Tyler R. Prestwood

Enhanced Infection of Liver Sinusoidal Endothelial Cells in a Mouse Model of Antibody-Induced Severe Dengue Disease

Dengue virus (DENV) causes disease ranging from dengue fever (DF), a self-limited febrile illness, to the potentially lethal dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). DHF/DSS usually occurs in patients who have acquired DENV-reactive antibodies prior to infection, either from a previous infection with a heterologous DENV serotype or from an immune mother. Hence, it has been hypothesized that subneutralizing levels of antibodies exacerbate disease, a phenomenon termed antibody-dependent enhancement (ADE). However, given the lack of suitable animal models for DENV infection, the mechanism of ADE and its contribution to pathology remain elusive. Here we demonstrate in mice that DENV-specific antibodies can sufficiently increase severity of disease so that a mostly nonlethal illness becomes a fatal disease resembling human DHF/DSS. Antibodies promote massive infection of liver sinusoidal endothelial cells (LSECs), resulting in increased systemic levels of virus. Thus, a subprotective humoral response may, under some circumstances, have pathological consequences.

A Protective Role for Dengue Virus-Specific CD8+ T Cells

Infection with one of the four serotypes of dengue virus (DENV1–4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8+ T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8+ T cell response, whereas viral replication and a robust CD8+ T cell response were observed after infection of IFN-α/βR−/− mice. Depletion of CD8+ T cells from IFN-α/βR−/− mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8+ T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-α/βR−/− mice. DENV-specific CD8+ T cells produced IFN-γ, TNF-α, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8+ T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8+ T cells in the host defense against DENV and demonstrate that the anti-DENV CD8+ T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

CD4+ T Cells Are Not Required for the Induction of Dengue Virus-Specific CD8+ T Cell or Antibody Responses but Contribute to Protection after Vaccination

The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8+ T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/βR−/− C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4+ T cells during primary DENV infection. Four I-Ab–restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4+ T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4+ T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4+ T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4+ T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8+ T cell response. However, immunization with the CD4+ T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4+ T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4+ T cell responses by vaccination may be beneficial.

A Mouse-Passaged Dengue Virus Strain with Reduced Affinity for Heparan Sulfate Causes Severe Disease in Mice by Establishing Increased Systemic Viral Loads

ABSTRACT The four serotypes of dengue virus (DENV1 to DENV4) cause extensive morbidity and mortality. A major obstacle to studying disease pathogenesis and developing therapies has been the lack of a small-animal model. We previously reported isolation of a DENV2 strain, obtained by passaging a clinical isolate between mosquito cells and mice, that caused severe DENV disease in mice and contained multiple mutations, including many in domain II of the envelope (E) protein. Here, we describe a recombinant virus, differing from the non-mouse-passaged virus by two mutations in the E protein, that induces vascular leakage and tumor necrosis factor alpha (TNF-α)-mediated lethality, while the non-mouse-passaged virus causes paralysis. This recombinant virus has a weaker affinity for heparan sulfate, resulting in an increased serum half-life, higher systemic viral loads, and high levels of TNF-α in the serum of infected mice. These results exemplify the role of the E protein in modulating virion clearance and connect the effect of clearance on the systemic viral loads responsible for severe disease manifestations.

Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity

Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

ABSTRACT We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8+ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8+ T cells in preventing DENV-induced CNS disease.

Cardif-Mediated Signaling Controls the Initial Innate Response to Dengue Virus In Vivo

ABSTRACT The role of Cardif-dependent signaling in controlling dengue virus (DENV) infection and regulating type I interferon (IFN) production in vivo was examined in Cardif-deficient mice. DENV RNA levels were significantly elevated in both the serum and lymphoid tissues of Cardif−/− mice at early times compared to those in wild-type animals. Type I IFN production was delayed in these locales of Cardif−/− mice until 18 h postinfection, indicating that Cardif regulates the initial type I IFN response in lymphoid tissues. In contrast, DENV viral loads in nonlymphoid tissues were similar between Cardif−/− and wild-type mice. These results reveal that RNA helicase-mediated sensing acts as a first line of innate defense against DENV infection in vivo and functions in a tissue-dependent manner.

Detection of Intestinal Cancer by Local, Topical Application of a Quenched Fluorescence Probe for Cysteine Cathepsins

Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

Trafficking and Replication Patterns Reveal Splenic Macrophages as Major Targets of Dengue Virus in Mice

ABSTRACT Human postmortem studies of natural dengue virus (DENV) infection have reported systemically distributed viral antigen. Although it is widely accepted that DENV infects mononuclear phagocytes, the sequence in which specific tissues and cell types are targeted remains uncharacterized. We previously reported that mice lacking alpha/beta and gamma interferon receptors permit high levels of DENV replication and show signs of systemic disease (T. R. Prestwood et al., J. Virol. 82:8411–8421, 2008). Here we demonstrate that within 6 h, DENV traffics to and replicates in both CD169+ and SIGN-R1+ macrophages of the splenic marginal zone or draining lymph node, respectively, following intravenous or intrafootpad inoculation. Subsequently, high levels of replication are detected in F4/80+ splenic red pulp macrophages and in the bone marrow, lymph nodes, and Peyers patches. Intravenously inoculated mice begin to succumb to dengue disease 72 h after infection, at which time viral replication occurs systemically, except in lymphoid tissues. In particular, high levels of replication occur in CD68+ macrophages of the kidneys, heart, thymus, and gastrointestinal tract. Over the course of infection, proportionately large quantities of DENV traffic to the liver and spleen. However, late during infection, viral trafficking to the spleen decreases, while trafficking to the liver, thymus, and kidneys increases. The present study demonstrates that macrophage populations, initially in the spleen and other lymphoid tissues and later in nonlymphoid tissues, are major targets of DENV infection in vivo.

Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.