Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.
Hepatitis A virus (HAV), the causative agent of type A viral hepatitis, is an ancient human virus that was first identified almost 35 years ago. It has several characteristics that make it unique among the Picornaviridae, particularly in terms of its mechanisms of polyprotein processing and virion morphogenesis, and which likely contribute to its pathobiology. Although efficacious vaccines containing formalin-inactivated virus produced in cell culture have been licensed in multiple countries, their use has been limited by cost considerations. Changes in public health sanitation and generally increasing standards of living are leading to a decreasing incidence of acute hepatitis A worldwide, with the result that the prevalence of preexisting immunity among adults is declining in many regions. These changes in the epidemiology of HAV may paradoxically enhance the disease burden, as greater numbers of individuals become infected at older ages when disease is more likely to be clinically evident, thus providing greater incentives for vaccine utilization. (HEPATOLOGY 2006;43:S164-S172.)
To develop a vaccine candidate against COVID-19, we generated a Lentiviral Vector (LV), eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, confers only partial protection, despite high levels of serum neutralizing activity. However, eliciting an immune response in the respiratory tract through an intranasal boost results in > 3 log10 decrease in the lung viral loads and reduces local inflammation. Moreover, both integrative and non-integrative LV platforms display strong vaccine efficacy and inhibit lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and closely mirror human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of the LV-based vaccination against SARS-CoV-2 and designate intranasal immunization as a powerful approach against COVID-19.
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