T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.

Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice.

BACKGROUND: Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. METHODS: The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored. RESULTS: Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy. CONCLUSION: Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models.

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis.

Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development.

Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology.

Immune escape of melanoma: first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway.

Sequence analyses of the transporter associated with antigen processing (TAP) in tumor cell lines with deficient MHC class I surface expression identified a bp deletion at position 1489 near the ATP-binding domain of Tap1, causing a frameshift in one melanoma cell line. The impaired TAP1 protein expression was associated with deficient TAP2 protein expression, peptide binding, translocation, and MHC class I surface expression. Stable TAP1 gene transfer reconstitutes the described defects, whereas lysis by HLA-A2-restricted CTLs was still abrogated. This was attributable to a 2-bp insertion at position 890 in the HLA-A2 gene and was corrected after HLA-A2 cotransfection. This study describes for the first time mutations in two distinct components of the MHC class I antigen processing pathway, suggesting an immune selection against CTLs recognizing both TAP-dependent and -independent T-cell epitopes.

Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.

Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.

Low prevalence of porcine circovirus type 2 infections in farrowing sows and corresponding pre-suckling piglets in southern German pig farms.

Porcine circovirus type 2 (PCV2) is the assumed causative agent of a number of different diseases summarized as porcine circovirus diseases (PCVD). The virus is shed via different se- and excretions of PCV2 infected pigs. Transmission of the virus occurs horizontally and vertically either by oronasal or diaplacental infection. Recent research emphasizes the importance of diaplacental PCV2 infection or the infection in early stages of the piglet's life attributable to excretion of PCV2 by the dams within the suckling period. To estimate the prevalence of intrauterine PCV2 infections under field conditions in Bavaria the PCV2 status of farrowing sows (n=198) and corresponding pre-suckling piglets (n=590) of 20 piglet producing farms was examined. PCV2 viral load and anti-PCV2 antibodies in the serum of the sows and piglets were examined at time of farrowing or before colostrum intake, respectively. PCV2 excretion of the sows via saliva, feces and urine was examined additionally. PCV2 specific antibodies in the serum of the sows were detectable on 11 farms with a mean in herd seroprevalence of 35.5% in these farms. Only 0.65% of all samples collected from 198 sows were positive for PCV2 DNA (serum: 1%; feces: 0.5%; saliva: 0.5%; urine: 0.6%). PCV2 DNA was detectable in sample material from seronegative sows as well as from seropositive sows. In none of the pre-suckling serum samples of the piglets IgG antibodies against PCV2 or PCV2 DNA were present. No correlation between the antibody- and viremia status of the sows and the PCV2 excretion was detectable. In contrast to reports about a high prevalence of viremic pre-suckling piglets in the suckling period in North America, the results of the present study reveal that diaplacental infection with PCV2 is comparatively rare in Southern Germany and infection of piglets within the suckling period seems to be more likely.

Establishing quality control ranges for temocillin following CLSI-M23-A3 guideline.

OBJECTIVES: This study aimed to establish acceptable quality control ranges for temocillin disk diffusion tests and Etest(®) minimal inhibitory concentrations. METHODS: According to Clinical and Laboratory Standards Institute (CLSI) guideline, a Tier 2 quality control study was performed and involves seven laboratories. Each of them tested 10 replicates of two quality control strains (Escherichia coli ATCC 25922 and E. coli ATCC 35218) on three different media lots and, for disk diffusion, two disk lots. RESULTS: Proposed zone diameter quality control ranges were 12-25 mm for E. coli ATCC 25922 and 19-28 mm for E. coli ATCC 35218. Proposed Etest quality control ranges were 3-24 mg/l for E. coli ATCC 25922 and 2-6 mg/l E. coli ATCC 35218. CONCLUSION: Based on our results, we would advise the use of E. coli ATCC 35218 as QC strain for temocillin susceptibility testing and Etest because ranges obtained are narrower than with E. coli ATCC 25922 and do not overlap temocillin breakpoint.

Detection of a new cluster of porcine circovirus type 2b strains in domestic pigs in Germany.

PCV2 can be divided into three different genotypes: PCV2a, PCV2b and PCV2c. Since 2004/2005 PCV2b has become the predominant genotype in the domestic pig population worldwide. In the years 2010 and 2012 PCV2b mutant strains (mPCV2), classified as PCV2b-1C strains, were detected in porcine circovirus diseases (PCVD) affected pigs in China and the United States, respectively. Within one year (April 2013-April 2014) newly emerging mPCV2 strains were isolated in seven German pig farms routinely vaccinating against PCV2. Histopathological, clinical and molecular biological findings including in-situ hybridization (ISH) and real-time PCR indicate PCVD in the affected animals. Characterized isolates from five farms were closely related to the PCV2b-1C reference strain BDH (GenBank no. HM038017), whereas strains from two other farms were only 99.1% and 99.0% identical (based on the nucleotide sequence of the complete genome) to mPCV2 strain BDH, respectively.

Stable expression of HIV-1 Nef induces changes in growth properties and activation state of human astrocytes.

OBJECTIVE: Nef was shown to be the predominant viral protein expressed in HIV-1-infected astrocytes in vivo and in vitro suggesting a distinct role of Nef in this cell type. Nef-induced activation of T cells is well described, whereas the functional activities of Nef in astrocytes are unknown. Our aim was to examine the effect of Nef on growth properties and activation of astrocytes. DESIGN: Human Nef-expressing astrocytic cell lines were established by stable transfection with different wild-type and mutant nef genes derived from laboratory isolates and brain tissue. METHODS: Nef-expressing astrocytes were characterized in terms of growth properties (proliferation, growth in soft agar, focus formation) and morphology. Apoptotic cell death and expression of activation markers were determined by fluorescent antibody cell sorting. RESULTS: Astrocytic cell lines revealed persistent Nef expression--detectable at the levels of mRNA and protein--and showed altered growth properties and morphology. Elevated expression of activation markers such as glial fibrillary acidic protein and CD88 (complement receptor C5a) was observed; these are regarded as markers for inflammatory processes in the brain. This effect was independent of the nef type or the expression level of the Nef protein. In contrast with previous reports no evidence for increased apoptotic cell death was found in astrocytes expressing Nef stably. CONCLUSIONS: Our findings suggest that Nef changes the cellular properties of astrocytes, thus contributing to astrocyte activation and induction of astrogliosis in the central nervous system of individuals with AIDS.

Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase.

Modified vaccinia virus Ankara (MVA), a host range restricted and highly attenuated vaccinia virus strain, is unable to multiply in human and most other mammalian cell lines. Since viral gene expression is unimpaired in non-permissive cells recombinant MVA viruses are efficient as well as exceptionally safe expression vectors. We constructed a recombinant MVA that expresses the bacteriophage T7 RNA polymerase and tested its usefulness for transient expression of recombinant genes under the control of a T7 promoter. Using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene, infection with MVA-T7pol allowed efficient synthesis of recombinant enzyme in mammalian cells. Despite the severe host restriction of MVA, enzyme activities induced by infection with MVA-T7pol were similar to those determined after infection with a replication-competent vaccinia-T7pol recombinant virus. Thus, MVA-T7pol may be used as a novel vaccinia vector to achieve T7 RNA polymerase-specific recombinant gene expression in the absence of productive vaccinia virus replication.

Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors.

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.

Transient host range selection for genetic engineering of modified vaccinia virus Ankara.

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.

Thromboxane receptor blockade in an animal model of ARDS.

Manipulation of arachidonic acid metabolism may be important in the prevention and treatment of the adult respiratory distress syndrome. This study evaluated a thromboxane receptor blocker, BM 13.177, in a sheep endotoxin model. Sheep with chronic lung lymph fistulas were pretreated with the blocker before being given endotoxin. The blocker attenuated the early increase in pulmonary artery pressure usually seen after endotoxin (blocker + endotoxin, 31.0 +/- 14.5 mm Hg; endotoxin alone, 42.1 +/- 9.6 mm Hg). There was no effect on lymph flows during the later (permeability) phase (at 6 hours: blocker + endotoxin, 574 +/- 287% baseline; endotoxin alone, 311 +/- 102% baseline). Administration of the blocker alone increased pulmonary artery pressure, arterial pressure, and central venous pressure and decreased heart rate and cardiac index. Thromboxane receptor blockade presumably has little effect on elements of arachidonic acid metabolism other than thromboxane. Thromboxane appears to be important in the early pulmonary hypertension of the sheep endotoxin model but is relatively unimportant in the development of increased pulmonary capillary permeability.

Rescue of measles virus using a replication-deficient vaccinia-T7 vector.

A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.

Recombinant Vaccinia Virus MVA for Generation and Analysis of T Cell Responses Against Tumor Associated Antigens.

Live attenuated viruses used as vaccines are known for their efficacy to elicit protective immunity against viral diseases. More recently, with an increasing number of tumor-associated antigens (TAA) being identified and molecularly cloned (1) the development of vaccines for cancer immunotherapy has gained considerable interest. In particular, live recombinant viral vectors seem to be appropriate delivery systems for efficient presentation of TAA to the immune system. The promise of viral vectors is likely to be founded on their capacity for high-level expression of target genes combined with their intrinsic property to activate immunological control systems mimicking an infection with a disease causing agent.

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.

Transferred triage to a level I trauma center in a mass catastrophe of patients; many of them with burns.

Three airplanes were involved in an airshow accident at the Ramstein military airbase on 28th of August 1988 causing immediate 45 dead and approximate 400 injured people. As a Level I Trauma facility we received 47 patients in different state of distress direct from airfield within an hour. The disaster plan was activated and sufficient personal and infrastructure could be mobilized.