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Background Preparation for an H5N1 influenza pandemic in humans could include priming the population in the pre-pandemic period with a vaccine produced from an existing H5N1 vaccine strain, with the possibility of boosting with a pandemic virus vaccine when it becomes available. We investigated the longevity of the immune response after one or two priming immunizations with a whole-virus H5N1 vaccine and the extent to which this can be boosted by later immunization with either a homologous or heterologous vaccine. Methods Mice received one or two priming immunizations with a Vero cell culture-derived, whole-virus clade 1 H5N1 vaccine formulated to contain either 750 ng or 30 ng hemagglutinin. Six months after the first priming immunization, mice received either a booster immunization with the same clade 1 vaccine or a heterologous clade 2.1 vaccine, or buffer. Humoral and cellular immune responses were evaluated before and at regular intervals after immunizations. Three weeks after booster immunization, mice were challenged with a lethal dose of wild-type H5N1 virus from clades 1, 2.1 or 2.2 and survival was monitored for 14 days. Results One or two priming immunizations with the 750 ng or 30 ng HA formulations, respectively, induced H5N1-neutralizing antibody titers which were maintained for ≥6 months and provided long-term cross-clade protection against wild-type virus challenge. Both humoral and cellular immune responses were substantially increased by a booster immunization after 6 months. The broadest protective immunity was provided by an immunization regimen consisting of one or two priming immunizations with a clade 1 vaccine and a boosting immunization with a clade 2.1 vaccine. Conclusions These data support the concept that pre-pandemic vaccination can provide robust and long-lasting H5N1 immunity which could be effectively boosted by immunization either with another pre-pandemic vaccine or with the pandemic strain vaccine.

Background Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness. Methods A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID50 assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus. Results The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus. Conclusions The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a beta-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this beta-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

Ross River Virus has caused reported outbreaks of epidemic polyarthritis, a chronic debilitating disease associated with significant long-term morbidity in Australia and the Pacific region since the 1920s. To address this public health concern, a formalin- and UV-inactivated whole virus vaccine grown in animal protein-free cell culture was developed and tested in preclinical studies to evaluate immunogenicity and efficacy in animal models. After active immunizations, the vaccine dose-dependently induced antibodies and protected adult mice from viremia and interferon α/β receptor knock-out (IFN-α/βR(-/-)) mice from death and disease. In passive transfer studies, administration of human vaccinee sera followed by RRV challenge protected adult mice from viremia and young mice from development of arthritic signs similar to human RRV-induced disease. Based on the good correlation between antibody titers in human sera and protection of animals, a correlate of protection was defined. This is of particular importance for the evaluation of the vaccine because of the comparatively low annual incidence of RRV disease, which renders a classical efficacy trial impractical. Antibody-dependent enhancement of infection, did not occur in mice even at low to undetectable concentrations of vaccine-induced antibodies. Also, RRV vaccine-induced antibodies were partially cross-protective against infection with a related alphavirus, Chikungunya virus, and did not enhance infection. Based on these findings, the inactivated RRV vaccine is expected to be efficacious and protect humans from RRV disease.

BackgroundA novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.MethodsAntibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine.ResultsThe whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.ConclusionsThe induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.

We have isolated a substance with anti-retroviral activity from the freshly prepared crude extract of Chelidonium majus L. (greater celandine) by 9-aminoacridine precipitation method and ion exchange chromatography using Dowex-50W/H+ resin followed by the gel filtration on Sephadex-75 column. Elemental and phenol/sulfuric acid method analyses as well as the mass spectrometry of the purified substance indicated that it may represent a low-sulfated poly-glycosaminoglycan moiety with molecular weight of approximately 3800 Da. The substance prevented infection of human CD4+ T-cell lines AA2 and H9 with HIV-1 at concentration of 25 microg/mL as well as the cell-to-cell virus spread in H9 cells continuously infected with HIV-1, as determined by the measurement of reverse transcriptase activity and p24 content in cell cultures. Furthermore, we have shown in a murine AIDS model that the treatment with purified substance significantly prevented splenomegaly and the enlargement of cervical lymph nodes in C57Bl/6 mice chronically infected with the pool of murine leukemia retroviruses. The mechanism(s) of anti-retroviral activity of this substance have to be elucidated.

Since we detected that donor dopamine pre-treatment ameliorates lung function after hypothermia and ischaemia/reperfusion in an isolated rat lung model we studied, whether other catecholamines have beneficial effects on lungs.Rats were treated with noradrenaline, adrenaline or dobutamine in different doses. Thereafter lungs were explanted, flushed with Perfadex® solution and stored at 4°C for different time periods. Oedema production was measured and inflammatory mediators were analysed after reperfusion and ventilation.Low-dose noradrenaline or dobutamine did not reduce tissue oedema after eight hours of hypothermia, whereas higher doses significantly reduced oedema formation. Low-dose catecholamines did not prevent the inflammatory response, whereas higher doses of beta-receptor-stimulating catecholamines significantly blunted inflammatory reaction.This study demonstrates that adrenergic-receptor-stimulating catecholamines have a protective dose-dependent effect on lungs after hypothermia and ischaemia/reperfusion. Although noradrenaline and dobutamine have similar dose-dependent organ-protective effects to dopamine, they have more side-effects.

BACKGROUND: The known biological activities of nitric oxide suggest a role in bone healing. We hypothesized that L-arginine, a source of nitric oxide, expedites the healing process of stabilized diaphyseal defects. TYPE OF STUDY: Prospective blinded animal study. METHODS: Using a guinea-pig model, a 7 mm diaphyseal and periosteal defect was produced in the right femur and splinted intramedullary with a 1.4 mm K-wire. The guinea pigs (n = 44) were treated orally in three parallel groups: two treatment groups received high doses of L-arginine (one group for 2 weeks and the other for 4 weeks) and a control group received vehicle only. After four weeks, all animals were killed and both femora explanted. Radiological, histological, histomorphometric and mechanical evaluation was performed blinded. RESULTS: Radiographs showed significantly more healings in the treatment groups (2 weeks, 10/15; 4 weeks, 11/15) than in the control group (3/14). The mechanical energy necessary for femur failure was significantly higher in the 4-week treatment group than in the control group (P < 0.05). Histology and histomorphometry showed significantly increased coverage of nonvascularized bone fragments with newly formed bone in the treatment groups (P ≤ 0.05). The contralateral uninjured femora did not show significant differences between groups. CONCLUSIONS: Oral L-arginine expedites healing in stabilized diaphyseal defects in guinea pigs without detrimentally affecting uninjured counterparts.

A double-inactivated, candidate whole virus vaccine against severe acute respiratory syndrome associated coronavirus (SARS-CoV) was developed and manufactured at large scale using fermenter cultures of serum protein free Vero cells. A two step inactivation procedure involving sequential formaldehyde and U.V. inactivation was utilised in order to ensure an extremely high safety margin with respect to residual infectivity. The immunogenicity of this double-inactivated vaccine was characterised in the mouse model. Mice that were immunised twice with the candidate SARS-CoV vaccine developed high antibody titres against the SARS-CoV spike protein and high levels of neutralising antibodies. The use of the adjuvant Al(OH)3 had only a minor effect on the immunogenicity of the vaccine. In addition, cell mediated immunity as measured by interferon-gamma and interleukin-4 stimulation, was elicited by vaccination. Moreover, the vaccine confers protective immunity as demonstrated by prevention of SARS-CoV replication in the respiratory tract of mice after intranasal challenge with SARS-CoV. Protection of mice was correlated to antibody titre against the SARS-CoV S protein and neutralising antibody titre.

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present “gold standard” of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.

BACKGROUND: The availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes. METHODS: Mice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively. RESULTS: MVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4(+) and CD8(+) T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4(+) T cell responses and NP-specific CD8(+) T cell responses were associated with increased protective efficacy. CONCLUSIONS: MVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4(+) and CD8(+) T cell responses.

Background For clinical development of a novel multivalent OspA vaccine against Lyme borreliosis, serological assays are required which can be used to establish immune correlates of protection against infection with Borrelia. Methods Four assays (an OspA IgG ELISA, a competitive inhibition (CI) ELISA, a Borrelia surface-binding (SB) assay and a Borrelia killing assay) were used to evaluate the correlation between immune responses induced by rOspA 1/2 (a chimeric immunogen containing protective epitopes from OspA serotypes 1 and 2), and protective immunity against infection by B. burgdorferi s.s. (OspA-1) and B. afzelii (OspA-2). Mice were immunized with OspA 1/2 doses ranging from 0.3 ng to 100 ng, to induce a range of OspA antibody titers, and exposed to needle challenge with B. burgdorferi s.s. or tick challenge with B. afzelii. Receiver operator characteristics (ROC) curves were constructed for each assay, and the area under the curve (AUC), sensitivity, specificity and Youden Index were calculated. Potential cutoff antibody titers which could be used as correlates of vaccine-induced protection were derived from the maximum Youden Index. Results Immunization with OspA-1/2 provided dose-dependent protection against infection with B. burgdorferi s.s. and B. afzelii. Antibody responses detected by all four assays were highly significantly correlated with protection from infection by either B. burgdorferi s.s. (p

Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin®, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin®-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2–8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin® by simple mixing. These preclinical data support further testing of Vaxfectin®-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans.

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

2-chloro-N(6) -cyclopentyladenosine (CCPA) was proven to be a protective factor in ischemic reperfusion injury. The purpose of this study was to determine how CCPA would affect the single tissue layers of the adipocutaneous flap.Seventy male Wistar rats were divided into 5 experimental groups. Samples were taken of the area of flap necrosis and the wound margin after classical or pharmacological preconditioning on the fifth postoperative day. All samples were fixed in formaldehyde, embedded in paraplast, and analyzed in 3- to 4-μm sections (hemalaun-eosin stain and light microscopy).In general, wound healing was alike and remained unaffected by the experimental design. The most sensitive part of the flap during preconditioning is the subcutis. The number of neutrophils and of plasma cells is reduced significantly (p.05).CCPA has an effect on each tissue layer of the flap. Subcutis became apparent as the most sensitive layer. CCPA influences complement pathway and neutrophils directly and indirectly.

Immune and inflammatory responses and the regulation of cellular events seem to be major factors in ischemia-reperfusion (I/R) injury. The inhibition of nuclear transcription factor κB (NF-κB) by pyrrolidine dithiocarbamate (PDTC) shows beneficial effects in animal models in various diseases and tissues with respect to I/R injury. If these results from other tissues could be transferred to adipocutaneous flaps in rats, a new approach to pharmacologic preconditioning could be defined for tissue transfer, and PDTC could be a solution as a trigger and effector to ameliorate the aftermath of the I/R injury.Fifty-six male WISTAR rats were divided into four experimental groups. An epigastric adipocutaneous flap was raised, and the average flap necrosis was assessed for all groups on the fifth postoperative day using planimetric software.The control group had a significantly lower flap necrosis than the ischemic control group (p.05). The PDTC nonischemic group had a significantly lower flap necrosis than the ischemic control group (p = .005). The ischemic PDTC group had a 10% reduction in flap necrosis that was not significant.Our data show that a significant reduction in flap necrosis can be achieved by intravenous application of PDTC.

BACKGROUND Donor treatment with dopamine (DA) is an effective modality to improve organ quality by reduction of hypothermic, ischemic and reperfusion (I/R) injury. It is unknown by which mechanism DA reduces oedema formation and inflammation. Therefore we tested the first time in an isolated rat lung model if dopaminergic or adrenergic receptors are involved. MATERIAL/METHODS Rats were treated for 1 hr with NaCl, DA or simultaneously with DA alpha- beta- D1- or D2-receptor blockers. Thereafter lungs were explanted, flushed with Perfadex-solution and stored at 4°C. Peak inspiratory pressure (PIP), pulmonary arterial pressure (PAP) and lung weight were measured during reperfusion of 3 hrs. Inflammatory mediators and the expression of adhesion molecules were measured after perfusion. RESULTS Up to 6 hours of hypothermia did not influence oedema formation or PIP and PAP during reperfusion time. However, hypothermia after 8 hrs significantly increased PIP, PAP and pulmonary oedema in NaCl, alpha- and beta-blocker treated lungs, but significantly not in DA, D1- or D2-blocker treated lungs. Perfusion and ventilation alone induced a strong upregulation of cytokine-induced neutrophil chemoattractant-1 and adhesion molecules in untreated, alpha- and beta-blocker treated lungs, while in DA, D1- and D2-blocker treated lungs significant lower levels were found. CONCLUSIONS Our study suggests that dopamine mediated protective effects on I/R damage and inflammation in donor lungs are most likely mediated via adrenergic receptors. These findings are highly relevant because new strategies for organ preservation are necessary in terms of long donation waiting lists.

Background Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge. Methods We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus. Results Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species. Conclusions These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.

A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borrelia afzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B. afzelii species was shown in a tick-challenge model using feral ticks. In both models, as little as .03 μg of antigen, when administered in a 2-dose immunization schedule with aluminum hydroxide as adjuvant, was sufficient to provide complete protection against the species targeted. This proof of principle study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines requiring fewer OspA antigens and suggests that this approach may facilitate the development of an OspA vaccine for global use.

Background The 2-chloro-N6-cyclopentyladenosine (CCPA) was proven to be a protective factor in ischemic reperfusion injury in myocardium and to reduce the infarct size in the heart. The purpose of this study was to determine whether flap necrosis could be reduced by intravenous administration of CCPA. Methods Fifty-six male Wistar rats were divided into 4 experimental groups. An epigastric adipocutaneous flap was raised, and the area of flap necrosis was assessed for all groups on the fifth postoperative day with planimetry software. Results The control group had a significantly lower rate of flap necrosis than the ischemic control group (p < .05). The nonischemic CCPA group had a significantly lower rate of flap necrosis than the nonischemic control group (p < .05). The ischemic CCPA group had a highly significant (p < .0001) rate of lower flap necrosis than the ischemic control group. Conclusion Our data show that reduction of flap necrosis can be achieved both with and without ischemic periods by intravenous administration of CCPA. © 2011 Wiley Periodicals, Inc. Head Neck, 2011

BACKGROUND: The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS: For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE: The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.

Recent findings indicate that seasonal influenza vaccination or infection of healthy humans may contribute to heterosubtypic immunity against new influenza A subtypes, such as H5N1. Here, we investigated whether seasonal influenza vaccination in a mouse model could induce any immunity against the H5N1 subtype. It could be demonstrated that, largely due to the H1N1 component strain A/NewCaledonia/20/99, parenteral immunization of mice with a trivalent seasonal influenza vaccine elicited heterosubtype H5-reactive antibodies able to confer partial protection against H5N1 influenza virus infection. Furthermore, the trivalent seasonal influenza vaccine was found to be compatible with a whole virus H5N1 vaccine in a heterologous prime-boost immunization regimen, achieving superior efficacy compared to a single immunization with an equivalent low-dose of the H5N1 vaccine.

Flap necrosis in ear, nose, and throat surgery, especially in high-risk groups, is not rare, but not all of the individual pathophysiological processes are known. The objective of this study was to establish an animal model to determine whether acute ischemic preconditioning, which has been reported to be successful in organ transplantation, will result in enhanced flap survival.Forty-two Wistar rats were divided into three experimental groups. An epigastric adipocutaneous flap, based on both superficial epigastric arteries and veins, was raised. The flap was either raised (control), clamped for 2 h (ischemic), or subjected to ischemia of 30 min, followed by 30 min of reperfusion and another 2 h of induced ischemia (IP). The mean flap necrosis area was assessed in all groups on the 5th postoperative day.All animals were doing well on the final day. The average necrosis in the ischemic group was significantly greater than in the control group. No significant superiority in the IP group was demonstrated.The data show that the experimental animal model is practicable and that additional approaches to ischemic preconditioning should be verified.

Preparation for an H5N1 influenza pandemic in humans may involve priming the population with a vaccine produced from an existing, available H5N1 strain. We have used a mouse challenge model to compare the immunogenicity and efficacy of inactivated, Vero cell-derived, whole virus H5N1 vaccines in single immunization and homologous or heterologous prime-boost regimes. A single immunization was sufficient to protect against a lethal challenge with strains from matched and unmatched H5N1 clades. Homologous and heterologous prime-boost regimes induced cross-neutralizing antibodies and cross-protection against representative viruses of H5N1 clade 1, clade 2.1, clade 2.2 and clade 2.3. Moreover, the results indicate that heterologous prime-boost immunization regimes might broaden the specificity of the anti-H5N1 antibody response.

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.

Ross River virus was grown in industrial facilities in vaccine-certified Vero cells in the absence of serum, inactivated using standard formalin-inactivation protocols, treated with Benzonase to digest host cell DNA and purified on a sucrose gradient. Mice given two subcutaneous injections of 0.625 microg of this vaccine or two doses of 0.156 microg vaccine with aluminium hydroxide adjuvant failed to develop a detectable viraemia after intravenous challenge with 10(6)TCID50 of the prototype strain of Ross River virus (T48). Guinea pigs immunised with one or two10 microg doses of vaccine with adjuvant also failed to develop a detectable viraemia following a similar challenge. The levels of neutralising antibody (neutralisation index 1.9-3.1) in the mice protected against challenge with 10(6)TCID50 Ross River virus were similar to those in 16 former epidemic polyarthritis patients (1.1-3.5) who had not experienced a second clinical infection with Ross River virus in the 20 years following their initial infection.

The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.

BACKGROUND: New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine. METHODOLOGY/PRINCIPAL FINDINGS: The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades. CONCLUSIONS/SIGNIFICANCE: The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine.

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.

Background Acute respiratory distress syndrome (ARDS) is the most common cause of respiratory failure among critically ill patients. Novel treatment strategies are required to address this common clinical problem. The application of exogenous adult stem cells was associated with a beneficial outcome in various pre-clinical models of ARDS. In the present study we evaluated the functional capacity and homing ability of bone marrow-derived progenitor cells (BMDPC) in vitro and investigated their potential as a treatment strategy in lipopolysaccharide (LPS)-induced ARDS. Results Evaluation of the BMDPC showed functional capacity to form endothelial outgrowth cell colonies, which stained positive for CD133 and CD31. Furthermore, DiI-stained BMDPC were demonstrated to home to injured lung tissue. Rats treated with BMDPC showed significantly reduced histopathological changes, a reduced expression of ICAM-1 and VCAM-1 by the lung tissue, an inhibition of proinflammatory cytokine synthesis, a reduced weight loss and a reduced mortality (p