Patent Application
20240350613
The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said.xml copy, created on Apr. 22, 2024 are named V409310005US02, and is 55,423 bytes in size.
The present disclosure relates to fusion proteins, and use of such fusion proteins as antigens, such as in immunizations against the antigens.
An antigen is a molecule, typically a protein or a polysaccharide, that is capable of triggering an immune response in an organism. These molecules are recognized by the immune system as foreign invaders, prompting the body to produce antibodies or activate specific immune cells to neutralize or eliminate them. Antigens can come from a variety of sources, including pathogens like bacteria, viruses, and parasites, as well as non-pathogenic substances such as pollen, dust, or certain foods. They possess specific molecular structures, known as epitopes, which interact with receptors on immune cells, initiating the complex cascade of events that constitutes the immune response. Understanding the nature and behavior of antigens is crucial in fields such as immunology, vaccine development, and diagnostics, as it underpins our ability to combat infectious diseases and maintain overall health.
Obtaining an antigen for a vaccine can be a challenging endeavor due to various factors. Identifying the specific antigen that will elicit a protective immune response against a particular pathogen requires extensive research and experimentation. Scientists must carefully study the pathogen's structure, life cycle, and interaction with the immune system to pinpoint the most effective target. Additionally, some pathogens mutate rapidly, making it difficult to find a stable antigen that remains consistent over time. Thus, there is a need for antigens for vaccines that can be used in immunizations.
In some embodiments, a fusion protein can include: a series of polypeptide domains starting from an N-terminus or C-terminus, the series of polypeptide domains comprising in order: an oligomerization domain having an oligomerization amino acid sequence that oligomerizes with other polypeptides having the oligomerization amino acid sequence; a first spacer domain having a first spacer amino acid sequence of at least 7 amino acids; an antigenic domain having an antigenic amino acid sequence of an antigen that is immunogenic, wherein the antigen is from one of: Domain III of an envelope glycoprotein of a flavivirus; hemagglutinin of a H5 influenza virus, or receptor binding domain of a SARS-COV-2 virus; a second spacer domain having a second spacer amino acid sequence of at least 6 amino acids; and optionally a purification tag having a purification amino acid sequence that binds with a purification substrate, thereby enabling purification. In some aspects, the fusion protein includes the purification tag. In some aspects, the fusion protein series of polypeptide domains start from the N-terminus. In some aspects, the fusion protein series of polypeptide domains start from the C-terminus.
In some embodiments, the oligomerization amino acid sequence includes SEQ ID NO: 1, with at least 95% identity thereof.
In some embodiments, the first spacer amino acid sequence or second spacer amino acid sequence independently includes SEQ ID NO: 2, SEQ ID NO: 49, or SEQ ID NO: 50, or at least 95% identity thereof.
In some embodiments, the purification tag includes the sequence of SEQ ID NO: 3 or at least 95% identity thereof.
In some embodiments, the fusion protein includes: SEQ ID NO: 4 or at least 95% identity thereof; SEQ ID NO: 5 or at least 95% identity thereof; SEQ ID NO: 7 or at least 95% identity thereof; SEQ ID NO: 8 or at least 95% identity thereof; SEQ ID NO: 10 or at least 95% identity thereof; SEQ ID NO: 11 or at least 95% identity thereof; SEQ ID NO: 13 or at least 95% identity thereof; SEQ ID NO: 14 or at least 95% identity thereof; SEQ ID NO: 16 or at least 95% identity thereof; SEQ ID NO: 17 or at least 95% identity thereof; SEQ ID NO: 19 or at least 95% identity thereof; SEQ ID NO: 20 or at least 95% identity thereof; SEQ ID NO: 22 or at least 95% identity thereof; SEQ ID NO: 23 or at least 95% identity thereof; SEQ ID NO: 25 or at least 95% identity thereof; SEQ ID NO: 26 or at least 95% identity thereof; SEQ ID NO: 28 or at least 95% identity thereof; SEQ ID NO: 29 or at least 95% identity thereof; SEQ ID NO: 31 or at least 95% identity thereof; SEQ ID NO: 32 or at least 95% identity thereof; SEQ ID NO: 34 or at least 95% identity thereof; SEQ ID NO: 35 or at least 95% identity thereof; SEQ ID NO: 37 or at least 95% identity thereof; SEQ ID NO: 38 or at least 95% identity thereof; SEQ ID NO: 40 or at least 95% identity thereof; SEQ ID NO: 41 or at least 95% identity thereof; SEQ ID NO: 43 or at least 95% identity thereof; SEQ ID NO: 44 or at least 95% identity thereof; SEQ ID NO: 46 or at least 95% identity thereof; or SEQ ID NO: 47 or at least 95% identity thereof.
In some embodiments, an immunological composition (e.g., vaccine) can include: a pharmaceutically acceptable carrier; and the particle having the oligomerized fusion proteins. The pharmaceutically acceptable carrier can be an aqueous composition that is suitable for administration to a subject, such as intramuscular injection. In some aspects, the immunological composition can include an adjuvant.
A method of immunizing a subject can include: providing the fusion protein of one of the embodiments (e.g., in oligomerized particle and/or vaccine); and administering the fusion protein to the subject in an amount sufficient to induce an immunological response against the fusion protein. In some aspects, the method can include administering an adjuvant with the fusion protein to the subject.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
The foregoing and following information as well as other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings.
The elements and components in the figures can be arranged in accordance with at least one of the embodiments described herein, and which arrangement may be modified in accordance with the disclosure provided herein by one of ordinary skill in the art.
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.
Generally, the present technology includes a fusion protein and methods of making the same, and use of the fusion protein in immunization protocols. The fusion protein includes in sequence from the N-terminus: (1) an oligomerization domain; (2) a spacer sequence; (3) an antigenic sequence; (4) a spacer sequence; and (5) a purification tag. This order is defined as the “Forward Order”. Alternatively, the fusion protein could also include in sequence from the N-terminus: (1) a purification tag, (2) a spacer sequence, (3) an antigenic sequence, (4) a spacer sequence, and (5) an oligomerization domain. This order is defined as the “Reverse Order”. Thus, the oligomerization domain can be present either at the N terminus or at the C terminus of the fusion protein.
The oligomerization domain allows the antigen to oligomerize (e.g., non-covalently self-assemble) into large proteins that can grow to the size of nanoparticles. This greatly enhances the immunogenicity of the antigens. Immunization in several animal models indicate strong antibody responses that are capable of neutralizing the viruses. Oligomerization is a process in which individual fusion peptides non-covalently self-assemble to form a larger structure called an oligomer, which can be a particle. The exact number of fusion proteins in an oligomer particle can vary depending on the context and the specific molecule. Oligomerization can significantly impact the function, stability, and activity of a protein. Many proteins exist naturally as oligomers, including enzymes, receptors, and structural proteins.
The spacer sequences flanking the virus-derived antigen sequences allow epitope separation, and facilitate recognition by B lymphocytes of relevant, antigen-derived epitopes. The spacer sequences can be any amino acid sequence that is not an antigen or oligomerization domain, or purification tag. The spacer sequence can be at least 6 amino acids, or at least 7 amino acids, up to 25 amino acids.
The purification tag can be an EPEA ((glutamic acid-proline-glutamic acid-alanine), SEQ ID NO: 3) tag at the C-terminus (or N-terminus) that allows the purification of the antigens using affinity chromatography.
In some embodiments, the sequences of the fusion proteins provided herein can omit the EPEA (SEQ ID NO: 3) tag or other purification tag. Thus, the fusion protein sequences can be devoid of a purification tag.
In some embodiments, the antigen is a sequence from Domain III of the Envelope Glycoprotein of a virus, such as flaviviruses. Domain III of the envelope glycoprotein (E-glycoprotein) of flaviviruses is a structural and functional domain that plays a key role in the virus's life cycle and interaction with host cells. The E-glycoprotein is a major surface protein of flaviviruses such as dengue, Zika, West Nile, and yellow fever viruses. It is involved in processes such as viral attachment, entry, and fusion with the host cell membrane. Domain III of the E-glycoprotein is the C-terminal domain and is structurally distinct from the other domains (I and II).
In some embodiments, the antigen is a sequence from hemagglutinin from a virus, such as H5 Influenza virus. Hemagglutinins are proteins that possess a specific affinity for certain sugar molecules. Since carbohydrate units exist in most animal cell membranes, the hemagglutinins may attach to these receptor groups.
In some embodiments, the antigen is a sequence from the receptor binding domain of a virus, such as SARS-COV-2 virus. The receptor binding domain (RBD) of SARS-CoV-2 is a critical region within the spike glycoprotein of the virus. This domain is responsible for binding to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), which facilitates viral entry into host cells. The RBD is located within the larger S1 subunit of the spike protein, which is the portion of the protein that interacts with the host cell.
In some embodiments, the fusion proteins can include:
The fusion protein can include an antigenic domain sequence from the Kyasanur Forest Disease virus, which fusion protein is referred to as NP-KFDV. The NP-KFDV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Kyasanur Forest Disease virus. The forward order of the NP-KFDV fusion protein includes:
Alternatively, the fusion protein having the Kyasanur Forest Disease virus antigenic domain can have the polypeptide domains arranged in the reverse order The reverse order of the polypeptide domains arranged in the NP-KFDV fusion protein (i.e., NP-KFDV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Kyasanur Forest Disease virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Powassan Virus, which fusion protein is referred to as NP-POWV. The NP-POWV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Powassan virus. The forward order of the polypeptide domains arranged in the NP-POWV fusion protein includes:
Alternatively, the fusion protein having the Powassan virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-POWV fusion protein (i.e., NP-POWV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Powassan virus is from the E-glycoprotein of Domain III and is:
The fusion protein can include an antigenic domain sequence from the Dengue Type 1 virus, which fusion protein is referred to as NP-DENV Type 1. The NP-DENV Type 1 includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Dengue Type 1 virus. The forward order of the polypeptide domains arranged in the NP-DENV Type 1 fusion protein includes:
Alternatively, the fusion protein having the Dengue Type 1 virus antigenic domain sequence can include the polypeptide regions in the reverse order. The reverse order of the polypeptide domains arranged in the NP-DENV Type 1 fusion protein (i.e., NP-DENV Type 1 Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Dengue Virus Type 1 virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Dengue Type 2 virus, which fusion protein is referred to as NP-DENV Type 2 (Canonical). The NP-DENV Type 2 (Canonical) includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Dengue Type 2 virus. The forward order of the polypeptide domains arranged in the NP-DENV Type 2 (Canonical) fusion protein includes:
Alternatively, the fusion protein having the Dengue Type 2 virus antigenic domain sequence can have the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-DENV Type 2 (Canonical) fusion protein (i.e., NP-DENV Type 2 (Canonical) Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Dengue Virus Type 2 (Canonical) virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Dengue Type 2 virus, which fusion protein is referred to as NP-DENV Type 2 (S16803). The NP-DENV Type 2 (S16803) includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Dengue Type 2 virus. The forward order of the polypeptide domains arranged in the NP-DENV Type 2 (S16803) fusion protein includes:
Alternatively, the fusion protein having the Dengue Type 2 virus antigenic sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-DENV Type 2 (S16803) fusion protein (i.e., NP-DENV Type 2 [S168031] Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Dengue Virus Type 2 (S16803) virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Dengue Type 3 virus, which fusion protein is referred to as NP-DENV Type 3. The NP-DENV Type 3 includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Dengue Type 3 virus. The forward order of the polypeptide domains arranged in the NP-DENV Type 3 fusion protein includes:
Alternatively, the fusion protein having the Dengue Type 3 virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-DENV Type 3 fusion protein (i.e., NP-DENV Type 3 Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Dengue Virus Type 3 virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Dengue Type 4 virus, which fusion protein is referred to as NP-DENV Type 4. The NP-DENV Type 4 includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Dengue Type 4 virus. The forward order of the polypeptide domains arranged in the NP-DENV Type 4 fusion protein includes:
Alternatively, the fusion protein having the Dengue Type 4 virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-DENV Type 4 fusion protein (i.e., NP-DENV Type 4 Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Dengue Virus Type 4 virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Zika virus, which fusion protein is referred to as NP-ZIKV. The NP-ZIKV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Zika virus. The forward order of the polypeptide domains arranged in the NP-ZIKV fusion protein includes:
Alternatively, the fusion protein having the Zika virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order The reverse order of the polypeptide domains arranged in the NP-ZIKV fusion protein (i.e., NP-ZIKV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Zika virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Yellow
Fever virus, which fusion protein is referred to as NP-YF. The NP-YF includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Yellow Fever virus. The forward order of the polypeptide domains arranged in the NP-YF fusion protein includes:
Alternatively, the fusion protein having the Yellow Fever virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-YF fusion protein (i.e., NP-YF Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Yellow Fever virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Japanese B Encephalitis virus, which fusion protein is referred to as NP-JEV. The NP-JEV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Japanese B Encephalitis virus. The forward order of the polypeptide domains arranged in the NP-JEV fusion protein includes:
Alternatively, the fusion protein having the Japanese B Encephalitis virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-JEV fusion protein (i.e., NP-JEV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Japanese B Encephalitis virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the West Nile virus, which fusion protein is referred to as NP-WNV. The NP-WNV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the West Nile virus. The forward order of the polypeptide domains arranged in the NP-WNV fusion protein includes:
Alternatively, the fusion protein having the West Nile virus antigenic domain sequence can included the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-WNV fusion protein (i.e., NP-WNV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the West Nile virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Murray Valley Encephalitis virus, which fusion protein is referred to as NP-MVEV. The NP-MVEV includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the Murray Valley Encephalitis virus. The forward order of the polypeptide domains arranged in the NP-MVEV fusion protein includes:
Alternatively, the fusion protein having the Murray Valley Encephalitis virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-MVEV fusion protein (i.e., NP-MVEV Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the Murray Valley Encephalitis virus is from the E-glycoprotein of Domain III is:
The fusion protein can include an antigenic domain sequence from the Influenza virus [H5N1], which fusion protein is referred to as NP-H5-RG71A/RG8A. The NP-H5-RG71A/RG8A includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the H5N1 Influenza virus. The forward order of the polypeptide domains arranged in the NP-H5-RG71A/RG8A fusion protein includes:
Alternatively, the fusion protein having the H5N1 Influenza virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-H5-RG71A/RG8A fusion protein (i.e., NP-H5-RG71A/RG8A Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the H5N1 Influenza virus (Type H5) is from the hemagglutinin region is:
The fusion protein can include an antigenic domain sequence from the Influenza virus [H5N1] from Vietnam, which fusion protein is referred to as NP-H5-VIET04. The NP-H5-VIET04 includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the H5N1 Influenza virus from Vietnam. The forward order of the polypeptide domains arranged in the NP-H5-VIET04 fusion protein includes:
Alternatively, the fusion protein having the H5N1 Vietnam Influenza Virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-H5-VIET04 fusion protein (i.e., NP-H5-VIET04 Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the H5N1 Vietnam Influenza virus (Type H5) is from the Hemagglutinin region is:
The fusion protein can include an antigenic domain sequence from the SARS-CoV-2 virus, which fusion protein is referred to as NP-SARS-COV-2. The NP-SARS-CoV-2 includes the oligomerization domain, first spacer domain and second spacer domain bounding the antigenic domain from the SARS-COV-2 virus. The forward order of the polypeptide domains arranged in the NP-SARS-COV-2 fusion protein includes:
Alternatively, the fusion protein having the SARS-COV-2 Virus antigenic domain sequence can include the polypeptide domains arranged in the reverse order. The reverse order of the polypeptide domains arranged in the NP-SARS-COV-2 fusion protein (i.e., NP-SARS-COV-2 Reverse Order) is:
These fusion proteins include the oligomerization domain of SEQ ID NO: 1; two spacer domains of SEQ ID NO: 2 flanking the antigenic domain; and the affinity tag of SEQ ID NO: 3. However, the affinity tag may be optional or omitted. The antigenic domain of the SARS-COV-2 virus is from the receptor binding domain is:
Particular viral proteins of interest include: an antigen from a virus in the flaviviridae family (genus flavivirus), such as from Kyasanur Forest Disease virus, Powassan virus, Yellow Fever virus, Zika virus, Japanese B Encephalitis virus, four serotypes of Dengue viruses, Tick-borne Encephalitis virus, West Nile virus, Murray Valley Encephalitis virus. These can be tick-borne or mosquito-borne viruses. Also, the antigen can be from Influenza virus, such as H5N1. The antigen can also be a universal SARS-COV-2 antigen.
The antigen can be the full sequences as shown herein. That is, there can be variations in the sequence so long as the sequence functions as described herein. Similarly, the oligomerization regions, spacers, or affinity tags may have variations so long as they retain functionality. The sequences in the sequence listing may vary by having at least 99% identity, at least 98% identity, at least 95% identity, at least 90% identity, at least 85% identity, or at least 80% identity.
The composition of the invention will typically, in addition to the components mentioned above, comprise one or more ‘pharmaceutically acceptable carriers’, which include any carrier that does not itself induce the production of antibodies. Such carriers are well known. Suitable carriers can include typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.
Compositions of the invention may be administered in conjunction with immunoregulatory agents. In particular, compositions will usually include one or more adjuvants.
Rabbits (n=4 per cohort) were immunized as follows: a prime dose on Day 0 and a single boost on Day 15. The antigen domain (20 micrograms/dose) used had the following H5 (Clade 2.3.3.4b) sequence: MANEGYHEEISDLSDETRDMHRAIVSLMEELEAVDWYNQRVDAAQDGDLKAIL AHNRDEEKEHAAMVLEWIRRKDPAFDKELKDYLFTEKPIAHSTGGGSGGGPGSG DLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANPANDLCYPGSLN DYEELKHLLSRINHFEKILIIPKSSWPNHETSLGVSAACPYQGAPSFFRNVVWLIKK NDAYPTIKISYNNTNREDLLILWGIHHSNNAEEQTNLYKNPTTYISVGTSTLNQRL VPKIATRSQVNGQRGRMDFFWTILKPDDAIHFESNGNFIAPEYAYKIVKKGDSTI MKSGVEYGGSGGGEPEA (SEQ ID NO: 40). The adjuvants used were either CQUIM-MA, or Alhydroxiquim-II, both at 20 micrograms imidazoquinoline content/dose. The serum from the immunization (e.g., Immune-1 sera) were obtained on Day 25, and were examined for neutralizing antibodies using a commercially-available, non-replicating GFP-expressing reporter virus.
The outline data points are for Experiment VV-45 Cohort B after Immunization-1, with fusion proteins NP-H5-RG71A/RG8A+NP-H5-VIET04, with the adjuvant being Alhydroxiquim-II. The outline square data is for rabbit experiment 4. VV45.VVX137.Imm1. The outline circle is for rabbit experiment 4.VV45.VVX138.Imm1. The outline up triangle is for rabbit experiment 4. VV45.VVX139.Imm1. The down triangle data is for rabbit experiment 4.VV45.VVX140.Imm1.
The data for the negative controls is minimal and the data for the positive controls matches the data from the immunization. Accordingly, these fusion proteins are shown to induce an immunological response.
Rabbits (n=4 per cohort) were immunized as follows: a prime dose on Day 0 and a single boost on Day 15. The antigen (20 micrograms/dose) used had the following H5 (Clade 1) sequence: MANEGYHEEISDLSDETRDMHRAIVSLMEELEAVDWYNQRVDAAQDGDLKAIL AHNRDEEKEHAAMVLEWIRRKDPAFDKELKDYLFTEKPIAHSTGGGSGGGPGSG DLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFN DYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKK NSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQR LVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTI MKSELEYGGSGGGEPEA (SEQ ID NO: 43). The adjuvants used were either CQUIM-MA, or Alhydroxiquim-II, both at 20 micrograms imidazoquinoline content/dose. The serum from the immunization (e.g., Immune-1 sera) were obtained on Day 25, and were examined for neutralizing antibodies using a commercially-available, non-replicating GFP-expressing reporter virus.
The outline data points are for Experiment VV-48 with Cohort B after Immunization-1, with fusion proteins NP-H5-RG71A/RG8A+NP-H5-VIET04, with the adjuvant being Alhydroxiquim-II. The outline circle data is for rabbit experiment 4.VV48.VVX189.Imm1. The outline up triangle is for experiment 4.VV48.VVX190.Imm1. The outline down triangle is for rabbit experiment 4. VV48.VVX191.Imm1. The outline diamond data is for rabbit experiment 4.VV48.VVX192.Imm1.
The data for the negative controls is minimal and the data for the positive controls matches the data from the immunization. Accordingly, these fusion proteins are shown to induce an immunological response.
Rabbits (n=4 per cohort) were immunized as follows: a prime dose on Day 0 and a single boost on Day 15. The antigen (20 micrograms/dose) used had the following West Nile Virus sequence: MANEGYHEPIDELTDETRDMHRAIISLMEELEAVDWYNQRVDACKDEALKAILA HNRDEEKEHAAMVLEWIRRKDPRFDKELKDYLFTEKPIAHKGGGSGGGTTYGV CSKAFKFAGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVN PFVSVATANSKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIGKAFTTTLKGA GGSGGGEPEA (SEQ ID NO: 34). The adjuvants used were either CQUIM-MA, or Alhydroxiquim-II, both at 20 micrograms imidazoquinoline content/dose. After the immunization, the serum (e.g., Immune-1 sera) were obtained on Day 25, and were examined for neutralizing antibodies using a commercially-available, non-replicating GFP-expressing reporter virus.
The outline data points are for Experiment VV-49 Cohort B after Immunization-1, with fusion protein NP-WNV, with the adjuvant being Alhydroxiquim-II. The outline square data is for rabbit experiment 4.VV49.VVX205.Imm1. The outline circle data is for rabbit experiment 4.VV49.VVX205.Imm1. The outline up triangle is for rabbit experiment 4.VV49.VVX207.Imm1. The outline down triangle is for rabbit experiment 4.VV49.VVX208.Imm1.
The data for the negative controls is minimal and the data for the positive controls matches the data from the immunization. Accordingly, these fusion proteins are shown to induce an immunological response.
Rabbits (n=4 per cohort) were immunized as follows: a prime dose on Day 0 and two boosts on Day 15 and Day 30. The antigen (20 micrograms/dose) used for Cohort A had the following Powassan Virus sequence: MANEGYHEPIDELTDETRDMHRAIISLMEELEAVDWYNQRVDACKDEALKAILA HNRDEEKEHAAMVLEWIRRKDPRFDKELKDYLFTEKPIAHKGGGSGGGTTYSM CDKTKFKWKRVPVDSGHDTVVMEVSYTGSDKPCRIPVRAVAHGVPTTNVAMLI TPNPTIETSGGGFIEMQLPLGDNIIYVGDLSQQWFQKGSTIGRMFGGSGGGEPEA (SEQ ID NO: 7). Cohorts B and C received a mixture of the above-described Powassan Virus antigen plus a West Nile Virus antigen of the following sequence: MANEGYHEPIDELTDETRDMHRAIISLMEELEAVDWYNQRVDACKDEALKAILA HNRDEEKEHAAMVLEWIRRKDPRFDKELKDYLFTEKPIAHKGGGSGGGTTYGV CSKAFKFAGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVN PFVSVATANSKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIGKAFTTTLKGA GGSGGGEPEA (SEQ ID NO: 34). Both antigens were at 20 micrograms per dose. The adjuvants used were either CQUIM-MA, or Alhydroxiquim-II, both at 20 micrograms imidazoquinoline content/dose. After immunization, the serum (e.g., Immune-2 sera) were obtained on Day 38, and were examined for neutralizing antibodies using live Powassan virus
The outline data points are for Experiment VV-46 Cohort B after Immunization-2, with fusion proteins NP-WNV+NP-POWV, with the adjuvant being Cquim-1 (MA). The outline square data is for rabbit experiment 3.VV46.VVX149.Imm2. The outline circle data is for rabbit experiment 3.VV46.VVX150.Imm2. The outline up triangle is for rabbit experiment 3.VV46.VVX151.Imm2. The outline down triangle is for rabbit experiment 3.VV46.VVX152.Imm2.
The light outline data points are for Experiment VV-46 Cohort C after Immunization-2, with fusion proteins NP-WNV+NP-POWV, with the adjuvant being Alhydroxiquim-II. The light outline square data is for rabbit experiment 4.VV46.VVX153.Imm2. The light outline circle data is for rabbit experiment 4.VV46.VVX154.Imm2. The light outline up triangle is for rabbit experiment 4.VV46.VVX155.Imm2. The light outline down triangle is for rabbit experiment 4.VV46.VVX156.Imm2.
The data for the negative controls is minimal and the data for the positive controls matches the data from the immunization. Accordingly, these fusion proteins are shown to induce an immunological response.
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.
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.
From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
This patent application claims priority to U.S. Provisional Application No. 63/497,656 filed Apr. 21, 2023, which provisional is incorporated herein by specific reference in its entirety.
This invention was made with government support under Contract HHSN272201800049C awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63497656 | Apr 2023 | US |
