Nir Paran

Detection of Zika virus RNA in whole blood of imported Zika virus disease cases up to 2 months after symptom onset, Israel, December 2015 to April 2016

Zika virus RNA presence in serum, whole-blood and urine samples from six Israeli travellers symptomatic for Zika virus disease was examined. Whole-blood samples were positive for as late as 2 months (58 days) post-symptom onset, longer than for urine (26 days) and serum (3 days). These findings suggest the utility of whole blood in Zika infection diagnosis.

Postexposure Immunization with Modified Vaccinia Virus Ankara or Conventional Lister Vaccine Provides Solid Protection in a Murine Model of Human Smallpox

BACKGROUND Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain. METHODS We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV). RESULTS In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses. CONCLUSIONS ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.

Clinical and Immune Responses after Revaccination of Israeli Adults with the Lister Strain of Vaccinia Virus

BACKGROUND During the winter of 2002-2003, the Israeli health authorities launched a campaign to vaccinate first responders against smallpox. METHODS In an open study, 159 healthy, preimmunized adults, 24-52 years old, who participated in the campaign were vaccinated with the Lister strain of vaccinia virus by the multipuncture technique. The safety, immunogenicity, and reactogenicity of the vaccine were assessed. RESULTS Successful vaccination rates were 61% and 56%, on the basis of clinical take and seroconversion, respectively. Adverse events among the vaccinees were minor. Seventy-nine (88%) of the 90 vaccinees with clinical take also seroconverted ( kappa =0.779). The level of preexisting antibodies inversely correlated with the rates of clinical take and seroconversion (P</=.0098). In the group of vaccinees with the lowest preexisting levels of antibodies, 89% and 86% developed clinical take or seroconverted, respectively. The time since last vaccination was significantly associated with the rates of clinical take and seroconversion (P</=0.001). CONCLUSIONS These rates of successful vaccination in previously immunized individuals are consistent with the historical experience of use of this vaccine in Israel. The rate of occurrence and the severity of local and other reactions in the vaccinees were within the expected range. Levels of preexisting antibodies and the time since last vaccination played a major role in determining success rates.

Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response

The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.

Smallpox vaccines New formulations and revised strategies for vaccination

Smallpox has been eradicated but stockpiles of the causative infectious agent, variola virus, have been maintained over decades. Today, the threat of accidental or intentional poxvirus release is accompanied by the fact that the existing licensed smallpox vaccines cause rare but severe adverse reactions yet are the only products with approved efficacy against smallpox. New safer vaccines and new strategies of immunization are to be developed to fit to a scenario of emergency smallpox vaccination. However, we still lack knowledge about the pathogen and the mechanisms involved in acquiring protective immunity. Here, we review the history of smallpox vaccines and recent achievements in the development of highly efficacious and safer vaccines and vaccine applications. These include i) assessment of adequate animal models to study pathogenesis and protective immunity, ii) characterization of the immunity elicited by next generation vaccines, and (iii) the investigation of the requirements for rapidly protective vaccination.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104–120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope’s critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Establishment of cell-based reporter system for diagnosis of poxvirus infection.

Poxvirus detection assays are based on morphology, viral antigens and specific nucleic acids, none of which indicates virus viability or infectious capacity. Determination of virus viability is achieved by propagation in cell cultures and subsequent analysis by the mentioned methods, a process that takes days. Thus, presented here the development of a new assay, named PILA (Poxvirus Infection Luciferase Assay), for rapid detection of infectious poxviruses which is a cell-based reporter assay. The assay is composed of two steps: (i) Transfection of cells with a poxvirus specific reporter vector which consists of the early 7.5-kDa-STR promoter, regulating the expression of luciferase gene; (ii) Infection with a poxvirus containing sample. Luciferase activity measured post infection, indicates the presence of infectious poxvirus in the sample. The assay can detect quantities as low as 100 PFU of VACV, six hours post infection. Orthopox virus universality was confirmed by detection of various Orthopoxviruses, and specificity was verified by using pox-specific neutralizing antibodies. The PILA is specific, rapid, simple, and suitable for detecting viable virus. The assay can be utilized for applications such as poxvirus titration, neutralizing assay and drug discovery. The assay was adjusted for live detection assay by using GFP as reporting gene.

TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

Induction, treatment and prevention of eczema vaccinatum in atopic dermatitis mouse models

Eczema vaccinatum is a severe and occasionally lethal complication of smallpox vaccine, characterized by systemic viral dissemination, distant from the initial inoculation site of the vaccine. A major risk factor for eczema vaccinatum is a background of atopic dermatitis, a chronic, common allergic, relapsing disorder, manifested by dry and inflamed skin, itchy rash, Th2 biased immune response and hypersensitivity to various antigens. Unlike the severe manifestations of eczema vaccinatum in humans, current models present only mild symptoms that limits examination of potential therapeutics for eczema vaccinatum. The atopic dermatitis and eczema vaccinatum models we present here, are the first to simulate the severity of the diseases in humans. Indeed, dermatitic mice display persistent severe dermatitis, characterized by dry and inflamed skin with barrier dysfunction, epidermal hyperplasia and significant elevation of serum IgE. By exposing atopic dermatitis mice to ectromelia virus, we generated eczema vaccinatum that mimic the human disease better than known eczema vaccinatum models. Similarly to humans, eczematous mice displayed enlarged and disseminated skin lesions, which correlated with elevated viral load. Cidofovir and antiviral antibodies conferred protection even when treatment started at a late eczematous stage. Moreover, we are the first to demonstrate that despite a severe background of atopic dermatitis, modified vaccinia Ankara virus (MVA) vaccination protects against lethal ectromelia virus exposure. We finally show that protection by MVA vaccination is dependent on CD4+ T cells and is associated with significant activation of CD8+ cytotoxic T cells and induction of humoral immunity.

First Diagnosed Case of Camelpox Virus in Israel

An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype.