Production and purification of chimeric HBc virus-like particles carrying influenza virus LAH domain as vaccine candidates

Background: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology. Results: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice. Conclusion: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component

X4 Human Immunodeficiency Virus Type 1 gp120 Promotes Human Hepatic Stellate Cell Activation and Collagen I Expression through Interactions with CXCR4

<div><h3>Background & Aims</h3><p>Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the <em>in vitro</em> impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the <em>in vivo</em> expression of gp120 in HIV/HCV coinfected livers.</p> <h3>Methods</h3><p>Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.</p> <h3>Results</h3><p>X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.</p> <h3>Conclusions</h3><p>X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.</p> </div