Impact of Recombinant VSV-HIV Prime, DNA-Boost Vaccine Candidates on Immunogenicity and Viremia on SHIV-Infected Rhesus Macaques.

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques. To improve the immunogenicity of these VSV-HIV Env vaccine candidates, we generated chimeric Envs containing the transmembrane and cytoplasmic tail of the simian immunodeficiency virus (SIV), which increases surface Env on the particle. Additionally, the Ebola virus glycoprotein was added to the VSV-HIV vaccine particles to divert tropism from CD4 T cells and enhance their replications both in vitro and in vivo. Animals were boosted with DNA constructs that encoded matching antigens. Vaccinated animals developed non-neutralizing antibody responses against both the HIV Env and the Ebola virus glycoprotein (EBOV GP) as well as systemic memory T-cell activation. However, these responses were not associated with observable protection against simian-HIV (SHIV) infection following repeated high-dose intra-rectal SHIV SF162p3 challenges.

Rhesus macaques show increased resistance to repeated SHIV intrarectal exposure following a heterologous regimen of rVSV vector vaccine expressing HIV antigen.

Despite the human immunodeficiency virus (HIV) pandemic continuing worldwide for 40 years, no vaccine to combat the disease has been licenced for use in at risk populations. Here, we describe a novel recombinant vesicular stomatitis virus (rVSV) vector vaccine expressing modified HIV envelope glycoproteins and Ebola virus glycoprotein. Three heterologous immunizations successfully prevented infection by a different clade SHIV in 60% of non-human primates (NHPs). No trend was observed between resistance and antibody interactions. Resistance to infection was associated with high proportions of central memory T-cell CD69 and CD154 marker upregulation, increased IL-2 production, and a reduced IFN-γ response, offering insight into correlates of protection.

A novel DNA platform designed for vaccine use with high transgene expression and immunogenicity.

The development of new, low-cost vaccines and effective gene therapies requires accurate delivery and high-level expression of candidate genes. We developed a plasmid vector, pIDV-II, that allows for both easy manipulation and high expression of exogenous genes in mammalian cells. This plasmid is based upon the pVax1 plasmid and shares a common structure with typical mammalian transcription units. It is composed of a chicken ß-actin promoter (CAG), followed by an intron and flanked by two restriction sites, and also includes a post-transcriptional regulatory element, followed by a transcriptional termination signal. While the modification of pVax1 elements either decreased eGFP expression levels or had no effect at all, replacement of the promoter, the poly-A signal, deletion of the T7 and AmpR promoters, and inversion of the ORI-Neo/Kan cassette, significantly increased in vitro eGFP expression with the modified plasmid called pIDV-II. To further evaluate our vector, expression levels of three viral antigens were compared in cell lines transfected either with pVax1 or pCAGGS backbones as controls. Higher transgene expression was consistently observed with pIDV-II. The humoral and cellular responses generated in mice immunized with pIDV-II vs pVax1 expressing each viral antigen individually were superior by 2-fold or more as measured by ELISA and ELISPOT assays. Overall these results indicate that pIDV-II induces robust transgene expression, with concomitant improved cellular and humoral immune responses against the transgene of interest over pVax1. The new vector, pIDV-II, offers an additional alternative for DNA based vaccination and gene therapy for animal and human use.

Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial.

BACKGROUND: Middle East respiratory syndrome (MERS) coronavirus causes a highly fatal lower-respiratory tract infection. There are as yet no licensed MERS vaccines or therapeutics. This study (WRAIR-2274) assessed the safety, tolerability, and immunogenicity of the GLS-5300 MERS coronavirus DNA vaccine in healthy adults. METHODS: This study was a phase 1, open-label, single-arm, dose-escalation study of GLS-5300 done at the Walter Reed Army Institute for Research Clinical Trials Center (Silver Spring, MD, USA). We enrolled healthy adults aged 18-50 years; exclusion criteria included previous infection or treatment of MERS. Eligible participants were enrolled sequentially using a dose-escalation protocol to receive 0·67 mg, 2 mg, or 6 mg GLS-5300 administered by trained clinical site staff via a single intramuscular 1 mL injection at each vaccination at baseline, week 4, and week 12 followed immediately by co-localised intramuscular electroporation. Enrolment into the higher dose groups occurred after a safety monitoring committee reviewed the data following vaccination of the first five participants at the previous lower dose in each group. The primary outcome of the study was safety, assessed in all participants who received at least one study treatment and for whom post-dose study data were available, during the vaccination period with follow-up through to 48 weeks after dose 3. Safety was measured by the incidence of adverse events; administration site reactions and pain; and changes in safety laboratory parameters. The secondary outcome was immunogenicity. This trial is registered at ClinicalTrials.gov (number NCT02670187) and is completed. FINDINGS: Between Feb 17 and July 22, 2016, we enrolled 75 individuals and allocated 25 each to 0·67 mg, 2 mg, or 6 mg GLS-5300. No vaccine-associated serious adverse events were reported. The most common adverse events were injection-site reactions, reported in 70 participants (93%) of 75. Overall, 73 participants (97%) of 75 reported at least one solicited adverse event; the most common systemic symptoms were headache (five [20%] with 0·67 mg, 11 [44%] with 2 mg, and seven [28%] with 6 mg), and malaise or fatigue (five [20%] with 0·67 mg, seven [28%] with 2 mg, and two [8%] with 6 mg). The most common local solicited symptoms were administration site pain (23 [92%] with all three doses) and tenderness (21 [84%] with all three doses). Most solicited symptoms were reported as mild (19 [76%] with 0·67 mg, 20 [80%] with 2 mg, and 17 [68%] with 6 mg) and were self-limiting. Unsolicited symptoms were reported for 56 participants (75%) of 75 and were deemed treatment-related for 26 (35%). The most common unsolicited adverse events were infections, occurring in 27 participants (36%); six (8%) were deemed possibly related to study treatment. There were no laboratory abnormalities of grade 3 or higher that were related to study treatment; laboratory abnormalities were uncommon, except for 15 increases in creatine phosphokinase in 14 participants (three participants in the 0·67 mg group, three in the 2 mg group, and seven in the 6 mg group). Of these 15 increases, five (33%) were deemed possibly related to study treatment (one in the 2 mg group and four in the 6 mg group). Seroconversion measured by S1-ELISA occurred in 59 (86%) of 69 participants and 61 (94%) of 65 participants after two and three vaccinations, respectively. Neutralising antibodies were detected in 34 (50%) of 68 participants. T-cell responses were detected in 47 (71%) of 66 participants after two vaccinations and in 44 (76%) of 58 participants after three vaccinations. There were no differences in immune responses between dose groups after 6 weeks. At week 60, vaccine-induced humoral and cellular responses were detected in 51 (77%) of 66 participants and 42 (64%) of 66, respectively. INTERPRETATION: The GLS-5300 MERS coronavirus vaccine was well tolerated with no vaccine-associated serious adverse events. Immune responses were dose-independent, detected in more than 85% of participants after two vaccinations, and durable through 1 year of follow-up. The data support further development of the GLS-5300 vaccine, including additional studies to test the efficacy of GLS-5300 in a region endemic for MERS coronavirus. FUNDING: US Department of the Army and GeneOne Life Science.

FXR1P is a GSK3ß substrate regulating mood and emotion processing.

Inhibition of glycogen synthase kinase 3ß (GSK3ß) is a shared action believed to be involved in the regulation of behavior by psychoactive drugs such as antipsychotics and mood stabilizers. However, little is known about the identity of the substrates through which GSK3ß affects behavior. We identified fragile X mental retardation-related protein 1 (FXR1P), a RNA binding protein associated to genetic risk for schizophrenia, as a substrate for GSK3ß. Phosphorylation of FXR1P by GSK3ß is facilitated by prior phosphorylation by ERK2 and leads to its down-regulation. In contrast, behaviorally effective chronic mood stabilizer treatments in mice inhibit GSK3ß and increase FXR1P levels. In line with this, overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional genetic polymorphisms affecting either FXR1P or GSK3ß gene expression interact to regulate emotional brain responsiveness and stability in humans. These observations uncovered a GSK3ß/FXR1P signaling pathway that contributes to regulating mood and emotion processing. Regulation of FXR1P by GSK3ß also provides a mechanistic framework that may explain how inhibition of GSK3ß can contribute to the regulation of mood by psychoactive drugs in mental illnesses such as bipolar disorder. Moreover, this pathway could potentially be implicated in other biological functions, such as inflammation and cell proliferation, in which FXR1P and GSK3 are known to play a role.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

Functional analysis of the fungal/plant class chitinase family in Aspergillus fumigatus.

A quintuple mutant was constructed to delete the entire family of the fungal/plant (class III) chitinases of Aspergillus fumigatus. Only a limited reduction in the total chitinolytic activity was seen for the different chitinase mutants including the quintuple mutant. In spite of this reduction in chitinolytic activity, no growth or germination defects were observed in these chitinase mutants. This result demonstrated that the fungal/plant chitinases do not have an essential role in the morphogenesis of A. fumigatus. A slight diminution of the growth during autolysis was seen for the quintuple mutant suggesting that class III chitinases may play only a nutritional role during this phase of the cycle, retarding fungal death.

Functional analysis of the superoxide dismutase family in Aspergillus fumigatus.

Reactive oxidant species produced by phagocytes have been reported as being involved in the killing of Aspergillus fumigatus. Fungal superoxide dismutases (SODs) that detoxify superoxide anions could be putative virulence factors for this opportunistic pathogen. Four genes encoding putative Sods have been identified in the A. fumigatus genome: a cytoplasmic Cu/ZnSOD (AfSod1p), a mitochondrial MnSOD (AfSod2p), a cytoplasmic MnSOD (AfSod3p) and AfSod4 displaying a MnSOD C-terminal domain. During growth, AfSOD1 and AfSOD2 were highly expressed in conidia whereas AfSOD3 was only strongly expressed in mycelium. AfSOD4 was weakly expressed compared with other SODs. The deletion of AfSOD4 was lethal. Delta sod1 and Delta sod2 mutants showed a growth inhibition at high temperature and a hypersensitivity to menadione whereas the sod3 mutant had only a slight growth delay at high temperature. Multiple mutations had only an additive effect on the phenotype. The triple sod1/sod2/sod3 mutant was characterized by a delay in conidial germination, a reduced conidial survival during storage overtime, the highest sensitivity to menadione and an increased sensitivity to killing by alveolar macrophage of immunocompetent mice. In spite of these phenotypes, no significant virulence difference was observed between the triple mutant and parental strain in experimental murine aspergillosis models in immunocompromised animals.

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium.

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and (1)H/(13)C NMR spectra. The corresponding structure is a 3-(O-alpha-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C(16:0), C(18:0), C(18:1), and C(18:2). This alpha-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Delta mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.

Galactofuranose attenuates cellular adhesion of Aspergillus fumigatus.

Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.

Transcriptomic analysis of the exit from dormancy of Aspergillus fumigatus conidia.

BACKGROUND: Establishment of aspergillosis is depending upon the exit from dormancy and germination of the conidia of Aspergillus fumigatus in the lung. To gain an understanding of the molecular mechanisms underlying the early steps of conidial germination, we undertook a transcriptomic analysis using macroarrays constructed with PCR fragments from > 3,000 genes (around one third of the annotated A. fumigatus genome). RESULTS: Major results of this analysis are the following: (i) conidia stored pre-packaged mRNAs transcripts (27% of genes have transcripts in the resting conidia; (ii) incubation at 37 degrees C in a nutritive medium induced up- and down-regulation of genes: 19% of the total number of genes deposited on the array were up-regulated whereas 22% of the genes with pre-packaged mRNA in the resting conidia were down-regulated; (iii) most modifications were seen during the first 30 min of germination whereas very little modification of gene expression occurred during the following hour; (iv) one-year old conidia and one-week old conidia behaved similarly at transcriptional level. CONCLUSION: Transcriptomic data indicate that the exit from dormancy is associated with a shift from a fermentative metabolism to a respiratory metabolism as well as a trend toward immediate protein synthesis.

Characterization of the SKN7 ortholog of Aspergillus fumigatus.

Reactive oxidant intermediates play a major role in the killing of Aspergillus fumigatus by phagocytes. In yeasts, SKN7 is a transcription factor contributing to the oxidative stress response. We investigated here the role of afSkn7p in the adaptation of A. fumigatus against oxidative stress. To analyze functionally the afSKN7 in A. fumigatus, we modified a quick PCR fusion methodology for targeted deletion in A. fumigatus. The afskn7Delta mutant was morphologically similar to the wild-type strain, but showed a growth inhibition phenotype associated with hydrogen peroxide and tert-butyl hydroperoxide. However, no significant virulence differences were observed between wild type, mutant and reconstituted strains in a murine model of pulmonary aspergillosis. This result indicated that an increased sensitivity of A. fumigatus to peroxides in vitro is not correlated with a modification of fungal virulence.