Long-Lasting Mucosal and Systemic Immunity against Influenza A Virus Is Significantly Prolonged and Protective by Nasal Whole Influenza Immunization with Mucosal Adjuvant N3 and DNA-Plasmid Expressing Flagellin in Aging In- and Outbred Mice.

Background: Vaccination is commonly used to prevent and control influenza infection in humans. However, improvements in the ease of delivery and strength of immunogenicity could markedly improve herd immunity. The aim of this pre-clinical study is to test the potential improvements to existing intranasal delivery of formalin-inactivated whole Influenza A vaccines (WIV) by formulation with a cationic lipid-based adjuvant (N3). Additionally, we combined WIV and N3 with a DNA-encoded TLR5 agonist secreted flagellin (pFliC(-gly)) as an adjuvant, as this adjuvant has previously been shown to improve the effectiveness of plasmid-encoded DNA antigens. Methods: Outbred and inbred mouse strains were intranasally immunized with unadjuvanted WIV A/H1N1/SI 2006 or WIV that was formulated with N3 alone. Additional groups were immunized with WIV and N3 adjuvant combined with pFliC(-gly). Homo and heterotypic humoral anti-WIV immune responses were assayed from serum and lung by ELISA and hemagglutination inhibition assay. Homo and heterotypic cellular immune responses to WIV and Influenza A NP were also determined. Results: WIV combined with N3 lipid adjuvant the pFliC(-gly) significantly increased homotypic influenza specific serum antibody responses (>200-fold), increased the IgG2 responses, indicating a mixed Th1/Th2-type immunity, and increased the HAI-titer (>100-fold). Enhanced cell-mediated IFNγ secreting influenza directed CD4+ and CD8+ T cell responses (>40-fold) to homotypic and heterosubtypic influenza A virus and peptides. Long-term and protective immunity was obtained. Conclusions: These results indicate that inactivated influenza virus that was formulated with N3 cationic adjuvant significantly enhanced broad systemic and mucosal influenza specific immune responses. These responses were broadened and further increased by incorporating DNA plasmids encoding FliC from S. typhimurum as an adjuvant providing long lasting protection against heterologous Influenza A/H1N1/CA09pdm virus challenge.

In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation.

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.

Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein.

Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1.

The adjuvant activity of alphavirus replicons is enhanced by incorporating the microbial molecule flagellin into the replicon.

Ligands of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) stimulate innate and adaptive immune responses and are considered as potent adjuvants. Combinations of ligands might act in synergy to induce stronger and broader immune responses compared to stand-alone ligands. Alphaviruses stimulate endosomal TLRs 3, 7 and 8 as well as the cytoplasmic PRR MDA-5, resulting in induction of a strong type I interferon (IFN) response. Bacterial flagellin stimulates TLR5 and when delivered intracellularly the cytosolic PRR NLRC4, leading to secretion of proinflammatory cytokines. Both alphaviruses and flagellin have independently been shown to act as adjuvants for antigen-specific antibody responses. Here, we hypothesized that alphavirus and flagellin would act in synergy when combined. We therefore cloned the Salmonella Typhimurium flagellin (FliC) gene into an alphavirus replicon and assessed its adjuvant activity on the antibody response against co-administered antigen. In mice immunized with recombinant alphavirus, antibody responses were greatly enhanced compared to soluble FliC or control alphavirus. Both IgG1 and IgG2a/c responses were increased, indicating an enhancement of both Th1 and Th2 type responses. The adjuvant activity of FliC-expressing alphavirus was diminished but not abolished in the absence of TLR5 or type I IFN signaling, suggesting the contribution of several signaling pathways and some synergistic and redundant activity of its components. Thus, we have created a recombinant adjuvant that stimulates multiple signaling pathways of innate immunity resulting in a strong and broad antibody response.

TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis.

Infection of macrophages by bacterial pathogens can trigger Toll-like receptor (TLR) activation as well as Nod-like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase-1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming-free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage-associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re-introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4-agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system.

DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant.

Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

Immunization with multiple vaccine modalities induce strong HIV-specific cellular and humoral immune responses.

Heterologous priming and boosting with antigens expressed by DNA, viral vectors, or as proteins, are experimental strategies to induce strong immune responses against infectious diseases and cancer. In a preclinical study we compared the ability of recombinant modified vaccinia Ankara encoding HIV antigens (MVA-CMDR), and/or recombinant gp140C (rgp140C), to boost responses induced by a multigene/multisubtype HIV DNA vaccine delivered by electroporation (EP). Homologous DNA immunizations augmented by EP stimulated strong cellular immune responses. Still stronger cellular immune responses were observed after DNA priming and MVA-CMDR boosting, which was superior to all other immunization schedules tested in terms of antigen-specific IFN-γ, IL-2, and bifunctional IFN-γ and IL-2 responses. For HIV Env-specific antibody responses, mice receiving repeated rgp140C immunizations, and mice boosted with rgp140C, elicited the highest binding titers and the highest numbers of antibody-secreting B cells. When considering both cellular and humoral immune responses, a combination of DNA, MVA-CMDR, and rgp140C immunizations induced the overall most potent immune responses and the highest avidity of HIV Env-specific antibodies. These data emphasize the importance of including multiple vaccine modalities that can stimulate both T and B cells, and thus elicit strong and balanced immune responses. The present HIV vaccine combination holds promise for further evaluation in clinical trials.

Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.

Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

Plasmid DNA vaccination using skin electroporation promotes poly-functional CD4 T-cell responses.

Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8(+) T-cell immune responses while limiting invasiveness of delivery. However, there is still only limited data available on the induction of CD4(+) T-cell immunity using this method. Here, we compare the ability of homologous prime/boost DNA vaccinations by skin EP and intramuscular (i.m.) injection to elicit immune responses by cytokine enzyme-linked immunosorbent spot (ELISPOT) assay, as well as study the complexity of CD4(+) T-cell responses to the human immunodeficiency virus antigen Gag, using multiparamater flow cytometry. We find that DNA vaccinations by skin EP and i.m. injection are capable of eliciting both single- and poly-functional vaccine-specific CD4(+) T cells. However, although DNA delivered by skin EP was administered at a five-fold lower dose it elicited significant increases in the magnitude of multiple-cytokine producers compared with i.m. immunization suggesting that the skin EP could provide greater poly-functional T-cell help, a feature associated with successful immune defense against infectious agents.

Tripeptidyl-peptidase II controls DNA damage responses and in vivo gamma-irradiation resistance of tumors.

Cellular responses to gamma-irradiation exposure are controlled by phosphatidylinositol 3-kinase-related kinases (PIKK) in the nucleus, and in addition, cytosolic PIKKs may have a role in such responses. Here, we show that the expression of tripeptidyl-peptidase II (TPPII), a high molecular weight cytosolic peptidase, required PIKK signaling and that TPPII was rapidly translocated into the nucleus of gamma-irradiated cells. These events were dependent on mammalian target of rapamycin, a cytosolic/mitochondrial PIKK that is activated by gamma-irradiation. Lymphoma cells with inhibited expression of TPPII failed to efficiently stabilize p53 and had reduced ability to arrest proliferation in response to gamma-irradiation. We observed that TPPII contains a BRCA COOH-terminal-like motif, contained within sequences of several proteins involved in DNA damage signaling pathways, and this motif was important for nuclear translocation of TPPII and stabilization of p53. Novel tripeptide-based inhibitors of TPPII caused complete in vivo tumor regression in mice in response to relatively low doses of gamma-irradiation (3-4 Gy/wk). This was observed with established mouse and human tumors of diverse tissue backgrounds, with no tumor regrowth after cancellation of treatment. These TPPII inhibitors had minor effects on tumor growth as single agent and had low cellular toxicity. Our data indicated that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways and that TPPII can be targeted for inhibition of tumor therapy resistance.

Dissection of the interaction of the human cytomegalovirus-derived US2 protein with major histocompatibility complex class I molecules: prominent role of a single arginine residue in human leukocyte antigen-A2.

Human cytomegalovirus encodes several proteins that interfere with expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The unique short protein 2 (US2) binds to many MHC class I allomorphs in the endoplasmic reticulum, preventing cell surface expression of the class I molecule in question. The molecular interactions underlying US2 binding to MHC class I molecules and its allele specificity have not been fully clarified. In the present study, we first compared the sequences and the structures of US2 retained versus non-retained human leukocyte antigen (HLA) class I allomorphs to identify MHC residues of potential importance for US2 binding. On the basis of this analysis, 18 individual HLA-A2 mutants were generated and the ability of full-length US2 to bind wild-type and mutated HLA-A2 complexes was assessed. We demonstrate that Arg181 plays a critical role in US2-mediated inhibition of HLA-A2 cell surface expression. The structural comparison of all known crystal structures of HLA-A2 either alone, or in complex with T cell receptor or the CD8 co-receptor, indicates that binding of US2 to HLA-A2 results in a unique, large conformational change of the side chain of Arg181. However, although the presence of Arg181 seems to be a prerequisite for US2 binding to HLA-A2, it is not sufficient for binding to all MHC class I alleles.

Activation of innate immunity, inflammation, and potentiation of DNA vaccination through mammalian expression of the TLR5 agonist flagellin.

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer.

Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines.

Pattern recognition receptors (PRR) play an important roll in immediate responses to different conserved molecules produced by microbes. In this paper we describe the cloning of the mouse homolog of Toll-like receptor (TLR) 3, and present an analysis of the expression of this gene in innate and adaptive immune cell lines. We also performed a broad expression study on these cells of other TLR, including TLR family members whose expression pattern is not known, i.e. TLR7. The analysis was done in order to understand, and possibly predict, how innate and adaptive immune cells respond to microbial pattern antigens. This first large-scale analysis of immune cell TLR expression in the mouse reveals that cells of the innate immune system express a broader number of TLR than cells of the adaptive immune system, supporting preconceptions concerning the hierarchy of immune cells involved in direct pathogen recognition. Additionally, the expression of TLR transcripts by mast cells, neutrophils and microglial cells observed here suggests that pathogen-associated molecular pattern molecules could induce activation of these cells through TLR. Finally, the mouse homolog of human TLR3 identified here may, like its human counterpart, be an exceptional TLR molecule due to its lack of a conserved proline residue seen to be involved in existing TLR signaling capabilities found in other TLR family members.