CN117860870A - Pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and use thereof - Google Patents
Pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and use thereof Download PDFInfo
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Abstract
The invention provides a pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and application thereof. The pharmaceutical composition comprises: (1) interferon- β and interferon- γ; or (2) a gene encoding interferon-beta, a gene encoding interferon-gamma, and a vector. The invention provides a pharmaceutical composition containing interferon-beta and interferon-gamma and a new application thereof, namely, the interferon-beta and the interferon-gamma can synergistically inhibit replication of vaccinia virus, which has important clinical application value for preventing and treating infection of poxviruses, in particular, monkey poxviruses and the like and new and sudden diseases caused by the infection.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and in particular relates to a pharmaceutical composition for preventing and/or treating poxvirus infection and diseases caused by poxvirus infection and application thereof.
Background
Poxviruses are the largest, most complex of all viruses that cause local or systemic suppurative skin lesions upon infection of humans and animals. Among poxviruses, orthopoxviruses include a variety of viral species that cause zoonotic diseases, such as smallpox, monkey pox, vaccinia, and orthopoxviruses are among the most serious genera of poxviruses.
Vaccinia virus is a large DNA virus, belongs to the genus orthopoxvirus of the family Poxvidae, has few cases of vaccinia virus infection in the world, and has been reported to find out 5 cases of epidemiologically related fever with unknown reasons in 2017 through report of clinicians and field investigation of disease control personnel. Cases are men with fever and pulmonary infection symptoms, one of which develops severe pneumonia; except for one example, skin pustules were found. Post-infection viral genome analysis, which is highly homologous to smallpox vaccine strain (Tiantan strain).
The monkey pox is caused by a monkey pox virus, which is one of the genera orthopoxvirus of the family Poxvidae. Monkey pox is a viral zoonotic disease, mainly occurring in tropical rainforest areas of middle and western africa, and occasionally exported to other areas. Monkey pox is clinically typical in terms of fever, rash and lymphadenectasis and can lead to a range of complications. Monkey pox is usually a self-limiting disease with symptoms lasting 2 to 4 weeks, with severe cases possibly occurring. In recent years, the mortality rate is about 3-6%. The monkey poxvirus is transmitted to humans by intimate contact with an infected human or animal, or with an object contaminated with the virus. Monkey poxviruses are transmitted from person to person by intimate contact with contaminated objects such as skin lesions, body fluids, respiratory droplets, bedding and the like.
The clinical manifestation of monkey pox is similar to smallpox, a related orthopoxvirus infection. The world health organization announced that smallpox was thoroughly eradicated on day 5 and 8 of 1980. In 1981, china announced that the vaccination with smallpox vaccine was stopped. The elimination of smallpox worldwide and the termination of vaccination with smallpox may lead to a reduced immunity of humans to monkey pox virus. Because monkey pox viruses can cause serious diseases in humans and have a great influence on human health, there is no choice but to find a medicament for preventing and treating monkey pox and other poxviruses. The infection of monkey pox is not as serious as that of smallpox, and the disease caused by the infection is not so serious, and an antiviral drug developed for treating smallpox has been licensed for treating the monkey pox. Vaccinia virus is of the same family as smallpox virus, which causes smallpox, and thus vaccination with a vaccine derived from vaccinia virus can prevent smallpox, monkey pox, and other orthopoxvirus infections. Although vaccination is dominant in the prevention and control of poxviruses, it is particularly important for drug treatment in individuals who are not vaccinated (with underlying diseases etc.) and who have been infected. Cidofovir and telitherama are currently marketed anti-poxvirus drugs, but they still have problems of low oral availability, viral resistance or nephrotoxicity.
Interferon (IFN) induces cells to produce antiviral immunity, which prevents or limits viral infection by interfering with viral gene transcription or translation of viral proteins, is currently the most prominent antiviral infection and antitumor biologic. Among them, the clinical application of IFN-alpha is most common, and is widely used for viral disease treatment and tumor treatment, such as treatment of chronic hepatitis, HPV infection, west Nile virus infection and the like, and cutaneous T-cell lymphoma, renal cell carcinoma, bladder carcinoma, ovarian carcinoma, cervical carcinoma, basal cell carcinoma, metastatic melanoma and the like; IFN- β has relatively few applications for viral therapy, and is used clinically primarily for the treatment of multiple sclerosis. Recently, it has been found that poxviruses are more sensitive to IFN- β stimulation than IFN- α. IFN-gamma is the only type II interferon, also Th1 cells, CD8+ T cells, gamma delta T cells, NKT cells, NK cells, dendritic cells and macrophages produced by the marker cytokines, it is involved in the antiviral, antitumor and immune regulation of cells. IFN-gamma was originally recognized for its antiviral activity, and at present IFN-gamma has been widely studied for its use in antitumor applications.
Interferon alone has a range of side effects, and the associated toxicity of IFN is dose-dependent, with the most common side effects including fever, chills, weakness, headache, myalgia, joint pain, and influenza-like. Rational use of interferon is necessary to achieve different therapeutic effects while reducing side effects, and little research has been done on the use of interferon combinations.
Disclosure of Invention
In response to the deficiencies of the prior art, the inventors of the present application have found in experiments that the use of human IFN- β and IFN- γ therapy can significantly reduce the production and release of vaccinia virus (Tiantan strain). IFN- β has been used clinically for many years for the treatment of multiple sclerosis with reliable safety. In addition, IFN- β has both antiviral and immunomodulating effects compared to purely antiviral drugs, thus suggesting that interferon- β may be a novel and safe therapeutic approach for the prevention and/or treatment of human poxvirus infection. The IFN-gamma can treat bacterial infection, rheumatoid arthritis and the like combined with chronic granulomatous diseases, and also has wide application and reliable safety.
The technical problems solved by the invention are as follows: the synergistic effect of IFN-beta and IFN-gamma combination is utilized to excite host resistance to inhibit poxvirus infection and replication, thereby providing an anti-poxvirus pharmaceutical composition.
In order to solve the technical problems, the invention provides the following technical scheme:
a pharmaceutical composition for preventing and/or treating poxvirus infection and/or a disease caused thereby, comprising:
(1) Interferon-beta and interferon-gamma; or alternatively
(2) A coding gene of interferon-beta, a coding gene of interferon-gamma and a vector.
According to some embodiments of the invention, the mass ratio of IFN- β to IFN- γ, or the mass ratio of interferon- β to interferon- γ expressed by the encoding gene of interferon- β and the encoding gene of interferon- γ, is 1:1 to 1:90, preferably 1:1 to 1:60, more preferably 1:30.
according to some embodiments of the invention, IFN- β is of mammalian origin, such as human or murine origin.
According to some preferred embodiments of the invention, the nucleotide sequence of the gene encoding IFN- β is shown as SEQ ID No.1 and/or the amino acid sequence of IFN- β is shown as SEQ ID No. 2.
According to some embodiments of the invention, IFN-gamma is IFN-gamma of mammalian origin, e.g., IFN-gamma of human or murine origin.
According to some preferred embodiments of the invention, the nucleotide sequence of the IFN-gamma encoding gene is shown in SEQ ID No.3 and/or the amino acid sequence of IFN-gamma is shown in SEQ ID No. 4.
According to some embodiments of the invention, a pharmaceutical composition comprises at least one vector comprising a gene encoding IFN- β having a nucleotide sequence as set forth in SEQ ID NO.1 and a gene encoding IFN- γ having a nucleotide sequence as set forth in SEQ ID NO. 3; or the pharmaceutical composition comprises at least two vectors, wherein the vectors respectively comprise a coding gene of interferon-beta with a nucleotide sequence shown as SEQ ID NO.1 and a coding gene of interferon-gamma with a nucleotide sequence shown as SEQ ID NO. 3. The vector may be selected from one or more of a plasmid vector, poxvirus vector, adenovirus vector, adeno-associated virus vector, simple virus vector, CMV vector, cellular vector or bacterial vector.
According to some embodiments of the invention, the pharmaceutical composition comprises a protein comprising IFN- β having an amino acid sequence as set forth in SEQ ID NO.2 and IFN- γ having an amino acid sequence as set forth in SEQ ID NO. 4. The protein can be an IFN-beta protein with an independently expressed amino acid sequence shown as SEQ ID NO.2 and an IFN-gamma protein with an independently expressed amino acid sequence shown as SEQ ID NO. 4; or fusion proteins, such as IFN-beta with the amino acid sequence shown as SEQ ID NO.2 and IFN-gamma with the amino acid sequence shown as SEQ ID NO.4 and the Fc fragment of the antibody, or IFN-beta with the amino acid sequence shown as SEQ ID NO.2 and IFN-gamma with the amino acid sequence shown as SEQ ID NO.4 and the signal peptide. Furthermore, the proteins may also be modified with PEG.
According to some embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
In a further aspect, the present invention provides the use of a pharmaceutical composition according to the invention for the manufacture of a medicament for the prevention and/or treatment of poxvirus infections and/or diseases caused thereby.
According to some embodiments of the invention, the medicament is an injection, an ointment or an aerosol, preferably an ointment or an aerosol, most preferably an aerosol.
According to some embodiments of the invention, the poxvirus is an orthopoxvirus poxvirus; preferably, the poxvirus is selected from one or more of vaccinia virus, monkey poxvirus, molluscum contagiosum virus, yata poxvirus or smallpox virus; more preferably, the poxvirus is a monkey poxvirus. The monkey poxvirus is a causative agent of monkey pox, belonging to the genus orthopoxvirus of the family poxviridae, which is highly serologically related to smallpox virus and vaccinia virus.
According to some embodiments of the invention, the poxvirus-induced disease is selected from one or more of monkey pox, smallpox-like, vaccinia, milker's nodule, or molluscum contagiosum.
According to some embodiments of the invention, the agent is administered prior to the poxvirus infection, after the poxvirus infection, or continuously or intermittently from the pre-infection to the post-infection poxvirus infection.
Accordingly, the present invention provides a method for the prevention and/or treatment of poxvirus infections and/or diseases caused thereby comprising administering to a subject in need thereof a pharmaceutical composition according to the present invention.
According to some embodiments of the invention, the poxvirus is an orthopoxvirus poxvirus; preferably, the poxvirus is selected from one or more of vaccinia virus, monkey poxvirus, molluscum contagiosum virus, yata poxvirus or smallpox virus; more preferably, the poxvirus is a monkey poxvirus.
According to some embodiments of the invention, the poxvirus-induced disease is selected from one or more of monkey pox, smallpox-like, a vaccinia milker nodule, or molluscum contagiosum virus.
According to some embodiments of the invention, the pharmaceutical composition is administered before the poxvirus infection, after the poxvirus infection, or continuously or intermittently from before the poxvirus infection to after the infection.
The inventor discovers that IFN-beta and IFN-gamma can jointly inhibit the replication of vaccinia virus and show good antiviral synergistic effect. The invention provides a pharmaceutical composition containing IFN-beta and IFN-gamma and a novel application thereof based on the inhibiting function of IFN-beta and IFN-gamma on vaccinia virus replication.
The invention has the beneficial effects that: compared with the prior art, the invention discovers the pharmaceutical composition of IFN-beta and IFN-gamma and the new application thereof, namely, the combination of IFN-beta and IFN-gamma can obviously inhibit the replication of vaccinia virus, which has important clinical application value for preventing and treating the new and sudden diseases caused by poxviruses such as vaccinia virus, monkey poxvirus and the like.
It is understood that within the scope of the present invention, the above technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Is limited to space limitations and will not be described in detail herein.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows IFN- β, α1, α2, γ and κ vs human thymus kinase deficient cell line TK143 at concentrations of 1ng/mL, 27ng/mL and 81ng/mL - Influence of the state of the cells.
FIG. 2 shows IFN-. Beta.,. Alpha.1,. Alpha.2,. Gamma.and. Kappa. In human thymus kinase deficient cell lines TK143, respectively, at concentrations of 1ng/mL, 3ng/mL, 9ng/mL, 27ng/mL and 81ng/mL - The inhibition of vaccinia virus (Tiantan strain) replication.
Fig. 3 shows that the effective target ratio is 0, 1: 1. 1: 2. 1:4 and 1:8, IFN- β alone, IFN- γ alone, and IFN- β and IFN- γ in combination inhibit vaccinia virus replication by T cells.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The cells, medium, kit, etc. used in the examples described below are commercially available products unless otherwise specified.
Example 1: comparison of inhibition of vaccinia Virus infection by several factors IFN- β, α1, α2, γ, κ
1.TK143 - Preparation of cells: TK143 in 24 well plate - Cells (purchased from ATCC), 1E5 cells per well, cell density up to 90% of the bottom area of 24 well plate in use;
2. the complete medium in the 24-well plate was discarded, replaced with fresh complete medium, and IFN- β, α1, α2, γ and κ (IFN- β, α1, α2, γ were purchased from Beijing-like Qyowa technologies Co., ltd., IFN- κ was purchased from near-shore protein technologies Co., suzhou) concentrations of 1ng/mL, 3ng/mL, 9ng/mL, 27ng/mL and 81ng/mL, respectively, were added for pre-stimulation.
After 3.24 hours, the complete medium in the 24-well plate was discarded, replaced with new D2 medium (complete medium with 2% fbs), and factor and vaccinia virus dilution (moi=0.02) were added at the same concentration as in step 2, respectively, and placed in an incubator for cultivation.
After 4.24 h several different IFNs were observed for their effect on the cell status and the number and size of formed viral plaques.
The results showed that IFN- β had a significant inhibitory effect on vaccinia virus replication at a concentration of 1ng/mL relative to the other four IFN factors, the number of plaques was reduced from 34 plaques to 8 in the control group without interferon, and the plaque size and fluorescence intensity were also significantly reduced, and the inhibitory effect on vaccinia virus was enhanced with increasing IFN- β concentration (see FIG. 2 and Table 1), and it was not significantly damaging to cells (see FIG. 1). Other factors have no significant inhibitory effect on vaccinia virus replication at the same concentrations.
TABLE 1 comparison of inhibition of vaccinia virus infection by several IFNs
Note that: the larger the number of plus signs, the larger the plaques, the more intense the fluorescence.
Example 2: IFN- β and IFN- γ proteins have synergistic effects on inhibition of vaccinia virus infection
1.TK143 - Preparation of cells: TK143 in 24 well plate - 1E5 cells per well, and the cell density reaches 90% of the bottom area of a 24-well plate in use;
2. the complete medium in the 24-well plate was discarded, and the plates were replaced with a new complete medium and divided into 8 groups, which were a blank control group, a T cell treatment group, an IFN- β alone treatment group, an IFN- β plus T cell treatment group, an IFN- γ alone treatment group, an IFN- γ plus T cell treatment group, an IFN- β combined IFN- γ treatment group, and an IFN- β combined IFN- γ plus T cell treatment group, respectively, wherein the working concentration of IFN- β added was 0.3ng/mL and the working concentration of IFN- γ was 9ng/mL.
After 3.24 hours, the complete medium in the 24-well plate was discarded, replaced with new D2 medium (complete medium with 2% fbs), and factor and vaccinia virus dilution (moi=0.02) were added at the same concentration as in step 2, respectively, and placed in an incubator for cultivation.
The effect on cell status and the number and size of plaques forming viruses was observed after 4.24 h for the different groups after infection with vaccinia virus.
The results show that the independent T cell group has obvious inhibition on virus replication, and the inhibition on vaccinia virus is more obvious with the increase of the number of the T cells; but the local target ratio (effector cells (T cells, important lymphocytes present in the human body) is compared with the target cells (vaccinia virus-infected cells, TK 143) - Cell line) is 1): 4 or 1:8, the effect of inhibiting viral replication is reduced, in which case inhibition of poxvirus replication is markedly increased by IFN- β and IFN- γ treatment. The effective target ratio is 1:4, the plaque number is reduced to 3 from 16 plaques of the untreated control group, and the plaque size and fluorescence brightness are also obviously reduced; in contrast, there were 40 IFN- β treated groups alone, 48 IFN- γ treated groups alone, 7 IFN- β plus T cell groups alone and 20 IFN- γ plus T cell groups alone. The effective target ratio is 1: at 8, the number of plaques of IFN- β+IFN- γ was reduced from 30 plaques in the untreated control group to 10 plaques; in contrast, there were 14 IFN- β plus T cell groups and 34 IFN- γ plus T cell groups (see FIG. 3 and Table 2). Thus, the IFN-beta and IFN-gamma combination has obvious synergistic effect and can obviously enhance the anti-vaccinia virus capability.
Comparing the results of FIG. 2 (Table 1) with that of FIG. 3 (Table 2), it is clear that IFN-. Gamma.alone does not inhibit vaccinia virus activity at a working concentration of 9ng/mL, whereas the effect of inhibiting IFN-. Beta.alone at a working concentration of 1ng/mL is comparable to that of IFN-. Beta.at a working concentration of 0.3ng/mL in combination with 9ng/mL, i.e., the dose of IFN-. Beta.can be reduced by a factor of 3. In general, the pharmaceutical compositions provided by the present invention can achieve an effective therapeutic goal even at a greatly reduced dosage of interferon, which is important for reducing side effects of interferon.
TABLE 2 IFN- β and IFN -γ Inhibition of vaccinia virus infection
Note that:
IFN-β：0.3ng/ml
IFN -γ ：9ng/ml
TK143 - and (3) cells: 1E5
The larger the number of plus signs, the larger the plaques, the more intense the fluorescence.
The foregoing has shown and described the basic 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 foregoing embodiments, and that the foregoing embodiments and description are merely illustrative of the principles of the invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, and these changes and modifications fall within the scope of the claimed invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A pharmaceutical composition for preventing and/or treating poxvirus infection and/or a disease caused thereby, comprising:
(1) Interferon-beta and interferon-gamma; or alternatively
(2) A coding gene of interferon-beta, a coding gene of interferon-gamma and a vector.
2. The pharmaceutical composition of claim 1, wherein the mass ratio of interferon- β to interferon- γ, or the mass ratio of interferon- β to interferon- γ expressed by the encoding gene of interferon- β and the encoding gene of interferon- γ is 1:1 to 1:90, preferably 1:1 to 1:60, more preferably 1:30.
3. the pharmaceutical composition according to claim 1 or 2, wherein the interferon- β is of mammalian origin, such as of human or murine origin;
preferably, the nucleotide sequence of the encoding gene of the interferon-beta is shown as SEQ ID No.1, and/or the amino acid sequence of the IFN-beta is shown as SEQ ID No. 2;
preferably, the interferon-gamma is of mammalian origin, such as interferon-gamma of human or murine origin;
preferably, the nucleotide sequence of the coding gene of the interferon-gamma is shown as SEQ ID No.3, and/or the amino acid sequence of the IFN-gamma is shown as SEQ ID No. 4.
4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises at least one vector comprising a gene encoding interferon- β having the nucleotide sequence shown in SEQ ID No.1 and a gene encoding interferon- γ having the nucleotide sequence shown in SEQ ID No. 3; or the pharmaceutical composition comprises two vectors, wherein the vectors respectively comprise a coding gene of interferon-beta with a nucleotide sequence shown as SEQ ID NO.1 and a coding gene of interferon-gamma with a nucleotide sequence shown as SEQ ID NO. 3;
preferably, the vector is selected from one or more of a plasmid vector, poxvirus vector, adenovirus vector, adeno-associated virus vector, simplicial virus vector, CMV vector, cellular vector or bacterial vector.
5. A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises a protein comprising IFN- β having the amino acid sequence shown in SEQ ID No.2 and IFN- γ having the amino acid sequence shown in SEQ ID No. 4;
preferably, the proteins are IFN-beta proteins with the amino acid sequences shown in SEQ ID NO.2 and IFN-gamma proteins with the amino acid sequences shown in SEQ ID NO.4 which are expressed independently; or fusion proteins, such as IFN-beta with the amino acid sequence shown as SEQ ID NO.2 and IFN-gamma with the amino acid sequence shown as SEQ ID NO.4 and the Fc fragment of the antibody, or IFN-beta with the amino acid sequence shown as SEQ ID NO.2 and IFN-gamma with the amino acid sequence shown as SEQ ID NO.4 and the signal peptide;
preferably, the protein is modified with PEG.
6. The pharmaceutical composition of any one of claims 1 to 5, further comprising a pharmaceutically acceptable adjuvant.
7. Use of a pharmaceutical composition according to any one of claims 1 to 5 for the preparation of a medicament for the prevention and/or treatment of poxvirus infections and/or diseases caused thereby;
preferably, the medicament is an injection, an ointment or an aerosol, preferably an ointment or an aerosol, most preferably an aerosol.
8. The use of claim 7, wherein the poxvirus is an orthopoxvirus poxvirus; preferably, the poxvirus is selected from one or more of vaccinia virus, monkey poxvirus, molluscum contagiosum virus, yata poxvirus or smallpox virus; more preferably, the poxvirus is a monkey poxvirus.
9. The use according to claim 7 or 8, wherein the poxvirus-induced disease is selected from one or more of monkey pox, smallpox, vaccinia, a milker's nodule, or molluscum contagiosum.
10. The use according to any one of claims 7 to 9, wherein the medicament is administered before, after, or continuously or intermittently from before to after the poxvirus infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211247894.5A CN117860870A (en) | 2022-10-12 | 2022-10-12 | Pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211247894.5A CN117860870A (en) | 2022-10-12 | 2022-10-12 | Pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and use thereof |
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| CN202211247894.5A Pending CN117860870A (en) | 2022-10-12 | 2022-10-12 | Pharmaceutical composition for preventing and treating poxvirus infection and diseases caused by poxvirus infection and use thereof |
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| Country | Link |
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| CN (1) | CN117860870A (en) |
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