Expanding the host range of hepatitis C virus through viral adaptation

Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes in vivo and in vitro Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes in vivo in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV's ability to enter murine hepatocytes in vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C. IMPORTANCE: At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV

Improved fabrication of zero-mode waveguides for single-molecule detection

Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics ͑circa a femtoliter͒. Observation volumes in the range of 10 −4 fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations ͑up to 20 M͒ of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beam lithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60-100 nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectroscopy measurements, which confirmed their geometry and optical confinement. Process throughput can be further increased using deep ultraviolet photolithography to replace electron beam lithography. This enables the production of aperture arrays in a high volume manufacturing environment

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria. Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. P. falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics

Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection

Behavioral and cognitive performance of humanized APOEε3/ε3 liver mice in relation to plasma apolipoprotein E levels

Abstract Plasma apolipoprotein E levels were previously associated with the risk of developing Alzheimer’s disease (AD), levels of cerebrospinal fluid AD biomarkers, cognition and imaging brain measures. Outside the brain, the liver is the primary source of apoE and liver transplantation studies have demonstrated that liver-derived apoE does not cross the blood–brain-barrier. How hepatic apoE may be implicated in behavioral and cognitive performance is not clear. In the current study, we behaviorally tested FRGN mice with humanized liver harboring the ε3/ε3 genotype (E3-human liver (HL)) and compared their behavioral and cognitive performance with that of age-matched ε3/ε3 targeted replacement (E3-TR) mice, the latter produces human apoE3 throughout the body whereas the E3-HL mice endogenously produce human apoE3 only in the liver. We also compared the liver weights and plasma apoE levels, and assessed whether plasma apoE levels were correlated with behavioral or cognitive measures in both models. E3-HL were more active but performed cognitively worse than E3-TR mice. E3-HL mice moved more in the open field containing objects, showed higher activity levels in the Y maze, showed higher activity levels during the baseline period in the fear conditioning test than E3-TR mice, and swam faster than E3-TR mice during training to locate the visible platform in the water maze. However, E3-HL mice showed reduced spatial memory retention in the water maze and reduced fear learning and contextual and cued fear memory than E3-TR mice. Liver weights were greater in E3-HL than E3-TR mice and sex-dependent only in the latter model. Plasma apoE3 levels were similar to those found in humans and comparable in female and male E3-TR mice but higher in female E3-HL mice. Finally, we found correlations between plasma apoE levels and behavioral and cognitive measures which were predominantly model-dependent. Our study demonstrates mouse-model dependent associations between plasma apoE levels, behavior and cognition in an ‘AD-neutral’ setting and suggests that a humanized liver might be sufficient to induce mouse behavioral and cognitive phenotypes