Compositions and methods for treating and preventing hepatitis a and e

Abstract

Disclosed herein are immunogenic compositions or product combinations of engineered hepatitis A virus (HAV) and / or hepatitis E virus (HEV) nucleic acids, genes, peptides, or proteins that can be used to elicit an immune response against a HAV and / or HEV infection. Also disclosed are methods of using the immunogenic compositions or product combinations in subjects to generate immune responses against HAV and / or by administering the compositions or combinations with a nucleic acid prime and polypeptide boost approach.

Description

SVF.010WO PATENT COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING HEPATITIS A AND E INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 590192, filed October 13, 2023, which is hereby expressly incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled SVF010WOseqlist, which was created on October 3, 2024and is 36,499 bytes in size. The information in the electronic Sequence Listing is hereby expressly incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Field of the Invention

[0003] Aspects of the present disclosure relate generally to immunogenic compositions or product combinations of engineered hepatitis A virus (HAV) and / or hepatitis E virus (HEV) nucleic acids, genes, peptides, or proteins that can be used to elicit an immune response against HAV and / or HEV. This immune response includes activation of cytotoxic immune cells and immune cells that produce neutralizing antibodies against HAV and / or HEV. The disclosure also relates generally to methods of using the immunogenic compositions or product combinations in subjects to generate immune responses against HAV and / or HEV by administering the compositions or combinations with a homologous or heterologous nucleic acid and / or polypeptide prime and nucleic acid and / or polypeptide boost approach. Further, the disclosure also relates generally to immunogenic compositions or product combinations and methods of using the same where the immunogenic compositions or product combinations are formulated to be delivered to a subject via virus- like particles (VLPs).Description of the Related Art

[0004] Hepatitis A and hepatitis E are viral infections of the liver caused by their respective pathogens, the hepatitis A virus (HAV) and hepatitis E virus (HEV). These viruses, which are endemic throughout most of Asia, Africa and Central and Southern America, are primarily transmitted via the oro-fecal route and are the cause of substantial morbidity and mortality: current estimates are that there are around 150 million HAV and 20 million HEV incident cases annually, causing 39,000 and 70,000 deaths (1, 2).

[0005] HAV was commonly known as a mainly asymptomatic childhood disease with an almost 100% seroprevalence at the age of 30. However, this is transitioning to infections at higher ages due to increased sanitary conditions and better water supply. As an example, in Thailand, the seroprevalence for HAV has dropped from 100% at 30 years of age in the 1970s to less than 20-30% in 2000s (3). Consequently, since it is well known that symptoms worsen with increasing age of HAV infection, it is likely that the prevalence of symptomatic or even severe HAV infections will increase in areas where HAV is still prevalent, but the HAV vaccine is not frequently used. HEV infection is more complicated as the virus is prevalent in both humans and animals. HEV is especially pathogenic in pregnant women, in whom the case fatality rate can be more than 10-fold higher than the general population.

[0006] Given the mode of transmission, outbreaks in endemic regions during humanitarian emergencies are frequently reported because of disrupted access to safe drinking water and sanitation, while sporadic outbreaks continue to be reported in non- endemic countries, with zoonotic transmission with HEV3 and HEV4 being especially important in industrialized countries.

[0007] Disclosed herein is a new immunization strategy for HAV and / or HEV with a single vaccine or immunogenic composition that prevents, inhibits, ameliorates or treats HAV and / or HEV infection. Such a vaccine or immunogenic composition can prevent or inhibit HAV and / or HEV infection in people living in endemic areas, prevent or inhibit outbreaks during humanitarian emergencies, and be used as a travel vaccine or immunogenic composition for those traveling to endemic areas.SUMMARY

[0008] Disclosed herein are several therapeutic agents useful in immunogenic compositions that prevent, inhibit, ameliorate or treat HAV and / or HEV infection or a sequela associated therewith, as well as methods of using these immunogenic compositions in subjects, such as humans infected with HAV and / or HEV or subjects at risk of being infected by HAV and / or HEV. Also described herein are heterologous prime-boost immunization approaches, which utilize a nucleic acid (DNA or RNA) prime and a polypeptide boost administration schedule.

[0009] In some embodiments, the immunogenic compositions or product combinations described herein comprise non-naturally occurring composite nucleic acids and / or polypeptides. In some embodiments, the nucleic acids are DNA or RNA. In some embodiments, the immunogenic compositions or product combinations are administered to an animal, such as a mammal, mouse, rabbit, cat, dog, or human, to induce an immunogenic response against HAV and / or HEV. In some embodiments, the immunogenic response comprises, consists essentially of, or consists of formation of active immune cells, such as cytotoxic T cells or immune cells that produce antibodies against the HAV and / or HEV, or any antigen, polypeptide, protein, nucleic acid, or genome component of HAV and / or HEV, preferably these antibodies are inactivating or provide protection to the subject. In some embodiments, the immunogenic compositions or product combinations are administered to subjects that are at risk of contracting HAV and / or HEV or subjects that are not currently infected with HAV and / or HEV. In some embodiments, the immunogenic compositions or product combinations disclosed herein provide lasting immunogenic protection against a HAV and / or HEV infection.

[0010] Some alternatives described herein concern nucleic acids comprising, consisting essentially of, or consisting of at least one HAV nucleic acid component. Some alternatives described herein concern nucleic acids comprising, consisting essentially of, or consisting of at least one HEV nucleic acid component. Some alternatives described herein concern nucleic acids comprising, consisting essentially of, or consisting of at least one HAV nucleic acid component and at least one HEV nucleic acid component. In some alternatives, the at least one HAV nucleic acid component comprises, consists essentially of, or consists of an VP2 protein sequence, and / or VP3 protein sequence. In some alternatives, the at least oneHEV nucleic acid component comprises, consists essentially of, or consists of an ORFgt1protein sequence, and / or ORFgt3 protein sequence. In some alternatives, the at least one HAV and / or HEV nucleic acid component contemplated for inclusion in the compositions and the methods described herein is a human codon optimized sequence of any one of the aforementioned wild-type sequences. In some alternatives, for example, the nucleic acids share at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19 or an amount of sequence identity to any one or more of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19 that is within a range defined by any two of the aforementioned percentages, wherein said nucleic acids encode an immunogenic or antigenic polypeptide, epitope, or fragment of HAV and / or HEV. In some alternatives, the nucleic acids referenced above are used for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith in a subject, such as a mammal, preferably a human. Accordingly, some alternatives include the use of a nucleic acid having at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to any one or more of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19 or an amount of sequence identity to any one or more of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19 that is within a range defined by any two of the aforementioned percentages, wherein said nucleic acids encode an immunogenic or antigenic polypeptide, epitope, or fragment of HAV and / or HEV, as a medicament, such as for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith, in a subject, such as a mammal, preferably a human, which may, optionally, be selected or identified to receive a medicament for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith. Such subjects can be selected or identified by clinical evaluation or diagnostic evaluation or both.

[0011] Some alternatives provided herein concern polypeptides comprising, consisting essentially of, or consisting of at least one HAV polypeptide component. Some alternatives provided herein concern polypeptides comprising, consisting essentially of, or consisting of at least one HEV polypeptide component. Some alternatives provided herein concern polypeptides comprising, consisting essentially of, or consisting of at least one HAVpolypeptide component and at least one HEV polypeptide component. In some alternatives, the at least one HAV polypeptide component comprises, consists essentially of, or consists of an VP2 protein sequence and / or VP3 protein sequence. In some alternatives, the at least one HEV polypeptide component comprises, consists essentially of, or consists of an ORFgt1 protein sequence and / or ORFgt3 protein sequence. In some embodiments, the polypeptides, which may be provided in a composition or method described herein share at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to any one or more of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20 or an amount of sequence identity to any one or more of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20 that is within a range defined by any two of the aforementioned percentages, wherein said polypeptides encode an immunogenic or antigenic portion, epitope, or fragment of HAV and / or HEV. In some alternatives, the polypeptides are used as a medicament, such as for the prevention, treatment, amelioration, or inhibition of HAV and / or HEV or a sequela associated therewith in a subject such as a mammal, preferably a human, which may, optionally, be selected or identified to receive a medicament for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith. Such subjects can be selected or identified by clinical evaluation or diagnostic evaluation or both. In some embodiments, the polypeptides are translated from the wild-type or codon optimized sequences referenced above. In some embodiments, the polypeptides are recombinantly expressed. In some embodiments, the polypeptides are recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell- free system. Accordingly, some alternatives include the use of a polypeptide having at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to any one or more of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20 or an amount of sequence identity to any one or more of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20 that is within a range defined by any two of the aforementioned percentages, wherein said nucleic acids encode an immunogenic or antigenic portion, epitope, or fragment of HAV and / or HEV as a medicament, such as for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith in a subject, such as a mammal, preferably a human, which may,optionally, be selected or identified to receive a medicament for the prevention, treatment, amelioration, or inhibition of HAV and / or HEV infection or a sequela associated therewith.

[0012] In some alternatives, the nucleic acids or polypeptides also comprise at least one autocatalytic peptide cleavage site. In some alternatives, the at least one autocatalytic peptide cleavage site is a P2A autocatalytic peptide cleavage site. In some alternatives, the polypeptide encoded by the at least one HAV and / or HEV nucleic acid component or the at least one HAV and / or HEV polypeptide component are separated by the at least one autocatalytic peptide cleavage site.

[0013] In some alternatives, the nucleic acids or polypeptides also comprise at least one linker sequence. In some alternatives, the polypeptide encoded by the at least one HAV and / or HEV nucleic acid component or the at least one HAV and / or HEV polypeptide component are separated by the at least one linker sequence. In some embodiments, the nucleic acids or polypeptides may comprise both at least one linker sequence and at least one autocatalytic peptide cleavage site. In some embodiments, the linker sequence may be any of the linker sequences described herein.

[0014] In some alternatives, at least one HAV sequence is provided in or encoded by the nucleic acids or polypeptides referenced above, such as a VP2 sequence, a VP3 sequence, or any combination thereof. In some alternatives, two HAV sequences are provided in or encoded by the nucleic acids or polypeptides referenced thereof. In some alternatives, there is one HAV sequence provided in or encoded by the nucleic acids or polypeptides. In some alternatives, the HAV sequences are provided in or encoded by the nucleic acids or polypeptides in tandem. In some alternatives, the HAV sequences are separated by peptide cleavage sites. In some alternatives, the HAV sequences are separated from the HEV sequences and / or additional HAV sequences with an autocatalytic peptide cleavage site. In some alternatives, the autocatalytic peptide cleavage site is a P2A autocatalytic peptide cleavage site. In some alternatives, the HAV sequences are separated from the HEV sequences and / or additional HAV sequences with linker sequence. In some embodiments, the linker sequence may be any of the linker sequences described herein.

[0015] In some alternatives, at least one HEV sequence is provided in or encoded by the nucleic acids or polypeptides referenced above, such as an ORFgt1sequence, an ORFgt3sequence, or any combination thereof. In some alternatives, two HEV sequences areprovided in or encoded by the nucleic acids or polypeptides referenced thereof. In some alternatives, there is one HEV sequence provided in or encoded by the nucleic acids or polypeptides. In some alternatives, the HEV sequences are provided in or encoded by the nucleic acids or polypeptides in tandem. In some alternatives, the HEV sequences are separated by peptide cleavage sites. In some alternatives, the HEV sequences are separated from the HAV sequences and / or additional HEV sequences with an autocatalytic peptide cleavage site. In some alternatives, the autocatalytic peptide cleavage site is a P2A autocatalytic peptide cleavage site. In some alternatives, the HEV sequences are separated from the HAV sequences and / or additional HEV sequences with linker sequence. In some embodiments, the linker sequence may be any of the linker sequences described herein.

[0016] In some alternatives, the immunogenic compositions or product combinations comprise, consist essentially of, or consist of a nucleic acid, described above (e.g., any one or more of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19), and / or a polypeptide, described above (e.g., any one or more of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20). In some alternatives, the immunogenic compositions or product combinations are administered to a subject in a heterologous prime-boost approach. In some alternatives, the prime dose comprises the nucleic acid and the boost dose comprises the polypeptide. In some alternatives, the prime dose comprises the polypeptide and the boost dose comprises the nucleic acid. In some alternatives, the prime dose comprises any one or more of the aforementioned polypeptides and the boost dose comprises any one or more of the aforementioned nucleic acids. In some alternatives, the immunogenic compositions or product combinations are administered to a subject as a homologous prime-boost approach. In some alternatives, the prime dose comprises any one or more of the aforementioned nucleic acids and the boost dose comprises either the same nucleic acid or a different nucleic acid. In some alternatives, the prime dose comprises any one or more of the aforementioned polypeptides and the boost dose comprises either the same polypeptide or a different polypeptide. In some alternatives, the immunogenic compositions or product combinations further comprise an adjuvant. In some embodiments, the adjuvant may comprise any of the adjuvants described herein. In some embodiments, the adjuvant is alum. In some alternatives, the nucleic acid is provided as a recombinant vector. In some alternatives, the recombinant vector is pVAX1. In some alternatives, the immunogenic compositions or productcombinations are used for the prevention, treatment, amelioration, or inhibition of HAV and / or HEV or a sequela associated therewith in a subject, such as a mammal, preferably a human, which may, optionally, be selected or identified to receive a medicament for the prevention, treatment, amelioration, or inhibition of a HAV and / or HEV infection or a sequela associated therewith. Such subjects can be selected or identified by clinical evaluation or diagnostic evaluation or both.

[0017] Some alternatives described herein concern methods of generating an immune response in a subject, preferably a human, using the immunogenic compositions, product compositions, nucleic acids, or polypeptides described above (e.g., any one or more of SEQ ID NO: 5-20. In some alternatives, the methods comprise a heterologous prime- boost approach. In some alternatives, at least one prime dose is administered to the subject and at least one boost dose is administered to the subject. In some alternatives, the at least one prime dose is a nucleic acid. In some alternatives, the at least one boost dose is a polypeptide. In some alternatives, the at least one boost dose comprises an adjuvant, such as alum. In some embodiments, the adjuvant may comprise any of the adjuvants described herein. In some alternatives, the at least one boost dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after the at least one prime dose is administered or within a range of time defined by any two of the aforementioned time points. In some alternatives, the methods comprise a homologous prime-boost approach.

[0018] Additional alternatives concern an injection device comprising any one or more of the compositions described herein, such as any one or more of the nucleic acids or polypeptides set forth in any one or more of SEQ ID NO: 5-20 Such injection devices can comprise a single dose of such nucleic acid or polypeptide and such injection devices can have modified needle designs configured to enhance delivery of the nucleic acid or polypeptide or both. Such injection devices can be used with or without electroporation. Contemplated injections devices, which can include any one or more of the nucleic acids or polypeptides of SEQ ID NO: 5-20 are described in U.S. Pat. App. Pub. No. 2016 / 0235928; PCT App. Pub. No. WO2014064534; U.S. Pat. Nos. 6,610,044; 6,132,419; 6,379,966; 6,897,068; 7,015,040; 7,214,369; 7,473,419; and 7,589,059, all of which are hereby expressly incorporated by reference in their entireties.

[0019] Embodiments provided herein are further described in the following enumerated aspects:

[0020] 1. A nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide.

[0021] 2. The nucleic acid of aspect 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1polypeptide, and a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

[0022] 3. The nucleic acid of aspect 2, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0023] 4. The nucleic acid of aspect 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP3 polypeptide, and a nucleic acid sequence encoding a HEV ORFgt1 polypeptide.

[0024] 5. The nucleic acid of aspect 4, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 9, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0025] 6. The nucleic acid of aspect 4, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding a linker sequence.

[0026] 7. The nucleic acid of aspect 6, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequenceidentity to SEQ ID NO: 11, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0027] 8. The nucleic acid of aspect 4, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

[0028] 9. The nucleic acid of aspect 8, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 7, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0029] 10. The nucleic acid of aspect 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1polypeptide, a nucleic acid sequence encoding a HEV ORFgt3polypeptide, at least one a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence, and at least one nucleic acid sequence encoding a linker sequence.

[0030] 11. The nucleic acid of aspect 10, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 13, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0031] 12. The nucleic acid of aspect 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1 polypeptide, and at least one a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

[0032] 13. The nucleic acid of aspect 12, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequenceidentity to SEQ ID NO: 15, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

[0033] 14. A polypeptide comprising at least one hepatitis A virus (HAV) polypeptide sequence and at least one hepatitis E virus (HEV) polypeptide sequence.

[0034] 15. The polypeptide of aspect 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, and a HEV ORFgt1polypeptide sequence.

[0035] 16. The polypeptide of aspect 15, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0036] 17. The polypeptide of aspect 14, wherein the polypeptide comprises an HAV VP3 polypeptide sequence, and a HEV ORFgt1polypeptide sequence.

[0037] 18. The polypeptide of aspect 17, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 10, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0038] 19. The polypeptide of aspect 17, wherein the polypeptide sequence further comprises a linker sequence.

[0039] 20. The polypeptide of aspect 19, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 12, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0040] 21. The polypeptide of aspect 17, wherein the polypeptide sequence further comprises a P2A autocatalytic cleavage sequence.

[0041] 22. The polypeptide of aspect 21, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 8, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0042] 23. The polypeptide of aspect 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, an HEV ORFgt1 polypeptide sequence, a HEV ORFgt3 polypeptide sequence, at least one P2A autocatalytic cleavage sequence, and at least one linker sequence.

[0043] 24. The polypeptide of aspect 23, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 14, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0044] 25. The polypeptide of aspect 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, an HEV ORFgt1 polypeptide sequence, and at least one P2A autocatalytic cleavage sequence.

[0045] 26. The polypeptide of aspect 25, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 16, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

[0046] 27. The nucleic acid of any one of aspects 1-13 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

[0047] 28. The polypeptide of any one of aspects 14-26 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

[0048] 29. The polypeptide of any one of aspects 14-26 or 28, wherein the polypeptide is recombinantly expressed.

[0049] 30. The polypeptide of any one of aspects 14-26 or 28-29, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0050] 31. An immunogenic composition or product combination comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and at least one HEV polypeptide.

[0051] 32. The immunogenic composition or product combination of aspect 31, wherein the nucleic acid comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

[0052] 33. The immunogenic composition or product combination of aspect 31 or 32, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, 7, 9, 11, 13, or 15,wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

[0053] 34. The immunogenic composition or product combination of any one of aspects 31-33, wherein the nucleic acid is codon optimized for expression in a human.

[0054] 35. The immunogenic composition or product combination of any one of aspects 31-34, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3polypeptide sequence; v) a P2A autocatalytic cleavage site polypeptide sequence; vi) a linker polypeptide sequence; or any combination thereof.

[0055] 36. The immunogenic composition or product combination of any one of aspects 31-35, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, 8, 10, 12, 14, or 16, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and HEV in a subject, such as a mammal, preferably a human.

[0056] 37. The immunogenic composition or product combination of any one of aspects 31-36, wherein the polypeptide is recombinantly expressed.

[0057] 38. The immunogenic composition or product combination of any one of aspects 31-37, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0058] 39. The immunogenic composition or product combination of any one of aspects 31-38, further comprising an adjuvant.

[0059] 40. The immunogenic composition or product combination of aspect 39, wherein the adjuvant is alum.

[0060] 41. The immunogenic composition or product combination of any one of aspects 31-40, wherein the nucleic acid is provided in a recombinant vector.

[0061] 42. A method of generating an immune response in a subject using the immunogenic composition or product combination set forth in any one of aspects 31-41, comprising: a) administering to the subject at least one prime dose comprising the nucleic acid; and b) administering to the subject at least one boost dose comprising the polypeptide; or c) administering to the subject at least one prime dose comprising the polypeptide; and d) administering to the subject at least one boost dose comprising the nucleic acid.

[0062] 43. The method of aspect 42, wherein the at least one boost dose further comprises an adjuvant.

[0063] 44. The method of aspect 43, wherein the adjuvant is alum.

[0064] 45. The method of any one of aspects 42-44, wherein the at least one boost dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeksafter the at least one prime dose is administered or within a range of time defined by any two of the aforementioned time points e.g., within 1-48 days or 1-48 weeks.

[0065] 46. The method of any one of aspects 42-45, wherein the administration is provided enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously or any combination thereof, and optionally with in vivo electroporation.

[0066] 47. The method of any one of aspects 42-46, wherein the administration is performed in conjunction with an antiviral therapy or an antiviral prodrug therapy.

[0067] 48. The method of aspect 47, wherein the antiviral therapy or antiviral prodrug therapy comprises administration of chloroquine, hydroxychloroquine, favipiravir, favilavir, remdesivir, tocilizumab, galidesivir, sarilumab, lopinavir, ritonavir, darunavir, ribavirin, interferon-Į, pegylated interferon-Į, interferon alfa-2b, a prodrug or precursor thereof, or any combination thereof.

[0068] 49. An immunogenic composition or product combination for use in the treatment or inhibition of hepatitis A virus (HAV) and / or hepatitis E virus (HEV), comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a HAV polypeptide and at least one nucleic acid sequence encoding a HEV polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and at least one HEV polypeptide.

[0069] 50. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 49, wherein the nucleic acid sequence comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide;iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

[0070] 51. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 49 or 50, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, 7, 9, 11, 13, or 15, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV.

[0071] 52. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 49 or 51, wherein the nucleic acid is codon optimized for expression in a human.

[0072] 53. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-52, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1polypeptide sequence; iv) a HEV ORF2gt3polypeptide sequence; v) a P2A autocatalytic polypeptide cleavage site sequence; vi) a linker polypeptide sequence or any combination thereof.

[0073] 54. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-53, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, 8, 10, 12, 14, or 16, wherein thepolypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitope or antigen of HAV and HEV.

[0074] 55. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-54, wherein the polypeptide is recombinantly expressed.

[0075] 56. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-55, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0076] 57. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-56, further comprising an adjuvant.

[0077] 58. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 57, wherein the adjuvant is alum.

[0078] 59. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 49-58, wherein the nucleic acid is provided in a recombinant vector.

[0079] 60. A nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide or at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide.

[0080] 61. The nucleic acid of aspect 60, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, and a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

[0081] 62. The nucleic acid of aspect 61, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 17, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV.

[0082] 63. The nucleic acid of aspect 60, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HEV ORFgt1polypeptide.

[0083] 64. The nucleic acid of any one of aspects 60-63, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 19, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HEV.

[0084] 65. A polypeptide comprising at least one hepatitis A virus (HAV) polypeptide sequence or at least one hepatitis E virus (HEV) polypeptide sequence.

[0085] 66. The polypeptide of aspect 65, wherein the HAV polypeptide sequence comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, and a P2A autocatalytic cleavage sequence.

[0086] 67. The polypeptide of aspect 66, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18, wherein the polypeptide comprises an epitope or antigen of HAV.

[0087] 68. The polypeptide of aspect 65, wherein the HAV polypeptide sequence comprises a HEV ORFgt1polypeptide sequence.

[0088] 69. The polypeptide of aspect 68, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequenceidentity to SEQ ID NO: 20, wherein the polypeptide comprises an epitope or antigen of HEV.

[0089] 70. The nucleic acid of any one of aspects 60-64 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

[0090] 71. The polypeptide of any one of aspects 65-69 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

[0091] 72. The polypeptide of any one of aspects 65-69 or 71, wherein the polypeptide is recombinantly expressed.

[0092] 73. The polypeptide of any one of aspects 65-69 or 71-72, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0093] 74. An immunogenic composition or product combination comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and / or at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and / or at least one HEV polypeptide.

[0094] 75. The immunogenic composition or product combination of aspect 74, wherein the nucleic acid comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide;v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

[0095] 76. The immunogenic composition or product combination of aspect 74 or 75, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 17 or 19, wherein the polypeptide comprises epitopes or antigens of HAV and / or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and / or HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

[0096] 77. The immunogenic composition or product combination of any one of aspects 74-76, wherein the nucleic acid is codon optimized for expression in a human.

[0097] 78. The immunogenic composition or product combination of any one of aspects 74-77, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3 polypeptide sequence; v) a P2A autocatalytic cleavage site polypeptide sequence; vi) a linker polypeptide sequence or any combination thereof.

[0098] 79. The immunogenic composition or product combination of any one of aspects 90-94, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18 or 20, wherein the polypeptide comprises epitopes or antigens of HAV and / or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and / orHEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

[0099] 80. The immunogenic composition or product combination of any one of aspects 74-79, wherein the polypeptide is recombinantly expressed.

[0100] 81. The immunogenic composition or product combination of any one of aspects 74-80, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0101] 82. The immunogenic composition or product combination of any one of aspects 74-81, further comprising an adjuvant.

[0102] 83. The immunogenic composition or product combination of aspect 82, wherein the adjuvant is alum.

[0103] 84. The immunogenic composition or product combination of any one of aspects 74-83, wherein the nucleic acid is provided in a recombinant vector.

[0104] 85. A method of generating an immune response in a subject using the immunogenic composition or product combination set forth in any one of aspects 74-84, comprising: a) administering to the subject at least one prime dose comprising the nucleic acid; and b) administering to the subject at least one boost dose comprising the polypeptide; or c) administering to the subject at least one prime dose comprising the polypeptide; and d) administering to the subject at least one boost dose comprising the nucleic acid.

[0105] 86. The method of aspect 85, wherein the at least one boost dose further comprises an adjuvant.

[0106] 87. The method of aspect 86, wherein the adjuvant is alum.

[0107] 88. The method of any one of aspects 85-87, wherein the at least one boost dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after the at least one prime dose is administered or within a range of time defined by any two of the aforementioned time points e.g., within 1-48 days or 1-48 weeks.

[0108] 89. The method of any one of aspects 85-88, wherein the administration is provided enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously or any combination thereof, and optionally with in vivo electroporation.

[0109] 90. The method of any one of aspects 85-89, wherein the administration is performed in conjunction with an antiviral therapy or an antiviral prodrug therapy.

[0110] 91. The method of aspect 90, wherein the antiviral therapy or antiviral prodrug therapy comprises administration of chloroquine, hydroxychloroquine, favipiravir, favilavir, remdesivir, tocilizumab, galidesivir, sarilumab, lopinavir, ritonavir, darunavir, ribavirin, interferon-Į, pegylated interferon-Į, interferon alfa-2b, a prodrug or precursor thereof, or any combination thereof.

[0111] 92. An immunogenic composition or product combination for use in the treatment or inhibition of hepatitis A virus (HAV) and / or hepatitis E virus (HEV), comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a HAV polypeptide or at least one nucleic acid sequence encoding a HEV polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide or at least one HEV polypeptide.

[0112] 93. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 92, wherein the nucleic acid sequence comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

[0113] 94. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 92 or 93, wherein the nucleic acid shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 17 or 19, wherein the polypeptide comprises epitopes or antigens of HAV or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV or HEV.

[0114] 95. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-94, wherein the nucleic acid is codon optimized for expression in a human.

[0115] 96. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-95, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3 polypeptide sequence; v) a P2A autocatalytic polypeptide cleavage site sequence; vi) a linker polypeptide sequenceor any combination thereof.

[0116] 97. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-96, wherein the polypeptide shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18 or 20, wherein the polypeptide comprises epitopes or antigens of HAV or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitope or antigen of HAV or HEV.

[0117] 98. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-97, wherein the polypeptide is recombinantly expressed.

[0118] 99. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-98, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

[0119] 100. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-99, further comprising an adjuvant.

[0120] 101. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of aspect 100, wherein the adjuvant is alum.

[0121] 102. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of aspects 92-101, wherein the nucleic acid is provided in a recombinant vector.

[0122] 103. The immunogenic composition or product combination of any one of aspects 31-41, 49-59, 74-84, 92-102, wherein the immunogenic composition or product combination is formulated to be delivered to a subject via virus-like particles (VLPs).

[0123] 104. The immunogenic composition or product combination of aspect 103, wherein the VLPs are based on one or more p239 fragments of the HEV pORF2 protein.

[0124] 105. The method of any one of aspects 42-48 or 85-91, wherein administering to the subject comprises delivery via virus-like particles (VLPs).

[0125] 106. The method of aspect 105, wherein the VLPs are based on one or more p239 fragments of the HEV pORF2 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] In addition to the features described above, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments and are not intended to be limiting in scope.

[0127] FIG. 1 depicts a schematic of the domains comprising the amino acid sequences for HAV and HEV immunogenic compositions SVF-004.1-SVF-004.8.

[0128] FIG. 2 depicts nucleotide sequences for BamHI, XbaI, Kozak sequence, and stop codon.

[0129] FIG. 3 depicts the schematic, nucleotide, and amino acid information for SVF-004.1 HAV and HEV immunogenic composition.

[0130] FIG. 4 depicts the schematic, nucleotide, and amino acid information for SVF-004.2 HAV and HEV immunogenic composition.

[0131] FIG. 5 depicts the schematic, nucleotide, and amino acid information for SVF-004.3 HAV and HEV immunogenic composition.

[0132] FIG. 6 depicts the schematic, nucleotide, and amino acid information for SVF-004.4 HAV and HEV immunogenic composition.

[0133] FIG. 7 depicts the schematic, nucleotide, and amino acid information for SVF-004.5 HAV and HEV immunogenic composition.

[0134] FIG. 8 depicts the schematic, nucleotide, and amino acid information for SVF-004.6 HAV and HEV immunogenic composition.

[0135] FIG. 9 depicts the schematic, nucleotide, and amino acid information for SVF-004.7 HAV immunogenic composition.

[0136] FIG. 10 depicts the schematic, nucleotide, and amino acid information for SVF-004.8 HEV immunogenic composition.

[0137] FIG. 11 depicts nucleotide and amino acid sequences for the P2A site and linker.

[0138] FIG.12 depicts an immunogenicity study design.

[0139] FIG. 13 depicts SVF-004.1-SVF-004.8 characterization by restriction enzyme digest.

[0140] FIG. 14A-14D depict the quantification of anti-ORF2 IgG titer in BALB / c mice immunized with SVF-004.1-SVF-004.8 DNA compositions. Serum from the mice were collected and tested by ELISA against the ORF2 HEV protein. Individual anti-sera (n=5) was tested in ELISA. Graph bars show the mean end anti-ORF2 titers for each group determined as the end last serum dilution giving an OD at 405 nm three times higher than the OD of non- immunized sera at the same dilution (FIG. 14A). The sera were titrated serially with six-fold dilutions starting at 1:60. Figure 14B-14D show individual anti-ORF2 titers at weeks 2, 5 and 8 weeks post first immunization respectively.

[0141] FIG. 15 depicts exemplary nucleic acid or polypeptide constructs comprising HAV and / or HEV antigens used herein. Eight constructs are provided, SVF- 004.1, SVF-004.2, SVF-004.3, SVF-004.4, SVF-004.5, SVF-004.6, SVF-004.7, and SVF- 004.8. Western blot confirms that at least polypeptide constructs SVF-004.1, SVF-004.5, and SVF-004.7 are properly expressed. Pooled serum from groups SVF-004.1, SVF-004.2, SVF- 004.3, SVF-004.4, SVF-004.5, SVF-004.6, SVF-004.7, SVF-004.8, and naïve mouse serum was used to detect HAV VP2 and / or VP3. The data indicates that at least constructs SVF- 004.1, SVF-004.5, and SVF-004.7 induced antibodies to HAV.DETAILED DESCRIPTION

[0142] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. Definitions

[0143] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are expressly incorporated by reference in their entireties unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0144] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0145] The terms “about” or “around” as used herein refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0146] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0147] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0148] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. If there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. The practice of the present disclosure will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration.

[0149] The terms “individual”, “subject”, or “patient” as used herein, means a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

[0150] The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0151] Some embodiments described herein relate to pharmaceutical compositions that comprise, consist essentially of, or consist of an effective amount of an oligonucleotide described herein and a pharmaceutically acceptable carrier, excipient, or combination thereof. A pharmaceutical composition described herein is suitable for human and / or veterinary applications.

[0152] The terms “function” and “functional” as used herein refer to a biological, enzymatic, or therapeutic function.

[0153] The terms “effective amount” or “effective dose” is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated. For example, an effective amount of compound can be the amount needed to alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated This response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of an effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein. The effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0154] The term “pharmaceutically acceptable salts” includes relatively non- toxic, inorganic and organic acid, or base addition salts of compositions, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include phosphates, hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N- methylpiperazine; morpholine; ethylenediamine; or N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0155] “Formulation”, “pharmaceutical composition”, and “composition” as used interchangeably herein are equivalent terms referring to a composition of matter for administration to a subject.

[0156] The term “pharmaceutically acceptable” means compatible with therapy for a subject, and in particular, a human.

[0157] The terms “agent” refers to an active agent that has biological activity and may be used in a therapy. Also, an “agent” can be synonymous with “at least one agent,” “compound,” or “at least one compound,” and can refer to any form of the agent, such as a derivative, analog, salt or a prodrug thereof. The agent can be present in various forms, components of molecular complexes, and pharmaceutically acceptable salts (e.g., hydrochlorides, hydrobromides, sulfates, phosphates, nitrates, borates, acetates, maleates, tartrates, or salicylates). The term “agent” can also refer to any pharmaceutical molecules or compounds, therapeutic molecules or compounds, matrix forming molecules or compounds, polymers, synthetic molecules and compounds, natural molecules and compounds, and any combination thereof.

[0158] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering one or more of the nucleic acids or polypeptides described herein or immunogenic compositions comprising said nucleic acids or polypeptides described herein are contemplated including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral, intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration. The pharmaceutical compositions described herein can also be administered to subjects along with other therapies, such as T cells, Natural Killer cells, B cells, macrophages, lymphocytes, stem cells, bone marrow cells, or hematopoietic stem cells.

[0159] The term “isolated” as used herein refers to material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated cell,” as used herein, includes a cell that has been purified from the milieu ororganisms in its naturally occurring state, a cell that has been removed from a subject or from a culture, for example, it is not significantly associated with in vivo or in vitro substances.

[0160] The pharmaceutical compound can also be administered in a local rather than systemic manner, for example, via injection of the compound directly into an organ, tissue, or infected area, often in a depot or sustained release formulation.

[0161] The pharmaceutical compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes. As described herein, compounds used in a pharmaceutical composition may be provided as salts with pharmaceutically compatible counterions.

[0162] As used herein, a “carrier” refers to a compound, particle, solid, semi- solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs. For example, without limitation, a lipid nanoparticle (LNP) is a type of carrier that can encapsulate an oligonucleotide to thereby protect the oligonucleotide from degradation during passage through the bloodstream and / or to facilitate delivery to a desired organ, such as to the liver.

[0163] As used herein, a “diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

[0164] The term “excipient” has its ordinary meaning as understood in light of the specification, and refers to inert substances, compounds, or materials added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. Excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodiumcitrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, dextran, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, methyl cellulose, hydroxypropyl methyl cellulose (hypromellose), glycerin, polyvinyl alcohol, povidone, propylene glycol, serum, amino acids, polyethylene glycol, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. The amount of the excipient may be found in a pharmaceutical composition at a percentage of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0165] The term “adjuvant” as used herein refers to a substance, compound, or material that stimulates the immune response and increase the efficacy of protective immunity and is administered in conjunction with an immunogenic antigen, epitope, or composition. Adjuvants serve to improve immune responses by enabling a continual release of antigen, up-regulation of cytokines and chemokines, cellular recruitment at the site of administration, increased antigen uptake and presentation in antigen presenting cells, or activation of antigen presenting cells and inflammasomes. Commonly used adjuvants, which can be included in any one or more of the formulations set forth herein include but are not limited to alum, aluminum salts, aluminum sulfate, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, potassium aluminum sulfate, oils, mineral oil, paraffin oil, oil-in-water emulsions, detergents, MF59®, squalene, AS03, Į-tocopherol, polysorbate 80, AS04, monophosphoryl lipid A, virosomes, nucleic acids, polyinosinic:polycytidylic acid, saponins, QS-21, proteins, flagellin, cytokines, chemokines, IL-1, IL-2, IL-12, IL-15, IL-21, imidazoquinolines, CpG oligonucleotides, lipids, phospholipids, dioleoyl phosphatidylcholine (DOPC), trehalose dimycolate, peptidoglycans, bacterial extracts, lipopolysaccharides, or Freund’s Adjuvant, or any combination thereof. In some embodiments, the immunogenic composition or product combination can further comprise an adjuvant. In some embodiments, the adjuvant of the immunogenic composition or product combination can comprise any of the adjuvants described herein.

[0166] The term “purity” of any given substance, compound, or material as used herein refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to side products, isomers, enantiomers, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. Purity can be measured technologies including but not limited to chromatography, liquid chromatography, gas chromatography, spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0167] Some embodiments disclosed herein related to selecting a subject or patient in need of receiving one or more of the nucleic acids or polypeptides described herein. In some embodiments, a patient or subject is selected who is in need of treatment or inhibition of a viral infection caused by hepatitis A virus (HAV), hepatitis E virus (HEV) or a sequela associated therewith. In some embodiments, a patient or subject is selected who has previously received a therapy for HAV and / or HEV In some embodiments, a patient or subject is selected who has previously received a therapy for being at risk of HAV and / or HEV infection or a sequela associated therewith. In some embodiments, a patient or subject is selected who has developed a recurrence of HAV and / or HEV infection. In some embodiments, a patient or subject is selected who has developed resistance to therapies for HAV and / or HEV infection or a sequela associated therewith. In some embodiments, a patient or subject is selected who may have any combination of the aforementioned selection criteria. Such selections can be made by clinical and diagnostic evaluation of the subject or a combination of both.

[0168] The terms “treat”, “treating”, “treatment”, “therapeutic”, or “therapy” as used herein has its ordinary meaning as understood in light of the specification, and do not necessarily mean total cure or abolition of the disease or condition. The term “treating” or “treatment” as used herein (and as well understood in the art) also means an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease,stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods comprise administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may comprise a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from or developing a disease or condition.

[0169] The term “inhibit” as used herein has its ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a viral infection, such as HAV and / or HEV. The reduction can be by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of an event, such as a viral infection, to a time which is later than would otherwise be expected. The delay can be a delay of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0170] The term “immunogenic composition” as used herein refers to a substance or mixture of substances, including but not limited to antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens, toxoids, inactivated organisms, or attenuated organisms, or any combination thereof, intended to elicit an immune response when administered to a host. The immune response includes both an innate and adaptive immune response, the latter of which establishes a lasting immunological memory through cells such as memory T cells and memory B cells. The antibodies created during the initial immune response to the immunogenic composition can be produced in subsequent challenges of the same antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens, toxoids, inactivated organisms, or attenuated organisms, or a live organism or pathogen that exhibits the antigens, epitopes, nucleic acids, peptides, polypeptides, proteins, polysaccharides, lipids, haptens, or toxoids or any combination thereof. In this manner, the immunogenic composition may serve as a vaccine against a specific pathogen. Immunogenic compositions may also include one or more adjuvants to stimulate the immune response and increase the efficacy of protective immunity. In some embodiments, the adjuvants may comprise any of the adjuvants described herein.

[0171] The term “product combination” as used herein refers to set of two or more individual compounds, substances, materials, or compositions that can be used together for a unified function. In some embodiments, a product combination comprises at least one nucleic acid composition and at least one polypeptide composition that are used together to elicit an immune response when administered to a host, optionally to a greater degree than would be elicited if only one composition type were to be administered.

[0172] The terms “nucleic acid” or “nucleic acid molecule” as used herein refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, or phosphoramidate. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, virus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and / or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0173] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein refers to a sequence being after the 3’-end of a previous sequence,on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein refers to a sequence being before the 5’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0174] The term “codon optimized” regarding a nucleic acid as used herein refers to the substitution of codons of the nucleic acid to enhance or maximize translation in a host of a particular species without changing the polypeptide sequence based on species-specific codon usage biases and relative availability of each aminoacyl-tRNA in the target cell cytoplasm. Codon optimization and techniques to perform such optimization is known in the art. Programs containing algorithms for codon optimization are known to those skilled in the art. Programs can include, for example, OptimumGene, GeneGPS® algorithms, etc. Additionally, synthetic codon optimized sequences can be obtained commercially for example from Integrated DNA Technologies and other commercially available DNA sequencing services. Those skilled in the art will appreciate that gene expression levels are dependent on many factors, such as promoter sequences and regulatory elements. As noted for most bacteria, small subsets of codons are recognized by tRNA species leading to translational selection, which can be an important limit on protein expression. In this aspect, many synthetic genes can be designed to increase their protein expression level.

[0175] The nucleic acids described herein comprise nucleobases. Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0176] The terms “peptide”, “polypeptide”, and “protein” as used herein refers to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein refers to a sequence being before the N-terminus of a subsequent sequence.

[0177] In some embodiments, the nucleic acid or peptide sequences presented herein and used in the examples are functional in various biological systems including but not limited to humans, mice, rabbits, E. coli, yeast, and mammalian cells. In other embodiments, nucleic acid or peptide sequences sharing at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, sequence identity, or homology, or any percentage within a range defined by any two of the aforementioned percentages similarity to the nucleic acid or peptide sequences presented herein and used in the examples can also be used with no effect on the function of the sequences in biological systems. As used herein, the term “similarity” refers to a nucleic acid or peptide sequence having the same overall order of nucleotide or amino acids, respectively, as a template nucleic acid or peptide sequence with specific changes such as substitutions, deletions, repetitions, or insertions within the sequence. In some embodiments, two nucleic acid sequences sharing as low as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity can encode for thesame polypeptide by comprising different codons that encode for the same amino acid during translation.

[0178] The term “recombinantly expressed” as used herein refers to the production of proteins in optimized or adapted biological systems. These systems provide advantages over protein expression in a natural host, including but not limited to high expression (overexpression), ease of purification, ease of transformation, inducibility, low cost, or stability of the protein. In some embodiments, proteins are expressed in mammalian, bacteria, yeast, insect, or cell-free recombinant expression systems. Each system has its own advantages or disadvantages. For example, bacterial expression systems are highly optimized for overexpression, but may cause misfolding or aggregation of the produced protein, yeast systems are useful when post-translational modifications are necessary, and insect and mammalian systems are useful for proper RNA splicing that occurs in higher-order organisms. In some embodiments, recombinant polypeptides are produced and purified from mammalian, human, primary, immortalized, cancer, stem, fibroblasts, human embryonic kidney (HEK) 293, Chinese Hamster Ovary (CHO), bacterial, Escherichia coli, yeast, Saccharomyces cerevisiae, Pichia pastoris, insect, Spodoptera frugiperda Sf9, or S. frugiperda Sf21 cells, or in a cell-free system. In some embodiments, expression genes, vectors, or constructs are delivered to the recombinant expression systems in the form of plasmids, bacteriophages, viruses, adeno-associated viruses (AAVs), baculovirus, cosmids, fosmids, phagemids, BACs, YACs, or HACs. For more discussion on recombinant expression systems, see Gomes et al. “An Overview of Heterologous Expression Host Systems for the Production of Recombinant Proteins” ((2016) Adv. Anim. Vet. Sci.4(7):346- 356), hereby expressly incorporated by reference in its entirety.

[0179] The terms “autocatalytic peptide cleavage site” or “2A peptide” as used herein refer to a peptide sequence that undergo cleavage of a peptide bond between two constituent amino acids, resulting in separation of the two proteins that flank the sequence. The cleavage is believed to be a result of a ribosomal “skipping” of the peptide bond formation between the C-terminal proline and glycine in the 2A peptide sequence. Four autocatalytic peptide cleavage site sequences identified to date have seen substantial use in biomedical research: foot-and-mouth disease virus 2A (F2A); equine rhinitis A virus (ERAV) 2A (E2A); porcine teschovirus-12A (P2A), and Thosea asigna virus 2A (T2A). Insome embodiments, the P2A autocatalytic peptide cleavage site nucleic acid (SEQ ID NO: 21) and polypeptide (SEQ ID NO: 22) sequences are used. In some embodiments, the P2A nucleic acid or polypeptide used can be substituted with an F2A, E2A, or T2A nucleic acid or polypeptide.

[0180] In some embodiments, the nucleic acids or peptides used herein comprise sequences that produce HAV and / or HEV antigens.

[0181] Hepatitis is a disease resulting in swelling and inflammation of the liver. This disorder is commonly caused by viruses, five types of which are currently known (hepatitis A, B, C, D, and E). The medical problem presented by hepatitis A and E:

[0182] Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. Although HAV infection is generally self-limited and resolves without treatment, it can cause significant morbidity and mortality in certain high-risk populations, such as elderly individuals and those with underlying liver disease. Although the HAV infection is mainly asymptomatic in children, the risk of a severe disease increases with age.

[0183] Acute HAV infection can cause a range of symptoms, including fever, fatigue, nausea, vomiting, abdominal pain, and jaundice. In rare cases, acute HAV infection can progress to fulminant hepatitis, which can be fatal.

[0184] HAV is primarily spread through the fecal-oral route, and outbreaks are often associated with poor sanitation and hygiene. Effective vaccination and public health measures, such as improved sanitation and hygiene, have led to a significant decline in the incidence of HAV infection in many developed countries.

[0185] While there is no specific antiviral therapy for acute HAV infection, supportive care can help manage symptoms and prevent complications. Vaccination against HAV is highly effective and recommended for individuals at increased risk of infection.

[0186] Hepatitis E virus (HEV) is a major cause of acute viral hepatitis mainly in developing countries due to a suboptimal cleaning of the water supply. Like HAV, it is predominantly spread by the oro-fecal route, either from contaminated water, or from eating undercooked meat products from animals, especially pigs, in which HEV also circulates. Inthe western world HEV infection is mainly related to undercooked meat but there is a risk for blood born transmission as the blood supply is not screened for HDV.

[0187] The worldwide distribution of hepatitis E virus (HEV) and its ability to cause major epidemics in low-income countries calls for a pressing need for a global HEV vaccine. Populations at risk of severe forms of the infection are well identified: patients with chronic liver disease - at risk of liver failure; pregnant women - at risk of fulminant hepatitis or obstetrical complications; and immunosuppressed patients, particularly those with solid organ transplants - at risk of chronic hepatitis and rapid progression to cirrhosis (4-7). Virology of hepatitis A virus:

[0188] The hepatitis A virus (HAV) is a small, non-enveloped, single-stranded RNA virus in the family Picornaviridae. The genome of HAV is approximately 7.5 kb in length and encodes a polyprotein that is cleaved into functional proteins.

[0189] HAV is relatively stable in the environment and can survive for long periods of time on surfaces and in water. The virus replicates in the liver and is shed in high concentrations in feces, which is the primary route of transmission. HAV can also be transmitted through contaminated food and water or person-to-person contact.

[0190] HAV infection typically results in acute hepatitis that is usually self- limited and resolves within a few weeks to months. However, in some cases, particularly in older adults or those with pre-existing liver disease, HAV infection can result in severe or even fatal disease.

[0191] There are currently two FDA-approved vaccines available for hepatitis A, which have been shown to be highly effective in preventing HAV infection and disease (8- 10). Virology of hepatitis E virus:

[0192] Hepatitis E virus (HEV) is a small, non-enveloped, single-stranded RNA virus in the family Hepeviridae and genus orthohepevirus. There are four major species of orthohepevirus, (A-D), Orthohepevirus A, which is the species most responsible for humandisease, has eight genotypes with distinct geographic distributions, host range and modes of transmission.

[0193] HEV is primarily transmitted through the fecal-oral route, either via contaminated food and water or person-to-person contact. The virus targets hepatocytes and can cause acute hepatitis, which is usually self-limiting and resolves within a few weeks to months. However, in some cases, particularly in pregnant women or those with pre-existing liver disease, HEV infection can lead to severe or fulminant hepatitis.

[0194] HEV is positive strand RNA virus, whose genome includes three open reading frames, one for non-structural proteins, one for the capsid protein, and one for a putative viroporin. The virus replicates primarily in the liver and is shed in high concentrations in feces, which is the primary route of transmission.

[0195] There are currently no FDA-approved vaccines available for hepatitis E, although several vaccine candidates are in development (11-14). Epidemiology of HAV:

[0196] Hepatitis A is a viral infection that primarily affects the liver and is caused by the hepatitis A virus (HAV). The epidemiology of hepatitis A varies widely depending on geographic region and socioeconomic factors.

[0197] In developing countries with poor sanitation and hygiene, HAV is often transmitted through contaminated water and food, leading to high rates of infection in childhood and low rates of symptomatic disease in adulthood. In contrast, in developed countries with improved sanitation, HAV infection is less common and tends to occur more frequently in young adults with risk factors such as international travel, injection drug use, and sexual contact with infected individuals.

[0198] The incidence of hepatitis A has declined substantially in many developed countries due to improved sanitation and the widespread use of vaccines. However, in some parts of the world, hepatitis A remains a significant public health concern. For example, in many countries in the Middle East and North Africa, hepatitis A continues to be a major cause of acute liver failure.

[0199] The global burden of hepatitis A is difficult to estimate accurately due to underreporting and varying rates of asymptomatic infection. However, it is estimated thatthere were over 1.4 million cases of acute hepatitis A worldwide in 2016, resulting in over 11,000 deaths (15-17). Epidemiology of HEV:

[0200] Hepatitis E is a viral infection that affects the liver and is caused by the hepatitis E virus (HEV). The epidemiology of hepatitis E varies widely depending on geographic region and the HEV genotype involved.

[0201] HEV is most common in developing countries with poor sanitation and hygiene, where the virus is transmitted primarily through contaminated water and food. In these settings, HEV is responsible for large outbreaks of acute hepatitis, especially in areas with limited access to clean drinking water and sanitation facilities.

[0202] In developed countries with good sanitation, HEV is primarily transmitted through the consumption of contaminated pork and other meats. While HEV infection is generally self-limited and resolves on its own, severe disease may occur in certain populations, such as pregnant women, those with pre-existing liver disease, and those with compromised immune systems. A problem that has been noted in the past 10 years is the risk of bloodborne HEV in patients receiving multiple blood transfusions. Here the probable cause is the consumption of undercooked pork or wild boar meat causing a subclinical infection and donation of blood at the time of viremia. In immune suppressed transplant patients this can cause a severe liver disease.

[0203] The global burden of hepatitis E is difficult to estimate accurately due to underreporting and varying rates of asymptomatic infection. However, it is estimated that there were over 20 million HEV infections worldwide in 2015, resulting in over 44,000 deaths (18-20). Genotype distribution:

[0204] Hepatitis E viruses (HEV) of the orthohepevirus A genus are classified into eight major genotypes (genotypes 1 to 8), which differ in their genetic and antigenic characteristics, as well as in their geographic distribution, clinical features, and modes of transmission.

[0205] Genotypes 1 and 2 are typically associated with large waterborne outbreaks in developing countries, particularly in Asia, Africa, and the Middle East. Thesegenotypes are transmitted through fecal-oral contamination of food and water, and typically cause acute, self-limiting hepatitis, which is usually asymptomatic but can occasionally progress to fulminant hepatic failure in pregnant women and people with pre-existing liver disease.

[0206] Genotypes 3 and 4 are found worldwide and are primarily transmitted through consumption of contaminated meat, particularly pork and wild boar. These genotypes are also associated with sporadic cases of hepatitis E, which can range from asymptomatic infection to severe liver disease, particularly in people with pre-existing liver disease and those who are immunocompromised.

[0207] Genotype 3 has been identified in several animal species, including domestic pigs, wild boars, deer, rabbits, and rodents, and can be transmitted from animals to humans through consumption of contaminated meat or contact with infected animals or their environment. Genotype 4 has been found primarily in humans and domestic pigs in China, Taiwan, and other parts of Southeast Asia. Genotypes 5 and 6 have been found only in wild boar, while genotypes 7 and 8 are found in camels (with one instance of a presumed zoonotic infection of human).

[0208] Orthohepeviruses from genera B, C and D are found predominantly in chickens, rats and other rodents, and bats respectively, although, some of these appear to also to have zoonotic potential and therefore pose a risk to human health.

[0209] In summary, the different species and genotypes of hepatitis E virus have distinct epidemiological, clinical, and transmission characteristics, which are important for understanding the global burden of the disease and developing effective prevention and control strategies (21-23). The HEV genome:

[0210] The genome of hepatitis E virus (HEV) is a single-stranded, positive-sense RNA molecule that is approximately 7.2 kb in length. It contains three open reading frames (ORFs) that are separated by non-coding regions.

[0211] The first ORF encodes a non-structural protein that is involved in viral replication, while the second and third ORFs encode structural proteins that are involved in virus assembly and release. The structural proteins include the capsid protein, which formsthe outer shell of the virus particle, and a small membrane protein that is embedded in the viral envelope.

[0212] HEV is genetically diverse and is classified into eight major genotypes based on nucleotide sequence variations, of which genotypes 1-4 are the most relevant for human disease. Genotypes 1 and 2 are primarily associated with waterborne epidemics in developing countries, while genotypes 3 and 4 are zoonotic and are primarily transmitted through the consumption of contaminated meat products.

[0213] Understanding the genomic structure of HEV is important for developing effective diagnostic tools, vaccines, and antiviral therapies (24-26). The HAV genome:

[0214] Hepatitis A virus (HAV) is a small, non-enveloped virus with a single- stranded, positive-sense RNA genome of approximately 7.5 kilobases in length. The genome contains a single open reading frame that encodes a polyprotein of about 2,200 amino acids. The polyprotein is subsequently cleaved by viral and cellular proteases into four structural proteins (VP1-VP4) and seven nonstructural proteins (2A-2C and 3A-3D).

[0215] The VP1 protein is the major structural protein that forms the viral capsid, which protects the viral RNA genome. The non-structural proteins are involved in viral replication, assembly, and release.

[0216] HAV has a high degree of genetic stability, with limited sequence variability within a given outbreak or geographic region. However, several different genotypes of HAV have been identified based on nucleotide sequence differences, which can vary between 10% and 25% among genotypes.

[0217] Understanding the genomic structure of HAV is important for the development of sensitive diagnostic tests, vaccines, and antiviral therapies (27-28). The mechanism of protection against HAV:

[0218] Protection against hepatitis A virus (HAV) is primarily mediated by neutralizing antibodies directed against the viral capsid protein, VP1. These antibodies prevent viral attachment and entry into host cells, thereby limiting viral replication and spread.

[0219] The development of protective immunity to HAV is associated with the induction of a strong humoral immune response, with production of HAV-specific immunoglobulin G (IgG) antibodies. The levels of HAV-specific IgG antibodies in serum correlate with protection against infection, and individuals with high levels of anti-HAV IgG are considered immune to HAV infection.

[0220] The duration of protective immunity following natural HAV infection or vaccination varies, but immunity is generally long-lasting and may persist for decades in many individuals. Booster doses of the HAV vaccine are not routinely recommended for healthy individuals (29-31). The mechanism of protection against HEV:

[0221] The mechanism of protection against hepatitis E infection is through the induction of virus-specific antibodies, particularly immunoglobulin G (IgG) antibodies, which can neutralize the virus and prevent its replication in the liver.

[0222] The hepatitis E vaccine (Hecolin), which is currently approved only in China and Pakistan, is based on a recombinant viral protein that is highly immunogenic and capable of inducing a strong antibody response. The vaccine is administered in three doses, with the second dose given 1 month after the initial dose and the third 6 months after the first. Studies have shown that the vaccine is highly effective in preventing symptomatic hepatitis E infection, with efficacy rates of up to 100% reported in clinical trials.

[0223] In addition to vaccination, other measures to prevent hepatitis E infection include improved sanitation and hygiene practices, particularly in areas where the disease is endemic (32-33). Current vaccines in development for HEV:

[0224] There are currently several hepatitis E virus (HEV) vaccine candidates in development, including:

[0225] HEV 239 vaccine: This is a recombinant vaccine produced by GlaxoSmithKline (GSK) that contains a portion of the viral capsid protein. Phase 2 clinical trials have shown the vaccine to be safe and immunogenic, with high levels of seroconversion in vaccinated individuals.

[0226] HEV 239 + AS03 vaccine: This is a vaccine produced by GSK that contains the same recombinant capsid protein as the HEV 239 vaccine, but with an adjuvant system called AS03 added. Phase 3 clinical trials have shown the vaccine to be highly effective in preventing HEV infection, with an efficacy of over 95%.

[0227] Hecolin® vaccine: This is a vaccine produced by Xiamen Innovax Biotech that contains virus-like particles (VLPs) derived from the viral capsid protein. The vaccine has been shown to be safe and immunogenic in clinical trials, with high levels of seroconversion in vaccinated individuals.

[0228] HEV-Bremon vaccine: This is a vaccine produced by Bharat Biotech that contains a recombinant protein derived from the viral capsid protein. Phase 1 and 2 clinical trials have shown the vaccine to be safe and immunogenic, with high levels of seroconversion in vaccinated individuals (34-37). HEV 239 vaccine (GSK):

[0229] The GSK HEV 239 vaccine, also known as Engerix HEV, is a hepatitis E virus (HEV) vaccine that contains a recombinant truncated ORF2 protein of HEV genotype 1. The ORF2 protein is a structural protein that forms the capsid of the virus. The truncated ORF2 protein used in the vaccine contains only the amino-terminal 239 amino acids of the full-length protein, hence the name HEV 239 vaccine.

[0230] The HEV 239 vaccine is produced using a recombinant expression system in Saccharomyces cerevisiae yeast. The truncated ORF2 protein is purified and formulated with an adjuvant system called AS04, which consists of aluminum hydroxide and a Toll-like receptor 4 agonist called MPL. The adjuvant system enhances the immune response to the vaccine and improves its effectiveness.

[0231] The GSK HEV 239 vaccine has been shown to be safe and immunogenic in clinical trials, with high levels of seroconversion in vaccinated individuals. It is administered in a three-dose schedule, with the second and third doses given at 1 and 6 months after the first dose, respectively (38-39). HEV 239 + AS03 vaccine (GSK):

[0232] The GSK HEV 239 + AS03 vaccine is a hepatitis E virus (HEV) vaccine that contains a recombinant truncated ORF2 protein of HEV genotype 1, similar to theEngerix HEV vaccine. The ORF2 protein is a structural protein that forms the capsid of the virus. The truncated ORF2 protein used in the vaccine contains only the amino-terminal 239 amino acids of the full-length protein.

[0233] In addition to the truncated ORF2 protein, the GSK HEV 239 + AS03 vaccine also contains an adjuvant system called AS03, which consists of squalene, tocopherol, and a Tween 80 emulsifier. The adjuvant system enhances the immune response to the vaccine and improves its effectiveness.

[0234] The GSK HEV 239 + AS03 vaccine has been shown to be safe and highly immunogenic in clinical trials, with high levels of seroconversion in vaccinated individuals. It is administered in a two-dose schedule, with the second dose given 21-56 days after the first dose (40-41). Hecolin® vaccine:

[0235] The Hecolin® vaccine is a hepatitis E virus (HEV) vaccine produced by Xiamen Innovax Biotech, and is the only hepatitis E vaccine currently approved, although only in China and Pakistan. It has proven to be effective and safe, however its accessibility is limited as well as data on its long-term efficacy and the duration of its protection. It contains virus-like particles (VLPs) derived from the viral capsid protein of HEV genotype 1. The VLPs are produced using a recombinant baculovirus expression system, and they closely mimic the native structure of the virus, which allows for a strong immune response.

[0236] The HEV capsid protein consists of three domains: S (shell), M (middle), and P (protruding). The VLPs used in the Hecolin® vaccine are composed of the S and P domains of the capsid protein. The P domain contains the major antigenic sites of the capsid protein and is responsible for inducing a neutralizing antibody response.

[0237] In addition to the HEV VLPs, the Hecolin® vaccine contains an adjuvant system called AS04, which consists of aluminum hydroxide and a Toll-like receptor 4 agonist called MPL. The adjuvant system enhances the immune response to the vaccine and improves its effectiveness.

[0238] The Hecolin® vaccine is administered in a three-dose schedule, administered intramuscularly into the deltoid muscle as a single injection on Days 1, 29, and 180. Clinical trials have shown the vaccine to be safe and immunogenic, with high levels of seroconversion in vaccinated individuals.

[0239] The Hecolin® vaccine is currently being tested in a phase III study in the USA (42-44). HEV-Bremon vaccine:

[0240] The HEV-Bremon vaccine is a recombinant protein subunit vaccine that contains the full-length open reading frame 2 (ORF2) protein of the HEV genotype 1. ORF2 encodes the capsid protein, which is responsible for forming the virus particle. The protein is produced using a recombinant expression system, purified and then adsorbed onto aluminum hydroxide as an adjuvant.

[0241] The HEV-Bremon vaccine is administered in two doses, with the second dose given at least six months after the first dose. It has been shown to be highly effective in preventing hepatitis E in clinical trials (45-46). Features of embodiments of the invention:

[0242] The present disclosure relates generally to the use of recombinant nucleic acids, DNA, RNA, proteins, polypeptides, or peptides comprising HAV and / or HEV antigens to induce immune responses, antibody production, immune protection, or immunity against HAV and / or HEV infections.

[0243] Proposed herein is a combined immunogenic composition or vaccine for HAV and HEV, which protects against the two principal form of oro-fecally transmitted viral hepatitis with a single course of vaccination. The combined immunogenic composition or vaccine has several advantages to a single therapeutic or prophylactic regimen because they simplify the immunization process and reduce the number of shots a person needs to receive. Combination immunogenic compositions or vaccines work by including multiple antigens in a single composition. The body develops an immune response to each antigen, providing protection or immunization against both diseases, HAV and HEV, included in the immunogenic composition or vaccine. The combination may also induce heterologous immunity where the immune response against one pathogen is beneficial also for the immunity of the other pathogen.

[0244] Development of a global vaccine against HEV requires protection against both genotype 1 and 3. A pan-genotypic vaccine design has therefore been applied for SVF- 004.5 by inclusion of HEV ORF2 gt1 and gt3 sequences.HAV components in embodiments of the vaccine compositions:

[0245] VP2 and VP3 are two structural proteins of the Hepatitis A virus (HAV), which are part of the viral capsid. The capsid is the outer layer of the virus, which encloses the genetic material (RNA) and protects it from the host immune system.

[0246] VP2 and VP3 are closely associated with each other and form a shell-like structure around the RNA genome of the virus. VP3 is located on the inner surface of the shell, while VP2 is on the outer surface. Together, they form the icosahedral capsid structure of the virus.

[0247] VP2 and VP3 are essential for the assembly and stability of the capsid, as well as for viral infectivity. In particular, VP3 plays a key role in the initiation of viral replication, while VP2 is involved in viral entry into host cells.

[0248] Studies have shown that VP2 and VP3 contain antigenic sites, which can be recognized by the host immune system and trigger the production of virus-specific antibodies. These antibodies can neutralize the virus and prevent further infection, making VP2 and VP3 important targets for the development of vaccines and antiviral therapies against hepatitis A (47). HEV components in embodiments of the vaccine compositions:

[0249] HEV has a positive-sense single-stranded RNA genome that contains three open reading frames (ORFs). ORF2 is the largest and encodes the viral capsid protein, also known as open reading frame 2 protein (ORF2p) or the viral protein 1 (VP1). This protein forms the outer shell of the HEV virus and is responsible for the binding, entry, and packaging of the viral RNA into the virion.

[0250] The ORF2 protein is divided into three domains: S, M, and P. The S domain forms the outermost part of the capsid and contains the immunodominant epitopes that are recognized by host neutralizing antibodies. The M domain forms the middle layer of the capsid and is involved in stabilizing the capsid structure. The P domain is the innermost part of the capsid and is involved in RNA binding and packaging.

[0251] The ORF2 protein is essential for viral replication and pathogenesis and is a major target for the host immune response to HEV infection. The S domain of ORF2 is the main target of neutralizing antibodies and is therefore an important component of HEVvaccines. Several vaccine candidates have been developed based on the ORF2 protein, including the Hecolin® vaccine, which is currently approved for use in China (48). Linker sequences in embodiments of the vaccine compositions:

[0252] Linker sequences are important in vaccine design for several reasons:

[0253] a. To improve the stability of the vaccine: Linker sequences can be used to connect different components of the vaccine, such as antigens and adjuvants, to improve the stability of the vaccine. By stabilizing the vaccine components, the vaccine can have a longer shelf life and be more effective.

[0254] b. To optimize immune responses: Linker sequences can be designed to optimize the immune responses generated by the vaccine. For example, linker sequences can be used to control the orientation and presentation of antigens to immune cells, which can improve the recognition and response to the vaccine.^

[0255] c. To reduce side effects: Linker sequences can be designed to minimize the side effects of the vaccine. For example, linker sequences can be used to separate antigens from toxic or immunogenic adjuvants, which can reduce the risk of adverse reactions.

[0256] In some compositions, we have included a linker sequence to optimize the immune responses. In some embodiments, the linker sequence can be any of the linker sequences described herein. Examples of commonly used linker sequences:

[0257] a. Flexible glycine (GGG) linker, is often used in combination with serine residues (GGGS). This linker is known for its flexibility, which allows it to accommodate different distances between the connected domains. The flexible glycine linker is often used in recombinant protein production to join two proteins or domains in a single construct. In vaccine design, the glycine-serine linker can be used to connect different antigenic domains or to link an antigen to an adjuvant or carrier protein. The use of a flexible linker like GGG or GGGS can increase the accessibility and immunogenicity of the antigen, leading to a stronger immune response.

[0258] b. Flexible glycine-serine (GGGS) linker: This linker is often used to connect two proteins or domains in a vaccine construct. The GGGS linker is flexible and canaccommodate different distances between the connected domains, which can optimize the immune response.

[0259] c. Protease cleavage sites: Protease cleavage sites are often used as linkers to connect vaccine components that need to be separated before they are presented to the immune system. For example, a protease cleavage site can be used to link an antigen to a toxin or adjuvant that needs to be removed before the vaccine is administered.^

[0260] d. Coiled-coil linkers: Coiled-coil linkers are composed of two or more alpha-helices that can interact with each other to form a stable coiled-coil structure. These linkers can be used to connect components and stabilize the comnposition.

[0261] e. Multiple antigen peptide (MAP) linkers: MAP linkers are used to display multiple copies of an antigen or epitope to the immune system. MAP linkers are composed of a scaffold that is modified with multiple copies of the antigen or epitope, which can increase the immunogenicity of the immunogenic composition or vaccine.

[0262] f. Tryptophan (W) linkers: Tryptophan linkers are often used to connect protein domains in a construct. The tryptophan amino acid has unique optical properties that can be used to monitor the conformation and stability of the linker. Self-cleaving peptides in the vaccine compositions:

[0263] In some immunogenic composition or vaccine compositions, we have included a P2Aself-cleaving peptide. P2A is a short (22 aa) peptide sequence, derived from the "Thosea asigna virus 2A" (T2A) peptide, that acts as a self-cleaving peptide, meaning it can catalyse its own cleavage without the need for additional enzymes or cofactors. The P2A peptide sequence can be used for the production of multiple proteins from a single mRNA transcript. P2A cleavage works by inducing a ribosomal skipping during translation, resulting in the separation of the polypeptide chain into two distinct proteins or functional domains. Embodiments of the invention:

[0264] The combined HAV / HEV immunogenic composition or vaccine can induce neutralizing antibodies and activate T cells responses. The immune responses can be broadly targeted to the capsid of HEV and cover multiple genotypes of HEV. The P2A self- cleaving peptide can be included to create multiple proteins from one composition.

[0265] The immunogenic composition or vaccine will be evaluated as a recombinant nucleic acids, DNA, RNA, proteins, polypeptides, or peptides comprising HAV and / or HEV antigens. The different immunogenic compositions or vaccine compositions can be used alone or in different combinations (prime-boost). Problems overcome by the invention. Combined immunogenic composition or vaccine:

[0266] Combining immunogenic compositions or vaccines for hepatitis A virus (HAV) and hepatitis E virus (HEV) could be beneficial in areas where both viruses are prevalent or where there is a high risk of exposure to both viruses, such as in regions with poor sanitation and hygiene, or in travelers to endemic regions. Vaccination against both HAV and HEV can help prevent illness and reduce the burden of these diseases on individuals and healthcare systems.

[0267] Hepatitis A and hepatitis E have similar modes of transmission, primarily through ingestion of contaminated food or water. Both viruses can cause acute hepatitis, but hepatitis E can also cause chronic infection in some individuals, particularly in those with underlying liver disease or compromised immune systems. In addition, hepatitis E has been associated with high mortality rates in pregnant women.

[0268] Vaccines for both HAV and HEV (only available in China and for people aged between 18-65 years) are currently available and have been shown to be safe and effective. Combining the immunogenic compositions or vaccines into a single product could simplify vaccination programs and increase vaccine uptake, particularly in areas with limited healthcare resources. Pan-genotypic immunogenic compositions or vaccines:

[0269] To ensure broad protection against all genotypes of HEV, an immunogenic composition or vaccine should ideally contain antigens from multiple genotypes. This can help ensure that the immunogenic composition or vaccine is effective against all circulating strains of the virus, even as new genotypes emerge. Including multiple genotypes in an immunogenic composition or vaccine can also help provide cross-protection against related strains, which can be important in areas where multiple genotypes are endemic.

[0270] Studies have shown that vaccines containing multiple HEV genotypes can induce broad and long-lasting immune responses in animal models.

[0271] Overall, including multiple genotypes in a HEV immunogenic composition or vaccine can be important for ensuring broad protection against all circulating strains of the virus and for providing long-lasting immunity. Combinatorial approaches using HAV and HEV

[0272] There have been studies investigating the use of hepatitis A virus (HAV) antigens in hepatitis E virus (HEV) virus-like particles (VLPs) for vaccine development.

[0273] One study reported the successful production of chimeric VLPs containing both HEV and HAV antigens. The researchers found that these chimeric VLPs induced immune responses against both viruses in mice, suggesting that they could be a promising candidate for immunization against both hepatitis E and A.

[0274] Another study described the design and production of a novel candidate using HAV antigens fused to the HEV VLP. The vaccine was found to elicit robust immune responses against both HAV and HEV in mice, indicating its potential as a dual vaccine against both viruses.

[0275] Other studies report incorporating HAV antigens into HEV VLPs to provide a dual vaccine against hepatitis E and A (49-51).

[0276] To our knowledge, there have not been any studies reported on the development of a hepatitis E virus (HEV) vaccine using hepatitis A virus (HAV) virus-like particles (VLPs).

[0277] An additional study described the development of a hepatitis E vaccine candidate using a fusion protein containing the HEV antigenic domain and the core domain of the bacteriophage Qȕ coat protein, which forms a VLP. The vaccine was found to induce immune responses in mice.

[0278] Another study reported the production of VLPs derived from insect cells, which were engineered to display the HEV antigen. The vaccine was found to induce immune responses in mice.

[0279] Further research is needed to evaluate the safety and efficacy of such compositions (52-54).Description of some preferred embodiments:

[0280] SVF-004.1-SVF-004.8 consists of different combinations of HAV (VP2, VP3) and HEV (ORF2gt1, ORF2gt2) sequences (FIG. 1).

[0281] We are not aware of any combined HAV / HEV vaccine based on;

[0282] a) hepatitis E virus (HEV) antigens incorporated into a hepatitis A virus (HAV) virus-like particles (VLPs)

[0283] b) covering multiple HEV genotypes

[0284] c) one vaccine for two infections. Components of HAV included in embodiments of a combined immunogenic composition or vaccine:

[0285] VP2 and VP3 is structural proteins of HAV that is a part of the viral capsid, the protective outer shell of the virus.

[0286] The HAV VP2 protein is approximately 63 kDa in size and contains 587 amino acids. It is composed of two domains, a shell (S) domain and a protruding (P) domain. The S domain forms the inner core of the capsid and is responsible for stabilizing the capsid structure, while the P domain forms the outer surface of the capsid and is involved in viral attachment and entry into host cells. The VP2 protein plays an essential role in the assembly and maturation of HAV virions, as well as in the induction of protective immune responses. It is also an important target for diagnostic assays and vaccine development against HAV infection. Antibodies against VP2 can provide protection against infections of the virus.

[0287] The HAV VP3 protein has a molecular weight of approximately 27 kDa and consists of 238 amino acids. It has a characteristic beta-barrel structure and forms 60 copies to assemble into the icosahedral capsid structure of the virus. The VP3 protein is involved in several key functions, including the assembly and stability of the viral capsid, and the protection of the viral RNA genome from environmental factors. The VP3 protein also plays a role in the attachment and entry of the virus into host cells. Presence of VP3 antibodies has been shown to correlate with long lasting protection against HAV infection.Components of HEV included in embodiments of a combined immunogenic composition or vaccine:

[0288] ORF2, or Open Reading Frame 2, is a region of the genome of Hepatitis E Virus (HEV) that encodes a viral protein called the capsid protein. The capsid protein is the main structural protein of the virus and is responsible for forming the outer shell, or capsid, of the viral particle. The capsid protein plays a critical role in protecting the viral RNA genome and facilitating viral entry into host cells.

[0289] The capsid protein encoded by ORF2 is translated from a single long open reading frame into a precursor protein, which then undergoes post-translational processing to yield the mature capsid protein. The mature capsid protein self-assembles into a symmetrical, quasi-icosahedral structure that forms the outer shell of the viral particle. The capsid protein is composed of multiple subunits arranged in a repeating pattern, and it has distinct antigenic regions that can trigger an immune response in the host.

[0290] ORF2 is an important target for diagnostics and vaccine development against HEV, as it is highly immunogenic and can induce protective immunity in infected individuals. Additionally, the capsid protein is involved in host cell recognition and viral entry, making it a key determinant of HEV infectivity. Combinatorial immunogenic composition or vaccine against HAV and HEV:

[0291] By combining HAV and HEV sequences in the immunogenic composition or vaccine composition an immune response can be achieved against one pathogen that indirectly affects the immune response against the other pathogen by altering the overall immune environment or immune cell functions (heterologous immunity). In this way immune responses against HAV can improve HEV immunity and vice versa. Methods of Therapy

[0292] The terms “prime” and “boost” as used herein related to separate immunogenic compositions used in a heterologous prime-boost immunization approach. Immunizations or vaccines commonly require more than one administration of an immunogenic composition to induce a successful immunity against a target pathogen in a host. Compared to this homologous approach where the same composition is provided for alladministrations, a heterologous prime-boost administration can be more effective in establishing robust immunity with greater antibody levels and improved clearing or resistance against some pathogens such as HAV and / or HEV. In a heterologous prime-boost administration, at least one prime dose comprising one type of immunogenic composition is first provided. After the at least one prime dose is provided, at least one boost dose comprising another type of immunogenic composition is then provided. Administration of the at least one boost dose is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after the at least one prime dose is administered or within a range of time defined by any two of the aforementioned time points e.g., within 1-48 days or 1-48 weeks. In some embodiments, the prime dose comprises a nucleic acid (e.g. DNA or RNA) that encodes for one or more antigens or epitopes, and the boost dose comprises a polypeptide that comprises one or more antigens or epitopes. In the host, the nucleic acid prime is translated in vivo to elicit an immune reaction and causes a greater response against the subsequent polypeptide boost. In some embodiments, the nucleic acid prime comprises, consists essentially of, or consists of sequences from HAV and / or HEV. In some embodiments, the sequences from HAV encode for VP2 and / or VP3 polypeptides. In some embodiments, the sequences from HEV encode for ORF2gt1and / or ORF2gt3polypeptides. In some embodiments, the nucleic acid sequences are codon optimized for expression in humans. In some embodiments, the polypeptide boost comprises, consists essentially of, or consists of polypeptides from HAV and / or HEV. In some embodiments, the polypeptides from HAV are VP2 and / or VP3 polypeptides. In some embodiments, the polypeptides from HEV are ORF2gt1and / or ORF2gt3polypeptides. In some embodiments, the prime dose is a polypeptide, and the boost dose is a nucleic acid. General information about heterologous prime-boost approaches can be found in PCT Publications WO 2006 / 013106, WO 2006 / 040334, WO 2008 / 094188, each of which are hereby expressly incorporated by reference for the purpose of describing prime-boost methods.

[0293] In some embodiments, administration of the nucleic acid prime and polypeptide boost comprising components of HAV and / or HEV in a subject (e.g. mouse, rabbit, monkey, human) results in greater anti-VP2, anti-VP3, anti-ORF2gt1, anti-ORF2gt3, anti-HAV and / or anti-HEV antibody titer at a ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 100000, or 1000000 or any ratiowithin a range defined by any two of the aforementioned ratios compared to nucleic acid- only or polypeptide-only immunized, or unimmunized control organisms, quantified by techniques known in the art such as ELISA. In some embodiments, administration of the nucleic acid prime and polypeptide boost comprising components of HAV and / or HEV in a subject results in serum that neutralizes the in vitro or in vivo infectivity of HAV and / or HEV more effectively and reduces the incidence of infection or multiplicity of infection (MOI) to a ratio of 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 or any ratio within a range defined by any two of the aforementioned ratios compared to sera from nucleic acid-only or polypeptide-only immunized, or unimmunized control organisms. In some embodiments, administration of the nucleic acid prime and polypeptide boost comprising components of HAV and / or HEV in a subject results in a greater number of interferon gamma (IFNȖ)- positive cells (e.g. T cells, macrophages, natural killer (NK) cells) at a ratio of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 5000, or 10000, or any ratio within a range defined by any two of the aforementioned ratios compared to nucleic acid-only or polypeptide-only immunized, or unimmunized control organisms.

[0294] In some embodiments, the immunogenic compositions or product combinations are administered with an adjuvant. In some embodiments, the adjuvant may comprise any of the adjuvants described herein. In some embodiments, the immunogenic compositions or product combinations are administered enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously or any combination thereof. In some embodiments, the immunogenic compositions or product combinations are administered in conjunction with an antiviral therapy or antiviral prodrug therapy having an effect against HAV and / or HEV, or side effects caused by HAV and / or HEV, including but not limited to chloroquine, hydroxychloroquine, favipiravir, favilavir, remdesivir, tocilizumab, galidesivir, sarilumab, lopinavir, ritonavir, darunavir, ribavirin, interferon-Į, pegylated interferon-Į, or interferon alfa-2b, a prodrug or precursor thereof, or any combination thereof.

[0295] The terms “in vivo electroporation”, “electroporation”, and “EP” as used herein refers to the delivery of genes, nucleic acids, DNA, RNA, proteins, or vectors intocells of living tissues or organisms using electrical currents using techniques known in the art. Electroporation can be used as an alternative to other methods of gene transfer such as viruses (transduction), lipofection, gene gun (biolistics), microinjection, vesicle fusion, or chemical transformation. Electroporation limits the risk of immunogenicity and detrimental integration or mutagenesis of the cell genome. DNA vectors such as plasmids are able to access the cell nucleus, enabling transcription and translation of constituent genes. In some embodiments, the genes, nucleic acids, DNA, RNA, proteins, or vectors are added to the target tissue or organism by subcutaneous, intramuscular, or intradermal injection. An electroporator then delivers short electrical pulses via electrodes placed within or proximal to the injected sample. As used herein, the term “im / EP” refers to in vivo electroporation of a sample delivered intramuscularly (“im”).

[0296] The term “transient immunosuppression” as used herein refers to a C57BL / 6 mouse model wherein transient immunosuppression is accomplished by administration of type I interferon (IFN-1) blocking antibody (mAb)-5A3 before challenge. The term “type I Interferon receptor (IFNAR- / -) mice” refers to a mouse model wherein the mice are engineered not to express the type I Interferon receptor. Other similar mouse models can be used as alternatives.

[0297] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments.

[0298] The term “% w / w” or “% wt / wt” as used herein has its ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0299] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.EXAMPLES

[0300] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims. Example 1: Exemplary HAV and / or HEV immunogenic constructs

[0301] The recombinant HAV and / or HEV constructs were assembled by combining one or more HAV sequences comprising VP2 and / or VP3, and / or one or more HEV sequences comprising ORFgt1 and / or ORFgt3, optionally with a P2A autocatalytic peptide cleavage site, and / or a linkage sequence. Schematics for eight exemplary recombinant constructs are shown in FIG. 1, and nucleotide and amino acid sequences comprising the constructs are depicted in FIG. 2-11. Alternatively, nucleotide and amino acid sequences for HAV and / or HEV constructs are also included in TABLE 1. TABLE 116Example 2: In vitro characterization

[0302] SVF-004.1-SVF-004.8 DNA / RNA and protein will be evaluated in vitro by transient transfection and western blot analysis to detect and quantify the expression level of the different proteins in the immunogenic compositions. 0.1-10 ug SVF-004.1-SVF-004.8 DNA / RNA and protein will be evaluated for transfection of different cell lines (HEK293, HeLa, CHO) using appropriate commercially available transfection reagents. Protein will be isolated from cell lysates and analyzed for protein expression using western blot. Example 3: In vivo characterization

[0303] Mice will be immunized intramuscularly and may be subjected to electroporation with 1-3 sequential doses of 5-50 ^g DNA / RNA comprising either SVF- 004.1-SVF-004.8, a polypeptide composition comprising (1-3 sequential does of 5-20 ^gprotein with adjuvant), or a DNA / RNA composition comprising SVF-004.1-SVF-004.8 followed by a polypeptide composition comprising SVF-004.1-SVF-004.8. two to four weeks after final administration of the compounds, blood serum will be tested for HAV / HEV specific antibodies, cross-reactivity between HEV genotypes, and PBMC will be tested for IFNȖ / IL-2 production in response to HAV / HEV antigens by ELISpot.

[0304] SVF-004.1-SVF-004.8 will be used for a DNA / RNA prime / protein boost immunization to build adaptive immunity and induce antibody production against HAV / HEV in vivo.

[0305] T cell responses will be evaluated by collecting splenocytes 1-4 week after the final vaccination and isolating IFN-Ȗ+ and IL-2+ T cells via ELISpot. Isolated T cells will be stimulated with peptide pools comprising the HAV / HEV segments. The proportion of IFN-Ȗ+ and IL-2+ T cells responding to the peptides of the HAV / HEV peptide pool will be assessed. Finally, Th1 and Th2 cytokine titers will be assessed in the sera of mice following the final vaccination. Example 4: Evaluation of Immunogenic Effect of HAV / HEV Vaccine Compositions

[0306] The immunogenic and protective effects of the HAV / HEV immunogenic compositions will be tested in mice immunized with the HAV / HEV immunogenic compositions comprising the protein and / or nucleotide sequences of SVF-004.1-8. The efficacy of the HAV / HEV immunogenic compositions will be assessed in two mouse strains (C57Bl / 6 and BALB / c). As a control, the Hecolin® vaccine, and one of the inactivated HAV vaccines like Havrix, Vaqta,, Avaxim, will be included as control. Example 5

[0307] Animals

[0308] Female BALB / c (H-2d) mice were obtained from Charles River Laboratories. All mice were 8-10 weeks old at the start of the experiments and maintained under standard conditions.

[0309] DNA Plasmids

[0310] Plasmids encoding for genotypes 1 and 3 of the HEV ORF2 and the HAV VP2 and VP3, were used in this study as fusion constructs, optionally cleaved by a P2A, consisting of different combinations of HAV and HEV sequences. Some constructs encode a linker sequence. All genes were cloned into the pVAX1 backbone (Invitrogen, Carlsbad, CA) using restriction sites BamHI and XbaI. Plasmids were grown in TOP10 E.coli cells (Life Technologies, Carlsbad, CA) and purified for in vivo injections using Qiagen Endofree DNA purification kit (Qiagen GmbH) following manufacturer´s instructions. The correct gene size was confirmed by restriction enzyme digests using ApaI and HindIII (Fast Digest, Thermo Fisher Scientific).

[0311] Immunization protocols for evaluating immunogenicity of HAV / HEV plasmids in mice and rabbits

[0312] To evaluate the immunogenicity of the constructs in vivo, mice were immunized three times with three weeks intervals between injections and sacrificed two weeks later for blood collection. In brief, female BALB / c mice (five per group) were immunized intramuscularly (i.m.) in the tibialis cranialis anterior (TA) muscle with 50 ^g plasmid DNA in a volume of 50 ^L in sterile PBS by regular needle (27G) injection followed by in vivo electroporation (EP) using the Cliniporator2 device (IGEA, Carpi, Italy). During in vivo electroporation, a 1 ms 600 V / cm pulse followed by a 400 ms 60 V / cm pulse pattern was used to facilitate better uptake of the DNA. Prior to vaccine injections, mice were given analgesic and kept under isoflurane anesthesia during the vaccinations.

[0313] Antibody detection by ELISA

[0314] Detection of mouse IgG against HEV ORF2 and HAV VP2 and VP3 (5- 10Pg / mL) was performed using protocols known in the art. Antibody titers determined as endpoint serum dilutions at which the OD value at 405 nm is at least twice the OD of the negative control (non-immunized or control animal serum) at the same dilution.

[0315] SDS-Page and Western blot

[0316] Western Blot was essentially performed as generally known in the art. HEK 293 cells were transfected with SVF-004.7 DNA plasmid (only containing HAV VP2and VP3), using Lipofectamine® 3000 Transfection Reagent (Thermo Fisher Scientific). For protein detection, pooled serum from SVF-004.1, SVF-004.2, SVF-004.3, SVF-004.4, SVF- 004.5, SVF-004.6, SVF-004.7, and SVF-004.8 vaccinated BALB / C mice diluted 1:1000 (polyclonal mouse serum) and rabbit anti-mouse Immunoglobulins HRP (Thermo Fischer Scientific) diluted 1:5000 (secondary antibody) was used. For Chemiluminescence detection, Pierce TM ECL Plus Western Blotting Substrate was used, and images were collected with Gel Doc XR+ System (Biorad). The data, as shown in FIG. 15 indicated that at least constructs SVF-004.1, SVF-004.5, and SVF-004.7 induced antibodies to HAV.

[0317] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0318] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0319] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodimentscontaining only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or claims, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0320] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0321] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0322] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0323] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. REFERENCES 1. Cao G, Jing W, Liu J, Liu M. The global trends and regional differences in incidence and mortality of hepatitis A from 1990 to 2019 and implications for its prevention. Hepatol Int. 2021 Oct;15(5):1068-1082. Doi: 10.1007 / s12072-021-10232-4. Epub 2021 Aug 3. PMID: 34345993; PMCID: PMC8514357. 2. Webb GW, Dalton HR. Hepatitis E: an underestimated emerging threat. Ther Adv Infect Dis. 2019 Apr 3;6:2049936119837162. Doi: 10.1177 / 2049936119837162. PMID: 30984394; PMCID: PMC6448100. 3. Khongviwatsathien S, Thaweerat W, Atthakitmongkol T, Chotiyaputta W, Tanwandee T. 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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide.

2. The nucleic acid of claim 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1polypeptide, and a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

3. The nucleic acid of claim 2, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

4. The nucleic acid of claim 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP3 polypeptide, and a nucleic acid sequence encoding a HEV ORFgt1 polypeptide.

5. The nucleic acid of claim 4, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 9, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

6. The nucleic acid of claim 4, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding a linker sequence.

7. The nucleic acid of claim 6, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identityto SEQ ID NO: 11, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

8. The nucleic acid of claim 4, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

9. The nucleic acid of claim 8, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 7, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

10. The nucleic acid of claim 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1polypeptide, a nucleic acid sequence encoding a HEV ORFgt3polypeptide, at least one a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence, and at least one nucleic acid sequence encoding a linker sequence.

11. The nucleic acid of claim 10, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 13, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

12. The nucleic acid of claim 1, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, a nucleic acid sequence encoding a HEV ORFgt1 polypeptide, and at least one a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

13. The nucleic acid of claim 12, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identityto SEQ ID NO: 15, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV and HEV.

14. A polypeptide comprising at least one hepatitis A virus (HAV) polypeptide sequence and at least one hepatitis E virus (HEV) polypeptide sequence.

15. The polypeptide of claim 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, and a HEV ORFgt1polypeptide sequence.

16. The polypeptide of claim 15, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

17. The polypeptide of claim 14, wherein the polypeptide comprises an HAV VP3 polypeptide sequence, and a HEV ORFgt1polypeptide sequence.

18. The polypeptide of claim 17, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 10, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

19. The polypeptide of claim 17, wherein the polypeptide sequence further comprises a linker sequence.

20. The polypeptide of claim 19, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 12, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

21. The polypeptide of claim 17, wherein the polypeptide sequence further comprises a P2A autocatalytic cleavage sequence.

22. The polypeptide of claim 21, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 8, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

23. The polypeptide of claim 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, an HEV ORFgt1 polypeptide sequence, a HEV ORFgt3 polypeptide sequence, at least one P2A autocatalytic cleavage sequence, and at least one linker sequence.

24. The polypeptide of claim 23, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 14, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

25. The polypeptide of claim 14, wherein the polypeptide comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, an HEV ORFgt1 polypeptide sequence, and at least one P2A autocatalytic cleavage sequence.

26. The polypeptide of claim 25, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 16, wherein the polypeptide comprises an epitope or antigen of HAV and HEV.

27. The nucleic acid of any one of claims 1-13 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

28. The polypeptide of any one of claims 14-26 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

29. The polypeptide of any one of claims 14-26 or 28, wherein the polypeptide is recombinantly expressed.

30. The polypeptide of any one of claims 14-26 or 28-29, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

31. An immunogenic composition or product combination comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and at least one HEV polypeptide.

32. The immunogenic composition or product combination of claim 31, wherein the nucleic acid comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

33. The immunogenic composition or product combination of claim 31 or 32, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, 7, 9, 11, 13, or 15, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein thenucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

34. The immunogenic composition or product combination of any one of claims 31-33, wherein the nucleic acid is codon optimized for expression in a human.

35. The immunogenic composition or product combination of any one of claims 31-34, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3 polypeptide sequence; v) a P2A autocatalytic cleavage site polypeptide sequence; vi) a linker polypeptide sequence; or any combination thereof.

36. The immunogenic composition or product combination of any one of claims 31-35, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, 8, 10, 12, 14, or 16, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and HEV in a subject, such as a mammal, preferably a human.

37. The immunogenic composition or product combination of any one of claims 31-36, wherein the polypeptide is recombinantly expressed.

38. The immunogenic composition or product combination of any one of claims 31-37, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

39. The immunogenic composition or product combination of any one of claims 31-38, further comprising an adjuvant.

40. The immunogenic composition or product combination of claim 39, wherein the adjuvant is alum.

41. The immunogenic composition or product combination of any one of claims 31-40, wherein the nucleic acid is provided in a recombinant vector.

42. A method of generating an immune response in a subject using the immunogenic composition or product combination set forth in any one of claims 31-41, comprising: a) administering to the subject at least one prime dose comprising the nucleic acid; and b) administering to the subject at least one boost dose comprising the polypeptide; or c) administering to the subject at least one prime dose comprising the polypeptide; and d) administering to the subject at least one boost dose comprising the nucleic acid.

43. The method of claim 42, wherein the at least one boost dose further comprises an adjuvant.

44. The method of claim 43, wherein the adjuvant is alum.

45. The method of any one of claims 42-44, wherein the at least one boost dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after the at least one prime dose is administered or within a range of time defined by any two of the aforementioned time points e.g., within 1-48 days or 1-48 weeks.

46. The method of any one of claims 42-45, wherein the administration is provided enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously or any combination thereof, and optionally with in vivo electroporation.

47. The method of any one of claims 42-46, wherein the administration is performed in conjunction with an antiviral therapy or an antiviral prodrug therapy.

48. The method of claim 47, wherein the antiviral therapy or antiviral prodrug therapy comprises administration of chloroquine, hydroxychloroquine, favipiravir, favilavir, remdesivir, tocilizumab, galidesivir, sarilumab, lopinavir, ritonavir, darunavir, ribavirin, interferon-Į, pegylated interferon-Į, interferon alfa-2b, a prodrug or precursor thereof, or any combination thereof.

49. An immunogenic composition or product combination for use in the treatment or inhibition of hepatitis A virus (HAV) and / or hepatitis E virus (HEV), comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a HAV polypeptide and at least one nucleic acid sequence encoding a HEV polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and at least one HEV polypeptide.

50. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 49, wherein the nucleic acid sequence comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1 polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3 polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

51. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 49 or 50, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 5, 7, 9, 11, 13, or 15, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and HEV.

52. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 49 or 51, wherein the nucleic acid is codon optimized for expression in a human.

53. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-52, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3 polypeptide sequence; v) a P2A autocatalytic polypeptide cleavage site sequence; vi) a linker polypeptide sequence or any combination thereof.

54. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-53, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 6, 8, 10, 12, 14, or 16, wherein the polypeptide comprises epitopes or antigens of HAV and HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitope or antigen of HAV and HEV.

55. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-54, wherein the polypeptide is recombinantly expressed.

56. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-55, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

57. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-56, further comprising an adjuvant.

58. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 57, wherein the adjuvant is alum.

59. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 49-58, wherein the nucleic acid is provided in a recombinant vector.

60. A nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide or at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide.

61. The nucleic acid of claim 60, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HAV VP2 polypeptide, a nucleic acid sequence encoding a HAV VP3 polypeptide, and a nucleic acid sequence encoding a P2A autocatalytic cleavage sequence.

62. The nucleic acid of claim 61, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identityto SEQ ID NO: 17, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HAV.

63. The nucleic acid of claim 60, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding a HEV ORFgt1 polypeptide.

64. The nucleic acid of any one of claims 60-63, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 19, wherein a polypeptide encoded by said nucleic acid comprises an epitope or antigen of HEV.

65. A polypeptide comprising at least one hepatitis A virus (HAV) polypeptide sequence or at least one hepatitis E virus (HEV) polypeptide sequence.

66. The polypeptide of claim 65, wherein the HAV polypeptide sequence comprises a HAV VP2 polypeptide sequence, a HAV VP3 polypeptide sequence, and a P2A autocatalytic cleavage sequence.

67. The polypeptide of claim 66, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18, wherein the polypeptide comprises an epitope or antigen of HAV.

68. The polypeptide of claim 65, wherein the HAV polypeptide sequence comprises a HEV ORFgt1 polypeptide sequence.

69. The polypeptide of claim 68, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 20, wherein the polypeptide comprises an epitope or antigen of HEV.

70. The nucleic acid of any one of claims 60-64 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

71. The polypeptide of any one of claims 65-69 for use in a medicament, such as for the prevention, treatment or inhibition of HAV and / or HEV in a subject, preferably a human.

72. The polypeptide of any one of claims 65-69 or 71, wherein the polypeptide is recombinantly expressed.

73. The polypeptide of any one of claims 65-69 or 71-72, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

74. An immunogenic composition or product combination comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a hepatitis A virus (HAV) polypeptide and / or at least one nucleic acid sequence encoding a hepatitis E virus (HEV) polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide and / or at least one HEV polypeptide.

75. The immunogenic composition or product combination of claim 74, wherein the nucleic acid comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1 polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3 polypeptide; v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

76. The immunogenic composition or product combination of claim 74 or 75, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 17 or 19, wherein the polypeptide comprises epitopes or antigens of HAV and / or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and / or HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

77. The immunogenic composition or product combination of any one of claims 74-76, wherein the nucleic acid is codon optimized for expression in a human.

78. The immunogenic composition or product combination of any one of claims 74-77, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1polypeptide sequence; iv) a HEV ORF2gt3polypeptide sequence; v) a P2A autocatalytic cleavage site polypeptide sequence; vi) a linker polypeptide sequence or any combination thereof.

79. The immunogenic composition or product combination of any one of claims 90-94, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18 or 20, wherein the polypeptide comprises epitopes or antigens of HAV and / or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV and / or HEV, which is optionally used in a medicament, such as for the prevention, treatment, or inhibition of HAV and / or HEV in a subject, such as a mammal, preferably a human.

80. The immunogenic composition or product combination of any one of claims 74-79, wherein the polypeptide is recombinantly expressed.

81. The immunogenic composition or product combination of any one of claims 74-80, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

82. The immunogenic composition or product combination of any one of claims 74-81, further comprising an adjuvant.

83. The immunogenic composition or product combination of claim 82, wherein the adjuvant is alum.

84. The immunogenic composition or product combination of any one of claims 74-83, wherein the nucleic acid is provided in a recombinant vector.

85. A method of generating an immune response in a subject using the immunogenic composition or product combination set forth in any one of claims 74-84, comprising: a) administering to the subject at least one prime dose comprising the nucleic acid; and b) administering to the subject at least one boost dose comprising the polypeptide; or c) administering to the subject at least one prime dose comprising the polypeptide; and d) administering to the subject at least one boost dose comprising the nucleic acid.

86. The method of claim 85, wherein the at least one boost dose further comprises an adjuvant.

87. The method of claim 86, wherein the adjuvant is alum.

88. The method of any one of claims 85-87, wherein the at least one boost dose is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or weeks after the atleast one prime dose is administered or within a range of time defined by any two of the aforementioned time points e.g., within 1-48 days or 1-48 weeks.

89. The method of any one of claims 85-88, wherein the administration is provided enterally, orally, intranasally, parenterally, subcutaneously, intramuscularly, intradermally, or intravenously or any combination thereof, and optionally with in vivo electroporation.

90. The method of any one of claims 85-89, wherein the administration is performed in conjunction with an antiviral therapy or an antiviral prodrug therapy.

91. The method of claim 90, wherein the antiviral therapy or antiviral prodrug therapy comprises administration of chloroquine, hydroxychloroquine, favipiravir, favilavir, remdesivir, tocilizumab, galidesivir, sarilumab, lopinavir, ritonavir, darunavir, ribavirin, interferon-Į, pegylated interferon-Į, interferon alfa-2b, a prodrug or precursor thereof, or any combination thereof.

92. An immunogenic composition or product combination for use in the treatment or inhibition of hepatitis A virus (HAV) and / or hepatitis E virus (HEV), comprising: (a) a nucleic acid comprising at least one nucleic acid sequence encoding a HAV polypeptide or at least one nucleic acid sequence encoding a HEV polypeptide; and (b) a polypeptide comprising at least one HAV polypeptide or at least one HEV polypeptide.

93. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 92, wherein the nucleic acid sequence comprises: i) a nucleic acid sequence encoding a HAV VP2 polypeptide; ii) a nucleic acid sequence encoding a HAV VP3 polypeptide; iii) a nucleic acid sequence encoding a HEV ORF2gt1polypeptide; iv) a nucleic acid sequence encoding a HEV ORF2gt3polypeptide;v) a nucleic acid sequence encoding a P2A autocatalytic polypeptide cleavage site; vi) a nucleic acid sequence encoding a linker polypeptide; or any combination thereof.

94. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 92 or 93, wherein the nucleic acid comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 17 or 19, wherein the polypeptide comprises epitopes or antigens of HAV or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitopes or antigens of HAV or HEV.

95. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-94, wherein the nucleic acid is codon optimized for expression in a human.

96. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-95, wherein the polypeptide comprises: i) a HAV VP2 polypeptide sequence; ii) a HAV VP3 polypeptide sequence; iii) a HEV ORF2gt1 polypeptide sequence; iv) a HEV ORF2gt3polypeptide sequence; v) a P2A autocatalytic polypeptide cleavage site sequence; vi) a linker polypeptide sequence or any combination thereof.

97. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-96, wherein the polypeptide comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or sequence identity to SEQ ID NO: 18 or 20, wherein the polypeptidecomprises epitopes or antigens of HAV or HEV and, wherein the nucleic acid encodes a polypeptide that comprises epitope or antigen of HAV or HEV.

98. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-97, wherein the polypeptide is recombinantly expressed.

99. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-98, wherein the polypeptide is recombinantly expressed in a mammalian, bacterial, yeast, insect, or cell-free system.

100. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-99, further comprising an adjuvant.

101. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of claim 100, wherein the is alum.

102. The immunogenic composition or product combination for use in the treatment or inhibition of HAV and / or HEV of any one of claims 92-101, wherein the nucleic acid is provided in a recombinant vector.

103. The immunogenic composition or product combination of any one of claims 31-41, 49-59, 74-84, 92-102, wherein the immunogenic composition or product combination is formulated to be delivered to a subject via virus-like particles (VLPs).

104. The immunogenic composition or product combination of claim 103, wherein the VLPs are based on one or more p239 fragments of the HEV pORF2 protein.

105. The method of any one of claims 42-48 or 85-91, wherein administering to the subject comprises delivery via virus-like particles (VLPs).

106. The method of claim 105, wherein the VLPs are based on one or more p239 fragments of the HEV pORF2 protein.

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