RU2635998C1 - Immunogen peptides and "epivakebol" vaccine against ebola fever with application of indicated peptides - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: immunogen peptide used as a component of the vaccine against Ebola fever, characterized by the amino acid sequence PQSLTTKPGPDNSTHNTPVYKLDISE (SEQ ID NO: 1), containing the antigenic epitopes of the GP protein of the Ebola virus, which have an ability to induce the formation of virus neutralizing antibodies to protect against the development of Ebola fever, is obtained. The second immunogen peptide used as a component of the vaccine against Ebola fever is characterized by the amino acid sequence HHQDTGEESASSGKLGLITNTIAG (SEQ ID NO: 2) containing the antigenic epitopes of the GP protein of the Ebola virus. The group of inventions also relates to a vaccine comprising a peptide-immunogen mixture having the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, wherein the said peptides in the mixture are conjugated to an immunogenic carrier and sorbed onto a pharmaceutically acceptable adjuvant.

EFFECT: application of this group of inventions allows to obtain such peptide immunogens which carry minimally necessary antigenic determinants for formation of a specific immune response and contain antigenic regions of the GP protein of the Ebola virus that are immunogenic and that induce protective immunity against Ebola infection.

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Description

The invention relates to the development of preventive drugs, namely to obtain a vaccine against Ebola, and can be used in medicine to prevent this disease in humans.

General characteristics of Ebola and Ebolaviruses

According to modern classification, Ebola fever is caused by ebolaviruses belonging to the genus Ebolavirus, family Filoviridae. The genus Ebolavirus includes 5 species that differ in antigenic structure, virulence, and distribution area: Bundibugyo ebolavirus (Bundibugyo ebolavirus, BDBV), Reston ebolavirus (Reston ebolavirus, RESTV), Sudan ebolavirus (Sudan ebolavirus, Tayvore Fayv Tayvf) ebolavirus, TAFV) and Zaire ebolavirus (Zaire ebolavirus, EBOV) [http://www.ictvonline.org/virusTaxonomy.asp]. The genome of ebavaviruses is represented by a single-stranded ribonucleic acid (RNA) molecule of negative polarity with a length of about 19,000 nucleotides, the molecular weight of RNA is 4.0 x 10 ⁶ Da. The virion is formed by 7 functional proteins: nucleoprotein (NP), membrane-associated protein (VP24), nucleocapsid proteins (VP30; VP35 cofactor polymerase), matrix protein (VP40), RNA-dependent RNA polymerase (L) and glycoprotein (GP) . Each of the genes encodes 1 protein, with the exception of the GP, which encodes sGPandssGP. Carbohydrates glycoproteins GP1 and GP2, RNA polymerase enzymes; transcriptase. The molecular weight of the virion is 300–600 MDa. The outer diameter of the particles is 70-80 nm. The length of viral particles, far exceeding their diameter, can reach 14,000 nm with an average value of 650 nm. Outside, the virion is covered with a lipoprotein membrane with a thickness of about 20 nm, on which spicules 7-10 nm long are located at a distance of 10 nm from each other.

The agent belongs to the pathogenicity group I according to the classification of Rospotrebnadzor and causes severe Ebola fever (Ebola Haemorrhagic Fever, EHF or Ebola virus disease, EVD, Lat. Ebola febris haemorrhagica). The disease begins acutely with the onset of fever, diarrhea, muscle pain, headache, sore throat and abdomen. Later, a dry cough and stitching pains in the chest area appear, dehydration of the body, vomiting develops, a hemorrhagic rash appears (in about half of the cases), along with a decrease in the functioning of the liver and kidneys. In 40-50% of cases, bleeding from the gastrointestinal tract, nose, vagina and gums begins. Lab tests reveal low levels of white blood cells and platelets along with elevated liver enzymes. The incubation period is in the range from 2 to 21 days, but may depend on the method of infection: 5-7 days when the virus enters through damaged skin and on average 9 days after the infected biological fluids reach the mucosa [Leligdowicz A., Fischer WA, Uyeki TMet al. Ebola virus disease and critical illness // Critical Care (2016) 20: 217, DOI 10.1186 / s13054-016-1325-2].

Mortality ranges from 25% to 90%. When infected with Zaire ebolavirus, up to 90% of patients die, Sudan ebavavirus - about 50%, Bundibuzhio ebolavirus - from 25 to 55%. Mortality among those infected with Ebavavirus Tai Forest is unknown. There are no reports of lethal outcomes of the disease caused by Reston ebolavirus. At the same time, it should be noted that the mortality rates are probably significantly overestimated, which is associated with insufficient identification of mild and asymptomatic forms of the disease, as well as difficulties in conducting differential diagnosis with diseases endemic on the territory of the African continent that have similar clinical symptoms. The presence of erased and asymptomatic forms of Ebola fever is evidenced by the presence of a wide immune layer in the population, as well as the identification of traces of the pathogen circulation among those living in areas where cases of Ebola fever have never been recorded.

As the disease progresses, unhindered replication of the virus leads to apoptosis of lymphocytes and the release of a large number of inflammatory mediators (“cytokine storm”). Excessive and uncontrolled inflammatory reaction causes a massive lesion of the vascular endothelium, exacerbating DIC and multiple organ failure.

Ebolaviruses, entering the body, trigger a chain of reactions leading to immunosuppression at the level of various parts of the immune system. For example, proteins VP35 and VP24 using different mechanisms block the production of interferon [Eric C. Dunham E.C., Banadyga L., Groseth A. et al. Assessing the contribution of interferon antagonism to the virulence of West African Ebola viruses // Nature Communications 6, Article number: 8000 (2015), doi: 10.1038 / ncomms9000]. The production of virus-neutralizing antibodies is delayed, and class G antibodies are rarely found in Ebola deceased [Broadhurst M.J., Brooks T.J.G., Pollock N.R. Diagnosis of Ebola virus disease: past, present, and future // Clin. Microbiol. Rev. 2016, 29: 773–93, doi: 10.1128 / CMR.00003-16].

Îáîëî ÷ å ÷ íûé ãëèêîïðîòåèí GP âèðóñà EBOV, îáåñïå ÷ èâàþùèé ðåöåïòîð-îáóñëîâëåííóþ ñâÿçü âèðóñà ñ êëåòêîé-ìèøåíüþ, ÿâëÿåòñÿ îñíîâíîé ìèøåíüþ ïðè ñîçäàíèè âàêöèí è èíäóêöèè íåéòðàëèçóþùèõ àíòèòåë. Ðåçóëüòàòû èññëåäîâàíèé óêàçûâàþò íà òî, ÷ òî ãóìîðàëüíûé îòâåò ó áîëüøèíñòâà âûæèâøèõ ïðè ëèõîðàäêå Ýáîëà íàèáîëåå âûðàæåí äëÿ ýïèòîïîâ ãëèêîïðîòåèíà GP (Becquart P etal. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus // PLoS One. 2014,10; 9 (6): e96360). Ýòîò áåëîê îðãàíèçîâàí â ôîðìå òðèìåðà, ñîñòîèò èç äâóõ ñóáúåäèíèö, à èìåííî âíåêëåòî ÷ íîé ñóáúåäèíèöû GP1, êîòîðàÿ ïðèêðåïëåíà ê îáîëî ÷ êå âèðèîíà ÷ åðåç ÿêîðü ñóáúåäèíèöû GP2. GP1 is to be used in conjunction with Âèðóñíûé áåëîê GP ýêñïðåññèðóåòñÿ íà ïîâåðõíîñòè âèðèîíà è íà ìåìáðàíå èíôèöèðîâàííûõ êëåòîê è ìîæåò, òàêèì îáðàçîì, ÿâëÿòüñÿ èäåàëüíîé ìèøåíüþ äëÿ íåéòðàëèçóþùèõ àíòèòåë.

The GP protein is known to contain at least five epitopes that are capable of inducing neutralizing antibodies. GP immunoreactive peptides are distributed in five regions located at positions 41-67, 93-127, 201-251, 301-359 and 381-411. Two of these regions belong to the domain located in the C-terminal part of GP1, which contains a highly N- and O-glycosylated mucin-like domain. This domain is extremely large and makes up more than half the molecular weight of the GP protein. Mucin-like domain neutralizing antibodies protect mice from lethal Ebola infections (Wilson JA, Hevey M., Bakken R. et al. Epitopes involved in antibody-mediated protection from Ebola virus // Science, 2000, 287: 1664–6) . Interestingly, the two immunogenic regions at positions 381-411 and 469-483 contain three linear epitopes (ao 401-417, 389-405 and 477-493), which are associated with protective antibodies. In addition, Becquart P. showed a large IgG specific immunodominant region in the mucin-like domain of GP, (amino acid residues from 301 to 359). The other three immunogenic regions in GP are located in GP1 on the "bowl" of the viral surface of the trimer formed by the three GP1 subunits. The IgG response covers a larger surface area of the "bowl" in asymptomatic patients than in survivors. Two of the three immunogenic regions are located in potential binding sites with host cells, including region 54-201 (ManicassamyB., WangJ., JiangH., RongL. ComprehensiveanalysisofebolavirusGP1 inviralentry // J. Virol.2005, 79: 4793–805; Brindley MA, Hughes L., Ruiz A. et al. Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events // J. Virol. 2007, 81: 7702–9).

It was also shown earlier that sera from individuals with an asymptomatic course of the disease react with three immunodominant domains of amino acid residues 189-207, 213-235 and 289-319 of the VP40 protein, while survivors' sera react much weaker with this protein. Most VP40 immunodominant domains are located inside the virion and are not exposed to specific receptors or antibodies externally, however, the results presented by Becquart P., 2014 indicate that the humoral response against these three key VP40 epitopes can help protect people against fever Ebola The protective effectiveness of the humoral response may be associated not only with viral neutralization, but also with the induction of antibody-dependent cellular cytotoxicity.

When developing whole-virionic inactivated or subunit recombinant Ebola vaccines, the researchers faced a number of problems that were caused by the pathogenesis of this disease, induced by Ebola virus proteins by immunosuppression and antibody-dependent increase in infection. Recent studies suggest that the humoral immune response is not sufficient to protect against this infection, therefore, its combination with the activation of CD4 + and CD8 + T-lymphocytes is necessary to ensure it. In addition, it was shown that some epitopes of the GP protein are able to induce an antibody-dependent increase in experimental infection in primates.

Ðåøèòü ýòè ïðîáëåìû âîçìîæíî ñ ïîìîùüþ ðàçëè ÷ íûõ ìåòîäîâ: îáðàòíîé ãåíåòèêè, ñîçäàíèåì ÄÍÊ-âàêöèí, ãåíåòè ÷ åñêèõ âàêöèí, âàêöèí íà îñíîâå ÂÏ ×, ïîëèýïèòîïíûõ âàêöèí. Îäíàêî ñîõðàíÿåòñÿ îäíà ïðîáëåìà äëÿ ðàçëè ÷ íûõ òèïîâ âàêöèí - âåðîÿòíîñòü èíäóöèðîâàòü èììóííûé îòâåò ñ ïàòîëîãè ÷ åñêèìè ñâîéñòâàìè, íàïðèìåð àíòèòåëîçàâèñèìîå óñèëåíèå èíôåêöèè, ÷ òî õàðàêòåðíî äëÿ íåêîòîðûõ âèðóñíûõ èíôåêöèé, âêëþ ÷ àÿ âèðóñ Ýáîëà. Àëüòåðíàòèâíûì ïîäõîäîì ê èììóíèçàöèè â ýòîì ñëó ÷ àå ìîæåò ñëóæèòü èììóíîãåí â âèäå ñèíòåòè ÷ åñêèõ ïåïòèäíûõ ýïèòîïîâ, êîòîðûå èíäóöèðóþò òðåáóåìûé èììóííûé îòâåò, à íåæåëàòåëüíûå ó ÷ àñòêè ïîñëåäîâàòåëüíîñòè, ñïîñîáíûå âûçâàòü ïîáî ÷ íûå ýôôåêòû, èñêëþ ÷ åíû èç ñîñòàâà ïåïòèäíîãî èììóíîãåíà. More options for your entertainment, including more options for your stay, as long as there is plenty of space for it. Increased syn- In case of direct use, the use of the device and the wheel drive are necessary. Îäíèì èç ðàñïðîñòðàíåííûõ ïðèåìîâ ïîâûøåíèÿ èììóíîãåííîñòè ïåïòèäíûõ àíòèãåíîâ è ñòàáèëèçàöèè èõ êîíôîðìàöèè ÿâëÿåòñÿ ïðèåì, îñíîâàííûé íà êîâàëåíòíîì ñâÿçûâàíèè ïåïòèäîâ ñ êðóïíûìè ìîëåêóëàìè, íàïðèìåð, àëüáóìèíîì (ìîë. Ìàññà 67 êÄà), èëè ãåìîöèàíèíîì (KLH, ìîë. Ìàññà 6 ÌÄà). Integrated, self-contained, high-quality, non-flammable fuel dispensers for industrial use. Long-lasting effective control of the high efficiency of the long-term simplicity. Selective power for quick, efficient and quick start (end). ÌÀÏ ñîñòîèò èç íåáîëüøîãî èììóíîëîãè ÷ åñêè èíåðòíîãî ÿäðà èç ðàäèàëüíî âåòâÿùèõñÿ àìèíîêèñëîòíûõ îñòàòêîâ ëèçèíà, ê êîòîðûì êîâàëåíòíî êðåïÿòñÿ ïåïòèäíûå àíòèãåíû. The product is used in conjunction with a quick-drive, but does not produce any Ñèíòåçèðîâàííûå ïåïòèäû ìîãóò áûòü ïðèñîåäèíåíû ê ëèïèäíûì ìèöåëëàì èëè ëèïîñîìàì, ÷ òî îáåñïå ÷ èâàåò âûñîêóþ èììóíîãåííîñòü è ðàçëè ÷ íûå ïóòè ïðîöåññèíãà èììóíîãåíà.

The advantage of peptide vaccines over vaccines containing full-sized proteins of the Ebola virus is that the presence of a full-sized protein in the vaccine leads to the fact that the vaccinated organism produces antibodies that do not have a protective function, which increases the load on the immune system and can affect the increase in the reactogenicity of the drug , including reactogenicity on own proteins. Another negative factor in the use of a full-sized GP protein may be the presence in it of a sequence homologous to the sequence of the immunosuppressive domain of retroviruses [Volchkov V.E., Blinov V.M., Netesov S.V. The envelope glycoprotein of Ebola virus contains an immunosuppressive-like domain similar to oncogenic retroviruses // FEBS Lett. 1992, 305 (3): 181-4].

Well-known analogues are recombinant vaccines against Ebola fever based on vesicular stomatitis virus (BBC) and adenovirus (ADV) with the embedding of the GP gene of the Ebola virus strain Zaire [Martins K.A., Jahrling P.B, Bavari S., Kuhn J.H. Ebola Virus Disease Candidate Vaccines Under Evaluation in Clinical Trials // Expert Review of Vaccines, 2016, DOI: 10.1080 / 14760584.2016.1187566]. Ebola Vaccine Based on BBC-rVSV-ZEBOV Manufactured by Merck Sharp & Dohme Corp. New Link Genetics, Public Health Agency of Canada [Kennedy S.B., Neaton J.D., Lane H.C. et al. Implementation of an Ebola virus disease vaccine clinical trial during the Ebola epidemic in Liberia: Design, procedures, and challenges // Clin Trials, 2016, 13 (1), 49-56; Henao-Restrepo A.M., Longini I.M., Egger M. et al. Efficacy and effectiveness of an rVSVvectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomized trial // Lancet, 2015, 386 (9996), 857-66] and Ebola vaccine based on ADV-Ad26.ZEBOV manufactured by Crucell Holland BV, Johnson & Johnson [Zahn R, Gillisen G, Roos A et al. Ad35 and ad26 vaccine vectors induce potent and cross-reactive antibody and T-cell responses to multiple filovirus species // PLoS One, 2012, 7 (12), e44115] when used once, they effectively protect against the disease, forming a specific humoral and cellular in vaccinees answer.

However, vaccines based on recombinant vectors are intended for use in the focus of Ebola infection by ring immunization methods, as can be used only once. After repeated administration, the immune response to the vector virus formed after the first vaccination blocks its reproduction in the body, and the immune response to the target antigen - GP protein ebolavirus is not enhanced. For this reason, vaccines based on recombinant viruses containing the ebolavirus GP gene are recommended in the prime booster immunization schedule with other vaccines: for example, primary administration is carried out with an air force or ADV vaccine, and secondary administration with vaccines based on the vaccine virus MVA-BN- Filo Bavarian Nordic GmbH / Crucell Holland BV, Johnson & Johnson [Tapia MD, Sow SO, Lyke KE etal.Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, singleblind, randomized trial, a phase 1b, open-label and double-blind , dose-escalation trial, and a nested, randomized, double-blind, placebo-controlled trial // Lancet Infect Dis, 2016, 16 (1), 31-42] or non-replicating human adenoviruses Ad5-EBOV manufactured by Tianjin CanSino Biotechnology and Beijing, Institute of Biotechnology [Zhu FC, Hou LH, Li JX et al. Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based Ebola vaccine in healthy adults in China: preliminary report of a randomized, double-blind, placebo-controlled, phase 1 trial // Lancet, 2015, 385 (9984), 2272-79] and chimpanzees ChAd3.EBOZ / ChAd3.EBO manufactured by Glaxo Smith Kline, NIAID [Martins KA, Jahrling PB, Bavari S., Kuhn JH Ebola Virus Disease Candidate Vaccines Under Evaluation in Clinical Trials // Expert Review of Vaccines, 2016, DOI: 10.1080 / 14760584.2016.1187566; Stanley D.A., Honko A.N., Asiedu C. et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge // Nat Med, 2014, 20 (10), 1126-29]. In addition, there are two more negative factors affecting the use of these vaccines. First: vaccines based on replicating recombinant vectors cause serious post-vaccination complications, for example, the introduction of the rVSV-ZEBOV vaccine causes arthritis, the number of such complications can reach 25%. Second: when using the Ad26.ZEBOV vaccine, it is important to take into account that adenovirus infection is registered on all continents, therefore antibodies to adenovirus are present in the blood serum of residents of many countries. Seroconversion for this indicator, for example, in sub-Saharan Africa (it is in this region of Africa that outbreaks and epidemics of Ebola are recorded) can reach 40-60%, i.e. in half of the inhabitants of this region, the reproduction efficiency of recombinant adenovirus can be reduced or completely suppressed, which will not allow them to form protective immunity against Ebola after administration of the drug.

The closest analogue (prototype) is a peptide for a vaccine against Ebola [International application WO 2016/059517, A1, "Peptide for Anti-Ebola Vaccine", publ. 04/21/2016]. The peptide for the Ebola vaccine is the LWQQWDR peptide of 7 amino acid residues, which was designed according to the results of a computer analysis of the amino acid sequences of the proteins of 45 strains of the Ebola virus and all human proteins so that they do not have 5-dimensional epitopes in common with human proteins, is a conservative element of VP30 protein among 45 strains of the Ebola virus. It is assumed that a vaccine containing a synthetic oligopeptide LWQQWDR will be immunogenic for humans, since it does not have common epitopes with human proteins, for the same reason it will not cause adverse cross-reactions of immunity during vaccination, and will have specificity for most Ebola virus strains.

However, in the prototype, a synthetic peptide carrying seven amino acid residues from VP30 protein is not able to induce neutralizing antibodies that interfere with the binding of the virus to the target cell via GP proteins. In the prototype, the peptide can induce only a cellular immune response directed to infected cells of the body. For the prototype, the ability to induce an immune response capable of exerting a protective effect against the Ebola virus is not shown.

The technical result of the invention is the production of such immunogen peptides that carry the minimum necessary antigenic determinants for the formation of a specific immune response and contain antigenic portions of the Ebola virus GP protein, which are immunogenic for mice, guinea pigs, primates and humans, inducing protective immunity in guinea pigs and primates against infection with the Ebola virus.

The specified technical result is achieved by the fact that the obtained peptide is an immunogen used as a component of the Ebola vaccine vaccine, characterized by the amino acid sequence PQSLTTKPGPDNSTHNTPVYKLDISE (SEQ ID NO: 1) containing antigenic epitopes of the Ebola virus GP protein that have the ability to induce anti-virus antibodies from the development of Ebola and do not cause the development of immunopathological conditions.

The indicated technical result is also achieved by the fact that a second immunogen peptide is obtained that is used as a component of the Ebola virus vaccine, characterized by the amino acid sequence HHQDTGEESASSGKLGLITNTIAG (SEQ ID NO: 2) containing antigenic epitopes of the Ebola virus GP protein, which have the ability to induce the formation of neutralizing antibodies to protect against the development of Ebola and do not cause the development of immunopathological conditions.

The specified technical result is also achieved by the fact that a vaccine against Ebola fever, including immunogen peptides according to the invention, is obtained, as immunogen peptides it contains a mixture of an immunogen peptide having the amino acid sequence: PQSLTTKPGPDNSTHNTPVYKLDISE (SEQ ID NO: 1), and the peptide an immunogen having the amino acid sequence HHQDTGEESASSGKLGLITNTIAG (SEQ ID NO: 2). Moreover, the above peptides in a mixture are conjugated to an immunogenic carrier and adsorbed onto a pharmaceutically acceptable adjuvant.

A mixture of immunogen peptides can have a 1: 1 ratio, it contains hemocyanin protein as an immunogenic carrier, and aluminum hydroxide as a pharmaceutically acceptable adjuvant.

Example 1. Obtaining immunogen peptides and a drug based on them

Ïðè ðàçðàáîòêå âàêöèíû, èñïîëüçóÿ ëèòåðàòóðíûå äàííûå î Ò- è Â-êëåòî ÷ íûõ ýïèòîïàõ ãëèêîïðîòåèíà âèðóñà Ýáîëà, áûëè ðàññ ÷ èòàíû ñòðóêòóðû 19 ïåïòèäîâ - àíàëîãîâ èììóíîãåííûõ ýïèòîïîâ áåëêîâ âèðóñà Ýáîëà, êîòîðûå çàòåì áûëè ñèíòåçèðîâàíû. Ïîñëåäîâàòåëüíîñòü ýòèõ ñèíòåòè ÷ åñêèõ ïåïòèäîâ, ìîäåëèðóþùèõ ôóíêöèîíàëüíî çíà ÷ èìûå ó ÷ àñòêè áåëêîâ âèðóñà Ýáîëà, áûëè ñïðîåêòèðîâàíû òàêèì îáðàçîì, ÷ òîáû èñêëþ ÷ èòü ïðèñóòñòâèå ýëåìåíòîâ, îòâåòñòâåííûõ çà ðàçâèòèå èììóíîïàòîëîãè ÷ åñêîãî ñîñòîÿíèÿ, íî ñîõðàíèòü ñïîñîáíîñòü èíäóöèðîâàòü îáðàçîâàíèå âèðóñíåéòðàëèçóþùèõ àíòèòåë è îáåñïå ÷ èòü çàùèòó îò ðàçâèòèÿ ëèõîðàäêè Ýáîëà. Synthesizing the method for measuring the length of 23 to 29 for the quick-change method in the case of a

In case of syn- In the case of a carrier, you can use the game for yourself (young man 6). Turn on the drive in order to use the license plate.

The synthesis of sequences is carried out using standard equipment and methods of solid-phase peptide synthesis and can be carried out using any other methods of synthesis of amino acid sequences, for example, such as liquid-phase peptide synthesis, synthesis using genetically modified microorganisms (technology for producing recombinant proteins), fragmentation of the native protein with subsequent selection of the corresponding fragments.

The following are the obtained peptides:

LPRDRFKRTSFFLWVIILFQRTD

RSVGLNLEGNGVATDVPSVTKRWGFR

HPKLRPILLPNKSGKKGNSADLTSPEK GVPPKVVNYEAGEWAENCYNLEIKKP

GIRGFPRCRYVHKVSGTGPCAGDFAFHKE

FSSHPLREPVNATEDPSSGYYSTTIR

RYQATGFGTNETEYLFEVDNLTYVQLESR

ETIYASGKRSNTTGKLIWKVNPEIDTT

PKYIGLDPVAPGDLTMVITQDCDTGHS

GEWAFWETKKNLTRKIRSEELSFT

ETNTTNEDHKIMASENSSAMVQVHSQGR

PQSLTTKPGPDNSTHNTPVYKLDISE

DNSTHNTPVYKLDISEATQVGQHHRRAD

DTPPATTAAGPLKAENTNTSKSADSLD

HHQDTGEESASSGKLGLITNTIAG

GRRTRREVIVNAQPKCNPNLHYWTTQDEG

CGLRQLANETTQALQLFLRATTELRT

RTFSILNRKAIDFLLQRWGGTCHILGPD

CIEPHDWTKNITDKIDQIIHDFVDKTLPD

To conjugate the peptide to a carrier protein, a 2.5 mg Sulfo SMCC sample is prepared, dissolved in 500 μl of distilled water. To 350 μl of carrier protein (10 mg / ml), 350 μl of Sulfo SMCC (5 mg / ml) is added, incubated at room temperature for one hour. The activated carrier protein is separated on a 10 ml Sepharose CL4B column (Pharmacia Fine Chemicals No. IB 28549) in FSB, the optical density is controlled at a wavelength of 280 nm, and the first peak is collected. Using calibration samples, a calibration curve is constructed and the concentration of the activated carrier protein after purification on the column is determined. 1 mg of the peptide is dissolved in 250 μl of FSB, after which the activated carrier protein is added, based on a certain concentration, so that 0.75 mg of the carrier protein falls per 1 mg of peptide. The mixture is incubated at room temperature for 2 hours, followed by dialysis. Peptide conjugates are dialyzed in saline overnight. The resulting conjugates of the peptide and the carrier protein are sorbed on aluminum hydroxide to obtain the finished dosage form at a final concentration of 200 μg of the peptide in 1.0 ml of the drug.

Example 2. Designing a test-simulator

Êàæäûì èç 19 ïåïòèäîâ, êîíúþãèðîâàííûì ñ áåëêîì ãåìîöèàíèíîì è ñîðáèðîâàííûì íà ãèäðîîêèñü àëþìèíèÿ, âíóòðèìûøå ÷ íî áûëè âàêöèíèðîâàíû 14-16-ãðàììîâûå ìûøè ICR è áåñïîðîäíûå ìîðñêèå ñâèíêè (âåñ 180-200 ã) ïî ñõåìå äâóêðàòíîé èììóíèçàöèè ñ èíòåðâàëîì 21 ñóò.

1àáëèöà 1

Immunogenicity and protectiveness of peptides - analogues of immunogenic epitopes of Ebola virus proteins during 2-fold intramuscular immunization of mice and guinea pigs with a dose of 50 μg / animal

Kind of animal Peptide Wed geome. titer of antipeptide antibodies in ELISA Wed geom. pH antibody titer Protection ratio (%) Mice LPRDRFKRTSFFLWVIILFQRTD <1:30 but.* but. RSVGLNLEGNGVATDVPSVTKRWGFR 1:83 but. but. HPKLRPILLPNKSGKKGNSADLTSPEK 1: 2786 but. but. GVPPKVVNYEAGEWAENCYNLEIKKP 1: 613 but. but. GIRGFPRCRYVHKVSGTGPCAGDFAFHKE 1: 140 but. but. FSSHPLREPVNATEDPSSGYYSTTIR 1: 630 but. but. RYQATGFGTNETEYLFEVDNLTYVQLESR 1: 29261 but. but. ETIYASGKRSNTTGKLIWKVNPEIDTT 1: 458 but. but. PKYIGLDPVAPGDLTMVITQDCDTGHS 1: 3940 but. but. GEWAFWETKKNLTRKIRSEELSFT 1: 476 but. but. ETNTTNEDHKIMASENSSAMVQVHSQGR <1:30 but. but. PQSLTTKPGPDNSTHNTPVYKLDISE 1: 528 but. but. DNSTHNTPVYKLDISEATQVGQHHRRAD 1: 1835 but. but. DTPPATTAAGPLKAENTNTSKSADSLD 1: 146 but. but. HHQDTGEESASSGKLGLITNTIAG 1: 1393 but. but. GRRTRREVIVNAQPKCNPNLHYWTTQDEG 1:42 but. but. CGLRQLANETTQALQLFLRATTELRT 1: 540 but. but. RTFSILNRKAIDFLLQRWGGTCHILGPD <1:30 but. but. CIEPHDWTKNITDKIDQIIHDFVDKTLPD <1:30 but. but. Placebo ** 1:45 but. but. Guinea pigs LPRDRFKRTSFFLWVIILFQRTD 1: 276 <1:10 0 RSVGLNLEGNGVATDVPSVTKRWGFR 1: 414 <1:10 0 HPKLRPILLPNKSGKKGNSADLTSPEK 1: 19700 twenty 0 GVPPKVVNYEAGEWAENCYNLEIKKP 1: 3066 <1:10 0 GIRGFPRCRYVHKVSGTGPCAGDFAFHKE 1: 800 <1:10 0 FSSHPLREPVNATEDPSSGYYSTTIR 1: 3184 <1:10 0 RYQATGFGTNETEYLFEVDNLTYVQLESR 1: 204804 <1:10 0 ETIYASGKRSNTTGKLIWKVNPEIDTT 1: 3212 <1:10 0 PKYIGLDPVAPGDLTMVITQDCDTGHS 1: 15222 34 25 GEWAFWETKKNLTRKIRSEELSFT 1: 3264 <1:10 0 ETNTTNEDHKIMASENSSAMVQVHSQGR 1: 219 <1:10 0 PQSLTTKPGPDNSTHNTPVYKLDISE 1: 2263 28 twenty DNSTHNTPVYKLDISEATQVGQHHRRAD 1: 12845 <1:10 0 DTPPATTAAGPLKAENTNTSKSADSLD 1: 824 <1:10 0 HHQDTGEESASSGKLGLITNTIAG 1: 9870 48 80 GRRTRREVIVNAQPKCNPNLHYWTTQDEG 1: 212 <1:10 0 CGLRQLANETTQALQLFLRATTELRT 1: 3271 <1:10 0 RTFSILNRKAIDFLLQRWGGTCHILGPD 1: 234 <1:10 0 CIEPHDWTKNITDKIDQIIHDFVDKTLPD 1: 247 <1:10 0 Placebo <1: 30 <1:10 0

Note:

* .. n.o - not determined;

** .. hemocyanin adsorbed on aluminum hydroxide was used as a placebo.

For each application, use the 50-meter / turn-key option (see table 1). 21 × åðåç ñóò ïîñëå âòîðîé èììóíèçàöèè ó æèâîòíûõ áûëè îòîáðàíû îáðàçöû êðîâè äëÿ îïðåäåëåíèÿ òèòðà ñïåöèôè ÷ åñêèõ àíòèòåë â ðåàêöèè èììóíîôåðìåíòíîãî àíàëèçà. Promoting the range of spark plugs if you need to have a quick shutter for the better Shorummy S-7 in the range 10-30 _{50 50} for you. Further Information in Table 1.

For further research, four peptides were selected that induce neutralizing antibodies and / or protect model animals from infection with the Ebola virus.

Example 3. Evaluation of the effect of peptides on the formation of immunity in combined use

For use in conjunction with the use of an instrument for

1Ï (HPKLRPILLPNKSGKKGNSADLTSPEK),

2Ï (PKYIGLDPVAPGDLTMVITQDCDTGHS),

3Ï (PQSLTTKPGPDNSTHNTPVYKLDISE) è

4Ï (HHQDTGEESASSGKLGLITNTIAG).

Èììóíèçàöèþ ïðîâîäèëè ëèáî ââîäÿ êàæäîìó æèâîòíîìó êàæäûé èç ïåïòèäîâ â 4 ðàçëè ÷ íûå òî ÷ êè òåëà æèâîòíîãî, ëèáî êàæäîìó æèâîòíîìó ñìåñü ïåïòèäîâ â 4 ðàçëè ÷ íûå òî ÷ êè òåëà æèâîòíîãî. Listed options for the 14-16-bit lower ICR and the latest minor version (all 150-200 ã).

2àáëèöà 2

The effects of peptides on the formation of immunity in combined use

Kind of animal Scheme of drug administration Peptide Reverse Wed geome. ELISA antipeptide antibody titer (CI ₉₅ ) ICR mice to 4 points of the animal’s body separately by each of the 4 peptides 1P 919 (336-2517) 2P 3200 (1258-8139) 3P 132 (79-221) 4P 2111 (873-5104) to 4 points of the animal’s body separately with a mixture of 4 peptides 1P 8063 (6342-10252) 2P 4222 (2300-7752) 3P 528 (266-1049) 4P 1056 (464-2403) Guinea pigs to 4 points of the animal’s body separately by each of the 4 peptides 1P 3805 (1182-12255) 2P 4525 (3501-5850) 3P 673 (260-1743) 4P 436 (207-920) to 4 points of the animal’s body separately with a mixture of 4 peptides 1P 6400 (1773-23104) 2P 9051 (5802-14120) 3P 617 (314-1213) 4P 2263 (930-5507)

The combined use of 4 peptides in mice by immunizing one animal in four different body parts near regional lymph nodes with one peptide immunogen indicates the immunodominant properties of 2P and 4P peptides and suppression of the immune response to 1P and 3P peptides. A mixture of 4 immunogens used in mice showed immunodomination of the peptide immunogen 1P, while the response to other peptides did not differ from that in the case of separate immunization (see table 1). For guinea pigs, the use of a mixture of 4 peptide immunogens demonstrates the immunodomination of peptides 1P and 2P over peptides 3P and 4P. Given the results of experiments to determine the titer of neutralizing antibodies and protective (see table. 1) the peptide 4P was included in the composition of the claimed vaccine. Considering the fact that immunodominant peptides 1P and 2P, which induce the highest level of peptide-specific antibodies and inhibit the action of two other peptides, do not provide protection (peptide 1P) or provide a low level of protection of 25% (peptide 2P), these peptides were excluded from the composition vaccines. Peptide 3P was included in the composition of the claimed vaccine, since it has the highest neutralization potential, is not immunodominant, and does not affect the induction of an immune response to peptide 4P.

Example 4. Determination of the optimal dose and immunization schedule

Äëÿ îïðåäåëåíèÿ îïòèìàëüíîé äîçû èììóíèçàöèè èñïîëüçîâàëàñü ñìåñü ïåïòèäîâ 3Ï è 4Ï â ñîîòíîøåíèè 1: 1, êîíúþãèðîâàííûõ ñ áåëêîì ãåìîöèàíèíîì è ñîðáèðîâàííûõ íà ãèäðîîêèñè àëþìèíèÿ â êîíå ÷ íûõ äîçàõ 10, 50, 100, 200 è ìêã ñìåñè ïåïòèäîâ / 0,5 ìë ïðåïàðàòà. A special way to automatically change the value of the maximum speed (180-200 ã) is provided. Î 180ûûûûûû (((ââââââââââûâââââââââââââââââââââââââââââââââ For the use of 1-, 2- and 3-short-circuit models, the operation of the unit is 21 in the case of a second. 21 × åðåç ñóò ïîñëå êàæäîé èììóíèçàöèè ó æèâîòíûõ áûëè îòîáðàíû îáðàçöû êðîâè äëÿ îïðåäåëåíèÿ òèòðà ñïåöèôè ÷ åñêèõ àíòèòåë â ðåàêöèè íåéòðàëèçàöèè in vitro è èììóíîôåðìåíòíîãî àíàëèçà. Ãðóïïû ìîðñêèõ ñâèíîê ïîñëå îäíîêðàòíîãî, äâóêðàòíîãî è òðåõêðàòíîãî ââåäåíèÿ ïðåïàðàòà áûëè çàðàæåíû âèðóñîì Ýáîëà øòàìì ÌÑ-7 â äîçå 30-100 ÁÎÅ / æèâîòíîå.

3àáëèöà 3

The choice of dose and dosage regimen for 1-, 2- and 3-fold intramuscular immunization of guinea pigs at doses of 10, 50, 100 and 200 μg / animal

Kind of animal Dose (mcg / animal) Wed geome. antipeptide antibody titer (95% confidence interval)
Protection coefficient (%) 1x 2x 3x 1 cr 2 cr 3 cr. Guinea pigs 10 5382
(2762-10486) 10763
(8466-13685) 18102
(9337-35096 0 thirty fifty fifty 3200
(1622–6312) 20319
(15398-26812) 9510
(4861-18607 fifty 80 80 one hundred 9510
(7357–12294) 12800
(7918-20692) 10500
(5411-20375) 60 one hundred one hundred 200 3592
(1011–12762) 11637
(6659-17769) 5382
(2908-9961) fifty one hundred 80 Placebo * <30 0

Note:

* hemocyanin adsorbed on aluminum hydroxide was used as a placebo

As follows from the data of table 3, the maximum - 100% level of protection was achieved with 2-fold administration of the drug at a dose of 100 μg of a mixture of peptides / 0.5 ml of the drug / animal. Using the same immunization scheme, an intense humoral immune response was formed in animals. An increase in the dose of the drug or the frequency of administration did not lead to a significant increase in the immune response, therefore, for the further studies, the following vaccination schedule and dose of the EpiVacEbola preparation were chosen: 2-fold administration at a dose of 100 μg / 0.5 ml.

Example 5. The study of the immunogenic and protective properties of the drug "EpiVakEbola" on monkeys

To determine the immunogenic and protective properties of the EpiVacEbola preparation, monkeys were immunized 3 times with a dose of 100 μg / 0.5 ml / animal. 21 days after the 2nd and 3rd immunization, blood samples were taken from animals to determine the titer of specific antibodies in the in vitro neutralization reaction and enzyme immunoassay. Groups of monkeys after double and triple administration of the drug were infected with the Ebola virus strain Zaire at a dose of 30-100 PFU / animal.

Section 4

The study of immunogenic and protective properties of the drug

"EpiVakEbola" on the monkeys

Multiplicity of drug administration Antibody titer in the reaction of viral neutralization Protection ratio (%) 2x 1: 40-1: 80 thirty 3x 1: 80-1: 320 75

Example 6. The study of the safety and effectiveness of the drug "EpiVacEbola" in the framework of preclinical and clinical studies

The EpiVacEbola series were used for preclinical and clinical trials.

As part of the EpiVakEbola pre-clinical study program for Ebola fever prophylaxis, its general and immunological toxicity was evaluated in mice, guinea pigs and rabbits in accordance with the recommendations (Guidelines for preclinical studies of drugs. Part 2. M .: Grif and K , 2012). The toxicological testing program included the study of the “acute” and chronic toxicity of the vaccine in mice and guinea pigs, an assessment of its effect on blood hematological and biochemical parameters, local irritant effects, immunotoxic and allergenic properties, pyrogenicity, and effects on the central nervous system function.

A toxicological study in mice and guinea pigs showed that a single intraperitoneal or subcutaneous administration of three EpiVacEbola vaccines in doses that are maximum possible for administration, as well as ten times subcutaneous administration in a vaccinating dose, did not lead to the death of animals, did not significantly affect appearance, body weight of animals, level of hematological and biochemical parameters, weight and structure of their internal organs. The vaccine preparation had a reversible local irritant effect comparable in intensity with the effect of its component, aluminum hydroxide. The EpiVacEbola vaccine did not show pronounced immunotoxic, allergenic and pyrogenic properties, and did not have a significant neurotoxic effect.

Based on the data obtained, a conclusion was drawn on the safety of the EpiVacEbola vaccine for the prevention of Ebola.

Volunteers were vaccinated as part of the EpiVacEbola clinical study program for the prevention of Ebola. Vaccination results showed that the use of the drug causes the development of mild local reactions in the form of hyperemia and the formation of infiltrate, which took place within 1-3 days, the use of medications was not required. Of the general reactions, only an increase in body temperature was observed. The reactions were mild (37.0 ° C, 37.3 ° C), observed during the first day. Only in 1 case did the temperature rise to 38.2 ° C. Biochemical, general clinical, as well as non-specific immunological reactions did not go beyond the norm. When studying the humoral response, it was shown that the EpiVacEbola vaccine induces in 100% of volunteers specific antibodies to the Ebola virus with a geometric mean titer in ELISA of 1: 577. Vaccination with the drug causes the formation of neutralizing antibodies in 95% of volunteers. When studying the cellular response, it was shown that the introduction of the EpiVacEbola vaccine leads to the activation of a specific cellular immunity reaction to Th2 type Ebola antigens in 100% of volunteers.

Thus, the mixture of the claimed peptides jointly induces a synergistic immune response that provides 100% protection. The introduction of such peptide immunogens leads to the formation of a cellular and humoral immune response in vaccinated people. To strengthen immunity, the adjuvant of aluminum hydroxide permitted in many countries of the world has been introduced into the vaccine. Preclinical trials of the EpiVacEbola vaccine have proven that immunization with the EpiVacEbola drug forms a cellular and humoral immune response in laboratory animals (mice, guinea pigs, primates) and protects guinea pigs and primates from lethal doses of the Ebola virus.

Claims (5)

1. An immunogen peptide used as a component of an Ebola vaccine vaccine, characterized by the amino acid sequence PQSLTTKPGPDNSTHNTPVYKLDISE (SEQ ID NO: 1) containing antigenic epitopes of the Ebola virus GP protein that are capable of inducing the formation of virus-neutralizing antibodies to protect against Ebola. 2. An immunogen peptide used as a component of an Ebola virus vaccine, characterized by the amino acid sequence HHQDTGEESASSGKLGLITNTIAG (SEQ ID NO: 2) containing antigenic epitopes of the Ebola virus GP protein, which have the ability to induce the formation of virus-neutralizing antibodies to protect against the development of fever. 3. An Ebola vaccine, including immunogen peptides, characterized in that as immunogen peptides it contains a mixture of the immunogen peptide according to claim 1, having the amino acid sequence: PQSLTTKPGPDNSTHNTPVYKLDISE (SEQ ID NO: 1), and the immunogen peptide according to p. 2, having the amino acid sequence HHQDTGEESASSGKLGLITNTIAG (SEQ ID NO: 2), wherein the above peptides in a mixture are conjugated to an immunogenic carrier and adsorbed onto a pharmaceutically acceptable adjuvant. 4. The Ebola vaccine according to claim 3, characterized in that the immunogen peptides according to claims. 1 and 2 in the mixture have a ratio of 1: 1. 5. The Ebola vaccine according to claim 3, characterized in that it contains hemocyanin protein as an immunogenic carrier, and aluminum hydroxide as a pharmaceutically acceptable adjuvant.