The continued emergence of hantaviruses: isolation of a Seoul virus implicated in human disease, United Kingdom, October 2012.

Following a suspected case of hantavirus in a patientsuffering from acute kidney injury, rodents fromthe patient's property in Yorkshire and the Humber,United Kingdom (UK) were screened for hantaviruses.Hantavirus RNA was detected via RT-PCR in two Rattusnorvegicus. Complete sequencing and phylogeneticanalysis established the virus as a Seoul hantavirus,which we have provisionally designated as strainHumber. This is the first hantavirus isolated from wildrodents in the UK and confirms the presence of a pathogenicSeoul virus in Europe.

The Phenotypic Spectrum of New-onset IBD in Canadian Children of South Asian Ethnicity: A Prospective Multi-Centre Comparative Study.

BACKGROUND: Canadian-born children of South Asian [SA] ethnicity develop inflammatory bowel disease [IBD] at similar rates to those among Caucasian children. We evaluated the variation in phenotypic spectrum of IBD in SA and Caucasian children in a national paediatric inception cohort of new-onset IBD. METHODS: Patients aged <17 years, enrolled in a Canadian nationwide inception cohort study, were included. Baseline demographic and IBD phenotypic features were compared between SA and Caucasian children. Longitudinal outcomes through 18 months of follow-up were compared matched by propensity scores. RESULTS: Of 1156 children enrolled over 2014 to 2019, 623 were Caucasian [98% and 88% parents Canadian born] and 114 SA [79% Canadian born, 87% parents SA born]. Fewer SAs have a first-degree relative with IBD, 6% vs 19% in Caucasians, p = 0.002. SAs present at a younger age, median age 11.4 years (interquartile range [IQR] 9.2-14.3) vs 13 years [IQR 10.9-15 years], p = 0.03 and more commonly with a UC/IBD-U [ulcerative colitis/IBD-unclassified] subtype [ratio of UC/IBD-U to CD 1.2:1 vs 1:1.8 for Caucasians, p <0.001]. Additionally, a greater proportion of SA CD patients present with colonic-only disease [colonic-only CD/UC/IBD-U in SAs 67% vs 57% for Caucasians, p = 0.001], and among those with CD, colonic CD in SAs 31% vs 23% in Caucasians, p = 0.20]. Perianal fistulising disease was also numerically more common in SAs (14 [27%] vs 64 [18%], p = 0.06]. Adjusting for differences in phenotypic presentation, anti-tumour necrosis factor [TNF] exposure, and time to initiation was similar, and two-thirds of children, whether anti-TNF exposed or naïve, were in corticosteroid-free clinical remission at 18 months irrespective of ethnicity. CONCLUSIONS: The phenotypic spectrum of new-onset IBD in SA children differs from that of Caucasian children, but treatment and clinical course are similar within phenotypic subgroups.

Characterisation of the T-cell response to Ebola virus glycoprotein amongst survivors of the 2013-16 West Africa epidemic.

Zaire ebolavirus (EBOV) is a highly pathogenic filovirus which can result in Ebola virus disease (EVD); a serious medical condition that presents as flu like symptoms but then often leads to more serious or fatal outcomes. The 2013-16 West Africa epidemic saw an unparalleled number of cases. Here we show characterisation and identification of T cell epitopes in surviving patients from Guinea to the EBOV glycoprotein. We perform interferon gamma (IFNγ) ELISpot using a glycoprotein peptide library to identify T cell epitopes and determine the CD4+ or CD8+ T cell component response. Additionally, we generate data on the T cell phenotype and measure polyfunctional cytokine secretion by these antigen specific cells. We show candidate peptides able to elicit a T cell response in EBOV survivors and provide inferred human leukocyte antigen (HLA) allele restriction. This data informs on the long-term T cell response to Ebola virus disease and highlights potentially important immunodominant peptides.

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

Survival and predictors of outcomes in non-HIV-infected patients with extensively drug-resistant tuberculosis.

SETTING: A tuberculosis (TB) referral hospital in South Korea. OBJECTIVE: To evaluate predictors of treatment outcomes and survival among non-human immunodeficiency virus (HIV) infected patients with extensively drug-resistant TB (XDR-TB). DESIGN: Patients who were diagnosed with XDR-TB at the National Masan Tuberculosis Hospital from January 2001 to December 2005 were included in this study. We conducted a retrospective review of their medical records and mortality data. RESULTS: A total of 176 non-HIV-infected patients with XDR-TB were included. TB-related mortality was 48% (84/176), and the median survival time from the diagnosis date of XDR-TB was 51 months (range 0-127, 95%CI 32.53-69.47). Cure and treatment completion were classified as favourable outcome and treatment failure, death during treatment and default as poor outcome. Previous TB treatment with second-line drugs (aOR 2.76, 95%CI 1.02-7.44) and cavitary disease (aOR 3.01, 95%CI 1.12-8.08) were independent risk factors for poor outcome. Use of linezolid (aOR 0.10, 95%CI 0.01-0.69) and surgical resection (aOR 0.18, 95%CI 0.04-0.78) were associated with favourable outcome. CONCLUSION: There was high mortality in non-HIV-infected patients with XDR-TB at a TB referral hospital in South Korea. Adjunctive surgical treatment and linezolid improved the outcome for selected patients with XDR-TB.

Vitamin D status and risk for sarcopenia in youth with inflammatory bowel diseases.

Suboptimal vitamin D (vitD) status and reduced lean body mass are highly prevalent in pediatric inflammatory bowel diseases (IBD). The study objective was to determine sarcopenia prevalence and associations with vitD status in newly diagnosed pediatric IBD. Children with Crohn's disease (CD; n = 58) and ulcerative colitis (UC; n = 27) were included. Primary outcomes included body composition (total/regional/percent fat mass (FM), fat-free mass (FFM), skeletal muscle mass (SMM)), and vitD status (serum 25(OH)D). Sarcopenia was defined as SMM-z < -2. Additional variables measured included serum CRP, ESR, anthropometric, Pediatric Crohn's Disease Activity Index (PCDAI), and the Pediatric Ulcerative Colitis Disease Activity index (PUCAI). Sarcopenia and suboptimal 25(OH)D levels (< 50 nmol/l) were found in 23.5% (n = 20) and 52% (n = 44) of children, respectively. Younger children (< 13 years) with CD with suboptimal 25(OH)vitD (< 50 nmol/l) had the greatest frequency of sarcopenia (57.1%) (p = 0.004). Sarcopenia was prevalent in newly diagnosed, young children with CD with vitD deficiency.

First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador.

Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador.

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen.

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.

Costimulation enhances the active immunotherapy effect of recombinant anticancer vaccines.

Activation of T lymphocytes in the absence of a costimulatory signal can result in anergy or apoptotic cell death. Two molecules capable of providing a costimulatory signal, B7-1 (CD80) and B7-2 (CD86), have been shown to augment the immunogenicity of whole-tumor cell vaccines. To explore a potential role for costimulation in the design of recombinant anticancer vaccines, we used lacZ-transduced CT26 as an experimental tumor and beta-galactosidase (beta-gal) as the model tumor antigen. Attempts to augment the function of a recombinant vaccinia virus (rVV) expressing beta-gal by admixture with rVV expressing murine B7-1 were unsuccessful. However, a double recombinant vaccinia virus engineered to express both B7-1 and the model antigen beta-gal was capable of significantly reducing the number of pulmonary metastases when administered to mice bearing tumors established for 3 or 6 days. Most important, the double recombinant vaccinia virus prolonged the survival of tumor-bearing mice. These effects were antigen specific. The related costimulatory molecule B7-2 was found to have a similar, although less impressive enhancing effect on the function of a rVV expressing beta-gal. Thus, the addition of B7-1 and, to a lesser extent, B7-2 to a rVV encoding a model antigen significantly enhanced the therapeutic antitumor effects of these poxvirus-based, therapeutic anticancer vaccines.

A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease.

Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model.

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Poxviruses as expression vectors.

Poxviruses are widely used for the cytoplasmic expression of recombinant genes in mammalian cells. Recent improvements allow high expression and simplify the integration of multiple foreign genes. Vaccinia virus mutants and other poxviruses that undergo abortive infection in mammalian cells are receiving special attention because of their diminished cytopathic effects and increased safety. New replicating and 'non-replicating' vectors, encoding the bacteriophage T7 RNA polymerase for transcription of recombinant genes, have been engineered.

Host range restricted, non-replicating vaccinia virus vectors as vaccine candidates.

Three model systems were used to demonstrate the immunogenicity of highly attenuated and replication-defective recombinant MVA. (1) Intramuscular inoculation of MVA-IN-Fha/np induced humoral and cell-mediated immune responses in mice and protectively immunized them against a lethal respiratory challenge with influenza virus. Intranasal vaccination was also protective, although higher doses were needed. (2) In rhesus macaques, an immunization scheme involving intramuscular injections of MVA-SIVenv/gag/pol greatly reduced the severity of disease caused by an SIV challenge. (3) In a murine cancer model, immunization with MVA-beta gal prevented the establishment of tumor metastases and even prolonged life in animals with established tumors. These results, together with previous data on the safety of MVA in humans, suggest the potential usefulness of recombinant MVA for prophylactic vaccination and therapeutic treatment of infectious diseases and cancer.

Evaluation of the safety and immunogenicity of a candidate tuberculosis vaccine, MVA85A, delivered by aerosol to the lungs of macaques.

Tuberculosis (TB) is a reemerging disease. The only available vaccine, Mycobacterium bovis BCG, is delivered intradermally and confers highly variable efficacy against pulmonary disease. There is an urgent need for improved vaccination strategies. Murine studies suggest that immunizations delivered directly to the respiratory mucosa might be a more effective route of vaccination. This study compared the immunogenicity of a leading candidate tuberculosis (TB) vaccine, modified vaccinia virus Ankara expressing antigen 85A (MVA85A), in rhesus macaques, delivered either as an aerosol or as an intradermal boost immunization 12 weeks after an intradermal BCG prime vaccine. Aerosol vaccination was well tolerated. MVA85A delivered by aerosol or by intradermal injection induced antigen-specific immune responses in the periphery and the lung, with a trend toward the highest response when the compartment and route of delivery were matched. The ability of poxvirus-vectored vaccines delivered by the systemic route to induce responses in the mucosal immune compartment in macaques is in contrast to the independent compartmentalization of mucosal and systemic immune systems described in mice. Unlike intradermal vaccination, aerosol vaccination did not induce a detectable serum anti-vector antibody response. The delivery of vaccines to the lungs might provide an immunization strategy that limits the induction of systemic anti-vector immunity, which would be extremely useful in the development of improved vaccine strategies. This is the first study to show a recombinant MVA-vectored vaccine to be highly immunogenic when delivered by the aerosol route to nonhuman primates. These results provide important safety and proof-of-concept data for further evaluation of this route of immunization for use in human clinical trials.

Frequency of adverse reactions to first- and second-line anti-tuberculosis chemotherapy in a Korean cohort.

OBJECTIVE: To determine the frequency of and risk factors for major adverse drug reactions (MADRs) associated with anti-tuberculosis treatment at a tuberculosis (TB) referral hospital in the Republic of Korea. METHODS: Data from an ongoing natural history cohort study were analyzed for permanent regimen changes due to adverse drug reactions and confirmed by chart review. RESULTS: Among 655 subjects, there were 132 MADRs in 112 (17%) subjects. The most common MADRs were gastrointestinal (n = 53), musculoskeletal (n = 22), psychiatric (n = 10), visual (n = 9) and peripheral neuropathic (n = 8). MADRs were more frequent in subjects being treated with second-line regimens (16%) compared to first-line regimens (2.5%). Drugs frequently associated with MADRs were amikacin (3/10, 30%), linezolid (8/29, 28%), para-aminosalicylic acid (47/192, 24%), pyrazinamide (31/528, 5.8%), macrolides (2/44, 4.5%) and cycloserine (12/272, 4.4%). Fluoroquinolones accounted for a single MADR (1/377, 0.003%), despite widespread usage. In multivariate analysis, infection with multi- or extensively drug-resistant disease and previous history of anti-tuberculosis treatment were risk factors for MADR, with adjusted hazard ratios of respectively 2.2 (P = 0.02) and 1.6 (P = 0.04). CONCLUSION: MADRs are common during anti-tuberculosis chemotherapy in this population, occurring in more than one in six subjects. New and less toxic agents to treat drug-resistant TB are urgently needed.

In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies.

Lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. We have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (EIAV), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. Here, we report on the application of EIAV vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. The therapeutic potential of a single chain antibody against the tumour-associated antigen, 5T4, fused to immune enhancer moieties has been demonstrated in vitro and here we evaluate the genetic delivery of a 5T4 scFv fused to B7.1 (scFvB7) using an EIAV vector. The kinetics and concentration of protein produced following both intravenous (i.v.) and intramuscular (i.m.) administration was determined in immune competent adult mice. In addition, the immune response to the EIAV vector and the transgene were determined. Here, we show that a single injection of EIAV expressing scFv-B7 can give rise to concentrations of protein in the range of 1-5 microg/ml that persist in the sera for more than 50 days. After a second injection, concentrations of scFv-B7.1 rose as high as 20 microg/ml and levels greater than 2 microg/ml were present in the sera of all mice injected i.v. after 210 days despite the detection of antibodies against both the transgene and viral envelope for the duration of this study. These results demonstrate the potential of EIAV as a gene therapy vector for long-term production of therapeutic recombinant proteins.

Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line.

Modified vaccinia virus Ankara (MVA), attenuated by over 500 passages in primary chick embryo fibroblasts (CEF), is presently being used as a safe expression vector. We compared the host ranges of MVA and the parental Ankara strain in CEF and 15 permanent cell lines. The cells could be grouped into three categories: permissive, semipermissive, and nonpermissive. For MVA, the permissive category consisted of primary CEF, a quail cell line derived from QT6, and the Syrian hamster cell line BHK-21. Only in BHK-21 cells did the virus yield approach that occurring in primary CEF. The semipermissive category included two African green monkey cell lines: BS-C-1 and CV-1. The nonpermissive category for MVA consisted of three human cell lines HeLa, 293, and SW 839; one rhesus monkey cell line FRhK-4; two Chinese hamster cell lines CHO and CHL; one pig cell line PK(15); and three rabbit cell lines RK13, RAB-9, and SIRC. The grouping for MVA with a restored K1L host range gene was similar except for the inclusion of RK13 cells among permissive lines. The grouping for the Ankara strain, however, was quite different with more permissive and semipermissive cell lines. Nevertheless, in cells that were permissive for MVA, the virus replicated to higher levels than Ankara, consistent with both positive and negative growth elements associated with the adaptation of MVA. The cell lines were also characterized according to their susceptibility to MVA-induced cytopathic effects, expression of a late promoter regulated reporter gene by an MVA recombinant, and stage at which virion morphogenesis was blocked. Finally, the permissive BHK-21 cell line was shown to be competent for constructing and propagating recombinant MVA, providing an alternative to primary CEF.

The metabolic cost of two ranges of arm position height with and without hand weights during low impact aerobic dance.

To determine the energy cost of low impact aerobic dance while varying arm movement height and the use of hand weights, 10 adults volunteered to participate in four choreographed trials. All trials consisted of identical leg movements. Arm movements, however, were performed above shoulder level both with and without 0.9-kg hand weights and below shoulder level both with and without 0.9-kg hand weights. Open circuit spirometry was employed throughout the 10-min videotape guided trials, and heart rate was measured by telemetry. Neither the use of hand weights nor the change in arm position height significantly altered the energy cost of low impact aerobic dance. However, heart rate responses were significantly different. Caution should be observed by aerobics instructors and participants as to the use of heart rate as an indicator of intensity for low impact aerobic dance.

Addition of the MSA1 signal and anchor sequences to the malaria merozoite surface antigen 1 C-terminal region enhances immunogenicity when expressed by recombinant vaccinia virus.

Genes encoding four different C-terminal fragments of a Plasmodium falciparum merozoite surface antigen were generated: MSA1C-(Si,A), containing signal and anchor regions of MSA1; MSA1C-(Si,nA), containing the signal but not the anchor; MSA1C-(nSi,A), containing the anchor but not the signal, and MSA1C-(nSi,nA) containing neither the signal nor the anchor region. Each gene was inserted into the thymidine kinase region of vaccinia virus, under the control of a synthetic strong early/ late promoter. When the plasmodial genes were expressed in cells infected by the recombinant vaccinia virus, the two proteins containing the signal region were transported to the surface of infected cells. Infection of mice and rabbits with the latter recombinant viruses stimulated C-terminal-specific antibody levels that were 10-80-fold higher than those induced by the two recombinant viruses without the signal region. The combination of the signal and anchor regions with the C-terminal MSA1 protein also generated the most effective neutralization in a P. falciparum invasion assay.