Andreas Suhrbier

Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.

H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies.

H‐2 class I‐negative, HLA‐A2.1‐transgenic HHD mice were used for a comparative evaluation of the immunogenicity of HLA‐A2.1‐restricted human tumor‐associated cytotoxic T lymphocyte (CTL) epitopes. A hierarchy was established among these peptides injected into mice in incomplete Freunds adjuvant which correlates globally with their capacity to bind and stabilize HLA‐A2.1 molecules. Co‐injection of a helper peptide enhanced most CTL responses. In contrast, classical HLA class I‐transgenic mice which still express their own class I molecules did not, in most cases, develop HLA‐A2.1‐restricted CTL responses under the same experimental conditions. Different monoepitope immunization strategies of acceptable clinical usage were compared in HHD mice. Recombinant Ty‐virus‐like particles, or DNA encoding epitopes fused to the hepatitis B virus middle envelope protein gave the best results. Using this latter approach and a melanoma‐based polyepitope construct, CTL responses against five distinct epitopes could be elicited simultaneously in a single animal. Thus, HHD mice provide a versatile animal model for preclinical evaluation of peptide‐based cancer immunotherapy.

Chikungunya Virus Arthritis in Adult Wild-Type Mice

ABSTRACT Chikungunya virus is a mosquito-borne arthrogenic alphavirus that has recently reemerged to produce the largest epidemic ever documented for this virus. Here we describe a new adult wild-type mouse model of chikungunya virus arthritis, which recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ). Infection with a virus isolate from the recent Reunion Island epidemic induced significantly more mononuclear infiltrates, proinflammatory mediators, and foot swelling than did an Asian isolate from the 1960s. Primary mouse macrophages were shown to be productively infected with chikungunya virus; however, the depletion of macrophages ameliorated rheumatic disease and prolonged the viremia. Only 1 μg of an unadjuvanted, inactivated, whole-virus vaccine derived from the Asian isolate completely protected against viremia and arthritis induced by the Reunion Island isolate, illustrating that protection is not strain specific and that low levels of immunity are sufficient to mediate protection. IFN-α treatment was able to prevent arthritis only if given before infection, suggesting that IFN-α is not a viable therapy. Prior infection with Ross River virus, a related arthrogenic alphavirus, and anti-Ross River virus antibodies protected mice against chikungunya virus disease, suggesting that individuals previously exposed to Ross River virus should be protected from chikungunya virus disease. This new mouse model of chikungunya virus disease thus provides insights into pathogenesis and a simple and convenient system to test potential new interventions.

Antitumor Activity of 3-Ingenyl Angelate: Plasma Membrane and Mitochondrial Disruption and Necrotic Cell Death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodessication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 micro g) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; n = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; n = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Foxn1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD(90) for PEP005 for a panel of tumor cell lines was 180-220 micro M. Electron microscopy showed that treatment with PEP005 both in vitro (230 micro M) and in vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. (51)Cr release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 micro M) treatment of tumor cells in vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Chikungunya Virus Nonstructural Protein 2 Inhibits Type I/II Interferon-Stimulated JAK-STAT Signaling

ABSTRACT Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms to circumvent the hosts antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2 (P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral pathogenesis.

Neutrophils are a key component of the antitumor efficacy of topical chemotherapy with ingenol-3-angelate.

Harnessing neutrophils for the eradication of cancer cells remains an attractive but still controversial notion. In this study, we provide evidence that neutrophils are required to prevent relapse of skin tumors following topical treatment with a new anticancer agent, ingenol-3-angelate (PEP005). Topical PEP005 treatment induces primary necrosis of tumor cells, potently activates protein kinase C, and was associated with an acute T cell-independent inflammatory response characterized by a pronounced neutrophil infiltrate. In Foxn1nu mice depleted of neutrophils and in CD18-deficient mice (in which neutrophil extravasation is severely impaired) PEP005 treatment was associated with a >70% increase in tumor relapse rates. NK cell or monocyte/macrophage deficiency had no effect on relapse rates. Both in vitro and in mice, PEP005 induced MIP-2/IL-8, TNF-α, and IL-1β, all mediators of neutrophil recruitment and activation. In vitro, PEP005 activated human endothelial cells resulting in neutrophil adhesion and also induced human neutrophils to generate tumoricidal-reactive oxygen intermediates. Treatment of tumors with PEP005 significantly elevated the level of anticancer Abs, which were able to promote neutrophil-mediated Ab-dependent cellular cytotoxicity (ADCC) in vitro. PEP005 treatment of tumors grown in SCID mice was also associated with >70% increase in tumor relapse rates. Taken together, these data suggest a central role for neutrophil-mediated ADCC in preventing relapse. PEP005-mediated cure of tumors therefore appears to involve initial chemoablation followed by a neutrophil-dependent ADCC-mediated eradication of residual disease, illustrating that neutrophils can be induced to mediate important anticancer activity with specific chemotherapeutic agents.

Clinical and pathologic aspects of arthritis due to Ross River virus and other alphaviruses

Purpose of reviewArthritogenic alphaviruses are globally distributed mosquito-borne RNA viruses causing epidemics of polyarthritis/arthralgia, with disease emerging or reemerging and increasingly being reported in travelers. This article summarizes the current knowledge of these diseases, focusing on recent developments in the understanding of Ross River virus disease. Recent findingsAlphaviral arthritides have often been blamed for protracted chronic illnesses. However, validated quality-of-life questionnaires and exhaustive searches for differential diagnoses showed that Ross River virus disease, although severe at onset, progressively resolved over 3 to 6 months. Many patients did experience long-term disease lasting more than 12 months, but in nearly all cases this was due to other conditions, primarily unrelated rheumatic conditions or depression. There is no indication that alphaviral arthritides predispose to other conditions; thus, patients whose Ross River virus disease has actually resolved may be underdiagnosed for other conditions. Ross River virus polyarthritis probably arises from inflammation associated with productive viral infections in synovial macrophages, which persist despite neutralizing antibodies and antiviral cytokine responses. Persistence may be facilitated by downregulation of cytokine responses by virus-antibody complexes binding to Fc receptors and induction of interleukin-10. How virus escapes neutralizing antibodies remains unclear but may involve phagocytosis of apoptotic virus-infected cells and infection of the phagocyte via the phagosome. SummaryDiagnosis of alphaviral arthritides is complicated by nonspecific symptoms and the lack of commercial serodiagnostic kits, except for Ross River and Barmah Forest virus infections in Australia. Differential diagnoses should be actively pursued, especially if symptoms persist. Treatment with nonsteroidal anti-inflammatory drugs appears largely effective, with no evidence of long-term sequelae or relapse.

Immune responses to ISCOM® formulations in animal and primate models

ISCOMs are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs will also be discussed.

Five new cytotoxic T cell epitopes identified within Epstein-Barr virus nuclear antigen 3.

Epstein-Barr virus (EBV) CD8+ cytotoxic T lymphocyte (CTL) epitopes are currently being considered for inclusion into subunit vaccines. Here we describe the characterization of five new CTL epitopes within EBV nuclear antigen 3 (EBNA3), confirming EBNA3 as a major target for CTL recognition.

Multi-epitope DNA vaccines.

The evolution of vaccine strategies has seen a move from whole organisms to recombinant proteins, and further towards the ultimate in minimalist vaccinology, the epitope. The epitope‐based approach is clearly compelling as only a relatively tiny, but immunologically relevant, sequence is often capable of inducing protective immunity against a large and complex pathogen. The post‐reductionist era in epitope‐based vaccinology has seen a quest to re‐construct complexity and design vaccines containing many epitopes. The hope is that such multi‐epitope vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. The ability of DNA vaccination to co‐deliver a series of antibody and/or CD4 T cell epitopes remains largely unexplored. Successful viral vector and DNA‐based experimental vaccines coding for multiple contiguous CDS CTL epitopes have, however, recently been described. This simple CTL poly‐epitope (or polytope) strategy may find application in the design of vaccines against several diseases including EBV, HIV and cancer.