WO2018146257A1

WO2018146257A1 - Hepatitis e virus vaccine

Info

Publication number: WO2018146257A1
Application number: PCT/EP2018/053291
Authority: WO
Inventors: Reginald CLAYTON; Peter GRETZ; André HABEL
Original assignee: Clayton Reginald; Gretz Peter; Habel Andre
Priority date: 2017-02-10
Filing date: 2018-02-09
Publication date: 2018-08-16
Also published as: GB201702193D0

Abstract

The present invention relates to a new HEV-specific vaccines as well as to methods of producing such vaccines, immunogenic polypeptides and nucleic acids encoding the same, and uses thereof in the treatment of and/or prevention from HEV infections.

Description

Hepatitis E Virus vaccine Field of the invention

The present invention relates, amongst others, to a vaccine for use in the treatment and/or prophylaxis of Hepatitis E Virus (HEY) infections, methods of preparing such vaccines, vectors for use in such vaccines, modified nucleic acid sequences and their use in the manufacture of the vaccine.

Technological Background

Hepatitis E virus (HEV) may be the source of hepatitis and it is one of five known human hepatitis viruses: A, B, C, D, and E. HEV is a positive-sense single-stranded non-enveloped RNA icosahedral virus and has a fecal-oral transmission route. Infection with this virus was first documented in 1955 during an outbreak in New Delhi, India. A preventative vaccine (HEV 239) has been developed and used in China. The hepatitis E virus causes around 20 million infections a year. These infections result in around three million acute illnesses and as of 2010, 57,000 deaths annually. It is particularly dangerous for pregnant women, who can develop an acute form of the disease that is lethal in about 25 percent of cases or more. The virus (HEV) is a major cause of illness and of death in the developing world and disproportionate cause of deaths among pregnant women. Hepatitis E is endemic in Central Asia, while Central America and the Middle East have reported outbreaks (cf. wiMpedia.org/wiki/Hepatitis_E and cited documents).

HEV often causes an acute and self-limiting infection with low mortality rates in the western world. However, it bears a high risk of developing chronic hepatitis in immunocompromised patients with substantial mortality rates. Organ transplant recipients who receive

immunosuppressive medication to prevent rejection are at risk of developing chronic hepatitis E. Furthermore, in healthy individuals during the duration of the infection, the disease severely impairs a person's ability to work, care for family members, and other daily activities. Hepatitis E occasionally develops into an acute, severe liver disease, and is fatal in about 2% of all cases. Clinically, the disease is comparable with hepatitis A, but in pregnant women the disease is often of greater severity and is associated with a clinical syndrome called fulminant liver failure. Pregnant women, especially those in the third trimester, suffer an elevated mortality rate from the disease. Hepatitis E infection affected about 28 million people in 2013 (idem). There is one known serotype of HEV and classification is based on the nucleotide sequences of the genome. Genotype 1 has been classified into five subtypes, genotype 2 into two subtypes and genotypes 3 and 4 have been classified into ten and seven subtypes respectively. Differences have been noted between the different genotypes. For genotype 1, the age at which the incidence of hepatitis E peaks is between IS and 35 years and mortality is about 1%. Genotype 3 and 4 are more often found in patients older than 60 years and the mortality is between 5 and 10%. Genotypes 1 and 2 are typically found in humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions. Genotypes 3 and 4 infect humans, pigs and other animal species and have been responsible for sporadic cases of hepatitis E in both developing and industrialized countries. In the United Kingdom the Department for Environment, Food and Rural Affairs (DEFRA) said that the number of reported human hepatitis E cases increased by 39% between 2011 and 2012. Hepatitis E is widespread in Southeast Asia, northern and central Africa, India, and Central America. It is spread mainly by the fecal-oral route due to fecal contamination of water supplies or food; person-to-person transmission is uncommon. Domestic animals have been reported as a reservoir for the hepatitis E virus, with some surveys showing infection rates exceeding 95% among domestic pigs. Replicating virus has been demonstrated in the small intestine, lymph nodes, colon and liver of experimentally infected pigs. Transmission after consumption of wild boar meat and uncooked deer meat has been reported, demonstrating zoonotic infections of humans. The rate of transmission to humans by this route and the public health importance of this is, however, still unclear (Idem).

While sanitation is an important measure in prevention of hepatitis E; mere is also need to provide medical treatment and prophylactic measures, particularly through the use of a vaccine that can be used in domestic animals and in humans. The vaccine should ideally enable protection against a broad range of known genotypes of the virus, should be safe and more efficient than previous vaccines that are based on selected HEV polypeptide fragments administered in order to induce immunity. Further, the HEV vaccine ideally should be stable upon lyophilization or other formulations stable during storage and easily restorable before use. It is of major importance, in particular in tropical and/or warmer zones on Earth, to provide a vaccine that is stable, easy to handle and at the same time does not contain any excipients that may cause adverse effects, particularly in geographic regions where medical care is costly and/or difficult to access. Brief description of the figures

Figure 1 shows the results of immunizations of mice with the inventive vector and controls, respectively.

Embodiments of the invention

Throughout the following description, embodiments are disclosed that occasionally refer to lists or groups of numbers or other members. Occasionally, embodiments refer to one or even more other embodiments that may also comprise one or more groups or lists comprising individual members. It is explicitly contemplated that any combination of members of lists referred to below in different embodiments constitutes a further individual embodiment with the proviso that such combination makes technical sense to a person skilled in the art.

In one embodiment, the invention relates to a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 , or a nucleic acid sequence encoding a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1. In the context of the present invention at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1 means that the polypeptide may have one or more substitutions, deletions, or additions under the proviso that at least 96% or 97% or 98% or 99% of the amino acid residues encoding SEQ ID NO: 1 are identical. The polypeptide sequence according to SEQ ID NO: 1 is preferably a consensus sequence that covers respective aligned sequences of the known four Hepatitis E Virus genotypes, i.e. hereinafter also referred to as pan-genotype consensus sequence. More preferably, the pan-consensus sequence is a balanced pan-genotype consensus sequence, which means that the presence of individual amino acids at given positions in said sequence is based on the maximum likelihood of occurrence when compared with other possible amino acids present at said position. In other word, when more than one amino acid (e.g. D, E, or K) is found at a given position of aligned HEV amino acid sequences, the amino acid with the highest percentage of occurrence is selected to be present in the balanced pan-genotype consensus sequence. SEQ ID NO: 1 is a part of the full-length balanced pan-genotype consensus sequence of the polypeptide encoded by open reading frame 2 (ORF2) of HEV, which is shown in SEQ ID NO: 5.

In further aspects, the present invention relates to a nucleic acid sequence comprising a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 or a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1. Some examples of nucleic acid sequence comprising a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 or a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1 are shown in SEQ ID NOs: 3, 5 and/or 11, e.g. a "balanced consensus" polypeptide sequences ("be") having a length of 678 amino acids (as depicted in SEQ ID NO: 5) and comprising a polypeptide sequence as depicted in SEQ ID NO: 1 or a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1, or a polypeptide sequence having at least 96% or 97% or 98% or 99% identity to the sequence in SEQ ID NO: 5. In another embodiment, the invention relates to a nucleic acid sequence encoding a polypeptide according to SEQ ED NO: 5, or a nucleic acid sequence encoding a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 5.

In another embodiment, the invention relates to a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 3, or a nucleic acid sequence encoding a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 3.

In another embodiment, the invention relates to a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 11 , or a nucleic acid sequence encoding a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 11.

In one embodiment, a sequence according to SEQ ID NO: 3 and/or SEQ ID NO: 5 comprise 678 amino acids, wherein the sequence comprises a sequence according to SEQ ID NO: 1 or a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1 and wherein a sequence stretch depicted in SEQ ID NO: 3 and/or SEQ ID NO: 5 at positions 1 to 14 corresponds to the following amino acid residues: MNNMFLCIAHGDAT.

In one embodiment, the sequence according to SEQ ID NO: 3 comprises 678 amino acids, wherein the sequence comprises SEQ ID NO: 1 or a polypeptide mat is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1 and wherein a sequence stretch depicted at positions 1 to 14 in SEQ ID NO: 3 has the following residues: MNNMFLCIAHGDAT.

Further, such sequence may have at least one or more mutations at amino acid positions selected from the group of mutations at amino acid 17, 20, 21, 22, 26, 27, 28, 29, 38, 51, 55, 81, 83, 85, 86, 87, 89, 91, 93, 97, 108, 112, 113, 114, 120, 121, 122, 126, 127, 135, 138, 164, 165, 167, 171, 179, 181, 206, 282, 331, 332, 354, 344, 350, 353, 382, 627, 632, 636, 650, 662, 670, and/or 678 of SEQ ID NO: 3. Some of the possible amino acids at the respective positions are shown in the Table 1 below. However, it is also possible that other mutations occur at respective positions. It is noted that the above expression "at least one or more mutations at amino acid positions selected from the group of mutations..." means that every possible mutation and/or combination of mutations is encompassed as understood by a person skilled in the art. The person skilled in the art would not exclude any mutation or combination of mutations from this disclosure unless technical reasons would prevent him/her from choosing a given mutation or combination of mutations. A skilled person understands that any individual amino acid of the above list may be selected, or that any combination selected from the above list is encompassed by the scope of the present invention, preferably with the proviso mat the percentage identity mentioned above is adhered to.

In one embodiment, the sequence according to SEQ ID NO: 5 comprises 678 amino acids, wherein the sequence comprises SEQ ID NO: 1 or a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1 and wherein a sequence stretch depicted at positions 1 to 14 in SEQ ID NO: 3 has the following residues: MNNMFLCIAHGDAT.

Further, such sequence may have at least one or more mutations at amino acid positions selected from the group of mutations at amino acid 17, 20, 21, 22, 26, 27, 28, 29, 38, 51, 55, 81, 83, 85, 86, 87, 89, 91, 93, 97, 108, 112, 113, 114, 120, 121, 122, 126, 127, 135, 138, 164, 165, 167, 171, 179, 181, 206, 282, 331, 332, 354, 344, 350, 353, 382, 627, 632, 636, 650, 662, 670, and/or 678 of SEQ ID NO: 5. Some of the possible amino acids at the respective positions are shown in the Table 1 below. However, it is also possible that other mutations occur at respective positions. It is noted that the above expression "at least one or more mutations at amino acid positions selected from the group of mutations..." means that every possible mutation and/or combination of mutations is encompassed as understood by a person skilled in the art. The person skilled in the art would not exclude any mutation or combination of mutations from this disclosure unless technical reasons would prevent him/her from choosing a given mutation or combination of mutations. A skilled person understands that any individual amino acid of the above list may be selected, or that any combination selected from the above list is encompassed by the scope of the present invention, preferably with the proviso that the percentage identity mentioned above is adhered to. TABLE 1 - Possible amino acid substitutions in SEQ ID NO: 3 and comparison with respective amino acid residues found in HEV-1, -2, -3, and -4

In another embodiment, the invention relates to a nucleic acid sequence according to the preceding embodiments, for example a nucleic acid sequence as depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding percentages representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, provided it encodes a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1.

In further aspects, the present invention relates to a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1. An example for such sequence is shown in SEQ ID NOs: 4 and 12, respectively.

In another embodiment, the invention relates to a nucleic acid sequence according to the preceding embodiments, for example a nucleic acid sequence as depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ID NO: 4, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding percentages representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ID NO: 4, provided it encodes a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 3.

In another embodiment, the invention relates to a nucleic acid sequence according to the preceding embodiments, for example a nucleic acid sequence as depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding percentages representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, provided it encodes a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 5.

In another embodiment, the invention relates to a nucleic acid sequence according to the preceding embodiments, for example a nucleic acid sequence as depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding percentages representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, provided it encodes a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 11.

In another embodiment, the invention relates to a synthetic nucleic acid construct comprising the nucleic acid sequence according to any one of the preceding embodiments, e.g. the synthetic nucleic acid construct comprises a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1. As used herein, the term "synthetic nucleic acid" indicates that the nucleic acid has been obtained as the result of molecular biologic genetic engineering work and is not found in nature prior to this invention. In another embodiment, the invention relates to a synthetic nucleic acid construct comprising the nucleic acid sequence according to any one of the preceding embodiments, e.g. the synthetic nucleic acid construct comprises a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ID NO: 4, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ID NO: 4, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 3.

In another embodiment, the invention relates to a synthetic nucleic acid construct comprising the nucleic acid sequence according to any one of the preceding embodiments, e.g. the synthetic nucleic acid construct comprises a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 5.

In another embodiment, the invention relates to a synthetic nucleic acid construct comprising the nucleic acid sequence according to any one of the preceding embodiments, e.g. the synthetic nucleic acid construct comprises a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 11.

In another embodiment, the invention relates to the above mentioned synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said synthetic nucleic acid construct is a vector. Accordingly, the vector nucleic acid construct may comprise a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 2 or a degenerate sequence of SEQ ID NO: 2, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1.

The vector nucleic acid construct may also comprise a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ID NO: 4, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 4 or a degenerate sequence of SEQ ED NO: 4, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 3. The vector nucleic acid construct may also comprise a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 6 or a degenerate sequence of SEQ ID NO: 6, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 5. The vector nucleic acid construct may comprise a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, or a nucleic acid sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (each of the preceding list of numbers being representing an individual embodiment) identical to the nucleic acid sequence depicted in SEQ ID NO: 12 or a degenerate sequence of SEQ ID NO: 12, provided it encodes a polypeptide comprising an amino acid sequence that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 11.

In another embodiment, the invention relates to synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of claims/embodiments, wherein said synthetic nucleic acid construct is a vector comprising a backbone derived from viral vectors selected from the group adenovirus vectors, adeno-associated virus vectors, measles virus vectors, parvovirus vectors, lenti virus vectors, retroviral vectors, pox virus vectors (e.g. Modified Vaccinia virus Ankara, MVA), arbovirus vectors (e.g. Vesicular Stomatitis Virus Indiana, VSIV) Herpes Simplex vectors or chimeric virus vectors. In the context of the present invention, chimeric virus vectors comprise genetic information from at least two different viruses (including genetic information from at least two different serotypes). For example, a chimeric virus vector may comprise the vector backbone derived from a first virus and regulatory elements, for example promoters or enhancers, derived from at least one other virus.

In one embodiment, the invention relates to the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said synthetic nucleic acid construct is a vector comprising a backbone derived from viral vectors selected from the group adenovirus vectors selected further from the group of primate adenovirus-based vectors.

In one embodiment, the invention relates to a synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vectors are adenovirus-based vectors, e.g. primate adenovirus-based vectors that are selected from the group comprising adenovirus 5 vectors, chimpanzee adenovirus 68 vector, [any other preferred ones? Ad35, Ad 2, Ad7, Ad 19a (= has been renamed Ad64).

In one embodiment, the invention relates to a synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said synthetic nucleic acid construct is a vector, further comprising at least one regulatory element.

In one embodiment, the invention relates to a vector polypeptide encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising a nucleic acid sequence according to any one of the preceding embodiments. In one embodiment, the invention relates to a vector encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments for use as medicament

In one embodiment, the invention relates to a vector encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments for use in the treatment and/or prevention/prophylaxis of infection with Hepatitis E Virus.

In one embodiment, the invention relates to a vaccine comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments.

In one embodiment, the invention relates to a vaccine comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments for use in the treatment and/or prevention of infection with Hepatitis E Virus. In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized or otherwise formulated. The vaccine may, however, also be provided as non-lyophilized, ready-for-use drug for injection.

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector encoded by a synthetic nucleic acid construct comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is adjuvant-free.

In another embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector encoded by a synthetic nucleic acid construct comprising the nucleic acid sequence according to the respective preceding embodiments, wherein said vaccine contains an adjuvant, which further boosts the immune system, but which per se does not raise a immune response that is specific exclusively for HEV.

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system. A solvent system is any liquid or combination of liquids that are suitable for dissolving the lyophilized vaccine, e.g. water for injection, saline, suitable buffers, etc. The respective solvent systems should not cause irritation at the site of injection, e.g. be free of preservatives. Further, the viscosity of the solvent system should be sufficiently high so mat the site of injection is not irritated or painful after injection. The solvent system should be suitable to dissolve the vaccine in such a way that the injection volume is small enough not to cause any discomfort for the vaccinated individual. The injection volume should therefore not exceed 500 μΐ, preferably it should not exceed 400 μΐ, and more preferably contain about SO μΐ to about 300 μΐ, e.g., about 100 μΐ, about ISO μΐ, about 200, or about 2S0 μΐ per injection, more preferably, the amount per injection should be about 100 to ISO μΐ. The injections may be repeated as required. In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system that is suitable for injection.

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments for use in the treatment and/or prevention of infection with Hepatitis E Virus in a mammal, preferably a human. In various embodiments the vaccine is for use in the treatment and/or prevention of an infection with HEV of immunocompromised patients, organ transplant recipients or individuals who are candidates for an organ transplantation, individuals receiving or destined to receive immunosuppressive medication, healthy individuals including pregnant women or women planning to get pregnant, for example, women preparing for, or women who are subject to in vitro fertilization programs, patients being treated for oncologic indications that may be under immune suppression or impaired immunocompetence, staff working at slaughterhouses or meat processing plants where an abundance of untreated animal material is present, patients with hepatic steatosis or non- alcoholic or alcoholic etiology, patients with fatty liver disease, patients with hepatic disorders.

In one embodiment, the invention relates to a method of treatment and/or prophylaxis of an infection of an individual with HEV, said method comprising administering a vaccine according to any one of the preceding embodiments to a mammal, preferably a human. In various embodiments this method of treatment and/or prophylaxis of an infection of an individual with HEV the vaccine is for administration to immunocompromised patients, organ transplant recipients or individuals who are candidates for an organ transplantation, individuals receiving or destined to receive immunosuppressive medication, healthy individuals including pregnant women or women planning to get pregnant, for example, women preparing for, or women who are subject to in vitro fertilization programs, patients being treated for oncologic indications that may be under immune suppression or impaired immunocompetence, staff working at slaughterhouses or meat processing plants where an abundance of untreated animal material is present, patients with hepatic steatosis or non- alcoholic or alcoholic etiology, patients with fatty liver disease, patients with hepatic disorders

In one embodiment, the invention relates to a use of the nucleic acid constructs according to any one of the preceding embodiments, the polypeptides according to any one of the preceding embodiments, the vectors according to any one of the preceding embodiments in methods of manufacturing a vaccine according to any one of the preceding embodiments for the treatment and/or prevention of infection with Hepatitis E Virus in a mammal, preferably in a human, e.g. immunocompromised patients, organ transplant recipients or individuals who are candidates for an organ transplantation, individuals receiving immunosuppressive medication, healthy individuals including pregnant women or women planning to get pregnant, for example, women preparing for, or women who are subject to in vitro

fertilizations, patients being treated for oncologic indications mat may be under immune suppression or impaired immunocompetence, staff working at slaughterhouses or meat processing plants where an abundance of untreated animal material is present, patients with hepatic steatosis or non-alcoholic or alcoholic etiology, patients with fatty liver disease, patients with hepatic disorders.

In anther embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising a vector polypeptide according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for

reconstitution with a solvent system, and wherein the vaccine is administered at a dose of about 10⁶ to about 10¹¹, e.g., at a dose of about 10⁷, 10⁸, 10⁹, or 10¹⁰ per inoculation (e.g. by injection.)

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system, and wherein the vaccine is administered at a dose of about 10⁶ to about 10^u, e.g., at a dose of about 10⁷, 10⁸, 10⁹, or 10¹⁰ per inoculation, and wherein at least one, at least two, at least three dosages are administered. Preferably, an individual vaccination regime does not comprise more than three administrations of the vaccine; preferably it comprises one first inoculation and one booster inoculation. An inoculation cycle or vaccination cycle means that not more than 6 months are required for the individual administrations of the vaccine, preferably, the vaccination regime comprises two vaccine administration separated by one, two, or three months. Occasionally, up to 6 months may expire between the first and the second, booster inoculation - depending, for example, on the health, age, and overall situation of the individual receiving the vaccine. Ideally, the interval between the initial and the booster injection(s) does not exceed about 4 weeks, about 6 weeks, about 8 weeks, or about 12 weeks. The inoculation may be orally or parenterally, e.g., per intramuscular injection.

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system, and wherein the vaccine is administered at a dose of about 10⁶ to about 10¹¹, e.g., at a dose of about 10⁷, 10⁸, 10⁹, or 10¹⁰ per inoculation (e.g. injection), and wherein one to two dosages are administered.

In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system, and wherein the vaccine is administered at a dose of about 10⁶ to about 10¹¹, e.g., at a dose of about 10⁷, 10⁸, 10⁹, or 10¹⁰ per inoculation (e.g. injection), and wherein one dosage is administered. In one embodiment, the invention relates to a vaccine according to any one of the preceding embodiments comprising the vector according to any one of the preceding embodiments encoded by the synthetic nucleic acid construct according to any one of the preceding embodiments comprising the nucleic acid sequence according to any one of the preceding embodiments, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system, and wherein the vaccine is administered at a dose of about 10⁶ to about 10¹¹, e.g., at a dose of about 10⁷, 10⁸, 10⁹, or 10¹⁰ per inoculation (e.g. injection), and wherein at least one to three dosages are administered, preferably two dosages are administered. In one embodiment, the invention relates to a cell system comprising the nucleic acid sequence according to any one of the preceding embodiments.

In one embodiment, the invention relates to a cell system comprising and translating the nucleic acid sequence according to any one of the preced ng embodiments into polypeptides.

In one embodiment, the invention relates to a method of determining / measuring / diagnosing / quantifying an infection with HEV, said method comprising:

- Using the cell system according to any one of the preceding embodiments

- Incubating the cell system with a sample obtained from a subject in the presence of an agent specifically indicating/signaling the presence of antibodies in the sample,

- Optionally comparing the level of signal obtained in step with a control.

It is also contemplated to use a threshold value based on methods known in the art to determine whether or not a sample comprises antibodies against HEV. The threshold value may be a threshold range that is chosen by a person skilled in the art taking into account mat signals obtained with methods of detennining / measuring / diagnosing / quantifying HEV must be both, specific and sensitive.

As used herein, the terms "sensitivity" and "specificity" are statistical measures of the performance of a binary classification test, also known in statistics as classification function: Sensitivity (also called the true positive rate, the recall, or probability of detection in some fields) measures the proportion of positives that are correctly identified as such (e.g., the percentage of individuals with HEV infection people who are correctly identified as carrying the virus). Specificity (also called the true negative rate) measures the proportion of negatives that are correctly identified as such (e.g., the percentage of healthy individuals who are correctly identified as not having an HEV). Thus sensitivity quantifies the avoidance of false negatives, as specificity does it for false positives. For any test, there is usually a trade-off between the measures. This trade-off can be represented graphically as a receiver operating characteristic curve. A perfect predictor would be described as 100% sensitive (e.g., all infected are identified as infected) and 100% specific (e.g., no HEV-free individuals are identified as infected); however, theoretically any predictor will possess a minimum error bound known as the Bayes error rate. The sensitivity and specificity of a diagnostic test or method of detennining whether or not HEV is present in a sample depends on more than just the analytical "equality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency, e.g., in "non-infected" and "infected" populations. Depending on the particular diagnostic question to be addressed, the reference group must not be necessarily "non-infected", but it might be a group of individuals from which the diseased group of interest shall be differentiated (e.g. individuals infected with another hepatitis virus). For any particular target in a sample (e.g. a protein or nucleic acid), a distribution of target levels for subjects with and without an infection will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, above which (or below which, depending on how a target changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results do not give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "infected" samples might be ranked according to degree (e.g. l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "non-infected" population, and a ROC curve created. These methods are well known in the art (See, e.g., Hanley et al., 1982. Radiology 143: 29-36). Preferably, a threshold is selected to provide a ROC curve area of greater than about 0.S, more preferably greater than about 0.7. The term "about" in this context refers to +/- 5% of a given measurement.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a given condition (e.g. infection with HEV). Thus, the area under the ROC curve can be used to determine the effectiveness of the test. Therefore, the person skilled in the art knows that a threshold range must be determined taking into account the individual assay ingredients, some of which may be more specific but less sensitive or vice versa. In another embodiment, the invention relates to the use of the polypeptide or a vector polypeptide or a derivative thereof according to any one of the preceding embodiments or of nucleic acids encoding such polypeptides in the detection of a HEV-specific immune response. In another embodiment, the invention relates to a kit of parts comprising at least one container comprising the vaccine according to any one of the preceding embodiments, further comprising a container with a solvent system, optionally further comprising instructions for use, and further optionally comprising a device for administration of the vaccine to a subject. In another embodiment, the present invention relates to a vaccine comprising an adenoviral vector, for example, chimpanzee adenovirus 68 encoding and expressing a polypeptide according to SEQ ID NO: 1, or a polypeptide that is at least 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 1, wherein said vaccine can be used in the treatment and/or prevention of an infection with Hepatitis E Virus in a human being.

Detailed description of the invention

The present invention will now be described in further detail. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Except as otherwise indicated, standard methods may be used for the construction of the recombinant adenovirus genomes, helper adenoviruses, measles virus vectors, parvovirus vectors, lenti virus vectors, retroviral vectors, pox virus vectors, arbovirus vectors (e.g. VSIV) Herpes Simplex vectors or chimeric virus vectors as well as packaging cells according to the present invention. Such techniques are known to those skilled in the art; see, e.g.,

SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York ).

The term "adenovirus" as used herein is intended to encompass all adenoviruses, including the Mastadenovirus and Avi adenovirus genera. To date, at least fifty-one human serotypes of adenoviruses have been identified (see, e.g., FIELDS VIROLOGY, chapter 63, p.23S6; 5th ed., Lippincott-Raven Publishers; Editors: Knipe, David M.; Howley, Peter M.). Preferably, the adenovirus is a sero group C adenovirus, still more preferably the adenovirus is serotype 2 (Ad2) or serotype S (AdS) or the adenovirus is a chimpanzee adenovirus, preferably serotype 68 (cAd68).

The various regions of the adenovirus genome have been mapped and are understood by those skilled in the art (see, e.g., FIELDS et al., VIROLOGY, volume 2, chapters 67 and 68 (3d ed., Lippincott-Raven Publishers). The genomic sequences of the various Ad serotypes, as well as the nucleotide sequence of the particular coding regions of the Ad genome, are known in the art and may be accessed, e.g., from GenBank and NCBI. Those skilled in the art will appreciate that the inventive adenovirus vectors may be modified or "targeted" as described in Douglas et al., (1996) Nature Biotechnology 14:1574; U.S. Patent No. 5,922,315 to Roy et al.; U.S. Patent No. 5,770,442 to Wickham et al.; and or U.S. Patent No. 5,712,136 to Wickham et al.. As used herein, the term "vector" may refer to a viral, e.g., an Ad particle mat functions as a gene delivery vehicle, and which comprises vDNA (i.e., the vector genome) packaged within an Ad capsid. Alternatively, the term "vector" may be used to refer to the vector

genome/vDNA when used as a gene delivery vehicle in the absence of the virion capsid. In the present context, the vector encodes a nucleic acid encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, which is a consensus sequence of HEV genotypes and comprises immunogenic epitopes. Accordingly, the vector encodes and expresses the immunogenic epitopes and is therefore suitable as active component of an anti- HEV vaccine. Said vaccine may be used in the treatment and/or prevention of an infection with HEV, particularly with any of the known genotypes of HEV, i.e. HEV-1, HEV-2, HEV- 3, and HEV-4.

An "Ad vector genome" refers to the viral genomic DNA, in either its naturally occurring or modified form. A "rAd vector genome" is a recombinant Ad genome (i.e., vDNA) that comprises one or more heterologous nucleotide sequence(s), e.g., the nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above. The Ad vector genome or rAd vector genome may comprise the Ad terminal repeat sequences and packaging signal. An "Ad particle" or "rAd particle" comprises an Ad vector genome or rAd vector genome, respectively, packaged within an Ad capsid. Generally, the Ad vector genome is most stable at sizes of about 28 kb to 38 kb (approximately 75% to 105% of the native genome size). In the case of an adenovirus vector containing large deletions and a relatively small transgene, "stuffer DNA" can be used to maintain the total size of the vector within the desired range by methods known in the art. A "heterologous nucleotide sequence" or "heterologous nucleic acid sequence" (also referred to as "transgene") will typically be a sequence mat is not naturally-occurring in the virus. Alternatively, a heterologous nucleotide or nucleic acid sequence may refer to a viral sequence that is placed into a non-naturally occurring environment (e.g., by association with a promoter with which it is not naturally associated in the virus). One preferred example of a heterologous nucleic acid sequence of the present invention is one encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above.

As used herein, the term "polypeptide" encompasses both peptides and proteins, unless indicated otherwise.

By "infectious", as used herein, it is meant that the virus, e.g., an adenovirus can enter the cell by natural transduction mechanisms and express the transgene therein, e.g. a transgene encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above.

Alternatively, an "infectious" virus, e.g., an adenovirus is one that can enter the cell by other mechanisms and express the transgene therein and/or produce progeny viruses. As one illustrative example, the vector can enter a target cell by expressing a ligand or binding protein for a cell-surface receptor in the adenovirus capsid or by using an antibody or antibodies directed against molecules on the cell-surface followed by internalization of the complex.

The term "replication", "viral replication" or "Ad replication" as used herein, refers specifically to replication of the Ad genome (i.e., making new copies of the virion DNA) including the heterologous nucleotide sequence.

The term "propagation" as used herein refers to a productive viral infection wherein the viral genome is replicated and packaged to produce new virions, which typically can "spread" by infection of cells beyond the initially infected cell. A "propagation-defective" virus is impaired in its ability to produce a productive viral infection and spread beyond the initially infected cell.

Adeno- associated viruses (AAV) have also been employed as gene delivery vectors. Their use is also contemplated in context of the present invention. AAV is a small, single-stranded DNA virus, in the dependovirus family of the Parvoviridae, and has a simple genomic organization. Two open reading frames encode a series of Rep and Cap polypeptides. Rep polypeptides (RepSO, Rep52, Rep68 and Rep78) are involved in replication, rescue and integration of the AAV genome, although significant activity may be observed in the absence of all four Rep polypeptides. The Cap proteins (VP1, VP2, and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends of the genome are 145 basepairs long inverted terminal repeats (ITRs), the first 125 basepairs of which are capable of forming Y- or T-shaped duplex structures. It has been shown that the ITRs represent the minimal cis sequences required for replication, rescue, packaging and integration of the AAV genome. Typically, in recombinant AAV vectors, the entire rep and cap coding regions are excised and replaced with a transgene of interest, e.g., preferably a nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 , or SEQ ID NOs: 3, 5, and/or 11 , or derivatives as defined above.

An immunogenic polypeptide, or immunogen, may be any polypeptide suitable for protecting the subject against a disease, i.e., hepatitis E, and is preferably a polypeptide according to SEQ ID NO: 1 or derivatives as defined above. It is possible to include further immunogenic regions into the vectors and/or vaccines as defined herein, e.g. polypeptide sequences according to SEQ ID NO. 3, 5 and/or 11. As mentioned above, in particular preferred embodiments of the invention, the heterologous nucleotide sequence encodes a polypeptide that is associated with hepatitis E. By "associated with hepatitis E", it is intended that the expressed polypeptide that is a causative agent in hepatitis E, preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above.

Those skilled in the art will appreciate that the above heterologous nucleotide sequence(s) are preferably operably associated with the appropriate expression control sequences. For example, the recombinant adenovirus vectors of the invention preferably contain appropriate transcription/translation control signals and polyadenylation signals operably associated with the heterologous nucleic acid sequences) to be delivered to the target cell. Those skilled in the art will appreciate that a variety of promoter/enhancer elements may be used depending on the expression level desired. The promoter can be constitutive or inducible (e.g., the metallothionine promoter or a hormone inducible promoter), depending on the pattern of expression desired. The promoter may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell(s) of interest. The use of a promoter should enable expression in a variety of cell types mat will be transduced, e.g. the CAR (coxsackie Adenovirus receptor) or other viral promoters that can be expressed in many cell types, and accordingly therefore an adenovirus vector will transduce a variety of cells, and then raise a response.

The heterologous nucleotide sequence(s), preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, may be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, HCMV immediate-early 1-2 promoter, an albumin promoter, an Elongation Factor 1-a (EFl-a) promoter, a ΡγΚ promoter, a MFG promoter, or a Rous sarcoma virus promoter. It has been speculated that driving heterologous nucleotide transcription with the CMV promoter results in down-regulation of expression in immunocompetent animals (see, e.g., Guo et al., (1996) Gene Therapy 3:802). Accordingly, it is also preferred to operably associate the heterologous nucleotide sequence(s), preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, with a modified CMV promoter mat does not result in this down-regulation of transgene expression.

In embodiments wherein there is more than one heterologous nucleotide sequence, preferably more than one nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, those skilled in the art will appreciate that the heterologous nucleotide sequences may be operatively associated with a single upstream promoter and one or more downstream internal ribosome entry site (IRES) sequences (e.g., the picornavirus EMC IRES sequence).

In embodiments of the invention in which the heterologous nucleotide sequence(s), preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, will be transcribed and then translated in the target cells, specific initiation signals are generally required for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. The methods of the present invention provide a means, e.g. a synthetic nucleic acid construct such as a vector according to the above disclosure, for delivering heterologous nucleotide sequences into host cells, including both dividing and non-dividing cells in vitro or in vivo. The vectors, methods, vaccines and formulations of the present invention are additionally useful in a method of administering a polypeptide to a subject in need thereof, as a method of treatment or otherwise. In this manner, the polypeptide may thus be produced in vivo in the subject. The subject may be in need of the polypeptide because the subject may impart some therapeutic effect, e.g. raise an immune response, as a method of treatment or otherwise, and as explained further below. The vector may be administered that encodes any therapeutic polypeptide as defined above.

As a further aspect, the present invention provides a method of producing an immune response in a subject, comprising administering an Ad vector carrying a nucleotide sequence encoding an immunogen to a subject, and an active immune response is mounted by the subject against the immunogen. Immunogens are as described hereinabove. Preferably, a protective immune response is elicited.

An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleulrin-2) from an actively immunized host to a non-immune host." A "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence or severity of disease. Further, the disease may still be manifest although sub- clinically. The vaccine may stop the progression of disease and resolve the disease before penetrance of symptoms. Alternatively, a protective immune response or protective immunity may be useful in the treatment of disease, in particular infections with HEV. The protective effects may be complete or partial, as long as the benefits of the treatment outweigh any disadvantages thereof.

The vector, e.g. the Ad vector expressing the immunogen, preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above may be administered directly to the subject, as described below.

Alternatively, the Ad vector may be administered to a cell ex vivo and the altered cell is administered to the subject. The heterologous nucleotide sequence is permitted to be introduced into the cell, and the cell is administered to the subject, where the heterologous nucleotide sequence encoding the immunogen is preferably expressed and induces an immune response in the subject against the immunogen. Preferably, the cell is an antigen presenting cell (e.g., a dendritic cell). According to the foregoing methods of inducing an immune response in a subject, it is preferred that the vector carrying the heterologous nucleotide sequence is administered in an immunogenically effective amount, as described below. As described in more detail below, the present invention also encompasses methods of treating HEV infection using immunotherapy by administration of vectors, e.g., Ad vectors, expressing HEV-specific antigens. In one particular embodiment, an immune response may be produced against a HEV- antigen in a subject by administering a vector, e.g., an Ad vector comprising a heterologous nucleotide sequence encoding the preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above, e.g. protect an individual from infection with HEV. The vector may be administered to a subject in vivo or by using cells expressing the vector and the immunogens, as described herein.

The present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. The term "mammal" as used herein includes, but is not limited to, humans, pigs, deer, dogs, etc. Human subjects are the most preferred. Human subjects include neonates, infants, juveniles, and adults.

In particular embodiments, the present invention provides a pharmaceutical composition comprising a virus vector of the invention in a pharmaceutically-acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and the like. For injection, the carrier will typically be a liquid. For other methods of

administration, the carrier may be either solid or liquid, such as sterile, pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. As an injection medium, it is preferred to use water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers. In general, a "physiologically acceptable carrier" is one that is not toxic or unduly detrimental to cells. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Physiologically-acceptable carriers include pharmaceutically-acceptable carriers. By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the viral vector without causing any undesirable biological effects. Thus, such a pharmaceutical composition can be used, for example, in transfection of a cell ex vivo or in administering a virus vector directly to a subject.

One aspect of the present invention is a method of transferring a nucleotide sequence to a cell or organism, preferably a nucleotide sequence encoding a polypeptide according to SEQ ID NO: 1 or derivatives as defined above. For example, the virus may be added to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of virus to administer can vary, depending upon the target cell type and the particular virus vector, and can be determined by those of skill in the art without undue experimentation. Typically, at least about 10³particles, at least about 10^s particles, at least about 10⁷ particles, at least about 10⁹ particles, or at least about 10¹¹ particles are administered to the cell or organism.

Alternatively, administration of a vector of the present invention can be accomplished by any other means known in the art. For example, vectors can be targeted to cells, including cells that are not normally competent for transduction by viruses, e.g. adenoviruses, using antibodies, e.g., as described in U.S. Patent No. 5,861,156 to George et al. ;U.S. Patent No., 5,521,291 to Curiel et al.. Alternatively, viruses, e.g., adenoviruses can be targeted to cell- surface proteins ( e.g., receptors) by expressing a binding protein or ligand on the surface of the adenovirus, e.g., as described by Douglas et al., (1996) Nature Biotechnology 14:1574; U.S. Patent No. 5,770,442 to Wickham et al. ;and U.S. Patent No. 5,712,136 to Wickham et al. Further, poly-cation conjugated adenovirus particles (e.g., polylysine conjugated particles) may be employed as described by Wu et al., (1989) J. Biol. Chem. 264:16985, Fisher et al. (1994) Biochem. J. 299:49; and U.S. Patent No.4,871,982.

Also embraced by the invention but given as illustrative example is a method of treating subjects in vivo with the inventive virus vectors or vaccines. Administration of the virus vectors, e.g., adenovirus vectors of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering virus vectors. The subject may be a mammalian subject, more particularly a human subject. Dosages will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and can be determined in a routine manner. Typically, with respect to viral particles, at least about 10³, at least about 10^s, at least about 10⁷, at least about 10⁹, or at least about 10¹¹ particles are administered to the subject per treatment Exemplary doses are virus titers of about 10⁷-10¹⁴ particles, about 10⁷- 10¹³ particles, or about 10⁸-10¹² particles.

A "therapeutically-effective" amount and a "prophylactically-effective" amount as used herein is an amount that provides sufficient expression of the heterologous nucleotide sequence delivered by the vector to provide some improvement, protection or other benefit to the subject. Alternatively stated, a "therapeutically-effective" amount or a "prophylactically- effective" amount is an amount that will provide some prevention, protection, alleviation, mitigation, or decrease in at least one clinical symptom associated with HEV infection in the subject. Those skilled in the art will appreciate that the therapeutic effects or prophylactic effects need not be complete or curative, as long as some benefit is provided to the subject.

More than one administration (e.g., two, or three, but occasionally also four, or rarely even more administrations) may be employed to achieve therapeutic or protective levels of gene expression.

Exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, inhalation, and, preferably, parenteral, e.g., intravenous, subcutaneous, intradermal, intramuscular (i.e., administration to cardiac, skeletal, diaphragm and/or smooth muscle). Also contemplated are intraarticular administration, and the like, as well as direct tissue (e.g., muscle) or organ injection (e.g., into the liver). Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the inventive virus vector in a local rather than systemic manner, for example, in a depot or sustained- release formulation.

Immunogenic compositions or vaccines of the present invention comprise an immunogenic amount of infectious virus vectors as disclosed herein, preferably in lyophilized form, for later reconstitution with a pharmaceutically-acceptable carrier. An "immunogenic amount" is an amount of the infectious virus vectors that is sufficient to evoke an immune response in the subject to which the immunogenic composition is administered. Typical doses of Adenovirus particles include an amount of from about lOMO¹⁴ particles, about 10⁷- 10¹³ particles, about 10⁸-10¹² particles, or about 10⁴-10⁸ particles, depending upon the age and species of the subject being treated, and the immunogen against which the immune response is desired. Other appropriate doses of the inventive virus vectors for producing a desired immune response may be routinely determined by those skilled in the art.

Further, it is also contemplated to administer a vector nucleotide sequence comprising a sequence encoding for a polypeptide according to SEQ ID NO: 1, or 3 or 11, and derivatives thereof according to the present invention, which is delivered to the liver of the subject.

Acmiinistration to the liver can be achieved by any method known in the art, including, but not limited to intravenous administration, intraportal administration, intrabiliary

administration, intra-arterial administration, and direct injection into the liver parenchyma. The vector nucleotide sequence enters target cells and may be translated into viral polypeptides comprising the heterologous polypeptide sequence described above.

Intramuscular delivery to skeletal muscle is also possible.

Experiments Development of a candidate antigen for eliciting protective immunity against HEV variants A genotye 1 ORF2 sequence (sp|Q68985|CAPSD_HEVHY) was searched against all non- redundant sequences associated with Hepatitis E virus (taxon id 12461) using the NCBI web blastp. This search returned 2680 HEV sequences of various lengths. These were downloaded as GenBank entries and further locally parsed and processed. File Databasis_07072016.xls lists relevant data extracted from these GenBank entries. Genotype assignment is only provided for a fraction of sequences. Due to the large number and in many cases short length an automated procedure has been included. All sequences are compared to template

(archetype) isolates using standard protein alignment metrics and assigned to the most similar archetype: Table 2

Where genotype assignments are provided this procedure works well, at least for larger sequences. For 12 of 2680 sequences no genotype could be assigned. The genotypes and numbers of sequences in Table 2 have been assigned when relying on predictions only for sequences with at least 80 amino acids length.

Table 3

Based on the available information, it seems that genotype 2 is rather rare so that a sequencing bias might exist, leading to either very few or very short genotype 2 sequences. This has to be taken into account when creating genotype-balanced consensus sequences. Consensus sequences were generated using a customarily developed tool to allow for nonstandard modifications where specified (simplified work with genotypes, optionally removal of rare insertions). Where applicable, generated protein consensus sequences were reverse translated using a reverse translation tool, the standard genetic code (which is applicable to the human genome) and human codon usage table. Consensus sequences - Global sequence consensus

Based on the alignment of all 2680 sequences the following consensus sequence as shown in SEQ ID NO: 11 has been generated. This sequence reflects the sequencing biases, meaning genotypes sequenced more frequently will tend to be over-represented. Naturally, identical variants sequenced multiple times will only be represented once, which may theoretically be a source of bias. On the other hand rare insertions will extend the consensus, even of only one sequence of the entire set contains the insertion. The Reverse translated HEV ORF2 - global consensus sequence is shown in SEQ ID NO: 12. Genotype specific consensus sequences

Furthermore, genotype-specific consensus sequences were generated, which are shown in SEQ ID NOs 7-10.

Pan-genotype consensus sequence

One straightforward way to create a consensus sequence representing all genotypes to an equal extent is to derive a consensus sequence from all four genotype specific consensus sequences by alignment. Pan-genotype consensus sequences are generated with the proviso that no genotype should dominate, and all should contribute equally to generate a

representation which is optimally centered between observed isolates, independent of interest (sequencing) biases. The pan-genotype sequence can be expected to be better centered within the sequence space of the four genotypes, at least given that differences are large enough to see relevant differences. Such a sequence contains potential rare insertions, which may not be present in the majority of all strains. However, these insertions create slightly chimeric sequences which could more efficiently cover different sequences without bias. The Pan- genotype consensus sequence is shown in SEQ ID NO: 5 and the reverse translated nucleotide sequence is shown in SEQ ID NO: 6.

Improved genotype frequency balanced consensus

The consensus of multiple consensus sequences helps when looking for a sequence in the center of evolutionary (sequence) space, in the case of four genotypes. However, it is possible that certain positions are not optimally centered. A sampling strategy can attempt to create a more optimally centered sequence. This may be explained using a particular amino acid position, when sequences of the four genotypes are considered. Aligned sequences at amino acid position 501 shows the following distribution (amino acid counts): Genotype 1: 170 D; 180 E; 30 K; consensus: E

Genotype 2: 1 D; consensus: D

Genotype 3: 476 D; 400 E; 20 R; 5 gaps; consensus: D

Genotype 4: 199 D; 220 E; 50 Q; consensus: E

In the pan-genotype alignment a choice can be made between E and D, which are equally strongly represented in the consensus sequences for individual genotypes. The consensus of consensus sequences could randomly choose between D and E. This may not perfectly represent the actual data situation. As an alternative, continuing the above hypothetical example, the total counts may instead be represented by relative percentages:

Genotype 1: 52.8% D; 44.4% E; 0.02% R; 0.01% gaps; consensus: D

Genotype 2: 100% D; consensus: D

Genotype 3 : 44.7% D; 47.3% E; 0.08% K; consensus: E

Genotype 4: 42.4% D; 46.9% E; 0.11% Q; consensus: E

Summing up relative percentages for individual amino acids leads to the following scores: score for D: 52.8 + 100 + 44.7 + 42.4 = 239.9 [139.9 without genotype 2]

score for E: 44.4 + 0 + 47.3 + 46.9 = 138.6

The overall score for D is higher than for E, suggesting it is better suited to represent this position in the consensus than E, while working to avoid genotype specific biases. It is also still a bit higher when omitting genotype 2, which, due to very low sequencing coverage, tends to provide extreme relative percentages (very few variants observed). Individual positions in the alignment show different degrees of variability, but are also supported by various degree of sequencing coverage. Based on such models, a pan-genotype consensus has been derived omitting genotype 2. The latter genotype was not sufficiently often sequenced so that no conclusive information on sequence variability within the genotype exists on ORF2 protein level. An additional optimization in comparison to previous consensus sequences may be the removal of rare insertions, which are primarily observed in few genotype 3 sequences.

The following sequence has been derived using the described strategy and is taken as pan- genotype consensus, albeit excluding genotype 2 for the described reasons. In addition, insertions featured by fewer than SO sequences have been automatically excluded to exclude the risk of incorporating rare and highly unusual phenotypes or rare host specific biases. The proposed genotype frequency balanced consensus sequence is shown in SEQ ID NO: 3 and comprises the fragment depicted in SEQ ID NO: l.The reverse translation of SEQ ID NO: 3 is found in SEQ ID NO: 4 and comprises SEQ ID NO: 2.

Construction of an adenovirus vector comprising a sequence encoding HEV-Orf2-fragment A nucleic acid sequence encoding the polypeptide sequence according to SEQ ID NO: 1 (GOI) as well as a polyadenylation signal and a cytomegalovirus (CMV) promoter is cloned into an adenovirus shuttle vector derived from human adenovirus 5 (AdS). The AdS-based shuttle vector comprises the 5' nucleic acids 1-448 of a wild-type AdS virus, followed by the CMV promoter and a polyadenylation signal. The construct is prepared by recombination of a p06AS-CMV vector and a BAC-AdS vector lacking the E1/E3 region and comprising a complete deletion of the E3 region comprising nucleotides 28598-30470 of the wild-type Ad5 virus.

Antibody production after immunization of BALB/c mice

In order to demonstrate that the adenovirus vector-based HEV vaccine can be used to raise an immune response, the vector expressing SEQ ID NO: 1 is tested in BALB/c mice. To this end, 20 female BALB/c mice (The Jackson Laboratory, Mouse Genome Database;

hr^://www.mfonnatics.jax.org/extemal/festmg/mouse/doc^ALB.shtml), aged 6-8 weeks, were raised and kept in a specified pathogen free environment complying with administrative regulations for scientific animal testing in the European Union (Directive 2010/63/EU). The vector will be delivered as ready-to-use suspension for use of one vial per item and application day. The preparations are intended for the intended application volume. All material data sheets will be stored with the raw data.

The test item will be administered two times (day 1 and day 15) to 10 female BALB/c mice by the intramuscular (i.m.) route. A total volume of 50uL will be injected (25 uL into each hind limb). Ten control mice are injected with buffer only, i.e. without vectors. During the in- life phase, viability, and general and clinical signs are being monitored. On day 29, blood will be taken from the retrobulbar vein plexus of each animal under anesthesia. Individual blood samples will be processed to serum. The parameters "Viability/mortality" and "General clinical signs" are being observed and documented during the in-life period on a daily basis. On day 29, 450-600 uL blood will be taken from the retrobulbar vein plexus of each animal under anesthesia. Individual blood samples will be processed to serum an approx. amount of 120-200uL immune serum per animal.

The presence of antibodies directed specifically against the HEV-derived polypeptide depicted in SEQ ID NO: 1 is tested in a specific HEV-ELISA.

The microtiter plate provided in this kit has been pre-coated with antigen. Samples are pipetted into the wells with anti-mouse IgG conjugated Horseradish Peroxidase (HRP). Any antibodies specific for the antigen present will bind to the pre-coated antigen. Following a wash to remove any unbound reagent, a substrate solution is added to the wells and color develops in proportion to the amount of mouse hepatitis E virus (HEV; Cusabio, Cat. -No.

CSB-EQ027394MO) antibody (IgG) bound in the initial step. The color development is stopped and the intensity of the color is measured. All the sera are tested in duplicated wells.

All the reagents are kept at RT 30 min before performing ELISA. The ELISA-protocol is conducted according to the manufacturer's instructions.

Data Analysis

For each sample (blank, positive and negative controls, test samples) a mean of OD value will be calculated from the duplicated wells. The mean background value (mean of blank) will subsequently subtracted to the mean of each OD.

Calculation of results

For calculate the valence of mouse hepatitis E virus (HEV) antibody (IgG), the mean of OD for each sample is compared with control. A cutoff value is defined as the average Negative control value plus 0,2. If the average value of OD negative<0.05, the value is assumed as 0.05. If mean of OD sample is < to cutoff value: sample is negative

If mean of OD sample is > to cutoff value : sample is positive

The results are shown in Figure 1. As can be seen, mice injected with the vector according to the present invention raised a powerful antibody response, while the control mice did not raise an immune response above the cutoff value. These results show that the vector comprising the consensus sequence as set out above is highly efficient as HEV vaccine. In figure 1, mice with numbers 1 to 10 are indicated with the symbol "0", whereas control mice can be identified by the symbol "Δ". The data are also shown in Table 4 below.

Table 4:

Claims

1. A nucleic acid sequence encoding a polypeptide according to SEQ ID NO: 1 , or a nucleic acid sequence encoding a polypeptide that is at least 96% or 97% or 98% or 99% identical to SEQ ID NO: 1.

2. A nucleic acid sequence according to claim 1 as depicted in SEQ ID NO: 2, or a degenerate sequence of SEQ ID NO: 2.

3. A synthetic nucleic acid construct comprising a nucleic acid sequence according to any one of claims 1 or 2.

4. The synthetic nucleic acid construct comprising a nucleic acid sequence according to any one of claims 1 or 2, wherein said synthetic nucleic acid construct is a vector.

5. The synthetic nucleic acid construct according to claim 4, wherein the vector comprises a backbone derived from viral vectors selected from the group adenovirus vectors, adeno-associated virus vectors, measles virus vectors, parvovirus vectors, lenti virus vectors, retroviral vectors, pox virus vectors, arbovirus vectors (e.g. VSIV) Herpes Simplex vectors or chimeric virus vectors.

6. The synthetic nucleic acid construct according to any one of claims 4 or 5, wherein the vector comprises a backbone derived from viral vectors selected from the group adenovirus vectors.

7. The synthetic nucleic acid construct according to any one of claims 4 to 6, wherein the adenovirus vector is selected from the group selected from the group of primate adenovirus- based vectors.

8. The synthetic nucleic acid construct according to any one of claims 4 to 7, wherein said primate adenovirus-based vector is selected from the group comprising human adenovirus 5 vectors, human adenovirus Ad35 vectors, human adenovirus Ad 2 vectors, human adenovirus Ad7 vectors, human adenovirus Ad64 vectors, and chimpanzee adenovirus 68 vector,

9. The synthetic nucleic acid construct according to any one of claims 4 to 8, wherein the vector further comprises at least one regulatory element.

10. A vector polypeptide encoded by the synthetic nucleic acid construct according to any one of claims 4 to 9.

11. The vector according to claim 10 for use as medicament.

12. The vector according to any one of claims 4 to 11 for use in the treatment and/or prevention or prophylaxis of infection with Hepatitis E Virus.

13. Use of a vector according to any one of claims 4 to 11 in the manufacture of a medicament for the treatment and/or prevention or prophylaxis of infection with Hepatitis E Virus.

14. A vaccine comprising the vector according to any one of claims 4 to 12.

15. The vaccine according to claim 14 for use in the treatment and/or prevention of infection with Hepatitis E Virus.

16. The vaccine comprising according to any one of claims 14 or 15, wherein said vaccine is lyophilized.

17. The vaccine according to any one of claims 14 to 16, wherein said vaccine is adjuvant- free.

18. The vaccine according to any one of claims 14 to 17, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system.

19. The vaccine according to any one of claims 14 to 18, wherein said vaccine is lyophilized and suitable for reconstitution with a solvent system that is suitable for injection.

20. The vaccine according to any one of claims 14 to 19, for use in the treatment and/or prevention or prophylaxis of an infection with Hepatitis E Virus in a mammal.

21. The vaccine for use according to claim 20, wherein the mammal is a human.

22. The vaccine for use according to any one claims 20 and 21 , wherein the vaccine is administered at a dose of about 10⁶ to about 10ⁿ viral particles.

23. The vaccine for use according to any one claims 20 to 22, wherein at least one, two or three dosages are administered.

24. The vaccine for use according to claim 23, wherein two dosages are administered.

25. The vaccine for use according to claim, wherein a single dosage is administered.

26. An in vitro cell system comprising the nucleic acid sequence or nucleic acid system according any one of claims 1 to 9.

27. The in vitro cell system expressing the polypeptide encoded by the nucleic acid sequence or nucleic acid system according any one of claims 1 to 9.

28. A method of determirimg/measurmg/diagnosmg/quantifying an infection with HEV, said method comprising:

a) Incubating the cell system according to any one of claims 26 or 27 with a sample obtained from a subject,

b) Adding an agent specifically indicating/signaling the presence of antibodies in the sample,

c) Optionally comparing the level of signal obtained in step b) with a control.

29. Use of the polypeptide or a vector polypeptide or a derivative thereof encoded by nucleic acid sequence or nucleic acid system according any one of claims 1 to 9 in the detection of a HEV-specific immune response.

A kit of parts for the immunization against HEV infections comprising a) a container comprising the vaccine according to any one of the preceding claims b) a container with a solvent system,

c) optionally further comprising instructions for use, and

d) optionally comprising a device for administration of the vaccine to a subject.

A method of producing a vaccine as defined in claims 14 to 25, comprising the steps: a) Providing a polynucleotide sequence as defined in claim 2,

b) Cloning the polynucleotide sequence of step a) into a vector suitable for

administration to mammals,

c) Expressing the vector of step b) to produce a vector polypeptide comprising a polypeptide sequence according to claim,

d) Formulating the vector polypeptide according to step c) as vaccine and/or

optionally subjecting the vector polypeptide to lyophilization.