nb-110 lead to a reduction in viral load in both the upper respiratory tract and brain of COVID-19-infected mice.²³⁷ Together, these findings underscore the versatility of intranasal nanobody delivery for addressing both respiratory and systemic viral diseases.

Challenges associated with the use of nanobodies as antivirals

Nanobodies, with their small size, high stability, and ease of production, offer numerous advantages over conventional mAbs for therapeutic applications.³ However, several challenges hinder their widespread application as therapeutics. Traditional methods for nanobody discovery, such as camelid immunization, are time-consuming and expensive.^3,238 Addressing this issue requires the development of efficient and cost-effective discovery and expression platforms, such as synthetic phage-displayed nanobody libraries,²³⁸ transgenic mice,³⁸ and the integration of AI-ML-based discovery platforms in the selection process.^45,46 Development of high-level expression systems is also important for scaling production.^49,54,59 Furthermore, several viral targets are localized in the host cell cytoplasm, necessitating the engineering of nanobodies that maintain stability and function in a reducing intracellular environment.²³⁹

Due to their small size (~12–15 kDa), nanobodies are rapidly cleared from circulation through renal filtration, necessitating frequent dosing.¹³ Strategies to extend their half-life include PEGylation, which increases hydrodynamic size and reduces renal clearance²⁴⁰ and fusion to albumin, which extends the circulation time due to long persistence of albumin in the human body.²⁴¹ Alternative approaches involve attaching nanobodies to anti-human serum albumin nanobodies,¹³⁵ or the Fc region of conventional mAbs, thereby engaging the neonatal Fc receptor (FcRn) to recycle complex back into the bloodstream.^242,243 Jia et al. described a nanobody engineering strategy involving modification with an intrinsically disordered protein, resulting in nanobodies with up to 15-fold longer half-lives compared to their unmodified counterparts.²⁴⁴

Unlike conventional mAbs, nanobodies lack an Fc domain, which limits their ability to mediate effector functions, such as complement activation and antibody-dependent cellular cytotoxicity (ADCC).²⁴⁵ To overcome this limitation, nanobody fusions with Fc regions, which restore Fc-mediated functions, have been described.^159,227,242 Schriek et al. demonstrated that fusing the anti-HIV nanobody J3 with the Fc domain restored Fc effector functions, including ADCC, trogocytosis, and natural killer cell activation.²⁴² Another approach involves attaching target-specific nanobodies to Fc gamma receptor-targeting nanobodies to engage FcγRIV and enhance their protection.¹⁹⁷

The non-human origin of nanobodies poses a risk of immunogenicity, potentially leading to anti-drug antibodies that compromise therapeutic efficacy and safety.^18,246,247 Humanization strategies, such as CDR grafting and resurfacing, aim to reduce immunogenicity while preserving antigen-binding affinity and structural integrity.^85,248,249 Computational tools can also be used to predict structural changes and identify residues contributing to immunogenicity of nanobodies. AbNatiV, a deep learning tool, can assess the nativeness of antibodies and nanobodies, and provide information about the probability of immunogenicity.²⁵⁰ Sang et al. described Llamanade, a tool for nanobody humanization developed using NGS datasets and high-resolution structural data of mammalian antibodies and nanobodies.²⁵¹ This tool enables the optimization of nanobody sequences and was applied to humanize nanobodies targeting SARS-CoV-2, yielding humanized versions with solubility and activity comparable to their non-humanized counterparts. However, even humanized nanobodies may elicit immune responses due to aggregation, high dosing, or the nature of the target antigen.⁸⁵ Therefore, balancing the reduced immunogenicity while preserving the antigen-binding affinity can be challenging.

The simplified structure of nanobodies may also limit their ability to bind to complex antigens or maintain their stability under extreme physiological conditions. Multivalent nanobody constructs prepared by fusing multiple nanobody domains can improve the binding avidity and functional versatility. These constructs can be highly effective against viruses such as SARS-CoV-2 and HIV, which have trimeric glycoproteins that facilitate host entry. Multimeric nanobodies targeting different epitopes can neutralize a broader range of viral variants.²⁵² Approved drugs such as ozoralizumab and sonelokimab, which are trimeric constructs, and caplacizumab, a homodimer, demonstrate the potential of such modifications to improve half-life and therapeutic outcomes.^17,253,254

Overall, while nanobodies hold great promise as antiviral therapeutics due to their favorable biochemical properties, optimizing their discovery, production, pharmacokinetics, and immunogenicity is essential for their effective clinical application.

Future directions and conclusion

With the approval of four nanobody-based therapies, nanobodies hold immense potential as antiviral agents, especially given the lack of vaccines and therapeutics for most viruses. Their high stability under extreme conditions makes them particularly effective against viruses that replicate in acidic environments, such as the gastrointestinal tract¹⁵ and vaginal environment.²²³ Nanobody-based intrabodies, capable of targeting viral antigens within the host cell cytoplasm, could inhibit viral replication and assembly.¹⁸² Engineered nanobody formats, such as multivalent constructs, biparatopic antibodies, and nanobody conjugates, can enhance the binding affinity,¹¹¹ neutralization breadth,^107,110 half-life,¹² and reduce the risk of viral escape mutants.⁹⁰

The development of efficient and safe technologies for nanobody delivery is important for the clinical success of nanobodies. Intranasal delivery has shown promise for respiratory viruses, while oral administration can be optimized for gastrointestinal infections. Due to their simple structure, nanobodies can also be easily conjugated with different moieties to achieve better treatment outcomes. Yang et al. developed nanobody peptide conjugates (NPCs) to effectively neutralize porcine reproductive and respiratory syndrome virus.²⁵⁵ Similarly, anti-CXCR4 has been used to deliver siRNA against viral infections.²⁵⁶ A nanobody-mediated drug delivery platform (SWCNTs-P-A-Nb) has shown potential for treating viral encephalopathy and retinopathy caused by the nervous necrosis virus (NNV).²⁵⁷ Nanobody-AAV conjugates have also been used to enhance gene delivery efficiency.²⁵⁸ Furthermore, nanobody-based CAR-T cells, effective in cancer therapies, are being explored for targeting virally infected cells, particularly in cases where conventional therapies fail to clear the infection.²⁵⁹ Nanobody conjugates with imaging agents, such as fluorophores, have facilitated the real-time visualization of viral infections.²⁶⁰ Their small size also enables nanobodies to cross the blood–brain barrier (BBB), offering potential for targeting neuroinvasive viruses such as West Nile virus.²⁶¹

While nanobody phage display is the most common technology used for the discovery of nanobodies, the application of other techniques is also gaining traction. Use of technologies, such as synthetic nanobody libraries, llama antibody encoding transgenic mice,³⁸ and computational tools for nanobody design and prediction is accelerating the discovery and optimization of nanobodies with improved properties.⁴⁵ A steady increase in publications describing the development of nanobodies for virus treatment (from 7% in 2018 to 33% in 2022), the entry of multiple nanobody-based therapies in clinical trials, and several approvals by regulatory bodies, underscores the growing appreciation of nanobody-based treatment strategies.¹¹

In conclusion, nanobodies have emerged as promising antiviral therapeutics due to their unique structural and functional properties, including high stability, solubility, and the ability to target conserved viral epitopes. They have demonstrated significant efficacy against a broad range of viral pathogens, including SARS-CoV-2, HIV, influenza, and RSV, with potential applications in both prophylactic and therapeutic settings. Key advancements, such as nanobody humanization, multivalent constructs, and Fc-fusion strategies, have enhanced binding affinity, half-life, and effector functions. Delivery innovations, including intranasal, oral, and AAV-based administration, further expand their clinical potential as antiviral therapeutics. Future research should focus on optimizing nanobody expression systems, minimizing immunogenicity, and improving pharmacokinetics to facilitate the development of safe and effective nanobody-based antiviral therapies.

Funding Statement

This work was supported by an institutional seed grant from Bennett University and a Start-up Research Grant by the Department of Science and Technology - Anusandhan National Research Foundation (DST-ANRF) (SRG/2022/000486) to Vaishali Verma.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Idea was conceived by VV; the literature search, data analysis and preparation of first draft of the manuscript was done by VV, NS and AR. Manuscript draft was edited by VV. All authors read and approved the final manuscript.