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Abstract Background 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. Results In vitro, 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. Conclusion 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.

Abstract 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.

Neutralizing antibodies targeting HIV-1 Env have been shown to protect from systemic infection. To explore whether these antibodies can inhibit infection of the first cells, challenge viruses based on simian immunodeficiency virus (SIV) were developed that use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env usage. Antibodies binding to Env of HIV-1, but not SIV, block HIV-1 Env-mediated infection events after rectal exposure of non-human primates to the switching challenge virus. After natural exposure to HIV-1, such a reduction of the number of first infection events should be sufficient to provide sterilizing immunity in the strictest sense in most of the exposed individuals. Since blocking infection of the first cells avoids the formation of latently infected cells and reduces the risk of emergence of antibody-resistant mutants, it may be the best mode of protection., Competing Interests: Declaration of interests D.R.B. is a paid consultant of IAVI. B.T.’s current affiliation is Department of Biochemistry and Pathobiochemistry, Systems Biochemistry, Ruhr-University Bochum, Bochum, Germany., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3-PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3-TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3-TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Gaussmann, Tippler, Ott, Popowicz, Schliebs, Sattler, Erdmann and Kalel.)

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.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma , including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.

Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Respiratory Syncytial Virus (RSV) and Influenza A Virus (IAV) are both two major causative agents of severe respiratory tract infections in humans leading to hospitalization and thousands of deaths each year. In this study, we evaluated the immunogenicity and efficacy of a combinatory DNA vaccine in comparison to the single component vaccines against both diseases in a mouse model. Intramuscular electroporation with plasmids expressing the hemagglutinin (HA) of IAV and the F protein of RSV induced strong humoral immune responses regardless if they were delivered in combination or alone. In consequence, high neutralizing antibody titers were detected, which conferred protection against a lethal challenge with IAV. Furthermore, the viral load in the lungs after a RSV infection could be dramatically reduced in vaccinated mice. Concurrently, substantial amounts of antigen-specific, polyfunctional CD8⁺ T-cells were measured after vaccination. Interestingly, the cellular response to the hemagglutinin was significantly reduced in the presence of the RSV-F encoding plasmid, but not vice versa. Although these results indicate a suppressive effect of the RSV-F protein, the protective efficacy of the combinatory vaccine was comparable to the efficacy of both single-component vaccines. In conclusion, the novel combinatory vaccine against RSV and IAV may have great potential to reduce the rate of severe respiratory tract infections in humans without increasing the number of necessary vaccinations.

Purpose: Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample., Experimental Design: The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications., Results: Gradient centrifugation resulted in an average of 125 μL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4) monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns., Conclusions and Clinical Relevance: A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.

Exosomes have been proposed as candidates for therapeutic immunization. The present study demonstrates that incorporation of the G protein of vesicular stomatitis virus (VSV-G) into exosome-like vesicles (ELVs) enhances their uptake and induces the maturation of dendritic cells. Targeting of VSV-G and ovalbumin as a model antigen to the same ELVs increased the cross-presentation of ovalbumin via an endosomal acidification mechanism. Immunization of mice with VSV-G and ovalbumin containing ELVs led to an increased IgG2a antibody response, expansion of antigen-specific CD8 T cells, strong in vivo CTL responses, and protection from challenge with ovalbumin expressing tumor cells. Thus, incorporation of VSV-G and targeting of antigens to ELVs are attractive strategies to improve exosomal vaccines.

Respiratory syncytial virus F-protein (RSV-F) is poorly expressed from DNA expression plasmids containing the wild type RSV-F open reading frame. By codon optimization, premature polyadenylation signals were deleted and a striking enhancement of RSV-F expression levels was achieved. Therefore, the immunogenicity and efficacy of wild type DNA vaccines were compared to codon optimized expression plasmids encoding full-length RSV-F or its ectodomain. Mice were immunized twice with the different DNA vaccines followed by an RSV challenge. Only codon optimized DNA vaccines and in particular the one encoding the ectodomain of RSV-F induced substantial antibody levels and reduced viral load 13-170-fold. Thus, codon optimization enhances the immunogenicity and efficacy of RSV encoding DNA vaccines.

We have previously reported that the serine protease plasmin generated during contact activation of human plasma triggers biosynthesis of leukotrienes (LTs) in human peripheral monocytes (PMs), but not in polymorphonuclear neutrophils (PMNs). We now show that purified plasmin acts as a potent chemoattractant on human monocytes, but not on PMNs. Human plasmin or plasminogen activated with urokinase, but not active site-blocked plasmin or plasminogen, elicited monocyte migration across polycarbonate membranes. Similarly, stimulation of monocytes with plasmin, but not with active site-blocked plasmin or plasminogen, induced actin polymerization. As assessed by checkerboard analysis, the plasmin-mediated monocyte locomotion was a true chemotaxis. The plasmin-induced chemotactic response was inhibited by the lysine analog trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which prevents binding of plasmin/ogen to the appropriate membrane binding sites. In addition, active site-blocked plasmin inhibited monocyte migration triggered by active plasmin. Further, plasmin-induced monocyte chemotaxis was inhibited by pertussis toxin (PTX) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG) and chelerythrine, two structurally unrelated inhibitors of protein kinase C (PKC). Plasmin, but not active site-blocked plasmin or plasminogen, triggered formation of cyclic guanosine monophosphate (cGMP) in monocytes. LY83583, an inhibitor of soluble guanylyl cyclase, inhibited both plasmin-induced cGMP formation and the chemotactic response. The latter effect could be antagonized by 8-bromo-cGMP. In addition, KT5823 and (Rp)-8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate [(Rp)-8-pCPT-cGMPs], two structurally unrelated inhibitors of cGMP-dependent protein kinase, inhibited plasmin-mediated monocyte chemotaxis. Thus, beyond being a stimulus for lipid mediator release, plasmin is a potent and specific chemoattractant for human monocytes acting via a cGMP-dependent mechanism. Therefore, plasmin represents a proinflammatory activator for human monocytes.

The objective of this study was to characterize the plasmin-induced stimulation of leukotriene (LT) B4 biosynthesis in human peripheral monocytes (PM). Plasmin up to 175 x 10(-3) CTA U/ml triggers a concentration-dependent release of 5-lipoxygenase-derived LTB4 while release of the cyclooxygenase products thromboxane (TX) B2 and prostaglandin (PG) E2 remained unaffected. The stimulatory effect appeared to be specific in as much as 1) it was found in PM, but not in polymorphonuclear neutrophils (PMN), 2) it requires the lysine binding sites of plasmin molecule since it was inhibited by the lysine analogues 6-aminohexanoic acid (6-AHA) and trans-4(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), 3) the intact catalytic center of plasmin is required since neither plasminogen nor catalytic center-blocked plasmin share the stimulatory effect of active plasmin, 4) other serine proteases such as alpha-chymotrypsin, human neutrophil elastase and cathepsin G did not stimulate release of detectable amounts of LTB4 from PM. In addition, catalytic center-blocked plasmin antagonized the stimulatory effect of active plasmin. Plasmin-mediated monocyte activation apparently proceeds via a pertussis toxin-sensitive G protein. Plasmin did not increase inositol (1,4,5) trisphosphate levels, but a time- and concentration-dependent stimulation of cyclic GMP formation was observed. The data show that plasmin is a specific stimulus for human peripheral monocytes. Plasmin may be an important link between the coagulation cascade and inflammatory reactions.

In gerbils pentylenetetrazole- or handling-induced seizures were accompanied by cerebral formation of small amounts of cysteinyl-leukotrienes (LT) but large amounts of prostaglandin (PG) F2 alpha. By contrast, in rats injected with pentylenetetrazole or bicuculline very large amounts of PGF2 alpha but no cysteinyl-LT could be detected in the brain tissues. The data indicate that at least in rats the extensive neuronal activity during tonic-clonic convulsions is not necessarily sufficient for the activation of the 5-lipoxygenase pathway. Apparently important species differences do exist.

In rats kainic acid-induced seizures were accompanied by time-dependent cerebral cysteinyl-leukotriene (LT) and prostaglandin (PG) F2 alpha formation. Cysteinyl-LT were identified in the rat brain tissue extracts by their immunoreactive properties and their retention times upon reversed phase HPLC profiling. In perfused blood-free brain tissue contents of LTC4-like material were significantly elevated in cortex, hippocampus, midbrain and hypothalamus at 3 h after kainic acid injection. PGF2 alpha tissue contents were significantly elevated in all brain areas studied with very large amounts in the hippocampus and smaller amounts in the cortex. The cyclooxygenase inhibitor indomethacin significantly inhibited formation of PGF2 alpha in whole brain tissue while leaving unaffected the production of cysteinyl-LT. A dose of indomethacin which nearly completely inhibited cyclooxygenase activity as monitored by cerebral PGF2 alpha contents also tended to aggravate behavioral changes and significantly increased the mortality. Phenidone, a lipoxygenase inhibitor, significantly and dose-dependently inhibited formation of cysteinyl-LT but did not significantly affect PGF2 alpha formation. Seizure activity tended to be attenuated by a higher dose of this compound. Dexamethasone which supposedly inhibits phospholipase A2 activity by induction of lipocortins, did not significantly reduce either cysteinyl-LT or PGF2 alpha biosynthesis. Flunarizine, trifluoperazine and diazepines protected a certain percentage of animals from kainic acid-induced seizures. In rats in which seizures occurred in spite of pretreatment with these compounds, the eicosanoid formation was not inhibited but in the case of flunarizine was even found to be somewhat enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)