WO2020236116A1

WO2020236116A1 - Universal crimean congo haemorrhagic fever virus (cchfv) vaccine

Info

Publication number: WO2020236116A1
Application number: PCT/TR2020/050428
Authority: WO
Inventors: Aykut ÖZDARENDELİ; Shaikh Terkis Islam PAVEL
Original assignee: Oezdarendeli Aykut
Priority date: 2019-05-20
Filing date: 2020-05-18
Publication date: 2020-11-26
Also published as: EP3941518A1; TR201907557A2

Abstract

This invention relates to universal Crimean Congo Haemorrhagic Fever Virus (CCHFV) vaccine. This vaccine comprising viral vector with Bioinformatically Generated Conserved Antigen (BGCA) of CCHFV which evoke immune responses against CCHFV, or diseases triggered by infection of CCHFV.

Description

UNIVERSAL CRIMEAN CONGO HAEMORRHAGIC FEVER VIRUS (CCHFV)

VACCINE

Field of the Invention

Background of the Invention

Crimean Congo Haemorrhagic Fever Virus (CCHFV) is a tick-borne infectious pathogen and causative agent of viral haemorrhagic fever. This disease was first described by a physician in 12^th century in Tadzhikistan. In modern medical science, it took its first medical entry when 200 soviet military personnel were infected during war in 1944-1945. In 1967, Chumakov and his colleagues first isolated the virus from newborn white mice. Between 1956 and 1965, a virus was isolated from human patients from the Congo and Uganda which was antigenically identical to the soviet strain.

Crimean Congo Haemorrhagic Fever Virus is a tripartite RNA genome virus with negative polarity of the genus Orthonairovirus and family Nairoviridae. The genome of this virus is composed of three different RNA segments: the small (S), The medium (M) and the Large (L). The small segment‘S’ encodes nucleoprotein (NP) which is around 1.6kb long. The medium segment‘M’ encodes glycoprotein (GPC) which is around 5.5kb long. The large segment‘L’ encodes RNA - dependent RNA polymerase (RdRp) which is around 12 kb long. The untranslated regions (UTRs) on 5' and 3' of the S, M and L segments necessary for viral transcription, replication and packaging. There are nine highly conserved nucleotides between nairoviruses which called terminal nucleotides (5'-TCTCAAAGA and 3'-AGAGTTTCT region) assist as viral promoter region.

The ticks from Hyalomma genus are predominantly responsible for the broad distribution of the virus. It is transmitted by the bite of infected ticks to human and livestock or by direct contact with the infected tissues or blood from humans and animals. Geographically, CCHFV is widespread in the areas of Western and Central Asia, the Middle East, South-Eastern Europe and Africa. The mortality rates of the Crimean Congo Haemorrhagic Fever (CCHF) is 5%-30%. Generally, the incubation period is less than 7 days though it depends on the mode of infection. Initially after infection, the patients are showing non-specific symptoms like high fever, fatigue, myalgia, vomiting and diarrhea. With progression of the incubation period, the patients are showing thrombocytopenia, high level of liver enzymes and several haemorrhagic appearances. Nosocomial CCHFV infection is also reported.

The early stage diagnosis is vital for Crimean Congo Haemorrhagic Fever (CCHF). Virus Culture, immunohistochemistry, antigen detection enzyme immunoassay (EIA) and reverse transcription- PCR are the diagnostic assays for acute Crimean-Congo Haemorrhagic Fever. The best method till date for checking virus infectivity are plaque assays or fluorescent focus assays. World health organization suggested nucleotide analog ribavirin to do treatment against CCHF because of its clinical advantages. Although recently favipiravir showed good efficacy result than ribavirin against two strains of CCHFV.

The viral vector is from human adenovirus type 5 (clontech). The genome size of adenoviral vector is 36000bp. This viral vector is easy to manipulate by recombinant DNA technology. The facilities of using adenoviral vector are as follows: adenoviral vector proliferates and remain stable in wide variety of mammalian cell types; the genome rearrangement rate is not so high during proliferation; the genome replicates higher titer in permissive cells.

The deletion of early El region from adenoviral genome make it replication incompetent as well as enable to insert foreign gene of interest. Likewise, the deletion of E3 together with El region of adenoviral genome enables to insert foreign gene of interest up to 8kb. Therefore, the adenoviral vector needs El complementing factor to replicate in mammalian cells. The vector also contains cytomegalovirus (CMV) at the early promoter region. Thus, if the vector remains inactive, the gene of interest will express independently.

Till date, there were no FDA or EMA approved vaccines against CCHFV. Aykut Ozdarendeli and his colleagues developed cell culture based inactivated vaccine which elicited significant level of protection against CCHFV Turkey Kelkit 06 strain in IFNAR a/b R mice; (Patent number W02014039021A1). Suckling mice brain inactivated vaccine against CCHFV is available in Bulgaria now but the result is not consistent in terms of neutralizing antibody titer. Additionally, protective efficacy result has not been reported in this case. Karen R Buttigieg et al showed modified vaccinia virus Ankara expressing CCHFV M segment from IbAr 10200 strain raised cellular and humoral immune responses (W02014/132013). Jorma Hinkula et al developed a DNA vaccine which showed 100% efficient preventive immunity against lethal CCHFV challenge. Additionally, Marko Zivcec et al demonstrated that adenovirus-based vaccine expressing CCHFV nucleoprotein (lb Ar 10200 strain) showed protective efficacy till 78% against IFNAR mice. However, they only showed efficacy result without any standard cellular immune responses report.

All the available vaccines against CCHFV elicit immune responses against a specific strain to a certain area in the globe. Therefore, it is badly in need to develop a universal CCHFV vaccine which elicit a broad antibody response against all the known CCHFV strain.

Summary of the Invention

The present invention accomplishes the demand of universal CCHFV vaccine by providing Bioinformatically Generated Conserved Antigen (BGCA) of CCHFV with viral vector. All the presently invented vaccine candidates illustrate enhanced efficacy and immunogenicity in IFNAR a/b R mouse model by evoking immune responses against CCHFV infection.

As used herein, antigenic fragment means amino acid sequence of protein from CCHFV BGCA glycoprotein (GPC) or BGCA nucleoprotein (NP) which possesses the ability to induce immune responses against CCHFV infection or CCHFV infection related diseases.

This present invention provides the method of several rounds of consensus sequence generation called Bioinformatically Generated Conserved Antigen (BGCA) of CCHFV. This method is based on determining most conserved amino acids of different CCHFV genotype groups wherein full length of consensus GPC sequence from group V of CCHFV named BGCA V GPC (Figure 3) and full length of consensus NP sequence from group V of CCHFV called BGCA V NP (Figure 2). Correspondingly, full length of consensus NP sequence from all strains of CCHFV in the world named BGCA I-VI NP (Figure 1).

In one embodiment, all the CCHFV strains glycoprotein (GPC) amino acid sequences from group V were downloaded from GenBank database. Likewise, all the CCHFV strains nucleoprotein (NP) amino acid sequences were downloaded from GenBank database. Phylogenetic subgroups designed to individual genotype groups by using MEGA bioinformatics tool based on geographic location. Then, the amino acid sequences (GPC and NP) align according to its individual group and determined the most conserved amino acids at respective positions as a primary consensus sequence in its group. The inventor named it as BGCA I, II, III, IV, V, VI and VII CCHFV antigen. After that, all the seven groups conserved amino acid sequences aligned and determined the most conserved amino acids at respective positions from all the groups as a I-VI consensus sequence. The inventor named it as BGCA I-VI CCHFV antigen. Later the amino acid sequences are reverse translate and codon optimize for expression in mammalian cells. These BGCA fragments were synthetically produced and inserted into pShuttle 2 expression vector.

In another embodiment, the BGCA V GPC (Europe/Turkey) amino acid sequences of CCHFV from group V (Figure 3) invented by following the methods that describe in the previous embodiment. Briefly, the amino acid sequences from the 25 different strains in group V were aligned and determined the most conserved amino acids at respective positions (Figure 3; Sequence ID No. 2). After that, the amino acid sequence is reverse translate and codon optimize for expression in mammalian cells. The name of the fragment is BGCA V GPC. BGCA V GPC was artificially synthesized and recombined into pShuttle 2 vector. In Addition, by applying BGCA method, the inventors generate BGCA group I GPC (W. Africa; Figure 4; Sequence ID No 9, 10 ), BGCA group III GPC (South Africa/ West Africa 2; Figure 5; Sequence ID No 11, 12 ), BGCA group IV GPC (Asia/Middle East; Figure 6; Sequence ID No 13, 14 ).

In such embodiment, there are 25 different strains or isolates in group V for GPC which inventor named BGCA V GPC (Figure 3). All the 25 different CCHFV strains contain 1688 amino acids. BGCA V GPC is 96 to 97% conserved to all Turkey CCHFV strains, 97.92% conserved to Kashmanov CCHFV strains, 97.92% conserved to ROS/HUVLV CCHFV strain, 97.27% conserved to Kosova/Hoti CCHFV strain, 96.20% conserved to Drosdov CCHFV strain, 97.03% conserved to VLG/TI29414 CCHFV strain, 96.62% conserved to Kosovo/9553/2001 CCHFV strain and 95.49% conserved to V42181/Bulgaria CCHFV strain (Figure 3).

In another embodiment, BGCA V GPC is 100% conserved to BGCA V (Europe/Turkey), 87.27% conserved to BGCA IV (Asia/Middle East), 86.96% conserved to BGCA III (South Africa/ West Africa), 75.36% conserved to BGCA I (West Africa), 76.25% conserved to BGCA VII (Mauritania), 74.17% conserved to BGCA VI (Greece) (Figure 3). In yet another embodiment, the BGCA I- VI NP amino acid sequence from all the groups are invented by following the methods that describe the earlier embodiment. Briefly, individual BGCA fragments determined from six different phylogenetic groups for NP. After that, I-VI BGCA NP fragment determined by using most conserved amino acids from all the BGCA groups. Later the amino acid sequence is reverse translate and codon optimize for expression in mammalian cells. The name of the fragment is BGCA I-VI NP (Figure 1; Sequence ID No.4). BGCA I-VI NP was artificially synthesized and recombined into pShuttle 2 vector.

In another embodiment, the BGCA V NP amino acid sequence of CCHFV from group V (Figure 2) invented by following the methods that describe the previous embodiment. Briefly, the amino acid sequences from the 29 different strains in group V are aligned and determined the most conserved amino acid at respective position (Figure 2; Sequence ID No.6). After that, the amino acid sequence is reverse translated, and codon optimized for expression in mammalian cells. The name of the fragment is BGCA V NP. BGCA V NP was artificially synthesized and recombined into pShuttle 2 vector.

In yet another embodiment, all the CCHFV nucleoprotein from group V has 482 amino acids (Sequence ID No. 6). The BGCA V NP is 98 to 99% conserved to all Turkey CCHFV strains, 99.79% conserved to Kashmanov CCHFV strains, 100% conserved to ROS/HUVLV CCHFV strain, 99.79% conserved to Kosova/Hoti CCHFV strain, 99.58% conserved to Drosdov CCHFV strain, 99.17% conserved to VLG/TI29414 CCHFV strain, 100% conserved to Kosovo/9553/2001 CCHFV strain and 99.17% conserved to V42181/Bulgaria CCHFV strain (Figure 2).

In another embodiment, BGCA V NP is 100% conserved to BGCA V (Europe/Asia), 98.88% conserved to BGCA IV (Asia/Middle East), 96.68% conserved to BGCA III (South Africa/West Africa), 94.81% conserved to BGCA I (West Africa), 92.53% conserved to BGCA VI (Greece) and 96.26% conserved to BGCA II (East Africa/Central Africa).

In such embodiment, CCHFV NP from all groups have 482 amino acids (Sequence ID No. 4). The BGCA I-VI NP is 97.92% conserved to BGCA V (Europe/Asia), 98.96% conserved to BGCA IV (Asia/Middle East), 98.34% conserved to BGCA III (South Africa/West Africa), 95.85% conserved to BGCA I (West Africa), 93.15% conserved to BGCA VI (Greece) and 97.92% conserved to BGCA II (East Africa/Central Africa). The present invention further provides replication incompetent adenoviral vector as viral vector comprising BGCA V GPC or BGCA V NP or BGCA I-VI NP to show protective immunity against CCHFV after immunization with different combination.

This invention provides the methods of constructing adenoviral vector with Bioinformatically Generated Conserved Antigen (BGCA) of GPC or NP which evoke immune responses against CCHFV infection.

This invention provides the methods of transfection of the adenoviral construct with BGCA segments (BGCA V GPC or BGCA V NP or BGCA I-VI NP) into HEK 293 cells.

In one embodiment, this HEK293 cells have El complementing factor which help to replicate the replication incompetent adenoviral vector inside the cells.

This invention provides the methods of harvesting recombinant adenovirus containing BGCA V GPC or BGCA V NP or BGCA I-VI NP from HEK293 cells after transfection.

This invention provides NP of CCHFV Turkey-Kelkit06 strain which is codon optimized for E- Coli based expression system (Sequence ID No. 8).

This invention also provides the method of constructing bacterial vector comprising codon optimized Turkey-Kelkit06 NP.

In one embodiment, pET28 used as bacterial vector for constructing pET28 Turkey-Kelkit06 NP.

In another embodiment, this bacterial vector with Turkey -Kelkit06 NP acts as antigenic fragment. Thus, it evokes immune responses against CCHFV infection in a subject.

This invention provides the nucleic acid sequence encoding BGCA V GPC has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 1 (Sequence ID No. 1).

In another embodiment, amino acid sequence encoding BGCA V GPC has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 2 (Sequence ID No. 2).

The invention provides the nucleic acid sequence encoding BGCA I-VI NP has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 3 (Sequence ID No.

3)·

In another embodiment, the amino acid sequence encoding BGCA I- VI NP has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the give sequence ID no 4 (Sequence ID No.

4)·

The invention provides the nucleic acid sequence encoding BGCA V NP has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 5 (Sequence ID No.

5).

In another embodiment, amino acid sequence encoding BGCA V NP has at least 70% (such as 70,

71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,

97, 98, 99 and 100%) sequence identity to the given sequence ID no 6 (Sequence ID No.6).

This invention provides nucleic acid sequence encoding CCHFV Turkey-Kelkit06 NP has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 7 (Sequence ID No 7)

In such embodiment, the amino acid sequence encoding CCHFV Turkey-Kelkit06 NP has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 8 (Sequence ID No 8)

In such embodiment, the amino acid sequence encoding BGCA I GPC has at least 70% (such as

70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 9 (Sequence ID No 9).

In such embodiment, the amino acid sequence encoding BGCA III GPC has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 11 (Sequence ID No 11).

In such embodiment, the amino acid sequence encoding BGCA IV GPC has at least 70% (such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%) sequence identity to the given sequence ID no 13 (Sequence ID No 13)

As used herein, the first-time administration of a vaccine candidate to a subject named prime and second time administration of a vaccine candidate to the same subject named prime boost. Prime boost vaccination may be homologous or heterologous combination.

As used herein, the term‘subject’ for administering of a vaccine. Subject can be animal or human.

The present invention also provides the amino acid sequence ID No. 2 for BGCA V GPC with adenoviral vector and sequence ID No. 4 for BGCA I-VI NP with adenoviral vector as viral vector mixed together for prime and prime boost vaccination. It provides adequate immune responses in a subject against CCHFV infection.

The present invention also provides the amino acid sequence ID no 2 for BGCA V GPC with adenoviral vector and sequence ID No. 4 for BGCA I-VI NP with adenoviral vector mixed together for prime vaccination. Similarly, the sequence ID No 2 for BGCA V GPC with adenoviral vector and sequence ID No 6 for BGCA V NP with adenoviral vector mixed together for prime boost vaccination provide adequate immune responses in a subject against CCHFV infection.

The present invention provides the amino acid sequence ID No. 4 for Turkey-Kelkit06 NP with pET28 as bacterial vector used as prime boost elicit enough immune responses against CCHFV infection.

In one embodiment, BGCA V GPC with adenoviral vector and BGCA I-VI NP with adenoviral vector mixed together as prime vaccination. pET28 comprising Turkey -Kelkit06 NP use as prime boost for the vaccination against CCHFV infection.

The inventors previously patented (Patent number WO2014039021A1) the cell culture based inactivated vaccine against CCHFV.

In one embodiment, BGCA V GPC with adenoviral vector and BGCA I-VI NP with adenoviral vector mixed for prime and cell culture based inactivated vaccine use as prime boost vaccination against CCHFV infection. This combination elicits adequate immune responses against CCHFV infection. This invention provides the delivery of antigenic fragments as BGCA V GPC or BGCA V NP or BGCA I-VI NP with adenoviral vector. This adenoviral vector is stable and express the BGCA V GPC or BGCA V NP or BGCA I-VI NP antigenic fragment independently which enhanced the immune responses in subject after vaccination.

This invention also provides the delivery of antigenic fragment as Turkey -Kelkit06 NP with pET28. This bacterial vector is stable and express the Turkey-Kelkit06 NP which enhanced the immune responses in a subject.

In one embodiment, The Xbal (TCTAGA) restriction site and BamHI (GGATCC) restriction site artificially combine to the 5' of the BGCA V GPC fragments. Similarly, The Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially combine to the 3' of the BGCA V GPC fragments (Sequence ID No. 1).

In another embodiment, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA V GPC fragments (Sequence ID No. 1).

In such embodiment, The Xbal (TCTAGA) restriction site and BamHI (GGATCC) restriction site artificially attach to the 5' of the BGCA I-VI NP fragments. Likewise, The Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially attach to the 3' of the BGCA I-VI NP fragments (Sequence ID No. 3).

In another embodiment, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA I-VI NP fragments (Sequence ID No. 3).

In yet another embodiment, The Xbal (TCTAGA) restriction site and BamHI (GGATCC) restriction site artificially merge to the 5' of the BGCA V NP fragments. Correspondingly, The Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially merge to the 3' of the BGCA V NP fragments (Sequence ID No. 5).

In another embodiment, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA V NP fragments (Sequence ID No. 5).

In certain embodiment, The BamHI (GGATCC) restriction site artificially combine to the 5' of the Turkey-Kelkit06 NP fragments. Likewise, The Xhol (CTCGAG) restriction site artificially attach to the 3' of the Turkey-Kelkit06 strain. This invention provides the composition of vaccine with adenoviral vector comprising BGCA V GPC or BGCA V NP or BCGA I- VI NP. During vaccination, sterile phosphate-buffered saline (PBS; pH- 7.2-7.4) use for dilution of that vaccine candidates. The composition of these vaccine candidates does not encompass any chemical adjuvants.

In one embodiment, the cell culture based inactivated vaccine mix with imject alum (Aluminum Hydroxide and Magnesium Hydroxide).

In such embodiment, pET28 comprising Turkey -Kelkit06 NP as a vaccine candidate mix with imject alum (Aluminum Hydroxide and Magnesium Hydroxide).

This invention provides adenoviral vector formulating with BGCA V GPC or BGCA V NP or BGCA I-VI NP administering to human or animal to evoke immune responses against CCHFV infections and diseases. This immune response may be T cell mediated or B cell mediated.

In such embodiment, pET28 as bacterial vector comprising Turkey-Kelkit06 NP administering to human or animal as prime boost evoke immune responses against CCHFV infections and diseases. These immune responses may be T cell mediated or B cell mediated.

In another embodiment, cell culture based inactivated vaccine administering to human or animal as prime boost evoke immune responses against CCHFV infections and diseases.

In certain embodiment, the immune responses embrace T cell and B cell responses.

This invention provides the strategy of vaccination with adenoviral vector. Adenoviral vector containing BGCA V GPC mixed with adenoviral vector containing BGCA I-VI NP just before vaccination to a subject. Adenoviral vector containing BGCA V M mixed with adenoviral vector comprising BGCA V NP just before vaccination to a subject. These assortments based on equal volume with equal infectious units per milliliter (ifu/ml).

The present invention provides the steps of administration of the adenoviral vector comprising BGCA V GPC mixed with adenoviral vector comprising BGCA I-VI NP used as prime and prime boost vaccination. This homologous prime-boost vaccination evokes adequate immune responses against CCHFV diseases and infections.

The present invention provides the steps of administration of adenoviral vector comprising BGCA V GPC mixed with adenoviral vector comprising BGCA I-VI NP used as prime vaccination. Adenoviral vector comprising BGCA V GPC mixed with adenoviral vector containing BGCA V NP used as prime boost vaccination. This heterologous prime boost vaccination evokes enough immune responses against CCHFV diseases and infections.

The present invention provides the steps of administration of adenoviral vector comprising BGCA

V GPC mixed with adenoviral vector comprising BGCA I- VI NP used as prime vaccination. Cell culture based inactivated vaccine used as prime boost vaccination. This heterologous prime boost vaccination evokes adequate immune responses against CCHFV diseases and infections.

V GPC mixed with adenoviral vector comprising BGCA I- VI NP used as prime vaccination. pET28 comprising Turkey -Kelkit06 NP used as prime boost vaccination. This heterologous prime boost vaccination evokes enough immune responses against CCHFV infections and diseases.

This present invention provides the schedules of administration of these vaccine candidates. These vaccine candidates are administering to a subject for multiple doses with 2-8 weeks interval. To evoke adequate immune responses, interval time is very important.

In one embodiment, the adenoviral vector comprising BGCA V GPC mixed with adenoviral vector containing BGCA I- VI NP used as prime and prime boost vaccination. There are 2-8 weeks interval time between prime and prime boost.

In another embodiment, the adenoviral vector comprising BGCA V GPC mixed with adenoviral vector comprising BGCA I-VI NP used as prime vaccination. Adenoviral vector containing BGCA

V GPC mixed with adenoviral vector containing BGCA V NP used as prime boost vaccination. There are 2-8 weeks interval time between prime and prime boost.

In yet another embodiment, the adenoviral vector comprising BGCA V GPC mixed with adenoviral vector comprising BGCA I-VI NP used as prime vaccination. Cell culture based inactivated vaccine used as prime boost vaccination. There are 2-8 weeks interval time between boost and prime boost.

In such embodiment, the adenoviral vector comprising BGCA V GPC mixed with adenoviral vector comprising BGCA I-VI NP used as prime vaccination. Bacterial vector pET28 comprising Turkey-Kelkit06 NP used as prime boost. There are 2-8 weeks interval time between boost and prime boost. The present invention provides the correct quantity of these vaccine candidates for administration to a subject. The exact quantity is very important to evoke adequate immune responses against any viral infection.

In such embodiment, the precise quantity of adenoviral vector vaccine candidates determined to induce adequate immune responses.

In one embodiment, the quantity of the Turkey-Kelkit06 NP with pET28 determined which evoke the adequate immune responses after administering as prime boost.

In another embodiment, the quantity of the cell culture based inactivated vaccine also determined which elicit the enough immune responses after administering as prime boost.

The present invention provides the route of administration for the vaccine. For adenoviral vector comprising BGCA V GPC or BGCA V NP or BGCA I- VI NP (whether it is prime or prime boost), the route of administration is intramuscular. For cell culture based inactivated vaccine as prime boost, the route of administration is intraperitoneal. Fikewise, for pET28 with Turkey-Kelkit06 NP, the route of administration is intraperitoneal.

As used herein, the viral challenge means injecting live CCHFV turkey/Kelkit 06 strain to a subject.

Brief Description of the Drawings

Figure 1. Phylogenetic analysis of 107 CCHFV strains/isolates based on CCHFV nucleoprotein.

Figure 2. Phylogenetic analysis of group V CCHFV strains/isolates based on CCHFV

nucleoprotein (NP).

Figure 3. Phylogenetic analysis of group V CCHFV strains/isolates based on CCHFV

glycoprotein (GPC).

Figure 4. Phylogenetic analysis of group I CCHFV strains/isolates based on CCHFV

glycoprotein (GPC).

Figure 5. Phylogenetic analysis of group III CCHFV strains/isolates based on CCHFV glycoprotein (GPC). Figure 6. Phylogenetic analysis of group IV CCHFV strains/isolates based on CCHFV glycoprotein (GPC).

Figure 7A. Experimental design for vaccine efficacy.

Figure 7B. Experimental design for vaccine efficacy.

Figure 8A. Immunoblotting for BGCA V GPC expression from adenoviral vector comprising BGCA V GPC. Lane 1 represents adenoviral vector comprising BGCA V GPC. Lane 2 represents adenoviral vector comprising Turkey/Kelkit GPC. Lane 3 represents peg precipitated CCHFV antigen from Turkey/Kelkit strain. Rabbit polyclonal sera against CCHFV used as primary antibody. Goat anti-rabbit HRP used as secondary antibody.

Figure 8B. Immunoblotting for BGCA I-VI NP expression from adenoviral vector containing BGCA I-VI NP. Lane 1 represents peg precipitated CCHFV antigen from Turkey/Kelkit strain. Lane 2 represent adenoviral vector comprising BGCA I-VI NP. Mouse monoclonal sera against CCHFV NP used as primary antibody. Goat anti-mouse HRP used as secondary antibody.

Figure 8C. Immunoblotting for Turkey-Kelkit06 NP expression from pET28 Turkey-Kelkit06 NP. Lane 1 represents pET28 comprising Turkey-Kelkit06 NP. Lane 2 represents peg precipitated CCHFV antigen from Turkey-Kelkit06 strain. Rabbit polyclonal sera against CCHFV used as primary antibody. Goat anti-rabbit HRP used as secondary antibody.

Figure 8D. Immunoblotting for BGCA V NP expression from adenoviral vector comprising BGCA V NP. Lane 1 represents adenoviral vector containing BGCA V NP. Lane 2 represents peg precipitated CCHFV antigen from Turkey-Kelkit06 strain. Mouse monoclonal sera against CCHFV NP used as primary antibody. Goat anti-mouse HRP used as secondary antibody.

Figure 9A. Animal survival percentage after CCHFV challenge. Comparison of survival percentages between following groups:

• Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime boost. · Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost.

• Ad empty vector. Figure 9B. Animal survival percentage after CCHFV challenge. Comparison of survival percentage between following groups:

• Adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as prime and cell culture based inactivated vaccine as prime boost.

• Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28 comprising Turkey-Kelkit06 NP as prime boost.

• Ad empty vector.

Figure 10A. Clinical signs and symptoms scores are showing on this graph. Clinical scores are based on Normal =0, Ruffling = 1, Ruffling and slight lethargy = 3, Lethargy = 3, labored breathing and lethargy = 4. Comparison of clinical signs and symptoms between different groups are as follows:

• Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime boost.

• Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost.

• Ad empty vector.

Figure 10B. Clinical signs and symptoms scores are showing on this graph. Clinical scores are based on Normal =0, Ruffling = 1, Ruffling and slight lethargy = 3, Lethargy = 3, labored breathing and lethargy = 4. Comparison of clinical signs and symptoms between different groups are as follows:

• Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and cell culture based inactivated vaccine as prime boost.

• Ad empty vector.

Figure 11 A. Temperature of all the vaccinated groups mice following CCHFV lethal challenge. Figure 11B. Temperature of all the vaccinated groups mice following CCHFV lethal challenge. Figure 12A. Weight change of vaccinated groups mice after CCHFV challenge.

Figure 12B. Weight change of vaccinated groups mice after CCHFV challenge. Figure 13A. Temperature change (°C) of vaccinated mice groups after CCHFV challenge.

Figure 13B. Temperature change (°C) of vaccinated mice groups after CCHFV challenge.

Figure 14 A. Temperature change (°C) after CCHFV challenge from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP- prime and prime boost vaccinated mice.

Figure 14B. Temperature change (°C) after CCHFV challenge from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost vaccinated mice.

Figure 14C. Temperature change (°C) after CCHFV challenge from adenoviral empty vector- prime and prime boost vaccinated mice.

Figure 14D. Temperature change (°C) after CCHFV challenge from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as prime with cell culture based inactivated vaccine as prime boost vaccinated mice.

Figure 14E. Temperature change (°C) after CCHFV challenge from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as prime with pET28 comprising Turkey-Kelkit06 NP as prime boost vaccinated mice.

Figure 15A. Weight change by percentage compared to the day of challenge.

Figure 15B. Weight change by percentage compared to the day of challenge.

Figure 16A. Weight change by percentage compared to the day of challenge from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP- prime and prime boost vaccinated mice.

Figure 16B. Weight change by percentage compared to the day of challenge from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost vaccinated mice.

Figure 16C. Weight change by percentage compared to the day of challenge from adenoviral empty vector- prime and prime boost vaccinated mice.

Figure 16D. Weight change by percentage compared to the day of challenge from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with cell culture based inactivated vaccine as prime boost vaccinated mice. Figure 16E. Weight change by percentage compared to the day of challenge from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with pET28 comprising turey- kelkit06 NP as prime boost vaccinated mice.

Figure 17A. Enzyme-linked immunosorbent assay (ELISA) used to analyze serum IgG responses against CCHFV antigens. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP (prime and prime boost) vaccinated mice sera used for this experiment. The sera were collected from Day 27 (before prime boost) and Day 55 (before CCHFV challenge). 1/400 dilution of mice sera used for this experiment. Adenoviral empty vector vaccinated mice sera used as negative control. The cut off value is calculated by mean + 2 standard deviation of negative controls. The A450 was measured in an ELISA reader.

Figure 17B. Enzyme-linked immunosorbent assay (ELISA) used to analyze serum IgG responses against CCHFV antigens. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and adenoviral vector containing BGCA V GPC and BGCA V NP as prime boost vaccinated mice sera used for this experiment. The sera were collected from Day 27 (before prime boost) and Day 55 (before CCHFV challenge). 1/400 dilution of mice sera used for this experiment. Adenoviral empty vector vaccinated mice sera used as negative control. The cut off value is calculated by mean + 2 standard deviation of negative controls. The A450 was measured in an ELISA plate reader.

Figure 17C. Enzyme-linked immunosorbent assay (ELISA) used to analyze serum IgG responses against CCHFV antigens. Adenoviral vector comprising BGCAV GPC and BGCAI-VI NP as prime and cell culture based inactivated vaccine as prime boost vaccinated mice sera used for this experiment. The sera were collected from Day 27 (before prime boost) and Day 41 (before CCHFV challenge). 1/400 dilution of mice sera used for this experiment. Adenoviral empty vector vaccinated mice sera used as negative control. The cut off value is calculated by mean + 2 standard deviation of negative controls. The A450 was measured in an ELISA reader.

Figure 17D. Enzyme-linked immunosorbent assay (ELISA) used to analyze serum IgG responses against CCHFV antigens. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28 comprising trkey-kelkit06 NP as prime boost vaccinated mice sera used for this experiment. The sera were collected from Day 27 (before prime boost) and Day 41 (before CCHFV challenge). 1/400 dilution of mice sera used for this experiment. Adenoviral empty vector vaccinated mice sera used as negative control. The cut off value is calculated by mean + 2 standard deviation of negative controls. The A450 was measured in an ELISA reader.

Figure 18. The endpoint serum IgG titers for each vaccinated group against CCHFV antigens. The vaccinated groups are as follows:

• Adenoviral vector comprising BGCA V GPC and BGCA I- VI NP- prime and prime boost.

• Adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as boost with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost.

Figure 19A. Neutralizing antibody titer study from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP (prime and prime boost) vaccinated mice sera. Sera were collected from Day 27 (before prime boost) and day 55 (before CCHFV challenge). 1/16, 1/32, 1/64 and 1/128 dilution of mice sera used for this experiment. The neutralizing activity from adenoviral empty vector vaccinated mice sera (collected from Day 27 and Day 55) used to compare with this group.

Figure 19B. Neutralizing antibody titer study from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost vaccinated mice sera. Sera were collected from Day 27 (before prime boost) and day 55 (before CCHFV challenge). 1/16, 1/32, 1/64 and 1/128 dilution of mice sera used for this experiment. The neutralizing activity from adenoviral empty vector vaccinated mice sera (collected from Day 27 and Day 55) used to compare with this group.

Figure 19C. Neutralizing antibody titer study from adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with cell culture based inactivated vaccine as prime boost vaccinated mice sera. Sera were collected from Day 27 (before prime boost) and day 55 (before CCHFV challenge). 1/16, 1/32, 1/64 and 1/128 dilution of mice sera used for this experiment. The neutralizing activity from adenoviral empty vector vaccinated mice sera (collected from Day 27 and Day 55) used to compare with this group. Figure 19D. Neutralizing antibody titer study from adenoviral vector comprising BGCA V GPC and BGCA I- VI NP as prime with pET28 comprising Turkey-Kelkit06 NP as prime boost vaccinated mice sera. Sera were collected from Day 27 (before prime boost) and day 55 (before CCHFV challenge). 1/16, 1/32, 1/64 and 1/128 dilution of mice sera used for this experiment. The neutralizing activity from adenoviral empty vector vaccinated mice sera (collected from Day 27 and Day 55) used to compare with this group.

Figure 20A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows Interferon-g (IFN-g) measurement from different vaccinated groups and the comparison between them.

Figure 21A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows tumor necrosis factor- a (TNF- a) measurement from different vaccinated groups and the comparison between them.

Figure 22A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows Interleukin- 12 pro70 (IL-12pro70) measurement from different vaccinated groups and the comparison between them.

Figure 23A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows Interleukin-2 (IL-2) measurement from different vaccinated groups and the comparison between them.

Figure 24A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows Interleukin-4 (IL-4) measurement from different vaccinated groups and the comparison between them.

Figure 25A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows Interleukin- 10 (IL-10) measurement from different vaccinated groups and the comparison between them.

Figure 26A, B. Pro-mouse cytokine assay Thl/Th2 panel used to analyze cytokine responses from vaccinated mice sera by using luminex bio-plex magpix. This graph shows granulocyte- macrophage colony-stimulating factor (GM-CSF) measurement from different vaccinated groups and the comparison between them.

Detailed Description of the Invention Sequence ID Details:

Sequence ID No: 1- Nucleic acid sequence of BGCA V GPC.

Sequence ID No: 2- Amino Acid Sequence of BGCA V GPC.

Sequence ID No: 3- Nucleic acid sequence of BGCA I- VI NP.

Sequence ID No: 4- Amino Acid sequence of BGCA I- VI NP.

Sequence ID No: 5- Nucleic acid sequence of BGCA V NP.

Sequence ID No: 6- Amino acid sequence of BGCA V NP.

Sequence ID No: 7- Nucleic acid sequence of Turkey-Kelkit06 NP

Sequence ID No: 8- Amino acid sequence of Turkey-Kelkit06 NP

Sequence ID No: 9- Nucleic acid sequence of BGCA I GPC

Sequence ID No: 10- Amino acid sequence of BGCA I GPC

Sequence ID No: 11- Nucleic acid sequence of BGCA III GPC

Sequence ID No: 12- Amino acid sequence of BGCA III GPC

Sequence ID No: 13- Nucleic acid sequence of BGCA IV GPC

Sequence ID No: 14- Amino acid sequence of BGCA IV GPC

The invention vaccine for use in inducing a specific immune response against Crimean Congo Haemorrhagic Fever Virus (CCHFV) or any other viruses in a subject, treating or reducing the risk of an CCHFV infection in a subject with a prime-boost immunization strategy, comprises a vector comprising at least one of the nucleic acid sequences selected from the group consisting of:

• nucleic acid sequence of SEQ ID NO: 1 encoding group V of Crimean Congo Haemorrhagic Fever Virus (CCHFV) Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA V GPC) or nucleic acid sequence that is at least 70% identical to SEQ ID NO: l,

• nucleic acid sequence of SEQ ID NO: 3 encoding group I- VI of CCHFV Bioinformatically Generated Conserved Nucleoprotein Antigen (BGCA I-VI NP) or nucleic acid sequence that is at least 70% identical to SEQ ID NO: 3, • nucleic acid sequence of SEQ ID NO: 5 encoding group V of CCHFV Bioinformatically Generated Conserved Nucleoprotein Antigen (BGCA V NP) or nucleic acid sequence that is at least 70% identical to SEQ ID NO:5,

• nucleic acid sequence of SEQ ID NO: 7 encoding Turkey-Kelkit06 Nucleoprotein (Turkey- Kelkit06 NP) or nucleic acid sequence that is at least 70% identical to SEQ ID NO:7,

• nucleic acid sequence of SEQ ID NO: 9 encoding group I of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA I GPC) or nucleic acid sequence that is at least 70% identical to SEQ ID NO: 9,

• nucleic acid sequence of SEQ ID NO: 11 encoding group III of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA III GPC) or nucleic acid sequence that is at least 70% identical to SEQ ID NO: 11 ,

• nucleic acid sequence of SEQ ID NO: 13 encoding group IV of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA IV GPC) or nucleic acid sequence that is at least 70% identical to SEQ ID NO: 13.

And the invention vaccine which contains said vector explained above, comprises a nucleic acid sequence encoding a Crimean Congo Haemorrhagic Fever Virus (CCHFV) glycoprotein or nucleoprotein antigen; and wherein said glycoprotein or nucleoprotein antigen comprises at least one of the amino acid sequences selected from the group consisting of;

• amino acid sequence of SEQ ID NO: 2 or having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2,

• amino acid sequence of SEQ ID NO: 4 or having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4,

• amino acid sequence of SEQ ID NO: 6 or having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 6,

• amino acid sequence of SEQ ID NO: 8 or having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8,

• amino acid sequence of SEQ ID NO: 10,

• amino acid sequence of SEQ ID NO: 12, _• amino acid sequence of SEQ ID NO: 14.

Detailed description of the present invention is given below with the examples,

Example 1: Constructing of recombinant adenoviral vector comprising BGCA V GPC

All the GPC amino acid sequences of known CCHFV strains were downloaded from GenBank databases. Seven phylogenetic subgroups were arranged according to their geographical location. Group V contained 25 CCHFV strains (Figure 3). These 25 CCHFV strains amino acid sequences were aligned together to determine consensus amino acids at their respective places (Seq ID No.2). The name of the consensus amino acid fragment is BGCA V GPC (Seq ID No.2). This amino acid sequence was reverse translate and codon optimized for expression in mammalian cells (Seq ID No. l).

The Xbal (TCTAGA) restriction site and Bamffl (GGATCC) restriction site artificially combine to the 5' of the BGCA V GPC fragments. Similarly, the Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially combine to the 3' of the BGCA V GPC fragments. Additionally, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA V GPC fragments.

BGCA V GPC was artificially synthesized and recombined into pShuttle 2 plasmid. The pShuttle 2 used to amplify BGCA V GPC fragment. This BGCA V GPC recombined with adenoviral vector (commercially available from Clontech) by infusion cloning reaction. The recombinant adenoviral vector comprising BGCA V GPC transfected into HEK-293 cells by using lipofectamine based transfection reagents. Several rounds of infections of recombinant adenoviral vector comprising BGCAV GPC into HEK-293 cells were performed to increase the titration (infectious unit/ml).

The expression of recombinant adenoviral vector containing BGCA V GPC was confirmed by immunoblot (Figure 8A). Rabbit polyclonal sera against CCHFV used as primary antibody for this experiment. Lane 3 represents peg precipitated CCHFV (Turkey/Kelkit 06 strain) where CCHFV GPC undergoes post-translational modification and expressed 75KDa product for Gc and 37KDa product for Gn (Figure 8A, lane 3). Lane 1 represents adenoviral vector containing BGCA V GPC. Lane number 2 represents adenoviral vector containing Turkey/Kelkit06 GPC. Adenoviral vector containing BGCA V GPC expressed several proteins. Some of them may be cross reaction with adenoviral vector protein. CCHFV GPC specific proteins were detected at 75KDa (red arrow) for Gc and 37KDa (blue arrow) for Gn from lane 1 (Figure 8A, lane 1). Likewise, same proteins were detected from lane 2. Thus, this recombinant construct of BGCA V GPC with adenoviral vector undergoes same post-translational modification and expressed same proteins (Figure 8A, lane 1) as native protein (Figure 8 A, lane 3).

Example 2: Constructing of recombinant adenoviral vector comprising BGCA I- VI NP

All the NP amino acid sequences of known CCHFV strains were downloaded from GenBank database. Six phylogenetic groups were arranged according to their geographical location (Figure 1). The consensus amino acid sequences were determined from each group separately. After getting all the 6 different consensus amino acid sequences from each group, arranged further alignment with these. The I-VIconsensus amino acid sequence was determined from that. This is called BGCA I- VI NP (Seq ID No. 4). This amino acid sequence was reverse translate and codon- optimized for expression in mammalian cells.

The Xbal (TCTAGA) restriction site and BamHI (GGATCC) restriction site artificially attch to the 5' of the BGCA I- VI NP fragments. Likewise, the Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially attach to the 3' of the BGCA I-VI NP fragments. Additionally, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA I-VI NP fragments.

BGCA I-VI NP was artificially synthesized and recombined into pShuttle 2 plasmid. The pShuttle 2 plasmid used to amplify BGCA I-VI NP fragment. This BGCA I-VI NP recombined with adenoviral vector (commercially available from Clontech) by infusion cloning reaction. This recombinant adenoviral vector comprising BGCA I-VI NP transfected in HEK293 cells by using lipofectamine based transfection reagents. Several infections of recombinant adenoviral vector comprising BGCA I-VI NP into HEK-293 cells were performed to increase the titration (infectious unit/ml).

The expression of recombinant adenoviral vector comprising BGCA I-VI NP was confirmed by immunoblot (Figure 8B). Mouse monoclonal sera against CCHFV NP used as primary antibody for this experiment. Lane 1 represents peg precipitated CCHFV Turkey/Kelkit 06 strain. Lane 2 represent adenoviral vector containing BGCA I-VI NP. Adenoviral vector comprising BGCA I- VI NP from lane 2 expressed a product at around 55KDa. Likewise, lane 1 expressed the same product as 55KDa. This 55KDa product indicated the expression of BGCA I-VI NP (Figure 8B, lane 2).

Example 3- Constructing recombinant adenoviral vector comprising BGCA V NP.

All the NP amino acid sequences of known CCHFV strains were downloaded from GenBank database. Six phylogenetic subgroups were arranged according to their geographical location (Figure 1). Group V contained 29 CCHFV strains (Figure 2). These 29 CCHFV strains NP amino acid sequences were aligned together to determined consensus amino acids at their respective places (Seq ID No.6). The name of the consensus amino acid fragment is BGCA V NP (Seq ID No.6). This amino acid sequence was reverse translate and codon optimized for expression in mammalian cells (Seq ID No.5).

The Xbal (TCTAGA) restriction site and Bamffl (GGATCC) restriction site artificially merge to the 5' of the BGCA V NP fragments. Correspondingly, the Xhol (CTCGAG) restriction site and Kpnl (GGTACC) restriction site artificially merge to the 3' of the BGCA V NP fragments. Additionally, KOZAC (GCCACC) sequence artificially add to the 5' of the BGCA V GPC fragments.

BGCA V NP was artificially synthesized and recombined into pShuttle 2 plasmid. The pShuttle 2 plasmid used to amplify BGCA V NP fragment. This BGCA V NP recombined with adenoviral vector (commercially available from Clontech) by infusion cloning reaction. The recombinant adenoviral vector comprising BGCA V NP transfected into HEK-293 cells by using lipofectamine based transfection reagents. Several infections of recombinant adenoviral vector comprising BGCA V NP into HEK-293 cells were performed to increase the titration (infectious unit/ml).

The expression of recombinant adenoviral vector comprising BGCA V NP was confirmed by immunoblot (Figure 8D). Mouse monoclonal sera against CCHFV NP used as primary antibody for this experiment. Lane 2 represents peg precipitated CCHFV Turkey/Kelkit 06 strain. Lane 1 represent adenoviral vector containing BGCA V NP. Adenoviral vector comprising BGCA V NP from lane 1 expressed a product at around 55KDa. Likewise, lane 2 expressed the same product as 55KDa. This 55KDa product indicated the expression of BGCA V NP (Figure 8D, lane 1).

Example 4- Constructing of recombinant pET28 plasmid comprising Turkey- Kelkit06 NP. Turkey-Kelkit06 NP nucleic acid sequence was downloaded from Genbank database. This Turkey- Kelkit06 NP fragment was codon optimized to express in E. Coli based expression system. After that it was artificially synthesized and cloned into pUC57 vector. Furthermore, it was recombined into pET28 expression vector.

The BamFH (GGATCC) restriction site artificially combine to the 5' of the Turkey -Kelkit06 NP fragments. Likewise, The Xhol (CTCGAG) restriction site artificially attach to the 3' of the Turkey-Kelkit06 strain.

The amplification of pET28 comprising Turkey -Kelkit06 NP was confirmed by immunoblot (Figure 8C). Rabbit polyclonal antibody against CCHFV used as primary antibody. Lane 1 represents pET28 comprising Turkey-Kelkit06 NP. Lane 2 represents peg precipitated CCHFV from Turkey/Kelkit 06 strain. pET28 comprising Turkey-Kelkit06 NP expressed product at around 55KDa. Likewise, Lane 1 has the same product. This 55KDa product confirmed the expression of Turkey-Kelkit06 NP.

Example 5- Preparation of cell culture based CCHFV inactivated vaccine

Preparation of cell culture based inactivated vaccine was followed the same protocol as described in the patent number W02014039021 A1 from the same inventors.

Example 6- Preparation of adenoviral empty vector

Adenoviral empty vector (clontech) directly transfected to HEK-293 cells according to manufactures instruction (clontech). After successful transfection, several rounds of infections were performed to increase the titration of adenoviral empty vector.

Example 7- Vaccine efficacy study in IFNAR a/b R mouse model by using these vaccine candidates (Example 1, 2, 3, 4, 5)

This vaccine efficacy study used type-1 interferon a and b receptor knockout mouse. This mouse model is susceptible to CCHFV infection and imitates lethal diseases.

Homologous and heterologous prime boost strategy used for this experiment. The first immunization as prime was always with adenoviral vector comprising BGCA V GPC and BGCA I- VI NP. For homologous prime boost, adenoviral vector comprising BGCA V GPC and BGCA I-VI NP used (Experimental protocol 4 A). For heterologous prime boost, three different groups used (Experimental protocol 4A, 4B):

1. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost.

2. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with cell culture based inactivated vaccine as prime boost.

3. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime with pET28 Turkey-Kelkit06 NP as prime boost.

For all the groups, 10 IFNAR a/b R mice used for immunization. 7 IFNAR a/b R mice also used for immunization with adenoviral empty vector as control. Mice were immunized intramuscularly with 10⁹ infectious unit per milliliter (ifu/ml) for all adenoviral recombinant vaccine. For cell culture based inactivated vaccine, 7.5ug used for intraperitoneal immunization as prime boost. Fikewise, for pET28 Turkey -Kelkit06 NP, 12.5ug used for intraperitoneal immunization as prime boost.

Adenoviral vector comprising BGCA V GPC was mixed with adenoviral vector comprising BGCA I-VI NP just before immunization by the number of 10⁹ifu/ml. 50ul of adenoviral vector comprising BGCA V GPC was mixed with 50ul of adenoviral vector comprising BGCA I-VI NP. The total volume of immunization dose was lOOul per mice which was divided into two intramuscular sites at 50ul each. Prime boost immunization was same for this group.

Adenoviral vector comprising BGCA V GPC was mixed with adenoviral vector comprising BGCA V NP just before prime boost immunization by the number of 10⁹ifu/ml. 50ul were mixed from both constructs. The total volume of immunization dose was lOOul per mice which was divided into two intramuscular sites at 50ul each.

As a prime boost immunization, 7.5ug of cell culture based inactivated vaccine used. Furthermore, 12.5ug of pET28 Turkey -Kelkit06 NP used as prime boost vaccination.

For adenoviral recombinant vaccine, each mouse received two immunization by 4 weeks interval. Mice receipt first immunization on day 0 as prime and second immunization on day 28 as prime boost. 4 weeks after 2^nd immunization (Day 56), mice were challenged with 100FFU of CCHFV. For cell culture based inactivated vaccine and pET28 Turkey -Kelkit06 NP as prime boost, each mouse received prime boost after 2 weeks of first immunization. Mice were challenged with CCHFV after 2 weeks of prime boost on Day 42.

Before 1 ^st immunization, 2^nd immunization (Day 27) and before CCHFV challenge (Day 55), blood was collected for serological assay and determining cytokine profile in serum. Blood was collected on Day 41 (before CCHFV challenge) from cell culture based inactivated vaccine and pET28 Turkey -Kelkit06 NP. From the day of CCHFV challenge till 14 days, body temperature and body weight were measured. After 14 days of CCHFV challenge, liver was collected to detect viral load analysis. Experimental protocol is showing on figure 4A and 4B.

• Animal survival ratio: Kaplan- Meier graph is showing the animal survival rate after CCHFV challenge. This vaccinated groups of mice were 100% protected from CCHFV lethal challenge (Figure 9A, B). Control group mice were dead after fifth day of CCHFV challenge (Figure 9 A, B). 40% mice were dead on day 4 after CCHFV challenge from control groups. On day 5, another 60% mice were dead from the same group (Figure 9A,

B).

• Clinical signs and symptoms: This vaccinated groups of mice did not show any CCHFV infection specific signs and symptoms (Figure 10A, B). In contrast, control group started showing CCHFV specific signs and symptoms after 2^nd days of CCHFV challenge (Figure 10A, B). Control group reached experimental endpoint on day 5 after CCHFV challenge.

• Temperature and body weight measurement: The graph of body temperature measurement remained stable after CCHFV challenge for all the vaccinated groups (Figure 11 A, B). In contrast, control group mice started decreasing body temperature sharply after 3^rd days of CCHFV challenge (Figure 11 A, B). The vaccinated mouse groups did not lose their body weight after CCHFV challenge (12A, B). On the other hand, control group started declining body weight after 2^nd days of CCHFV challenge (Figure 12A, B).

Example 8- Detection of viral load from vaccinated and control group mice liver

4 vaccinated groups and 1 control group mice liver was used for this experiment:

1. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime

boost- group 1 2. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost-group 2

3. Adenoviral vector comprising BGCAY GPC and BGCA I-VI NP as prime and cell

culture based inactivated vaccine as prime boost-group 3

4. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28 Turkev-Kelkit06 NP as prime boost- group 4.

5. Adenoviral empty vector - prime and prime boost- control group.

Livers were collected on day 14 after CCHFV challenge from vaccinated groups (group 1, 2, 3, 4). Liver was collected on day 3 after CCHFV challenge from control group as they all died within 5 days of CCHFV challenge. RNA was extracted by using RNeasy midi kit (qiagen) and qRT- PCR was performed to detect the viral load by using real star CCHFV RT-PCR kit (altona). There was no detectable CCHFV in vaccinated mice group (group 1, 2, 3, 4). In contrast, CCHFV was detected in liver from control group mice.

Example 9- Serological study by using the sera from vaccinated and control groups.

Enzyme Linked Immunosorbent Assay (ELISA)

1. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime

boost.

2. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and

adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost.

The serum IgG responses against CCHFV antigens were studied by ELISA for this group. Sera were collected before first immunization, on day 27 (before 2^nd immunization as prime boost) and on day 55 (before challenge). After first immunization as prime, mice developed some CCHFV specific IgG antibody (Figure 17 A, B). The level is significantly increased after prime boost immunization (Figure 17A, B). Homologous and heterologous prime boost approaches showed significantly increased of IgG against CCHFV. A Mann-whitney test was performed for group- wise contrast where P value of <0.01 was considered significant.

3. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and cell culture based inactivated vaccine as prime boost. 4. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28

Turkev-Kelkit06 NP as prime boost.

Sera were collected before first immunization, on day 27 (before 2^nd immunization as prime boost) and on day 41 (before CCHFV challenge). After 1^st immunization as prime, mice developed IgG antibody against CCHFV antigens (Figure 17 C, D). For cell culture based inactivated vaccine as prime boost, the level is significantly increased (Figure 17 C). However, for pET28 Turkey- Kelkit06 as prime boost, the IgG antibody against CCHFV antigen did not increase as other groups (Figure 17D).

Therefore, the endpoint titer of IgG against CCHFV for group 1, 2 and 3 is higher than the group 4 (Figure 18).

Neutralizing antibody assay

Sera were pooled from each vaccinated group. There are 10 mice from each group. There are 4 vaccinated mice groups:

1. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime boost-group 1

2. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and

adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost-group 2

3. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and cell culture based inactivated vaccine as prime boost- group 3

4. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28

Turkev-Kelkit06 NP as prime boost- group 4

Adenoviral empty vector vaccinated mice sera used as control group. Sera were collected on day 27 (before 2^nd immunization) and on day 55 (before CCHFV challenge) for group 1 and 2. Additionally, sera were collected on day 27 (before 2^nd immunization) and on day 41 (before CCHFV challenge) for group 3 and 4. Control group sera were collected on day 27 and day 55.

Group 1 and 2 developed neutralizing antibodies after 1^st immunization (Figure 19A, B). To compare with control group, day 27 collected sera contained higher neutralizing antibody titer (Figure 19A, B). Thus, after 2^nd immunization as prime boost, mice developed higher neutralizing antibodies than day 27 collected sera (Figure 19A, B). Group 3 immunized mice developed higher neutralizing antibodies than control group (Figure 19C). After prime boost with cell culture based inactivated vaccine on day 41, the neutralizing antibody titer was higher than day 27 for this group (Figure 19C).

Group 4 is pET28 BGCA I- VI NP prime boost vaccinated group. After adenoviral vector comprising BGCAV GPC and BGCA I-VI NP immunization as prime, mice developed some neutralizing antibodies like previous groups (Figure 19D). Though, after prime boost with pET28 Turkey-Kelkit06 NP, there were no development of neutralizing antibodies (Figure 19D).

Example 10- Cytokine profiling using a pro-mouse cytokine assay Thl/Th2 panel from vaccinated mouse sera.

Sera were collected from the following groups for cytokine profiling:

1. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP- prime and prime

boost- group 1.

2. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and

adenoviral vector comprising BGCA V GPC and BGCA V NP as prime boost-group 2 3. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and cell culture based inactivated vaccine as prime boost- group 3

4. Adenoviral vector comprising BGCA V GPC and BGCA I-VI NP as prime and pET28 Turkev-Kelkit06 NP as prime boost-group 4

Adenoviral empty vector vaccinated mice sera (day 55 collected) and pre-immunized mice sera used for this experiment as control. For group 1 and 2, day 55 (before CCHFV challenge) collected sera used for cytokine profiling. For group 3 and 4, day 41 (before CCHFV challenge) collected sera used for cytokine profiling. A Mann-whitney test was performed for group-wise contrast where P value of <0.001 was considered significant.

• Interferon g (IFN-g)- Group 1, 2, 3 and 4 has significantly higher level of IFN-g

compare to control group (Figure 20 A, B).

• Tumor Necrosis Factor-a (TNF-a)- TNF-a level is significantly higher in vaccinated groups than in control groups (Figure 21A, B).

• Interleukin 12p70 (IL-12p70)- Group 1 and 2 collected sera contained significantly higher IL-12p70 level than control groups (Figure 22 A). Cell culture based inactivated vaccine as prime boost and pET28 Turkey -Kelkit06 NP prime boost vaccinated mice sera did not contain higher IL-12p70 than adenoviral empty vector vaccinated mice sera (Figure 22B). However, group 3 and 4 has significantly higher IL-12p70 than pre- immunized mice sera (Figure 22B).

• Interleukin 2 (IL-2)- Group 1 and 2 collected sera showed significantly higher level of IF-4 than control groups (Figure 23 A). pET28 Turkey-Kelkit06 NP prime boost (group 4) vaccinated mice sera showed significantly higher level of IF-4 than group 3 and control groups (Figure 23 B). Fikewise, group 3 collected sera showed pointedly higher-level IF- 4 than in control groups (Figure 23 B).

• Interleukin 4 (IL-4)- Groups 1 , 2 and 3 showed significantly higher level of IF-2 than control groups (Figure 24 A). pET28 Turkey-Kelkit06 NP (group 4) prime boost vaccinated mice sera did not show higher IF-2 level than control groups (Figure 24 B).

• Interleukin 10- (IL-10)- IF-10 level was significantly higher in group 1, 2 and 3 compare to control groups (Figure 25 A, B). Nevertheless, group 4 did not show higher IF-10 level than control groups (Figure 25 A, B).

• Granulocyte Macrophage- Colony Stimulating Factor (GM-CSF)- Group 1, 2 and 3 showed significantly higher GM-CSF than control groups (Figure 26 A, B). Group 4 did not show any higher level of GM-CSF then control groups (Figure 26 A, B).

Claims

1. A vaccine; wherein the vaccine comprises a vector comprising at least one of the nucleic acid sequences selected from the group consisting of:

• nucleic acid sequence encoding group V of Crimean Congo Haemorrhagic Fever Virus (CCHFV) Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA V GPC) that is at least 70% identical to SEQ ID NO: 1,

• nucleic acid sequence encoding group I- VI of CCHFV Bioinformatically Generated Conserved Nucleoprotein Antigen (BGCA I- VI NP) that is at least 70% identical to SEQ ID NO: 3,

• nucleic acid sequence encoding group V of CCHFV Bioinformatically Generated Conserved Nucleoprotein Antigen (BGCA V NP) that is at least 70% identical to SEQ ID NO: 5,

• nucleic acid sequence encoding Turkey-Kelkit06 Nucleoprotein (Turkey-Kelkit06 NP) that is at least 70% identical to SEQ ID NO: 7,

• nucleic acid sequence encoding group I of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA I GPC) that is at least 70% identical to SEQ ID NO: 9,

• nucleic acid sequence encoding group III of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA III GPC) that is at least 70% identical to SEQ ID NO: 11,

• nucleic acid sequence encoding group IV of CCHFV Bioinformatically Generated Conserved Glycoprotein Antigen (BGCA IV GPC) that is at least 70% identical to SEQ ID NO: 13.

2. The vaccine according to claim 1 , wherein the vaccine comprises a vector comprising at least one of the nucleic acid sequences selected from the group consisting of SEQ ID NO: 1,

SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l 1 and SEQ ID

NO: 13.

3. The vaccine according to claim 1 or 2, wherein said vector comprises a nucleic acid sequence encoding a Crimean Congo Haemorrhagic Fever Virus (CCHFV) glycoprotein or nucleoprotein antigen; and wherein said glycoprotein or nucleoprotein antigen comprises at least one of the amino acid sequences selected from the group consisting of;

• having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2,

• having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 4,

• having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 6,

• having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8,

• amino acid sequence of SEQ ID NO: 10,

• amino acid sequence of SEQ ID NO: 12,

_• amino acid sequence of SEQ ID NO: 14.

4. The vaccine according to any one of claims 1 to 3, wherein the said vector is a viral or a bacterial vector.

5. The vaccine according to any one of claims 1 to 4 for use in a prime-boost immunization strategy, comprising a prime vaccine and a boost vaccine, wherein the said prime-boost immunization is homologous or heterologous prime-boost immunization.

6. The vaccine for use according to claim 5, wherein; the prime vaccine and boost vaccine comprises a viral vector containing BGCA V GPC nucleic acid sequence of SEQ ID NO: 1 and a viral vector containing BGCA I-VI NP nucleic acid sequence of SEQ ID NO:3.

7. The vaccine for use according to claim 5, wherein;

• the prime vaccine comprises the viral vector containing BGCA V GPC nucleic acid sequence of SEQ ID NO: 1 and the viral vector containing BGCA I-VI NP nucleic acid sequence of SEQ ID NO: 3.

• the boost vaccine comprises viral vector containing BGCA V GPC nucleic acid sequence of SEQ ID NO: l and viral vector containing BGCA V NP nucleic acid sequence of SEQ ID NO:5.

8. The vaccine for use according to claim 5, wherein; • the prime vaccine comprises the viral vector containing BGCA V GPC nucleic acid sequence of SEQ ID NO: l and the viral vector containing BGCA I- VI NP nucleic acid sequence of SEQ ID NO: 3,

• the boost vaccine comprises bacterial vector containing Turkey-Kelkit06 NP nucleic acid sequence of SEQ ID NO: 7.

9. The vaccine for use according to claim 5, wherein the administration of the prime vaccine is intramuscular or intraperitoneal.

10. The vaccine for use according to claim 5, wherein the administration of the boost vaccine is intramuscular or intraperitoneal.

11. The vaccine according to any one of claims 1 to 4 for use in inducing a specific immune response against Crimean Congo Haemorrhagic Fever Virus (CCHFV) in a subject.

12. The vaccine according to any one of claims 1 to 4 for use in treating or reducing the risk of an CCHFV infection in a subject.