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KR-20260066127-A - Novel Hepatitis C Virus Immunogens and Related Uses

KR20260066127AKR 20260066127 AKR20260066127 AKR 20260066127AKR-20260066127-A

Abstract

Hepatitis C virus (HCV) soluble fusion immunogens (sE1E2) are provided, and the HCV soluble fusion immunogens (sE1E2) are derived from engineered or redesigned HCV sE1 polypeptides and sE2 polypeptides and configured to form a natural-like E1E2 interface that may be used in vaccine formulations. Related vaccine compositions are also provided for displaying engineered sE1E2 immunogens after cleavage into an sE1E2 protein having a natural E1E2 conformation on a self-assembling nanoparticle scaffold. Additionally, methods of using the immunogens and vaccine compositions in methods for the prevention or therapy of HCV infections are provided.

Inventors

  • 허, 린링
  • 주, 지앙
  • 로, 만선
  • 퍼스트, 토마스, 알.
  • 오펙, 길라드, 아디
  • 토스, 에릭, 에이.

Assignees

  • 더 스크립스 리서치 인스티튜트
  • 유니버시티 오브 매릴랜드, 칼리지 파크

Dates

Publication Date
20260512
Application Date
20240906
Priority Date
20230906

Claims (20)

  1. (1) an engineered E1 ectodomain having a deletion of the C-terminal membrane proximal external region (MPER), (2) a cleavage site for protease, and (3) an engineered E2 ectodomain having a deletion of the C-terminal membrane proximal external region (MPER); said engineered E1 ectodomain and said engineered E2 ectodomain each fused to two interacting motifs of a protein heterodimerization system, a hepatitis C virus (HCV) soluble E1E2 (sE1E2) immunogen polypeptide.
  2. In Article 1, The above-mentioned engineered E1 ectododomain further comprises (1) deletion of an unstructured hydrophobic loop motif or (2) replacement of said loop motif with a spacer composed of 1 to about 3 non-polar, hydrophilic, or charged amino acid residues, and said loop motif corresponds to residues 264 to 293 (SEQ No. 32) of the HCV H77 isolate polyprotein sequence represented by SEQ No. 1, an HCV sE1E2 immunogen polypeptide.
  3. In Article 2, An HCV sE1E2 immunogen polypeptide further comprising the substitution of sequence motifs corresponding to residues 257 to 263 of the HCV H77 isolated polyprotein into a peptide sequence comprising seven or more residues that does not contain cystines or any stretch of two or more hydrophobic residues.
  4. In Article 2, HCV sE1E2 immunogen polypeptide having a spacer and containing or composed of glycine residues.
  5. In Article 1, HCV sE1E2 immunogen polypeptide, the cleavage site for protease is a furin cleavage site.
  6. In Article 5, The above purine cleavage site is an HCV sE1E2 immunogen polypeptide comprising RRRRRR (SEQ No. 6).
  7. In Article 1, HCV sE1E2 immunogen polypeptide, wherein the above-mentioned engineered E1 ectodomain and its fused interaction motif are located at the N-terminus for the above-mentioned engineered E2 ectodomain and its fused interaction motif.
  8. In Article 1, The above protein heteromerization system is a coiled coil (CC) system, HCV sE1E2 immunogen polypeptide.
  9. In Article 8, HCV sE1E2 immunogen polypeptide, each engineered ectodomain fused at the C-terminus to a coiled coil via a PGG tripeptide linker.
  10. In Article 8, The two interaction motifs of the above-described coiled system each comprise SEQ NOs 20 and 21, SEQ NOs 22 and 23, SEQ NOs 24 and 25, or SEQ NOs 26 and 27, an HCV sE1E2 immunogen polypeptide.
  11. In Article 1, The above protein heteromerization system is an HCV sE1E2 immunogen polypeptide, which is a SpyTag/SpyCatcher system.
  12. In Article 11, The SpyCatcher above is an HCV sE1E2 immunogen polypeptide comprising a cleavage of the unstructured N-terminal sequence.
  13. In Article 11, The above SpyTag motif and the above SpyCatcher motif are each an HCV sE1E2 immunogen polypeptide comprising (1) sequence number 7 and sequence number 8, or (2) sequence number 7 and sequence number 43.
  14. In Article 13, The above-mentioned engineered E1 ectodomain is an HCV sE1E2 immunogen polypeptide fused at the C-terminus to SpyTag via a GSGS (SEQ No. 34) linker.
  15. In Article 13, The above-mentioned engineered E2 ectodomain is an HCV sE1E2 immunogen polypeptide fused to the SpyCatcher at the C-terminus via an AS dipeptide linker.
  16. In Article 1, The deletion of the MPER in E1 comprises a cleavage of the E1 C-terminal sequence after residue H312, and the deletion of the MPER in E2 comprises a cleavage of the E2 C-terminal sequence after residue Y701; and the amino acid numbering is based on the sequence of the HCV H77 isolated polyprotein (SEQ No. 1), HCV sE1E2 immunogen polypeptide.
  17. In Article 1, The deletion of the MPER in E1 comprises a cleavage of the E1 C-terminal sequence after residues P310, G311, I313, or T314, and the deletion of the MPER in E2 comprises a cleavage of the E2 C-terminal sequence after residues L702, Y703, G704, or V705; and the amino acid numbering is based on the sequence of the HCV H77 isolated polyprotein (SEQ No. 1), HCV sE1E2 immunogen polypeptide.
  18. In Article 1, The deletion of the MPER in E1 comprises a cleavage of the E1 C-terminal sequence after residues P310, G311, I313, or T314, and the deletion of the MPER in E2 comprises a cleavage of the E2 C-terminal sequence after residue Y701; and the amino acid numbering is based on the sequence of the HCV H77 isolated polyprotein (SEQ No. 1), HCV sE1E2 immunogen polypeptide.
  19. In Article 1, The deletion of the MPER in E1 comprises a cleavage of the E1 C-terminal sequence after residue H312, and the deletion of the MPER in E2 comprises a cleavage of the E2 C-terminal sequence after residues L702, Y703, G704, or V705; and the amino acid numbering is based on the sequence of the HCV H77 isolated polyprotein (SEQ No. 1), HCV sE1E2 immunogen polypeptide.
  20. In Article 1, An HCV sE1E2 immunogen polypeptide comprising, from the N-terminus to the C-terminus, a modified E1 ectodomain shown in SEQ No. 9, a first PGG tripeptide spacer, a first coiled coil of a coiled coil dimerization system, a furin cleavage site, a modified E2 ectodomain shown in SEQ No. 10, a second PGG tripeptide spacer, and a second coiled coil of the coiled coil dimerization system.

Description

Novel Hepatitis C Virus Immunogens and Related Uses Government support statement The present invention was made with the support of the government under AI168251 granted by the National Institutes of Health. The government holds specific rights to the present invention. Sequence list The present application includes a sequence list submitted in .xml format, the entirety of which is incorporated herein by reference. The .xml file is named "2213_1PC_Sequence Listing", was created on September 4, 2024, and has a size of 43.6 KB bytes. Hepatitis C virus (HCV) is a small, enveloped, single-stranded, positive-sense RNA virus of the genus Hepacivirus in the family Flaviviridae. HCV comprises seven major genotypes, which can be further subdivided into subtypes where the number of subtypes depends on the genotype. The RNA genome of HCV encodes a single polyprotein that is processed by host and viral proteases into three structural proteins and seven non-structural proteins. This RNA genome is a single strand with positive polarity and is enveloped by a lipid bilayer containing two glycoproteins (GPs), E1 and E2, to be packaged by a core protein and form a virion. E1 and E2 envelope glycoproteins form a heterodimer that mediates HCV infection, during which E2 attaches to CD81 and SR-B1 coreceptors to initiate a multi-step entry process into hepatocytes. Although HCV is classified as a flavivirus, E1 and E2, it does not appear to share sequence or structural features with other flavivirus type II fusion glycoproteins. Both E1 and E2 are single-pass transmembrane proteins having 160 and 330 residues of N-terminal ectodomains, respectively, and approximately 30 residues of C-terminal transmembrane domains. Similar to human immunodeficiency virus-1 (HIV-1), HCV glycoproteins carry immunogenic variable regions (VRs) and dense glycan shields to evade immune responses. While more than 60% of the E2 core domain (E2c) consists of loops or is disordered, N-linked glycans constitute 50% of the molecular mass of E1 and E2, possessing 5 to 6 and 9 to 11 N -linked glycosylation sites in E1 and E2, respectively. HCV infects approximately 1% of the global population, and it is estimated that about 58 million people live with HCV. Roughly 75% of HCV infections become chronic and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Direct-acting antivirals (DAAs) offer a possible cure for the infection but cannot prevent reinfection. Furthermore, due to medical and financial constraints, DAA treatment is accessible to less than 10% of the population infected with HCV. Given these limitations, vaccination remains the most cost-effective method to control HCV infections and protect the blood supply from viral contamination in countries where HCV nucleic acid tests (NATs) are not readily available. However, the development of a prophylactic HCV vaccine remains a challenge due to the significant variability and hypervariability of this RNA virus. Methods for inducing a broadly neutralizing antibody (bNAb) response pose significant challenges for HCV vaccines. Genetic diversity, glycan shielding, and the preparation of homogeneous E1E2 antigens all contribute to the difficulty of generating protective, cross-genotype neutralizing antibodies (NAbs). NAb and bNAb responses to HCV infection target epitopes in E1, E2, or E1E2 heterodimers. A panel of bNAbs has been identified to indicate several key vulnerable sites on HCV glycoproteins. Among these bNAbs, the AR3 series, HEPC3/74 series, HC84 series, HCV1, and HC33 series target the neutralizing face (NF) on E2, while AR4A and AR5A target the quaternary structure near the E1E2 interface. In passive transfer experiments, these bNAbs provide protection against xenogeneic HCV infection in humanized mouse models and highlight the importance of inducing bNAb responses to HCV vaccines. However, membrane-bound E1E2 formulations failed to induce bNAb responses in chimpanzees and humans, showing only strain-specific NAb responses to immunodominant VR loops in E2. For example, there is a need in the industry for more effective and potent HCV vaccine immunogens based on E1E2 heteromers to prevent HCV infection. The present disclosure addresses these and other unmet needs in the industry. Cross-reference regarding related applications This application claims priority to U.S. Provisional Patent Application No. 63/580,740 filed September 6, 2023, the entire disclosure of which is incorporated herein by reference. In one embodiment, the present disclosure provides engineered hepatitis C virus (HCV) soluble E1E2 (sE1E2) fusion immunogen polypeptides. Each of these fusion immunogen polypeptides comprises (1) a first fusion partner comprising an engineered E1 ectodomain (sE1) having a deletion of the C-terminal membrane proximal external region (MPER), (2) a peptide spacer comprising a cleavage site for a host cell protease, and (3) a second fusion partner comprising an engin