OA18489A - Immunobiological agent and utilization method thereof for inducing specific immunity against Ebola virus (variants). - Google Patents

Immunobiological agent and utilization method thereof for inducing specific immunity against Ebola virus (variants). Download PDF

Info

Publication number
OA18489A
OA18489A OA1201700364 OA18489A OA 18489 A OA18489 A OA 18489A OA 1201700364 OA1201700364 OA 1201700364 OA 18489 A OA18489 A OA 18489A
Authority
OA
OAPI
Prior art keywords
pfu
mouse
vsv
seq
ebola virus
Prior art date
Application number
OA1201700364
Inventor
Denis Jurievich LOGUNOV
Maksim Mikhailovich SHMAROV
Irina Leonidovna TUTYKHINA
Olga Vadimovna ZUBKOVA
Dmitry Viktorovich SCHEBLYAKOV
Dmitry Nikolaevich SCHERBININ
Inna Vadimovna DOLZHIKOVA
Alina Shakhmirovna DZHARULLAEVA
Natalya Mikhailovna ARTEMICHEVA
Amir Ildarovich TUKHVATULIN
Vladimir Borisovich PANTJUKHOV
Svetlana Ivanovna SYROMYATNIKOVA
Irina Viktorovna SHATOKHINA
Boris Savelievich NARODITSKY
Aleksandr Leonidovich GINTSBURG
Sergey Vladimirovich Borisevich
Original Assignee
Federalnoe Gosudarstvennoe Bjudzhetnoe Uchrezhdenie "Federalny Nauchno-Issledovatelsky Tsentr Epidemiologii I Mikrobiologii Imeni Pochetnogo Akademika N.F. Gamalei" Ministerstva Zdravookhranenia Rossiiskoi Federatsii
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Federalnoe Gosudarstvennoe Bjudzhetnoe Uchrezhdenie "Federalny Nauchno-Issledovatelsky Tsentr Epidemiologii I Mikrobiologii Imeni Pochetnogo Akademika N.F. Gamalei" Ministerstva Zdravookhranenia Rossiiskoi Federatsii filed Critical Federalnoe Gosudarstvennoe Bjudzhetnoe Uchrezhdenie "Federalny Nauchno-Issledovatelsky Tsentr Epidemiologii I Mikrobiologii Imeni Pochetnogo Akademika N.F. Gamalei" Ministerstva Zdravookhranenia Rossiiskoi Federatsii
Publication of OA18489A publication Critical patent/OA18489A/en

Links

Abstract

The invention relates to immunology and virology, and can be used as a specific prophylactic agent against diseases caused by the Ebola virus. An immunobiological agent created based on recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 1). There is also an immunobiological agent created based on recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 2). There is also an immunobiological agent created based on recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus/H. sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 with a sequence SEQ ID No: 3 and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebolavirus/H.sapiens-wt/SLE/ 2014 /Makona-EM124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 taken in effective proportions (variant 3). A utilization method of variant 1 immunobiological agent: it is administered to mammals in an effective amount to induce specific immunity against the Ebola virus. A utilization method of variant 2 immunobiological agent: it is administered to mammals in an effective amount to induce specific immunity against the Ebola virus. A utilization method of variant 3 immunobiological agent: it is administered to mammals in an effective amount to induce specific immunity against the Ebola virus. A utilization method of variant 1 and variant 2 immunobiological agent: consequential administration to mammals with >lweek interval to induce specific immunity against the Ebola virus. A utilization method of variant 1 and variant 3 immunobiological agent: consequential administration to mammals with >lweek interval to induce specific immunity against the Ebola virus.

Description

Field of the Invention
The invention relates to immunology and virology, and can be used as a spécifie prophylactic agent against diseases caused by the Ebola virus.
Background of the Invention
Ebola haemorrhagic fever is a highly contagîous acute viral disease caused by the Ebola virus, which mortality rate cornes up to 90%. The virus is transmitted to people from wîld animais and then spreads from person to person. The disease was first identified in 1976. Since that time, there were more than 10 outbreaks of Ebola fever, the largest of which was in 2014 and !ed to an épidémie sweeping the major cities and rural areas of five countries in West and Centra! Africa, and had killed more than nine thousand people. Currently, there are no vaccinesto protect against Ebola virus disease licensed for use in humans approved.
One of the most promising developments in this area is a type of vaccines based on the vira! vectors containing genes of the Ebola virus protective antigens.
There is a solution according to the patent WO 2011130627 A2, which suggests to induce an immune response to fïloviruses (Ebola virus, Marburg virus) by using chimpanzee adenoviruses that contain genes of different virus proteins including the protein of the Ebola virus GP.
There is a solution according to the patent CA 2821289 Al, which suggests to induce an immune response against fïloviruses by using recombinant adénoviral vector based on adenovirus of 26 and 35 serotype which are expressing filovirus antigens.
There is a solution according to the patent US20100047282 Al, which suggests to use recombinant adenovirus containing glycoprotein gene of the Ebola virus (GP) as a vaccine against Ebola hémorrhagie fever.
There is a solution according to the patent CA 2493142 C, which suggests to stimulate the immune response to the Ebola virus by using recombinant vesicular stomatitis virus (VSV) containing the gene of glycoprotein selected from the group consisting of Lassa virus glycoprotein, Marburg virus glycoprotein, and Ebola virus glycoprotein inserted into the vira! genome in such a way that this gene sequence replaces the original gene of VSV glycoprotein.
There is an agent, application US 20060269572 Al, containing two recombinant human adenoviruses of serotype 5, wherein the first adenovirus contains the GP gene ofthe Ebola virus, and the second adenovirus contains the NP gene of the Ebola virus. A method of individual immunization using such agent is also described.
However, when using unmodified nucléotide sequences a réduction of immunogenicity is observed comparing to the proteins produced using the modified nucléotide sequence. As a resuit they get an agent with low spécifie immunogenicity.
The authors of the claimed invention chose as a prototype the technical solutions according to the patents US20100047282 Al and CA 2493142 C as the most similar considering the content and method of administration. Due to the fact that at the date of this applications on the national level, 2 Augjist 2004 and 26 July 2002, there were no available information about the strain of Ebola/H.sapiens-wt/SLE/2014/Makona-EM 124.1 GenBank ID KM233045.1, since this pathogen was first isolated and characterized in July 2014, the nucléotide sequence of its genes could not been used to construct recombinant viruses. Accordingly, the applicants hâve not been able to create a construction with the characterized cultural-morphologîcal features, antigen expression, and immunogenic properties. This is a significant drawback of these structures, since it is not possible to use them to produce spécifie immunîty to the virus Ebola/H.sapienswt/SLE/2014/Makona-EM 124.1 GenBank ID KM233045.1.
Disclosure of the Invention
The aim of each variant ofthe présent invention is to create an immunobiologicaï agent for induction of immune response effective against the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1.
To solve this problem, an immunobiologicaï agent has been created based on recombinant adenovirus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 1).
There is also created an immunobiologicaï agent based on recombinant vesicular stomatîtis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /MakonaEM 124.1 GenBank ID KM233045.Ï with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 2).
There is also created an immunobiologicaï agent based on recombinant vesicular stomatîtis virus containing the non-modified GP gene of the Ebola virus/H.sapienswt/1976/Mayinga/ZaïreGenBank ID AF086833.2 with a sequence SEQ ID No:3 and the recombinant vesîcular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124. lGenBank ID KM233045.I with a sequence selected from SEQ ID No:I, SEQ ID No:2 taken in effective ratios (variant 3).
A utilization method of variant 1 immunobiological agent: it is administered to mammals in an effective amount to înduce spécifie immunity against the Ebola virus.
A utilization method of variant 2 immunobiological agent: it is administered to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
A utilization method of variant 3 immunobiological agent: it is administered to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
A utilization method of variant 1 and variant 2 immunobiological agent: consequential administration to mammals with >l-week interval to induce spécifie immunity against the Ebola virus.
A utilization method of variant 1 and variant 3 immunobiological agent: consequential administration to mammals with >l-week interval to induce spécifie immunity against the Ebola virus.
Description of the sequences listing
SEQ ID No:l is a nucléotide sequence of GP1 gene which is sequence of the GP gene of the Ebola virus/H.sapiens-wt/SLE/2014 /Makona-EM 124.1 GenBank ID KM233045.1 modified by addîng a single adenine to a sequence. A detailed description of the modification is presented in claim “The implémentation of the invention”.
SEQ ID No:2 is a nucléotide sequence of GP2 gene which is the sequence of the GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 modified by replacement of codons which are rarely présent in human body with more common codons. A detailed description of the modification is presented in claim “The implémentation of the invention”.
SEQ ID No:3 is a nucléotide sequence of GP3 gene which is a non-modified sequence of the GP gene of the Ebola virus/H.sapiens-wt/1976/Mayinga/ZaîreGenBank ID AF086833.2.
SEQ ID No:4 is an non-modified nucléotide sequence of the GP gene of the Ebola virus/.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1.
SEQ ID No:5 is a nucléotide sequence which is an expression cassette containing the GP gene sequence of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 modified by adding a single adenine to a sequence.
SEQ ID No:6 is a nucléotide sequence which is an expression cassette containing the GP gene sequence of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID
KM233045.I modified by adding a single adenine to a sequence.
Short description of the figures
Figure 1 illustrâtes the results of quality testing of prepared immunobiological agent based on recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapienswt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I with a sequence selected from SEQ ID No:l, SEQ ID No:2 by determining GP expression in HEK 293 cells after addition of the mentioned immunobiological agent.
— cell lysate, to which phosphate bufiered saline was added — cell lysate, to which Ad-null was added — cell lysate, to which Ad-wt was added — cell lysate, to which Ad-GP 1 was added — cell lysate, to which Ad-GP2 was added
Figure 2 illustrâtes the results of quality testing of prepared immunobiological agent based on recombinant vesicular stomatitis containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I with a sequence selected from SEQ ID No:l, SEQ ID No:2 by determining GP expression in HEK 293 cells after addition ofthe mentioned immunobiological agent.
— cell lysate, to which phosphate buffered saline was added — cell lysate, to which VSV-null was added — cell lysate, to which VSV-wt was added — cell lysate, to which VSV-GP1 was added — cell lysate, to which VSV-GP2 was added
Figure 3 illustrâtes the results of quality testing of prepared immunobiological agent based on recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus /H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 SEQ ID No:3 and the recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No: 1, SEQ ID No:2 by determining GP expression in HEK 293 cells after addition of the corresponding immunobiological agent.
— cell lysate, to which phosphate buffered saline was added — cell lysate, to which VSV-null was added — cell lysate, to which VSV-wt was added — cell lysate, to which VSV-GP1 was added — cell lysate, to which VSV-GP2 was added — cell lysate, to which VSV-GP3 was added
Figure 4 illustrâtes the results of immunization efficacy testing of prepared immunobiological agent based on recombinant adenovirus containîng the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 by analysis of lymphoprolifération.
Y-axis — the number of proliferatîng cells, %
X-axis — different groups of animais treated with
1. Phosphate buffer (100 μΐ)
2. Ad-null 10® PFU/mouse
3. Ad-GPl 108 PFU/mouse
4. Ad-GP2 108 PFU/mouse
Figure 5 illustrâtes the results of immunization efficacy testing of prepared immunobiological agent based on recombinant vesicular stomatitis virus containîng the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 by analysis of lymphoprolifération.
Y-axîs — the number of proliferatîng cells, %
X-axis — different groups of animais treated with
1. Phosphate buffer (100 μΐ)
2. VSV-null 107 PFU/mouse
3. VSV-GP1 107 PFU/mouse
4. VSV-GP2 107 PFU/mouse
Figure 6 illustrâtes the results of immunization efficacy testing of prepared immunobiological agentbased on recombinant vesicular stomatitis virus containîng the nonmodified GP gene of the Ebola virus /H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombinant vesicular stomatitis virus containîng the modified
GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM124.J GenBank ID
KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 by analysis of lymphoprolifération.
Y-axis — the number of proliferating cells, %
X-axis — different groups of animais treated with
1. Phosphate buffer (100 μΐ)
2. VSV-null 107 PFU/mouse
3. VSV-GP1 107 PFU/mouse, VSV-GP3 I07 PFU/mouse
4. VSV-GP1 106 PFU/mouse, VSV-GP3 109 PFU/mouse
5. VSV-GP1 109 PFU/mouse, VSV-GP3 106 PFU/mouse
6. VSV-GP2 I07 PFU/mouse, VSV-GP3 107 PFU/mouse
7. VSV-GP2 106 PFU/mouse, VSV-GP3 109 PFU/mouse
8. VSV-GP2 I09 PFU/mouse, VSV-GP3 106 PFU/mouse
The implémentation of the invention
Glycoprotein GP of the Ebola virus is one of the most promising vaccine antigens; induction of immune response with this antigen provides protection against Ebola fever. In order to achieve the most effective induction of immune response, the authors modified the nucléotide sequence of the GP of Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1. Two types of modified gene were obtained: GP1 (SEQ ID No:l) and GP2 (SEQ ID No:2).
GP1 (SEQ ID No:l) was modified by replacing the sequence “ACTAAAAAAACCT” with “ACTAAAAAAAACCT’ which differ in one nucléotide (adenine). The GP gene sequence was changed according to data available in the literature that at this point a loop is formed which is a complex structure for the passage of L-polymerase of the Ebola virus; unlike the polymerase of eukaryotic cells, additional nucléotide is inserted at this site which shifts the reading frame and changes the amino acid sequence of protein. Consequently, in order to get the fui! version of GP protein in eukaryotic cells it is necessary to add one nucléotide in the mentioned sequence.
GP2 (SEQ ID No:2) has been obtained using the same method as for GP1 — by replacing the sequence “ACTAAAAAAACCT” with “ACTAAAAAAAACCT’, and then additionally optimized for expression in human cells. Optimization of the nucléotide sequence is a process in which codons rarely présent in human body are replaced with common codons. The more common codon is, the greater the amount of corresponding tRNA in cells, and amino acids will be faster transported to the site of protein synthesis. Therefore, optimization of the sequence provides a higher rate of protein synthesis.
Then the authors hâve developed several designs based on fiflh serotype of human adenovirus and vesicular stomatitis virus in order to efiîciently deliver modified GP genes into mammalian cells.
Variant 1 invention is the recombinant adenovirus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2.
This viral vector is one of the safest and most effective vectors for humans (VanKampenK.R. et al., Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous înfluenza vaccines in humans, Vaccine, 2005, No.23 (8) p. 1029-1036). Recombinant adenoviruses hâve the following advantages: not able to replicate in human cells, able to penetrate both dividing and nondividing cells, induce both cellular and humoral immune response, provide a high level ofexpression ofthe target antigen.
Variant 2 invention is the recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapîens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2.
In this invention the glycoprotein gene in the genome of a recombinant vesicular stomatitis virus (VSV) was replaced with the glycoprotein gene of Ebola virus. When assembling VSV virus, the glycoprotein of Ebola virus is incorporated in a viral particle shell and exposed on the surface, and therefore it is effectively recognized by the immune system. Moreover, when a virus enters a cell this protein is expressed on its membrane. Such cells are recognized as infected by the immune system, and immune response against the GP protein develops.
In general, it can be said that perfect viral vector does not exist, and choice of the vector type primarily based on the target population specificity. Conduction of additional tests may be useful before immunizatîon to détermine the titer of antibodies to spécifie viral vector.
Variant 3 invention is also based on the vesicular stomatitis virus and includes two vectors, one of which contains the GP gene of Ebola virus/H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 with a sequence SEQ ID No:3, another one contains the modified GP gene of Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No: 1, SEQ ID No:2 taken in effective proportions.
Thus, this immunobiological agent provides simultaneous expression of two types of glycoproteins in mammalian body derived from different strains of the Ebola virus. Therefore, it is more versatile and induces immune responses against two most dangerous strains of the Ebola virus.
The authors hâve developed several utilization methods of these immunobiological agents. Utilization methods of inventions presented hereinafter in claims 4, 5 and 6 involve administration of immunobiological agents for each ofthe 3 variants in mammals individually in an effective amount to induce spécifie immunity against the Ebola virus. This method is suitable for induction of immune response in the general population.
However, there are categories of people (healthcare workers, people living within the area of épidémie) with increased risks of Ebola virus infection. For this category there were developed methods described in claims 7 and 8 which involves sequential immunization (at intervals more than 1 week) with immunobiological agents of variant 1 and variant 2 (claims 1 and 2), or variant 1 and variant 3 (claims 1 and 3). These methods are very effective since immunization with various viral vectors induces strong immune response. However, in tum, immunization of general population by this method may be economically impractical.
Examples.
Example 1.
Création of different nucléotide sequences of Ebola virus glycoprotein.
GP is the most immunogenic protein of the Ebola virus and the most promîsing vaccine antigen. Authors developed variants of immunobiological agent which include modified sequence of the GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 selected from SEQ ID No:l (GP1), SEQ ID No:2 (GP2) or nonmodified sequence /H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 SEQ ID No:3 (GP3).
Modification of sequence was performed as described above. AH nucléotide sequences were obtained by synthesis.
Example 2.
Obtaining of immunobiological agent based on recombinant adenovirus containing the modified GP gene ofthe Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:I, SEQ ID No:2 (variant 1).
Two plasmid constructions including different variants of the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I: pShuttleGPI (contains SEQ ID No:l sequence), pShuttle-GP2 (contains SEQ ID No:2 sequence) and one plasmid construction which contains original non-modîfied control pShuttle-GPwt, SEQ ID No:4 sequence were constructed to obtain genetically engineered recombinant adenovirus.
Then the above-described plasmid constructions were processed with the restriction endonucleases Pacl and BstII07I to extract expression cassettes. cAd5-EGFP cosmid was also processed with Pacl and BstII07l endonucleases. Hydrolysis products were lîgated. The obtained DNA contained in the ligation mixture was packaged into phage heads. Then DH5a competent cells were transduced with phage heads. Transformed cells were selected by their growth in LB solid medium with a sélective antibiotic. Plasmid DNA were isolated from the grown cells. Thus 3 cosmîds were obtained. cAd5-GPl contains recombinant human adenovirus genome of 5th serotype with an expression cassette (SEQ ID No:5) containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I SEQ ID No:l. cAd5-GP2 contains recombinant human adenovirus genome of 5th serotype with an expression cassette SEQ ID No:6 containing the modified GP gene ofthe Ebola virus/H.sapienswt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 SEQ IDNo:2. cAd5-GPwt contains recombinant human adenovirus genome of 5th serotype with an expression cassette containing the non-modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I
Next step included hydrolysis of obtained cosmides with endonucleases of Swal and Pacl restriction in order to remove the plasmid part and transfection of HEK 293 eukaryotic cells. On Day 5 after transfection, material blind passages were conducted for recombinant adenoviruses multiplication. After détection of viral cytopathic effect (mîcroscopy data) cells with culture medium were overfreezed three rimes to disrupt cells and release virus. As a resuit, biomaterial was obtained which was subsequently used for accumulation of préparative amounts of recombinant adenoviruses.
Thus, as the resuit, 3 recombinant adenoviruses were obtained: Ad-GPI, Ad-GP2, AdGPwt.
Example 3.
Obtaining of immunobiologîcal agent based on the recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /MakonaEM124.1 GenBank ID KM233045.I with a sequence selected from SEQ ID No:I, SEQ ID No:2 (variant 2).
VSV-deltaG-GFP plasmid was used to obtain recombinant vesicular stomatitis virus, which is a cDNA complementary to genomîc RNA of vesicular stomatitis virus with removed glycoproteîn gene. Two plasmid vectors were obtained using genetic engineering and included the sequence ofthe modified GP gene: pVSV-deltaG-GPl (contains SEQ ID No:I sequence), pVSV-deltaG-GP2 (contains SEQ ID No:2 sequence), and one plasmid construction which contains control initial non-modified sequence: pVSV-deltaG-GPwt (contains SEQ ID No:4 sequence).
Transfection of CV-I cells was performed in order to obtain préparations of recombinant vesîcular stomatîtis viruses using 4 plasmids: pdVSV-N (contains nucleocapsid protein genes), VSV-P (contains phosphoprotein gene), VSV-L (contains large subunit of polymerase gene), and vector plasmid selected from pVSV-deltaG-GPl, pVSV-deltaG-GP2, pVSV-deltaG-GPwt. Transfection was performed usîng Lipofectamine-2000 reagent 10 (LifeTechnologies) according to manufacturer's instructions.
Thus, three agents of recombinant vesîcular stomatîtis virus were obtained: VSV-GP1, VSV-GP2, VSV-GPwt.
Example 4.
Obtaining of immunobiological agents based on the recombinant vesîcular stomatîtis virus contaîning the * non-modified GP gene of the Ebola virus /H.sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and vesîcular stomatîtis virus contaîning the modified GP gene of Ebola /H.sapîens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant
3).
Recombinant vesîcular stomatîtis viruses contaîning the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/2014 /Makona-EM 124.1 GenBank ID KM233045.1 (VSV-deltaG-GPI, VSV-deltaG -GP2) and vesîcular stomatîtis virus contaîning the non-modified GP gene of the Ebola virus/H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 (VSV-deltaG-GP3) were received for this immunobiological préparation. Virus contaîning the non-modified sequence of Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I(VSV-deltaG-GPwt) was obtained as a control. Ail viruses were obtained by the procedure described in Example 2.
To obtain the immunobiological agent the following components were mixed:(i) the recombinant vesîcular stomatîtis virus contaîning the non-modified GP gene of the Ebola virus/H.sapiens30 wt/1976/Mayinga/ZaireGenBank ID AF086833.2. and (ii)the recombinant vesîcular stomatîtis virus contaîning the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /MakonaEM124.1 GenBank ID KM233045.1 selected from the VSV-deltaG-GPI, VSV-deltaG-GP2.
At 1:1000, 1:100,1:10, 1:1, 10:1, 100:1,1000:1 ratios.
As a resuit of this work, various versions of immunobiologica! agent were received according to claim 3.
Example 5.
Détermination of GP protein expression in cells after addition of immunobiological agent based on recombinant adenovirus containing the modifïed GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2.
HEK 293 cells were cultured in DMEM medium containing 10% fêtai calf sérum in incubator at 37 °C and 5% CO2. Cells were seeded into 35 mm2 Pétri dishes and incubated for 24 hours until reaching 70% confluence. Then recombinant adenoviruses (Ad-GPl, Ad-GP2,) and control samples (Ad-null — recombinant adenovirus containing no inserts, Ad-GPwt — recombinant adenovirus containing not optimized sequence GP) were added to the cells at 100 PFU/cell. Phosphate buffer was used as a négative control. 24 hours later expression of Ebola virus antigens was determined by immunoblotting. For this purpose, the medium was removed from cells, the cells were washed with stérile PBS and then lysed using RIPA buffer (Pierce) according to manufacturer^ protocol.
After cell lysis a total protein concentration was measured in the samples using Bradford (Sigma) reagent according to manufacturées instructions. Aliquots of prepared samples normalized byprotein concentration were mixed with Laemmli Sample Buffer (Sigma) and incubated for 5 minutes at 95 °C.
Then electrophoresis was performed under dénaturaiing conditions in 10% SDSpolyacrylamide gel SPRINT NEXT GEL® 10% (Amresco) in NEXT GEL® Running Buffer, 20X (Amresco) using Mini-PROTEAN® Tetra Cell system (Bio-Rad) for 20 minutes at 250V. After electrophoresis, proteins were transferred onto nitrocellulose membrane Trans-Blot® Turbo ™ Mini Nitrocellulose Transfer Packs (Bio-Rad) in Tris-CAPS buffer (Bio-Rad) using a transfer system Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad) for 15 minutes at 25V. Then the nonspecific signal was blocked. For this purpose, the membrane was incubated in solution of 3% nonfat milk in PBS with 0.05% Twin20 (T-PBS) for one hour at room température. The membrane was then incubated for 16 hours at 4 °C with antibodies against GP protein in 3% solution of nonfat milk in T-PBS. Then washing was performed with T-PBS solution, and membrane was treated with a solution of secondary antibodies conjugated to horseradish peroxidase in 3% nonfat milk solution in T-PBS at 37 °C on a shaker for 1 hour. The membrane was then washed thoroughly with T-PBS solution. Further détection was performed using Clarity™ Western ECL Substrate (Bio-Rad) kit and the Hyperfilm® ECL™ (Amersham) film.
The same protocol was used for détection of comparing GAPDH protein using anti- GAPDH antibodies.
The results are shown in Figure 2.
— cell lysate, to which phosphate buffered saline was added — cell lysate, to which Ad-null was added — cell lysate, to which Ad-wt was added — cell lysate, to which Ad-GPl was added — cell lysate, to which Ad-GP2 was added
As seen from the obtained data, in ail cells transduced with recombinant adenoviruses containing both modified and non-modified GP gene of Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM124.1 GenBank ID KM233045.I with a sequence selected from SEQ ID No:l, SEQ ID No:2, expression of target GP protein was observed. In the cells treated with recombinant adenoviruses containing modified GP genes (SEQ ID No:l, SEQ ID No:2) there were higher levels of expression than in cells transduced with adenovirus expressing the non-modified GP gene (SEQIDNo:4).
Example 6.
The purpose of this experiment was to compare the immunogenicïty of recombinant proteins GPI and GP2 obtained after administration of the modified GP gene sequence into cells-producers (selected from SEQ ID No:l, SEQ ID No:2, respectively) with immunogenicity of GP3 protein obtained after administration of the non-modified GP gene sequence into cellsproducers (SEQ ID No:3). Proteins were isoiated from the cells-producers and purified using chromatography methods.
Immunogenicity of proteins was assessed by the formation of spécifie antibodies after two immunizations in mice BALB/c (dose 10 pg/mouse) at intervals of 3 weeks. One week after the last immunization, the blood was collected from the animais. Further, the tubes with blood were kept for 30 minutes in a thermostat at 37 *C, and then the sérum was collected. The titer of spécifie antibodies was determined by ELISA. Sérum samples were diluted by two-fold dilution method starting from 1:200. A total of 8 dilutions of each sample was prepared, each or which in amount of 50 pl was added to the wells of the plate with pre-adsorbed GP protein. Then it was încubated for I hour at 37 °C.
After incubation, the wells were washed and the secondary antibodies against mouse immunoglobulins were added conjugated with biotin. After incubation and washing a conjugale of horseradish peroxîdase and streptavidin was added to the wells. In the final phase, after washing the wells, a solution of TMB was added which is a horseradish peroxîdase substrate and, as a resuit of reaction, is converted into a colored compound. The reaction was stopped after 15 minutes by the addition of sulfuric acid. Next, the solution absorbance was measured using a spectrophotometer (OD) in each well with wavelength of 450 nm. Antibodies titer was determined as the last dilution at which the optical density of the solution was two-fold higher than the optical density in the négative control group (intact animais). The results are presented in Table 1.
Table 1— titer of spécifie antibodies against GP protein in mouse sérum
Protein which was used for immunization Averange titer of spécifie antibodies
GPI 1:800
GP2 1:400
GP3 1:200
As shown in Table 1, immunization with ail proteins resulted in formation of spécifie antibodies. The highest titers of antibodies to the GP were detected in animais immunized with modified proteins GPI, GP2 (compared to the unmodified protein GP3).
Thus, the results presented in this example show that modification of the nucléotide sequence of GP gene leads to increased immunogenîcity of the protein itself independently of its expression levels (ali proteins hâve been taken in équivalent amounts).
Example 7.
Détermination of GP protein expression in cells after addition of immunobiological agent based on the recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/S LE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2. HEK 293 cells were cultured in DMEM medium containing 10% fêtai calf sérum in incubator at 37 °C and 5% CO2. Cells were seeded into 35 mm2 Pétri dishes and incubated for 24 hours until reaching 70% confluence. Then préparations of recombinant vesicular stomatitis virus (VSV-GP1, VSV-GP2) and control préparations (VSV-null — recombinant vesicular stomatitis virus, containing no inserts, VSVGPwt — recombinant vesicular stomatitis virus, containing non-optimized sequence of GP) were added to the cells in amount 10 PFU/cell.Phosphate buffer was used as a négative control. A day later expression of antigens was determined by immunoblotting of Ebola virus, similar to the protocol described in Example 4.
The results are shown in Figure 3.
— cell lysate, to which phosphate buffered saline was added — cell Iysate, to which VSV-null was added — cell Iysate, to which VSV-GPwt was added — ce!! Iysate, to which VSV-GP1 was added — ce!! Iysate, to which VSV-GP2 was added
Experimental results showed that al! cells transduced by recombinant vesicular stomatitis viruses containing both modified and non-modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233O45.I. express GP proteins. Furthermore, those cells which were treated with recombinant vesicular stomatitis viruses containing the modified GP genes had higher expression levels.
Example 8.
Détermination of GP protein expression in cells after addition of immunobiological preparation based on recombinant vesicular stomatitis virus containing the non-modified GP gene /H.sapiens-wt/I976/Mayinga/ZaireGenBank ID AFO86833.2 (SEQ ID No:3) and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045 with a sequence selected from SEQ ID No:I, SEQ ID No:2.
HEK 293 cells were cultured in DMEM medium with containing 10% fêtai calf sérum in incubator at 37 °C and 5% CO2. Cells were seeded into 35 mm2 Pétri dishes and incubated for 24 hours until reaching 70% confluence. Then recombinant vesicular stomatitis viruses, which were the components ofthe developed immunobiological préparations (VSV-deltaG-GPl, VSVdeltaG-GP2, VSV-deltaG-GP3, VSV-deltaG-GP4, VSV-de!taG-GP5) and control préparations (VSV-deltaG-null — recombinant adenovirus containing no înserts, VSV-deltaG-GPwt — recombinant adenovirus containing non-optimîzed GP sequence) were added to the cells (10 PFU/cell). Phosphate buffer was used as a négative control. A day later expression of antigens was determined by immunoblotting, similar to the protocol described in Example 4.
The results are shown in Figure 3 — cell Iysate, to which phosphate buffered saline was added — cell Iysate, to which VSV-null was added — cell Iysate, to which VSV-GPwt was added — cell Iysate, to which VSV-GP1 was added — cell Iysate, to which VSV-GP2 was added — cell Iysate, to which VSV-GP3 was added
The experimental results showed that ail cells transduced with recombinant vesicular stomatitis viruses containing GP gene had expression of Ebola virus glycoprotein. Maximum expression was observed in the groups 4 and 5 in which recombinant vesîcular stomatitis viruses were added carrying the modified GP genes of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 (SEQ ID No:l, SEQ ID No:2).
Example 9.
A utîlization method of immunobiological agent based on the recombinant adenovirus containing the modified GP gene of the Ebola vi rus/H.sapiens-wt/S LE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:I, SEQ ID No:2 by administering to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
The developed immunobiological agent based on the recombinant adenovirus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l (GP1), SEQ ID No:2 (GP2) is used by administering to mammals by any method known to be used for virus vector administration (subcutaneously, întramuscularly, intravenously, intranasally). This way mammalian develops an immune response to the target protein of Ebola virus glycoprotein.
One of the main characteristics of immunization efïîcacy is the antibody titer. Example présents data relating to changes in the titer of antibodies against the Ebola virus glycoprotein after twice (with an interval of 1 week) subcutaneous immunization of animais with immunobiological agent comprising the recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 selected from GP1 (SEQ ID No:l, SEQ ID No:2).
Mammals used in the experiment — mice Balb/c, female 18 g. Ail the animais were divided into 13 groups, 3 animais each, injected întramuscularly:
1) Ad-GPl 107 PFU/mouse
2) Ad-GPl 10s PFU/mouse
3) Ad-GPl 109 PFU/mouse
4) Ad-GPI 1010 PFU/mouse
5) Ad-GP2 107 PFU/mouse
6) Ad-GP2 10 PFU/mouse
7) Ad-GP2 109 PFU/mouse
8) Ad-GP2 1010 PFU/mouse
9) Ad-null 107 PFU/mouse
10) Ad-null 10R PFU/mouse
11) Ad-null 109 PFU/mouse
12) Ad-null 1O'° PFU/mouse
13) phosphate buffered saline
Three weeks later the blood samples were taken from the tail vein of animais, and blood sérum was isolated. Antibodies titer was determined by ELISA following this protocol:
1) Protein (GP) was adsorbed onto wells of 96-well ELISA plate for 16 hours at + 4°C.
2) Sérum samples from immunized mice were diluted using 2-fold dilution method. A total of 8 dilutions of each sample were prepared.
3) 50 μΐ ofeach diluted sérum sample was added to the wells.
4) Then it was incubated for 1 hour at 37 °C.
5) After incubation the wells were washed three times with phosphate buffer.
6) Then secondary antibodies against mouse immunoglobulins conjugated with biotin were added.
7) Then it was incubated for 1 hour at 37 °C.
8) After incubation the wells were washed three times with phosphate buffer.
9) Then streptavidin conjugate and horseradîsh peroxidase were added to each weil.
10) Then it was incubated for 30 minutes at 37 °C.
11) After incubation the wells were washed three times with phosphate buffer.
12) Then TMB solution was added which is a substrate for horseradîsh peroxidase and is converted into a colored compound by the reaction. The reaction was stopped after 15 minutes by the addition of sulfuric acid. Next, the optical density (OD) of obtained solution was measured using a spectrophotometer in each weil with wavelength of450nm.
Antibody titer was determined as the last dilution at which the optical density of the solution was two-fold higher than in the négative control group. The results are presented in Table 2.
Table 2 — Titer of spécifie antibodies against GP protein in mouse blood.
Averange titer of anti-GP antibodies
Ad-GPl (107 PFU/mouse) 1:6400
Ad-GPl (10s PFU/mouse) 1:12800
Ad-GPl ( 10y PFU/mouse) 1:25600
Ad-GPl (10,ü PFU/mouse) 1:25600
Ad-GP2 (107 PFU/mouse) 1:6400
Ad-GP2 ( 10K PFU/mouse) 1:12800
Ad-GP2 (10y PFU/mouse) 1:12800
Ad-GPl (10lü PFU/mouse) 1:25600
Ad-null (107 PFU/mouse) 0
Ad-null (108 PFU/mousë) 0
Ad-null (104 PFU/mouse) 0
Ad-null (10lu PFU/mouse) 0
Phosphate buffered saline 0
The results of experiment hâve shown that immunobiological agent developed according to claim 1 induces immune response to Ebola virus in mammalian organism in ail selected dose range. It is obvious that dose escalation will resuit in antibody titer increase in mammalian blood till the toxic effect occurs.
Example 10.
A utilization method of immunobiological agent based on the recombinant vesicular stomatîtis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:I, SEQ ID No:2 by administering to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
The developed immunobiological agent based on the recombinant vesicular stomatîtis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /MakonaEM 124.1 GenBank ID KM233O45.I with a sequence selected from SEQ ID No:l (GP1), SEQ ID No:2 (GP2) is administered to mammals by any method known to be used for virus vector administration (subcutaneously, intramuscularly, intravenously, intranasally). This way mammalian develops an immune response to the target protein of Ebola virus glycoprotein.
At this stage, we hâve evaluated the efficacy of immunization with the developed immunobiological agent by détermination of an antibody titer against the Ebola virus glycoprotein after 2-tîmes (with an interval of 1 week) subeutaneous immunization of animais with immunobiological agent comprising recombinant vesicular stomatîtis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233O45.1 selected from GP 1 (SEQ ID No:l, SEQ ID No:2), GP2 (SEQ ID No:2). The experiment was performed according to the scheme similar to that described in Example 9.
Mammals used in the experiment — mice Balb/c, female 18 g. Ail the animais were divided into 13 groups, 3 animais each, injected intramuscularly:
1) VSV-GP1 106PFU/mouse
2) VSV-GP1 107PFU/mouse
3) VSV-GP1 108PFU/mouse
4) VSV-GP1 109PFU/mouse
5) VSV-GP2 106PFU/mouse
6) VSV-GP2 107PFU/mouse
7) VSV-GP2108PFU/mouse
8) VSV-GP2 109PFU/mouse
9) VSV-null 106PFU/mouse
10) VSV-null 107PFU/mouse ) VSV-null 108PFU/mouse
12) VSV-null 109PFU/mouse
13) phosphate bufTered saline
Three weeks later the blood samples were taken from the tail vein of animais, and blood sérum was isolated. Antibodies titer was determined by ELISA according to the protocol described in Example 8. The results of experiment are provided in Table 3.
Table 3. Titer of spécifie antibodies against GP protein in mouse blood, determined after administering the immunobiologicaï agent based on recombinant vesicular stomatîtis virus.
Group Averange titer of anti-GP antibodies
VSV-GP1 (I06PFU/mouse) 1:12800
VSV-GP1 (107PFU/mouse) 1:25600
VS V-GP1 ( 108PFU/mouse) 1:51200
VS V-GP 1 ( 109PFU/mouse) 1:25600
VSV-GP2 (106PFU/mouse) 1:25600
VSV-GP2 (107PFU/mouse) 1:25600
VSV-GP2 (10KPFU/mouse) 1:51200
VSV-GP2 (109PFU/mouse) 1:51200
VSV-null (106PFU/mouse) 0
VSV-null (107PFU/mouse) 0
VSV-null (10HPFU/mouse) 0
VSV-null (I09PFU/mouse) 0
Phosphate buffered saline 0
The data of the experiments conducted hâve shown that immunobiological agent developed according to claim 2 induces immune response to Ebola virus in mammalian organism in ail selected dose range. It is obvious that dose escalation will resuit in antibody titer increase in mammalian blood till the toxic effect occurs.
Example 11.
A utilization method of immunobiological agent based on the recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus/H.sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombînant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 by administering to mammals in effective amount to induce spécifie immunity against the Ebola virus.
An immunobiological agent based on the recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus /H.sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombînant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 is administered to mammals by any method known to be used for virus vector administration (subcutaneously, intramuscularly, intravenously, intranasally). Such usage of mentioned immunobiological agent results to formation of immune response to the target protein. One of the method for assessment the efficacy of immunization is to détermine the antibody titer against the target protein (GP).
The purpose of this experiment is to détermine the antibody titer against the Ebola virus glycoprotein after 2-times (with an interval of 1 week) immunization of animais with developed immunobiological agent based on recombinant vesicular stomatitis virus containing the nonmodified GP gene of the EboIavirus/H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 taken in effective proportions. The viral vectors hâve been used at the following ratios: 1:1000,1:100, 1:10,1:1,10:1, 100:1, 1000:1, The experiment was performed according to the scheme similar to that described in Example 10.
Mammals used in the experiment — mice Balb/c, female 18 g. ΑΠ the animais were divided into 97 groups, 3 animais each, injected intramuscularly:
1. VS V-GP1 106 PFU/mouse, VS V-GP3 106 PFU/mouse (1:1)
2. VS V-GP1 106 PFU/mouse, VS V-GP3 107 PFU/mouse (1:10)
3. VSV-GP1 106 PFU/mouse, VSV-GP3 108 PFU/mouse (1:100)
4. VSV-GP 1 106 PFU/mouse, VSV-GP3 109 PFU/mouse (1:1000)
5. VSV-GP 1 107 PFU/mouse, VSV-GP3 106 PFU/mouse ( 10:1 )
6. VSV-GP 1 107 PFU/mouse, VSV-GP3 107 PFU/mouse (1:1)
7. VSV-GP 1 107 PFU/mouse, VSV-GP3 108 PFU/mouse (1:10)
8. VSV-GP 1 107 PFU/mouse, VSV-GP3 109 PFU/mouse (1:100)
9. VSV-GP1 108 PFU/mouse, VSV-GP3 106 PFU/mouse (100:1 )
10. VSV-GP1 108 PFU/mouse, VSV-GP3 107 PFU/mouse (10:1 )
11. VSV-GP1 108 PFU/mouse, VSV-GP3 107 PFU/mouse (1:1)
12. VSV-GP1 10® PFU/mouse, VS V-GP3 109 PFU/mouse (1:10)
13. VSV-GP 1 109PFU/mouse, VSV-GP3 106PFU/mouse (1000:1 )
14. VSV-GP1 109PFU/mouse, VSV-GP3 107PFU/mouse (100:1)
15. VSV-GP 1 10’PFU/mouse, VSV-GP3 108PFU/mouse (10:1)
16. VSV-GP 1 10’PFU/mouse, VSV-GP3 109PFU/mouse (1:1)
17. VSV-GP1 106PFU/mouse, VSV-null 106PFU/mouse
18. VSV-GP1 106PFU/mouse, VSV-null 107PFU/mouse
19. VSV-GP1 106PFU/mouse, VSV-null 10gPFU/mouse
20. VSV-GP1 106PFU/mouse, VSV-null 109PFU/mouse
21. VSV-GP 1 107PFU/mouse, VSV-null 106PFU/mouse
22. VSV-GP 1 107PFU/mouse, VSV-null 107PFU/mouse
23. VSV-GP 1 107PFU/mouse, VSV-null 108PFU/mouse
24. VSV-GP 1 107PFU/mouse, VSV-null 109PFU/mouse
25. VSV-GP 1 10gPFU/mouse VSV-null 106PFU/mouse
26. VSV-GP1 10gPFU/mouse, VSV-null 107PFU/mouse
27. VSV-GP1 108PFU/mouse, VSV-null 10gPFU/mouse
28. VSV-GP 1 108PFU/mouse, VSV-null 109PFU/mouse
29. VSV-GP 1 109PFU/mouse VSV-null 106PFU/mouse
30. VSV-GP 1 109PFU/mouse, VSV-null 107PFU/mouse
31. VSV-GP1 109PFU/mouse, VSV-null 108PFU/mouse
32. VSV-GP 1 109PFU/mouse, VSV-null 109PFU/mouse
33. VSV-GP2 106PFU/mouse, VSV-GP3 106PFU/mouse (1:1)
34. VSV-GP2 106PFU/mouse, VSV-GP3 107PFU/mouse (1:10)
35. VSV-GP2 106PFU/mouse, VSV-GP3 1 OgPFU/mouse ( 1:100)
36. VSV-GP2 106PFU/mouse, VSV-GP3 109PFU/mouse (1:1000)
37. VSV-GP2 107PFU/mouse, VSV-GP3 106PFU/mouse (10:1)
38. VSV-GP2 107PFU/mouse, VSV-GP3 107PFU/mouse (1:1)
39. VS V-GP2 107PFU/mouse, VS V-GP3 1 08PFU/mouse (1:10)
40. VSV-GP2 107PFU/mouse, VSV-GP3 109PFU/mouse (1:100)
41. VSV-GP2 108PFU/mouse, VSV-GP3 106PFU/mouse (100:1)
42. VSV-GP2 108PFU/mouse, VSV-GP3 107PFU/mouse (10:1)
43. VSV-GP2 108PFU/mouse, VS V-GP3 108PFU/mouse( 1:1)
44. VSV-GP2 108PFU/mouse, VSV-GP3 109PFU/mouse (1:10)
45. VSV-GP2 109PFU/mouse, VSV-GP3 106PFU/mouse ( 1000:1)
46. VS V-GP2 109PFU/mouse, VSV-GP3 107PFU/mouse(l 00:1 )
47. VS V-GP2 109PFU/mouse, VSV-GP3 108PFU/mouse( 10:1)
48. VSV-GP2 109PFU/mouse, VSV-GP3 109PFU/mouse (1:1)
49. VSV-GP2 106PFU/mouse, VS V-nuIl 10fiPFU/mouse
50. VSV-GP2 106PFU/mouse, VSV-null 107PFU/mouse
51. VSV-GP2 106PFU/mouse, VSV-null 108PFU/mouse
52. VSV-GP2 106PFU/mouse, VSV-null 109PFU/mouse
53. VSV-GP2 107PFU/mouse, VSV-null 106PFU/mouse
54. VSV-GP2 107PFU/mouse, VSV-null 107PFU/mouse
55. VSV-GP2 107PFU/mouse, VSV-null 108PFU/mouse
56. VSV-GP2 107PFU/mouse, VSV-null 109PFU/mouse
57. VSV-GP2 108PFU/mouse, VSV-null 106PFU/mouse
58. VSV-GP2 108PFU/mouse, VSV-null 107PFU/mouse
59. VSV-GP2 108PFU/mouse, VSV-null 108PFU/mouse
60. VSV-GP2 108 PFU/mouse, VSV-null 109 PFU/mouse
61. VSV-GP2 10’ PFU/mouse, VSV-null 10e PFU/mouse
62. VSV-GP2 10’PFU/mouse, VSV-null 107PFU/mouse
63. VSV-GP2 10’PFU/mouse, VSV-null 108PFU/mouse
64. VSV-GP2 109PFU/mouse, VSV-null 109PFU/mouse
65. VSV-GP3 106PFU/mouse, VSV-null 106PFU/mouse
66. VSV-GP3 10fiPFU/mouse, VSV-null 107PFU/mouse .
67. VSV-GP3 106 PFU/mouse, VSV-null 108 PFU/mouse
68. VSV-GP3 10e PFU/mouse, VSV-null 109 PFU/mouse
69. VS V-GP3 107 PFU/mouse, VSV-null 106 PFU/mouse
70. VSV-GP3 107 PFU/mouse, VSV-nulI 107 PFU/mouse
71. VSV-GP3 107 PFU/mouse, VSV-null 108 PFU/mouse
72. VSV-GP3 107 PFU/mouse, VSV-null 109 PFU/mouse
73. VSV-GP3 108 PFU/mouse, VSV-null 106 PFU/mouse
74. VSV-GP3 108 PFU/mouse, VSV-null 107 PFU/mouse
75. VSV-GP3 108 PFU/mouse, VSV-null 108 PFU/mouse
76. VSV-GP3 108PFU/mouse, VSV-null 109PFU/mouse
77. VSV-GP3 109 PFU/mouse, VSV-null 106 PFU/mouse
78. VSV-GP3 109 PFU/mouse, VSV-null 107 PFU/mouse
79. VSV-GP3 109 PFU/mouse, VSV-null 108 PFU/mouse
80. VSV-GP3 109 PFU/mouse, VSV-null 109 PFU/mouse
81. VSV-null 106 PFU/mouse, VSV-null 106 PFU/mouse
82. VSV-null 106 PFU/mouse, VSV-null 107 PFU/mouse
83. VSV-null 106 PFU/mouse, VSV-null 108 PFU/mouse
84. VSV-null 106 PFU/mouse, VSV-null 109 PFU/mouse
85. VSV-null 107 PFU/mouse, VSV-null 106 PFU/mouse
86. VSV -null 107PFU/mouse, VSV -null 107PFU/mouse
87. VSV-null 107PFU/mouse, VSV-null 108PFU/mouse
88. VSV-null 107PFU/mouse, VSV-null 109PFU/mouse
89. VSV-null 108PFU/mouse, VSV-null 106PFU/mouse
90. VSV-null 108PFU/mouse, VSV-null 107PFU/mouse
91. VSV-null 108PFU/mouse, VSV-null 108PFU/mouse
92. VSV-null 108PFU/mouse, VSV-null 109PFU/mouse
93. VSV-null 109PFU/mouse, VSV-null 106PFU/mouse
94. VSV-null 109PFU/mouse, VSV -null 107PFU/mouse
95. VSV-null 109PFU/mouse, VSV-null 108PFU/mouse
96. VSV-null 109PFU/mouse, VSV-null 109PFU/mouse
97. phosphate buffered saline
The results of experiment are presented in Table 4.
Table 4. Average antibody titer against GP protein after administration of the immunobiological agent based on the recombinant vesicular stomatitis viruses.
VSV-GP3 VSV-null
Dose, PFU/ 106 107 108 109 106 107 108 109
mous e
vsv -GPI 106 1:12800 1:25600 1:25600 1:51200 1:12800 1:6400 1:1280 0 1:1280 0
107 1:25600 1:25600 1:25600 1:51200 1:25600 1:12800 1:1280 0 1:2560 0
10” 1:25600 1:25600 1:51200 :102400 1:25600 1:25600 1:2560 0 1:5120 0
109 1:51200 1:25600 1:51200 1:102400 1:51200 1:51200 1:2560 0 1:5120 0
VSV -GP2 106 1:12800 1:25600 1:25600 1:51200 1:12800 1:12800 1:1280 0 1:1280 0
107 1:25600 1:25600 1:25600 1:51200 1:25600 1:12800 1:2560 0 1:5120 0
10’ 1:25600 1:25600 1:25600 1:51200 1:51200 1:51200 1:2560 0 1:5120 0
109 1:25600 1:25600 1:25600 :102400 1:51200 1:25600 1:5120 0 1:5120 0
VSV -null 106 1:6400 1:25600 1:25600 1:51200 0 0 0 0
107 1:12800 1:12800 1:51200 1:51200 0 0 0 0
108 1:12800 1:25600 1:25600 1:51200 0 0 0 0
109 1:12800 1:25600 1:25600 1:51200 0 0 0 0
The experimental results hâve shown that immunobiological agent developed according to claim 3 when introduced into a mammalian body induces an immune response to Ebola virus in the entire range of doses and ratios selected.
Example 12.
Testing of efficacy of immunization with immunobiological agent developed based on the recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapienswt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 by assessment of proliferating lymphocytes portion.
Lymphocyte prolifération assay aîlows to assess the ability of lymphocytes to undergo an active prolifération when stimuîated by an antigen. Prolifération was determined by the authors using staining of lymphocytes with fluorescent dye CFSE. This dye binds to the cellular proteins and is maintained for a long time and, thus, is never spread into neighboring cells in the population. However, the fluorescent label is passed onto the daughter cells. The concentration of the label, and, consequently, the fluorescence intensity is decreased precisely 2 times in daughter cells in comparison to patent. Therefore dividing cells can be easily traced by reducing of their fluorescence intensity.
Balb/c mice were used in the experiment. ΑΠ the animais were divided into 4 groups, 3 animais each, injected intramuscularly:
1) phosphate buffer (100 μΐ)
2) Ad-nuII 108 PFU/mouse
3) Ad-GPI 108 PFU/mouse
4) Ad-GP2 108 PFU/mouse
The doses of recombinant adenoviruses were selected based on the data obtained from antibody titer analysis.
Two weeks after the animais were euthanîzed. Lymphocytes were isoiated from the spleen by Ficoll-Urografin density gradient centrifugation. Then the isoiated cells were stained with CFSE according to the procedure (Monitoring lymphocyte prolifération in vitro and in vivo with the intracellular fluorescent dyecarboxy fluorescent diacetate succinimidy lester/ Quah BJ, Warren HS, Parish CR Nat Protoc. 2007; 2(9), ρ:2049·2056) and were cultured in the presence of antigen. The cells were analyzed b y flow cytometry. The obtained results are shown in histogram form (Figure 4).
Results of the experiment suggest that immunobiological agent developed according to the claim 1 greatly stimulâtes prolifération of lymphocytes in the dose used.
Exampîe 13.
Détermination of the immunization efficacy using immunobiological agent developed based on vesicular stomatitis virus containing the modified gene GP of the Ebola virus /H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 as assessed by the percentage of proliferating lymphocytes.
The purpose of this experiment was to détermine the proliférative activity of lymphocytes isoiated from mice C57/BI6 (18 g) treated with the following:
1. phosphate buffer ( 100 μ I)
2. VSV-null 107 PFU/mouse
3. VSV-GP1 107 PFU/mouse
4. VSV-GP2 107 PFU/mouse
The doses of recombinant vesicular stomatitis virus were selected based on the data obtained from antibody titer analysis.
The experiment was performed according to the protocol provided in Example 12.
The experiment results are shown in Figure 5.
These results suggest that immunobiological agent developed according to the claim 2 greatly stimulâtes prolifération of lymphocytes in the dose used.
Example 14.
Détermination of the immunization efficacy using immunobiological agent developed based on the recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus/H.sapiens-wt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 as assessed by the percentage of proliferating lymphocytes.
In this experiment the analysis of proliferating lymphocytes isolated from mice was performed; mice were injected with
1. Phosphate buffer (100 μΐ)
2. VSV-null 107PFU/mouse '
3. VSV-GP1 I07PFU/mouse, VSV-GP3 107PFU/mouse (1:1)
4. VSV-GPI 106PFU/mouse, VSV-GP3 109PFU/mouse (1:1000)
5. VSV-GP1 109PFU/mouse, VSV-GP3 106PFU/mouse (1000:1)
6. VSV-GP2 107PFU/mouse, VSV-GP3 107PFU/mouse(l:l)
7. VSV-GP2 106PFU/mouse, VSV-GP3 10qPFU/mo use (1:1000)
8. VSV-GP2 109PFU/mouse, VSV-GP3 I06PFU/mouse (1000:1)
The doses of recombinant vesicular stomatitis virus were selected based on the data obtained from antibody titer analysis.
Experiment was performed according to the protocol presented in Example 11. The experiment results are shown in Figure 6.
As seen from the experiment results, immunobiological agent developed according to claim 3 eflectively stimulâtes lymphocyte prolifération with the dose used.
Example 15.
A utilization method of immunobiological agents based on the recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.I with a sequence selected from SEQ ID No:l, SEQ ID No:2 and immunobiological agents based on recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 including the sequential administration to mammals with an interval more than one week to induce a spécifie immunity against Ebola virus.
As seen from the results of experiments described in Examples 9-11, developed variants of immunobiological agents based on recombinant adenoviruses and vesicular stomatitis viruses resuit in high titers of antibodies in the blood of immunized animais. The use of these variants is appropriate for inducing an immune response against Ebola virus in the vast majority of population.
However, there are categories of population (healthcare workers, people living in the épidémie areas) who are at risk: employées of infections departments of hospitals, healthcare workers going to épidémie area, people living in the épidémie area etc. For these categories, methods according to claims 7 and 8 were developed including sequential immunization (at intervals longer than ! week) using immunobiological agents according to claims 1 and 2 or claims I and 3.
Method of immune response induction when one recombinant viral vector is administered, followed by another, will be more effective than administration of the same virus vectors. This is due to the fact that after first immunization, the antibodies in the body are produced not only to the target antigen (GP of Ebola virus), but also to vector’s proteins. Therefore, after booster immunization using the same viral vector type antibodies prevent the pénétration of the viral particles into the cells. Therefore, expression of target protein will also be reduced, and immune response will be weaker.
This example represents the experimental data showing that sequential immunization of animais using different viral vectors: at first immunobiological agents according to claim 1. (including recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with the sequence selected from SEQ ID No:I), and within a week immunobiological agent according to claim 2 (including recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with the sequence selected from SEQ ID No:l, SEQ ID No:2) or otherwise results in a stronger induction of antibodies against the target protein (GP) than immunization performed the same way, but using the same îmmunobiological agent.
The experiment was performed according to the protocol described in Example 9.
Ail the animais were divided into 11 groups, 3 animais each, injected intramuscularly:
1) Ad-GPl 108PFU/mouse, one week later VS V-GP1 l07PFU/mouse
2) Ad-GP2 108PFU/mouse, one week later VSV-GP1 107PFU/mouse
3) Ad-GPl 10*PFU/mouse, one week later VS V-GP2 107PFU/mouse
4) Ad-GP2 108PFU/mouse, one week later VS V-GP2 107PFU/mouse
5) Ad-GP 1 108PFU/mouse, one week later Ad-GP 1 108PFU/mouse
6) Ad-GP2 108PFU/mouse, one week later Ad-GP2 108PFU/mouse
7) VSV-GP1 107PFU/mouse, one week later VSV-GP1 107PFU/mouse
8) VS V-GP2 107PFU/mouse, one week later VS V-GP2 107PFU/mouse
9) Ad-null 108PFU/mouse, one week later Ad-null 108PFU/mouse
10) VSV-null 107PFU/mouse, one week later VSV-null 107PFU/mouse ) Phosphate buffer, one week later — phosphate buffer
The results are presented in Table 5.
Table 5. Titers of antibodies
Group of animais Titer of antibodies
“Ï) Ad-GPl 108 PFU/mouse, VSVGP1 107 PFU/mouse 1:51200
~2) Ad-GP2 10* PFU/mouse VSV-GP 1 107PFU/mouse 1:51200
“3) Ad-GPl 10s PFU/mouse, VSVGP2 107 PFU/mouse 1:102400
~4) Ad-GP2 108 PFU/mouse, VSVGP2 107 PFU/mouse 1:51200
“5) Ad-GPl 108 PFU/mouse, Ad-GPl 108 PFU/mouse 1:12800
~6) Ad-GP2 10s PFU/mouse, Ad-GP2 10® PFU/mouse 1:12800
~7) VSV-GPl 107 PFU/mouse VSV-GP1 107PFU/mouse 1:25600
“8) VSV-GP2 10' PFU/mouse, VSV- GP2 107 PFU/mouse 1:25600
9) Ad-nu!l 10sPFU/mouse, Ad -null 108PFU/mouse 0
10) VSV-null lO'PFU/mouse, VSVnull 107PFU/mouse 0
11) Phosphate buffer, phosphate buffer 0
When administering immunostimulating agents according to claim 1 and claim 2 in reverse order (first agent according to claim 2, then agents according to claim 1) the same results were obtained.
Thus, the results of experiment hâve fully confirmée! that sequential immunization with developed immunobiological agents according to claim 1 and claim 2, including various viral vectors, would cause more powerful induction of an immune response than immunization by the same schedule using only one vector type.
Example 16.
A utilization method of immunobiological agents (claim 1) based on the recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:I, SEQ ID No:2 and immunobiological agent based on the recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus/H.sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 (SEQ ID No:3) and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 including the sequential administration to mammals with an interva! more than one week to induce a spécifie immunity against Ebola virus.
The purpose of this experiment is to ensure that immunization of animais with different viral vectors:
1) First with immunobiological agent (claim I) (with recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l), and 1 week later — immunization with immunobiological agent (claim 3) (with recombinant vesicular stomatitis virus containîng the non-modified GP gene of the Ebola virus/H.sapiens-wt/I976/Mayinga/ZaireGenBank ID AF0S6S33.2 with a sequence SEQ ID No:3 and recombinant vesicular stomatitis virus containîng the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBanklD KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2, taken in effective proportion) or vice versa lead to more powerful induction of antibodies against the target protein (GP) than immunization performed the same way, but with the only one type of immunobiological agent.
The experiment was performed according to the protocol described in Example 9.
Ail the animais were divided into 11 groups, 3 animais each, injected intramuscularly:
1) Ad-GPl 108PFU/mouse, one week later — VSV-GP1 107PFU/mouse, VSV-GP3
107 PFU/mouse
2) Ad-GP2 IOsPFU/mouse, one week later VSV-GP1 107PFU/mouse, VSV-GP3
107PFU/mouse
3) Ad-GPl 108PFU/mouse, one week later VSV-GP2 107PFU/mouse, VSV-GP3
107PFU/mouse
4) Ad-GP2 108PFU/mouse, one week later VSV-GP2 107PFU/mouse, VSV-GP3
107PFU/mouse
5) Ad-GPl 108PFU/mouse, one week later Ad-GPl 108PFU/mouse
6) Ad-GP2 I08PFU/mouse, one week later Ad-GP2 I08PFU/mouse
7) VSV-GP1 107PFU/mouse, VSV-GP3 107PFU/mouse, one week later — VSVGP1 107PFU/mouse, VSV-GP3 107 PFU/mouse
8) VSV-GP2 107PFU/mouse, VSV-GP3 107PFU/mouse, one week later VSV-GP2 107PFU/mouse, VSV-GP3 107PFU/mouse
9) Ad-null 108PFU/mouse, one week later — Ad-null 108PFU/mouse
10) VSV-null 2* 107PFU/mouse, one week later VSV-null 2* ! 07PFU/mouse ) Phosphate buffer, one week later — phosphate buffer
The results are presented in Table 6.
Table 6. Titers of antibodies
Group of animais . Titer of antibodies
I. Ad-GPl 10sPFU/mouse, one week later VSV-GP1 107PFU/mouse, VSV-GP3 107PFU/mouse 1:102400
2. Ad-GP2 10sPFU/mouse, one week later VSV-GP] 107PFU/mouse, VSV-GP3 107PFU/mouse 1:102400
3. Ad-GPl 108PFU/mouse, one week later VSV-GP2 107PFU/mouse, VSV-GP3 lO’PFU/mouse 1:102400
4. Ad-GP2 10a PFU/mouse, one week later VS V-GP2 107PFU/mouse, VSV-GP3 107PFU/mouse 1:102400
5. Ad-GPl 10aPFU/mouse, one week later Ad-GPl 108PFU/mouse 1:12800
6. Ad-GP2 10a PFU/mouse, one week later Ad-GP2 10sPFU/mouse 1:12800
Ύ VSV-GP 1 107 PFU/mouse, VSV-GP3 107 PFU/mouse, one week later — VSV-GP 1 107 PFU/mouse, VSV-GP3 107 PFU/mouse 1:25600
T VSV-GP2 10' PFU/mouse, VSV-GP3 107 PFU/mouse, one week later VSV-GP2 107 PFU/mouse, VSV-GP3 107 PFU/mouse 1:51200
9. Ad-null 108PFU/mouse, one week later — Ad-null 10*PFU/mouse 0
10. VSV-null 2* 10'PFU/mouse, one week later VSV-null 2* 107PFU/mouse 0
11. Phosphate buffer, one week later — phosphate buffer 0
When administering immunostimulating agents according to claim 1 and claim 3 in reverse order (first agent according to claim 3, then agents according to claim 1), the same results were obtained.
Thus, the results of experiment hâve fully confirmed that sequential immunization with deveioped immunobiological agents according to claim 1 and claim 3, including various viral vectors, would cause stronger induction of an immune response than immunization by the same schedule with the only one type of viral vector.
Thus, it can be concluded that deveioped original immunobiological agent (variants) is 10 “new”, fully original and has proper “inventive level”, and it was first used by the authors for the effective induction of spécifie immunity against Ebola virus.
Industrial Applicability
Ail these examples confirm the fulfillment of technïcal task, as well as industrial utility of developed technical solution.

Claims (8)

  1. Claims
    1. Immunobiological agent based on recombinant adenovirus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 1).
  2. 2. Immunobiological agent based on recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM 124.1 GenBank ID KM233045.1 with a sequence selected from SEQ ID No:l, SEQ ID No:2 (variant 2)·
  3. 3. Immunobiological agent based on recombinant vesicular stomatitis virus containing the non-modified GP gene of the Ebola virus/H.sapienswt/1976/Mayinga/ZaireGenBank ID AF086833.2 with a sequence SEQ ID No:3 and recombinant vesicular stomatitis virus containing the modified GP gene of the Ebola virus/H.sapiens-wt/SLE/ 2014 /Makona-EM124.1 GenBank ID KM233O45.I with a sequence selected from SEQ ID No:l, SEQ ID No:2 taken in effective ratios (variant 3).
  4. 4. A utîlization method of immunobiological agent (claim 1): it is administered to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
  5. 5. A utîlization method of immunobiological agent (claim 2): it is administered to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
  6. 6. A utîlization method of immunobiological agent (claim 3): it is administered to mammals in an effective amount to induce spécifie immunity against the Ebola virus.
  7. 7. A utilization method of immunobiological agent according to claim 1 and claim 2: consequential administration to mammals with >l-week interval to induce spécifie immunity against the Ebola virus.
  8. 8. A method of using of immunobiological agent according to claim 1 and claim 3: consequential administration to mammals with >l-week interval to induce spécifie immunity against the Ebola virus.
OA1201700364 2015-03-31 2016-02-18 Immunobiological agent and utilization method thereof for inducing specific immunity against Ebola virus (variants). OA18489A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
RU2015111368 2015-03-31

Publications (1)

Publication Number Publication Date
OA18489A true OA18489A (en) 2018-12-04

Family

ID=

Similar Documents

Publication Publication Date Title
US11612649B2 (en) HIV pre-immunization and immunotherapy
CN105980570B (en) CMV vaccines
JP7153332B2 (en) HIV vaccination and immunotherapy
CN110951699A (en) Recombinant rabies virus for expressing structural protein of canine distemper virus and application thereof
US5882650A (en) Cross-reactive influenza A immunization
EP3202411A1 (en) Compositions and methods
Stubbs et al. Vesicular stomatitis virus chimeras expressing the Oropouche virus glycoproteins elicit protective immune responses in mice
US20230190917A1 (en) Viral vaccine vector for immunization against a betacoronavirus
CN105530956A (en) Hendra and Nipah virus G glycoprotein immunogenic compositions
CN107614515A (en) For AntiHIV1 RT activity (human immunodeficiency virus) therapy and/or the T20 constructs of vaccine
RU2578160C1 (en) Immunobiological agent and method for use thereof to induce specific immunity against ebola virus (versions)
OA18489A (en) Immunobiological agent and utilization method thereof for inducing specific immunity against Ebola virus (variants).
CN115094088A (en) pSFV-flag X-CMV replicon plasmid, preparation method and cocktail mixed vaccine
KR102211077B1 (en) A pseudo type rabies virus vaccine using virus-like particles
RU2701953C1 (en) Method of producing a polyvalent influenza vaccine
CA2431938A1 (en) A novel vaccine formulation consisting of dna vaccine inactivated virus
CN113185586B (en) T cell epitope polypeptide derived from SARS-CoV-2 coding protein and application thereof
CN109022456A (en) A kind of preparation method of viral vaccine that expressing Plasmodium ovale AMA1 albumen
WO2024055273A1 (en) Rabies mrna vaccine and preparation and use thereof
WO2022195900A1 (en) Recombinant measles virus
RU2706191C1 (en) Multivalent influenza vaccine
RU2681439C1 (en) Influenza virus the virus-like particle and its obtaining method
JPS62501973A (en) Improvements to influenza vaccines
WO2021173965A1 (en) Identification of variable influenza residues and uses thereof
WO2016130047A1 (en) Immunobiological drug and method for using same for inducing specific immunity against the ebola virus