Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles

AbstractThe simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77–93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly

Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10–20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund’s adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40%of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBcderived virus-like particles as the biological component

A VLP library of C-terminally truncated Hepatitis B core proteins: correlation of RNA encapsidation with a Th1/Th2 switch in the immune responses of mice.

An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens

Electron microscopy of the HBc VLPs before and after formulation with adjuvants.

<p><b>A</b> – HBc VLPs without adjuvant, <b>B</b> – Silica-adjuvanted HBc VLPs<b>, C</b> – Alhydrogel-adjuvanted HBc VLPs. EM pictures of the initial non-loaded Silica and Alhydrogel are shown on the appropriate insets. Scale bar, 100 nm.</p

Proliferative response and cytokine production after immunisation of BALB/c mice with HBc VLPs formulated with different adjuvants in the presence or absence of MPL and splenocyte stimulation <i>in vitro</i>.

<p><b>A</b> – Stimulation indexes (SI), <b>B</b> – interferon (IFN)-γ level in culture media, <b>C</b> – IL-2 level in culture media. Splenocytes were isolated at day 10 after subcutaneous immunisation and stimulated with HBc VLPs. SI values are presented as the mean stimulation indexes for triplicate wells ± standard deviation (SD). The cytokine production values are gained from duplicate wells ± SD. MPL (alone) – control group, mice immunised with 10 µg of MPL in PBS. Naive – control group or unstimulated <i>in vivo</i> mice.</p

Induction of the humoral anti-HBc response in BALB/c mice by HBc VLPs formulated with different adjuvants in combination with MPL.

<p><b>A</b> – total anti-HBc titers, <b>B</b> – ratio of anti-HBc IgG2a/IgG1 isotype titers. HBc VLPs were adjuvanted with Silica and Alhydrogel in the presence or absence of MPL or adjuvanted with MPL alone. Mice were bled after subcutaneous (s.c.) injection on day 10. The results represent anti-HBc titers or the ratios of the IgG2a/IgG1 titers as the means from five mice ± standard deviation (SD).</p