WO2023156532A1 - ISOLATED PLASMA MEMBRANE-DERIVED VESICLES (PMdV) FOR USE IN THE TREATMENT OF VIRAL INFECTIONS - Google Patents

Abstract

The present invention refers to isolated plasma membrane-derived vesicles, such as microsomes, for use in the treatment of viral infections, wherein the plasma membrane- derived vesicles are able to capture and eliminate viral particles from the bloodst

Description

ISOLATED PLASMA MEMBRANE-DERIVED VESICLES (PMdV) FOR USE IN THE TREATMENT OF VIRAL INFECTIONS

FIELD OF THE INVENTION

The present invention refers to the medical field. Particularly, the present invention refers to isolated plasma membrane-derived vesicles, such as microsomes, for use in the treatment of viral infections, wherein the plasma membrane-derived vesicles can capture viral particles from the bloodstream.

FIELD OF THE INVENTION

Epidemics of emerging and re-emerging viral diseases such as the current pandemic by the SARS-CoV-2 pose a real threat to global health. In most cases there are no specific drugs for the treatment of viral infections causing epidemics or pandemics, including new influenza virus (IV) types, SARS-CoV-2, Ebola virus (EV), Zika virus (ZKV), dengue virus (DENV), West Nile virus (WNV), Chikungunya virus (CHIKV), human adenovirus (HAdV) or herpes viruses, such as HH8 or HH6. For those such as vaccinia virus (VACV), and HAdV, whose clinical impact stands out especially in immunosuppressed patients, the lack of effective treatments is also a global health problem. In this line, the World Health Organization (WHO) released in 2020 its list of 13 urgent, global health challenges that must be addressed for the next decade, being to stop infectious diseases one of them (https://www.who.int/). In addition to human viral diseases, those affecting domestic animals are one of the major threats to cattle farming and livestock and can cause considerable damage at local, regional, and even at the international level both in industrialized and in developing countries (World Organization for Animal Health). Examples of viruses causing these diseases are the foot- and-mouth disease virus (FMDV) and the African swine fever virus (ASFV), which global impact on farms and economics suppose a great concern.

Approximately 90 new antiviral drugs have been approved in the last 50 years, 29 of them in the last 6 years, in most cases intended for the selective treatment of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. There is a clear increase in R&D aimed at developing antiviral drugs but, unlike antibiotics, their spectrum of activity is generally limited to specific virus groups. For many viruses, especially those highly prevalent in developing countries, these drugs are not available, as well as for different coronavirus, such as SARS-CoV-2. Many synthetic small molecules with biological activity have led to the identification of new classes of antiviral drugs. Piperazinone derivatives have previously shown their usefulness as a source of antiviral molecules with different mechanisms of action. There are a great variety of examples belonging to different libraries of tri substituted piperazinones, which have shown antiviral activity against HAdV and Flavivirus. In addition, the repurposing of drugs is gaining increasing interest as a more efficient, cheaper, and faster alternative to the generation of new antiviral therapies. An example of this is niclosamide, an anthelmintic drug approved by the regulatory agencies that has been shown to have significant antiviral in vitro activity against a wide variety of viruses including IV, CHIKV, WNV, DENV, VACV, CMV, and HAdV. However, the poor aqueous solubility of niclosamide, as well as its low oral bioavailability and moderate cytotoxicity, pose important limitations that must be addressed before it can be used in clinic as a broad-spectrum antiviral agent. There is no doubt that R&D aimed at the generation of new antimicrobial molecules specifically directed at pathogens must continue, but it is also true that new additional approaches are necessary.

Despite the great clinical impact of outbreaks and epidemics caused by emergent and re- emerging viruses, there are no specific antiviral treatments for many of them. Currently, in severe cases off-label antivirals are used without satisfactory results. Furthermore, most of the antivirals used nowadays in clinical practice show side effects which also limit their use.

So, in summary, there is an unmet medical need of finding reliable antiviral strategies particularly focused on eliminating the presence of pathogens in the bloodstream of healthy individuals, thus avoiding serious infectious diseases such as sepsis, a potentially fatal systemic disease characterized by generalized inflammation in response to microbial invasion.

The present invention is focused on solving this problem and a reliable strategy has been designed aimed at controlling most of the viral diseases for which nowadays there is not a therapeutic or prophylactic alternative available.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention refers to isolated extracellular vesicles, preferably plasma membrane- derived vesicles, more preferably microsomes, for use in the treatment of viral infections, wherein the vesicles are able to capture viral particles in the bloodstream. In a further step, virus-vesicle complexes are removed from the patient, for instance by plasmapheresis or similar methods.

According to the results provided in the present invention, plasma membrane-derived vesicles, like microsomes, which are membranous structures containing the same composition of the plasma membrane, can be used as “tools” for the "capture" of viral pathogens. Particularly, after the homogenization of a cell extract, lipid membranes that make up the different cell compartments are recircularized, constituting microsomes, which are plasma membrane-derived vesicles covered by a lipid bilayer of cellular origin and which therefore have exactly the same composition as they have in their original compartment.

The use plasma membrane-derived vesicles, such as microsomes, to capture viral particles from the bloodstream is combined with an external device to allow the collection of these plasma membrane-derived vesicles containing the viral particles. Particularly, the external device is able to retain the plasma membrane-derived vesicles (loaded with viral particles) by means of plasmapheresis, because the plasma membrane-derived vesicles have been previously magnetised (filled with magnetic nanoparticles).

So, according to the results provided by the present invention, plasma membrane-derived vesicles, such as microsomes, originated from the plasma membrane of cells, contain the perfect binding motifs to attach viral pathogens (see Figure 1 and Figure 2), and their combination with an extracorporeal filtration device, for the elimination of viral pathogens from the bloodstream.

The results provided by the present invention (see Examples below) show, as proof-of- concept, how the infection of adenovirus (HAdV) has been blocked using a crude extract of microsomes preincubated with viral particles. However, the same strategy could be used to capture any viral particle from the bloodstream.

So, the first embodiment of the present invention refers to isolated plasma membrane-derived vesicles for use in the treatment of viral infections, characterized in that the plasma membrane-derived vesicles are able to capture viral particles from the bloodstream. Alternatively, the present invention refers to a method for the treatment of viral infections which comprises the administration of plasma membrane-derived vesicles to the patient, wherein the plasma membrane derived vesicles are able to capture viral particles from the bloodstream. In a preferred embodiment of the invention, the plasma membrane-derived vesicles are microsomes.

In a preferred embodiment of the invention, the plasma membrane-derived vesicles are in the form of a crude extract.

In a preferred embodiment of the invention, the plasma membrane-derived vesicles are obtained by means of a purification process, which comprises: 1) Cell extract homogenization, 2) Centrifugation and fractioning to obtain the microsomal fraction.

In a preferred embodiment of the invention, the plasma membrane-derived vesicles are derived from A549 human lung carcinoma cell line.

In a preferred embodiment of the invention, an adenovirus infection is treated.

In a preferred embodiment of the invention, an adenovirus infection caused by Human adenovirus C serotype 5 (HAdV-5) is treated.

In a preferred embodiment of the invention, an adenovirus infections caused by Human adenovirus C serotype 5 (HAdV-5) is treated and the plasma membrane-derived vesicles are derived from A549 human lung carcinoma cell line.

In a preferred embodiment of the invention, the treatment is a therapeutic treatment or prophylactic a treatment.

In a preferred embodiment of the invention, the plasma membrane-derived vesicles are magnetized because they are filled with magnetic nanoparticles.

In a preferred embodiment of the invention, the magnetised plasma membrane-derived vesicles are used in combination with an extracorporeal filtration device, for collecting the plasma membrane-derived vesicles loaded with viral pathogens from the bloodstream.

The second embodiment of the present invention refers to a composition (preferably a pharmaceutical composition) which comprises isolated plasma membrane-derived vesicles, such as microsomes and, optionally, pharmaceutical acceptable excipients or carriers.

In a preferred embodiment of the invention, the composition comprises a crude extract of plasma membrane-derived vesicles. In a preferred embodiment of the invention, the composition comprises plasma membrane- derived vesicles coming from A549 human lung carcinoma cell line or from any other target cell line of the viruses to be captured.

According to the present invention, the vesicles of the invention containing the viral particles can be eliminated from the bloodstream by both natural or non-natural processes. Among the natural processes, and as it happens for the extracellular vesicles, the vesicles of the invention containing the viral particles will likely be cleared from blood mostly by the mononuclear phagocyte system [Biomaterials. 2021 Aug;275: 121000. doi: 10.1016/j. biomaterials.2021.121000], On the other hand, among the non-natural processes, the vesicles of the invention containing the viral particles could be eliminated for instance by apheresis (which is a procedure in which blood is collected, part of the blood comprising vesicles is taken out, and the rest of the blood is returned to the donor) or any other device such as a system suitable for treating viral infections by capturing and eliminating viral particles from the bloodstream, which comprises: a. A composition comprising magnetized plasma membrane-derived vesicles, such as microsomes, for capturing viral particles from the bloodstream; and b. Extracorporeal filtration device for collecting and eliminating the plasma membrane-derived vesicles loaded with viral pathogens from the bloodstream.

For the purpose of the present invention the following terms are defined:

• The term "comprising" means including, but not limited to, whatever follows the word "comprising". Thus, the use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

• By "consisting of’ it is meant including, and limited to, whatever follows the expression “consisting of’. Thus, the expression "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

• “Plasma membrane-derived vesicles” (PMdV) are sub-membrane fragments of variable size, in the nanometre range, shed from the plasma membrane of cells during cell growth, activation, proliferation, senescence, apoptosis and when stimulated.

• “Microsomes” are closed vesicles of between 50 and 300 nm that result from the fragmentation of the endoplasmic reticulum of liver cells obtained by ultracentrifugation of liver homogenates, which contain oxidase function that depends on cytochrome P450 and where metabolic transformations are catalysed, which are widely used for the study of drug metabolism. After the homogenization of a cell extract, the lipid membranes that make up the different cell compartments are recircularized, constituting microsomes, spherical vesicles covered by a lipid bilayer of cellular origin, and which therefore have a composition exactly the same as what they would have in their original compartment. There are two types of microsomes according to their origin: those that come from the rough endoplasmic reticulum are lined with ribosomes and are called rough microsomes, and the so-called smooth microsomes, coming from parts of the smooth endoplasmic reticulum and from vesicular fragments of the plasma membrane, the Golgi apparatus, and mitochondria.

Description of the figures

Figure 1. Microsome purification protocol.

Figure 2. Infectivity assay in the presence of A549-derived microsomes.

Figure 3. Workflow of the approach.

Figure 4. TEM images of vesicles and their association with HAdV.

Detailed description of the invention

The present invention is illustrated by means of the Examples set below without the intention of limiting its scope of protection.

Example 1. Material and methods

Example 1.1. Microsome purification protocol

Whole cells will be recovered from the culture flasks and spin down at 2000 x g for 5 min. Then, cell pellet will be washed with PBS and spin again. The pellet will be resuspended in a salt-free, sucrose-free buffer (10 mM Tris ph 7.4, 5 mM EDTA and protease inhibitors). Cells will be then subjected to several cycles of freeze-thaw (liquid nitrogen and water bath at 37°C) to break cells. The number of freeze/thaw cycles may affect the size of the PMdV, so we will optimize this step to generate a homogeneous size of vesicles in our final solution.

Sucrose will be added then to the lysed cells to 30% final and spin at low speed (5 min, 200 x g, using less than 0.3 ml/tube) to remove nuclei (pellet). Supernatant will be spin again for 2 min at 200 x g, to get rid of any rest of nuclei. Then, the supernatant will be diluted with an equal volume of water (final sucrose concentration 15%), and the total volume will be divided into tubes with <0.3 ml/tube and spin at top speed (16000-21000 x g) for 30 min. At this point, the pellet will contain our total vesicles solution that will be washed twice with 0.5 ml Tris/EDTA buffer and spin for 10 min top speed.

Example 1.2. Generation of PMdV pre-filled with nanoparticles of magnetite.

Nanoparticles of magnetite stabilized with anionic coating (EMG 707) will be purchased from FerroTec (Bedford, NH, USA). The particles will have a nominal diameter of 10 nm, determined by TEM, a coefficient of viscosity of less than 5 mPa s at 27°C, and a 1.8% volume content in magnetite. These nanoparticles will be added to the solution of whole cells during the process for PMdV generation protocol, just before the cycles of freeze-thaw (liquid nitrogen + water bath at 37°C) to break cells. That way, when we crash the cells and let the PM fragments to form microsomes, these will end up recirculate keeping inside them the medium containing the nanoparticles of magnetite.

Example 1.3. Recovery of the PMdV pre-filled with magnetite and virus complexes.

To evaluate our capacity to recover the magnetized PMdV/virus complexes, we first will run an antiviral evaluation, using the combinations that generated antiviral effect but, in this case using these magnetized PMdV. If the antiviral effect observed previously keep seeing with these magnetized PMdV, we will evaluate our capacity to remove these complexes from the cell culture. In other to do that, we will use a strong neodymium-iron-boron (Nd2Fel2B) magnet (1.2 T) obtained from Halde GAC (Barcelona, Spain). This magnet will be placed under a 96-well plate containing a solution of the magnetized PMdV/virus complexes. After 5 min of incubation with the magnet, the solution will be removed, added to a cell culture, and incubated for 72 h to evaluate its infectivity.

Example 1.4. Design and development of an extracorporeal device to assess the recovery of the prefilled with nano-magnetic particles PMdV.

We will design and generate an extracorporeal device based in microfluidic technology that will be able to recover the viral particles attached to the PMdV, to evaluate the feasibility of the proposed approach (Figure 3).

Example 2. Results

Example 2.1. Adenovirus assay

A549 human lung carcinoma cell line was used to generate a crude microsome solution. The pathogen used was HAdV5-GFP, which has the coxsackie-adenovirus (CAR) receptors as specific receptors, present on the plasma membrane of A549 cells. After purification of the microsomes, a solution containing the isolated microsomes was pre-incubated at 37°C for 1 h with the HAdV particles. The preincubated solution with the virus was subsequently added to a cell culture plate at a MOI of 2000 pv/cell. In parallel, as an infection control, the same concentration of viral particles was used in the same diluent where the generated microsomes were resuspended and subjected to the same incubation. After 24 h of infection, and as can be seen in Figure 2, the cell culture infected with the virus pre-incubated with the microsomes solution showed a practically total reduction in infection compared to the positive control.

These experiment implies that there must be an interaction of the virus particles with the generated microsomes and we assess two different scenarios for this interaction: On the one hand, to explain the block of the infection, the microsome, by containing specific receptors for this virus in its membrane, could act as an anchoring surface for HAdV, so that they would interfere competitively with the cells of the cell culture in their uptake. Alternatively, the virus could stay anchored to the microsomal surface and, in this situation, the subsequent binding and internalization processes in the host cells may be altered because of the structural impairment resulting from this conformation. Moreover, the interaction of the virus particles with the microsomes could result in the stable capture and internalization of the viral particle inside this vesicle after anchoring to its cell receptors, thus avoiding the infection of the cell culture by the viral particles "captured” inside the microsomes.

The management of disseminated viral infections using microsomes is presented as an easily accessible tool, taking into account the simplicity of the protocol for its generation and its origin, cell cultures. Moreover, the use of microsomes to control viral infections has the potential to be applied to any viral pathogen since in each case microsomes will be generated from the optimal target cells of these viruses. In addition, microsomes filled with nano- magnetic particles can be generated, which will allow their recovery from the bloodstream once the viral particles have been stably bound to them, or with an antiviral drug, to improve its pharmacokinetics.

Example 2.2. Extracorporeal filtration device

According to the present invention, a specific system or device can be used for capturing and eliminating viral PMdV (loaded with viral particles) from the bloodstream.

For instance, an extracorporeal microfluidic device incorporating a flow channel design, can be used to cleanse pathogens from the flowing blood of patients with viral infections when combined with magnetized PMdV. The microfluidic device comprises two adjacent, rectangular fluidic channels. One acts like a vascular channel that contains flowing blood, and the other one contains saline under intermittent or slow flow. The device contains a series of open rectangular slits that will provide direct access between the blood channel and the saline-filled sinusoid channel.

Claims

1. Isolated plasma membrane-derived vesicles for use in the treatment of viral infections, characterized in that the plasma membrane-derived vesicles are able to capture viral particles from the bloodstream.

2. Isolated plasma membrane-derived vesicles for use, according to claim 1, wherein the plasma membrane-derived vesicles are magnetized, and they are collected from the bloodstream by using an extracorporeal filtration device.

3. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, characterized in that they are microsomes.

4. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, characterized in that they are in the form of a crude extract.

5. Isolated plasma membrane-derived vesicles, for use, according to any of the previous claims, wherein the plasma membrane-derived vesicles are obtained by means of a purification process, which comprises: 1) Cell extract homogenization, 2) Centrifugation and fractioning to obtain the microsomal fraction.

6. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, wherein the plasma membrane-derived vesicles come from A549 human lung carcinoma cell line or from any other target cell line of the viruses to be captured.

7. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, in the treatment of adenovirus infections.

8. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, in the treatment of adenovirus infections caused by Human adenovirus C serotype 5 (HAdV-5).

9. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, in the treatment of adenovirus infections wherein the plasma membrane- derived vesicles are derived from A549 human lung carcinoma cell line.

10. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, in the treatment of adenovirus infections caused by Human adenovirus C serotype 5 (HAdV-5), wherein plasma membrane-derived vesicles are derived from A549 human lung carcinoma cell line. Isolated plasma membrane-derived vesicles for use, according to any of the previous claims, characterized in that the treatment is a therapeutic treatment or prophylactic a treatment. Antiviral composition comprising isolated plasma membrane-derived vesicles and, optionally, pharmaceutical acceptable excipients or carriers. Antiviral composition, according to claim 12, comprising a crude extract of membrane-derived vesicles. Antiviral composition, according to any of the claims 12 or 13, comprising plasma membrane-derived vesicles coming from A549 human lung carcinoma cell line or from any other target cell line of the viruses to be captured. System, suitable for treating broad-spectrum viral infections by capturing and eliminating viral particles from the bloodstream, which comprises: a. A composition comprising magnetized plasma membrane-derived vesicles, such as microsomes, for capturing viral particles from the bloodstream; and b. Extracorporeal filtration device for collecting and eliminating plasma membrane-derived vesicles loaded with viral particles from the bloodstream.