Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity

<p>Abstract</p> <p>Background</p> <p>Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel.</p> <p>Results</p> <p><it>In vitro</it>, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies.</p> <p>Conclusion</p> <p>The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.</p

Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides

BACKGROUND: The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism. RESULTS: Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1. CONCLUSION: Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved

Molecular basis of the glycosomal targeting of PEX11 and its mislocalization to mitochondrion in trypanosomes

PEX19 binding sites are essential parts of the targeting signals of peroxisomal membrane proteins (mPTS). In this study, we characterized PEX19 binding sites of PEX11, the most abundant peroxisomal and glycosomal membrane protein from Trypanosoma brucei and Saccharomyces cerevisiae. TbPEX11 contains two PEX19 binding sites, one close to the N-terminus (BS1) and a second in proximity to the first transmembrane domain (BS2). The N-terminal BS1 is highly conserved across different organisms and is required for maintenance of the steady-state concentration and efficient targeting to peroxisomes and glycosomes in both baker’s yeast and Trypanosoma brucei. The second PEX19 binding site in TbPEX11 is essential for its glycosomal localization. Deletion or mutations of the PEX19 binding sites in TbPEX11 or ScPEX11 results in mislocalization of the proteins to mitochondria. Bioinformatic analysis indicates that the N-terminal region of TbPEX11 contains an amphiphilic helix and several putative TOM20 recognition motifs. We show that the extreme N-terminal region of TbPEX11 contains a cryptic N-terminal signal that directs PEX11 to the mitochondrion if its glycosomal transport is blocked

Divergence of Primary Cognate B- and T-Cell Proliferative Responses to Subcutaneous and Intravenous Immunization with Virus-Like Particles

A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL) on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes

The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity

The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5'coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5 ' gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-2

GM-CSF-ovalbumin fusion protein (GM-OVA). One week after a single injection or the second injection the percentage of OT-I tetramer positive CD8cells (A) and IFN-γ and CD107a double-positive CD8+ cells after stimulation with the OT-I peptide (B) were determined. All percentages are percent of CD8+ lymphocytes. Mean percentages with SEM of three independent experiments each with three animals per group are shown. Only statistically significant differences between immunization regimens expressing ovalbumin or GM-OVA in the one-way ANOVA test are indicated.<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8 T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity-8

K vaccinated (PBS). Two weeks after the adenoviral vector booster immunization, mice were challenged with 3000 SFFU of FV. 11 days post challenge FV-specific immune responses were monitored by MHC I (A) and MHC II (C) tetramer staining. Mean percentages and SEM (n = 6) of TetCD8(A) and CD4(C) cells are shown. Splenocytes of an uninfected animal were used as negative control (naiv). The viral load in the blood was detected by AB34cells via flow cytometry (B). In addition the numbers of infectious centers per spleen were determined (D).<p><b>Copyright information:</b></p><p>Taken from "Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity"</p><p>http://www.biomedcentral.com/1471-2172/9/13</p><p>BMC Immunology 2008;9():13-13.</p><p>Published online 11 Apr 2008</p><p>PMCID:PMC2324072.</p><p></p