Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

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To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies.

Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. IMPORTANCEA prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development. I t is estimated that over 2% of the world's population is chronically infected with hepatitis C virus (HCV) (1). Although recently approved direct-acting antiviral (DAA) drugs (2) have greatly improved upon the curing efficacy of the previous interferon (IFN)-based regimen, these new therapies are very expensive and thus unaffordable for the majority of HCV-infected individuals who live in developing countries, where most new infections occur. Since the approval of these highly effective DAAs, the number of chronic HCV carriers has not significantly declined. Furthermore, there is little evidence that patients cured of their chronic infections with DAAs retain antiviral immunity that is protective against future HCV exposures. Therefore, the...

Uptake of hepatitis C virus (HCV) into hepatocytes is an orchestrated process, involving numerous host factors, virion-associated lipoproteins and a growing number of cell-associated factors. Several of these factors likely contribute to the hepatotropism and limited host range of this virus. Discerning the minimal set of human-specific factors required for viral uptake into non-human cells has facilitated the development of small animal models with inheritable HCV susceptibility. This review summarizes current knowledge of host factors required for HCV entry, the molecular mechanisms underlying HCV entry into hepatocytes, and aspects of viral entry contributing to HCV host tropism.

Infectious diseases are the second leading cause of death worldwide. Although the host multitropism of some pathogens has rendered their manipulation possible in animal models, the human-restricted tropism of numerous viruses, bacteria, fungi, and parasites has seriously hampered our understanding of these pathogens. Hence, uncovering the genetic basis underlying the narrow tropism of such pathogens is critical for understanding their mechanisms of infection and pathogenesis. Moreover, such genetic dissection is essential for the generation of permissive animal models that can serve as critical tools for the development of therapeutics or vaccines against challenging human pathogens. In this review, we describe different experimental approaches utilized to uncover the genetic foundation regulating pathogen host tropism as well as their relevance for studying the tropism of several important human pathogens. Finally, we discuss the current and future uses of this knowledge for generating genetically modified animal models permissive for these pathogens.

The study of interactions between hepatitis C virus (HCV) with its mammalian host, along with the development of more effective therapeutics and vaccines has been delayed by the lack of a suitable small animal model. HCV readily infects only humans and chimpanzees, which poses logistic, economic and ethical challenges with analyzing HCV infection in vivo. Progress has been made in understanding the determinants that dictate HCV’s narrow host range providing a blueprint for constructing a mouse model with inheritable susceptibility to HCV infection. Indeed, genetically humanized mice were generated that support viral uptake, replication and production of infectious virions – albeit at low levels. These efforts are complemented with attempts to select for viral variants that are inherently more capable of replicating in non-human species. In parallel, engraftment of relevant human tissues into improved xenorecipients is being continuously refined. Incorporating advances in stem-cell-biology and tissue engineering may allow the generation of patient-specific humanized mice. Construction of such mouse “avatars” may allow analyzing functionally patient-specific differences with respect to susceptibility to infection, disease progression and responses to treatment. In this review, we discuss the three, before mentioned approaches to overcome current species barriers and generate a small animal model for HCV infection, i.e. genetic modification of mice to increase their susceptibility to the virus; genetic modification of HCV, to increase its pathogenicity for mice; and the introduction of human liver and immune cells into immunodeficient mice, to create “humanized” mice. Although in the foreseeable future there will not be a single model that perfectly mimics the natural course of HCV in humans there is reason for optimism. The spectrum of murine animal models for hepatitis C provides a broad arsenal for analyzing the disease. These models may play an important role by prioritizing vaccine candidates and possibly refining combination anti-viral drug therapies. This article forms part of a symposium in Anti-viral Research on “Hepatitis C: next steps toward global eradication.”

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo.IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, smallanimal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry.KEYWORDS animal models, hepatitis C virus, viral entry, viral hepatitis H epatitis C virus (HCV) is a positive-sense, single-stranded RNA virus belonging to the Flaviviridae family, genus Hepacivirus (1). HCV progresses to persistent infection in 70 to 80% of those individuals who become acutely infected (2). Chronic carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) if they remain untreated. Over a few years, very potent directly acting antivirals (DAAs) which can cure