Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Plant-derived H7 VLP vaccine elicits protective immune response against H7N9 influenza virus in mice and ferrets.

In March 2013, the Chinese Centre for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A H7N9 virus. Infection with this virus often caused severe pneumonia and acute respiratory distress syndrome resulting in a case fatality rate >35%. The risk of pandemic highlighted, once again, the need for a more rapid and scalable vaccine response capability. Here, we describe the rapid (19 days) development of a plant-derived VLP vaccine based on the hemagglutinin sequence of influenza H7N9 A/Hangzhou/1/2013. The immunogenicity of the H7 VLP vaccine was assessed in mice and ferrets after one or two intramuscular dose(s) with and without adjuvant (alum or GLA-SE™). In ferrets, we also measured H7-specific cell-mediated immunity. The mice and ferrets were then challenged with H7N9 A/Anhui/1/2013 influenza virus. A single immunization with the adjuvanted vaccine elicited a strong humoral response and protected mice against an otherwise lethal challenge. Two doses of unadjuvanted vaccine significantly increased humoral response and resulted in 100% protection with significant reduction of clinical signs leading to nearly asymptomatic infections. In ferrets, a single immunization with the alum-adjuvanted H7 VLP vaccine induced strong humoral and CMI responses with antigen-specific activation of CD3(+) T cells. Compared to animals injected with placebo, ferrets vaccinated with alum-adjuvanted vaccine displayed no weight loss during the challenge. Moreover, the vaccination significantly reduced the viral load in lungs and nasal washes 3 days after the infection. This candidate plant-made H7 vaccine therefore induced protective responses after either one adjuvanted or two unadjuvanted doses. Studies are currently ongoing to better characterize the immune response elicited by the plant-derived VLP vaccines. Regardless, these data are very promising for the rapid production of an immunogenic and protective vaccine against this potentially pandemic virus.

Isolation and evaluation of novel adeno-associated virus sequences from porcine tissues.

High antigenic compatibility and low toxicity is associated with xenograft transplantation of porcine tissues in immunodeficient human recipients. We hypothesized that adeno-associated viruses (AAVs) of porcine origin could be highly compatible to human tissues and thus of good efficiency and low toxicity for in vivo gene transfer. Porcine tissues were screened by PCR for the presence of AAV using primers designed to bind conserved regions and amplify variable regions of an alignment of several AAV sequences available on GenBank. We isolated new AAV capsid sequences from porcine tissues and successfully generated a recombinant AAV2/po1 vector by transfection. The AAV2/po1 vector was not cross-neutralized by antisera generated against all other commonly used AAVs (serotype 1, 2, 3, 4, 5, 7 and 8) indicating a distinct antigenic profile. Preexisting immunity to AAVpo1 could not be detected in the human sera evaluated. In mice, AAV2/po1 particles expressing beta-galactosidase or green fluorescent protein demonstrated high transduction efficiency in muscle fibers and the retina after intramuscular or intraocular administration. Biodistribution experiments following systemic administration showed efficient gene transfer exclusively in muscle fibers. Novel AAVs derived from porcine tissues may contribute to the generation of new preventive or curative clinical modalities acceptable for human use.

Gene transfer in human skin with different pseudotyped HIV-based vectors.

Pseudotyping lentiviral vector with other viral surface proteins could be applied for treating genetic anomalies in human skin. In this study, the modification of HIV vector tropism by pseudotyping with the envelope glycoprotein from vesicular stomatitis virus (VSV), the Zaire Ebola (EboZ) virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), Rabies or the rabies-related Mokola virus encoding LacZ as a reporter gene was evaluated qualitatively and quantitatively in human skin xenografts. High transgene expression was detected in dermal fibroblasts transduced with VSV-G-, EboZ- or MuLV-pseudotyped HIV vector with tissue irregularities in the dermal compartments following repeated injections of EboZ- or LCMV-pseudotyped vectors. Four weeks after transduction, double-labeling immunofluorescence of beta-galactosidase and involucrin or integrin beta1 demonstrated that VSV-G-, EboZ- or MuLV-pseudotyped HIV vector effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies. Among the six different pseudotyped HIV-based vectors evaluated, VSV-G-pseudotyped vector was found to be the most efficient viral glycoprotein for cutaneous transduction as demonstrated by the highest level of beta-galactosidase expression and genome copy number evaluated by TaqMan PCR.

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.

Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo.

Traditional gene therapy vectors have demonstrated limited utility for treatment of chronic lung diseases such as cystic fibrosis (CF). Herein we describe a vector based on a Filovirus envelope protein-pseudotyped HIV vector, which we chose after systematically evaluating multiple strategies. The vector efficiently transduces intact airway epithelium from the apical surface, as demonstrated in both in vitro and in vivo model systems. This shows the potential of pseudotyping in expanding the utility of lentiviral vectors. Pseudotyped lentiviral vectors may hold promise for the treatment of CF.

HIV-1 Vpr-chloramphenicol acetyltransferase fusion proteins: sequence requirement for virion incorporation and analysis of antiviral effect.

The human immunodeficiency virus type 1 Vpr is a virion-associated protein that is incorporated in trans into viral particles, presumably via an interaction with the p6 domain of the Gag polyprotein precursor. Recently, several studies demonstrated that Vpr fusion proteins could be used as intravirion inactivating agents. In this study, we compared different Vpr-chloramphenicol acetyltransferase (CAT) fusion proteins for their virion incorporation ability and their effect on the infectivity of HIV viruses. Our deletion analysis indicates that both the N-terminal alpha-helical domain and the leucine/isoleucine-rich (LR) domain located in the middle region of Vpr are required for optimal virion incorporation of Vpr-CAT fusion proteins. The C-terminal basic region, associated with Vpr's ability to mediate cell cycle arrest in G2, was not required for virion incorporation, thus allowing the development of Vpr-based chimeric proteins devoid of any effect on cell growth. The fusion of Vpr at the N- or C-terminus of CAT targeted with equal efficiency the chimeric protein into virions. While the virion incorporation of most Vpr-CAT fusion proteins tested in this study was dependent on the presence of an intact p6 domain, fusion proteins containing only the N-terminal alpha-helical domain of Vpr (amino acid 1 to 42) were incorporated into virions in a p6-independent manner. Virion incorporation of Vpr-CAT fusion proteins was shown to decrease viral infectivity. Moreover, the insertion of HIV protease-cleavage sites between Vpr and CAT not only efficiently delivered and released the cleaved CAT product into HIV viral particles, but also greatly potentiated the inhibition of progeny virion infectivity. Overall, our study: (1) defines the Vpr sequence requirement and configuration necessary for the specific and optimal incorporation of Vpr fusion protein into HIV particles; (2) shows that Vpr fusion proteins have the ability to suppress HIV infectivity by targeting multiple steps of viral morphogenesis.

Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene.

The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells. Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity. To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter. Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells. Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines. This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production. Similar results using the third generation psi CRIP packaging cell line confirmed these findings. Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line. These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.