Functionalized nanoparticles for virus clearance

Abstract

This invention relates to functionalized nanoparticles or microparticles of suitable size and shape, comprising nanoparticles or microparticles and at least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of the nanoparticles or microparticles. These nanoparticles or microparticles form aggregates with targeted viral particles to initiate phagocytosis, thereby achieving viral clearance. This invention also relates to the use of said functionalized nanoparticles or microparticles in virus detection, treatment, and diagnosis.

Description

[0001] This invention relates to functionalized nanoparticles or microparticles of suitable size and shape, comprising nanoparticles or microparticles and at least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of the nanoparticles or microparticles. These nanoparticles or microparticles form aggregates with targeted viral particles to initiate phagocytosis, thereby achieving viral clearance. This invention also relates to the use of said functionalized nanoparticles or microparticles in virus detection, treatment, and diagnosis. Background Technology

[0002] Viral infectious diseases are a significant factor in both human and veterinary health care. The transmission, spread, and especially the pathophysiology of viruses are highly pathogen-specific and largely depend on the adaptation of viral particles to the host, making the development of effective targeted therapies against viral strains challenging.

[0003] Airborne viruses, in particular, can be highly infectious and sometimes lead to exponentially threatening spread, including large outbreaks and even pandemics. A prominent example is the global pandemic associated with SARS-CoV-2, which belongs to the coronavirus family. Beta coronaviruses (β-CoV or Beta-CoV) are one of four genera of coronaviruses in the subfamily Orthocoronavirinae of the family Coronaviridae in the order Nidovirales. They are zoonotic, enveloped, positive-sense, single-stranded RNA viruses. The need for treatment, prevention, and diagnosis of these viruses is growing.

[0004] Several liposome-based viral therapies are available. Epixal®, a vaccine formulated with formalin-inactivated HAV (strain RG-SB) virions adsorbed onto the surface of specific liposomes (virions), has been developed (Bovier PA. Epixal: a virosomal vaccine to prevent hepatitis A infection. Expert RevVaccines. 2008;7(8):1141-1150. doi:10.1586 / 14760584.7.8.1141). In addition, Inflexal® virion (150 nm liposome) vaccines, which mimic natural antigen presentation, have been marketed for some time. Here, liposomes mimic the structure of natural viruses, thereby allowing cell entry and membrane fusion (the retention of natural presentation of antigens on the surface of liposomes provides high immunogenicity. Herzog C, Hartmann K, Künzi V, et al. Eleven years of Inflexal V-avirosomal adjuvanted influenza vaccine. Vaccine. 2009;27(33):4381-4387. doi:10.1016 / j.vaccine.2009.05.029).

[0005] Singh et al. (in: The role of nanotechnology in the treatment of viral infections. Ther Adv Infect Dis. 2017;4(4):105-131. doi:10.1177 / 2049936117713593) provided a broad overview of the application of nanoscale materials in the treatment of common viral infections.

[0006] Virus-mimicking cell-interaction nanoparticles (NPs) have been described as another valuable strategy to enhance the specificity of NPs for therapeutic and diagnostic applications (Maslanka Figueroa S, et al. Influenza Avirus mimetic nanoparticles trigger selective cell uptake. Proc Natl Acad SciU S A. 2019;116(20):9831-9836. doi:10.1073 / pnas.1902563116).

[0007] US 2013-0337066 describes nanoparticles comprising: a) a core containing non-cellular material; and b) an outer surface containing a cell membrane derived from a cell or a membrane derived from a virus. The core may comprise a biocompatible or synthetic material selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine, and polyglutamic acid, and the cell membrane may be derived from blood cells, tumor cells, cancer cells, immune cells, stem cells, endothelial cells, exosomes, secretory vesicles, or synaptic vesicles. The nanoparticles may contain a releaseable carrier, such as a therapeutic agent, prophylactic agent, diagnostic agent or marker, prognostic agent, or combination thereof.

[0008] During infection and replication of pathogenic viruses, they typically hijack intracellular uptake mechanisms, such as pinocytosis and clathrin-mediated or clathrin-independent endocytosis, by utilizing their host cell recognition sites (receptor domains). Intracellular uptake is crucial for viral infection because viral replication inherently depends on cellular machinery such as the ribosome complex to provide all the building blocks and enzymes required for viral particle assembly and release. While current antiviral therapies primarily target intracellular viral enzymatic pathways, only a few compounds actively and effectively prevent viral entry (e.g., enfuvirtide).

[0009] A very promising concept for inhibiting viral internalization is the competitive saturation of viral envelope proteins, such as the spike protein of SARS-CoV-2, which is crucial for host cell contact to prevent effective protein-protein interactions. However, viral envelope saturation may only slow the infection process because the virus is not actively neutralized by the immune system.

[0010] Cagno et al. (in: Broad-Spectrum Non-Toxic Antiviral Nanoparticles with aVirucidal Inhibition Mechanism. Nat. Mater. 2018, 17, 195-203. 10.1038 / nmat5053) designed antiviral nanoparticles with long and flexible junctions mimicking HSPGs, allowing for efficient viral association. The inventors simulated and found that the binding to the VAL repeating unit is strong and multivalent, generating a force (approximately 190 pN) that ultimately leads to irreversible viral deformation. These nanoparticles were active in vitro in human cervical and vaginal tissue cultures infected with HSV-2 and in vivo in RSV-infected mice.

[0011] Han et al. (in: Han Y, Král P. Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2. ACS Nano. 2020;14(4):5143-5147. doi:10.1021 / acsnano.0c02857) described a potential peptide inhibitor against the SARS-CoV-2 coronavirus, which is primarily formed by two consecutive self-supporting α-helices (bundles) extracted from the protease domain (PD) of angiotensin-converting enzyme 2 (ACE2), which bind to the SARS-CoV-2 receptor-binding domain. Molecular dynamics simulations showed that the α-helical peptide retains its secondary structure and provides highly specific and stable binding (blocking) to SARS-CoV-2. The proposed small peptide (“minibinder”) could be used as an inhaled therapeutic agent for local lung delivery, providing an effective way to combat COVID-19. They also described early attempts to block SARS-CoV, investigating short peptide inhibitors and implementing amino acid mutations in the SARS-CoV S protein. However, the proposed peptides were too short (8 residues) to maintain secondary structure and therefore could not block the entire SARS-CoV binding surface. Broad-spectrum antiviral nanoparticles and cyclodextrins were designed, mimicked, and implemented in the blocking of other viruses. These are class 2 or 3 inhibitors, but their applicability to SARS-CoV-2 is unknown.

[0012] Han et al. proposed that many such peptides could attach to the surface of nanoparticle carriers to provide multivalent binding to the SARS-CoV-2 receptor. However, these nanoparticles, dendritic polymers, and clusters are designed solely to improve binding with inhibitors.

[0013] APEIRON Biologics AG has begun further development of APN01 for the treatment of patients with severe COVID-19; the soluble APN01 mimics the receptor ACE2, thus providing a dual treatment approach.

[0014] Cao L, et al. (in: De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science. 2020 Oct 23;370(6515):426-431. doi: 10.1126 / science.abd9909. Epub 2020 Sep 9. PMID: 32907861) described a promising therapeutic strategy targeting the interaction between the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the human angiotensin-converting enzyme 2 (ACE2) receptor. They designed inhibitors using two de novo design approaches. Computer-generated scaffolds were constructed around ACE2 helices that interact with the receptor-binding domain (RBD), or docked to the RBD to recognize novel binding modes, and their amino acid sequences were engineered to optimize target binding, folding, and stability. Ten designs incorporate RBD with affinities ranging from 100 picomoles to 10 nanomoles and achieve half-maximal inhibitory concentrations (IC50) of 24 picomoles to 35 nanomoles. 50 The SARS-CoV-2 infection in Vero E6 cells was blocked by the 56th and 64th residues. The most potent (with a novel binding mode) proteins were those with an IC50 value. 50 (Approximately 0.16 ng / mL). The cryo-electron microscopy structure of these microbinding agents, which are complexed with the SARS-CoV-2 spike extracellular domain trimer (in which all three RBDs are bound), is almost identical to the computational model. These ultrastable microbinding agents provide a starting point for SARS-CoV-2 treatment.

[0015] Case et al. (in: Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease. Cell Host Microbe. 2021 Jul 14;29(7):1151-1161.e5. doi: 10.1016 / j.chom.2021.06.008. Epub 2021 Jun 24.PMID: 34192518) described the ability of a modified form of the leader miniprotein LCB1 to protect against SARS-CoV-2-mediated lung disease in mice. Systemic administration of LCB1-Fc reduced viral load, decreased immune cell infiltration and inflammation, and provided complete protection against lung disease and pathological conditions. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lungs when administered up to 5 days prior to or within 2 days after viral inoculation. Importantly, LCB1v1.3 provides in vivo protection against historical strains (WA1 / 2020), emerging strain B.1.1.7, and strains encoding replacements of the critical E484K and N501Y spike proteins. These data support the development of LCB1v1.3 for the prevention or treatment of SARS-CoV-2 infection.

[0016] WO 2022 / 165081 describes nanoparticles comprising a biodegradable polymer core and a lipid coating layer, the lipid coating layer being functionalized with pathogen-binding receptors (e.g., angiotensin-converting enzyme 2 (ACE2) receptor protein) and / or pathogen-binding antibodies or their antigen-binding fragments (e.g., virus-binding antibodies or their antigen-binding fragments). The nanoparticles are further functionalized by phagocytic cell-specific ligands (e.g., lipids containing phosphatidylserine contained in the lipid coating layer) to facilitate the clearance of pathogens bound to the nanoparticles. Methods for using nanoparticles to treat or prevent pathogen infections (e.g., coronavirus infections) are also described. They pointed out that controlling particle size can modulate phagocytosis efficacy and provide reproducible mechanical properties and material biocompatibility (refer to Chunbai He, Yiping Hu, Lichen Yin, Cui Tang, Chunhua Yin, Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles, Biomaterials, Vol. 31, No. 13, 2010, pp. 3657-3666, ISSN 0142-9612, https: / / doi.org / 10.1016 / j.biomaterials.2010.01.0653, which indicates that NPs with a slightly negative charge and a particle size of 150 nm tend to accumulate more efficiently in tumors. These results can serve as a guide for the rational design of drug nanocarriers with maximum therapeutic efficacy and predictable in vivo properties, where the control of particle size and surface charge is of great importance; and Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric microspheres. Pharm Res. 2008 Aug;25(8):1815-21. doi: 10.1007 / s11095-008-9562-y. Epub 2008 Mar 29. PMID: 18373181; PMCID: PMC2793372, which indicates that particles with a diameter of 2 to 3 μm exhibit the greatest phagocytosis and adhesion. However, the internalization rate is not significantly affected by particle size. It is assumed that the maximum adhesion of 2 to 3 μm microspheres originates from the characteristic feature of membrane folds in macrophages. The hydrolyzed diameter of functionalized nanoparticles was measured by dynamic light scattering, and it increased with the addition of each molecule. See also Figure 1B. The diameter of the core can be from about 200 to about 1200 nanometers, optionally about 500 nanometers. Figure 2 A showed that after 48 hours, up to 50% uptake was achieved for 500 nm and larger particles; however, 1200 nm particles were uptaken more slowly, and smaller particles (200 nm) were uptaken significantly less.

[0017] US 2022 / 041660 describes a composition for sequestering target viral particles in the respiratory tract of an object, comprising an effective amount of sequestering particles having a protein binder on its surface, wherein the protein binder binds to capsid proteins on the surface of the viral particles, thereby causing the sequestering particles and viral particles to form aggregates. The protein binder may be an ACE2 peptide. The term "microparticle" as used refers to a particle with a diameter of at least 750 nm or 1 μm. The term "nanoparticle" as used refers to a particle with a diameter less than 750 nm or 1 μm. Particle size can be used to control the deposition site of the sequestering particles. The described sequestering particles may have a diameter of 1 to 10 μm. The sequestering particles may have a diameter of 5 to 10 μm. That is, the particle size used is 1000 to 10000 nm, preferably greater than 5000 nm. The involvement of phagocytosis is not described.

[0018] WO 2023 / 044494 discloses a pharmaceutical composition for the prevention of SARS-CoV-2 infection, comprising *Staphylococcus lugdunensis* or *Streptococcus salivarius* M18 functionalized with a SARS-CoV-2 binding peptide translated, produced, and released by bacteria. Virus-binding small molecules immobilized on the surface of nanoparticles or microparticles are not disclosed. Staphylococcus species typically have a diameter of about 0.5 to 1.0 micrometers (µm). Streptococcus bacteria are very small, with a diameter of 0.5 to 2.0 micrometers.

[0019] Despite advances in antiviral therapy and vaccination, the search remains for effective and reliable agents and methods for the prevention and treatment of viral infections that are unlikely to induce viral resistance and are well-tolerated by patients. Therefore, one object of this invention is to provide effective agents for the prevention and treatment of viral infections, particularly coronavirus infections. Other objects and advantages will become apparent to those skilled in the art upon studying this specification of the invention.

[0020] In its first aspect, according to the invention, the aforementioned objective is achieved by functionalized nanoparticles or microparticles for the prevention and / or treatment of target viral infection in mammalian subjects, said functionalized nanoparticles or microparticles comprising: a) at least one nanoparticle or microparticle, and b) at least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of said nanoparticle or microparticle, wherein the virus-binding peptide and / or virus-binding small molecule binds to at least one target virus with nanomolar to picomolar affinity, wherein the overall diameter of the functionalized nanoparticle or microparticle is at least about 400 nm, preferably about 400 to 2000 nm, and more preferably about 600 to 700 nm. The invention will provide an overall complex that allows for active phagocytosis, which requires a particle size of about > 400 nm.

[0021] Preferably, the functionalized nanoparticles or microparticles used in the application according to the present invention are substantially spherical in shape.

[0022] Further preferred are functionalized nanoparticles or microparticles according to the present invention, wherein the virus-binding peptide is selected from microbinding proteins, host cell receptors, sialylated polysaccharides such as SA and gangliosides containing SA, cell adhesion molecules (CAMs) such as igSF members including CD4, JAM-A, CAR, integrins such as αvβ3, PtdSer receptors, cellular immunoglobulins and mucin domains (TIMs), Tyro3, ​​Axl, Mer (TAM), and ACE2 receptors, preferably wherein the virus-binding peptide is selected from LCB1 and AHB2.

[0023] In its second aspect, according to the invention, the aforementioned objective is achieved by aggregates for the prevention and / or treatment of target viral infection in mammalian subjects, said aggregates comprising at least one functionalized nanoparticle or microparticle according to the application described in the invention and at least one target virus, wherein said prevention and / or treatment comprises phagocytosis carried out by cells in said mammalian subjects, particularly macrophage-mediated phagocytosis.

[0024] Preferably, the application is a functionalized nanoparticle or microparticle according to the invention, or an aggregate according to the invention, wherein the at least one target virus is selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV), and Lassa virus. Virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S and SARS-CoV-2.

[0025] In its third aspect, according to the invention, the above-mentioned objective is achieved by a pharmaceutical composition for the prevention and / or treatment of target viral infection in mammalian subjects, the pharmaceutical composition comprising functionalized nanoparticles or microparticles or aggregates according to the invention, wherein the pharmaceutical composition further comprises suitable excipients and / or adjuvants.

[0026] Preferred are pharmaceutical compositions according to the invention, wherein the composition is formulated for intravenous injection, as a drinking solution / suspension, as a solution / suspension suitable for aerosol generation and inhalation, or as a dry powder for inhalation. More preferably are pharmaceutical compositions according to the invention, wherein the functionalized nanoparticles or microparticles have a size of about 400 to 700 nm and are optionally suitable for administration by inhalation.

[0027] In its fourth aspect, according to the invention, the above-mentioned objective is achieved by a method for detecting a target virus and / or a target virus-specific antiviral antibody in a biological sample obtained from a mammalian object, the method comprising contacting a functionalized nanoparticle or microparticle according to the invention with the biological sample and detecting the binding of the target virus and / or the target virus-specific antiviral antibody to the microparticle and / or detecting the formation of aggregates of the target virus with the functionalized nanoparticle or microparticle, wherein the binding and / or the formation of aggregates indicate the target virus and / or the target virus-specific antiviral antibody in the biological sample.

[0028] Preferably, the method according to the invention further includes the step of quantifying the amount of target virus and / or target virus-specific antiviral antibody in a biological sample obtained from a mammalian object based on the formation of binding and / or aggregates in the biological sample.

[0029] In its fifth aspect, according to the invention, the above-mentioned objective is achieved by a kit for performing the method according to the invention, the kit comprising at least one functionalized nanoparticle or microparticle for the application according to the invention, and suitable adjuvants.

[0030] In its sixth aspect, according to the invention, the above-mentioned objective is achieved by using the kit according to the invention for detecting or quantifying target viruses and / or target virus-specific antiviral antibodies in biological samples obtained from mammalian subjects.

[0031] In its seventh aspect, according to the invention, the above-mentioned objective is achieved by a method for preventing and / or treating target virus infection in mammalian subjects, the method comprising applying functionalized nanoparticles or microparticles, aggregates, or pharmaceutical compositions according to the invention to mammalian subjects requiring prevention and / or treatment of target virus infection.

[0032] Preferably, the method according to the present invention refers to the target virus selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV); Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

[0033] As described above, the present invention provides functionalized nanoparticles or microparticles for the prevention and / or treatment of target viral infections in mammalian subjects. At least one nanoparticle or microparticle is functionalized with at least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of the nanoparticle or microparticle. These virus-binding peptides and / or virus-binding small molecules bind to at least one target virus with nanomolar to picomolar affinity. Finally, the overall diameter of the functionalized nanoparticle or microparticle is at least about 400 nm, preferably about 400 to 2000 nm, and more preferably about 600 to 700 nm. Typically, nanoparticles or microparticles with a size of about 100 to about 10,000 nm are recognized by the host immune system and may be removed by phagocytosis.

[0034] In a particularly preferred embodiment of the invention, the functionalized nanoparticles or microparticles of the application have a size of about 400 to 700 nm and are optionally suitable for application to mammalian subjects by inhalation.

[0035] Behbahanipour M, et al. (in: OligoBinders: Bioengineered Soluble Amyloid-like Nanoparticles to Bind and Neutralize SARS-CoV-2. ACS Appl Mater Interfaces. 2023 Mar 8;15(9):11444-11457. doi: 10.1021 / acsami.2c18305. Epub2023 Feb 22. PMID: 36890692; PMCID: PMC9969896) describe a nanoparticle capable of neutralizing SARS-CoV-2. For this purpose, the inventors utilized a modular self-assembly strategy to modify OligoBinders (soluble oligomeric nanoparticles) with two previously described small proteins that bind to the S protein receptor-binding domain (RBD) with high affinity. The multivalent nanostructures compete with the RBD-ACE2r interaction and exhibit an IC50 in the pM range. 50 The SARS-CoV-2 virus-like particles (SC2-VLP) are neutralized, preventing SC2-VLP from fusing with the membranes of cells expressing ACE2r. Furthermore, OligoBinders are biocompatible and remarkably stable in plasma. The particles used comprise the peptide LCB1 and have a size of approximately 20 nm.

[0036] In contrast, the particles used according to the present invention actively utilize phagocytosis, which requires the complex to be larger than about 400 nm—if the particles are too small, they can only bind a limited number of viral particles. This weakens the neutralizing effect or requires higher doses of nanoparticles. Furthermore, detectability in cells and tissues is crucial for determining therapeutic efficacy in vivo and in vitro. This becomes significantly more difficult when the complex size is less than 400 nm.

[0037] Small particles can cross the mucosal barrier and thus be lost. This should be avoided in the context of this invention. If particles cross or enter the mucosal barrier after application, particle / virus contact is inhibited / blocked. This leads to reduced therapeutic effect. The use of PEG-connectors also improves mucosal penetration; therefore, particles with a size of about 600 to 700 nm are preferred.

[0038] Finally, significantly larger particles (greater than 900 nm) elicited an undesirable immune response, which is undesirable in the context of peptide modification and subsequent viral interactions.

[0039] In the context of this invention, nanoparticles or microparticles may be made of any material suitable for application to a target and / or suitable for diagnostic uses as disclosed herein. The material must also be suitable for providing functionalized particles. In the context of this invention, the term "functionalized" should refer to modification of the particle surface used in the method according to the invention. According to the invention, particle functionalization includes at least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of the nanoparticle or microparticle.

[0040] In a preferred embodiment of the invention, the selection of the combination of materials comprising nanoparticles or microparticles with virus-binding peptides and / or virus-binding small molecules can be coordinated as desired, thereby enabling a modular design of functionalized nanoparticles or microparticles. In this modular design according to the invention, polymeric organic or inorganic materials compatible with the desired route of administration and functionalization are selected to constitute the nanoparticles or microparticles. Preferably, particulate materials sufficiently stable to host metabolism but not completely inert are used to achieve adequate clearance.

[0041] Particle selection can also be driven by toxicological parameters and local tolerance. The polymer material can be, for example, PLA, PLGA, PLGH, polyglutamic acid, styrene-maleic acid copolymer, or PE, or nanospheres composed of membranes and / or lipids, such as liposomes.

[0042] In a preferred embodiment of the invention, the polymer material comprises poly(lactic-co-glycolic acid) (PLGA), which is frequently used due to its well-documented biocompatibility and biodegradability. The monomers lactic acid and glycolic acid can be metabolized by the body, and the degradation rate can be affected by changing the monomer ratio. In another preferred embodiment of the invention, the polymer material further comprises a block copolymer containing PLGA and poly(ethylene glycol) (PEG) as a stealth polymer. During formulation, the hydrophilic PEG should be located on the outer side of the particles; therefore, ω-terminal groups should be present on the surface of the particles to facilitate any surface modifications.

[0043] In one relevant embodiment of the invention, the polymer material comprises PLGA-Mal, PLGA-PEG-Mal, PLGA-DY550, PLGA-PEG, PLGA-PEG-DBCO, and / or combinations thereof. In another preferred embodiment of the invention, the nanoparticles or microparticles comprise any polymer composition according to Table 1.

[0044] Table 1: Preferred polymer compositions.

[0045]

[0046] Preferably, the nanoparticles or microparticles comprise suitable organic or inorganic materials, such as metals, plastics, polymers such as PLA, PLGA, PLGH, polyglutamic acid, styrene-maleic acid copolymer or PE, or nanospheres composed of membranes and / or lipids, such as liposomes. Preferably, the nanoparticles or microparticles comprise PLGA-Mal, PLGA-PEG-Mal, PLGA-DY550, PLGA-PEG, PLGA-PEG-DBCO and / or combinations thereof.

[0047] In addition to the specific functionalization and size of the functionalized nanoparticles or microparticles used according to the invention, the shape of the functionalized nanoparticles or microparticles has been found to be relevant to their application. Particle geometry can help regulate the internalization of particles or aggregates into cells via phagocytosis (see below). Different local particle shapes at the cell attachment point produce different angles between the membrane and the particle. This contact angle has a significant impact on the ability of macrophages to internalize particles via actin-driven macrophage membrane movement. Preferably, the particle shape is substantially spherical, in contrast to, for example, oblate spheroids, elongated ellipsoids, elliptical disks, rectangular disks, and UFOs. Elongated particles are known to be less conducive to phagocytosis.

[0048] Compared to existing technologies, another important feature of this invention is the highly specific binding of virus-binding peptides and / or virus-binding small molecules on the surface of nanoparticles or microparticles to the target virus. In some specific embodiments, the functionalized nanoparticles or microparticles compete with host cell receptors for binding to the virus to a) prevent infection-related interactions with natural host cell receptors, and b) trigger uptake via macrophages.

[0049] After functionalizing nanoparticles or microparticles with the high-affinity virus-binding peptides and / or virus-binding small molecules of the present invention, viruses can be selectively captured, making them directly accessible for macrophage-mediated phagocytosis. Therefore, a particularly preferred embodiment of the present invention is a binding of at least nanomolar to picomolar amounts between the virus-binding peptides and / or virus-binding small molecules and the target virus.

[0050] Using structural entities that bind to target viral particles with nanomolar to picomolar affinity is advantageous because it allows nanoparticles or microparticles to bind to the virus over natural receptors. For example, in the case of the SARS-CoV-2 spike protein, the viral binder on the surface of the nanoparticle needs to be able to interact with the wild-type (wt) spike RBD with nanomolar affinity to overcome the natural ACE2 receptor.

[0051] The virus-binding small molecule can be selected from small molecule virus binders, such as elbasvir, grazoprevir, sovaprevir, hesperidin, pamaqueside, diosmin, and sitogluside, and those skilled in the art will know that there are many more different high-affinity binders to virus particles in the prior art.

[0052] The virus-binding peptide can be selected from microbinding proteins, host cell receptors, sialylated glycans such as SA and SA-containing gangliosides, cell adhesion molecules (CAMs) such as igSF members including CD4, JAM-A, CAR, integrins such as αvβ3, PtdSer receptors, cellular immunoglobulin and mucin domains (TIMs), Tyro3, ​​Axl, Mer (TAM), and ACE2 receptors. Preferably, functionalized nanoparticles or microparticles according to the present invention are used, wherein the virus-binding peptide is selected from LCB1 and AHB2.

[0053] Virus-binding peptides and microbinding agents can be successfully designed according to methods in the art (see, for example, Cao, L., Coventry, B., Goreshnik, I. et al. Design of protein-binding proteins from the target structure alone. Nature 605, 551-560 (2022). https: / / doi.org / 10.1038 / s41586-022-04654-9; [8];

[26] ).

[0054] As described above, the preferred applications according to the present invention are functionalized nanoparticles or microparticles, wherein the binding between the virus-binding peptide and / or virus-binding small molecule and at least one target virus is superior to the binding of the target virus to its host cell receptor.

[0055] Therefore, according to the present invention, the affinity between the virus-binding peptides and / or virus-binding small molecules of the functionalized nanoparticles or microparticles used is in the nanomolar to picomolar range, preferably wherein the dissociation constant (K) is [missing information]. d The value is less than 100 nM, preferably about 1 pM to 20 nM, more preferably about 1 pM to 100 pM.

[0056] Typically, any suitable host cell receptor, i.e., the "entry point" for viral infection in mammalian subjects, can be used to design viral-binding peptides and / or viral-binding small molecules. Preferably, the receptor is selected from cell adhesion molecules (CAMs) that function in cell-to-cell and cell-to-extracellular matrix interactions. The broad CAM family includes selectins, cadherins, integrins, and IgSF members. The ubiquitous expression and multifactorial functions of CAMs in ligand binding, endocytosis, and signal transduction provide numerous possibilities for viral conjugation to CAMs. Viruses including HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, and Coxsackievirus B (CVB) utilize IgSF members as receptors. Additionally, integrins serve as receptors for reovirus, rotavirus, adenovirus, West Nile virus (WNV), human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), and herpes simplex virus (HSV), as well as human cytomegalovirus (HCMV) and human herpesvirus-8.

[0057] In another embodiment of the invention, the host cell receptor is selected from IgSF members, which have become receptors for a wide range of viruses, including enveloped and non-enveloped viruses with DNA or RNA genomes, including reovirus, adenovirus, Coxsackievirus, rabies virus, measles virus, and HIV.

[0058] In another embodiment of the invention, the host cell receptor is selected from integrins, which are receptors that mediate multiple functions, such as viral entry and activation of signal transduction events. For example, hMPV (an enveloped, negative-sense single-stranded RNA paramyxovirus and a cause of respiratory infections, particularly in children) utilizes integrin αvβ1 and α5β1 receptors to mediate infection through interaction with viral fusion (F) proteins.

[0059] In another embodiment of the invention, the host cell receptor is selected from PtdSer receptors, including T-cell immunoglobulin and mucin domains (TIMs) and the TYRO3, AXL, and MERTK receptor tyrosine kinase families (TAMs), which have been shown to act as receptors for enveloped viruses from multiple viral families and viruses within the same viral family. PtdSer receptors have reportedly mediated viral entry for many enveloped viruses, including filoviruses EBOV and Marburg virus (MARV); flaviviruses WNV, dengue virus (DENV), and Zika virus (ZIKV); and arecaviruses such as lassa virus and poxvirus.

[0060] In another embodiment of the invention, the host cell receptor is selected from SA receptors, which mediate binding and / or entry onto the surface of host cells. SV40, BKPyV, and PyV all utilize ganglioside receptors containing α2,3 and α2,8-linked SA to mediate viral attachment to their respective host cells.

[0061] In a particularly preferred embodiment of the invention, at least one virus-binding peptide is selected from angiotensin-converting enzyme 2 (ACE2), which is used by SARS-CoV-2 as a cell entry receptor.

[0062] Clearly, virus-binding peptides and / or virus-binding small molecules must be designed to functionally inhibit, or even surpass, the binding of the target virus to its target receptors / proteins on mammalian cells (such as those naturally targeted by the target virus).

[0063] In the context of this invention, the virus-binding peptide may be suitably modified, for example, to be immobilized on particles, and thus may contain at least one terminal cysteine ​​residue. The cysteine ​​residue may optionally be modified to carry an azide group. In its azide form, the virus binder is no longer readily dimerized and can be covalently bound to the surface of nanoparticles or microparticles. The virus-binding azide can, for example, react with alkynes attached to a surface (e.g., the surface of a nanoparticle core) in a strain-promoted azide-alkyne click reaction.

[0064] Furthermore, in a preferred embodiment, the present invention utilizes the advantages of click chemistry to rapidly and conveniently allow for combinations between modified particles, linkers, and virus-binding particles. This multimodal possibility set makes the modification and replacement of linkers and virus-binding peptides relatively simple. This enables rapid adaptation to new variants of interest in the event of viral changes, such as in the case of SARS-CoV-2 or different viruses.

[0065] Technicians can easily apply the click chemistry illustrated above with cysteine ​​and the use of DCBO to other binding groups and pairs.

[0066] According to the present invention, the immobilization of virus-binding peptides or virus-binding small molecules onto the surface of nanoparticles or microparticles can be direct or indirect, for example, through conjugation, physical adsorption, linkers, and / or covalent bonding. In a preferred embodiment of the functionalized nanoparticles or microparticles used according to the present invention, the immobilization is a succinimide thioether bond or a bibenzocyclooctyne (DBCO) azide link. The peptide-virus binder or virus-binding protein can be attached to or linked to nanoparticles or microparticles covalently or via physical adsorption using chemical coupling strategies known to those skilled in the art.

[0067] In a preferred embodiment of the invention, a peptide-virus binding agent or virus-binding protein is immobilized on the surface of nanoparticles or microparticles through non-covalent interactions, particularly wherein the nanoparticles or microparticles contain PLGA.

[0068] In a preferred embodiment of the functionalized nanoparticles or microparticles used in the application according to the present invention, the functionalized nanoparticles or microparticles further comprise at least one antiviral drug. Preferably, the drug is contained within the particle, or if the particle comprises a membrane, it is integrated into the membrane therein. The antiviral drug is selected, for example, from at least one of the following: neuraminidase inhibitors (e.g., oseltamivir, zanamivir), favipiravir, remdesivir, ribavirin (tribavirin), interferon α-2b / ribavirin (systemic), interferon α2a or α2b (including any pegylated form), chloroquine or hydroxylated chloroquine (administered in combination with azithromycin), dolutegravir + rilpivirine (JULUCA®), dolutegravir + lamivudine (lamivudine). The following combinations are available: ritonavir (DOVATO®), lopinavir (Kaletra®), bictegravir (BIKTARVY®), tenofovir alafenamide (BIKTARVY®), bacavir (TRIUMEQ®), elvitegravir (GENVOYA®), cobicistat (GENVOYA®), and tenofovir disoproxil fumarate (STRIBILD®).

[0069] For diagnostic purposes, the functionalized nanoparticles or microparticles used according to the present invention may also contain at least one detectable marker or biomarker, such as an enzyme, antigen, weight, and / or fluorescent biomarker.

[0070] In the immune system of multicellular organisms, phagocytosis, as a type of endocytosis, is the primary mechanism for removing pathogens and cellular debris. Ingested material is subsequently digested in the phagosome. Bacteria, dead tissue cells, and small mineral particles are examples of objects that can be phagocytosed. This invention provides a method for virus clearance utilizing the phagocytic pathway. This offers advantages compared to prior art methods, where nanoparticles containing viral components are prepared solely to elicit a targeted immune response against a specific target virus.

[0071] Innate immune cells (such as macrophages, monocytes, neutrophils, and dendritic cells), as well as epithelial and endothelial cells, eliminate pathogens or particles through a process called phagocytosis. While these cells do play a role in the immunopathobiology of viral infection, many virions are not easily phagocytosed due to their small size. In this invention, the phagocytic process can be utilized for viral clearance, which effectively neutralizes and removes viruses before they can interact with their corresponding host cells.

[0072] According to the present invention, aggregation is driven by high-affinity binding between a virus binder on the surface of a nanoparticle or microparticle and the virus body. One such functionalized nanoparticle or microparticle can capture multiple virus bodies. Each target virus can interact with the nanoparticle through multiple binding events, thereby enhancing the high binding affinity and reducing the likelihood of complex dissociation. Furthermore, the particle-virus complex is large enough to be phagocytosed by the aforementioned cells, allowing for viral digestion in mature phagolysosomes.

[0073] Therefore, another aspect of the present invention relates to aggregates for the prevention and / or treatment of target virus infection in mammalian subjects, comprising at least one functionalized nanoparticle or microparticle according to the application described in the present invention and at least one target virus, wherein the prevention and / or treatment comprises phagocytosis in the mammalian subject, such as macrophage-mediated phagocytosis.

[0074] Preferably, the application is a functionalized nanoparticle or microparticle or an aggregate according to the invention, wherein the at least one target virus is selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV), Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

[0075] Then, another aspect of the invention relates to pharmaceutical compositions for the prevention and / or treatment of target viral infections in mammalian subjects, comprising functionalized nanoparticles or microparticles or aggregates according to the application of the invention, wherein the pharmaceutical composition further comprises suitable excipients and / or adjuvants.

[0076] Preferred pharmaceutical compositions according to the invention are formulated as intravenous injection, drinking solutions / suspensions, solutions / suspensions suitable for aerosol generation and inhalation, or dry powders for inhalation. The pharmaceutical compositions can be formulated for administration via intravenous injection, drinking solutions / suspensions, or inhalation. Therefore, the pharmaceutical compositions can be tablets, capsules, granules, powders, sachets, reconfigurable powders, dry powder inhalers, and / or chewable tablets. Such solid dosage forms may also contain suitable amounts of excipients and other ingredients, such as cellulose, microcrystalline cellulose, povidone, particularly FB povidone, and / or magnesium stearate.

[0077] Preferably, the pharmaceutical composition for inhalation is formulated according to the present invention, wherein the functionalized nanoparticles or microparticles have a size of about 400 to 700 nm for diffusion through the lung mucus layer. In this case, since the drug is applied directly to the site of action, i.e., the respiratory tract, the applied dose can preferably be reduced.

[0078] The pharmaceutical composition according to the present invention may optionally include a pharmaceutically acceptable carrier. Pharmaceutical carriers or excipients include diluents (fillers, fillers, such as lactose, microcrystalline cellulose), disintegrants (such as sodium starch glycolate, croscarmellose sodium), binders (such as PVP, HPMC), lubricants (such as magnesium stearate), flow aids (such as colloidal SiO2), solvents / cosolvents (such as aqueous carriers, propylene glycol, glycerin), buffers (such as citrate, gluconate, lactate), preservatives (such as sodium benzoate, parabens (Me, Pr and Bu), BKC), antioxidants (such as BHT, BHA, ascorbic acid), wetting agents (such as polysorbate, dehydrated sorbitan ester), thickeners (such as methylcellulose or hydroxyethylcellulose), sweeteners (such as sorbitol, saccharin, aspartame, acesulfame), flavoring agents (such as peppermint, lemon oil, butterscotch, etc.), and humectants (such as propylene glycol, glycerin, sorbitol). Other suitable pharmaceutically acceptable excipients are described in particular in Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5th edition, Govi-Verlag-Frankfurt (1997). Those skilled in the art will recognize suitable formulations for cilengitide and / or its derivatives and will be readily able to select appropriate pharmaceutically acceptable carriers or excipients based, for example, on the formulation and route of administration of the pharmaceutical composition.

[0079] It should be understood that the pharmaceutical compositions according to the present invention are intended for mammalian subjects. The pharmaceutical compositions of the present invention may be used alone or as part of a co-treatment approach, i.e., co-administered with other drugs or pharmaceuticals and / or any other therapeutic agents that may be beneficial in the context of the methods of the present invention.

[0080] In another aspect, the above-mentioned objective is achieved by providing a pharmaceutical composition for the prevention or treatment of viral infections, the pharmaceutical composition comprising functionalized nanoparticles or microparticles according to the invention, wherein the pharmaceutical composition optionally further comprises suitable excipients and / or adjuvants as described herein.

[0081] In another aspect, the above-mentioned objective is achieved by providing a method for detecting target viruses and / or target virus-specific antiviral antibodies in biological samples obtained from mammalian subjects. The method comprises contacting functionalized nanoparticles or microparticles according to the application described in the invention with the biological sample and detecting the binding of the target virus and / or target virus-specific antiviral antibodies to the particles and / or detecting the formation of aggregates of the target virus with the functionalized nanoparticles or microparticles, wherein the binding and / or the formation of aggregates indicate the presence of target viruses and / or target virus-specific antiviral antibodies in the biological sample. Methods and strategies for performing this method are available from relevant literature and can be readily modified for specific detections.

[0082] In one relevant embodiment of the invention, the functionalized nanoparticles or microparticles further comprise at least one detectable marker or label, such as a fluorescent tag, enabling the detection of aggregates formed between the functionalized nanoparticles or microparticles and the target virus.

[0083] Preferably, the method according to the invention further includes a step of quantifying the amount of target virus and / or target virus-specific antiviral antibody in a biological sample obtained from a mammalian object based on the formation of binding and / or aggregates in the biological sample. Here, the number of viral particles bound to functionalized nanoparticles or microparticles can be determined. Those skilled in the art are familiar with different techniques for detecting or quantifying labeled particles. For example, flow cytometry can be used to detect recombinant wt-SARS-CoV-2 RBD fluorescently labeled with Alexa Fluor 488 dye (RBDAF488) incubated with functionalized nanoparticles or microparticles carrying LCB1 microbinding agents.

[0084] In the context of this invention, any suitable sample, such as a biological sample, may be used for the determination of this invention, for example, a blood or serum sample derived from a mammal, or a pooled sample from a group of mammalian subjects.

[0085] The object of the present invention is also achieved by a diagnostic composition for detecting target viruses and / or specific antiviral antibodies in samples obtained from mammalian subjects, comprising the steps of: contacting the biological sample with functionalized nanoparticles or microparticles according to the present invention, and detecting the binding of the virus and / or the specific antibody to the particles, thereby detecting the target virus and / or the antibody in the biological sample.

[0086] In this aspect of the invention, nanoparticles or microparticles are used as diagnostic tools, wherein target virus particles or antibodies bound to the nanoparticles or microparticles are detected as part of the diagnosis. Preferably, the diagnostic composition according to the invention comprises a detectable marker or biomarker as described above.

[0087] Another aspect of the invention then relates to a kit for performing the method according to the invention, such as a diagnostic kit, comprising at least one functionalized nanoparticle or microparticle for the application according to the invention, or the diagnostic composition described above, and suitable adjuvants. The kit may contain materials for performing the method according to the invention in one or more separate containers, optionally together with adjuvants for performing the method and / or instructions.

[0088] Preferred applications are those of the diagnostic kit according to the invention for detecting target viruses and / or antiviral antibodies in biological samples obtained from mammalian subjects, particularly in the context of β-coronavirus infections such as COVID-19. Further preferred applications are those of the kit according to the invention for detecting or quantifying target viruses and / or target virus-specific antiviral antibodies in biological samples obtained from mammalian subjects.

[0089] Another aspect of the invention relates to a method for preventing and / or treating target virus infection in mammalian subjects, comprising applying functionalized nanoparticles or microparticles, aggregates, or pharmaceutical compositions according to the invention to mammalian subjects requiring prevention and / or treatment of target virus infection.

[0090] Preferably, the method according to the present invention refers to the target virus selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV); Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

[0091] "Treatment" means any treatment of a disease or condition in an object (e.g., a mammal or a human), including: prevention or protection from the disease or condition, so that the clinical symptoms of the disease do not develop; suppression of the disease, i.e., preventing or inhibiting the development of clinical symptoms; and / or relief of the disease, i.e., causing the remission of clinical symptoms. "Improvement" means the prevention, reduction, or alleviation of a state, or an improvement in the state of an object; improvement of stress is the offsetting of the negative aspects of stress. Improvement includes, but does not require, complete restoration or complete prevention of stress.

[0092] In summary, the combination of competition with host cell receptors and the binding of virions to functionalized nanoparticles or microparticles to form phagocytosed aggregates will reduce viral load in the host and thus enable rapid recovery in mammalian subjects with viral infections, particularly COVID-19.

[0093] According to the present invention, the mammalian object can be selected from rodents, cattle, pets, and humans. The object can be a patient.

[0094] In the context of this invention, the term "target virus" refers to a virus in the form of a virus particle or virion.

[0095] The method according to the invention can be performed in vivo or in vitro.

[0096] In another aspect, the present invention provides a method for producing functionalized nanoparticles or microparticles according to the invention, the method comprising the steps of: suitably providing nanoparticles or microparticles as described herein, wherein the overall diameter of said functionalized nanoparticles or microparticles is at least about 400 nm, preferably about 400 to 2000 nm, and more preferably about 600 to 700 nm; suitably providing at least one virus-binding peptide and / or virus-binding small molecule that binds to at least one target virus with nanomolar to picomolar affinity as described herein; and suitably immobilizing at least one virus-binding peptide and / or virus-binding small molecule on the surface of said nanoparticles or microparticles.

[0097] In another aspect, the present invention provides a method for generating aggregates comprising at least one functionalized nanoparticle or microparticle according to an application described herein, comprising the steps of: generating nanoparticles or microparticles as described herein, and binding the functionalized nanoparticles or microparticles of the application to at least one target virus to form aggregates. The aggregates are suitable for phagocytosis, i.e., they are greater than 400 nm, for example, about 400 to 5000 µm.

[0098] In another embodiment of the method according to the invention, the material used to form the nanoparticles or microparticles according to the invention may be additionally prepared with suitable functional groups for high-affinity conjugation of at least one virus-binding peptide and / or virus-binding small molecule in a subsequent fixation step. This functional group may be particularly suitable for use in click chemistry reactions, especially maleimide groups or dibenzylcyclooctynyl (DBCO) groups.

[0099] As an example, these functional groups can be added to polymers through suitable chemical processes known in the art (see also...). Figure 18 and Figure 19 To attach maleimide groups, for example, polymer materials containing nanoparticles or microparticles are esterified with N-hydroxysuccinimide (NHS), followed by amidation with maleimide-amine or PEG-maleimide-amine. Alternatively, to attach DBCO groups, as another example, the polymer material is first amidated with NH2-PEG-COOH via NHS ester activation, and then the DBCO groups are attached in an NHS ester activation reaction with DBCO amine.

[0100] Those skilled in the art will recognize various methods for forming nanoparticles or microparticles. In a preferred embodiment of the method according to the invention, a monoemulsion technique is used to form nanoparticles and / or microparticles. For example, the material forming the particles can be dissolved in ethyl acetate to form an organic solution. The organic solution can be mixed with an aqueous phase containing poly(vinyl alcohol) (PVA). When the organic solvent evaporates, nanoparticles or microparticles are formed and can be purified by ultrafiltration.

[0101] Then, another embodiment of the invention relates to a method for producing at least one virus-binding peptide using *Escherichia coli*, comprising expressing a recombinant plasmid encoding the desired virus-binding peptide in a suitable *E. coli* strain. The strain may be cultured to overexpress the virus-binding peptide, and / or the peptide may be expelled into a culture medium. The peptide can then be isolated from the cells or culture medium using conventional techniques.

[0102] In another relevant embodiment of the method according to the invention, the virus-binding peptide can be expressed using a suitable purification tag, such as an N-terminal His6-tag. In one specific example, the virus binder with the His6-tag can be purified using Co-chelation chromatography, and in a subsequent step, the tag can be removed by digestion with a tobacco etch virus (TEV) protease followed by size exclusion chromatography.

[0103] In another embodiment of the method according to the invention, the resulting virus-binding peptide carries at least one terminal cysteine ​​residue. For example, the resulting virus-binding peptide may be side-joined with a short Ser and Gly sequence carrying a Cys residue at the C-terminus. This terminal cysteine ​​residue can be used for covalent linkage with nanoparticles or microparticles in subsequent steps.

[0104] In another preferred embodiment of the method according to the invention, the binding affinity of the virus-binding peptide to the target virus particle can be verified to, for example, optimize the composition of the virus binding agent. Techniques such as mutagenesis and surface plasmon resonance (SPR) spectroscopy can be used for this purpose.

[0105] In the context of this invention, unless otherwise stated, the term "about" shall mean a deviation of + / - 10% from a given value.

[0106] The objective of this invention is to establish novel therapeutic methods for binding, neutralizing, and clearing, in particular, SARS-CoV-2 virions using nanoparticles. These particles are modified with a high-affinity microbinding agent that surpasses the natural SARS-CoV-2∙hACE2 interaction by binding to the SARS-CoV-2 spike RBD with higher specificity and affinity than hACE2. Furthermore, the inventors have demonstrated that by compositing several virions with nanoparticles, the size of the virions is increased, which is necessary for their degradation through phagocytosis and intracellular processing by host cells.

[0107] The inventors have demonstrated that nanoparticles equipped with covalently bound microbinding agents, such as compounds P, 28A, and 29A, can bind VSVΔG-S virions in vitro and guide them to phagocytosis and intracellular degradation. Therefore, the inventors have identified a novel approach to achieving viral clearance, with potential applicability in treating all types of viral infections in humans. In principle, this method is applicable not only to SARS-CoV-2 but also to other airborne viruses small enough to evade phagocytosis, such as influenza, provided that small proteins with sufficient affinity to bind the virus are available to attach them to the particle surface.

[0108] In addition to promoting phagocytosis, nanoparticles equipped with covalently bound microbinding agents can block and neutralize viruses, as shown in SARS-CoV-2 neutralization assays. In these assays, not only those VBKs with covalently bound microbinding agents on their surfaces showed neutralizing activity, but also those particles with only microbinding agents adsorbed showed neutralizing activity. This suggests that nanoparticles can also act as delivery carriers for free microbinding agents, which have been successfully used in animal models of SARS-CoV-2 infection

[28] .

[0109] In addition to their significant in vitro virus-neutralizing activity, VBK does not affect cell viability or penetrate cell membranes.

[0110] This invention relates to the following items.

[0111] Project 1. Functionalized nanoparticles or microparticles for the prevention and / or treatment of target viral infection in mammalian subjects, comprising: a) at least one nanoparticle or microparticle, and b) at least one virus-binding peptide and / or at least one virus-binding small molecule immobilized on the surface of the nanoparticle or microparticle, wherein the virus-binding peptide and / or virus-binding small molecule binds to at least one target virus with nanomolar to picomolar affinity, and wherein the overall diameter of the functionalized nanoparticle or microparticle is at least about 400 nm, preferably about 400 to 2000 nm, and more preferably about 600 to 700 nm.

[0112] Project 2. Functionalized nanoparticles or microparticles for the application described in Project 1, wherein the binding between the virus-binding peptide and / or virus-binding small molecule and the at least one target virus is superior to the binding between the target virus and its host cell receptor.

[0113] Project 3. Functionalized nanoparticles or microparticles for applications according to Project 1 or 2, wherein the shape of the functionalized nanoparticles or microparticles is substantially spherical.

[0114] Project 4. Functionalized nanoparticles or microparticles for any of the applications described in Projects 1 to 3, wherein the nanoparticles or microparticles comprise suitable organic or inorganic materials, such as metals, plastics, polymers such as PLA, PLGA, PLGH, polyglutamic acid, styrene-maleic acid copolymer or PE, or nanospheres composed of membranes and / or lipids, such as liposomes, preferably wherein the nanoparticles or microparticles comprise PLGA-Mal, PLGA-PEG-Mal, PLGA-DY550, PLGA-PEG, PLGA-PEG-DBCO and / or combinations thereof.

[0115] Project 5. Functionalized nanoparticles or microparticles for any of the applications described in Projects 1 to 4, wherein the virus-binding peptide is selected from microbinding proteins, host cell receptors, sialylated polysaccharides such as SA and gangliosides containing SA, cell adhesion molecules (CAMs) such as igSF members including CD4, JAM-A, CAR, integrins such as αvβ3, PtdSer receptors, cellular immunoglobulin and mucin domains (TIMs), Tyro3, ​​Axl, Mer (TAM), and ACE2 receptors.

[0116] Project 6. Functionalized nanoparticles or microparticles for the application described in Project 5, wherein the virus-binding peptide is selected from LCB1 and AHB2.

[0117] Project 7. Functionalized nanoparticles or microparticles for applications according to Project 5 or 6, wherein the virus-binding peptide comprises at least one terminal cysteine ​​residue, the cysteine ​​residue optionally being modified to carry an azide group.

[0118] Project 8. Functionalized nanoparticles or microparticles for any of the applications described in Projects 1 to 7, wherein the immobilization of the virus-binding peptide or the virus-binding small molecule on the surface of the nanoparticles or microparticles is direct or indirect, for example, through conjugation, physical adsorption and / or covalent bonding.

[0119] Project 9. Functionalized nanoparticles or microparticles for the application described in Project 8, wherein the fixation is a succinimide thioether bond or a dibenzocyclooctylene (DBCO) azide linker.

[0120] Item 10. Functionalized nanoparticles or microparticles for any of the applications described in Items 1 to 9, wherein the functionalized nanoparticles or microparticles further comprise at least one antiviral drug.

[0121] Item 11. Functionalized nanoparticles or microparticles for any of the applications described in Items 1 to 10, wherein the functionalized nanoparticles or microparticles further comprise at least one detectable marker or label.

[0122] Item 12. An aggregate for the prevention and / or treatment of target viral infection in mammalian subjects, comprising at least one functionalized nanoparticle or microparticle according to any one of Items 1 to 11 and at least one target virus, wherein the prevention and / or treatment comprises phagocytosis in the mammalian subject, particularly macrophage-mediated phagocytosis.

[0123] Project 13. Functionalized nanoparticles or microparticles for the application according to any one of Projects 1 to 11, or aggregates for the application according to Project 12, wherein the at least one target virus is selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV), Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

[0124] Item 14. A pharmaceutical composition for the prevention and / or treatment of target viral infection in mammalian subjects, comprising functionalized nanoparticles or microparticles for the application according to any one of Items 1 to 11 or 13, or aggregates for the application according to Item 12 or 13, wherein the pharmaceutical composition further comprises suitable excipients and / or adjuvants.

[0125] Item 15. The pharmaceutical composition according to Item 14, wherein the composition is formulated for intravenous injection, as a drinking solution / suspension, as a solution / suspension suitable for aerosol generation and inhalation, or as a dry powder for inhalation.

[0126] Item 16. A pharmaceutical composition for use according to Item 14 or 15, wherein the functionalized nanoparticles or microparticles have a size of about 400 to 700 nm and are optionally suitable for administration by inhalation.

[0127] Item 17. A method for detecting a target virus and / or a target virus-specific antiviral antibody in a biological sample obtained from a mammalian object, comprising contacting the biological sample with functionalized nanoparticles or microparticles according to any one of Items 1 to 11, and detecting the binding of the target virus and / or the target virus-specific antiviral antibody to the microparticles and / or detecting the formation of aggregates of the target virus with the functionalized nanoparticles or microparticles, wherein the binding and / or the formation of aggregates indicate the presence of the target virus and / or the target virus-specific antiviral antibody in the biological sample.

[0128] Item 18. The method according to Item 17, wherein the functionalized nanoparticles or microparticles further comprise at least one detectable marker or label.

[0129] Item 19. The method according to Item 17 or 18, wherein the sample is a blood or serum sample derived from a mammal, or a pooled sample from a group of subjects.

[0130] Item 20. The method according to any one of Items 17 to 19, further comprising the step of quantifying the amount of target virus and / or target virus-specific antiviral antibody in the biological sample obtained from the mammalian object based on the formation of binding and / or aggregates in the biological sample.

[0131] Item 21. A kit for performing the method according to any one of Items 17 to 20, comprising at least one functionalized nanoparticle or microparticle for the application according to any one of Items 1 to 11, and suitable adjuvants.

[0132] Item 22. Use of the kit described in Item 21 for detecting or quantifying target viruses and / or target virus-specific antiviral antibodies in biological samples obtained from mammalian subjects.

[0133] Item 23. A method for preventing and / or treating target virus infection in mammalian subjects, comprising applying a functionalized nanoparticle or microparticle according to any one of Items 1 to 11 or 13, an aggregate according to Item 12 or 13, or a pharmaceutical composition according to any one of Items 14 to 16 to a mammalian subject requiring prevention and / or treatment of target virus infection.

[0134] Project 24. The method according to Project 23, wherein the target virus is selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV); Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

[0135] The invention will now be further described in the following examples with reference to the accompanying drawings and sequence listing; however, the invention is not limited thereto. For the purposes of this invention, all references cited herein are incorporated herein by reference in their entirety.

[0136] Figure 1 The purification of recombinant microbinding agents LCB1(A) and AHB2(B) is shown. SDS-PAGE analysis of fractions obtained by cobalt chelate affinity chromatography, TEV protease treatment, and size exclusion chromatography (SEC) is presented.

[0137] Figure 2 SPR-based binding analyses of monomeric RBD and dimeric RBD-mFc fusion constructs with hACE2, LCB1, and AHB2 are shown. Interactions with SARS-CoV-2 HH01 variants (A and D) and omicron SARS-CoV-2 RBD variants BA.1 and BA.5 (B, C, and E) were measured. Sensing plots of RBD and RBD-mFc binding with hACE2 (column 1), LCB1 (column 2), and AHB2 (column 3) are shown. The kinetic titration series binding responses (black lines) indicating RBD and RBD-mFc concentrations overlap with the best fit (gray lines) derived from the 1:1 interaction model (including the mass transfer term). Dissociation constant (Ki) is also shown. D The values ​​are shown in the figure. The decrease in affinity compared to wt-RBD or dimer wt-RBD is given in parentheses.

[0138] Figure 3 The flow cytometry analysis of particles in MS is shown with and without pre-incubation with fluorescently labeled RBD (dark gray).

[0139] Figure 4The neutralizing titer of VBK MT is shown. VBK MT was incubated with VSVΔG particles (VSVΔG-S) pseudotyped with SARS-CoV-2 spike surface proteins at 37°C for 1 hour and then added to Vero cells. GFP-positive cells were counted 16 hours post-infection.

[0140] Figure 5 The LDH release assay following 48 hours of co-incubation of Calu-3 cells with VBK is shown. Calu-3 cells were treated with eight different concentrations of VBK, and at 48 hours post-treatment, the release of LDH, representing cell membrane damage, was measured using a prepared LDH release assay (Promega). Untreated cells served as a negative control (Ctrl). Cell cytotoxicity (%) is shown as the mean ± SD from a single biological replicate (performed in technical triplicate).

[0141] Figure 6 The MTT assay of Calu-3 cells after 48 hours of co-incubation with VBK is shown. Calu-3 cells were treated with eight different concentrations of VBK, and metabolic activity was measured using a prepared cell proliferation assay (Promega) 48 hours after treatment. Untreated cells served as a negative control. Cell viability (% of control) is shown as mean ± SD from a biological replicate (performed in technical triplicate).

[0142] Figure 7 Inhibition of SARS-CoV-2, SARS-CoV, and MERS-CoV replication by VBK is demonstrated. Calu-3 cells were infected with the corresponding viral strains at an MOI of 0.01 and treated with a single dose of VBK at 10 µg / ml. Untreated cells served as a negative control (Ctrl). Remdesivir (Remd, 10 µM) (a pan-CoV inhibitor) served as a positive control. Infectious viral titers in cell culture supernatants were determined by plaque assay 48 hours post-infection. Viral titers are shown as mean log10 plaque-forming units / ml ± SD from a single biological replicate (performed in technical triplicate). Detection limits are indicated by dashed lines.

[0143] Figure 8Inhibition of SARS-CoV-2 replication by VBK is demonstrated. Calu-3 cells were infected with SARS-CoV-2 HH01 at MOI 0.01 and treated with eight different concentrations of VBK. Untreated cells served as a negative control (control). Infectious viral titers in cell culture supernatants were determined by plaque assay at 48 hours post-infection. A) Viral titers are shown as mean log10 plaque-forming units / ml ± SD from a biological replicate (performed in technical triplicate). B) For VBK_P, IC 50 and IC 90 Values ​​were determined using nonlinear regression with adjustable concentration ranges from 1 to 10 µg / ml. Data are presented as mean ± SD of viral replication inhibition (% of control). Detection limits are indicated by dashed lines.

[0144] Figure 9 shows the evaluation of the phagocytic ability of the virus-nanoparticle complex. A) with VSVΔG-S Atto643 Incubate with the virus or with VSVΔG-S Atto643 CLSM micrographs of bright-field images of RAW264.7 cells co-incubated with VBK_P or VBK_S. VSVΔG-S Atto643 Virisomes appear as red spots. The membrane, stained with CTB-Alexa Fluor 555 via sphingolipid GM1, appears green. VSVΔG-S is captured in intracellular vesicles (such as phagosomes) of macrophages. Atto643 Virisomes are marked with arrows and shown as superimposed yellow signals of the two stains used. Scale bar 10 µm B) Viral uptake is quantified by signal colocalization.

[0145] Figure 10 A comparison of SPR analysis of RBD-mFc (SARS-CoV-2 HH01 variant) with sensor-immobilized LCB1 and LCB1-azide binding is shown. Data are attached. Figure 1 The figure illustrates this. The affinity was found to be too high to be accurately determined by SPR spectroscopy because the dissociation rate exceeded instrumental limitations. Therefore, the difference in affinity for RBD-mFc was not significant.

[0146] Figure 11The diagram shows different concentrations of LCB1-azides competing with ACE2 (7.5 nM) for binding to the trimer spikes of the wt variant (blue) and the omicron BA.1 variant (black). For the wt-spike, assays were performed using two different batches of LCB1-azides, each evaluated in duplicate (light gray triangles, dark gray pentagons, and hexagons). Assays involving the omicron BA.1 spike were performed in duplicate (black). Red symbols represent outliers excluded from the fitting procedure.

[0147] Figure 12 The half-maximal inhibitory concentration (IC50) of different VBKs relative to VSVΔG virions equipped with spike proteins of different SARS-CoV-2 variants is shown. 50 ).

[0148] Figure 13 The antiviral activity of VBK 28A-C and 29A-B (including their respective blank controls) against SARS-CoV-2 HH01, Delta, and Omicron BA.1 variants is demonstrated. Human Calu-3 cells were infected with the corresponding viral strains at an MOI of 0.01 and treated with a single dose of VBK at 10 µg / ml. Untreated cells served as a negative control (Ctrl). Purified microbinding agents LCB1 and LCB1 叠氮化物 (100 nM) was used as a positive control. The infectious viral titer in the cell culture supernatant was determined by a plaque assay 48 hours post-infection. Viral titers are shown as the mean log10 plaque-forming units / ml ± SD from a biological replicate (performed in technical triplicate). Detection limits are indicated by dashed lines.

[0149] Figure 14 The IC50 values ​​of VBK 28A-C and 29A-B in SARS-CoV-2 and in assays are shown. 90 Determination of viral load. Calu-3 cells were infected with SARS-CoV-2 HH01 at an MOI of 0.01 and treated with eight different concentrations of VBK (range 0.001 to 10 µg / ml). Untreated cells served as a negative control. Infectious viral titers in cell culture supernatants were determined by plaque assay at 48 hours post-infection. Data are presented as mean ± SD of the inhibition (% of control) of viral replication from two independent biological replicates (each performed in technical triplicate). 90 The value is determined through nonlinear regression.

[0150] Figure 15 The microbinding agents LCB1 and LCB1 in SARS-CoV-2 and assays are shown.叠氮化物 IC 90 The determination was made by infecting Calu-3 cells with SARS-CoV-2 HH01 at an MOI of 0.01 and then with eight different concentrations (range 0.01 to 500 nM) of LCB1 and LCB1. 叠氮化物 Therapeutic treatment was performed. Untreated cells served as a negative control. Infectious viral titers in cell culture supernatants were determined by plaque assay 48 hours post-infection. Data are presented as mean ± SD of the inhibition (% of control) of viral replication from a single biological replicate (performed in technical triplicate). 90 The value is determined through nonlinear regression.

[0151] Figure 16 The inhibition of SARS-CoV-2 HH01 replication by adding VBK 28A-C and 29A-B at different time points post-infection is illustrated. Calu-3 cells were infected with SARS-CoV-2 HH01 at an MOI of 0.01 and treated with a single dose of 5 µg / ml VBK at 0 h (i.e., immediately after viral infection), 1 h, 6 h, and 24 h post-infection. Untreated cells served as a negative control (control). The infectious viral titer in the cell culture supernatant was determined by a plaque assay at 48 h post-infection. Viral titers are shown as mean log10 plaque-forming units / ml ± SD from a single biological replicate (performed in technical triplicate). Detection limits are indicated by dashed lines.

[0152] Figure 17 This study demonstrates the inhibition of SARS-CoV-2 HH01 replication on primary human lung cells by VBK 28A and 29A. Human bronchial epithelial primary cells (HBEpC) were differentiated in an air-liquid interface (ALI) system and infected from the apical region with SARS-CoV-2 HH01 at an MOI of 1. Cells were subsequently treated in basal medium with a single dose of VBK 28A and 29A at 5 µg / ml. Untreated cells served as a negative control. The infectious viral titer in the apical cell culture supernatant was determined by a plaque assay 48 hours post-infection. Viral titers are shown as mean log10 plaque-forming units / ml ± SD from a single biological replicate (n = 12 technical replicates per condition). Detection limits are indicated by dashed lines.

[0153] Figure 18 A schematic diagram of the synthesis of PLGA-modified nanoparticles is shown.

[0154] Figure 19 Modification of nanoparticle surfaces via thiol-maleimide click reaction and azide-alkyne click reaction is shown.

[0155] SEQ ID NO: 1 shows the N-terminal extended amino acid sequence of the recombinant core LCB1 and AHB2 microbinding agents. .

[0156] SEQ ID NO: 2 shows the C-terminal extension amino acid sequence of the recombinant core LCB1 and AHB2 microbinding agents. . Example

[0157] method

[0158] Production and purification of recombinant SARS-CoV-2 spike protein RBD microbinding agents

[0159] Codon-optimized synthetic genes encoding LCB1 and AHB2 were cloned into the vector pET28H6TEV (a modified pET28b vector encoding an N-terminal 6×His tag followed by a tobacco etch virus (TEV) protease site). Recombinant core LCB1 and AHB2 microbinding agents were generated in *E. coli* BL21(DE3) cells (New England Biolabs) at 25°C via autoinduction (overnight expression on immediate TB medium, Novagen), carrying the N-terminal (… (SEQ ID NO: 1) and C-end ( (SEQ ID NO: 2) extension. Bacterial cells were then harvested by centrifugation (10,500 × g, 20 min at 16 °C) and stored at -80 °C. The frozen bacterial cells were resuspended in lysis buffer (100 mM HEPES, 300 mM NaCl, 1 mM AEBSF, pH 7.5) and destroyed using a high-pressure homogenizer (Emulsiflex C5, Avestin) at 100 bar. After centrifugation (48,000 × g, 20 min at 4 °C), the His-labeled protein was purified by metal chelate affinity chromatography using a 20 ml TALON Superflow column (Clontech). The protein was eluted from a TALON column with 20 mM HEPES, 300 mM NaCl, and 200 mM imidazole (pH 7.5), incubated overnight at room temperature with TEV protease (internal preparation, 4 µg TEV protease / 1 mg microbinding agent), and further purified by size exclusion chromatography (SEC) in 50 mM HEPES and 150 mM NaCl (pH 7.5) on a Superdex 75 preparative grade 26 / 600 column (Cytiva). The SEC fraction containing pure LCB1 and AHB2 was rapidly frozen in liquid nitrogen and stored at -80°C.

[0160] Synthesis of LCB1-Azide

[0161] To generate LCB1-azides, 546 µl of TCEP stock solution (273 µmol, 0.5 M, adjusted to pH 7 with NaOH) was added to 10 mL of LCB1 solution (2.17 mg / mL, 273 µM, 2.73 µmol, in 50 mM HEPES buffer (pH 7.5) containing 150 mM NaCl). After a brief vortex and a reaction time of 30 min, size exclusion chromatography (SEC) was performed under anaerobic conditions using four Cytiva sephadex PD-10 size exclusion columns with buffer exchange in anaerobic sodium borate buffer (50 mM, pH 9). Four fractions of LCB1 solution in borate buffer were obtained and combined. 27 mg (79.6 µmol) of bromoacetamide-PEG3-N3 (Sigma Aldrich, Prod.no. QBD11217, Merck KGaA, Darmstadt, Germany; M = 339.19 g / mol; protein:linker = 1:29.2) was added to the LCB1 solution. The reaction mixture was vortexed at 500 rpm for 2.5 h at 25 °C. The solution was then divided into five 2.5 mL fractions, each added to an SEC column pretreated with deionized sterile water. The protein was eluted from the columns by adding 3 mL of MiliQ water to each column. The protein fractions from all three columns were combined. A solution of LCB1-azide (138.9 µM, 2.086 µmol, 17.1 mg) in 15 mL of MiliQ water was obtained. The protein was rapidly frozen and lyophilized.

[0162] Surface Plasmon Resonance Measurement

[0163] Real-time analysis was performed on a Biacore T200 system using a CM5 sensor chip (Cytiva) at 25 °C. Prior to ligand immobilization, the sample and reference flow cell surfaces were activated by injecting a 1:1 mixture of 0.4 M EDC and 0.1 M NHS at a flow rate of 10 µl / min. Human ACE2 (Sino Biological 10108-H08H) was diluted in 10 mM sodium acetate (pH 4.5) (5 µg / ml) and covalently coupled to approximately 500 response units (RU). Excess reactive succinimide ester was inactivated with 1 M ethanolamine-HCl (pH 8.5). Following amine coupling with 0.1 M ethylenediamine in 0.1 M sodium borate (pH 8.5), the reduced (100-fold molar excess of TCEP) and diluted microbinding agents LCB1 and AHB2 (5 µg / ml in 10 mM sodium acetate (pH 4.0)) were fixed to approximately 50 RU levels via standard maleimide coupling using 50 mM sulfo-GMBS (N-γ-maleimide butyryl-oxysulfosuccinimide ester, Thermo Scientific) in 0.1 M sodium borate (pH 8.5). The excess maleimide groups were blocked with 50 mM cysteine ​​and 1 M NaCl in 0.1 M sodium acetate (pH 4.0).

[0164] Recombinant spike protein RBDs from SARS-CoV-2 and VOCs were purchased from Sino Biological (WH-1 RBD, 40592-V08B; WH-1 RBD-mFc, 40592-V05H; BA.1 RBD, 40592-V08H121; BA.1 RBD-mFc, 40592-V05H3; BA.5 RBD, 40592-V08H131). These were reconstituted in water and transferred to HBS-EP+ assay buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) surfactant P20, pH 7.4) using a PD MiniTrap G-25 desalting column (Cytiva). Serial 2-fold dilutions of each RBD were injected at a flow rate of 30 µl / min in single-cycle mode. The flow cell loaded with microbinding agents was regenerated with a 30-second pulse of 6 M guanidine hydrochloride after each single cycle. The raw data, minus the reference, were processed and fitted to a 1:1 interaction model using Biacore evaluation software 3.2.0.5.

[0165] To immobilize the LCB1-azide onto the sensor chip, the following modifications are required. Prior to immobilization, the EDC / NHS-activated flow cell was treated with an amine-functionalized cyclooctyne derivative (N-[(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl]-1,8-diamino-3,6-dioxane (BCN-amine, Sigma). BCN-amine (0.3 mM in 10 mM maleic acid (pH 6.0)) was injected and covalently coupled to approximately 400 RU, followed by deactivation of the remaining reactive succinimide ester with 1 M ethanolamine-HCl (pH 8.5). The LCB1-azide (10 µg / ml in 10 mM sodium acetate (pH 4.0)) was then bound via click coupling to approximately 60 RU.

[0166] Preparation of particulate suspension group 1

[0167] The NP formulation was performed using the T 10 basic ULTRA-TURRAX® (IKA, Staufen, Germany) monoemulsion technology. The mixture of each polymer was dissolved in ethyl acetate at a concentration of 10 mg / ml. 2 ml of the organic solution was mixed with 1 ml of an aqueous phase containing 1% (w / v) poly(vinyl alcohol) (PVA) at approximately stage 4.5 for five minutes. To stabilize the particle suspension, 14 ml of 10 mM HEPES buffer at pH 6 was added. The suspension was allowed to stand at room temperature with continuous stirring at 800 rpm for at least four hours to evaporate the organic solvent.

[0168] The particles were purified by ultrafiltration using a 5804 R centrifuge (Eppendorf, Hamburg, Germany). They were then transferred to Amicon. ® Ultra 15 ml centrifuge filter (100,000 g mol) −1 MWCO), and centrifuged at 4000 × g for 20 min in a swing bucket rotor at 4 °C. Then add the remaining volume of sample and centrifuge the suspension again for 30 min until the lysate volume in the filter is equal to or less than 1500 µl. Resuspend the nanoparticles (NPs) and add 5 ml of 10 mM HEPES buffer pH 6 to the NP lysate suspension. This washing step is repeated four times, with centrifugation for 15 min and addition of 5 ml HEPES buffer after each centrifugation. For the final purification step, centrifuge the NPs for 19 min until the lysate volume is 500 µl. Collect the NP suspension. To minimize NP loss, Amicon... ®The filter membrane was washed with HEPES buffer to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 2 ml, and the polymer concentration was 10 mg / ml. The total volume of wash water was collected after each purification step.

[0169] To remove DTT from the microbinding agent LCB1, a buffer exchange of 50 mM NaH₂PO₄ and 150 mM NaCl (pH 7.5) was performed using a PD MidiTrap G-25 (Cytiva) and a PD-10 desalting column (Cytiva) according to the manufacturer's instructions. The absorbance of the microbinding agent LCB1 was measured at 280 nm using a NanoDrop plate, and the peptide concentration was calculated using the molar extinction coefficient (ε) and the Lambert-Beer law. A 100-fold molar excess of TCEP (pH 7.0) was added to the microbinding agent, and the mixture was incubated at room temperature for 20 minutes to reduce potential disulfide bonds.

[0170] For maleimide coupling, the calculated amount of micro-binding agent LCB1 (in a ratio of 2:1 (n) 马来酰亚胺部分聚合物 :n 微结合剂 Add to 1 ml of purified NP suspension and incubate at 200 rpm and room temperature for 20 hours. The amount of microbinding agent in control sample S is set to be the same as that in particle M.

[0171] Similar to the first purification step, the NP suspension was purified by ultrafiltration and transferred to Amicon. ® Ultra 15 ml centrifuge filter (100,000 g / mol MWCO) and centrifuge in a bucket rotor at 4000 × g for 7 min at 4 °C. After resuspending NP, add 5 ml of 10 mM HEPES buffer pH 6. This washing step is repeated twice, with centrifugation for 15 min and 5 ml of HEPES buffer added after each centrifugation. For the final purification step, centrifuge NP for 21 min until the osmotic volume is 500 µL. Collect the NP suspension. To minimize NP loss, use Amicon... ® The filter membrane was washed with HEPES buffer to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 5 ml, and the polymer concentration was 2 mg / ml. The total volume of wash water was collected after each purification step.

[0172] Preparation of particulate suspension group 2 - VBK 28 AC

[0173] The NPs were prepared using the T 10 basic ULTRA-TURRAX® (IKA, Staufen, Germany) monoemulsion technology. The mixture of each polymer was dissolved in ethyl acetate to a final concentration of 10 mg / ml. 1 ml of the aqueous phase containing 1% (w / v) poly(vinyl alcohol) (PVA) was mixed with 2 ml of organic solution at approximately 4.5% for 5 minutes. To stabilize the particulate suspension, 14 ml of 10 mM HEPES buffer at pH 6 was added. To evaporate the organic solvent, the suspension was allowed to stand at room temperature with continuous stirring at 800 rpm for at least four hours.

[0174] The particles were purified by ultrafiltration using a 5804 R centrifuge (Eppendorf, Hamburg, Germany). They were then transferred to Amicon. ® The sample was centrifuged using an Ultra 15 ml centrifuge filter (100,000 g / mol MWCO) at 2000 × g for 20 min in a bucket rotor at 4 °C. The remaining volume of sample was then added, and the mixture was centrifuged again for 33 min until the lysate volume in the filter was equal to or less than 1500 µl. The NP was resuspended, and 5 ml of 10 mM HEPES buffer (pH 6) was added to the NP suspension. This washing step was repeated a total of six times. The NP was centrifuged for 23, 15, and two 20 min intervals, always resuspended in 5 ml of HEPES buffer after each centrifugation, until it was finally used for the final purification step. The mixture was centrifuged for 30 min until the lysate volume was 500 µL. The NP suspension was collected. To minimize NP loss, Amicon was used... ® The filter membrane was washed with HEPES buffer to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 2 ml, and the polymer concentration was 10 mg / ml. The total volume of wash water was collected after each purification step.

[0175] For maleimide coupling, the calculated amounts of micro-binding agent LCB1 (in a ratio of 4:1 (n) 马来酰亚胺部分聚合物 :n 微结合剂 Add to 1 ml of purified NP suspension and incubate at 200 rpm and room temperature for 20 hours. The amount of microbinding agent in control samples 28B and 28C was set to be the same as that in particle 28A.

[0176] Similar to the first purification step, the NP suspension was purified by ultrafiltration and transferred to Amicon. ®Ultra 15 ml centrifuge filter (100,000 g / mol MWCO) and centrifuge at 2000 × g for 8 min at 4 °C in a bucket rotor. After resuspending NP, add 5 ml of Milli-Q water. Repeat this washing step a total of four times. Centrifuge NP for 16 and 12 min, always resuspending with 5 ml of Milli-Q water, until it is finally used for the final purification step. Centrifuge for 12 min until the osmotic volume is 500 µl. Collect the NP suspension. To minimize NP loss, use Amicon... ® The filter membrane was rinsed with Milli-Q water to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 5 ml, and the polymer concentration was 2 mg / ml. The total volume of wash water was collected after each purification step.

[0177] Preparation of particulate suspension group 2 - VBK 29A-B

[0178] The NP formulation was performed using the T 10 basic ULTRA-TURRAX® (IKA, Staufen, Germany) monoemulsion technology. The mixture of each polymer was dissolved in ethyl acetate to a final concentration of 10 mg / ml. 1 ml of the aqueous phase containing 1% (w / v) poly(vinyl alcohol) (PVA) was mixed with 2 ml of organic solvent at approximately 4.5% concentration for 5 minutes. To stabilize the particulate suspension, 14 ml of Milli-Q water was added. To evaporate the organic solvent, the suspension was allowed to stand at room temperature with continuous stirring at 800 rpm for at least four hours.

[0179] The particles were purified by ultrafiltration using a 5804 R centrifuge (Eppendorf, Hamburg, Germany). They were then transferred to Amicon. ® The sample was centrifuged using an Ultra 15 ml centrifuge filter (100,000 g / mol MWCO) at 2000 × g for 20 min in a bucket rotor at 4 °C. The remaining volume of sample was then added, and the mixture was centrifuged again for 13 min until the tretinoin volume in the filter was equal to or less than 1500 µL. The NP was resuspended, and 5 ml of Milli-Q water was added to the NP suspension. This washing step was repeated a total of six times. The NP was centrifuged three times for 8 min, followed by 7 min, and resuspended with 5 ml of Milli-Q water after each centrifugation step until it was finally used for the final purification step, centrifuging for 7 min until the tretinoin volume was 500 µL. The NP suspension was collected. To minimize NP loss, Amicon... ®The filter membrane was rinsed with Milli-Q water to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 2 ml, and the polymer concentration was 10 mg / ml. The total volume of wash water was collected after each purification step.

[0180] For alkyne coupling, the calculated amount of micro-binding agent LCB1-azide (ratio of 4:1 (n) 马来酰亚胺部分聚合物 :n 微结合剂 The microbinding agent was added to 1 ml of purified NP suspension and incubated at 200 rpm and room temperature for 20 hours. The amount of microbinding agent in control sample 29B was set to be the same as that in particle 29A.

[0181] Similar to the first purification step, the NP suspension was purified by ultrafiltration and transferred to Amicon. ® Ultra 15 ml centrifuge filter (100,000 g / mol MWCO) and centrifuge at 2000 × g for 8 min at 4 °C in a bucket rotor. After resuspending NP, add 5 ml of Milli-Q water. Repeat this washing step a total of four times. Centrifuge NP for 16 and 12 min, always resuspending with 5 ml of Milli-Q water, until it is finally used for the final purification step. Centrifuge for 12 min until the osmotic volume is 500 µL. Collect the NP suspension. To minimize NP loss, use Amicon... ® The filter membrane was rinsed with Milli-Q water to collect the NPs adhering to the membrane and transferred to an NP suspension. The volume of the NP suspension was adjusted to a final volume of 5 ml, and the polymer concentration was 2 mg / ml. The total volume of wash water was collected after each purification step.

[0182] Scanning Electron Microscopy (SEM)

[0183] Another method for studying NPs is scanning electron microscopy (SEM). For imaging the particles, a Sigma VP field emission scanning electron microscope (Carl-Zeiss, Jena, Germany) with an Inlens detector and an accelerating voltage of 5–6 kV was used. Therefore, the samples were pre-coated with a 4 nm platinum layer using a CCU-010 HV sputtering system (Safematic, Zizers, Switzerland). ImageJ was used to evaluate the NP size.

[0184] Diquinoline carboxylic acid assay (BCA assay)

[0185] The concentrations of LCB1 and LCB1-azide in the NP formulation were quantified using the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc.). The assay was performed in VWR according to the manufacturer's protocol and microplate procedure. ® Measurements were performed in 96-well F plates for tissue culture. Absorbance was measured at λ = 562 nm using an Infinite M200 Pro reader (Tecan Group, Männedorf, Switzerland), with multiple readouts of 3 × 3 per well and a well boundary of 2000 μm. Calibration curves were constructed for LCB1 from 963.71 to 0.47 μg / mL and LCB1-azide from 1250.02 to 0.31 μg / mL. Blanks for calibration curves, NP suspension, and wash water were provided, depending on the aqueous phase.

[0186] Dynamic light scattering (DLS)

[0187] Particle size and polydispersity were characterized by DLS measurements using a Zetasizer Nano ZS (Malvern Panalytical GmbH, Malvern, UK) with a laser wavelength of λ = 633 nm. Each measurement of size and polydispersity index (PDI) was performed in a UV cuvette made of polystyrene (Brand GmbH + Co KG, Wertheim, Germany), with five 30-second runs performed after a 30-second equilibration time, while the backscattering angle was set to 173°. The hydrodynamic diameter (d) of the particles was collected based on the size distribution according to the intensity. H DLS measurements were performed after NP preparation, after each purification step, and after storage at 4 to 8°C for different times.

[0188] PVA assay

[0189] To quantify PVA residues in the NP formulation, PVA was determined by UV-Vis spectroscopy. Due to the formation of a complex between PVA and iodine in Lugol solution, absorption can be measured at λ = 650 nm. A lyophilized aliquot was used and resuspended in 1 ml of pure water. 90 µL of the resuspended suspension was transferred to a VWR. ®Tissue culture was performed in 96-well F plates. Subsequently, 20 μL of 1 M sodium hydroxide was added to each well, and the mixture was incubated at room temperature for 15 minutes at 850 rpm using a BioShaker to degrade the particles via hydrolysis of the NP matrix. Neutralization was then performed by adding 20 µL of 1 M hydrochloric acid. Recombination was initiated after adding 60 µL of 0.65 M boric acid and 10 µL of Lugol solution. Next, sample absorbance was measured using a plate reader over 15 minutes. The setup of the relevant calibration curve was similar to that of the NP samples. Determinations were performed using different NP suspensions.

[0190] cell

[0191] Human lung adenocarcinoma epithelial cells (Calu-3; ATCC-HTB-55) were cultured in Eagles Minimum Essential Medium (EMEM) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). African green monkey kidney epithelial cells (VeroE6; ATCC-CCL-81) were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (v / v) FBS, 1% (w / v) L-glutamine (L-Glu), and 1% (w / v) P / S. Human bronchial epithelial primary cells (HBEpC) were purchased from Promocell and cultured in intact airway epithelial cell growth medium (Promocell). The mouse macrophage cell line RAW264.7 (ATCC TIB-71) was cultured in DMEM (Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS) (GE Healthcare Life Sciences), 1% (w / v) ultraglutamine (Gibco), and 27.5 µg / ml gentamicin sulfate (Gibco) in a humidified chamber at 37°C and 5% (v / v) CO2.

[0192] All cell lines were maintained in a temperature-controlled incubator (37°C, 5% (v / v) CO2, in a humidified atmosphere) and periodically verified to be mycoplasma negative.

[0193] Virus

[0194] Wild-type SARS-CoV-2 HH01 (SARS-CoV-2 / Germany / HPI06-n / 2020, HH01) was isolated from nasal swabs of SARS-CoV-2 infected patients being treated in the ICU of the University Medical Campus Hamburg-Eppendorf, Germany. Delta and Omicron BA.1 SARS-CoV-2 isolates (SARS-CoV-2 / B.1.617.2 / Germany / Hamburg / 01 / 2021 and SARS-CoV-2 / B.1.1.529 / Germany / Hamburg / 01 / 2021, respectively) were obtained from the University Medical Campus Hamburg-Eppendorf, Germany. SARS-CoV (Frankfurt 1, FFM) was a generous donation from Dr. BL Haagmans (Erasmus Medical Center, Rotterdam, The Netherlands), and MERS-CoV (EMC-2012) was a generous donation from Professor Dr. Christian Drosten (Charité Universitätsmedizin Berlin, Berlin, Germany). All coronavirus stocks used in this study were produced by propagation on VeroE6 cells, and viral titers were determined using plaque assays according to established protocols.

[0195] Therapeutic agents

[0196] VBK, used to test its ability to inhibit SARS-CoV-2 replication in Calu-3 and HBEpC cells, was stored at 4°C (particles M to T) or -20°C (particles 28A-C and 29A-B). Remdesivir was obtained from Hölzel Diagnostika Handels GmbH (Cologne, Germany). Remdesivir stock solutions were prepared in DMSO and stored at -80°C. Repeated freeze-thaw cycles were avoided whenever possible for all therapeutic agents.

[0197] Air-fluid interface (ALI) differentiation of human bronchial epithelial primary cells (HBEpC)

[0198] HBEpC cells were seeded onto a 24-well transwell insert (0.4 µM, polyester (PET) membrane) at a density of 0.25 × 10⁶ cells per well. 5Cells were exposed to culture medium from both the apical and basal regions and cultured at 37°C (5% (v / v) CO2, 96% rH) for 5 to 7 days until they reached 100% confluence (as estimated by microscopic analysis). At this point, the culture medium at the apical region was removed, and the cells were exposed to air from the apical region and to culture medium (intact air-liquid interface medium; Promocell) from the basal region to initiate differentiation. The culture medium in the basal chamber was changed every 2 to 3 days. After approximately two weeks of differentiation, the cells were also washed twice weekly with phosphate-buffered saline (PBS) to remove mucus. Simultaneously, transepithelial electrical resistance (TEER) measurements were performed weekly to assess the integrity of the cell monolayer. Four to five weeks after air exposure, differentiation was complete (as indicated by high TEER values ​​and significant mucus production), and the ALI cultures were then subjected to SARS-CoV-2 infection.

[0199] Cell viability assay

[0200] Calu-3 cells were cultured at a concentration of 3.5 × 10⁻⁶. 5 Cells / well were seeded in 96-well plates for 24 hours. The test compound or VBK solvent (as a positive control) was serially diluted in growth medium to obtain a 5-fold desired final concentration. The growth medium was removed from the cells and replaced with 80 µl / well of fresh growth medium. Subsequently, for each concentration, 20 µl of the diluted compound was added in quadruplicate (i.e., a 5-fold dilution to achieve the final concentration). Cells were incubated at 37°C (5% (v / v) CO2, 96% rH) for 48 hours. 48 hours after treatment, cell viability was measured using a CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega) on a Tecan Safire 2 plate reader according to the manufacturer's instructions.

[0201] Cell cytotoxicity assay

[0202] Calu-3 cells were seeded in 96-well plates and treated with the VBK to be tested (or its solvent as a control) as described above (“Cell Viability Assay”). Forty-eight hours after treatment, cytotoxicity of the cells was measured using a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) on a Tecan Safire 2 plate reader according to the manufacturer’s instructions. This assay is based on intracellular lactate dehydrogenase (LDH) released into the cell culture supernatant when the cell membrane is damaged.

[0203] Phagocytosis assay

[0204] RAW264.7 cells were cultured at 2 × 10⁻⁶ cells one day in advance. 5 RAW264.7 cells were seeded at a density of 10 cells / well in 24-well plates with coverslips. The following day, prior to infection, RAW264.7 cells were pre-stained with CTB-Alexa 555 (Invitrogen) to label sphingolipid GM1, which is part of both the cytoplasmic and intracellular membranes. Macrophages were then seeded with 10 µg / ml VBK and VSVΔG-S at an MOI of 0.1. Atto643 Infection, or only using VSVΔG-S Atto643 Infection. After infection synchronization, co-incubation continued for two or four hours, and the samples were fixed with 3.7% (v / v) formaldehyde / PBS for ten minutes. The samples were observed under a microscope using an LSM 780 (Zeiss) and the images were analyzed using Zen software.

[0205] Flow cytometry

[0206] To examine the ability to bind SARS-CoV-2 RBD, formulated VBK was tested by flow cytometry using labeling with recombinant SARS-CoV-2RBD-Alexa Fluor 647 (R&D Systems). Samples with unstained nanoparticles and nanoparticles without microbinding agents were analyzed. For sample preparation, 100 µg / ml VBK was incubated with 1 µg SARS-CoV-2 RBD-Alexa Fluor 647 / PBS for 30 min. After washing by centrifugation (250 × g, 5 min at room temperature), the particles were resuspended in PBS (Gibco, Thermo Fisher Scientific Inc.) and measured using BD LSRFortessa. The obtained data were evaluated using FlowJo.

[0207] Viral replication dynamics

[0208] All experiments involving live SARS-CoV-2 isolates were conducted at the Leibniz Institute of Virology (Hamburg, Germany) under BSL-3 conditions according to standard operating procedures.

[0209] Calu-3 cells were cultured at a concentration of 3.5 × 10⁻⁶. 5 Cells / well were seeded in 24-well plates for 24 hours. The VBK to be tested was diluted to the final concentration in infection medium (DMEM supplemented with 2% (v / v) FBS, 1% (w / v) L-Glu, and 1% (w / v) P / S). The growth medium was removed from the cells, the cells were washed once with PBS, and then seeded with SARS-CoV-2 with an MOI (multiple of infection) of 0.01 prepared in infection medium. 45 minutes after viral particles attached to the cells, the inoculum was removed, the cells were washed twice with 1× PBS, and infection medium containing the test compound was added (1 ml / well). For delayed treatment experiments, the desired concentration of VBK was added directly to the cells (“0 h” time point), or directly to the medium at 1 h, 6 h, or 24 h post-infection. Since SARS-CoV-2 replication peaks approximately 48 h post-infection (pi), this time point was chosen for all subsequent analyses. At pi 48 hours, the supernatant was collected from the infected cells and stored at -80°C.

[0210] HBEpC cells (24-well configuration) differentiated from ALI were infected with SARS-CoV-2 at an MOI of 1 from the top portion according to the procedure described above. ALI medium containing the test compound was added to the basal chamber. At 48 hours post-incubation, 100 µl of ALI medium was added to the top portion, and the cells were incubated at 37°C for 15 minutes to harvest viral particles present in the mucus. The supernatant was stored at -80°C. The viral titer in all cell culture supernatants was determined by plaque assay on VeroE6 cells as described below.

[0211] Plaque test

[0212] The viral titer in the supernatant collected from SARS-CoV-2-infected cells was determined by a plaque assay on VeroE6 cells. In short, VeroE6 cells were seeded in 12-well plates (2.5 × 10⁻⁶ cells per well). 5Cells / well, for 24 hours. Cell culture supernatant was serially diluted 10-fold in PBS. Growth medium was removed from the cells, cells were washed once with PBS, and diluted supernatant (150 µl / well) was added. 30 minutes after seeding, covering medium (1.5 ml / well) of double-concentrated minimum essential medium (MEM; supplemented with 2% (w / v) L-glu, 2% (w / v) P / S, and 0.4% (w / v) bovine serum albumin (BSA)) was added to the cells. Cells were then incubated at 37°C for 72 hours. After 72 hours, covering medium was removed from the cells, and cells were fixed with 4% (v / v) paraformaldehyde (PFA) at 4°C for at least 30 minutes after washing with PBS. Subsequently, the PFA solution was removed, and the cells were counterstained with crystal violet solution to reveal virus-induced plaques in the cell layer. The number of plaques at a given dilution was used to calculate the viral titer in log10 plaque formation units / ml (PFU / ml).

[0213] Colorimetric competitive assay of spike inhibitors

[0214] The assay was performed according to the protocol of PBS bioscience (San Diego, CA, USA) product #78365, “Spike Trimer (S1+S2) (B.1.1.529 BA.1, Omicron Variant) (SARS-CoV-2): ACE2 Inhibitor Screening Colorimetric Method”. To obtain results for the wt-spike protein, the wt-spike mutant (product #100728; BPS bioscience, San Diego, CA, USA) was used instead of the omicron BA.1 variant (product #101343; BPS bioscience, San Diego, CA, USA) originally included in the assay.

[0215] Using spike trimer solution (4 µg / ml in PBS, 50 µl / well; wt variant or omicron BA.1 variant), coat clear polypropylene (PP) flat-bottom 96-well plates with spike trimer by incubation at 5°C for 20 hours. After removing the spike solution and washing three times with 100 µl / well of immunobuffer (supplied by the manufacturer), block each well by incubation with blocking buffer (supplied by the manufacturer) for 1 hour. Remove the blocking buffer and tap the plate upside down on a clean paper towel to remove excess liquid. Create a four-fold dilution series (4 µM to 1 pM) of LCB1-azide in blocking buffer and administer 50 µl of the diluted microbinding agent to each well labeled “Test Inhibitor”. Add 50 µl of blocking buffer to the wells labeled “Blank” and “Positive Control”. Incubate the plate at 22°C with gentle agitation for 1 hour. Add 50 µl of biotin-labeled ACE2 (ACE2-Biotin; 1.5 µg / ml in blocking buffer) to each well labeled “Test Inhibitor” or “Positive Control”. The final concentration of LCB1-azide is 2 µM to 0.5 pM, and the final concentration of ACE2-Biotin is 0.75 µg / ml, which corresponds to approximately 7.5 nM. Add 50 µl of blocking buffer to each well labeled “Blank”. After incubating at 22°C with gentle agitation for 1 hour, discard the solution and wash the wells three times with 100 µl of immunobuffered buffer. Remove excess buffer by gently tapping the plate upside down on a clean paper towel. Dilute the streptavidin-HRP solution provided by the manufacturer 1000-fold with blocking buffer. Add 50 µl of this diluted streptavidin-HRP solution to each well. After a 45-minute incubation period, wash the plate three times with 100 µl of immunobuffered buffer. Add substrate (50 µl) to all wells. Substrate conversion was rapid and completed in about two minutes, as determined by measurements at 450 nm every other PC and blank well using a Tecan spark plate reader (Tecan Group AG, Männedorf, Switzerland).

[0216] In experiments involving the omicron BA.1, the reaction was terminated after two minutes by adding 100 µl of 1 M HCl to each well, resulting in a transient color change from pale blue to deep yellow. The absorbance of each well was measured at 450 nm using a Tecan Spark plate reader.

[0217] Unexpectedly, in the wt-spike-related experiments, the reaction did not terminate until 12 minutes later. Adding 1 M HCl (100 µl) to each well caused a transient color change from light blue to deep yellow. The absorbance at 450 nm in each well was too strong to be accurately determined using a Tecan Spark plate reader. Therefore, aliquots were removed from each well and pipetted to the corresponding wells of a new flat-bottomed 96-well plate. After dilution with water 16.7-fold, the absorbance of each well in the diluted plate was measured at 450 nm using a Tecan Spark plate reader.

[0218] The data is processed by blank subtraction and normalization, so that 1 represents no suppression and 0 represents complete suppression. A generalized logic function is fitted to the data points to determine the IC. 50 value.

[0219] Statistical analysis

[0220] All data were analyzed using Microsoft Excel and GraphPad Prism software. Nonlinear regression was performed to determine the inhibitory concentrations (IC50 and 90) of 8 doses of treatment on Calu-3 cells (IC50 and 90, respectively). 50 and IC 90 ).

[0221] Production and biophysical characterization of microbinding agents targeting SARS-CoV-2

[0222] The use of a structural entity that binds to the SARS-CoV-2 spike protein with nanomolar to picomolar affinity is crucial to the success of the inventors' strategy, as the nanoparticles need to outperform the hACE2 receptor to bind the virion. The microbinding agent on the nanoparticle surface needs to be able to interact with the wild-type (wt) spike RBD with nanomolar affinity. Initially, the inventors considered a short peptide similar to the hACE2 amino acid sequence, but thorough research showed that the affinity of the peptide for the spike RBD was generally too low for the intended use.

[0223] Therefore, the inventors turned to so-called “ultra-stable microbinding agents” [8, 28], which consist of small proteins designed to inhibit the interaction between spike RBD and hACE2 by blocking RBD binding sites with picomolar affinity and high specificity. Two of these microbinding agents, LCB1 and AHB2 [8], are produced in E. coli and have an N-terminal His6-tag. After purification by Co-chelation chromatography, the His6-tag was removed by tobacco etch virus (TEV) protease and subsequently size exclusion chromatography was performed to obtain microbinding agents LCB1 and AHB2 in yields of 109 mg and 40 mg per liter of culture, respectively. Figure 1Both microbinding agents have short Ser and Gly sequences attached to their ends and Cys residues at their C-terminus for covalent bonding with nanoparticles.

[0224] Surface plasmon resonance (SPR) spectroscopy was used to evaluate the affinity of the microbinding agent for the spike RBDs of wild-type SARS-CoV-2 and omicron (PANGO lineage B.1.1.529) variants BA.1 and BA.5. Since the microbinding agent needs to be immobilized on the nanoparticle surface, the inventors decided to use the microbinding agent as a ligand, i.e., immobilized on the surface of the SPR chip. To connect the microbinding agent, the chip was modified with a cysteine ​​reactive group. In addition to using monomeric RBDs as analytes, the inventors also used dimer RBDs fused together via crystallizable fragment (Fc) regions of mouse (m) immunoglobulin G1 (IgG1) antibodies. Monomeric RBDs have the advantage of interacting with the microbinding agent in a monovalent manner, which is easy to interpret, but they exhibit rather poor solubility and a tendency to aggregate. In contrast, the mFc-labeled dimer RBD (RBD-mFc) is highly soluble, but in principle allows for divalent interactions when the chip surface is densely coated with the microbinding agent. Since divalent interactions are more difficult to fit and interpret, SPR chips are loaded with relatively low-density micro-binding agents to minimize divalent interactions. Typically, dimer RBDs are found to have similar or slightly higher affinity for immobilized micro-binding agents than their monomer counterparts. When comparing the affinity of micro-binding agents LCB1 and AHB2 for monomeric wt-RBDs, LCB1 exhibits a higher affinity for wt-RBDs (K0). D ≈ 5 pM) is AHB2 (K D The affinity of LCB1 to microbinding particles is approximately 10 times higher than that of BA.1 and BA.5 RBD (≈ 50 pM). LCB1 and AHB2 exhibit comparable affinity for micron BA.1 and BA.5 RBD, falling within the low nanomolar range. Due to its extremely high affinity, LCB1 microbinding agent was chosen for the experimental functionalization of nanoparticles.

[0225] Synthesis of functional PLGA

[0226] The nanoparticles formulated in this invention are made of poly(lactic acid-co-glycolic acid) (PLGA), which is frequently used due to its well-documented biocompatibility and biodegradability. The monomers lactic acid and glycolic acid can be metabolized by the body, and the degradation rate can be affected by changing the monomer ratio. Therefore, degradation can be slowed down as the PLA content increases. In addition, PLGA is FDA approved and has been used in authorized drugs [9]. In this invention, molar mass (M) is used. wPLGA, with a concentration of 7000 to 17000 g / mol and a PLA to PGA ratio of 50:50, is also used in nanoparticles due to its opsonizing effects and potential recognition by the immune system. In addition to PLGA, block copolymers containing PLGA and polyethylene glycol (PEG) as a stealth polymer are also used (see [link to article]). Figure 18 PEG with a molar mass of 1 to 5 kDa is often used to exert stealth effects, particularly on the surface of nanoparticles, which led to the selection of 2.1 kDa PEG in this invention

[10] . In formulation, the hydrophilic PEG should be located on the outer side of the particle, and therefore, the ω-terminus should be present on the surface of the particle to facilitate modification with functional peptides. Modification of the ω-terminus of PLGA / PEG-PLGA is accomplished by ester activation using N-hydroxysuccinimide (NHS), followed by amidation using maleimide-amine or PEG-maleimide-amine (see [link to documentation]). Figure 18 The reactive dibenzylcyclooctyne (DBCO) functionality is achieved through the following linkages: i) amidation of PLGA with NH2-PEG-COOH via NHS ester activation, followed by conversion with DBCO amine via NHS ester activation again. The peptide can then be linked via click reactions (azide-alkyne or thiol-maleimide click) (see [link to product]). Figure 19 ).

[0227] Formulation and characterization of reactive nanoparticles for microbinding fixation

[0228] Using a monoemulsion technique with polyvinyl alcohol (PVA) as the surfactant, a first group of eight different particles of similar size (Table 1) were formulated from (PEG-)maleimide-modified and unmodified PLGA in mixtures or as pure products. The nanoparticles were co-incubated with LCB1 in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to achieve covalent bonding of LCB1 to the surface of the nanoparticles. Particle S was included as a control to examine whether non-covalent bonding of LCB1 to the particle could occur.

[0229] Table 1: Virus binding conjugates (VBKs) loaded with LCB1.

[0230]

[0231] a) Average diameter of LCB1-modified particles as determined by DLS. b) Polydispersity index as determined by DLS. c) DY550 is a fluorescent dye to allow visualization by FACS or confocal microscopy.

[0232] To quantify the micro-binding agents that bind to polymer particles, Pierce was used. ®Experiments were conducted using the Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific Inc.) and the Micro BCA® Protein Assay Kit (Thermo Fisher Scientific Inc.). However, none of these assays showed conclusive results. Residual PVA from the formulations interfered with the Bradford assay. Therefore, the amount of PVA in each formulation was subsequently determined by a PVA assay. Due to the sensitivity of the Bradford assay to detergents and the amount of residual PVA in the nanoparticle suspension, a new detergent-compatible Bradford assay kit (Pierce) was developed. ® The detergent-compatible Bradford assay kit (Thermo Fisher Scientific Inc.) was used for all upcoming Bradford assays. The results of this assay remain inconclusive. Similarly, residual TCEP used during preparation to reduce disulfide bonds affected the results of the BCA assay. Furthermore, the use of the reducing agent-compatible Pierce kit… ® The BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Fisher Scientific Inc.) failed to provide conclusive results in overcoming the aforementioned problem. Therefore, for subsequent preparations, the use of 50 mM HEPES, 150 mM NaCl, and MilliQ water as the solvent for LCB1 protein from the outset omits the need for TCE as a reducing agent and the rebuffering of LCB1 during preparation.

[0233] Additionally, attempts were made to visualize and quantify surface-bound proteins using SDS-PAGE. Due to their size, particle-bound proteins could not migrate through the gel in an electric field. On the other hand, surface-adsorbed LCB1 could be separated from the particles and identified by comparison with the LCB1 used in formulation. Although attempts were made to quantify this amount by measuring fluorescence intensity using image processing, the exact amount could not be determined due to the low protein concentration. However, changes in the mass of LCB1 and subsequently LCB1-azide were observed after maleimide-thiol and azide-alkyne reacted with small water-soluble polymers, respectively, indicating that the reactions themselves were identified.

[0234] Flow cytometry analysis of viral conjugates (VBK) in the presence of fluorescently labeled SARS-CoV-2 RBD.

[0235] To quantify the amount of LCB1 bound, the particles were mixed with Alexa Fluor 488 dye (RBD). AF488The recombinant wt-SARS-CoV-2 RBD labeled with fluorescent markers was incubated together and subsequently analyzed by flow cytometry. The obtained spectra show a comparison of the detection signals of unstained VBK with its stained analogues. Although the particle MR was not compared with RBD, the signal was significantly different. AF488 No fluorescence was observed during pre-incubation, but as expected, fluorescence was observed with RBD. AF488 After incubation, the fluorescence intensity distribution shifted towards higher intensities. The strongest shift was observed for VBK_P (PLGA-PEG-Mal / PLGA, 5:5). All were treated with RBD. AF488 Approximately 88% of the treated particles P exhibited higher fluorescence intensity than the untreated particles. In contrast, when compared with RBD... AF488 During pre-incubation, compared with untreated particles, particles S (composed of PLGA alone) that cannot covalently bind LCB1 showed only a slightly broadened fluorescence intensity distribution.

[0236] Virus neutralization using microbinding-functionalized particles

[0237] To assess whether VBK could inhibit infection in Vero cells, replication-deficient spike pseudotyped vesicular stomatitis virus (strain Indiana; VSVΔG-S) virions displaying a delta variant (B.1.617.2) spike protein on their surface were generated. These virions are able to infect cells displaying the ACE2 receptor on their surface, similar to SARS-CoV-2, due to the spike protein on their surface

[11] . The genome of the VSVΔG-S virions used here is missing the VSV surface protein G and instead contains a gene encoding green fluorescent protein (GFP), which allows for fluorescence-based viral infection readout. Neutralization of the VSVΔG-S virions used by different viral conjugates (VBKs) resulted in reduced uptake by Vero cells and thus a decrease in the number of GFP-positive cells after co-incubation. The half-maximal inhibitory concentration (IC50) of each VBK was determined by incubating VSVΔG-S with serially diluted VBKs for 1 hour and then seeding Vero cells. 50 () Figure 4 After 16 hours of co-incubation, GFP-positive cells were counted. Although all particles were found to reduce infection via VSVΔG-S virions, particles P and S were the most effective. This inhibitory activity of VBK is specific to spike-carrying virions, as infection with VSVΔG particles pseudotyped with the VSV endogenous surface protein G (VSVΔG-G; as a negative control) was unaffected by VBK.

[0238] In vitro neutralizing effect of VBK on live SARS-CoV-2

[0239] Following demonstrations that VBK could reduce infection caused by VSVΔG-S virion, its antiviral activity against “live” SARS-CoV-2 was evaluated in assays dependent on the human lung cancer-derived Calu-3 epithelial cell line. The toxicity of the particles and their effect on Calu-3 cell viability were assessed prior to testing the neutralizing capacity of VBK MT. Particle toxicity to Calu-3 cells was measured by the release of lactate dehydrogenase (LDH). Compared to untreated Calu-3 cells (negative control), all tested particles showed no significant effect on cell membrane integrity after 48 hours of incubation. Figure 5 This indicates that these VBKs are not toxic.

[0240] The effect of granules on cell viability was observed by reducing the pale yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to purple methyl... The MTT assay was measured and found to be directly proportional to the metabolic activity of cells in the culture. The MTT assay showed that, compared to the untreated control, there was no decrease in metabolic activity after 48 hours of VBK exposure.

[0241] In contrast to VSV-based infection assays that rely on pre-incubation of virions with nanoparticles, infection assays involving a clinical isolate of SARS-CoV-2 (SARS-CoV-2 HH01 strain) were performed using a therapeutic method. Calu-3 cells were infected with SARS-CoV-2 for 45 minutes, followed by a single dose of VBK (10 µg / ml). The entire experimental setup included untreated Calu-3 cells infected with SARS-CoV-2 as a negative control and cells pretreated with the antiviral inhibitor remdesivir (Remd) as a positive control. Infectious viral particles in the supernatant 48 hours post-infection were quantified by plaque assay. At the concentration of 10 µg / ml used, particles M, Q, and T did not neutralize SARS-CoV-2, while particles N, O, P, R, and S showed a strong reduction in viral titer by several orders of magnitude after two days. Figure 7 (Left figure). To examine specificity, in the same setting as the aforementioned infection assay, targeting SARS-CoV ( Figure 7 (Image in Chinese) and related Middle East respiratory syndrome coronavirus (MERS-CoV); Figure 7 (See right figure) All VBKs were tested. However, VBKs showed no activity against SARS-CoV and MERS-CoV, further demonstrating the specificity of microbinding-modified particles for SARS-CoV-2.

[0242] To further evaluate the antiviral activity of VBK N, O, P, R, and S, the SARS-CoV-2 infection assay was repeated, but using different concentrations of particles (1 pg / ml to 10 µg / ml) to determine their respective inhibitory concentrations of 50 and 90 (IC50, IC50, and IC50, respectively). 50 and IC 90 Surprisingly, in this assay, particles N, O, and R showed no activity, even at the highest dose of 10 µg / ml—at which they had previously shown activity. Figure 7 This may be due to the decreased stability of VBK N, O, and R at 4 °C. In contrast, compounds P and S reduced viral titers at 10 µg / ml, but not at 1 µg / ml or lower. Repeat determinations were performed with compound P at varying concentration ranges (1 µg / ml to 10 µg / ml), and the IC50 values ​​were obtained. 50 and IC 90 The values ​​were 4.86 µg / ml and 4.99 µg / ml, respectively, indicating a narrow range of therapeutic activity, which is also reflected in the steep Hill slope of the nonlinear regression curve.

[0243] Phagocytosis of virus-nanoparticle complexes

[0244] Besides effectively reducing viral titers by simply blocking the viral spike protein RBD, the phagocytic activity of the SARS-CoV-2 nanoparticle complex is one of the key inventions of this inventor's project. Therefore, mouse RAW264.7 macrophages were combined with fluorescently labeled VSVΔG-S (VSVΔG-S... Atto643 The virus can be incubated alone or with VSVΔG-S. Atto643 Virisomes and particles P (covalently bound microbinding agents) or S (adsorbed microbinding agents) were co-incubated to examine whether particles induced viral aggregation and phagocytosis. Prior to infection, RAW264.7 cells were pre-stained with cholera toxin subunit B Alexa Fluor 555 (CTB-AF555) to reveal sphingolipid GM1. Sphingolipids (such as GM1) are part of the cell membrane and intracellular membrane and are enriched in lipid raft microstructures that play an important role in phagosome maturation

[12] . Phagocytic events were monitored using confocal laser scanning microscopy (CLSM) after two or four hours. VSVΔG-S alone was used to monitor phagocytic events. Atto643 Virosome infection of RAW264.7 macrophages showed major association of virions attached to the cell membrane. Virosome uptake was observed only occasionally in these macrophage-like cells (Fig. 9). Using VSVΔG-S...Atto643 Infection of RAW264.7 macrophages with VBK_P resulted in increased virion internalization, especially after four hours of co-incubation, and some virions remained bound to the cell membrane. In contrast, infection with VSVΔG-S... Atto643 Less uptake due to particulate infection with VBK_S, and with VSVΔG-S Atto643 Intake was similar in control groups. Quantification was based on VSVΔG-S. Atto643 The signal colocalizes with the intracellular signal stained by CTB-AF555.

[0245] In the context of this invention, two microbinding agents, LCB1 and AHB2, were successfully produced in E. coli, purified, and their affinity for the RBDs of various SARS-CoV-2 variants was analyzed. Although LCB1 has been reported to have a higher affinity for the wt-spike RBD than AHB2, the inventors also included AHB2 in their study because the two microbinding agents are structurally different, which could affect their binding affinity for other SARS-CoV-2 variants of interest [8]. While LCB1 was designed de novo, AHB2 is based on helix 1 of hACE2, which forms the major part of the hACE2 RBD binding interface [8]. Due to the de novo design process, LCB1 has a different binding site than hACE2, although both bind to the same surface of the viral spike RBD. The difference between LCB1 and hACE2 could lead to the evolution of viral escape mutants that bind hACE2 but not LCB1. Since AHB2 and hACE2 have similar binding sites, the evolution of escape mutants that bind hACE2 but not AHB2 is unlikely. As expected, LCB1 exhibits a two-order-of-magnesium affinity for omicron RBD compared to wt-RBD. In contrast, the stability of the omicron RBD∙AHB2 complex is only about one order of magnitude lower than that of the wt-RBD∙AHB2 complex. Nevertheless, their affinity for omicron RBD is comparable. In summary, LCB1 is superior to AHB2 because it can be produced in higher yields and is smaller in size, while showing similar affinity for highly mutated omicron RBD.

[0246] Several factors were considered regarding the size of the nanoparticles used. First, the efficiency of spherical nanoparticles and microparticles being internalized by cells is size-dependent

[13] ,

[14] . However, depending on the particle size, cellular uptake of particles can lead to a pro-inflammatory immune response

[15] . Another aspect of the particle size to be considered is the requirement for the particles to penetrate the mucus layer of airway surface fluid in the lungs, as the inventors envision the particles being administered by inhalation. Particles with a diameter of 200 nm or less can diffuse through mucus, while particles with an increased diameter of 500 nm or more obviously cannot

[16] . However, it should be noted that particles formulated with PEG-PLGA, as in this invention, show increased mucus diffusion. It can be shown that nanoparticles with a diameter of 200 to 500 nm can diffuse rapidly through the human mucus layer

[17] .

[0247] For the method described here, quantitative detection of particles via CLSM is crucial, and particles with a diameter less than 500 nm are difficult to detect using this technique. Therefore, a group of eight different particles with an average diameter of approximately 500 nm was formulated. This size represents a trade-off between mucus permeability, effective phagocytosis, and detectability via CLSM. This particle group was formulated with different mixtures of thiol reactivity and inert PLGA to adjust the density of the maleimide moiety on the particle surface.

[0248] Determining the amount of microbinding agent attached to particles has proven quite difficult. BCA or Bradford assays, as well as analyses by SDS-PAGE, have yielded unreliable results. However, flow cytometry analysis by VBK clearly showed that only particles S, which cannot covalently bind microbinding agents, insufficiently bind to the fluorescent SARS-CoV-2 spike RBD. In contrast, other particles M to R bind to the spike RBD, as indicated by the signal shift associated with increased fluorescence intensity.

[0249] Surprisingly, the number of thiol reactive groups incorporated into VBK and the amount of ipso facto covalently bound microbinding agents were not correlated with the results of the virus inhibition assay using pseudotyped VSVΔG-S. Except for particle P (which contains a PLGA core, PEG linker, and covalently bound microbinding agents), particle S (which does not carry covalently bound microbinding agents) showed the highest activity in the VSV-based assay. In contrast, particle M, containing the highest amount of maleimide groups and therefore exhibiting the most microbinding agents on its surface, had very low VSVΔG-S inhibitory efficacy. The antiviral activity of particle S can be explained by the presence of a large amount of non-covalently bound microbinding agents adsorbed on the particle surface. In the virus inhibition assay, it was assumed that the adsorbed microbinding agents were released into the culture medium that blocked (covered) the viral spike protein.

[0250] The results of the SARS-CoV-2 inhibition assay were comparable to those of the VSV-based inhibition assay, making the latter a good approximation of SARS-CoV-2 inhibition. This is consistent with previous research that found that the pseudotyped VSV-based assay provided results comparable to those of virus inhibition assays involving clinical SARS-CoV-2 isolates

[18] . In this SARS-CoV-2 neutralization assay, not only particles M and T, but also particles Q failed to inhibit SARS-CoV-2 infection in Calu-3 cells at a particle concentration of 10 µg / ml. One aspect in this context is that the flexibility of the PEG connector is required for the attached microbinding agent, which is necessary to allow the microbinding agent to be properly oriented to bind to the viral spike RBD. However, particles S, which do not have PEG connectors on their surface, were able to inhibit SARS-CoV-2 infection in Calu-3 cells. In repeat experiments, particles N, O, and R also showed no activity against SARS-CoV-2 at 10 µg / ml. For those particles, a concentration of 10 µg / ml can be below the activity threshold, and therefore small changes in VBK concentration or initial viral concentration have a strong effect on whether activity is observed. The half-maximum inhibitory concentration (IC50) of VBK_P was determined to be approximately IC50. 50 = 4.9 µg / ml, and the IC50 of compound S is 4.9 µg / ml. 50 Observations were made within the same order of magnitude, from 1 µg / ml to 10 µg / ml. When applied to the same assay, the IC50 of free microbound LCB1 was observed. 50 The range is from 10 nM to 100 nM.

[0251] As expected from the high specificity of the microbinding agent against the SARS-CoV-2 spike RBD[8], no inhibition of SARS-CoV and MERS-CoV was observed via VBK M-T. Furthermore, free microbinding agent LCB1 and particle M-T were found to be non-toxic to Calu-3 epithelial cells. It should be noted that virus neutralization assays (VSV-based and SARS-CoV-2-based) only inform of the ability of the particles used to block the spike RBD, but do not provide information on the formation of viral aggregates that promote phagocytosis by macrophages. Probable modes of action of VBK include (1) releasing previously adsorbed microbinding agents into the culture medium to block the spike RBD and / or (2) the interaction of VBK with the virion, which blocks its spike RBD and impairs its ability to enter host cells.

[0252] To assess whether VBK can induce phagocytosis of virions, it was applied to mouse RAW264.7 macrophages and VSVΔG-S cells. Atto643In the phagocytosis model of virions. Since these virions cannot replicate, they are a harmless alternative to highly infectious SARS-CoV-2 and can be handled without the need for biosafety level 3 facilities

[18] . Bullet-shaped VSVΔG-S with dimensions of approximately 220 nm (length) by 83 nm (diameter)

[19] Atto643 The virions are only slightly larger than SARS-CoV-2 virions. Besides their similar size, they also display SARS-CoV-2-derived spike proteins on their surface, making them ideally suited for representing SARS-CoV-2 virions in phagocytosis assays to elucidate the cellular action patterns of VBK. Notably, when mouse RAW264.7 macrophages were infected with VSVΔG-S… Atto643 In the viral phase, the presence of particle P significantly increased the number of observed phagocytic events compared to the control experiment without VBK, thus fully supporting the inventors' initial hypothesis. If macrophages displaying the human ACE2 receptor on their surface are used to efficiently bind SARS-CoV-2 aggregates, the number of phagocytic events is likely to be further increased. The VSVΔG-S used herein... Atto643 Low virion titers (multiple of infection = 0.1) are also a key factor in in vitro studies, limiting the evaluation of different infection conditions, such as the infectious dose or the ratio of virions to VBK. Despite these limitations in the assays used herein, it is not only possible to demonstrate virion neutralization in the presence of VBK, but the inventors have also provided evidence of increased virion uptake by immune cells.

[0253] References

[0254]

Claims

1. Functionalized nanoparticles or microparticles for the prevention and / or treatment of target viral infections in mammalian subjects, comprising, a) at least one nanoparticle or microparticle, and b) At least one virus-binding peptide and / or virus-binding small molecule immobilized on the surface of the nanoparticles or microparticles, wherein the virus-binding peptide and / or virus-binding small molecule binds to at least one target virus with nanomolar to picomolar affinity, and The overall diameter of the functionalized nanoparticles or microparticles is at least about 400 nm, preferably about 400 to 2000 nm, and more preferably about 600 to 700 nm. Preferably, the binding between the virus-binding peptide and / or virus-binding small molecule and the at least one target virus is superior to the binding between the target virus and its host cell receptor.

2. The functionalized nanoparticles or microparticles of claim 1, wherein the shape of the functionalized nanoparticles or microparticles is substantially spherical.

3. Functionalized nanoparticles or microparticles for the application according to claim 1 or 2, wherein the nanoparticles or microparticles comprise suitable organic or inorganic materials, such as metals, plastics, polymers such as PLA, PLGA, PLGH, polyglutamic acid, styrene-maleic acid copolymer or PE, or nanospheres composed of membranes and / or lipids, such as liposomes, preferably wherein the nanoparticles or microparticles comprise PLGA-Mal, PLGA-PEG-Mal, PLGA-DY550, PLGA-PEG, PLGA-PEG-DBCO and / or combinations thereof.

4. Functionalized nanoparticles or microparticles for any of the applications described in any one of claims 1 to 3, wherein the virus-binding peptide is selected from microbinding proteins, host cell receptors, sialylated polysaccharides such as SA and gangliosides containing SA, cell adhesion molecules (CAMs) such as igSF members including CD4, JAM-A, CAR, integrins such as αvβ3, PtdSer receptors, cellular immunoglobulins and mucin domains (TIMs), Tyro3, ​​Axl, Mer (TAM), and ACE2 receptors, wherein the virus-binding peptide is preferably selected from LCB1 and AHB2.

5. Functionalized nanoparticles or microparticles for the application of claim 4, wherein the virus-binding peptide comprises at least one terminal cysteine ​​residue, the cysteine ​​residue optionally being modified to carry an azide group.

6. Functionalized nanoparticles or microparticles for any of claims 1 to 5, wherein the fixation of the virus-binding peptide or virus-binding small molecule to the surface of the nanoparticles or microparticles is direct or indirect, for example, by conjugation, physical adsorption and / or covalent bonding, wherein preferably the fixation is a succinimide thioether bond or a dibenzocyclooctylene (DBCO) azide linker.

7. The functionalized nanoparticles or microparticles for any one of claims 1 to 6, wherein the functionalized nanoparticles or microparticles further comprise at least one antiviral drug.

8. Functionalized nanoparticles or microparticles for any one of claims 1 to 7, wherein the functionalized nanoparticles or microparticles further comprise at least one detectable marker or label.

9. An aggregate for the prevention and / or treatment of target viral infection in mammalian subjects, comprising at least one functionalized nanoparticle or microparticle according to any one of claims 1 to 8 and at least one target virus, wherein the prevention and / or treatment comprises phagocytosis in the mammalian subject, particularly cell-mediated phagocytosis.

10. Functionalized nanoparticles or microparticles for the application according to any one of claims 1 to 8, or aggregates for the application according to claim 9, wherein the at least one target virus is selected from the group consisting of: HIV, measles virus, reovirus, rhinovirus, adenovirus, poliovirus, Coxsackievirus B, reovirus, rotavirus, adenovirus, West Nile virus, human metapneumovirus (hMPV), foot-and-mouth disease virus (FMDV), herpes simplex virus (HSV), HPV, human cytomegalovirus (HCMV), human herpesvirus-8, rabies virus, hMPV, paramyxovirus, filovirus, Ebola virus (EBOV), Marburg virus (MARV), flavivirus, dengue virus (DENV), Zika virus (ZIKV), Lassa virus, poxvirus, SV40, BKPyV, PyV, influenza A virus (IAV), orthomyxoviridae, MERS-CoV, β-coronavirus, SARS-S, and SARS-CoV-2.

11. A pharmaceutical composition for the prevention and / or treatment of target viral infection in mammalian subjects, comprising functionalized nanoparticles or microparticles for the application according to any one of claims 1 to 8 or 10, or aggregates for the application according to claim 9 or 10, wherein the pharmaceutical composition further comprises suitable excipients and / or adjuvants.

12. The pharmaceutical composition of claim 11, wherein the functionalized nanoparticles or microparticles have a size of about 200 to 500 nm and are optionally suitable for administration by inhalation.

13. A method for detecting a target virus and / or a target virus-specific antiviral antibody in a biological sample obtained from a mammalian subject, comprising contacting a functionalized nanoparticle or microparticle of any one of claims 1 to 8 with the biological sample and detecting the binding of the target virus and / or the target virus-specific antiviral antibody to the microparticle and / or detecting the formation of aggregates of the target virus with the functionalized nanoparticle or microparticle, wherein the binding and / or the formation of aggregates indicates the target virus and / or the target virus-specific antiviral antibody in the biological sample, wherein preferably the functionalized nanoparticle or microparticle further comprises at least one detectable marker or biomarker, and wherein preferably the sample is a blood or serum sample derived from the mammal, or a pooled sample from a group of subjects.

14. The method of claim 13, further comprising the step of quantifying the amount of target virus and / or target virus-specific antiviral antibody in the biological sample obtained from the mammalian object based on the formation of the binding and / or aggregates in the biological sample.

15. A kit for performing the method according to claim 13 or 14, comprising at least one functionalized nanoparticle or microparticle for the application according to any one of claims 1 to 8, and a suitable adjuvant.

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