Efficient bone formation by gene transfer of human LIM mineralization protein-3

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Transduction of human dendritic cells with adenovirus encoding anti-PD-1 reduces PD-1 expression on co-cultured T cells

A potent tumor-specific T cell response is an important part of antitumor immunity. Thus, enhancing T cell responses against tumor cells is a major focus in cancer immunotherapy. Dendritic cells (DC) play a critical role in the induction of T cell responses not only against pathogens but also against tumor cells. Studies have shown that DC-based vaccines are capable of presenting antigens via MHC Class I and MHC Class II molecules resulting in tumor antigen-specific T cell activation in vitro and in vivo. However, T cell responses against tumor antigens can be negatively regulated. For example, PD-1, which is up-regulated in activated T cells, can bind to PD-L1 or PD-L2 expressed in tumor cells as part of the immune suppressive tumor environment and thus inhibiting T cell activation. We wished to determine whether addition of anti-PD-1 to DC vaccines would result in enhanced tumor antigen-specific T cell responses. DCs transduced with recombinant adenovirus (AdV) encoding anti-PD1 (Ad5.hPD1Ab) secreted anti-PD1 in the supernatants which was able to bind to PD-1 expressed on the surface of HEK-293 cells via stable transfection. We examined the expression of surface markers associated with DC function and maturation 48 hours after transduction via flow cytometry. Our results show that Ad5.hPD1Ab transduced DCs had similar expression levels of antigen presentation molecules MHC Class I and II, costimulatory, and maturation related molecules CD40, CD80, CD83 and CD86 compared to DCs that were transduced with adenovirus encoding tumor antigens for hepatocellular carcinoma and melanoma (AdVhAFP and AdVTMM2, respectively). Furthermore, surface expression of inhibitory molecules PD-L1 and PD-L2 were also comparable among the three groups. Cytokine analysis show that 24-48 hours after transduction, Ad5.hPD1Ab transduced DCs secrete more IL-7, IL-15 and IP-10 than AdVhAFP and AdVTMM2 transduced DCs. Importantly, autologous T cells co-cultured with Ad5.hPD1Ab transduced DC results in reduced expression of not only surface PD-1 but also surface CTLA-4 inhibitory molecules in both CD4+ and CD8+ T cells

Initial Considerations Before Designing a Promoter Construct.

Before designing a synthetic promoter, it can be helpful to think about its final application. Is the study purely an in vitro exercise in monitoring short-term promoter activity from an episomal vector, or does the promoter eventually need to be permanently active and be integrated into the genome or perhaps even to function in vivo? The final application will have a bearing on promoter design and vector of choice from the start of the study. In this chapter I highlight some of the vector attributes to consider and features that should be thought about

A Candidate H1N1 Pandemic Influenza Vaccine Elicits Protective Immunity in Mice

BackgroundIn 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model.MethodsWe generated two adenovirus(Ad5)-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA) from the recently emerged swine influenza isolate A/California/04/2009 (H1N1)pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFN? Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus.Conclusions/SignificanceA single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunizatio

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development

Background: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed

Influenza A H5N1 and HIV co-infection: case report

<p>Abstract</p> <p>Background</p> <p>The role of adaptive immunity in severe influenza is poorly understood. The occurrence of influenza A/H5N1 in a patient with HIV provided a rare opportunity to investigate this.</p> <p>Case Presentation</p> <p>A 30-year-old male was admitted on day 4 of influenza-like-illness with tachycardia, tachypnea, hypoxemia and bilateral pulmonary infiltrates. Influenza A/H5N1 and HIV tests were positive and the patient was treated with Oseltamivir and broad-spectrum antibiotics. Initially his condition improved coinciding with virus clearance by day 6. He clinically deteriorated as of day 10 with fever recrudescence and increasing neutrophil counts and died on day 16. His admission CD4 count was 100/μl and decreased until virus was cleared. CD8 T cells shifted to a CD27⁺CD28^-phenotype. Plasma chemokine and cytokine levels were similar to those found previously in fatal H5N1.</p> <p>Conclusions</p> <p>The course of H5N1 infection was not notably different from other cases. Virus was cleared despite profound CD4 T cell depletion and aberrant CD8 T cell activation but this may have increased susceptibility to a fatal secondary infection.</p

Haemophilus parasuis (Glaesserella parasuis) as a Potential Driver of Molecular Mimicry and Inflammation in Rheumatoid Arthritis

Background: Haemophilus parasuis (Hps; now Glaesserella parasuis) is an infectious agent that causes severe arthritis in swines and shares sequence similarity with residues 261–273 of collagen type 2 (Coll261−273), a possible autoantigen in rheumatoid arthritis (RA). Objectives/methods: We tested the presence of Hps sequencing 16S ribosomal RNA in crevicular fluid, synovial fluids, and tissues in patients with arthritis (RA and other peripheral arthritides) and in healthy controls. Moreover, we examined the cross-recognition of Hps by Coll261−273-specific T cells in HLA-DRB1*04pos RA patients, by T-cell receptor (TCR) beta chain spectratyping and T-cell phenotyping. Results: Hps DNA was present in 57.4% of the tooth crevicular fluids of RA patients and in 31.6% of controls. Anti-Hps IgM and IgG titers were detectable and correlated with disease duration and the age of the patients. Peripheral blood mononuclear cells (PBMCs) were stimulated with Hps virulence-associated trimeric autotransporter peptide (VtaA10755−766), homologous to human Coll261−273 or co-cultured with live Hps. In both conditions, the expanded TCR repertoire overlapped with Coll261−273 and led to the production of IL-17. Discussion: We show that the DNA of an infectious agent (Hps), not previously described as pathogen in humans, is present in most patients with RA and that an Hps peptide is able to activate T cells specific for Coll261−273, likely inducing or maintaining a molecular mimicry mechanism. Conclusion: The cross-reactivity between VtaA10755−766 of a non-human infectious agent and human Coll261−273 suggests an involvement in the pathogenesis of RA. This mechanism appears emphasized in predisposed individuals, such as patients with shared epitope

The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex