0000000000117885
AUTHOR
Dace Skrastina
Signal sequences modulate the immunogenic performance of human hepatitis C virus E2 gene
Abstract Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic s…
N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus
The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized.We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro.The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies bi…
Stop codon insertion restores the particle formation ability of hepatitis B virus core-hantavirus nucleocapsid protein fusions.
In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBcΔ) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBcΔ mosaic particles based on a read-through mechanism in an <i>Escherichia coli</i> suppressor strain [J Gen Virol 1997;78:2049–2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocap…
Recombinant virus-like particles as a carrier of B- and T-cell epitopes of hepatitis C virus (HCV)
The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3' terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete "mature" VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants …
Behavior of a Short preS1 Epitope on the Surface of Hepatitis B Core Particles
The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody M…
Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment
Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alp…
An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice
AbstractPreviously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out …
Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies
Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particl…
Mosaic hepatitis B virus core particles presenting the complete preS sequence of the viral envelope on their surface
The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc–preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium- and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particle…
Evaluation of HBs, HBc, and frCP virus-like particles for expression of human papillomavirus 16 E7 oncoprotein epitopes.
<i>Objectives:</i> In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35–98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). <i>Methods:</i> The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. <i>Results:</i> The HBs*-E7(35–54) protein expressing E7 residues 35–54 between residues 139 and 142 of the HBs carrier formed HBs-like particles…
A hantavirus nucleocapsid protein segment exposed on hepatitis B virus core particles is highly immunogenic in mice when applied without adjuvants or in the presence of pre-existing anti-core antibodies.
Hepatitis B virus (HBV) core particles carrying the amino-terminal 120 amino acids (aa) of the nucleocapsid (N) protein of the hantaviruses Dobrava, Hantaan or Puumala have been demonstrated to be highly immunogenic in mice when complexed with adjuvants. Here we demonstrate that even without adjuvant, these chimeric particles induced high-titered, and strongly cross-reactive N-specific antibody responses in BALB/c and C57BL/6 mice. The induced N-specific antibodies represented all IgG subclasses. Pre-existing core-specific antibodies did not abrogate the induction of an N-specific immune response by a hantavirus N insert presented on core particles. Therefore, chimeric core particles should…