Joshua A. Horwitz

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.

HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice

Significance Treatment of HIV-1 infection in humans is achieved using combinations of highly effective antiretroviral therapy (ART) drugs to potently suppress viral replication and prevent the emergence of drug-resistant viruses. However, ART drugs must be taken indefinitely owing to rapid return of viremia upon termination of treatment. Highly potent broadly neutralizing antibodies (bNAbs) present a new potential therapeutic modality in the treatment of HIV-1 infection. Because of their comparatively longer half-lives relative to ART drugs and their ability to eliminate infected cells, bNAbs may alleviate some aspects of the lifelong treatment adherence burden of ART. Here we show that lowering the initial viral load with ART enables single bNAbs to effectively control an established HIV-1 infection in humanized mice. Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1–infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.

HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption

Interruption of combination antiretroviral therapy (ART) in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117, a broad and potent neutralizing antibody (bNAb) against the CD4 binding site of HIV-1 Env, in the setting of analytical treatment interruption (ATI) in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Two or four 30 mg/kg infusions of 3BNC117, separated by 3 or 2 weeks, respectively, were generally well tolerated. The infusions were associated with a delay in viral rebound for 5-9 weeks after 2 infusions, and up to 19 weeks after 4 infusions, or an average of 6.7 and 9.9 weeks respectively, compared with 2.6 weeks for historical controls (p=<1e-5). Rebound viruses arose predominantly from a single provirus. In most individuals, emerging viruses showed increased resistance indicating escape. However, 30% of participants remained suppressed until antibody concentrations waned below 20 μg/ml, and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks. We conclude that administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during ATI in humans.

Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

A subset of broadly neutralizing anti-HIV antibodies inhibits cell to cell transmission of the virus.

Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies

Mutating anti–HIV-1 broadly neutralizing antibodies increases their breadth and reduces pathways for viral escape through mutation.

Computational analysis of anti–HIV-1 antibody neutralization panel data to identify potential functional epitope residues

Advances in single-cell antibody cloning methods have led to the identification of a variety of broadly neutralizing anti–HIV-1 antibodies. We developed a computational tool (Antibody Database) to help identify critical residues on the HIV-1 envelope protein whose natural variation affects antibody activity. Our simplifying assumption was that, for a given antibody, a significant portion of the dispersion of neutralization activity across a panel of HIV-1 strains is due to the amino acid identity or glycosylation state at a small number of specific sites, each acting independently. A model of an antibody’s neutralization IC50 was developed in which each site contributes a term to the logarithm of the modeled IC50. The analysis program attempts to determine the set of rules that minimizes the sum of the residuals between observed and modeled IC50 values. The predictive quality of the identified rules may be assessed in part by whether there is support for rules within individual viral clades. As a test case, we analyzed antibody 8ANC195, an anti-glycoprotein gp120 antibody of unknown specificity. The model for this antibody indicated that several glycosylation sites were critical for neutralization. We evaluated this prediction by measuring neutralization potencies of 8ANC195 against HIV-1 in vitro and in an antibody therapy experiment in humanized mice. These experiments confirmed that 8ANC195 represents a distinct class of glycan-dependent anti–HIV-1 antibody and validated the utility of computational analysis of neutralization panel data.

Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants

Klein et al. find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively transferred bNAbs to prevent the emergence of bNAb viral escape variants.

Biological performance of biodegradable amino acid-based poly(ester amide)s: Endothelial cell adhesion and inflammation in vitro.

Functionalized amino-acid-based poly(ester-amide)s (PEA) are a new family of synthetic biodegradable polymers consisting of three naturally occurring building blocks (amino acids, diols, and dicarboxylic acids) that have been suggested to be promising biomaterials for therapeutic use. However, little is known about their cytotoxicity, ability to support cell growth, inflammatory properties, or mechanical properties, key aspects to most biomaterials designed for in vivo implantation and tissue engineering applications. In this study, we investigated the ability of two functionalized PEA materials (amino-functionalized and carboxylic acid functionalized) and a neutral PEA control to support endothelial cell viability, proliferation, and adhesion. Additionally, we investigated the inflammatory response elicited by these functionalized PEA materials using a macrophage cell model. Our results indicate that all forms of PEA were noncytotoxic and noninflammatory in vitro. The amino-functionalized PEA bests supports endothelial cell adhesion, growth, and monolayer formation. Mechanical testing indicates that the elastic moduli of these materials are strongly dependent on the charge formulation, but do exhibit linearly elastic behavior at small strains (<10%). Our data suggest that PEA may be a viable biomaterial for use in tissue engineering applications, particularly for use as a vascular graft.

Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines.

Hepatitis C virus (HCV) remains a major medical problem. In-depth study of HCV pathogenesis and immune responses is hampered by the lack of suitable small animal models. The narrow host range of HCV remains incompletely understood. We demonstrate that the entire HCV life-cycle can be recapitulated in mouse cells. We show that antiviral signaling interferes with HCV RNA replication in mouse cells. We were able to infect mouse cells expressing human CD81 and occludin (OCLN)-the minimal set of entry factor factors required for HCV uptake into mouse cells. Infected mouse cells sustain HCV RNA replication in the presence of miR122 and release infectious particles when mouse apoE is supplied. Our data demonstrate that the barriers of HCV interspecies transmission can be overcome by engineering a suitable cellular environment and provide a blue-print towards constructing a small animal model for HCV infection.