CN114917242A - Application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus through nose dropping and application thereof - Google Patents

Abstract

The invention discloses application and application of synthetic double-stranded RNA analogs in preventing respiratory tract transmitted viruses by nasal drip, wherein the natural immune defense function of an upper respiratory tract is enhanced by locally stimulating nasopharynx with polyinosinic acid cytosine nucleotide poly (I: C) so as to prevent the infection of the respiratory tract transmitted viruses including new coronavirus; the invention has the advantages that the local infusion of poly (I: C) in the nasopharynx activates the interferon reaction of natural immune cells of the upper respiratory tract, and enhances the first line of defense against respiratory tract virus infection; poly (I: C) activation of local type I and type III interferon responses in the upper respiratory tract can effectively and relatively long-term prevent viral infection, reduce viral replication, spread to the lower respiratory tract or bloodstream, and generalized organ function impairment due to an expanded inflammatory response.

Description

Application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus through nasal drip and application thereof

Technical Field

The invention relates to the technical field of respiratory tract virus infection prevention, in particular to application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus through nasal drip and application thereof.

Background

Biological products for vaccination used to prevent infection by various pathogenic microorganisms, which are collectively referred to as vaccines, act by activating the body's innate and adaptive immune responses to protect against or reduce the risk of infection by pathogens prior to infection. Among these, the body's natural (innate) immune response is the first line of defense against pathogen infection, and the mucosal epithelial and phagocytic systems constitute the natural immune barrier of the respiratory tract. The innate immune cells of the upper respiratory tract are essential for a timely and effective Interferon (IFN) response against viral infections transmitted through the respiratory tract.

The innate immune cells of the upper respiratory tract recognize pathogens that invade the body by sensing pathogen-associated molecular patterns (PAMPs), including viruses, through various Pattern Recognition Receptors (PRRs). Viral PAMPs are typically unique molecular structures not found in the host cell, such as unique nucleic acid structures of the viral genome or viral replication intermediates. In the absence of a particular PRR and its signaling pathway, the organism's susceptibility to viruses is increased. It has been reported that natural immune cells recognize the activation of Type I and Type III IFN responses following viral infection as having antiviral effects (Park Annsea, Iwasaki Akiko, Type I and Type III interference-indication, Signaling, Evasion, and Application to Commat COVID-19 [ J ] Cell Host Microbe,2020,27: 870-. Among them, type I IFN (human IFN-. alpha., IFN-. beta., IFN-. epsilon., IFN-. kappa., IFN-. mu.) binds to type I IFN receptors (IFNAR) widely expressed in various cells in an autocrine and paracrine manner to activate a strong antiviral defense system consisting of hundreds of interferon-stimulated genes (ISG) which can interfere with each step of virus replication; type III interferons (IFN λ s) bind to type III interferon receptors (IFNLRs), which are predominantly expressed in epithelial and certain myeloid cells. Timely and effective type I and type III IFN responses are critical to host defense against viral infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Insufficient intensity of respiratory IFN responses in the first few days of viral infection may be responsible for the massive replication and systemic dissemination of the virus, causing excessive inflammatory responses in the lungs or in the body.

However, in a series of studies on SARSCoV-2, it was found that IFN-I or IFN-III transcript levels induced after SARSCoV-2 infection are very low and the associated ISG response is limited; but causes the expression of a large number of chemokine and proinflammatory cytokine genes, which indicates that the antiviral IFN response of the body is relatively weak and the systemic inflammatory response is strengthened after the body is infected with the SARSCoV-2. This phenomenon may be attributed to the "anti-interferon mechanism" developed by the virus to evade and suppress the antiviral effects of the body's IFN response, including (1) evading recognition by natural immune cells; (2) inhibiting IFN production; (3) interference with IFN signaling; (4) inhibiting ISG effector function. That is, once the virus infects and the body fails to effectively suppress its replication in time, the virus may evade the antiviral action of the innate immune cells by suppressing the body's IFN response through the "anti-interferon mechanism" described above. Therefore, it is necessary to try to properly activate the IFN response of innate immune cells prior to viral infection to achieve prevention and control of viral infection, in view of the evolutionary nature of sarclov-2.

Exogenous supplementation with IFNs such as recombinant or pegylated IFN- α and IFN- β has been used to treat a variety of diseases, including multiple sclerosis and viral hepatitis. Recombinant IFN- λ has not been approved for any indication, but is undergoing clinical trials for viral hepatitis. In the field of exogenous IFN supplementation via the upper respiratory tract (intranasal administration or inhalation), test results have supported the protective role of early IFN- λ in SARS-CoV-2 infection (Chong Zhenlu, Karl Cortney E, Halfmann Peter J et al, Nasally-delayed interferon- λ protection microorganism against SARS-CoV-2variant infection. [ J ]. bioRxiv,2022, undefined: undefined.). Prophylactic administration of IFN- β to mice accelerates viral clearance without causing weight loss or excessive inflammatory response. Notably, early administration of IFN- β therapy before viral peak has been reached has protective effects, while late administration of IFN- β may fail to control the virus and result in an inflammatory response that is exacerbated and even fatal pneumonia. These studies have together underscored the importance of the induction time of the IFN response for controlling virus replication, i.e., early IFN induction or administration provides good infection prevention and antiviral effects (Major Jack, Crotta Stefania, Llorian Mirian et al.Type I and III interference dispersion viral infection [ J ]. Science,2020,369: 712-. Thus, the host is more likely to benefit from exogenous supplementation with IFN early in the course of the disease. However, the direct supplementation of exogenous IFN also brings about some side effects, such as cytokine storm and related tissue damage caused by inhaled IFN directly entering the lower respiratory tract; since IFNs have the characteristics of reduced proliferation and pro-apoptosis, the regeneration of lung epithelium is hindered by the direct action of long-term excess exogenous IFNs, thereby disrupting respiratory barrier function, resulting in bacterial co-infection (Major Jack, Crotta Stefania, Lloyan Miriam et al.Type I and III interferons disturbing epithelial repair reducing recovery from viral infection [ J ]. Science,2020,369: 712-; since IFNAR is expressed on almost all cells, type I IFNs may produce serious systemic side effects; exogenous IFN activates AhR in airway epithelial cells to produce mucus, which increases the thickness of the blood-gas barrier and prevents gas diffusion from causing hypoxia in the body (Liu Yuying, Lv Jiandi, Liu Jianning et al. culture production stimulated by IFN-AhR signaling trigger of COVID-19.[ J ] Cell Res,2020,30: 1078-.

Disclosure of Invention

The invention provides application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus through nose dropping and application thereof.

The application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus via nasal drip.

Preferably, the nose drops are local instillations.

Preferably, the respiratory transmitted virus for prevention comprises a new coronavirus and a respiratory transmitted virus.

As a preferred embodiment, the synthetic double-stranded RNA analog is a double-stranded RNA analog capable of activating the IFN response of natural immune cells, including but not limited to polyinosinic acid cytosine nucleotide poly (I: C).

The invention also discloses the use of the synthetic double-stranded RNA analogue of claim 1 in local respiratory tract.

As a preferred technical scheme, the synthetic double-stranded RNA analogue is used for enhancing the effect of local natural immune cells of the upper respiratory tract on inducing the cellular interferon response after nasal drip, and the synthetic double-stranded RNA analogue is poly (I: C).

As a preferred technical scheme, the synthetic double-stranded RNA analogue prevents the effects of new coronavirus and respiratory spread virus through local nasal drops at the nasopharynx, and the synthetic double-stranded RNA analogue is poly (I: C).

Preferably, the synthetic double-stranded RNA analogue is poly (I: C) and has the effect of reducing the amount of virus in the lower respiratory tract after infection with a new coronavirus or a respiratory tract-transmitted virus by nasal drip.

As a preferred technical scheme, the double-stranded RNA analogue is transnasal-drip-prevented from inducing the lower respiratory tract or the aggravation of the systemic inflammatory response caused by the cell interferon response systemically.

By adopting the technical scheme, the application and the application of the synthetic double-stranded RNA analogue for preventing the virus from being spread to the respiratory tract by nasal dropping, and the application of the synthetic double-stranded RNA analogue for preventing the virus from being spread to the respiratory tract by nasal dropping are provided.

The invention has the following advantages:

1) the present invention demonstrates that type I and type III IFN responses to native mouse respiratory immune cells can be efficiently activated by the use of poly (I: C) nasal drops.

2) The invention demonstrates that the viral load of the lung of a mouse after subsequent infection with virus can be effectively reduced by using poly (I: C) nasal drops;

3) the invention develops the new discovery of poly (I: C) function, and the IFN reaction of local natural immune cells of the upper respiratory tract is activated by low-concentration poly (I: C) nasal drops, thereby reducing the side effect of the whole body, having wider virus coverage and wide application prospect.

4) The present invention explores the provision of a new concept for preventing viral infections transmitted via the respiratory tract by locally activating the upper respiratory IFN response to enhance the innate immune response.

Local instillation of poly (I: C) in the nasopharynx activates the Interferon (IFN) response of natural immune cells of the upper respiratory tract-the first line of defense to enhance protection against respiratory viral infection; poly (I: C) activation of local type I and type III interferon responses in the upper respiratory tract can effectively and relatively long-term prevent viral infection, reduce viral replication, spread to the lower respiratory tract or bloodstream, and generalized organ function impairment due to an exaggerated inflammatory response.

The invention adopts poly (I: C) to locally stimulate the nasopharynx part to increase the IFN reaction of natural immune cells of the upper respiratory tract, and is used as a novel vaccine for local application to enhance the natural immune function to resist viruses. Poly (I: C) is a synthetic double-stranded RNA (dsRNA) analogue, and can effectively induce the IFN reaction of natural immune cells of the upper respiratory tract by activating a non-specific IFN reaction through PRR (mainly RLRs and TLR3), and avoid a series of side effects caused by direct exogenous IFN supplementation. Cell experiments and animal in vivo experiments show that poly (I: C) can effectively activate I type and III type IFN reactions of natural immune cells so as to reduce the virus load of the cells after the cells are infected with viruses; poly (I: C) local stimulation of mouse nasopharynx also effectively enhances mouse ability to resist viral infection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a graph of the pre-stimulation of the example poly (I: C) to enhance macrophage interferon response and inhibit viral replication; after pre-stimulation of macrophages with poly (I: C) (10. mu.g/ml) for 12h, VSV was re-stimulated, rtq-PCR for IFN β (FIG. 1A), IFN λ production (FIG. 1B) and viral load (FIG. 1C);

FIG. 2 is a graph of the time window for the pre-stimulation of the poly (I: C) enhanced macrophage interferon response of the example; (FIG. 2A) after macrophage stimulation by poly (I: C) (10. mu.g/ml) for 12h, VSV was re-stimulated for 6h at different time points, and viral load was measured by rtq-PCR; (FIG. 2B) after different time points of macrophage stimulation by poly (I: C), VSV was re-stimulated for 6h, rtq-PCR for viral load;

FIG. 3 is a graphical representation of the reasonable concentrations of the example poly (I: C) pre-stimulation to enhance macrophage interferon response and efficacy in preventing RNA and DNA viral infections. (FIG. 3A) after macrophage stimulation for 12h with different concentrations of poly (I: C), VSV was re-stimulated for 6h, rtq-PCR to detect viral load; (FIG. 3B) after macrophage stimulation for 12h with various concentrations of poly (I: C), VSV, SeV and HSV (MOI:1) were re-stimulated for 6h, and TCID50 was tested for viral load;

FIG. 4 is a schematic view of: poly (I: C) pre-stimulates viral infection and decreases pulmonary viral load profiles that protect human and mouse lung epithelial cells; (FIG. 4A) after 12h stimulation of human lung carcinoma cells (A549) and mouse tracheal epithelial cells (MLE12) with poly (I: C) (5. mu.g/ml), VSV was re-stimulated for 6h and viral load was measured by Q-PCR; (FIG. 4B) mice were nasally infected with VSV 12h after nasal instillation of poly (I: C) (25ng in 5ul) on both sides, and lung viral load was measured at different time points rtq-PCR.

Detailed Description

In order to make up for the above deficiencies, the present invention provides the use of synthetic double-stranded RNA analogs for preventing respiratory transmitted viruses by nasal drip and the use thereof to solve the above problems in the background art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

An application of synthetic double-stranded RNA analog in preventing respiratory tract transmitted virus by nasal drip.

The nose drops are local drip drops.

The respiratory transmitted viruses to be prevented include new corona viruses and respiratory transmitted viruses.

The synthetic double-stranded RNA analogs are double-stranded RNA analogs that can activate the natural immune cell IFN response, including but not limited to polyinosinic acid cytosine nucleotide poly (I: C).

Polyinosinic acid cytosine nucleotide poly (I: C) induces cell Interferon (IFN) reaction by locally applying poly (I: C) in nasopharynx, activates natural immune response of upper respiratory tract before virus infection, and effectively prevents respiratory tract virus infection including SARS-CoV-2.

In a first aspect of the invention, there is provided a possibility of enhancing local innate immune cell IFN responses in the upper respiratory tract following nasal instillation with poly (I: C).

In a second aspect, the invention provides the feasibility of applying poly (I: C) nasal drops in the prevention of respiratory viral infections including SARS-CoV-2.

In a third aspect of the invention, the feasibility of applying nasal drops of poly (I: C) to reduce the viral load of the lower respiratory tract following viral infection of respiratory tracts including SARS-CoV-2 is provided.

In a fourth aspect of the invention, there is provided a method of administration by local infusion of poly (I: C) to avoid exacerbation of lower respiratory or systemic inflammatory response to systemic IFN response.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example one, Experimental method

1. Preparation of reagents (including viruses)

The cell RNAfast200 extraction kit is purchased from Shanghai Feijie Biotechnology GmbH. RNA reverse transcription reagents and fluorescent quantitative PCR reagents were purchased from TOYOBO. Poly (I: C) was purchased from Sigma (St. Louis, MO), Vesicular Stomatitis Virus (VSV) (Indiana Strain), herpes simplex Virus 1(herpes simplex Virus-1, HSV-1) (Kos Strain) Sendai Virus (Sendai Virus, SeV) supplied by the university of military medical science and microbiology laboratory. RPMI1640 medium and DMEM medium were purchased from PAN-Biotech. Fetal Bovine Serum (FBS), antimicrobial-antimicrobial (15240) was purchased from Gibco.

2. Mouse primary alveolar macrophage extraction

C57BL/6J mice were purchased from Shanghai Bikeley laboratory animal center and were all bred in SPF-level environment. After anesthetizing, the neck of the mouse is preserved, disinfected, cut into skin, the subcutaneous tissue is separated layer by layer, and a scalp needle is inserted into the lung after reaching the trachea and is fixed by silk threads. Slowly injecting 1ml of RPMI1640 culture solution, slowly pumping back and injecting into a centrifuge tube after 1min, repeating for 10 times, centrifuging the obtained liquid (2000r/min) for 10min, discarding the supernatant, uniformly mixing single cell suspension, counting and plating, wherein the cell concentration is about 1.5 multiplied by 10/ml. Adding 7ml of RPMI1640 culture solution and 1ml of fetal calf serum into each 2ml of single cell suspension, mixing well, and adding 5% CO ₂ And/or aseptically culturing at 37 ℃ in an incubator for 3h, recovering adherent cells after the macrophages adhere to the wall, and adjusting the cell concentration to 1.5 multiplied by 10 per ml.

3. Cell lines

Human lung cancer cell line (A549) and mouse airway epithelial cell line (MLE12) were purchased from ATCC (American Type Culture Collection) and stored by laboratory expansion.

ELISA detection

According to the instruction of the corresponding kit, sequentially adding a detection buffer solution and a sample to be detected, and incubating at room temperature for 1-2 h; washing the detection hole by using the washing solution provided by the kit, and then adding a second antibody for incubation for 1-2h at room temperature; and (3) adding washing liquor to clean the detection hole, adding a horseradish peroxidase (HRP) -coupled anti-secondary antibody, incubating at room temperature for 1h, then cleaning the detection hole with the washing liquor, adding a reaction substrate solution, reacting for 15-30min in a dark place, adding a stop solution to terminate the reaction, measuring the OD value with an enzyme-linked immunosorbent assay, and finally converting the OD value into the corresponding protein amount according to a standard curve.

rt-qPCR assay

Tissue and cell total RNA was extracted with Trizol. Collecting the tissue or cell after experiment stimulation, adding 1ml Trizol reagent, fully blowing (cell) or polishing (tissue), adding 200 μ l chloroform, fully reversing and uniformly mixing, and standing at room temperature for 5 min; centrifuging at 12,000g for 10min at 4 deg.C; carefully sucking the liquid of the upper aqueous phase into a new 1.5ml EP tube (taking care not to suck solid protein), adding 0.5ml isopropanol, fully reversing and uniformly mixing, standing at room temperature for 10min, centrifuging for 10min at 4 ℃ at 12,000g, discarding the supernatant, adding 75% ethanol to wash and precipitate once, centrifuging for 5min at 4 ℃ at 12,000g, discarding the supernatant, adding an appropriate amount of DEPC water to blow and dissolve after the precipitate is dried, measuring the concentration and identifying the purity, storing at-80 ℃ or performing reverse transcription PCR to obtain cDNA, wherein the reverse transcription system and conditions are as follows:

the RT-PCR reverse transcription system is as follows:

Reaction Component Volume(μl)

ReverTra Ace*1μl

5×buffer 4μl

1mM dNTPs 2μl

10μM Olig dT 1μl

Total RNA template 1μg

DEPC H2O Total to 20μl

*ReverTra Ace(TOYOBO，Code No：TRT-101)

RT-PCR reverse transcription reaction conditions: 42 ℃ for 25 min; 99 ℃ for 5 min; 4 ℃ and infinity.

TCID50 detection

Diluting the virus solution to be tested, inoculating the diluted virus solution to a 96-well plate according to a concentration gradient, carrying out aseptic incubation for 1h in an incubator at 55CO2/37 ℃, adding 200 mu l of maintenance solution into each well after the virus solution is absorbed, and continuously culturing in the incubator at 5% CO2/37 ℃. After 48h, the cells were observed under a microscope, and the TCID50 of the virus solution was calculated by the Spearman-Karber method.

Second, experimental results

Pre-stimulation of macrophages with low concentrations of poly (I: C) enhances their IFN response and inhibits replication following subsequent viral infection for a long period of time. In ex vivo cell experiments, cells were re-stimulated for VSV using poly (I: C) (10. mu.g/ml) pre-stimulation of macrophages for 12h, with increased IFN-. beta. (FIG. 1A) and IFN-. lambda. (FIG. 1B) production and decreased VSV viral load (FIG. 1C). That is, poly (I: C) pre-stimulation causes macrophages to enhance the antiviral type I and type III IFN response and thereby significantly inhibit viral replication. The results show that poly (I: C) pre-stimulated macrophages enhance type I and type III IFN responses following viral infection and significantly inhibit viral replication;

further, the time window for poly (I: C) pre-stimulation to enhance the macrophage IFN response was examined. After 12h of macrophage stimulation with poly (I: C) (10. mu.g/ml), VSV was re-stimulated at various time points for 6h, and the results showed that within 1-96h, poly (I: C) pre-stimulation could significantly suppress the viral load, i.e., poly (I: C) pre-stimulation could provide protection for macrophages for more than 96h (FIG. 2A); the results show that the viral load can be significantly inhibited within 1-96h after poly (I: C) stimulation for 12 h. The poly (I: C) pre-stimulation can last for over 96 hours; in addition, the VSV loading can be obviously inhibited after 0.5h of pre-stimulating macrophages by poly (I: C). poly (I: C) pre-stimulation for 1h-12h, no difference exists in the inhibition effect on the viral load, which indicates that the protection force can be obtained by pre-stimulation for 1h (figure 2B); the results show that VSV loading can be significantly inhibited after 0.5h of poly (I: C) pre-stimulation. The poly (I: C) is pre-stimulated for 1h to 12h, and has no difference on the inhibition effect of the viral load, which indicates that the protective force can be obtained after the pre-stimulation for 1 h.

Further, reasonable concentrations of poly (I: C) pre-stimulation to enhance macrophage interferon response and efficacy in preventing RNA and DNA viral infection were explored. In terms of the concentration of poly (I: C), the viral load was significantly inhibited after 12h of stimulation with 1-50. mu.g/ml of poly (I: C). Poly (I: C) pre-stimulation at concentrations of 5. mu.g/ml and higher, was consistent in viral load control. Indicating that 5. mu.g/ml is a reasonable concentration of poly (I: C) protective power (FIG. 3A); the results show that 1-50. mu.g/ml of poly (I: C) can significantly inhibit viral load after 12h of stimulation. Poly (I: C) pre-stimulation at concentrations of 5. mu.g/ml and higher, was consistent in viral load control. Indicating that 5. mu.g/ml is an effective concentration of poly (I: C) to produce protection. After macrophage stimulation is carried out for 12h by poly (I: C) with different concentrations, VSV, SeV and HSV (MOI:1) are stimulated for 6h again, and the result of TCID50 shows that the VSV, SeV and HSV viral load can be remarkably inhibited after poly (I: C) with 5-10 mu g/ml is stimulated for 12 h. Poly (I: C) pre-stimulation at concentrations of 5. mu.g/ml and higher, was consistent in viral load control. Indicating that 5. mu.g/ml is a reasonable concentration of poly (I: C) protection, with equal protection against RNA and DNA viruses (FIG. 3B); the results show that poly (I: C) of 5-10 mug/ml can remarkably inhibit VSV, SeV and HSV viral load after 12h of stimulation. Poly (I: C) pre-stimulation at concentrations of 5. mu.g/ml and higher, was consistent in viral load control. Indicating that 5. mu.g/ml is a reasonable concentration of poly (I: C) protection, with equal protection for RNA and DNA viruses.

Poly (I: C) pre-stimulation protects human and mouse lung epithelial cells from viral infection.

After poly (I: C) (5. mu.g/ml) pre-stimulated human lung cancer cells (A549) and mouse tracheal epithelial cells (MLE12) for 12h, VSV re-stimulated for 6h, and the viral load of the poly (I: C) pre-stimulated cells was found to be significantly lower than that of the control group, indicating that poly (I: C) pre-stimulation can protect viral infection of respiratory epithelial cells of human and mouse (FIG. 4A); the results show that the viral load can be significantly inhibited after poly (I: C) pre-stimulation for 12 h. Indicating that poly (I: C) pre-stimulation can protect human and mouse lung epithelial cell viral infection.

And thirdly, the virus load of the lung after subsequent respiratory virus infection can be reduced by using poly (I: C) nasal drops for the mice.

Poly (I: C) (25ng in 5. mu.l) was added to the nasal mice for 12h, then the mice were infected with VSV through the nose, and the virus load of VSV in the lungs of the mice was measured at different time points, which indicated that poly (I: C) nasal drops could significantly reduce the virus load in the lungs after infection with virus. See FIG. 4B; the results show that pulmonary viral replication is significantly inhibited in poly (I: C) nasal drip mice.

Third, discuss

The variant strain Omicron of SARS-CoV-2 is currently prevalent worldwide, causing a serious social and economic burden. As a virus transmitted through the respiratory tract, Omicron is characterized by weak pathogenicity but strong infectivity, and thus it may be more effective in preventing infection by Omicron to directly enhance the antiviral ability of natural upper respiratory immune cells, as compared to the conventional acquired immunity established by intravenous vaccination. Cell experiments prove that poly (I: C) can effectively enhance the interferon response of mouse macrophages, thereby resisting subsequent virus infection. The same effect was also demonstrated in human and mouse lung epithelial cell lines. In mouse experiments, poly (I: C) nasal drops can effectively reduce the viral load in the lung after viral infection. On the one hand, it is possible that poly (I: C) enhances the interferon response in the upper respiratory tract to directly inhibit local infection of the virus, and reduces virus replication and dissemination to the lower respiratory tract; on the other hand, the virus has a mechanism for inhibiting the interferon response, but the local poly (I: C) induction action stimulates a certain degree of interferon response before the virus infection, so the natural immune suppression effect caused by the virus is avoided, and the anti-virus capability of the organism is enhanced. In addition, the interferon effect of poly (I: C) activation is non-specific. Non-specificity is manifested not only in the type of interferon activated, but also in the ability to defend against different viruses. First, poly (I: C) pre-stimulation simultaneously upregulates macrophage type I interferon (IFN-. beta.) and type III interferon (IFN-. lambda.). Both type I interferon and type III interferon have significant antiviral effects, but direct use of exogenous interferon has been reported to produce adverse effects such as impairment of lung epithelial regeneration, disruption of respiratory barrier function, concomitant bacterial infection, systemic inflammatory response, and the like. Secondly, experiments demonstrate that poly (I: C) pre-stimulation can simultaneously reduce the replication capacity of RNA viruses (VSV and SeV) and DNA viruses (HSV) after invading cells, thereby coping with the highly variable characteristics of the viruses.

Because the interferon response of natural immune cells and viruses have the characteristic of mutual antagonism, namely the interferon response has the function of antivirus, but the interferon response can be weakened by various mechanisms after the viruses infect cells. Therefore, the timing of the activation of the interferon response of innate immune cells is very important, and the activation of a certain degree of interferon response before the virus infects innate immune cells can significantly reduce the replication load after subsequent viral infection. Therefore, poly (I: C) nasal drops can pre-activate the interferon response of natural immune cells of the upper respiratory tract, block viruses in a first line of defense, and reduce the invasion to the lower respiratory tract and the systemic severe reaction caused by the expansion of inflammatory reaction.

The application of local instillation rather than inhalation in terms of route selection is intended to avoid the risk of drug entry into the lower respiratory tract. The literature reports that the upper respiratory tract is dominated by the type III interferon response of epithelial cells, while the lower respiratory tract further activates the type I interferon response. Since the type I interferon receptor IFNAR is expressed on almost all cells, a strong type I interferon response may produce serious systemic side effects. Therefore, the application selects local instillation of poly (I: C) for administration, generates moderate interferon response in the form of local natural immune cell response, and can generate certain antiviral effect to play a role in prevention and avoid systemic side effect.

The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (9)

1. The application of synthetic double-stranded RNA analogue in preventing respiratory tract transmitted virus via nasal drip.

2. The use of a synthetic double stranded RNA analog according to claim 1 for nasal drip prevention of respiratory transmitted viruses, wherein: the nose drop is local drip.

3. The use of a synthetic double stranded RNA analog according to claim 1 for nasal drip prevention of respiratory transmitted viruses, wherein: the respiratory transmitted viruses to be prevented include new corona viruses and respiratory transmitted viruses.

4. The use of a synthetic double stranded RNA analog according to claim 1 for nasal drip prevention of respiratory transmitted viruses, wherein: the synthetic double-stranded RNA analogs are double-stranded RNA analogs that can activate the natural immune cell IFN response, including but not limited to poly (I: C).

5. Use of the synthetic double stranded RNA analog of claim 1 locally in the respiratory tract.

6. Use of a synthetic double stranded RNA analog according to claim 5 for topical application to the respiratory tract, wherein: the synthetic double-stranded RNA analogue enhances the effect of local natural immune cells of the upper respiratory tract on inducing the reaction of the cell interferon after being dripped through the nasal pharynx part, and the synthetic double-stranded RNA analogue is poly (I: C).

7. Use of a synthetic double stranded RNA analog according to claim 5 for topical application to the respiratory tract, wherein: the synthetic double-stranded RNA analogue prevents the effects of new coronavirus and respiratory spread virus through nasal drops, and the synthetic double-stranded RNA analogue is poly (I: C).

8. Use of a synthetic double stranded RNA analog according to claim 5 for topical application to the respiratory tract, wherein: the synthetic double-stranded RNA analogue is used for reducing the virus planting amount of the lower respiratory tract after the infection of new coronavirus and respiratory tract transmitted virus by nasal drip, and the synthetic double-stranded RNA analogue is poly (I: C).

9. Use of a synthetic double stranded RNA analog according to claim 5 for topical application to the respiratory tract, wherein: the double-stranded RNA analogue is dripped through the nose to avoid the aggravation of lower respiratory tract or systemic inflammatory reaction caused by systemic induction of cell interferon reaction.

Images