Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory Factor as a Molecular Redox Switch.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased ß-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

A vero cell-derived whole-virus H5N1 vaccine effectively induces neuraminidase-inhibiting antibodies.

A Vero cell-derived whole-virus H5N1 influenza vaccine has been shown to induce neutralizing antibodies directed against the hemagglutinin (HA) protein of diverse H5N1 strains in animal studies and clinical trials. However, neuraminidase-inhibiting (NAi) antibodies can reduce viral spread and may be of particular importance in the event of an H5N1 pandemic, where immunity due to HA antibodies is likely absent in the general population. Here we demonstrate the effective induction of NAi antibody titers after H5N1 vaccination in humans. In contrast to the immune response directed toward HA, a single vaccine dose induced a strong NAi response that was not significantly boosted by a second dose, most probably due to priming by previous vaccination or infection with seasonal influenza viruses. After 2 immunizations, seroconversion rates based on antibody titers against HA and NA were similar, indicating the induction of equally strong immune responses against both proteins by this H5N1 vaccine.

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.

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) OVA(LOW) 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 OVA(LOW) mice, were generated by this regimen. These responses were associated with the development of autoimmunity and increased protection to tumor challenge in the RIP OVA(LOW) 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 OVA(LOW) 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.

LY6G6D is a selectively expressed colorectal cancer antigen that can be used for targeting a therapeutic T-cell response by a T-cell engager.

Colorectal cancer (CRC) is one of the most common cancers worldwide and demands more effective treatments. We sought to identify tumor selective CRC antigens and their therapeutic potential for cytotoxic T-cell targeting by transcriptomic and immunohistochemical analysis. LY6G6D was identified as a tumor selectively expressed CRC antigen, mainly in the microsatellite stable (MSS) subtype. A specific anti LY6G6D/CD3 T cell engager (TcE) was generated and demonstrated potent tumor cell killing and T cell activation in vitro. Ex vivo treatment of primary patient-derived CRC tumor slice cultures with the LY6G6D/CD3 TcE led to IFNγ secretion in LY6G6D positive tumor samples. In vivo, LY6G6D/CD3 TcE monotherapy demonstrated tumor regressions in pre-clinical mouse models of engrafted human CRC tumor cells and PBMCs. Lastly, 2D and 3D cocultures of LY6G6D positive and negative cells were used to explore the bystander killing of LY6G6D negative cells after specific activation of T cells by LY6G6D positive cells. LY6G6D/CD3 TcE treatment was shown to lyse target negative cells in the vicinity of target positive cells through a combined effect of IFNγ, TNFα and Fas/FasL. In summary, LY6G6D was identified as a selectively expressed CRC antigen that can be utilized to potently re-direct and activate cytotoxic T-cells to lyse LY6G6D expressing CRC using a TcE. This effect can be spread to target negative neighboring tumor cells, potentially leading to improved therapeutic efficacy.

CD206+ tumor-associated macrophages cross-present tumor antigen and drive antitumor immunity.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for antitumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self-Ag and non-self-Ag, including tumor-associated Ag (TAA), as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable with cross-presenting CLEC9A+ DCs (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

Nonreplicating vaccinia virus vectors expressing the H5 influenza virus hemagglutinin produced in modified Vero cells induce robust protection.

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.

Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics.

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain-L1-4, L66, L79-and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.

Identification of Helicobacter pylori surface proteins by selective proteinase K digestion and antibody phage display.

Five surface proteins of Helicobacter pylori were identified by proteinase K treatment of live H. pylori followed by proteome analysis. One of the identified proteins, HopQ, is also recognized by an antibody selected by phage display screening of intact H. pylori.

A cell culture-derived whole-virus H5N1 vaccine induces long-lasting cross-clade protective immunity in mice which is augmented by a homologous or heterologous booster vaccination.

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.

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.

Comparison of single, homologous prime-boost and heterologous prime-boost immunization strategies against H5N1 influenza virus in a mouse challenge model.

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.

Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine.

BACKGROUND: An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41. METHODS: In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [(3)H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-gamma) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays. RESULTS: More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-gamma CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema. CONCLUSION: Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.

Seasonal influenza vaccine elicits heterosubtypic immunity against H5N1 that can be further boosted by H5N1 vaccination.

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.

A pandemic influenza H1N1 live vaccine based on modified vaccinia Ankara is highly immunogenic and protects mice in active and passive immunizations.

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.

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.

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.

A comparison of murine and human immunoproteomes of Helicobacter pylori validates the preclinical murine infection model for antigen screening.

Preclinical mouse infection models are widely used for Helicobacter vaccine development, but how well such models mimic important aspects of human infections is unknown. A comparison of Helicobacter pylori immunoproteomes of infected mice with previously reported patient data reveals a high agreement in the antigens recognized, suggesting that H. pylori in vivo protein composition and recognition by the host immune system are comparable in mice and humans. Murine Helicobacter models may thus be valid to screen antigens for human vaccination.

Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori.

Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.