Crimean-congo haemorrhagic fever virus antigenic composition

This application is a National Stage Application of PCT/GB2013/053174, filed 29 Nov. 2013, which claims benefit of Serial No. 1303406.1, filed 26 Feb. 2013 in Great Britain and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.

BACKGROUND

The present invention relates to viral vectors and bacterial vectors comprising Crimean-Congo Haemorrhagic Fever Virus (CCHFV) antigens and their use in immunogenic and antigenic compositions.

Crimean-Congo Haemorrhagic fever (CCHF) is a viral haemorrhagic fever caused by a virus of the Nairovirus group (CCHFV). CCHF is a zoonosis, and infects a range of domestic and wild animals. It is spread via the bite of an infected tick. CCHF was first described in the Crimea in 1944 among soldiers and agricultural workers, and in 1969 it was recognised that the virus causing the disease was identical to a virus isolated from a child in the Congo in 1956.

CCHF virus is endemic in many countries in Africa, the Middle East, Eastern Europe and Asia, and outbreaks have been recorded in Russia, Turkey, Iran, Kazakhstan, Mauritania, Kosovo, Albania, Pakistan, and southern Africa in recent years. The first clinical case of CCHF in Greece was reported in July 2008. The global distribution of cases corresponds to those areas where the ticks are found.

The virus is spread by the bite of infected Ixodidae ticks, and the most efficient and common vectors appear to be members of the Hyalomma genus, which commonly infest livestock and other animals. Immature ticks acquire the virus by feeding on infected small animals. Once infected, the tick carries the virus for life, and passes it to animals or humans when it bites them. Domestic ruminants such as cattle, sheep and goats carry the virus for around one week after becoming infected. Most birds are thought to be relatively resistant to infection with CCHF virus, however, many bird species carry Hyalomma ticks, and human cases have occurred in those working with ostriches in the past.

Humans may be infected with CCHF by the bite of an infected tick, contamination with tick body contents, or direct contact with the blood, tissues or body fluids of infected humans or animals. The majority of cases occur in those living in tick infested areas with occupational exposure to livestock, including farmers, veterinarians, slaughter-house workers, livestock owners and others working with animals. Cases also occur in healthcare workers or others caring for infected persons without taking adequate infection control precautions.

CCHF outbreaks are generally associated with a change in situation such as war, population and animal movements, or climatic and vegetation changes which produce more ground cover for small mammals which act as hosts for ticks. These conditions can lead to explosions in tick populations, and allow increased tick/human contact.

The incubation period of CCHF appears to vary according to the mode of acquisition of the virus. If a patient has been infected by a tick bite, the incubation period is usually 1-3 days, and up to 9 days. Infection via contact with infected blood or tissues leads to an incubation period of 5-6 days, and the maximum recorded incubation period is 13 days. The illness begins abruptly, with fever, muscle aches, dizziness, neck pain and stiffness, backache, headache, sore eyes and photophobia (sensitivity to light). Nausea, vomiting and sore throat may also occur, with diarrhoea and abdominal pain. Over the next few days the patient may experience mood swings, confusion and aggression, followed by sleepiness, depression and liver enlargement. More severe symptoms may follow, including petechial rash (a rash caused by bleeding into the skin), bruising and generalised bleeding of the gums and orifices. In severe cases patients develop failure of the liver, kidneys and lungs, and become drowsy and comatose after 5 days. Approximately 30% of cases are fatal.

Diagnosis of CCHF requires highly specialised, high biosafety level laboratory facilities. Antibodies may be detected in serum by about day six of illness. The virus may be isolated from blood or tissue specimens in the first five days of illness, and grown in cell culture, and nucleic acid detection methods may also be used to detect the viral genome. Patients with fatal disease do not usually develop a detectable antibody response, and in these individuals, and those in the early stages of infection, diagnosis is by virus detection. General supportive therapy is given, including replacement of blood components, balancing fluids and electrolytes, and maintaining oxygen status and blood pressure. There is evidence that CCHF responds to treatment with the antiviral drug ribavirin, in both oral and intravenous formulations.

A vaccine based on inactivated CCHF virus has been used in Eastern Europe, however this vaccine is not licensed by the EMA or FDA, and there is no literature to demonstrate its efficacy. There is therefore at present no safe and effective, commercially-available vaccine against CCHFV.

There is therefore a need for new vaccine compositions that demonstrate improved immunogenicity when used in the prevention and treatment of CCHFV infections, in particular in human subjects.

SUMMARY AND DETAILED DESCRIPTION

The present invention addresses one or more of the above problems by providing viral vectors and bacterial vectors encoding CCHFV glycoproteins or antigenic fragments thereof, together with corresponding compositions and uses of said vectors and compositions in the prevention and treatment of CCHFV infection.

The vectors and compositions of the invention enable an immune response against CCHFV to be stimulated (i.e. induced) in an individual (i.e. a subject), and provide improved immunogenicity and efficacy.

In one aspect, the invention provides a viral vector or bacterial vector, said vector comprising a nucleic acid sequence encoding a Crimean-Congo Haemorrhagic Fever Virus (CCHFV) glycoprotein or antigenic fragment thereof; wherein said vector is capable of inducing an immune response in an individual. The present inventors have found that highly effective immune responses against CCHFV can be generated in an individual by using a viral vector or bacterial vector to deliver to the subject nucleic acid sequences encoding CCHFV glycoproteins (or antigenic fragments thereof), as described above.

In a preferred embodiment, the vector of the invention is a viral vector.

The CCHFV glycoproteins are two proteins G.sub.N and G.sub.C, which are encoded by the M segment of the CCHFV genome. A CCHFV virion carries its genome as three single-stranded, negative sense RNA segments, S (small), M (medium) and L (large), which encode a nucleoprotein, a polyprotein, and an RNA-dependent RNA polymerase, respectively. The M segment, which is approximately 5.4 kb in length, encodes a single open reading frame of approximately 1,400 residues which is translated into a polyprotein. This polyprotein is processed by a complex set of proteolytic cleavage events to release a total of four separate proteins: two glycoproteins G.sub.N and G.sub.C and two other domains, a mucin-like domain and a GP35 domain. The CCHFV glycoproteins G.sub.N and G.sub.C are incorporated into the envelope of mature CCHFV virions, and influence host range, cell tropism and pathogenicity. These glycoprotein ecto-domains are the primary CCHFV proteins exposed to the immune system of an infected individual. The remaining two proteins produced from the M segment polyprotein remain in the Golgi apparatus of an infected cell and play a role in chaperoning the maturation of G.sub.N and G.sub.C. The CCHFV glycoproteins contain approximately 80 cysteine residues, suggesting the presence of a large number of disulphide bonds and a complex secondary structure. The G.sub.N precursor protein (Pre-G.sub.N) contains a highly variable domain at its amino terminus that contains a high proportion of serine, threonine and proline residues, and is predicted to be heavily glycosylated, thus resembling a mucin-like domain present in other viral glycoproteins.

As used herein, the term "antigenic fragment" means a peptide or protein fragment of a CCHFV glycoprotein which retains the ability to induce an immune response in an individual, as compared to the reference CCHFV glycoprotein. An antigenic fragment may therefore include at least one epitope of the reference protein. By way of example, an antigenic fragment of the present invention may comprise (or consist of) a peptide sequence having at least 10, 20, 30, 40 or 50 amino acids, wherein the peptide sequence has at least 80% sequence homology over a corresponding peptide sequence of (contiguous) amino acids of the reference protein. An antigenic fragment may comprise (or consist of) at least 10 consecutive amino acid residues from the sequence of the reference protein (for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 177, 200, 250, 300, 350, 400, 450, 500, 600, 750, 1000, 1250, or 1500 consecutive amino acid residues of said reference protein).

An antigenic fragment of a reference protein may have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the reference protein. For example, an antibody capable of binding to an antigenic fragment of a reference protein would also be capable of binding to the reference protein itself. By way of further example, the reference protein and the antigenic fragment thereof may share a common ability to induce a "recall response" of a T lymphocyte (e.g. CD4+, CD8+, effector T cell or memory T cell such as a TEM or TCM), which has been previously exposed to an antigenic component of a CCHFV infection.

In one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3.

In one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3. In a further embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3.

The present inventors have found that the CCHFV glycoproteins encoded by the nucleic acid sequences of SEQ ID NOs: 1, 2, and 3 can be used to generate effective immune responses in individuals against CCHFV. In particular, the inventors have found that a highly effective immune response against CCHFV is obtained when one or more members of this group of glycoproteins is delivered to the subject using a bacterial vector or a viral vector, such as a non-replicating poxvirus vector or an adenovirus vector.

The nucleic acid sequence of SEQ ID NO: 1 represents the full-length M segment open reading frame, encoding both G.sub.N and G.sub.C glycoproteins. Thus, in one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises (or consists of) a CCHFV M segment. In one embodiment, a vector of the invention can be used to deliver a full-length M segment to a target cell in a subject, thus leading to production of the polyprotein encoded by the M segment, subsequent processing of which produces both G.sub.N and G.sub.C glycoproteins, thus stimulating an immune response against both G.sub.N and G.sub.C glycoproteins.

The nucleic acid sequence of SEQ ID NO: 2 encodes the G.sub.N glycoprotein. The nucleic acid sequence of SEQ ID NO: 3 encodes the G.sub.C glycoprotein. Thus, in one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein encodes a CCHFV G.sub.N or a CCHFV G.sub.C glycoprotein. Accordingly, in one embodiment, a vector of the invention can be used to deliver a nucleic acid sequence encoding a CCHFV G.sub.N glycoprotein to a target cell in a subject, stimulating an immune response against said CCHFV G.sub.N glycoprotein. In another embodiment, a vector of the invention can be used to deliver a nucleic acid sequence encoding a CCHFV G.sub.C glycoprotein to a target cell in a subject, stimulating an immune response against said CCHFV G.sub.C glycoprotein.

An immune response against both G.sub.N and G.sub.C glycoproteins may also be generated using a vector comprising a nucleic acid sequence that encodes both G.sub.N and G.sub.C glycoproteins, but which sequence is not the full-length M segment. Thus, in one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein or antigenic fragment thereof comprises a first nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 2, and a second nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 3.

In one embodiment, the vector of the invention (as described above) comprises a nucleic acid sequence encoding a CCHFV protein, wherein said CCHFV protein comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 represents the amino acid sequence of the polyprotein encoded by the full-length CCHFV M segment. As said polyprotein encodes G.sub.C and G.sub.N, the polyprotein may also be considered to be a CCHFV glycoprotein.

In one embodiment, the vector of the invention (as described above) encodes a CCHFV glycoprotein or antigenic fragment thereof, wherein said CCHFV glycoprotein or antigenic fragment thereof comprises (or consists of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 5 and 6. The amino acid sequence of SEQ ID NO: 5 represents the amino acid sequence of the CCHFV G.sub.N protein. The amino acid sequence of SEQ ID NO: 6 represents the amino acid sequence of the CCHFV G.sub.C protein.

In one embodiment, the vector comprises a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 7.

Vectors are tools which can be used as vectors for the delivery of genetic material into a target cell. By way of example, viral vectors serve as antigen delivery vehicles and also have the power to activate the innate immune system through binding cell surface molecules that recognise viral elements. A recombinant viral vector can be produced that carries nucleic acid encoding a given antigen. The viral vector can then be used to deliver the nucleic acid to a target cell, where the encoded antigen is produced and then presented to the immune system by the target cell's own molecular machinery. As "non-self", the produced antigen generates an immune response in the target subject.

Viral vectors suitable for use in the present invention include poxvirus vectors (such as non-replicating poxvirus vectors), adenovirus vectors, and influenza virus vectors.

In certain embodiments, a "viral vector" may be a virus-like particle (VLP). VLPs are lipid enveloped particles which contain viral proteins. Certain viral proteins have an inherent ability to self-assemble, and in this process bud out from cellular membranes as independent membrane-enveloped particles. VLPs are simple to purify and can, for example, be used to present viral antigens. VLPs are therefore suitable for use in immunogenic compositions, such as described below. In certain embodiments, a viral vector is not a virus-like particle.

Bacterial vectors can also be used as antigen delivery vehicles. A recombinant bacterial vector can be produced that carries nucleic acid encoding a given antigen. The recombinant bacterial vector may express the antigen on its surface. Following administration to a subject, the bacterial vector colonises antigen-presenting cells (e.g. dendritic cells or macrophages). An antigen-specific immune response is induced. The immune response may be a cellular (T cell) immune response, or may comprise both humoral (e.g. B cell) and cellular (T cell) immune responses. Examples of bacteria suitable for use as recombinant bacterial vectors include Escherichia coli, Shigella, Salmonella (e.g. S. typhimurium), and Listeria bacteria. In one embodiment, the vector of the invention is a bacterial vector, wherein the bacterium is a Gram-negative bacterium. In one embodiment, the vector of the invention is a bacterial vector selected from an Escherichia coli vector, a Shigella vector, a Salmonella vector and a Listeria vector.

Without wishing to be bound by any one particular theory, the inventors believe that antigen delivery using the vectors of the invention stimulates, amongst other responses, a T cell response in the subject. Thus, the inventors believe that one way in which the present invention provides for protection against CCHFV infection is by stimulating T cell responses and the cell-mediated immunity system. In addition, humoral (antibody) based protection can also be achieved.

A viral vector of the invention may be a non-replicating viral vector.

As used herein, a non-replicating viral vector is a viral vector which lacks the ability to productively replicate following infection of a target cell. Thus, the ability of a non-replicating viral vector to produce copies of itself following infection of a target cell (such as a human target cell in an individual undergoing vaccination with a non-replicating viral vector) is highly reduced or absent. Such a viral vector may also be referred to as attenuated or replication-deficient. The cause can be loss/deletion of genes essential for replication in the target cell. Thus, a non-replicating viral vector cannot effectively produce copies of itself following infection of a target cell. Non-replicating viral vectors may therefore advantageously have an improved safety profile as compared to replication-competent viral vectors. A non-replicating viral vector may retain the ability to replicate in cells that are not target cells, allowing viral vector production. By way of example, a non-replicating viral vector (e.g. a non-replicating poxvirus vector) may lack the ability to productively replicate in a target cell such as a mammalian cell (e.g. a human cell), but retain the ability to replicate (and hence allow vector production) in an avian cell (e.g. a chick embryo fibroblast, or CEF, cell).

A viral vector of the invention may be a non-replicating poxvirus vector. Thus, in one embodiment, the viral vector encoding a CCHFV glycoprotein or antigenic fragment thereof is a non-replicating poxvirus vector.

In one embodiment, the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. MVA and NYVAC are both attenuated derivatives of vaccinia virus. Compared to vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.

In a preferred embodiment, the non-replicating poxvirus vector is an MVA vector.

A viral vector of the invention may be an adenovirus vector. Thus, in one embodiment, the viral vector encoding a CCHFV glycoprotein or antigenic fragment thereof is an adenovirus vector.

In one embodiment, the adenovirus vector is a non-replicating adenovirus vector (wherein non-replicating is defined as above). Adenoviruses can be rendered non-replicating by deletion of the E1 or both the E1 and E3 gene regions. Alternatively, an adenovirus may be rendered non-replicating by alteration of the E1 or of the E1 and E3 gene regions such that said gene regions are rendered non-functional. For example, a non-replicating adenovirus may lack a functional E1 region or may lack functional E1 and E3 gene regions. In this way the adenoviruses are rendered replication incompetent in most mammalian cell lines and do not replicate in immunised mammals. Most preferably, both E1 and E3 gene region deletions are present in the adenovirus, thus allowing a greater size of transgene to be inserted. This is particularly important to allow larger antigens to be expressed, or when multiple antigens are to be expressed in a single vector, or when a large promoter sequence, such as the CMV promoter, is used. Deletion of the E3 as well as the E1 region is particularly favoured for recombinant Ad5 vectors. Optionally, the E4 region can also be engineered.

In one embodiment, the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

In one embodiment, wherein the vector is a viral vector, the virus (i.e. viral vector) is not a pseudotyped virus. Thus, in one embodiment, the envelope of the viral vector does not comprise foreign glycoproteins (i.e. glycoproteins that are not native to said viral vector).

In one embodiment, wherein the vector is a viral vector, the vector is not a retrovirus vector.

In one embodiment, wherein the vector is a viral vector, the vector is not a murine leukaemia virus (MLV) vector (for example, a Moloney murine leukaemia virus (MoMLV) vector).

In one embodiment, wherein the vector is a viral vector, the vector is not a Newcastle disease virus (NDV) vector.

In one embodiment, wherein the vector is an adenovirus vector, the adenovirus is not a human adenovirus serotype 5 (AdHu5).

Thus, in one embodiment, wherein the vector is a viral vector, the vector is not a retrovirus vector, a Newcastle disease virus vector, or a human adenovirus serotype 5 vector.

In one embodiment, wherein the vector is an adenovirus vector (such as a human adenovirus serotype 5 (AdHu5) vector), the nucleic acid sequence encoding a Crimean-Congo Haemorrhagic Fever Virus (CCHFV) glycoprotein or antigenic fragment thereof does not comprise a CCHFV M segment. In one embodiment, wherein the vector is an adenovirus vector, the vector is stable, expresses a CCHFV glycoprotein product, and induces a protective immune response in a subject.

The nucleic acid sequences as described above may comprise a nucleic acid sequence encoding a CCHFV glycoprotein wherein said glycoprotein comprises a fusion protein. The fusion protein may comprise a CCHFV glycoprotein polypeptide fused to one or more further polypeptides, for example an epitope tag, another antigen, or a protein that increases immunogenicity (e.g. a flagellin).

In one embodiment, the nucleic acid sequence encoding a CCHFV glycoprotein (as described above) further encodes a Tissue Plasminogen Activator (tPA) signal sequence, and/or a V5 fusion protein sequence. In certain embodiments, the presence of a tPA signal sequence can provide for increased immunogenicity; the presence of a V5 fusion protein sequence can provide for identification of expressed protein by immunolabeling.

In one embodiment, the vector (as described above) further comprises a nucleic acid sequence encoding an adjuvant (for example, a cholera toxin, an E. coli lethal toxin, or a flagellin).

A bacterial vector of the invention may be generated by the use of any technique for manipulating and generating recombinant bacteria known in the art.

In another aspect, the invention provides a nucleic acid sequence encoding a viral vector, as described above. Thus, the nucleic acid sequence may encode a non-replicating poxvirus vector as described above. Alternatively, the nucleic acid sequence may encode an adenovirus vector as described above.

The nucleic acid sequence encoding a viral vector (as described above) may be generated by the use of any technique for manipulating and generating recombinant nucleic acid known in the art.

In one aspect, the invention provides a method of making a viral vector (as described above), comprising providing a nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a vector (as described above); transfecting a host cell with the nucleic acid; culturing the host cell under conditions suitable for the propagation of the vector; and obtaining the vector from the host cell.

As used herein, "transfecting" may mean any non-viral method of introducing nucleic acid into a cell. The nucleic acid may be any nucleic acid suitable for transfecting a host cell. Thus, in one embodiment, the nucleic acid is a plasmid. The host cell may be any cell in which a vector (e.g. a non-replicating poxvirus vector or an adenovirus vector, as described above) may be grown. As used herein, "culturing the host cell under conditions suitable for the propagation of the vector" means using any cell culture conditions and techniques known in the art which are suitable for the chosen host cell, and which enable the vector to be produced in the host cell. As used herein, "obtaining the vector", means using any technique known in the art that is suitable for separating the vector from the host cell. Thus, the host cells may be lysed to release the vector. The vector may subsequently be isolated and purified using any suitable method or methods known in the art.

In one aspect, the invention provides a host cell comprising a nucleic acid sequence encoding a viral vector, as described above. The host cell may be any cell in which a viral vector (e.g. a non-replicating poxvirus vector or an adenovirus vector, as described above) may be grown or propagated. In one embodiment, the host cell is selected from: a 293 cell (also known as a HEK, or human embryonic kidney, cell), a CHO cell (Chinese Hamster Ovary), a CCL81.1 cell, a Vero cell, a HELA cell, a Per.C6 cell, a BHK cell (Baby Hamster Kidney), a primary CEF cell (Chick Embryo Fibroblast), a duck embryo fibroblast cell, a DF-1 cell, or a rat IEC-6 cell.

The present invention also provides compositions comprising vectors as described above.

In one aspect, the invention provides a composition comprising a vector (as described above) and a pharmaceutically-acceptable carrier.

Substances suitable for use as pharmaceutically-acceptable carriers are known in the art. Non-limiting examples of pharmaceutically-acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage. Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 7.4).

In addition to a pharmaceutically-acceptable carrier, the composition of the invention can be further combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/or antimicrobial compound.

The composition may be formulated as a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

In one embodiment, the composition (as described above) further comprises at least one CCHFV polypeptide antigen (i.e. an antigen present in the composition in the form of a polypeptide). Thus, the composition may comprise both vector and polypeptide. In one embodiment, the polypeptide antigen is a CCHFV glycoprotein, such as CCHFV G.sub.N or CCHFV G.sub.C. In one embodiment, the presence of a polypeptide antigen means that, following administration of the composition to a subject, an improved simultaneous T cell and antibody response can be achieved. In one embodiment, the T cell and antibody response achieved surpasses that achieved when either a vector or a polypeptide antigen is used alone.

In one embodiment, the polypeptide antigen is not bonded to the vector. In one embodiment, the polypeptide antigen is a separate component to the vector. In one embodiment, the polypeptide antigen is provided separately from the vector.

In one embodiment, the polypeptide antigen is a variant of the antigen encoded by the vector. In one embodiment, the polypeptide antigen is a fragment of the antigen encoded by the vector. In one embodiment, the polypeptide antigen comprises at least part of a polypeptide sequence encoded by a nucleic acid sequence of the vector. Thus, the polypeptide antigen may correspond to at least part of the antigen encoded by the vector.

In one embodiment, the polypeptide antigen is a CCHFV glycoprotein comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 5 and 6.

The polypeptide antigen may be the same as (or similar to) that encoded by a nucleic acid sequence of the vector of the composition. Thus, administration of the composition comprising a vector and a polypeptide antigen may be used to achieve an enhanced immune response against a single antigen, wherein said enhanced immune response comprises a combined T cell and an antibody response, as described above.

In one embodiment, a composition of the invention (as described above) further comprises at least one naked DNA (i.e. a DNA molecule that is separate from, and not part of, the viral vector of the invention) encoding a CCHFV glycoprotein or antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3. In one embodiment, the naked DNA encodes a CCHFV glycoprotein comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 5 and 6.

In one embodiment, a composition of the invention (as described above) further comprises an adjuvant. Non-limiting examples of adjuvants suitable for use with compositions of the present invention include aluminium phosphate, aluminium hydroxide, and related compounds; monophosphoryl lipid A, and related compounds; outer membrane vesicles from bacteria; oil-in-water emulsions such as MF59; liposomal adjuvants, such as virosomes, Freund's adjuvant and related mixtures; poly-lactid-co-glycolid acid (PLGA) particles; cholera toxin; E. coli lethal toxin; and flagellin.

The vectors and compositions of the invention (as described above) can be employed as vaccines. Thus, a composition of the invention may be a vaccine composition.

As used herein, a vaccine is a formulation that, when administered to an animal subject such as a mammal (e.g. a human, bovine, porcine, ovine, caprine, equine, cervine, canine or feline subject; in particular a human subject), stimulates a protective immune response against an infectious disease. The immune response may be a humoral and/or a cell-mediated immune response. Thus, the vaccine may stimulate B cells and/or T cells.

The term "vaccine" is herein used interchangeably with the terms "therapeutic/prophylactic composition", "immunogenic composition", "formulation", "antigenic composition", or "medicament".

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in medicine.

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of inducing an immune response in a subject. The immune response may be against a CCHFV antigen (e.g. a CCHFV glycoprotein) and/or a CCHFV infection. Thus, the vectors and compositions of the invention can be used to induce an immune response in a subject against a CCHFV glycoprotein (for example, as immunogenic compositions or as vaccines).

In one embodiment, the immune response comprises a T cell response.

In one embodiment, the method of inducing an immune response in a subject comprises administering to a subject an effective amount of a vector (as described above) or a composition (as described above).

In one aspect, the invention provides a vector (as described above) or a composition (as described above) for use in a method of preventing or treating a CCHFV infection in a subject.

As used herein, the term "preventing" includes preventing the initiation of CCHFV infection and/or reducing the severity of intensity of a CCHFV infection. Thus, "preventing" encompasses vaccination.

As used herein, the term "treating" embraces therapeutic and preventative/prophylactic measures (including post-exposure prophylaxis) and includes post-infection therapy and amelioration of a CCHFV infection.

Each of the above-described methods can comprise the step of administering to a subject an effective amount, such as a therapeutically effective amount, of a vector or a compound of the invention.

In this regard, as used herein, an effective amount is a dosage or amount that is sufficient to achieve a desired biological outcome. As used herein, a therapeutically effective amount is an amount which is effective, upon single or multiple dose administration to a subject (such as a mammalian subject, in particular a human subject) for treating, preventing, curing, delaying, reducing the severity of, ameliorating at least one symptom of a disorder or recurring disorder, or prolonging the survival of the subject beyond that expected in the absence of such treatment.

Accordingly, the quantity of active ingredient to be administered depends on the subject to be treated, capacity of the subject's immune system to generate a protective immune response, and the degree of protection required. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be particular to each subject.

Administration to the subject can comprise administering to the subject a vector (as described above) or a composition (as described above) wherein the composition is sequentially administered multiple times (for example, wherein the composition is administered two, three or four times). Thus, in one embodiment, the subject is administered a vector (as described above) or a composition (as described above) and is then administered the same vector or composition (or a substantially similar vector or composition) again at a different time.

In one embodiment, administration to a subject comprises administering a vector (as described above) or a composition (as described above) to a subject, wherein said composition is administered substantially prior to, simultaneously with, or subsequent to, another immunogenic composition.

Prior, simultaneous and sequential administration regimes are discussed in more detail below.

In certain embodiments, the above-described methods further comprise the administration to the subject of a second vector, wherein the second vector comprises a nucleic acid sequence encoding a CCHFV glycoprotein. Preferably, the second vector is a vector of the invention as described above (such as a viral vector, for example a non-replicating poxvirus vector or an adenovirus vector as described above).

In one embodiment, the first and second vectors encode the same CCHFV glycoprotein(s). In one embodiment, the first and second vectors encode different glycoprotein(s) or different CCHFV antigens.

In one embodiment, the first and second vectors are of the same vector type. In on embodiment, the first and second vectors are of different vector types. In one embodiment, the first vector is an adenovirus vector (as described above) and the second vector is a non-replicating poxvirus vector (as described above). In one embodiment, the first vector is a non-replicating poxvirus vector (as described above) and the second vector is an adenovirus vector (as described above).

In one embodiment, the first and second vectors are administered sequentially, in any order. Thus, the first ("1") and second ("2") vectors may be administered to a subject in the order 1-2, or in the order 2-1.

As used herein, "administered sequentially" has the meaning of "sequential administration", as defined below. Thus, the first and second vectors are administered at (substantially) different times, one after the other.

In one embodiment, the first and second vectors are administered as part of a prime-boost administration protocol. Thus, the first vector may be administered to a subject as the "prime" and the second vector subsequently administered to the same subject as the "boost". Prime-boost protocols are discussed below.

In one embodiment, each of the above-described methods further comprises the step of administration to the subject of a CCHFV polypeptide antigen. In one embodiment, the CCHFV polypeptide antigen is a CCHFV glycoprotein (or antigenic fragment thereof) as described above. In one embodiment, the CCHFV polypeptide antigen is a CCHFV glycoprotein comprising an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 5 and 6.

In one embodiment, the polypeptide antigen is administered separately from the administration of a vector; preferably the polypeptide antigen and a vector are administered sequentially, in any order. Thus, in one embodiment, the vector ("V") and the polypeptide antigen ("P") may be administered in the order V-P, or in the order P-V.

In one embodiment, each of the above-described methods further comprises the step of administration to the subject of a naked DNA encoding a CCHFV glycoprotein or antigenic fragment thereof. In one embodiment, the naked DNA comprises (or consists of) a nucleic acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 1, 2, and 3. In one embodiment, the naked DNA encodes a CCHFV glycoprotein comprising (or consisting of) an amino acid sequence having at least 70% (such as at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 5 and 6.

In one embodiment, the naked DNA is administered separately from the administration of a vector; preferably the naked DNA and a vector are administered sequentially, in any order. Thus, in one embodiment, the vector ("V") and the naked DNA ("D") may be administered in the order V-D, or in the order D-V.

In one embodiment, a naked DNA (as described above) is administered to a subject as part of a prime-boost protocol.

Heterologous prime-boosting approaches can improve immune responses, by allowing repeated vaccinations without increasing anti-vector immunity. A CCHFV glycoprotein (GP) or an antigenic fragment thereof can be serially delivered via different vectors (as described above) or naked DNA vectors (as described above). In any heterologous prime-boost vaccination regime, GP-specific antibody response is increased, GP-specific T-cell response is increased, and/or clinical illness is reduced, as compared to use of a single vector. Suitable combinations of vectors include but are not limited to:

DNA prime, MVA boost

DNA prime, Fowlpox boost

Fowlpox prime, MVA boost

MVA prime, Fowlpox boost

DNA prime, Fowlpox boost, MVA boost

MVA prime, Adenovirus boost

As used herein, the term polypeptide embraces peptides and proteins.

In certain embodiments, the above-described methods further comprise the administration to the subject of an adjuvant. Adjuvant may be administered with one, two, three, or all four of: a first vector, a second vector, a polypeptide antigen, and a naked DNA.

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be given in a single dose schedule (i.e. the full dose is given at substantially one time). Alternatively, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be given in a multiple dose schedule.

A multiple dose schedule is one in which a primary course of treatment (e.g. vaccination) may be with 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example (for human subjects), at 1-4 months for a second dose, and if needed, a subsequent dose(s) after a further 1-4 months.

The dosage regimen will be determined, at least in part, by the need of the individual and be dependent upon the judgment of the practitioner (e.g. doctor or veterinarian).

Simultaneous administration means administration at (substantially) the same time.

Sequential administration of two or more compositions/therapeutic agents/vaccines means that the compositions/therapeutic agents/vaccines are administered at (substantially) different times, one after the other.

For example, sequential administration may encompass administration of two or more compositions/therapeutic agents/vaccines at different times, wherein the different times are separated by a number of days (for example, 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150 or 200 days).

For example, in one embodiment, the vaccine of the present invention may be administered as part of a `prime-boost` vaccination regime.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention can be administered to a subject such as a mammal (e.g. a human, bovine, porcine, ovine, caprine, equine, cervine, ursine, canine or feline subject) in conjunction with (simultaneously or sequentially) one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL-12), and/or cytokines (e.g. IFN.gamma.).

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) may contain 5% to 95% of active ingredient, such as at least 10% or 25% of active ingredient, or at least 40% of active ingredient or at least 50, 55, 60, 70 or 75% active ingredient.

The immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.

Administration of immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) is generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral administration; for example, a subcutaneous or intramuscular injection.

Accordingly, immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.

The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents.

Generally, the carrier is a pharmaceutically-acceptable carrier. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long term storage.

Examples of buffering agents include, but are not limited to, sodium succinate (pH 6.5), and phosphate buffered saline (PBS; pH 6.5 and 7.5).

Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

It may be desired to direct the compositions of the present invention (as described above) to the respiratory system of a subject. Efficient transmission of a therapeutic/prophylactic composition or medicament to the site of infection in the lungs may be achieved by oral or intra-nasal administration.

Formulations for intranasal administration may be in the form of nasal droplets or a nasal spray. An intranasal formulation may comprise droplets having approximate diameters in the range of 100-5000 .mu.m, such as 500-4000 .mu.m, 1000-3000 .mu.m or 100-1000 .mu.m. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 .mu.l, such as 0.1-50 .mu.l or 1.0-25 .mu.l, or such as 0.001-1 .mu.l.

Alternatively, the therapeutic/prophylactic formulation or medicament may be an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol particles is relevant to the delivery capability of an aerosol. Smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli. In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1-50 .mu.m, preferably 1-25 .mu.m, more preferably 1-5 .mu.m.

Aerosol particles may be for delivery using a nebulizer (e.g. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/or surfactant.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention comprise a pharmaceutically acceptable carrier, and optionally one or more of a salt, excipient, diluent and/or adjuvant.

In one embodiment, the immunogenic compositions, therapeutic formulations, medicaments, pharmaceutical compositions, and prophylactic formulations (e.g. vaccines) of the invention may comprise one or more immunoregulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g. IL-2, IL-12), and/or cytokines (e.g. IFN.gamma.).

The present invention encompasses polypeptides that are substantially homologous to polypeptides based on any one of the polypeptide antigens identified in this application (including fragments thereof). The terms "sequence identity" and "sequence homology" are considered synonymous in this specification.

By way of example, a polypeptide of interest may comprise an amino acid sequence having at least 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 99 or 100% amino acid sequence identity with the amino acid sequence of a reference polypeptide.

There are many established algorithms available to align two amino acid sequences.

Typically, one sequence acts as a reference sequence, to which test sequences may be compared. The sequence comparison algorithm calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of amino acid sequences for comparison may be conducted, for example, by computer implemented algorithms (e.g. GAP, BESTFIT, FASTA or TFASTA), or BLAST and BLAST 2.0 algorithms.

The BLOSUM62 table shown below is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; incorporated herein by reference). Amino acids are indicated by the standard one-letter codes. The percent identity is calculated as:

.times..times..times..times..times..times..times..times..times..times..ti- mes..times..times..times..times..times..times..times..times..times..times.- .times..times..times..times..times..times..times..times..times..times..tim- es..times..times..times..times..times..times..times..times..times..times..- times..times..times..times..times. ##EQU00001##

TABLE-US-00001 BLOSUM62 table A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

In a homology comparison, the identity may exist over a region of the sequences that is at least 10 amino acid residues in length (e.g. at least 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 685 amino acid residues in length--e.g. up to the entire length of the reference sequence.

Substantially homologous polypeptides have one or more amino acid substitutions, deletions, or additions. In many embodiments, those changes are of a minor nature, for example, involving only conservative amino acid substitutions. Conservative substitutions are those made by replacing one amino acid with another amino acid within the following groups: Basic: arginine, lysine, histidine; Acidic: glutamic acid, aspartic acid; Polar: glutamine, asparagine; Hydrophobic: leucine, isoleucine, valine; Aromatic: phenylalanine, tryptophan, tyrosine; Small: glycine, alanine, serine, threonine, methionine. Substantially homologous polypeptides also encompass those comprising other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of 1 to about 30 amino acids (such as 1-10, or 1-5 amino acids); and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and do not imply any length restriction. As used herein, the terms "nucleic acid" and "nucleotide" are used interchangeably. The terms "nucleic acid sequence" and "polynucleotide" embrace DNA (including cDNA) and RNA sequences.

The polynucleotide sequences of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.

The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.

The polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

When applied to a nucleic acid sequence, the term "isolated" in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.

In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:

TABLE-US-00002 Amino Degenerate Acid Codons Codon Cys TGC TGT TGY Ser AGC AGT TCA TCC TCG TCT WSN Thr ACA ACC ACG ACT ACN Pro CCA CCC CCG CCT CCN Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN Asn AAC AAT AAY Asp GAC GAT GAY Glu GAA GAG GAR Gln CAA CAG CAR His CAC CAT CAY Arg AGA AGG CGA CGC CGG CGT MGN Lys AAA AAG AAR Met ATG ATG Ile ATA ATC ATT ATH Leu CTA CTC CTG CTT TTA TTG YTN Val GTA GTC GTG GTT GTN Phe TTC TTT TTY Tyr TAC TAT TAY Trp TGG TGG Ter TAA TAG TGA TRR Asn/Asp RAY Glu/Gln SAR Any NNN

One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.

A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 99% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.

Alternatively, a "variant" nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the "variant" and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30.degree. C., typically in excess of 37.degree. C. and preferably in excess of 45.degree. C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST.

One of ordinary skill in the art appreciates that different species exhibit "preferential codon usage". As used herein, the term "preferential codon usage" refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferential. Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.

Thus, in one embodiment of the invention, the nucleic acid sequence is codon optimized for expression in a host cell.

A "fragment" of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide. By way of example, a "fragment" of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950 or 1000 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. Example MVA Vector Construction.

FIG. 1A. Cassette used in plasmid pLW44. Green Fluorescent Protein (GFP) is driven by poxvirus promoter p11. A multi-cloning site is downstream of poxvirus promoter mH5. MVA flanks L and R allow recombination with MVA genome.

FIG. 1B. Cassette used in plasmid pLW44 following modification (renamed as plasmid pDEST44-TPA-V5).

FIG. 1C. Recombination between pDEST44-TPA-V5 and pENTR-GP results in pTP-GP, containing the depicted cassette (SEQ ID NO: 7).

FIG. 2. MVA-GP immunogenicity in Balb/c mice. IFN-.gamma. ELISpot on splenocytes 8 or 14 days post-boost showed T-cell immunogenicity of GP

FIG. 3. Experimental protocol for Example 2.

FIG. 4A-B. IFN-.gamma. ELISPOT results.

FIG. 5A-B. IFN-.gamma. ELISPOT results subdivided into peptide pools (1 for TPA/V5, 7 for GP and 2 for NP).

FIG. 6. Animal survival rates following CCHFV challenge (Example 3).

FIG. 7A-B. Weight loss in study animals (Example 3).

FIG. 8A-B. Temperature rise in study animals (Example 3).

FIG. 9. Experimental protocol for Example 4.

FIG. 10. Animal survival rates following CCHFV challenge (Example 4).

FIG. 11. Animal clinical scores (Example 4).

FIG. 12. Animal temperature scores (Example 4).

FIG. 13. Animal weight change scores (Example 4).

FIG. 14A-B. Animal immune responses (Example 4).

FIG. 15.

Western Blot Analysis of Glycoproteins Expressed by MVA-GP and CCHFv.

(A) Western blotting of MVA-GP with anti-V5 antibody indicates a protein of approximately 75 kDa, confirming recombinant protein expression, and consistent with cleavage of the predicted 76.6 kDa G.sub.C.about.V5 fusion protein.

(B) Western blotting of MVA-1974 (lane 1), MVA-GP (lane 2), CCHFv-infected SW13 cells (lane 3) and uninfected SW13 cells (lane 4) with polyclonal serum from rabbits vaccinated with peptides derived from the CCHF viral glycoprotein. Major products expressed by MVA-GP correspond with those expressed by CCHFv, indicating the recombinant protein undergoes similar post-translational cleavages as the native protein.

FIG. 16.

Antibody Responses from Vaccinated A129 and 129Sv/Ev Mice.

Sera from vaccinated mice were tested for reactivity with CCHFv-infected (lane 3) or uninfected (lane 2) SW13 cells by Western blotting. Lane 1 shows a molecular weight marker. Blots show proteins reactive with serum from representative individual animals 7 days after booster vaccination (A-E) or representative pooled serum 14 days after booster vaccination (F). Secondary antibody used was specific for mouse IgG (A-C, F), or mouse IgG, IgA and IgM (D-E). Arrows indicate CCHFv-specific proteins, indicating specific antibody responses in both mouse strains.

(A) 129Sv/Ev mouse vaccinated with MVA-GP. (B, E) A129 mouse vaccinated with MVA-GP. (C) A129 mouse vaccinated with MVA 1974. (D) A129 mouse vaccinated with MVA-GP. (F) Pooled serum from A129 mice vaccinated with MVA-GP.

FIG. 17.

Tissue Histology of A129 Mice, 4 Days after Challenge with CCHFv.

A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A-B) or MVA-GP (C-D). Four days after challenge, sections of spleen (A, C) and liver (B, D) were fixed, HE stained, and examined for pathology. More severe pathology was found in mice that received MVA 1974, compared to those that received MVA-GP. (A) Marked lymphocyte loss with prominent apoptotic bodies, and infiltration by macrophages. (B) Marked, multifocally extensive hepatocyte necrosis (arrows). (C) A single infiltration of macrophages in the white pulp (asterisk) (scored minimal). (D) Scattered, multifocal areas of hepatocellular necrosis with a mixed inflammatory cell infiltrate (arrows) (scored moderate).

FIG. 18.

Immunohistochemistry of Tissues from A129 Mice, 4 Days after Challenge with CCHFv.

A129 mice were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA 1974 (A-C) or MVA-GP (D-E). Four days after challenge, sections of spleen (A, D) and liver (B-C, E) were fixed, immunohistochemically stained with CCHFv-specific antibody, and examined microscopically. Tissues in panels A, B, D and E were from the same individuals as shown in FIGS. 5A, 5B, 5C and 5D, respectively. A diffuse staining pattern of viral proteins was found in tissues from animals that received the MVA 1974 negative control. However, in MVA-GP vaccinated animals, the only staining found was of a minimal degree, in liver from one individual.

(A) A few, scattered cells with cytoplasmic staining within the parenchyma. (B) Frequent, diffuse, positively stained hepatocytes. (C) Scattered, small, elongated cells consistent with Kupffer cells, with cytoplasmic staining. (D) Normal parenchyma. (E) A few, positively stained cells within an inflammatory cell focus.

FIG. 19.

Viral Load Analysis of CCHFv RNA Copy Number by RT-PCR.

A129 mice (n=9) were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA-GP, MVA 1974, or saline. Four days post-challenge (day 32), 3 randomly selected animals from each group were killed humanely and analysed by RT-PCR for copy number of CCHFv S segment in various tissues. Fourteen days post-challenge (day 42), all surviving animals were killed humanely and also analysed. Each bar represents the mean.+-.standard deviation in an individual animal.

In all tissues tested, viral load was significantly lower in MVA-GP vaccinated mice than in control groups. Within the MVA-GP group, there was no significant difference in viral load between 4 days and 14 days post-challenge. (A) Blood. (B) Spleen. (C) Liver.

FIG. 20.

Normalised Viral Load Analysis of CCHFv RNA by RT-PCR.

A129 mice (n=9) were challenged with double the minimum lethal dose of CCHFv, 14 days after booster vaccination with MVA-GP, MVA 1974, or saline. Four days post-challenge (day 32), 3 randomly selected animals from each group were killed humanely and analysed by RT-PCR for CCHFv gene expression, normalised to mouse HPRT gene expression. Fourteen days post-challenge (day 42), all surviving animals were killed humanely and also analysed. Each point represents the mean value of triplicate measurements in an individual animal. Lines show mean.+-.standard deviation.

In all tissues tested, viral load was significantly lower in MVA-GP vaccinated mice than in control groups. Within the MVA-GP group, there was no significant difference in viral load between 4 days and 14 days post-challenge. (A) Blood. (B) Spleen. (C) Liver.

KEY TO SEQ ID NOS

SEQ ID NO: 1 Full length CCHFV M segment open reading frame nucleic acid sequence (from Accession Number U39455).

SEQ ID NO: 2 CCHFV G.sub.N glycoprotein nucleic acid sequence.

SEQ ID NO: 3 CCHFV G.sub.C glycoprotein nucleic acid sequence.

SEQ ID NO: 4 Amino acid sequence of protein encoded by CCHFV M segment.

SEQ ID NO: 5 CCHFV G.sub.N glycoprotein amino acid sequence.

SEQ ID NO: 6 CCHFV G.sub.C glycoprotein amino acid sequence.

SEQ ID NO: 7 MVA-GP nucleic acid sequence (representing the section shown in FIG. 1C).

Sequences

TABLE-US-00003 SEQ ID NO: 1 atgcatatatcattaatgtatgcaatcctttgcctacagctgtgtggtct gggagagactcatggatcacacaatgaaactagacacaataaaacagaca ccatgacaacacacggtgataacccgagctctgaaccgccagtgagcacg gccttgtctattacacttgacccctccactgtcacacccacaacaccagc cagtggattagaaggctcaggggaagtctacacatcccctccgatcacca ccgggagcttgcccctgtcggagacaacaccagaactccctgttacaacc ggcacagacaccttaagcgcaggtgatgtcgatcccagcacgcagacagc cggaggcacctccgcaccaacagtccgcacaagtctacccaacagcccta gcacaccatctacaccacaagacacacaccatcctgtgagaaatctactt tcagtcacgagtcctgggccagatgaaacatcaacaccctcgggaacagg caaagagagctcagcaaccagtagccctcatccagtctccaacagaccac caacccctcctgcaacagcccagggacccactgaaaatgacagtcacaac gccactgaacaccctgagtccctgacacagtcagcaaccccaggcctaat gacctctccaacacagatagtccacccacaaagtgccacccccataaccg ttcaagacacacatcccagtccaacgaacaggtctaaaagaaaccttaag atggaaataatcttgactttatctcagggtttaaaaaagtactatgggaa aatattaaggcttctgcaactcaccttagaggaggacactgaaggtctac tggaatggtgtaagagaaatcttggtcttgattgtgatgacactttcttt caaaagagaattgaagaattctttataactggtgagggccattttaatga agttttacaatttagaacgccaggcacgttgagcaccacagagtcaacac ctgctgggctgccaacagctgaaccttttaagtcctacttcgccaaaggc ttcctctcgatagattcaggttactactcagccaaatgttactcaggaac atccaattcagggcttcaattgattaacattacccgacattcaactagaa tagttgacacacctgggcctaagatcactaacctaaagaccatcaactgc ataaacttgaaggcatcgatcttcaaagaacatagagaggttgaaatcaa tgtgcttctcccccaagttgcagttaatctctcaaactgtcacgttgtaa tcaaatcacatgtctgtgactactctttagacattgacggtgcggtgagg cttcctcacatttaccatgaaggagttttcatcccaggaacttacaaaat agtgatagataaaaaaaataagttgaatgacagatgcaccttatttaccg actgtgtgataaaaggaagggaggttcgtaaaggacagtcagttttgagg cagtacaagacggaaatcaggattggcaaggcatcaaccggctttagaag attgctttcagaagaacccagtgatgactgtgtatcaagaactcaactat taaggacagagactgcagagatccacggcgacaactatggtggcccgggt gacaaaataaccatctgcaatggctcaactattgtagaccaaagactggg cagtgaactaggatgctacaccatcaatagagtgaggtcattcaagctat gcgaaaacagtgccacagggaagaattgtgaaatagacagtgtcccagtt aaatgcaggcagggttattgcctaagaatcactcaggaagggaggggcca cgtaaaattatctaggggctcagaggttgtcttagatgcatgcgatacaa gctgtgaaataatgatacctaagggcactggtgacatcctagttgactgt tcaggtgggcagcaacattttctaaaggacaatttgatagatctaggatg ccccaaaattccattattgggcaaaatggctatttacatttgcagaatgt caaaccaccccaaaacaaccatggctttcctcttctggttcagctttggc tatgtaataacctgcatactttgcaaggctattttttacttgttaataat tgttggaacactagggagaaggctcaagcagtatagagagttgaaacctc agacttgcaccatatgtgagacaactcctgtaaatgcaatagatgctgag atgcatgacctcaattgcagttacaacatttgtccctactgtgcatctag actaacctcagatgggcttgctaggcatgtgatacaatgccctaagcgga aggagaaagtggaagaaactgaactgtacttgaacttagaaagaattcct tgggttgtaagaaagctgttgcaggtgtcagagtcaactggtgtggcatt gaaaagaagcagttggctgattgtgctgcttgtgctattcactgtttcat tatcaccagttcaatcagcacccattggtcaagggaagacaattgaggca taccgggccagggaagggtacacaagtatatgcctctttgtactaggaag tatcctatttatagtttcttgcctaatgaaagggctggttgacagtgttg gcaactccttcttccctggactgtccatttgcaaaacgtgctccataagc agcattaatggctttgaaattgagtcccataagtgctattgcagcttatt ctgttgcccctattgtaggcactgctctaccgataaagaaattcataagc tgcacttgagcatctgcaaaaaaaggaaaacaggaagtaatgtcatgttg gctgtctgcaagctcatgtgtttcagggccaccatggaagtaagtaacag agccctgtttatccgtagcatcatcaacaccacttttgttttgtgcatac tgatactagcagtttgtgttgttagcacctcagcagtggagatggaaaac ctaccagcagggacctgggaaagagaagaagacctaacaaatttctgtca tcaggaatgccaggttacagagactgaatgcctctgcccttatgaagctc tagtactcagaaagcctttattcctagatagtacagctaaaggcatgaaa aatctgctaaattcaacaagtttagaaacgagtttatcaattgaggcacc atggggagcaataaatgttcagtcaacctacaaaccaactgtgtcaactg caaacatagcactcagttggagctcagtggaacacagaggcaataagatc ttggtttcaggcagatcagaatcaattatgaagctggaagaaaggacagg aatcagctgggatctcggtgtagaagatgcctctgaatctaaactgctta cagtatctgtcatggacttgtctcagatgtactctcctgtcttcgagtac ttatcaggggacagacaggtggaagagtggcccaaagcaacttgcacagg tgactgcccagaaagatgtggctgcacatcatcaacctgtttgcacaaag aatggcctcactcaagaaattggagatgcaatcccacttggtgctggggt gtagggactggctgcacctgttgtggattagatgtgaaagacctttttac agattatatgtttgtcaagtggaaagttgaatacatcaagacagaggcca tagtgtgtgtagaacttactagtcaggaaaggcagtgtagcttgattgaa gcgggcacaaggttcaatttaggtcctgtgaccatcacactgtcagaacc aagaaacatccaacaaaaactccctcctgaaataatcacactgcatccta ggatcgaagaaggtttttttgacctgatgcatgtgcaaaaggtgttatcg gcaagcacagtgtgtaagttgcagagttgcacacatggtgtgccaggaga cctacaggtctaccacatcggaaatttattaaaaggggataaggtaaatg gacatctaattcataaaattgagccacacttcaacacctcctggatgtcc tgggatggttgtgacctagactactactgcaacatgggagattggccttc ttgcacatacacaggggtcacccaacacaatcatgcttcatttgtaaact tactcaacattgaaactgattacacaaagaacttccactttcactctaaa agggtcactgcacacggagatacaccacaactagatcttaaggcaagacc aacctatggtgcaggcgagatcactgttctggtagaagttgctgacatgg agttacatacaaagaagattgaaatatcaggcttaaaatttgcaagctta gcttgcacaggttgttatgcttgtagctctagcatctcatgcaaagttag aattcatgtggatgaaccagatgaacttacagtacatgttaaaagtgatg atccagatgtggttgcagctagctcaagtctcatggcaaggaagcttgaa tttggaacagacagtacatttaaagctttctcggccatgcctaaaacttc tctatgtttctacattgttgaaagagaacactgtaagagctgcagtgaag aagacacaaaaaaatgtgttaacacaaaacttgagcaaccacaaagcatt ttgatcgaacacaagggaactataatcggaaagcaaaacagcacttgcac ggctaaggcaagttgctggttagagtcagtcaagagttttttttatggcc taaagaacatgcttagtggcatttttggcaatgtctttatgggcattttc ttgttccttgcccccttcatcctgttaatactattctttatgtttgggtg gaggatcctattctgctttaaatgttgtagaagaaccagaggcctgttca agtatagacacctcaaagacgatgaagaaactggttatagaaggattatt gaaaaactaaacaataaaaaaggaaaaaacaaactgcttgatggtgaaag acttgctgatggaagaattgccgaactgttctctacaaaaacacacattg gctag SEQ ID NO: 2 agaagattgctttcagaagaacccagtgatgactgtgtatcaagaactca actattaaggacagagactgcagagatccacggcgacaactatggtggcc cgggtgacaaaataaccatctgcaatggctcaactattgtagaccaaaga ctgggcagtgaactaggatgctacaccatcaatagagtgaggtcattcaa gctatgcgaaaacagtgccacagggaagaattgtgaaatagacagtgtcc cagttaaatgcaggcagggttattgcctaagaatcactcaggaagggagg ggccacgtaaaattatctaggggctcagaggttgtcttagatgcatgcga tacaagctgtgaaataatgatacctaagggcactggtgacatcctagttg actgttcaggtgggcagcaacattttctaaaggacaatttgatagatcta ggatgccccaaaattccattattgggcaaaatggctatttacatttgcag aatgtcaaaccaccccaaaacaaccatggctttcctcttctggttcagct ttggctatgtaataacctgcatactttgcaaggctattttttacttgtta ataattgttggaacactagggagaaggctcaagcagtatagagagttgaa acctcagacttgcaccatatgtgagacaactcctgtaaatgcaatagatg ctgagatgcatgacctcaattgcagttacaacatttgtccctactgtgca tctagactaacctcagatgggcttgctaggcatgtgatacaatgccctaa gcggaaggagaaagtggaagaaactgaactgtacttgaacttagaaagaa ttccttgggttgtaagaaagctgttg SEQ ID NO: 3 agaaagcctttattcctagatagtacagctaaaggcatgaaaaatctgct aaattcaacaagtttagaaacgagtttatcaattgaggcaccatggggag caataaatgttcagtcaacctacaaaccaactgtgtcaactgcaaacata gcactcagttggagctcagtggaacacagaggcaataagatcttggtttc

aggcagatcagaatcaattatgaagctggaagaaaggacaggaatcagct gggatctcggtgtagaagatgcctctgaatctaaactgcttacagtatct gtcatggacttgtctcagatgtactctcctgtcttcgagtacttatcagg ggacagacaggtggaagagtggcccaaagcaacttgcacaggtgactgcc cagaaagatgtggctgcacatcatcaacctgtttgcacaaagaatggcct cactcaagaaattggagatgcaatcccacttggtgctggggtgtagggac tggctgcacctgttgtggattagatgtgaaagacctttttacagattata tgtttgtcaagtggaaagttgaatacatcaagacagaggccatagtgtgt gtagaacttactagtcaggaaaggcagtgtagcttgattgaagcgggcac aaggttcaatttaggtcctgtgaccatcacactgtcagaaccaagaaaca tccaacaaaaactccctcctgaaataatcacactgcatcctaggatcgaa gaaggtttttttgacctgatgcatgtgcaaaaggtgttatcggcaagcac agtgtgtaagttgcagagttgcacacatggtgtgccaggagacctacagg tctaccacatcggaaatttattaaaaggggataaggtaaatggacatcta attcataaaattgagccacacttcaacacctcctggatgtcctgggatgg ttgtgacctagactactactgcaacatgggagattggccttcttgcacat acacaggggtcacccaacacaatcatgcttcatttgtaaacttactcaac attgaaactgattacacaaagaacttccactttcactctaaaagggtcac tgcacacggagatacaccacaactagatcttaaggcaagaccaacctatg gtgcaggcgagatcactgttctggtagaagttgctgacatggagttacat acaaagaagattgaaatatcaggcttaaaatttgcaagcttagcttgcac aggttgttatgcttgtagctctagcatctcatgcaaagttagaattcatg tggatgaaccagatgaacttacagtacatgttaaaagtgatgatccagat gtggttgcagctagctcaagtctcatggcaaggaagcttgaatttggaac agacagtacatttaaagctttctcggccatgcctaaaacttctctatgtt tctacattgttgaaagagaacactgtaagagctgcagtgaagaagacaca aaaaaatgtgttaacacaaaacttgagcaaccacaaagcattttgatcga acacaagggaactataatcggaaagcaaaacagcacttgcacggctaagg caagttgctggttagagtcagtcaagagttttttttatggcctaaagaac atgcttagtggcatttttggcaatgtctttatgggcattttcttgttcct tgcccccttcatcctgttaatactattctttatgtttgggtggaggatcc tattctgctttaaatgttgtagaagaaccagaggcctgttcaagtataga cacctcaaagacgatgaagaaactggttatagaaggattattgaaaaact aaacaataaaaaaggaaaaaacaaactgcttgatggtgaaagacttgctg atggaagaattgccgaactgttctctacaaaaacacacattggctag SEQ ID NO: 4 MHISLMYAILCLQLCGLGETHGSHNETRHNKTDTMTTHGDNPSSEPPVST ALSITLDPSTVTPTTPASGLEGSGEVYTSPPITTGSLPLSETTPELPVTT GTDTLSAGDVDPSTQTAGGTSAPTVRTSLPNSPSTPSTPQDTHHPVRNLL SVTSPGPDETSTPSGTGKESSATSSPHPVSNRPPTPPATAQGPTENDSHN ATEHPESLTQSATPGLMTSPTQIVHPQSATPITVQDTHPSPTNRSKRNLK MEIILTLSQGLKKYYGKILRLLQLTLEEDTEGLLEWCKRNLGLDCDDTFF QKRIEEFFITGEGHFNEVLQFRTPGTLSTTESTPAGLPTAEPFKSYFAKG FLSIDSGYYSAKCYSGTSNSGLQLINITRHSTRIVDTPGPKITNLKTINC INLKASIFKEHREVEINVLLPQVAVNLSNCHVVIKSHVCDYSLDIDGAVR LPHIYHEGVFIPGTYKIVIDKKNKLNDRCTLFTDCVIKGREVRKGQSVLR QYKTEIRIGKASTGFRRLLSEEPSDDCVSRTQLLRTETAEIHGDNYGGPG DKITICNGSTIVDQRLGSELGCYTINRVRSFKLCENSATGKNCEIDSVPV KCRQGYCLRITQEGRGHVKLSRGSEVVLDACDTSCEIMIPKGTGDILVDC SGGQQHFLKDNLIDLGCPKIPLLGKMAIYICRMSNHPKTTMAFLFWFSFG YVITCILCKAIFYLLIIVGTLGRRLKQYRELKPQTCTICETTPVNAIDAE MHDLNCSYNICPYCASRLTSDGLARHVIQCPKRKEKVEETELYLNLERIP WVVRKLLQVSESTGVALKRSSWLIVLLVLFTVSLSPVQSAPIGQGKTIEA YRAREGYTSICLFVLGSILFIVSCLMKGLVDSVGNSFFPGLSICKTCSIS SINGFEIESHKCYCSLFCCPYCRHCSTDKEIHKLHLSICKKRKTGSNVML AVCKLMCFRATMEVSNRALFIRSIINTTFVLCILILAVCVVSTSAVEMEN LPAGTWEREEDLTNFCHQECQVTETECLCPYEALVLRKPLFLDSTAKGMK NLLNSTSLETSLSIEAPWGAINVQSTYKPTVSTANIALSWSSVEHRGNKI LVSGRSESIMKLEERTGISWDLGVEDASESKLLTVSVMDLSQMYSPVFEY LSGDRQVEEWPKATCTGDCPERCGCTSSTCLHKEWPHSRNWRCNPTWCWG VGTGCTCCGLDVKDLFTDYMFVKWKVEYIKTEAIVCVELTSQERQCSLIE AGTRFNLGPVTITLSEPRNIQQKLPPEIITLHPRIEEGFFDLMHVQKVLS ASTVCKLQSCTHGVPGDLQVYHIGNLLKGDKVNGHLIHKIEPHFNTSWMS WDGCDLDYYCNMGDWPSCTYTGVTQHNHASFVNLLNIETDYTKNFHFHSK RVTAHGDTPQLDLKARPTYGAGEITVLVEVADMELHTKKIEISGLKFASL ACTGCYACSSSISCKVRIHVDEPDELTVHVKSDDPDVVAASSSLMARKLE FGTDSTFKAFSAMPKTSLCFYIVEREHCKSCSEEDTKKCVNTKLEQPQSI LIEHKGTIIGKQNSTCTAKASCWLESVKSFFYGLKNMLSGIFGNVFMGIF LFLAPFILLILFFMFGWRILFCFKCCRRTRGLFKYRHLKDDEETGYRRII EKLNNKKGKNKLLDGERLADGRIAELFSTKTHIG SEQ ID NO: 5 RRLLSEEPSDDCVSRTQLLRTETAEIHGDNYGGPGDKITICNGSTIVDQR LGSELGCYTINRVRSFKLCENSATGKNCEIDSVPVKCRQGYCLRITQEGR GHVKLSRGSEVVLDACDTSCEIMIPKGTGDILVDCSGGQQHFLKDNLIDL GCPKIPLLGKMAIYICRMSNHPKTTMAFLFWFSFGYVITCILCKAIFYLL IIVGTLGRRLKQYRELKPQTCTICETTPVNAIDAEMHDLNCSYNICPYCA SRLTSDGLARHVIQCPKRKEKVEETELYLNLERIPWVVRKLL SEQ ID NO: 6 RKPLFLDSTAKGMKNLLNSTSLETSLSIEAPWGAINVQSTYKPTVSTANI ALSWSSVEHRGNKILVSGRSESIMKLEERTGISWDLGVEDASESKLLTVS VMDLSQMYSPVFEYLSGDRQVEEWPKATCTGDCPERCGCTSSTCLHKEWP HSRNWRCNPTWCWGVGTGCTCCGLDVKDLFTDYMFVKWKVEYIKTEAIVC VELTSQERQCSLIEAGTRFNLGPVTITLSEPRNIQQKLPPEIITLHPRIE EGFFDLMHVQKVLSASTVCKLQSCTHGVPGDLQVYHIGNLLKGDKVNGHL IHKIEPHFNTSWMSWDGCDLDYYCNMGDWPSCTYTGVTQHNHASFVNLLN IETDYTKNFHFHSKRVTAHGDTPQLDLKARPTYGAGEITVLVEVADMELH TKKIEISGLKFASLACTGCYACSSSISCKVRIHVDEPDELTVHVKSDDPD VVAASSSLMARKLEFGTDSTFKAFSAMPKTSLCFYIVEREHCKSCSEEDT KKCVNTKLEQPQSILIEHKGTIIGKQNSTCTAKASCWLESVKSFFYGLKN MLSGIFGNVFMGIFLFLAPFILLILFFMFGWRILFCFKCCRRTRGLFKYR HLKDDEETGYRRIIEKLNNKKGKNKLLDGERLADGRIAELFSTKTHIG SEQ ID NO: 7 GTTGGTGGTCGCCATGGATGGTGTTATTGTATACTGTCTAAACGCGTTAG TAAAACATGGCGAGGAAATAAATCATATAAAAAATGATTTCATGATTAAA CCATGTTGTGAAAAAGTCAAGAACGTTCACATTGGCGGACAATCTAAAAA CAATACAGTGATTGCAGATTTGCCATATATGGATAATGCGGTATCCGATG TATGCAATTCACTGTATAAAAAGAATGTATCAAGAATATCCAGATTTGCT AATTTGATAAAGATAGATGACGATGACAAGACTCCTACTGGTGTATATAA TTATTTTAAACCTAAAGATGCCATTCCTGTTATTATATCCATAGGAAAGG ATAGAGATGTTTGTGAACTATTAATCTCATCTGATAAAGCGTGTGCGTGT ATAGAGTTAAATTCATATAAAGTAGCCATTCTTCCCATGGATGTTTCCTT TTTTACCAAAGGAAATGCATCATTGATTATTCTCCTGTTTGATTTCTCTA TCGATGCGGCACCTCTCTTAAGAAGTGTAACCGATAATAATGTTATTATA TCTAGACACCAGCGTCTACATGACGAGCTTCCGAGTTCCAATTGGTTCAA GTTTTACATAAGTATAAAGTCCGACTATTGTTCTATATTATATATGGTTG TTGATGGATCTGTGATGCATGCAATAGCTGATAATAGAACTTACGCAAAT ATTAGCAAAAATATATTAGACAATACTACAATTAACGATGAGTGTAGATG CTGTTATTTTGAACCACAGATTAGGATTCTTGATAGAGATGAGATGCTCA ATGGATCATCGTGTGATATGAACAGACATTGTATTATGATGAATTTACCT GATGTAGGCGAATTTGGATCTAGTATGTTGGGGAAATATGAACCTGACAT GATTAAGATTGCTCTTTCGGTGGCTGGGTACCAGGCGCGCCTTTCATTTT GTTTTTTTCTATGCTATAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCG GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAG TTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCC TCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGAC CACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTA CAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACG GCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTG CAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGT GCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAG ACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCC GCCGGGATCACTCTCGGCATGCACGAGCTGTACAAGTAAGCGGCCGCTGG TACCCAACCTAAAAATTGAAAATAAATACAAAGGTTCTTGAGGGTTGTGT

TAAATTGAAAGCGAGAAATAATCATAAATAAGCccggtGCCACCATGgat gcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagtctt cgtttcgcccagccaggaaatccatgcccgattcagaagaggagccagat ctcccATCAAACAAGTTTGTACAAAAAAGCAGGCTcatatatcattaatg tatgcaatcctttgcctacagctgtgtggtctgggagagactcatggatc acacaatgaaactagacacaataaaacagacaccatgacaacacacggtg ataacccgagctctgaaccgccagtgagcacggccttgtctattacactt gacccctccactgtcacacccacaacaccagccagtggattagaaggctc aggggaagtctacacatcccctccgatcaccaccgggagcttgcccctgt cggagacaacaccagaactccctgttacaaccggcacagacaccttaagc gcaggtgatgtcgatcccagcacgcagacagccggaggcacctccgcacc aacagtccgcacaagtctacccaacagccctagcacaccatctacaccac aagacacacaccatcctgtgagaaatctactttcagtcacgagtcctggg ccagatgaaacatcaacaccctcgggaacaggcaaagagagctcagcaac cagtagccctcatccagtctccaacagaccaccaacccctcctgcaacag cccagggacccactgaaaatgacagtcacaacgccactgaacaccctgag tccctgacacagtcagcaaccccaggcctaatgacctctccaacacagat agtccacccacaaagtgccacccccataaccgttcaagacacacatccca gtccaacgaacaggtctaaaagaaaccttaagatggaaataatcttgact ttatctcagggtttaaaaaagtactatgggaaaatattaaggcttctgca actcaccttagaggaggacactgaaggtctactggaatggtgtaagagaa atcttggtcttgattgtgatgacactttctttcaaaagagaattgaagaa ttctttataactggtgagggccattttaatgaagttttacaatttagaac gccaggcacgttgagcaccacagagtcaacacctgctgggctgccaacag ctgaaccttttaagtcctacttcgccaaaggcttcctctcgatagattca ggttactactcagccaaatgttactcaggaacatccaattcagggcttca attgattaacattacccgacattcaactagaatagttgacacacctgggc ctaagatcactaacctaaagaccatcaactgcataaacttgaaggcatcg atcttcaaagaacatagagaggttgaaatcaatgtgcttctcccccaagt tgcagttaatctctcaaactgtcacgttgtaatcaaatcacatgtctgtg actactctttagacattgacggtgcggtgaggcttcctcacatttaccat gaaggagttttcatcccaggaacttacaaaatagtgatagataaaaaaaa taagttgaatgacagatgcaccttatttaccgactgtgtgataaaaggaa gggaggttcgtaaaggacagtcagttttgaggcagtacaagacggaaatc aggattggcaaggcatcaaccggctttagaagattgctttcagaagaacc cagtgatgactgtgtatcaagaactcaactattaaggacagagactgcag agatccacggcgacaactatggtggcccgggtgacaaaataaccatctgc aatggctcaactattgtagaccaaagactgggcagtgaactaggatgcta caccatcaatagagtgaggtcattcaagctatgcgaaaacagtgccacag ggaagaattgtgaaatagacagtgtcccagttaaatgcaggcagggttat tgcctaagaatcactcaggaagggaggggccacgtaaaattatctagggg ctcagaggttgtcttagatgcatgcgatacaagctgtgaaataatgatac ctaagggcactggtgacatcctagttgactgttcaggtgggcagcaacat tttctaaaggacaatttgatagatctaggatgccccaaaattccattatt gggcaaaatggctatttacatttgcagaatgtcaaaccaccccaaaacaa ccatggctttcctcttctggttcagctttggctatgtaataacctgcata ctttgcaaggctattttttacttgttaataattgttggaacactagggag aaggctcaagcagtatagagagttgaaacctcagacttgcaccatatgtg agacaactcctgtaaatgcaatagatgctgagatgcatgacctcaattgc agttacaacatttgtccctactgtgcatctagactaacctcagatgggct tgctaggcatgtgatacaatgccctaagcggaaggagaaagtggaagaaa ctgaactgtacttgaacttagaaagaattccttgggttgtaagaaagctg ttgcaggtgtcagagtcaactggtgtggcattgaaaagaagcagttggct gattgtgctgcttgtgctattcactgtttcattatcaccagttcaatcag cacccattggtcaagggaagacaattgaggcataccgggccagggaaggg tacacaagtatatgcctctttgtactaggaagtatcctatttatagtttc ttgcctaatgaaagggctggttgacagtgttggcaactccttcttccctg gactgtccatttgcaaaacgtgctccataagcagcattaatggctttgaa attgagtcccataagtgctattgcagcttattctgttgcccctattgtag gcactgctctaccgataaagaaattcataagctgcacttgagcatctgca aaaaaaggaaaacaggaagtaatgtcatgttggctgtctgcaagctcatg tgtttcagggccaccatggaagtaagtaacagagccctgtttatccgtag catcatcaacaccacttttgttttgtgcatactgatactagcagtttgtg ttgttagcacctcagcagtggagatggaaaacctaccagcagggacctgg gaaagagaagaagacctaacaaatttctgtcatcaggaatgccaggttac agagactgaatgcctctgcccttatgaagctctagtactcagaaagcctt tattcctagatagtacagctaaaggcatgaaaaatctgctaaattcaaca agtttagaaacgagtttatcaattgaggcaccatggggagcaataaatgt tcagtcaacctacaaaccaactgtgtcaactgcaaacatagcactcagtt ggagctcagtggaacacagaggcaataagatcttggtttcaggcagatca gaatcaattatgaagctggaagaaaggacaggaatcagctgggatctcgg tgtagaagatgcctctgaatctaaactgcttacagtatctgtcatggact tgtctcagatgtactctcctgtcttcgagtacttatcaggggacagacag gtggaagagtggcccaaagcaacttgcacaggtgactgcccagaaagatg tggctgcacatcatcaacctgtttgcacaaagaatggcctcactcaagaa attggagatgcaatcccacttggtgctggggtgtagggactggctgcacc tgttgtggattagatgtgaaagacctttttacagattatatgtttgtcaa gtggaaagttgaatacatcaagacagaggccatagtgtgtgtagaactta ctagtcaggaaaggcagtgtagcttgattgaagcgggcacaaggttcaat ttaggtcctgtgaccatcacactgtcagaaccaagaaacatccaacaaaa actccctcctgaaataatcacactgcatcctaggatcgaagaaggtttCt ttgacctgatgcatgtgcaaaaggtgttatcggcaagcacagtgtgtaag ttgcagagttgcacacatggtgtgccaggagacctacaggtctaccacat cggaaatttattaaaaggggataaggtaaatggacatctaattcataaaa ttgagccacacttcaacacctcctggatgtcctgggatggttgtgaccta gactactactgcaacatgggagattggccttcttgcacatacacaggggt cacccaacacaatcatgcttcatttgtaaacttactcaacattgaaactg attacacaaagaacttccactttcactctaaaagggtcactgcacacgga gatacaccacaactagatcttaaggcaagaccaacctatggtgcaggcga gatcactgttctggtagaagttgctgacatggagttacatacaaagaaga ttgaaatatcaggcttaaaatttgcaagcttagcttgcacaggttgttat gcttgtagctctagcatctcatgcaaagttagaattcatgtggatgaacc agatgaacttacagtacatgttaaaagtgatgatccagatgtggttgcag ctagctcaagtctcatggcaaggaagcttgaatttggaacagacagtaca tttaaagctttctcggccatgcctaaaacttctctatgtttctacattgt tgaaagagaacactgtaagagctgcagtgaagaagacacaaaaaaatgtg ttaacacaaaacttgagcaaccacaaagcattttgatcgaacacaaggga actataatcggaaagcaaaacagcacttgcacggctaaggcaagttgctg gttagagtcagtcaagagtttCttttatggcctaaagaacatgcttagtg gcatttttggcaatgtctttatgggcattttcttgttccttgcccccttc atcctgttaatactattctttatgtttgggtggaggatcctattctgctt taaatgttgtagaagaaccagaggcctgttcaagtatagacacctcaaag acgatgaagaaactggttatagaaggattattgaaaaactaaacaataaa aaaggaaaaaacaaactgcttgatggtgaaagacttgctgatggaagaat tgccgaactgttctctacaaaaacacacattggcACCCAGCTTTCTTGTA CAAAGTGGTTCGATggggatctagagggcccgcggttcgaaggtaagcct atccctaaccctctcctcggtctcgattctacgtaaGTCGACCTGCAGGG AAAGTTTTATAGGTAGTTGATAGAACAAAATACATAATTTTGTAAAAATA AATCACTTTTTATACTAATATGACACGATTACCAATACTTTTGTTACTAA TATCATTAGTATACGCTACACCTTTTCCTCAGACATCTAAAAAAATAGGT GATGATGCAACTTTATCATGTAATCGAAATAATACAAATGACTACGTTGT TATGAGTGCTTGGTATAAGGAGCCCAATTCCATTATTCTTTTAGCTGCTA AAAGCGACGTCTTGTATTTTGATAATTATACCAAGGATAAAATATCTTAC GACTCTCCATACGATGATCTAGTTACAACTATCACAATTAAATCATTGAC TGCTAGAGATGCCGGTACTTATGTATGTGCATTCTTTATGACATCGCCTA CAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATT GTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTAC ACATTCACCAGAAACTAGTT

EXAMPLES

Example 1. Preparation of an Example MVA-GP (Glycoprotein) Vector (FIG. 1A-1C)

The sequence of the CCHFV M segment was taken from published sequence data on the IbAr10200 CCHFV strain.

The M segment sequence was modified for improved compatibility with MVA expression. M segment untranslated regions were deleted, start and stop codons were deleted, and 2.times.TTTTTNT sequences (poxvirus transcription stop signals) silently mutated. attB1 and attB2 sequences were added for compatibility with the proprietary cloning system used (Gateway Cloning, by Invitrogen).

Tissue Plasminogen Activator (tPA) signal sequence (for increased immunogenicity and intracellular transport) and V5 fusion protein sequence (for identification of expressed protein by immunolabeling) were added.

The construct was synthesised and recombined into a pDONR vector to generate Entry Clone plasmid (pENTR-GP).

The plasmid was then recombined with Destination vector pDEST44-TPA-V5 to generate pTP-GP plasmid.

This resulted in the gene for tPA-GP-V5 fusion protein downstream of the poxvirus mH5 promoter. This promoter was chosen for increased stability and strong early expression, to drive a cytotoxic T lymphocyte response.

pTP-GP was transfected into MVA-infected cells. Recombination occurs between the MVA flanks on the plasmid and in the MVA genome, inserting the GFP and GP cassette into the MVA genome. The MVA strain used was MVA 1974/NIH clone 1.

MVA-GP was plaque-purified based on GFP expression and underwent quality checks: confirmation of purity by PCR, confirmation of GP expression by Western blot (FIG. 15A), and sequencing of the insertion site.

SDS-PAGE of MVA-GP and Western blotting with anti-V5 antibody (FIG. 15A) indicated a single protein of approximately 75 kDa, confirming expression and consistent with cleavage of the predicted 76.6 kDa Gc-V5 fusion protein from the GP precursor at RKPL sequence.

SDS-PAGE and Western blotting with anti-glycoprotein polyclonal serum were performed, in order to compare glycoprotein expression by MVA-GP with CCHFv (FIG. 15B). Several products were expressed by CCHFv (lane 3). Some of these were also detected in uninfected SW13 cells (lane 4), suggesting a possible cross-reaction with cellular proteins. CCHFv-specific products were detected at approximately 109, 60, 34 and <20 kDa. Similarly, sucrose cushion-purified MVA-GP expressed major proteins at approximately 92-136, 64 and 35 kDa (lane 2). Sucrose cushion-purified MVA-1974 (a negative control) did not show any products of a similar size (lane 1), confirming all products in lane 2 as specific to the recombinant vaccine.

The appropriate clone of MVA-GP was amplified, purified by sucrose cushion centrifugation and titrated by plaque assay.

Example 2. MVA-GP Immunogenicity in Balb/c Mice

11 Balb/c mice were injected intramuscularly with 10.sup.7 plaque-forming units (pfu) per animal of MVA-GP, prepared according to Example 1. A volume of 100 .mu.l was delivered, split into two sites at 50 .mu.l each. Animals received 2 vaccinations, spaced 2 weeks apart. Control animals (n=9) received saline.

Eight days after the final vaccination, 5 (MVA-GP) or 4 (saline) mice were sacrificed for T cell immunogenicity testing. The remaining animals were sacrificed 14 days after the final vaccination.

Animals immunised with MVA-GP showed a significant antigen response to CCHF glycoprotein peptides as compared to control animals (FIG. 2).

Examples 3 and 4 (described below) utilise an animal model that replicates lethal disease in mice that are infected with CCHF virus. In vaccine efficacy experiments, all animals that were vaccinated with MVA-GP survived a lethal challenge dose of CCHFV. Those animals that received a mock immunisation with (i) saline or (ii) a simple MVA preparation all succumbed to disease and met humane clinical endpoints by five days post-challenge.

Example 3. MVA-GP Vaccine Efficacy Study in Mouse Model (Mouse Strains A129 and 129Sv/Ev)

This in vivo efficacy study used both type-1 interferon receptor knockout (A129) and wild-type (129Sv/Ev) mice which were immunised with MVA containing CCHF virus glycoprotein (MVA-GP), construct alone (MVA-empty) and saline. A129 mice are susceptible to CCHF virus infection, and the 129Sv/Ev are the wild-type parent strain. Mice were immunised at day 0, and then boosted at day 14. 7 days after the last immunisation, 5 animals from each group were used to assess immune response. 14 days after the last immunisation, A129 mice from each immunisation group were challenged with 10.sup.2 TCID.sub.50 CCHF virus, delivered intradermally. 6 challenged animals from each group were observed for up to 10 days post-challenge for survival studies.

Experimental protocol is depicted in FIG. 3.

Results: IFN-.gamma. ELISPOT results showed that MVA-GP immunised mice generated immune responses specific to the CCHFv glycoproteins that they were immunised with. Statistically similar results were observed between both strains after MVA-GP immunisation (P<0.05, Mann-Whitney statistical test). T-cell responses to peptides derived from the TPA/V5 fusion proteins, or from an irrelevant antigen (NP) were negligible, indicating specificity of the response. (FIG. 4).

When the responses shown in FIG. 4 were subdivided into the peptide pools (1 for TPA/V5, 4 for GP, 3 for GC and 2 for NP) the same pools were optimal between strains. Some peptide pools were consistently non-immunogenic. (FIG. 5).

After challenge with CCHF virus, control groups of saline and MVA-empty showed 66% survival and 33% survival, respectively. 100% survival was observed with 2.times.MVA-GP group. (FIG. 6).

Weight loss was seen in animals that reached humane clinical endpoints in control groups 2 and 3. Some of the vaccinated animals in the 100% survival group showed evidence of weight loss, indicative of disease/illness. (FIG. 7).

Animals that reached humane clinical endpoints in groups 2 (MVA-empty) and 3 (saline control) also exhibited a rise in temperature at days 5-7 post-challenge. Temperatures fluctuated in the MVA-GP immunised animals, but no trend was observed. (FIG. 8).

Conclusions: MVA-GP is immunogenic, producing protein-specific immune responses. No differences were observed in the immune response to MVA-GP between wild-type mice (129Sv/Ev) and those with a knockout in the type-1 interferon receptor (A129). All MVA-GP immunised animals survived a challenge with 10.sup.2 TCID.sub.50 CCHF virus (strain IbAr10200). Animals which reached humane clinical endpoints in the MVA-empty and saline groups exhibited a rise in temperature and weight loss.

Example 4. MVA-GP Vaccine Efficacy Study in Mouse Model (Mouse Strain A129)

The study described above in Example 3 found 10.sup.2 TCID.sub.50/100 .mu.l delivered intradermally (i.d.) to have 34% lethality in saline `vaccinated` 10 week old IFN-.alpha./.beta.R.sup.-/- mice. Therefore this study used 2.times.10.sup.2 TCID.sub.50/100 .mu.l of strain IbAr10200 delivered i.d. as the challenge dose to IFN-.alpha./.beta.R.sup.-/- mice.

Prior to challenge, animals were vaccinated with MVA-GP derived from strain IbAr10200. Vaccinations were given intramuscularly (i.m.) into the caudal thigh at a dose of 10.sup.7 plaque-forming units (pfu) per animal. A volume of 100 .mu.l was delivered, split into two sites at 50 .mu.l each. Animals received 2 vaccinations, spaced 2 weeks apart. Two weeks elapsed between final vaccination and challenge.

Control groups were vaccinated with saline (groups 1 & 6), or MVA-empty (group 2), which is unmodified, non-recombinant MVA.

Representatives from each vaccination group were sacrificed 7 days post-final vaccination, for histology and immunogenicity (T-cell and antibody) testing. Further animals were sacrificed 4 days post-challenge, for histology and viral load testing. After challenge, animals were weighed and temperature monitored daily, and observed for signs of illness twice daily.

Animals showing moderate symptoms (such as loss of 10% body weight, shaking or paralysis) were euthanised. Spleen and liver samples were collected from animals that reached humane clinical endpoints. At 14 days post-challenge, all remaining animals were culled and samples collected for histology and viral load testing.

Experimental protocol is depicted in FIG. 9.

Results--Survival. 100% protection was achieved with MVA-GP (survival 2 weeks post-challenge). All other groups died 4-5 days post-challenge. (FIG. 10).

Results--Clinical Data. MVA-GP showed no signs of disease. Control groups showed severe illness and were euthanized. (FIG. 11). MVA-GP temperature was stable whilst other groups were succumbing, but slight spike in temperature at 9 days post-challenge. Control groups showed spike in temperature, then sharp reduction as succumbed to disease. (FIG. 12). MVA-GP showed some weight loss at days 4-5, then stable, but did not regain peak weight. Control groups lost 5-10% of body weight. (FIG. 13).

Results--Immune Responses (ELISpot) IFN-.gamma. ELISpot on splenocytes 7 days post-boost showed T-cell immunogenicity of GP. Low immunogenicity from TPA & V5 fusion tags (FIG. 14A). GP\ peptide pools showed that density of epitopes varied across the GP protein. (FIG. 14B).

Western Blotting

Immunised animals (including those used in Example 3) were also tested for induction of a CCHFv-specific humoral response by MVA-GP, using Western Blotting. IgG antibody reacting with a protein of approximately 114 kDa was detected in 3 out of 5 vaccinated animals' sera from 129Sv/Ev mice collected on day 21 of the vaccination schedule (FIG. 16A). In A129 mice, a CCHFv-specific IgG antibody response was detectable by Western Blot in only 1 out of 8 individual animals, which recognised a 79 kDa protein (FIG. 16B). A randomly selected A129 mouse that received the MVA-1974 negative control was tested, and no CCHFv-specific antibody response was seen (FIG. 16C).

Sera from the 8 A129 animals vaccinated with MVA-GP were also assessed by Western Blot for an early phase immune response, using a detector antibody specific for mouse IgG, IgA and IgM. Antibodies specific for a 79 kDa CCHFv protein were detected in the same individual animal in this assay, as had been detected by anti-IgG only (FIG. 16E). Broadening the sensitivity to additional antibody subclasses, detected antibodies specific for a protein of approximately 112 kDa (FIG. 16D) in a further 4 animals.

Histopathology

Histopathological findings in immunised, CCHFv challenged mice are shown in FIGS. 17-18 and Tables 1-2 (below).

TABLE-US-00004 TABLE 1 Severity of microscopic lesions in HE stained tissues from vaccinated A129 mice, challenged with CCHFv. Group Saline MVA 1974 MVA-GP MVA-GP Severity (day 32-33) (day 32-33) (day 32) (day 42) Spleen Normal 0 0 1 6 Minimal 1 1 2 0 Mild 2 3 0 0 Moderate 2 1 0 0 Marked 4 4 0 0 Liver Normal 0 1 2 6 Minimal 0 0 0 0 Mild 2 0 0 0 Moderate 3 3 1 0 Marked 4 5 0 0 Numbers of animals in each group, according to severity rating of histological lesions.

TABLE-US-00005 TABLE 2 Frequency of immunohistochemically stained cells in tissues from selected vaccinated A129 mice, challenged with CCHFv. Group Saline MVA 1974 MVA-GP MVA-GP Frequency (day 32-33) (day 32-33) (day 32) (day 42) Spleen Normal 0 0 3 1 Minimal 4 4 0 0 Moderate 2 3 0 0 Marked 1 0 0 0 Liver Normal 0 0 2 1 Minimal 1 2 1 0 Moderate 3 1 0 0 Marked 3 4 0 0 Numbers of animals in each group, according to frequency of cells stained by immunohistochemistry.

Viral Load Analysis

Viral load was analysed by RT-PCR of CCHFv S segment in blood, spleen, and liver from 3 animals per group at day 32, and all surviving animals at day 42. CCHFv copy number was calculated by use of a standard curve. At day 32, CCHFv copy number was significantly lower in MVA-GP vaccinated animals compared to control groups in blood, spleen and liver (p=0.05). In the blood, it was detected in only 2 of 3 animals. At day 42, CCHFv levels were not statistically different in any tissue to those in vaccinated animals at the earlier timepoint. It was detectable in the blood in only 1 out of 5 vaccinated mice (FIG. 19).

Alternatively, CCHFv expression was normalised to expression of the HPRT reference gene (FIG. 20). Normalised CCHFv expression was significantly lower in MVA-GP vaccinated animals compared to control groups in blood, spleen and liver (p=0.05). There was no statistically significant difference between saline and MVA 1974 control groups, or between day 32 and day 42 samples from MVA-GP vaccinated animals.

Conclusions: MVA-GP is immunogenic, producing protein-specific immune responses. MVA-GP is protective, protecting 100% of mice from a fully lethal challenge of homologous strain CCHF. Animals which reached humane clinical endpoints in the MVA-empty and saline groups exhibited signs of illness, a rise in temperature and weight loss. No such clinical signs were observed in MVA-GP vaccinated animals.

Example 5. Heterologous Prime-Boost Study

Heterologous prime-boosting approaches improve immune responses by allowing repeated vaccinations without increasing anti-vector immunity. A CCHFV glycoprotein (GP) or an antigenic fragment thereof is serially delivered via different viral or DNA vectors.

In a heterologous regime where the prime vaccination is delivered by a DNA vector, and the boost vaccination is delivered by a Fowlpox virus vector, GP-specific antibody response is increased, GP-specific T-cell response is increased, and/or clinical illness is reduced, as compared to where the prime and boost are delivered by the same vector.

The following heterologous combinations of vectors are provided for use in prime-boosting approaches:

DNA prime, MVA boost

Fowlpox prime, MVA boost

MVA prime, Fowlpox boost

DNA prime, Fowlpox boost, MVA boost

MVA prime, Adenovirus boost

Example 6. Immunogenicity Studies in Non-Human Primates

Prior to use in clinical trials, immune responses to a CCHF vaccine are tested in a non-human primate model.

Non-human primates (e.g. rhesus macaques or cynomolgous macaques) are inoculated with the CCHF vaccine expressing the glycoprotein gene or functional fragment thereof. Animals receive either a single dose, or multiple doses in a prime-boost regime, by a parenteral route. Subsequent immunological analysis indicates that they have generated a CCHF-specific immune response. The latter is confirmed by one or more of the following methods:

CCHF-specific antibodies present in serum

Neutralising antibodies present in serum

Cellular response to peptides derived from the CCHF glycoprotein

Cellular response to virally-infected cells.

A cellular response is characterised by an increase in one or more of: proliferation, production of cytokines or chemokines, phagocytosis, and/or release of granzymes.

Example 7. Preparation of an Example Adenovirus Vector

A non-replicating adenovirus is engineered to express a CCHF glycoprotein or partial fragment thereof. The genetic sequence for the CCHF glycoprotein is inserted into the genome of the adenovirus vector. Expression of the glycoprotein is indicated by reactivity between a glycoprotein-specific antibody and products from the adenovirus by Western blotting or ELISA as follows:

Cellular lysate of cells infected with the recombinant adenovirus, subjected to SDS-PAGE and Western blotting with an antibody specific for the CCHF glycoprotein, show a specific reactivity compared to negative controls.

Alternatively, products from cells infected with the recombinant adenovirus are used to coat an ELISA plate. CCHF-specific antibodies bind to the coating and are detected via a chemical reaction.

Example 8. CCHF Vaccine Provides Cross-Strain Protection

A vaccine expressing the glycoprotein gene or functional fragment thereof, in an adenovirus or non-replicating poxvirus vector, is delivered via a parenteral route into mice that are susceptible to disease caused by CCHF virus. They are challenged with a lethal dose of CCHF virus, from a strain other than that on which the vaccine is based. The challenged animals show no or mild clinical signs of illness, and do not require euthanasia. Control animals which received the same challenge dose of CCHF, but did not receive the vaccine, show severe signs of illness, reach humane clinical endpoints and require euthanasia.

Example 9. MVA-GP Immunogenicity in A129 Mice

Twenty-five A129 mice were injected intramuscularly with 10.sup.7 pfu per animal of MVA-GP, prepared according to Example 1. A volume of 100 .mu.l was delivered, split into two sites at 50 .mu.l each. Animals received 2 vaccinations, spaced 2 weeks apart. Control animals (n=25) received 10.sup.7 pfu per animal of non-recombinant MVA 1974/NIH clone 1 according to the same regime.

Fourteen days after the final vaccination, all mice were sacrificed for antibody testing. Sera were prepared, heat-inactivated and pooled; each pool contained sera from 5-6 animals that received the same treatment as each other. Pools were subjected to Western blot analysis against lysate of cells infected with CCHF virus.

In 4 out of 5 pools from animals that received MVA-GP, an IgG antibody response specific for a CCHF virus protein of approximately 114 kDa was detected (FIG. 16F).

Example 10. Preparation and Efficacy of a Recombinant Influenza Virus Vector

Reverse genetics are used to construct a recombinant influenza virus that carries a protective epitope of CCHFv glycoprotein in the neuraminidase stalk. CCHFv specific cytotoxic T lymphocytes (CTLs) are induced in mice after intranasal or parenteral administration. These CTLs provide a reduction in viral load and clinical illness after challenge with CCHFv.

Example 11. Preparation and Efficacy of a Recombinant Bacterial Vector

The CCHFv glycoprotein gene, or functional fragment thereof, is expressed on the surface of genetically attenuated, gram-negative bacteria. After intranasal or parenteral administration to mice, the bacterial vector colonises antigen-presenting cells (e.g. dendritic cells or macrophages). A humoral and cellular CCHFv-specific immune response is induced. These immune responses provide a reduction in viral load and clinical illness after challenge with CCHFv.

SEQUENCE LISTINGS

1

715055DNACrimean-Congo hemorrhagic fever virus 1atgcatatat cattaatgta tgcaatcctt tgcctacagc tgtgtggtct gggagagact 60catggatcac acaatgaaac tagacacaat aaaacagaca ccatgacaac acacggtgat 120aacccgagct ctgaaccgcc agtgagcacg gccttgtcta ttacacttga cccctccact 180gtcacaccca caacaccagc cagtggatta gaaggctcag gggaagtcta cacatcccct 240ccgatcacca ccgggagctt gcccctgtcg gagacaacac cagaactccc tgttacaacc 300ggcacagaca ccttaagcgc aggtgatgtc gatcccagca cgcagacagc cggaggcacc 360tccgcaccaa cagtccgcac aagtctaccc aacagcccta gcacaccatc tacaccacaa 420gacacacacc atcctgtgag aaatctactt tcagtcacga gtcctgggcc agatgaaaca 480tcaacaccct cgggaacagg caaagagagc tcagcaacca gtagccctca tccagtctcc 540aacagaccac caacccctcc tgcaacagcc cagggaccca ctgaaaatga cagtcacaac 600gccactgaac accctgagtc cctgacacag tcagcaaccc caggcctaat gacctctcca 660acacagatag tccacccaca aagtgccacc cccataaccg ttcaagacac acatcccagt 720ccaacgaaca ggtctaaaag aaaccttaag atggaaataa tcttgacttt atctcagggt 780ttaaaaaagt actatgggaa aatattaagg cttctgcaac tcaccttaga ggaggacact 840gaaggtctac tggaatggtg taagagaaat cttggtcttg attgtgatga cactttcttt 900caaaagagaa ttgaagaatt ctttataact ggtgagggcc attttaatga agttttacaa 960tttagaacgc caggcacgtt gagcaccaca gagtcaacac ctgctgggct gccaacagct 1020gaacctttta agtcctactt cgccaaaggc ttcctctcga tagattcagg ttactactca 1080gccaaatgtt actcaggaac atccaattca gggcttcaat tgattaacat tacccgacat 1140tcaactagaa tagttgacac acctgggcct aagatcacta acctaaagac catcaactgc 1200ataaacttga aggcatcgat cttcaaagaa catagagagg ttgaaatcaa tgtgcttctc 1260ccccaagttg cagttaatct ctcaaactgt cacgttgtaa tcaaatcaca tgtctgtgac 1320tactctttag acattgacgg tgcggtgagg cttcctcaca tttaccatga aggagttttc 1380atcccaggaa cttacaaaat agtgatagat aaaaaaaata agttgaatga cagatgcacc 1440ttatttaccg actgtgtgat aaaaggaagg gaggttcgta aaggacagtc agttttgagg 1500cagtacaaga cggaaatcag gattggcaag gcatcaaccg gctttagaag attgctttca 1560gaagaaccca gtgatgactg tgtatcaaga actcaactat taaggacaga gactgcagag 1620atccacggcg acaactatgg tggcccgggt gacaaaataa ccatctgcaa tggctcaact 1680attgtagacc aaagactggg cagtgaacta ggatgctaca ccatcaatag agtgaggtca 1740ttcaagctat gcgaaaacag tgccacaggg aagaattgtg aaatagacag tgtcccagtt 1800aaatgcaggc agggttattg cctaagaatc actcaggaag ggaggggcca cgtaaaatta 1860tctaggggct cagaggttgt cttagatgca tgcgatacaa gctgtgaaat aatgatacct 1920aagggcactg gtgacatcct agttgactgt tcaggtgggc agcaacattt tctaaaggac 1980aatttgatag atctaggatg ccccaaaatt ccattattgg gcaaaatggc tatttacatt 2040tgcagaatgt caaaccaccc caaaacaacc atggctttcc tcttctggtt cagctttggc 2100tatgtaataa cctgcatact ttgcaaggct attttttact tgttaataat tgttggaaca 2160ctagggagaa ggctcaagca gtatagagag ttgaaacctc agacttgcac catatgtgag 2220acaactcctg taaatgcaat agatgctgag atgcatgacc tcaattgcag ttacaacatt 2280tgtccctact gtgcatctag actaacctca gatgggcttg ctaggcatgt gatacaatgc 2340cctaagcgga aggagaaagt ggaagaaact gaactgtact tgaacttaga aagaattcct 2400tgggttgtaa gaaagctgtt gcaggtgtca gagtcaactg gtgtggcatt gaaaagaagc 2460agttggctga ttgtgctgct tgtgctattc actgtttcat tatcaccagt tcaatcagca 2520cccattggtc aagggaagac aattgaggca taccgggcca gggaagggta cacaagtata 2580tgcctctttg tactaggaag tatcctattt atagtttctt gcctaatgaa agggctggtt 2640gacagtgttg gcaactcctt cttccctgga ctgtccattt gcaaaacgtg ctccataagc 2700agcattaatg gctttgaaat tgagtcccat aagtgctatt gcagcttatt ctgttgcccc 2760tattgtaggc actgctctac cgataaagaa attcataagc tgcacttgag catctgcaaa 2820aaaaggaaaa caggaagtaa tgtcatgttg gctgtctgca agctcatgtg tttcagggcc 2880accatggaag taagtaacag agccctgttt atccgtagca tcatcaacac cacttttgtt 2940ttgtgcatac tgatactagc agtttgtgtt gttagcacct cagcagtgga gatggaaaac 3000ctaccagcag ggacctggga aagagaagaa gacctaacaa atttctgtca tcaggaatgc 3060caggttacag agactgaatg cctctgccct tatgaagctc tagtactcag aaagccttta 3120ttcctagata gtacagctaa aggcatgaaa aatctgctaa attcaacaag tttagaaacg 3180agtttatcaa ttgaggcacc atggggagca ataaatgttc agtcaaccta caaaccaact 3240gtgtcaactg caaacatagc actcagttgg agctcagtgg aacacagagg caataagatc 3300ttggtttcag gcagatcaga atcaattatg aagctggaag aaaggacagg aatcagctgg 3360gatctcggtg tagaagatgc ctctgaatct aaactgctta cagtatctgt catggacttg 3420tctcagatgt actctcctgt cttcgagtac ttatcagggg acagacaggt ggaagagtgg 3480cccaaagcaa cttgcacagg tgactgccca gaaagatgtg gctgcacatc atcaacctgt 3540ttgcacaaag aatggcctca ctcaagaaat tggagatgca atcccacttg gtgctggggt 3600gtagggactg gctgcacctg ttgtggatta gatgtgaaag acctttttac agattatatg 3660tttgtcaagt ggaaagttga atacatcaag acagaggcca tagtgtgtgt agaacttact 3720agtcaggaaa ggcagtgtag cttgattgaa gcgggcacaa ggttcaattt aggtcctgtg 3780accatcacac tgtcagaacc aagaaacatc caacaaaaac tccctcctga aataatcaca 3840ctgcatccta ggatcgaaga aggttttttt gacctgatgc atgtgcaaaa ggtgttatcg 3900gcaagcacag tgtgtaagtt gcagagttgc acacatggtg tgccaggaga cctacaggtc 3960taccacatcg gaaatttatt aaaaggggat aaggtaaatg gacatctaat tcataaaatt 4020gagccacact tcaacacctc ctggatgtcc tgggatggtt gtgacctaga ctactactgc 4080aacatgggag attggccttc ttgcacatac acaggggtca cccaacacaa tcatgcttca 4140tttgtaaact tactcaacat tgaaactgat tacacaaaga acttccactt tcactctaaa 4200agggtcactg cacacggaga tacaccacaa ctagatctta aggcaagacc aacctatggt 4260gcaggcgaga tcactgttct ggtagaagtt gctgacatgg agttacatac aaagaagatt 4320gaaatatcag gcttaaaatt tgcaagctta gcttgcacag gttgttatgc ttgtagctct 4380agcatctcat gcaaagttag aattcatgtg gatgaaccag atgaacttac agtacatgtt 4440aaaagtgatg atccagatgt ggttgcagct agctcaagtc tcatggcaag gaagcttgaa 4500tttggaacag acagtacatt taaagctttc tcggccatgc ctaaaacttc tctatgtttc 4560tacattgttg aaagagaaca ctgtaagagc tgcagtgaag aagacacaaa aaaatgtgtt 4620aacacaaaac ttgagcaacc acaaagcatt ttgatcgaac acaagggaac tataatcgga 4680aagcaaaaca gcacttgcac ggctaaggca agttgctggt tagagtcagt caagagtttt 4740ttttatggcc taaagaacat gcttagtggc atttttggca atgtctttat gggcattttc 4800ttgttccttg cccccttcat cctgttaata ctattcttta tgtttgggtg gaggatccta 4860ttctgcttta aatgttgtag aagaaccaga ggcctgttca agtatagaca cctcaaagac 4920gatgaagaaa ctggttatag aaggattatt gaaaaactaa acaataaaaa aggaaaaaac 4980aaactgcttg atggtgaaag acttgctgat ggaagaattg ccgaactgtt ctctacaaaa 5040acacacattg gctag 50552876DNACrimean-Congo hemorrhagic fever virus 2agaagattgc tttcagaaga acccagtgat gactgtgtat caagaactca actattaagg 60acagagactg cagagatcca cggcgacaac tatggtggcc cgggtgacaa aataaccatc 120tgcaatggct caactattgt agaccaaaga ctgggcagtg aactaggatg ctacaccatc 180aatagagtga ggtcattcaa gctatgcgaa aacagtgcca cagggaagaa ttgtgaaata 240gacagtgtcc cagttaaatg caggcagggt tattgcctaa gaatcactca ggaagggagg 300ggccacgtaa aattatctag gggctcagag gttgtcttag atgcatgcga tacaagctgt 360gaaataatga tacctaaggg cactggtgac atcctagttg actgttcagg tgggcagcaa 420cattttctaa aggacaattt gatagatcta ggatgcccca aaattccatt attgggcaaa 480atggctattt acatttgcag aatgtcaaac caccccaaaa caaccatggc tttcctcttc 540tggttcagct ttggctatgt aataacctgc atactttgca aggctatttt ttacttgtta 600ataattgttg gaacactagg gagaaggctc aagcagtata gagagttgaa acctcagact 660tgcaccatat gtgagacaac tcctgtaaat gcaatagatg ctgagatgca tgacctcaat 720tgcagttaca acatttgtcc ctactgtgca tctagactaa cctcagatgg gcttgctagg 780catgtgatac aatgccctaa gcggaaggag aaagtggaag aaactgaact gtacttgaac 840ttagaaagaa ttccttgggt tgtaagaaag ctgttg 87631947DNACrimean-Congo hemorrhagic fever virus 3agaaagcctt tattcctaga tagtacagct aaaggcatga aaaatctgct aaattcaaca 60agtttagaaa cgagtttatc aattgaggca ccatggggag caataaatgt tcagtcaacc 120tacaaaccaa ctgtgtcaac tgcaaacata gcactcagtt ggagctcagt ggaacacaga 180ggcaataaga tcttggtttc aggcagatca gaatcaatta tgaagctgga agaaaggaca 240ggaatcagct gggatctcgg tgtagaagat gcctctgaat ctaaactgct tacagtatct 300gtcatggact tgtctcagat gtactctcct gtcttcgagt acttatcagg ggacagacag 360gtggaagagt ggcccaaagc aacttgcaca ggtgactgcc cagaaagatg tggctgcaca 420tcatcaacct gtttgcacaa agaatggcct cactcaagaa attggagatg caatcccact 480tggtgctggg gtgtagggac tggctgcacc tgttgtggat tagatgtgaa agaccttttt 540acagattata tgtttgtcaa gtggaaagtt gaatacatca agacagaggc catagtgtgt 600gtagaactta ctagtcagga aaggcagtgt agcttgattg aagcgggcac aaggttcaat 660ttaggtcctg tgaccatcac actgtcagaa ccaagaaaca tccaacaaaa actccctcct 720gaaataatca cactgcatcc taggatcgaa gaaggttttt ttgacctgat gcatgtgcaa 780aaggtgttat cggcaagcac agtgtgtaag ttgcagagtt gcacacatgg tgtgccagga 840gacctacagg tctaccacat cggaaattta ttaaaagggg ataaggtaaa tggacatcta 900attcataaaa ttgagccaca cttcaacacc tcctggatgt cctgggatgg ttgtgaccta 960gactactact gcaacatggg agattggcct tcttgcacat acacaggggt cacccaacac 1020aatcatgctt catttgtaaa cttactcaac attgaaactg attacacaaa gaacttccac 1080tttcactcta aaagggtcac tgcacacgga gatacaccac aactagatct taaggcaaga 1140ccaacctatg gtgcaggcga gatcactgtt ctggtagaag ttgctgacat ggagttacat 1200acaaagaaga ttgaaatatc aggcttaaaa tttgcaagct tagcttgcac aggttgttat 1260gcttgtagct ctagcatctc atgcaaagtt agaattcatg tggatgaacc agatgaactt 1320acagtacatg ttaaaagtga tgatccagat gtggttgcag ctagctcaag tctcatggca 1380aggaagcttg aatttggaac agacagtaca tttaaagctt tctcggccat gcctaaaact 1440tctctatgtt tctacattgt tgaaagagaa cactgtaaga gctgcagtga agaagacaca 1500aaaaaatgtg ttaacacaaa acttgagcaa ccacaaagca ttttgatcga acacaaggga 1560actataatcg gaaagcaaaa cagcacttgc acggctaagg caagttgctg gttagagtca 1620gtcaagagtt ttttttatgg cctaaagaac atgcttagtg gcatttttgg caatgtcttt 1680atgggcattt tcttgttcct tgcccccttc atcctgttaa tactattctt tatgtttggg 1740tggaggatcc tattctgctt taaatgttgt agaagaacca gaggcctgtt caagtataga 1800cacctcaaag acgatgaaga aactggttat agaaggatta ttgaaaaact aaacaataaa 1860aaaggaaaaa acaaactgct tgatggtgaa agacttgctg atggaagaat tgccgaactg 1920ttctctacaa aaacacacat tggctag 194741684PRTCrimean-Congo hemorrhagic fever virus 4Met His Ile Ser Leu Met Tyr Ala Ile Leu Cys Leu Gln Leu Cys Gly 1 5 10 15 Leu Gly Glu Thr His Gly Ser His Asn Glu Thr Arg His Asn Lys Thr 20 25 30 Asp Thr Met Thr Thr His Gly Asp Asn Pro Ser Ser Glu Pro Pro Val 35 40 45 Ser Thr Ala Leu Ser Ile Thr Leu Asp Pro Ser Thr Val Thr Pro Thr 50 55 60 Thr Pro Ala Ser Gly Leu Glu Gly Ser Gly Glu Val Tyr Thr Ser Pro 65 70 75 80 Pro Ile Thr Thr Gly Ser Leu Pro Leu Ser Glu Thr Thr Pro Glu Leu 85 90 95 Pro Val Thr Thr Gly Thr Asp Thr Leu Ser Ala Gly Asp Val Asp Pro 100 105 110 Ser Thr Gln Thr Ala Gly Gly Thr Ser Ala Pro Thr Val Arg Thr Ser 115 120 125 Leu Pro Asn Ser Pro Ser Thr Pro Ser Thr Pro Gln Asp Thr His His 130 135 140 Pro Val Arg Asn Leu Leu Ser Val Thr Ser Pro Gly Pro Asp Glu Thr 145 150 155 160 Ser Thr Pro Ser Gly Thr Gly Lys Glu Ser Ser Ala Thr Ser Ser Pro 165 170 175 His Pro Val Ser Asn Arg Pro Pro Thr Pro Pro Ala Thr Ala Gln Gly 180 185 190 Pro Thr Glu Asn Asp Ser His Asn Ala Thr Glu His Pro Glu Ser Leu 195 200 205 Thr Gln Ser Ala Thr Pro Gly Leu Met Thr Ser Pro Thr Gln Ile Val 210 215 220 His Pro Gln Ser Ala Thr Pro Ile Thr Val Gln Asp Thr His Pro Ser 225 230 235 240 Pro Thr Asn Arg Ser Lys Arg Asn Leu Lys Met Glu Ile Ile Leu Thr 245 250 255 Leu Ser Gln Gly Leu Lys Lys Tyr Tyr Gly Lys Ile Leu Arg Leu Leu 260 265 270 Gln Leu Thr Leu Glu Glu Asp Thr Glu Gly Leu Leu Glu Trp Cys Lys 275 280 285 Arg Asn Leu Gly Leu Asp Cys Asp Asp Thr Phe Phe Gln Lys Arg Ile 290 295 300 Glu Glu Phe Phe Ile Thr Gly Glu Gly His Phe Asn Glu Val Leu Gln 305 310 315 320 Phe Arg Thr Pro Gly Thr Leu Ser Thr Thr Glu Ser Thr Pro Ala Gly 325 330 335 Leu Pro Thr Ala Glu Pro Phe Lys Ser Tyr Phe Ala Lys Gly Phe Leu 340 345 350 Ser Ile Asp Ser Gly Tyr Tyr Ser Ala Lys Cys Tyr Ser Gly Thr Ser 355 360 365 Asn Ser Gly Leu Gln Leu Ile Asn Ile Thr Arg His Ser Thr Arg Ile 370 375 380 Val Asp Thr Pro Gly Pro Lys Ile Thr Asn Leu Lys Thr Ile Asn Cys 385 390 395 400 Ile Asn Leu Lys Ala Ser Ile Phe Lys Glu His Arg Glu Val Glu Ile 405 410 415 Asn Val Leu Leu Pro Gln Val Ala Val Asn Leu Ser Asn Cys His Val 420 425 430 Val Ile Lys Ser His Val Cys Asp Tyr Ser Leu Asp Ile Asp Gly Ala 435 440 445 Val Arg Leu Pro His Ile Tyr His Glu Gly Val Phe Ile Pro Gly Thr 450 455 460 Tyr Lys Ile Val Ile Asp Lys Lys Asn Lys Leu Asn Asp Arg Cys Thr 465 470 475 480 Leu Phe Thr Asp Cys Val Ile Lys Gly Arg Glu Val Arg Lys Gly Gln 485 490 495 Ser Val Leu Arg Gln Tyr Lys Thr Glu Ile Arg Ile Gly Lys Ala Ser 500 505 510 Thr Gly Phe Arg Arg Leu Leu Ser Glu Glu Pro Ser Asp Asp Cys Val 515 520 525 Ser Arg Thr Gln Leu Leu Arg Thr Glu Thr Ala Glu Ile His Gly Asp 530 535 540 Asn Tyr Gly Gly Pro Gly Asp Lys Ile Thr Ile Cys Asn Gly Ser Thr 545 550 555 560 Ile Val Asp Gln Arg Leu Gly Ser Glu Leu Gly Cys Tyr Thr Ile Asn 565 570 575 Arg Val Arg Ser Phe Lys Leu Cys Glu Asn Ser Ala Thr Gly Lys Asn 580 585 590 Cys Glu Ile Asp Ser Val Pro Val Lys Cys Arg Gln Gly Tyr Cys Leu 595 600 605 Arg Ile Thr Gln Glu Gly Arg Gly His Val Lys Leu Ser Arg Gly Ser 610 615 620 Glu Val Val Leu Asp Ala Cys Asp Thr Ser Cys Glu Ile Met Ile Pro 625 630 635 640 Lys Gly Thr Gly Asp Ile Leu Val Asp Cys Ser Gly Gly Gln Gln His 645 650 655 Phe Leu Lys Asp Asn Leu Ile Asp Leu Gly Cys Pro Lys Ile Pro Leu 660 665 670 Leu Gly Lys Met Ala Ile Tyr Ile Cys Arg Met Ser Asn His Pro Lys 675 680 685 Thr Thr Met Ala Phe Leu Phe Trp Phe Ser Phe Gly Tyr Val Ile Thr 690 695 700 Cys Ile Leu Cys Lys Ala Ile Phe Tyr Leu Leu Ile Ile Val Gly Thr 705 710 715 720 Leu Gly Arg Arg Leu Lys Gln Tyr Arg Glu Leu Lys Pro Gln Thr Cys 725 730 735 Thr Ile Cys Glu Thr Thr Pro Val Asn Ala Ile Asp Ala Glu Met His 740 745 750 Asp Leu Asn Cys Ser Tyr Asn Ile Cys Pro Tyr Cys Ala Ser Arg Leu 755 760 765 Thr Ser Asp Gly Leu Ala Arg His Val Ile Gln Cys Pro Lys Arg Lys 770 775 780 Glu Lys Val Glu Glu Thr Glu Leu Tyr Leu Asn Leu Glu Arg Ile Pro 785 790 795 800 Trp Val Val Arg Lys Leu Leu Gln Val Ser Glu Ser Thr Gly Val Ala 805 810 815 Leu Lys Arg Ser Ser Trp Leu Ile Val Leu Leu Val Leu Phe Thr Val 820 825 830 Ser Leu Ser Pro Val Gln Ser Ala Pro Ile Gly Gln Gly Lys Thr Ile 835 840 845 Glu Ala Tyr Arg Ala Arg Glu Gly Tyr Thr Ser Ile Cys Leu Phe Val 850 855 860 Leu Gly Ser Ile Leu Phe Ile Val Ser Cys Leu Met Lys Gly Leu Val 865 870 875 880 Asp Ser Val Gly Asn Ser Phe Phe Pro Gly Leu Ser Ile Cys Lys Thr 885 890 895 Cys Ser Ile Ser Ser Ile Asn Gly Phe Glu Ile Glu Ser His Lys Cys 900 905 910 Tyr Cys Ser Leu Phe Cys Cys Pro Tyr Cys Arg His Cys Ser Thr Asp 915 920 925 Lys Glu Ile His Lys Leu His Leu Ser Ile Cys Lys Lys Arg Lys Thr 930 935 940 Gly Ser Asn Val Met Leu Ala Val Cys Lys Leu Met Cys Phe Arg Ala 945 950 955 960 Thr Met Glu Val Ser Asn Arg Ala Leu Phe Ile Arg Ser Ile Ile Asn 965 970 975 Thr Thr Phe Val Leu Cys Ile Leu Ile Leu Ala Val Cys Val Val Ser 980 985 990 Thr Ser Ala Val Glu Met Glu Asn Leu Pro Ala Gly Thr Trp Glu Arg 995 1000 1005 Glu Glu Asp Leu Thr Asn Phe Cys His Gln Glu Cys Gln Val Thr 1010 1015 1020 Glu Thr Glu Cys Leu Cys Pro Tyr Glu Ala Leu Val Leu Arg Lys 1025 1030 1035 Pro Leu Phe Leu Asp Ser Thr Ala Lys Gly Met Lys Asn Leu Leu 1040 1045 1050 Asn Ser

Thr Ser Leu Glu Thr Ser Leu Ser Ile Glu Ala Pro Trp 1055 1060 1065 Gly Ala Ile Asn Val Gln Ser Thr Tyr Lys Pro Thr Val Ser Thr 1070 1075 1080 Ala Asn Ile Ala Leu Ser Trp Ser Ser Val Glu His Arg Gly Asn 1085 1090 1095 Lys Ile Leu Val Ser Gly Arg Ser Glu Ser Ile Met Lys Leu Glu 1100 1105 1110 Glu Arg Thr Gly Ile Ser Trp Asp Leu Gly Val Glu Asp Ala Ser 1115 1120 1125 Glu Ser Lys Leu Leu Thr Val Ser Val Met Asp Leu Ser Gln Met 1130 1135 1140 Tyr Ser Pro Val Phe Glu Tyr Leu Ser Gly Asp Arg Gln Val Glu 1145 1150 1155 Glu Trp Pro Lys Ala Thr Cys Thr Gly Asp Cys Pro Glu Arg Cys 1160 1165 1170 Gly Cys Thr Ser Ser Thr Cys Leu His Lys Glu Trp Pro His Ser 1175 1180 1185 Arg Asn Trp Arg Cys Asn Pro Thr Trp Cys Trp Gly Val Gly Thr 1190 1195 1200 Gly Cys Thr Cys Cys Gly Leu Asp Val Lys Asp Leu Phe Thr Asp 1205 1210 1215 Tyr Met Phe Val Lys Trp Lys Val Glu Tyr Ile Lys Thr Glu Ala 1220 1225 1230 Ile Val Cys Val Glu Leu Thr Ser Gln Glu Arg Gln Cys Ser Leu 1235 1240 1245 Ile Glu Ala Gly Thr Arg Phe Asn Leu Gly Pro Val Thr Ile Thr 1250 1255 1260 Leu Ser Glu Pro Arg Asn Ile Gln Gln Lys Leu Pro Pro Glu Ile 1265 1270 1275 Ile Thr Leu His Pro Arg Ile Glu Glu Gly Phe Phe Asp Leu Met 1280 1285 1290 His Val Gln Lys Val Leu Ser Ala Ser Thr Val Cys Lys Leu Gln 1295 1300 1305 Ser Cys Thr His Gly Val Pro Gly Asp Leu Gln Val Tyr His Ile 1310 1315 1320 Gly Asn Leu Leu Lys Gly Asp Lys Val Asn Gly His Leu Ile His 1325 1330 1335 Lys Ile Glu Pro His Phe Asn Thr Ser Trp Met Ser Trp Asp Gly 1340 1345 1350 Cys Asp Leu Asp Tyr Tyr Cys Asn Met Gly Asp Trp Pro Ser Cys 1355 1360 1365 Thr Tyr Thr Gly Val Thr Gln His Asn His Ala Ser Phe Val Asn 1370 1375 1380 Leu Leu Asn Ile Glu Thr Asp Tyr Thr Lys Asn Phe His Phe His 1385 1390 1395 Ser Lys Arg Val Thr Ala His Gly Asp Thr Pro Gln Leu Asp Leu 1400 1405 1410 Lys Ala Arg Pro Thr Tyr Gly Ala Gly Glu Ile Thr Val Leu Val 1415 1420 1425 Glu Val Ala Asp Met Glu Leu His Thr Lys Lys Ile Glu Ile Ser 1430 1435 1440 Gly Leu Lys Phe Ala Ser Leu Ala Cys Thr Gly Cys Tyr Ala Cys 1445 1450 1455 Ser Ser Ser Ile Ser Cys Lys Val Arg Ile His Val Asp Glu Pro 1460 1465 1470 Asp Glu Leu Thr Val His Val Lys Ser Asp Asp Pro Asp Val Val 1475 1480 1485 Ala Ala Ser Ser Ser Leu Met Ala Arg Lys Leu Glu Phe Gly Thr 1490 1495 1500 Asp Ser Thr Phe Lys Ala Phe Ser Ala Met Pro Lys Thr Ser Leu 1505 1510 1515 Cys Phe Tyr Ile Val Glu Arg Glu His Cys Lys Ser Cys Ser Glu 1520 1525 1530 Glu Asp Thr Lys Lys Cys Val Asn Thr Lys Leu Glu Gln Pro Gln 1535 1540 1545 Ser Ile Leu Ile Glu His Lys Gly Thr Ile Ile Gly Lys Gln Asn 1550 1555 1560 Ser Thr Cys Thr Ala Lys Ala Ser Cys Trp Leu Glu Ser Val Lys 1565 1570 1575 Ser Phe Phe Tyr Gly Leu Lys Asn Met Leu Ser Gly Ile Phe Gly 1580 1585 1590 Asn Val Phe Met Gly Ile Phe Leu Phe Leu Ala Pro Phe Ile Leu 1595 1600 1605 Leu Ile Leu Phe Phe Met Phe Gly Trp Arg Ile Leu Phe Cys Phe 1610 1615 1620 Lys Cys Cys Arg Arg Thr Arg Gly Leu Phe Lys Tyr Arg His Leu 1625 1630 1635 Lys Asp Asp Glu Glu Thr Gly Tyr Arg Arg Ile Ile Glu Lys Leu 1640 1645 1650 Asn Asn Lys Lys Gly Lys Asn Lys Leu Leu Asp Gly Glu Arg Leu 1655 1660 1665 Ala Asp Gly Arg Ile Ala Glu Leu Phe Ser Thr Lys Thr His Ile 1670 1675 1680 Gly 5292PRTCrimean-Congo hemorrhagic fever virus 5Arg Arg Leu Leu Ser Glu Glu Pro Ser Asp Asp Cys Val Ser Arg Thr 1 5 10 15 Gln Leu Leu Arg Thr Glu Thr Ala Glu Ile His Gly Asp Asn Tyr Gly 20 25 30 Gly Pro Gly Asp Lys Ile Thr Ile Cys Asn Gly Ser Thr Ile Val Asp 35 40 45 Gln Arg Leu Gly Ser Glu Leu Gly Cys Tyr Thr Ile Asn Arg Val Arg 50 55 60 Ser Phe Lys Leu Cys Glu Asn Ser Ala Thr Gly Lys Asn Cys Glu Ile 65 70 75 80 Asp Ser Val Pro Val Lys Cys Arg Gln Gly Tyr Cys Leu Arg Ile Thr 85 90 95 Gln Glu Gly Arg Gly His Val Lys Leu Ser Arg Gly Ser Glu Val Val 100 105 110 Leu Asp Ala Cys Asp Thr Ser Cys Glu Ile Met Ile Pro Lys Gly Thr 115 120 125 Gly Asp Ile Leu Val Asp Cys Ser Gly Gly Gln Gln His Phe Leu Lys 130 135 140 Asp Asn Leu Ile Asp Leu Gly Cys Pro Lys Ile Pro Leu Leu Gly Lys 145 150 155 160 Met Ala Ile Tyr Ile Cys Arg Met Ser Asn His Pro Lys Thr Thr Met 165 170 175 Ala Phe Leu Phe Trp Phe Ser Phe Gly Tyr Val Ile Thr Cys Ile Leu 180 185 190 Cys Lys Ala Ile Phe Tyr Leu Leu Ile Ile Val Gly Thr Leu Gly Arg 195 200 205 Arg Leu Lys Gln Tyr Arg Glu Leu Lys Pro Gln Thr Cys Thr Ile Cys 210 215 220 Glu Thr Thr Pro Val Asn Ala Ile Asp Ala Glu Met His Asp Leu Asn 225 230 235 240 Cys Ser Tyr Asn Ile Cys Pro Tyr Cys Ala Ser Arg Leu Thr Ser Asp 245 250 255 Gly Leu Ala Arg His Val Ile Gln Cys Pro Lys Arg Lys Glu Lys Val 260 265 270 Glu Glu Thr Glu Leu Tyr Leu Asn Leu Glu Arg Ile Pro Trp Val Val 275 280 285 Arg Lys Leu Leu 290 6648PRTCrimean-Congo hemorrhagic fever virus 6Arg Lys Pro Leu Phe Leu Asp Ser Thr Ala Lys Gly Met Lys Asn Leu 1 5 10 15 Leu Asn Ser Thr Ser Leu Glu Thr Ser Leu Ser Ile Glu Ala Pro Trp 20 25 30 Gly Ala Ile Asn Val Gln Ser Thr Tyr Lys Pro Thr Val Ser Thr Ala 35 40 45 Asn Ile Ala Leu Ser Trp Ser Ser Val Glu His Arg Gly Asn Lys Ile 50 55 60 Leu Val Ser Gly Arg Ser Glu Ser Ile Met Lys Leu Glu Glu Arg Thr 65 70 75 80 Gly Ile Ser Trp Asp Leu Gly Val Glu Asp Ala Ser Glu Ser Lys Leu 85 90 95 Leu Thr Val Ser Val Met Asp Leu Ser Gln Met Tyr Ser Pro Val Phe 100 105 110 Glu Tyr Leu Ser Gly Asp Arg Gln Val Glu Glu Trp Pro Lys Ala Thr 115 120 125 Cys Thr Gly Asp Cys Pro Glu Arg Cys Gly Cys Thr Ser Ser Thr Cys 130 135 140 Leu His Lys Glu Trp Pro His Ser Arg Asn Trp Arg Cys Asn Pro Thr 145 150 155 160 Trp Cys Trp Gly Val Gly Thr Gly Cys Thr Cys Cys Gly Leu Asp Val 165 170 175 Lys Asp Leu Phe Thr Asp Tyr Met Phe Val Lys Trp Lys Val Glu Tyr 180 185 190 Ile Lys Thr Glu Ala Ile Val Cys Val Glu Leu Thr Ser Gln Glu Arg 195 200 205 Gln Cys Ser Leu Ile Glu Ala Gly Thr Arg Phe Asn Leu Gly Pro Val 210 215 220 Thr Ile Thr Leu Ser Glu Pro Arg Asn Ile Gln Gln Lys Leu Pro Pro 225 230 235 240 Glu Ile Ile Thr Leu His Pro Arg Ile Glu Glu Gly Phe Phe Asp Leu 245 250 255 Met His Val Gln Lys Val Leu Ser Ala Ser Thr Val Cys Lys Leu Gln 260 265 270 Ser Cys Thr His Gly Val Pro Gly Asp Leu Gln Val Tyr His Ile Gly 275 280 285 Asn Leu Leu Lys Gly Asp Lys Val Asn Gly His Leu Ile His Lys Ile 290 295 300 Glu Pro His Phe Asn Thr Ser Trp Met Ser Trp Asp Gly Cys Asp Leu 305 310 315 320 Asp Tyr Tyr Cys Asn Met Gly Asp Trp Pro Ser Cys Thr Tyr Thr Gly 325 330 335 Val Thr Gln His Asn His Ala Ser Phe Val Asn Leu Leu Asn Ile Glu 340 345 350 Thr Asp Tyr Thr Lys Asn Phe His Phe His Ser Lys Arg Val Thr Ala 355 360 365 His Gly Asp Thr Pro Gln Leu Asp Leu Lys Ala Arg Pro Thr Tyr Gly 370 375 380 Ala Gly Glu Ile Thr Val Leu Val Glu Val Ala Asp Met Glu Leu His 385 390 395 400 Thr Lys Lys Ile Glu Ile Ser Gly Leu Lys Phe Ala Ser Leu Ala Cys 405 410 415 Thr Gly Cys Tyr Ala Cys Ser Ser Ser Ile Ser Cys Lys Val Arg Ile 420 425 430 His Val Asp Glu Pro Asp Glu Leu Thr Val His Val Lys Ser Asp Asp 435 440 445 Pro Asp Val Val Ala Ala Ser Ser Ser Leu Met Ala Arg Lys Leu Glu 450 455 460 Phe Gly Thr Asp Ser Thr Phe Lys Ala Phe Ser Ala Met Pro Lys Thr 465 470 475 480 Ser Leu Cys Phe Tyr Ile Val Glu Arg Glu His Cys Lys Ser Cys Ser 485 490 495 Glu Glu Asp Thr Lys Lys Cys Val Asn Thr Lys Leu Glu Gln Pro Gln 500 505 510 Ser Ile Leu Ile Glu His Lys Gly Thr Ile Ile Gly Lys Gln Asn Ser 515 520 525 Thr Cys Thr Ala Lys Ala Ser Cys Trp Leu Glu Ser Val Lys Ser Phe 530 535 540 Phe Tyr Gly Leu Lys Asn Met Leu Ser Gly Ile Phe Gly Asn Val Phe 545 550 555 560 Met Gly Ile Phe Leu Phe Leu Ala Pro Phe Ile Leu Leu Ile Leu Phe 565 570 575 Phe Met Phe Gly Trp Arg Ile Leu Phe Cys Phe Lys Cys Cys Arg Arg 580 585 590 Thr Arg Gly Leu Phe Lys Tyr Arg His Leu Lys Asp Asp Glu Glu Thr 595 600 605 Gly Tyr Arg Arg Ile Ile Glu Lys Leu Asn Asn Lys Lys Gly Lys Asn 610 615 620 Lys Leu Leu Asp Gly Glu Arg Leu Ala Asp Gly Arg Ile Ala Glu Leu 625 630 635 640 Phe Ser Thr Lys Thr His Ile Gly 645 77620DNAArtificial SequenceMVA-GP nucleic acid sequence 7gttggtggtc gccatggatg gtgttattgt atactgtcta aacgcgttag taaaacatgg 60cgaggaaata aatcatataa aaaatgattt catgattaaa ccatgttgtg aaaaagtcaa 120gaacgttcac attggcggac aatctaaaaa caatacagtg attgcagatt tgccatatat 180ggataatgcg gtatccgatg tatgcaattc actgtataaa aagaatgtat caagaatatc 240cagatttgct aatttgataa agatagatga cgatgacaag actcctactg gtgtatataa 300ttattttaaa cctaaagatg ccattcctgt tattatatcc ataggaaagg atagagatgt 360ttgtgaacta ttaatctcat ctgataaagc gtgtgcgtgt atagagttaa attcatataa 420agtagccatt cttcccatgg atgtttcctt ttttaccaaa ggaaatgcat cattgattat 480tctcctgttt gatttctcta tcgatgcggc acctctctta agaagtgtaa ccgataataa 540tgttattata tctagacacc agcgtctaca tgacgagctt ccgagttcca attggttcaa 600gttttacata agtataaagt ccgactattg ttctatatta tatatggttg ttgatggatc 660tgtgatgcat gcaatagctg ataatagaac ttacgcaaat attagcaaaa atatattaga 720caatactaca attaacgatg agtgtagatg ctgttatttt gaaccacaga ttaggattct 780tgatagagat gagatgctca atggatcatc gtgtgatatg aacagacatt gtattatgat 840gaatttacct gatgtaggcg aatttggatc tagtatgttg gggaaatatg aacctgacat 900gattaagatt gctctttcgg tggctgggta ccaggcgcgc ctttcatttt gtttttttct 960atgctataaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1020gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1080gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1140tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1200cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1260accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1320gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1380ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1440cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1500cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1560gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1620cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat gcacgagctg 1680tacaagtaag cggccgctgg tacccaacct aaaaattgaa aataaataca aaggttcttg 1740agggttgtgt taaattgaaa gcgagaaata atcataaata agcccggtgc caccatggat 1800gcaatgaaga gagggctctg ctgtgtgctg ctgctgtgtg gagcagtctt cgtttcgccc 1860agccaggaaa tccatgcccg attcagaaga ggagccagat ctcccatcaa acaagtttgt 1920acaaaaaagc aggctcatat atcattaatg tatgcaatcc tttgcctaca gctgtgtggt 1980ctgggagaga ctcatggatc acacaatgaa actagacaca ataaaacaga caccatgaca 2040acacacggtg ataacccgag ctctgaaccg ccagtgagca cggccttgtc tattacactt 2100gacccctcca ctgtcacacc cacaacacca gccagtggat tagaaggctc aggggaagtc 2160tacacatccc ctccgatcac caccgggagc ttgcccctgt cggagacaac accagaactc 2220cctgttacaa ccggcacaga caccttaagc gcaggtgatg tcgatcccag cacgcagaca 2280gccggaggca cctccgcacc aacagtccgc acaagtctac ccaacagccc tagcacacca 2340tctacaccac aagacacaca ccatcctgtg agaaatctac tttcagtcac gagtcctggg 2400ccagatgaaa catcaacacc ctcgggaaca ggcaaagaga gctcagcaac cagtagccct 2460catccagtct ccaacagacc accaacccct cctgcaacag cccagggacc cactgaaaat 2520gacagtcaca acgccactga acaccctgag tccctgacac agtcagcaac cccaggccta 2580atgacctctc caacacagat agtccaccca caaagtgcca cccccataac cgttcaagac 2640acacatccca gtccaacgaa caggtctaaa agaaacctta agatggaaat aatcttgact 2700ttatctcagg gtttaaaaaa gtactatggg aaaatattaa ggcttctgca actcacctta 2760gaggaggaca ctgaaggtct actggaatgg tgtaagagaa atcttggtct tgattgtgat 2820gacactttct ttcaaaagag aattgaagaa ttctttataa ctggtgaggg ccattttaat 2880gaagttttac aatttagaac gccaggcacg ttgagcacca cagagtcaac acctgctggg 2940ctgccaacag ctgaaccttt taagtcctac ttcgccaaag gcttcctctc gatagattca 3000ggttactact cagccaaatg ttactcagga acatccaatt cagggcttca attgattaac 3060attacccgac attcaactag aatagttgac acacctgggc ctaagatcac taacctaaag 3120accatcaact gcataaactt gaaggcatcg atcttcaaag aacatagaga ggttgaaatc 3180aatgtgcttc tcccccaagt tgcagttaat ctctcaaact gtcacgttgt aatcaaatca 3240catgtctgtg actactcttt agacattgac ggtgcggtga ggcttcctca catttaccat 3300gaaggagttt tcatcccagg aacttacaaa atagtgatag ataaaaaaaa taagttgaat 3360gacagatgca ccttatttac cgactgtgtg ataaaaggaa gggaggttcg taaaggacag 3420tcagttttga ggcagtacaa gacggaaatc aggattggca aggcatcaac cggctttaga 3480agattgcttt cagaagaacc cagtgatgac tgtgtatcaa gaactcaact attaaggaca 3540gagactgcag agatccacgg cgacaactat ggtggcccgg gtgacaaaat aaccatctgc 3600aatggctcaa ctattgtaga ccaaagactg ggcagtgaac taggatgcta caccatcaat 3660agagtgaggt cattcaagct atgcgaaaac agtgccacag ggaagaattg tgaaatagac 3720agtgtcccag ttaaatgcag gcagggttat tgcctaagaa tcactcagga agggaggggc 3780cacgtaaaat tatctagggg ctcagaggtt gtcttagatg catgcgatac aagctgtgaa 3840ataatgatac ctaagggcac tggtgacatc ctagttgact gttcaggtgg gcagcaacat 3900tttctaaagg acaatttgat agatctagga tgccccaaaa ttccattatt gggcaaaatg 3960gctatttaca tttgcagaat gtcaaaccac cccaaaacaa ccatggcttt cctcttctgg 4020ttcagctttg gctatgtaat aacctgcata ctttgcaagg ctatttttta cttgttaata 4080attgttggaa cactagggag aaggctcaag cagtatagag agttgaaacc tcagacttgc 4140accatatgtg agacaactcc tgtaaatgca atagatgctg agatgcatga cctcaattgc 4200agttacaaca tttgtcccta ctgtgcatct agactaacct cagatgggct tgctaggcat 4260gtgatacaat gccctaagcg gaaggagaaa gtggaagaaa ctgaactgta cttgaactta 4320gaaagaattc cttgggttgt aagaaagctg ttgcaggtgt cagagtcaac tggtgtggca 4380ttgaaaagaa gcagttggct gattgtgctg cttgtgctat tcactgtttc attatcacca 4440gttcaatcag cacccattgg tcaagggaag

acaattgagg cataccgggc cagggaaggg 4500tacacaagta tatgcctctt tgtactagga agtatcctat ttatagtttc ttgcctaatg 4560aaagggctgg ttgacagtgt tggcaactcc ttcttccctg gactgtccat ttgcaaaacg 4620tgctccataa gcagcattaa tggctttgaa attgagtccc ataagtgcta ttgcagctta 4680ttctgttgcc cctattgtag gcactgctct accgataaag aaattcataa gctgcacttg 4740agcatctgca aaaaaaggaa aacaggaagt aatgtcatgt tggctgtctg caagctcatg 4800tgtttcaggg ccaccatgga agtaagtaac agagccctgt ttatccgtag catcatcaac 4860accacttttg ttttgtgcat actgatacta gcagtttgtg ttgttagcac ctcagcagtg 4920gagatggaaa acctaccagc agggacctgg gaaagagaag aagacctaac aaatttctgt 4980catcaggaat gccaggttac agagactgaa tgcctctgcc cttatgaagc tctagtactc 5040agaaagcctt tattcctaga tagtacagct aaaggcatga aaaatctgct aaattcaaca 5100agtttagaaa cgagtttatc aattgaggca ccatggggag caataaatgt tcagtcaacc 5160tacaaaccaa ctgtgtcaac tgcaaacata gcactcagtt ggagctcagt ggaacacaga 5220ggcaataaga tcttggtttc aggcagatca gaatcaatta tgaagctgga agaaaggaca 5280ggaatcagct gggatctcgg tgtagaagat gcctctgaat ctaaactgct tacagtatct 5340gtcatggact tgtctcagat gtactctcct gtcttcgagt acttatcagg ggacagacag 5400gtggaagagt ggcccaaagc aacttgcaca ggtgactgcc cagaaagatg tggctgcaca 5460tcatcaacct gtttgcacaa agaatggcct cactcaagaa attggagatg caatcccact 5520tggtgctggg gtgtagggac tggctgcacc tgttgtggat tagatgtgaa agaccttttt 5580acagattata tgtttgtcaa gtggaaagtt gaatacatca agacagaggc catagtgtgt 5640gtagaactta ctagtcagga aaggcagtgt agcttgattg aagcgggcac aaggttcaat 5700ttaggtcctg tgaccatcac actgtcagaa ccaagaaaca tccaacaaaa actccctcct 5760gaaataatca cactgcatcc taggatcgaa gaaggtttct ttgacctgat gcatgtgcaa 5820aaggtgttat cggcaagcac agtgtgtaag ttgcagagtt gcacacatgg tgtgccagga 5880gacctacagg tctaccacat cggaaattta ttaaaagggg ataaggtaaa tggacatcta 5940attcataaaa ttgagccaca cttcaacacc tcctggatgt cctgggatgg ttgtgaccta 6000gactactact gcaacatggg agattggcct tcttgcacat acacaggggt cacccaacac 6060aatcatgctt catttgtaaa cttactcaac attgaaactg attacacaaa gaacttccac 6120tttcactcta aaagggtcac tgcacacgga gatacaccac aactagatct taaggcaaga 6180ccaacctatg gtgcaggcga gatcactgtt ctggtagaag ttgctgacat ggagttacat 6240acaaagaaga ttgaaatatc aggcttaaaa tttgcaagct tagcttgcac aggttgttat 6300gcttgtagct ctagcatctc atgcaaagtt agaattcatg tggatgaacc agatgaactt 6360acagtacatg ttaaaagtga tgatccagat gtggttgcag ctagctcaag tctcatggca 6420aggaagcttg aatttggaac agacagtaca tttaaagctt tctcggccat gcctaaaact 6480tctctatgtt tctacattgt tgaaagagaa cactgtaaga gctgcagtga agaagacaca 6540aaaaaatgtg ttaacacaaa acttgagcaa ccacaaagca ttttgatcga acacaaggga 6600actataatcg gaaagcaaaa cagcacttgc acggctaagg caagttgctg gttagagtca 6660gtcaagagtt tcttttatgg cctaaagaac atgcttagtg gcatttttgg caatgtcttt 6720atgggcattt tcttgttcct tgcccccttc atcctgttaa tactattctt tatgtttggg 6780tggaggatcc tattctgctt taaatgttgt agaagaacca gaggcctgtt caagtataga 6840cacctcaaag acgatgaaga aactggttat agaaggatta ttgaaaaact aaacaataaa 6900aaaggaaaaa acaaactgct tgatggtgaa agacttgctg atggaagaat tgccgaactg 6960ttctctacaa aaacacacat tggcacccag ctttcttgta caaagtggtt cgatggggat 7020ctagagggcc cgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct 7080acgtaagtcg acctgcaggg aaagttttat aggtagttga tagaacaaaa tacataattt 7140tgtaaaaata aatcactttt tatactaata tgacacgatt accaatactt ttgttactaa 7200tatcattagt atacgctaca ccttttcctc agacatctaa aaaaataggt gatgatgcaa 7260ctttatcatg taatcgaaat aatacaaatg actacgttgt tatgagtgct tggtataagg 7320agcccaattc cattattctt ttagctgcta aaagcgacgt cttgtatttt gataattata 7380ccaaggataa aatatcttac gactctccat acgatgatct agttacaact atcacaatta 7440aatcattgac tgctagagat gccggtactt atgtatgtgc attctttatg acatcgccta 7500caaatgacac tgataaagta gattatgaag aatactccac agagttgatt gtaaatacag 7560atagtgaatc gactatagac ataatactat ctggatctac acattcacca gaaactagtt 7620