A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

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

H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model

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 Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

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.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-γ-secreting (IFN-γ) 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.The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza

Construction and Use of an <i>ipb</i> DNA Module To Generate <i>Pseudomonas</i> Strains with Constitutive Trichloroethene and Isopropylbenzene Oxidation Activity

ABSTRACT Pseudomonas sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway ( ipbA1 , ipbA2 , ipbA3 , ipbA4 , ipbB , and ipbC ) and identified the initial IPB dioxygenase (IpbA1A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967–3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of ipbA1 . The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the −35 region of a putative ς 70 promoter were found. These repeats exhibit significant sequence similarity to the operator-promoter region of the xyl meta operon in Pseudomonas putida , which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an ipb DNA module devoid of this ipb operator-promoter region and the stable insertion of this DNA module into the genomes of different Pseudomonas strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities. Constitutive TCE oxidation of two such Pseudomonas hybrid strains, JR1A:: ipb and CBS-3:: ipb , was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A:: ipb resulted in a significant increase in rates of TCE degradation. </jats:p

Induction of Homologous Rather than Heterologous Antigen-Specific CD4 T Cell Responses Is Critical for Functional CD8 T Cell Responses in Mice Transgenic for a Foreign Antigen

Abstract The development of a successful cancer vaccine requires the ability to break immunological tolerance to self-Ags expressed on tumor cells. The transgenic rat insulin promoter (RIP) OVALOW mouse model has been reported to be hyporesponsive for both OVA-specific CD4 and CD8 T cell responses. The experiments described in the current study show that this hyporesponsiveness can be overcome by inclusion of GM-CSF and the TLR7 agonist imiquimod as adjuvants in a DNA immunization regimen with OVA-encoding plasmids. High frequencies of OVA-specific CD8 and CD4 T cells, including a response to a CD4 T cell epitope seen only in the RIP OVALOW mice, were generated by this regimen. These responses were associated with the development of autoimmunity and increased protection to tumor challenge in the RIP OVALOW mice. Heterologous CD4 T cell help has been shown to improve functional CD8 T cell responses, and we confirmed that inclusion of the CD4 T cell epitope pan HLA-DR–binding epitope improved CD8 T cell responses compared with self-Ag alone. Addition of GM-CSF and imiquimod, however, resulted in dominance of the pan HLA-DR–binding epitope-specific response over the OVA-specific CD4 T cell responses, decreased OVA-specific CD8 T cell numbers and function in tolerant RIP OVALOW mice, and failure to induce diabetes. The results of this study suggest that the use of heterologous help needs to be evaluated carefully in the context of specific immunization regimes and that a preferable approach may be adjuvantization of DNA vaccines.</jats:p

Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane

Abstract 3385: Cancerous tissue can be identified by the presence of oxMIF, the oxidized form of macrophage migration inhibitory factor

Abstract Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and is pathologically associated with several types of cancer. We identified oxMIF, an immunological and conformationally distinct isoform of MIF that can be mimicked in vitro by mild oxidation of recombinant MIF. Fully human monoclonal antibodies that specifically target oxMIF were shown to inhibit signaling pathways associated with tumor proliferation and progression in vitro and in vivo in syngeneic or xenogeneic animal models. To evaluate the use of oxMIF as a biomarker of malignant tissue, we acquired plasma and tumor tissues from patients with colorectal cancer (CRC), ovarian, and non-small cell lung cancer (NSCLC), as well as control plasma and control tissue from healthy donors, i.e. in a normal state of health, with no apparent signs of disease. We developed a novel immunohistochemistry (IHC) method to detect oxMIF in malignant tissues and used a specific new ELISA to detect oxMIF in plasma. In patients with CRC, we detected oxMIF by IHC in primary tumors (31 of 39) and in liver, lung and lymph-node metastases (n = 10). Staining intensities showed variation between different tumor cells, as well as in the microenvironment. Similarly, we detected oxMIF by IHC in most types of ovarian cancer (n = 42), but not in controls (n = 4), and observed some variations in the localization and intensities of staining. We also detected oxMIF in liver metastases from patients with ovarian cancer (n = 2). In patients with NSCLC (n = 32), 22 samples showed a strong staining for oxMIF. In tissues from healthy donors (4 colon, 4 ovary and 3 lung), oxMIF was not or barely detectable, whereas total MIF (sum of reduced and oxidized MIF) was widely expressed in normal and tumor tissues and showed a more diffuse spatial distribution than oxMIF. In commercially acquired plasma samples from patients with CRC (n = 46) and NSCLC (n = 15), we did not observe a significant increase of oxMIF levels (median = 0 for both) compared with the plasma from healthy donors (median = 0; n = 69). In patients with ovarian cancer, significant increased levels of oxMIF were measured in plasma (median = 3.5ng/mL, n = 42), and more specifically from patients with papillary serous cystadenocarcinoma or serous cystadenocarcinoma compared with clear cell adenocarcinoma (medians = 3.5, 4.0, and 0 ng/mL respectively). Our studies demonstrated that oxMIF is a disease-related isoform of MIF that can be specifically detected in tissues from various types of cancer. We therefore suggest that oxMIF reflects a new biomarker in solid tumors and has potential diagnostic and prognostic value. A phase 1 clinical study of a novel human antibody that selectively targets oxMIF is currently ongoing in patients with solid malignancies (ClinicalTrials.gov identifier: NCT01765790). Citation Format: Nicolas Sabarth, Dirk Völkel, Michael Thiele, Alexander Schinagl, Patrice Douillard, Friedrich Scheiflinger, Randolf Kerschbaumer. Cancerous tissue can be identified by the presence of oxMIF, the oxidized form of macrophage migration inhibitory factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3385. doi:10.1158/1538-7445.AM2015-3385</jats:p