Development of a vaccine production platform for poultry diseases in Africa: Newcastle Disease Virus non-replicative adenovirus-vectored vaccine

Poultry are a vital village livestock playing an important economic and nutritional role in the livelihoods of poor rural households in developing countries, including the Sub-Saharan Africa. Poultry production in Africa is threatened by infectious diseases such as Newcastle Disease (ND), which is highly contagious and endemic, with recurrent outbreaks that provoke heavy losses every year. ND is caused by the Newcastle disease virus (NDV), a negative-sense single-stranded RNA virus from the genus Avulavirus, family Paramyxoviridae. In particular, ND is one of the major problems in village chickens in most parts of Ethiopia where commercial poultry is routinely vaccinated with inactivated or live vaccines. Available ND vaccines are produced in specific pathogen free chicken embryonated eggs, whose supply is expensive and imported from Europe. The development and execution of the present project, funded by the Canadian International Development Research Center and presently in its initial phase, aims towards the implementation at the National Veterinary Institute (NVI), Ethiopia, of a technological platform for the production of veterinary vaccines based on the development of recombinant non-replicating adenoviral vectors, using the human adenovirus serotype 5 (Ad5). The ND adenovirus vaccine proposed, expressing protective antigens from ND virus (NDV), will provide an efficient and cost-effective system to address the limitations associated with the current vaccines such as efficacy and virus shedding in flocks of vaccinated birds. The key success factor of the project relies on the development of a robust and cost-effective production platform using serum-free suspension HEK293 adapted cells expressing maximized rAd5 product yields. This will be achieved by augmenting the production cell mass and the cellular productivity beyond cell densities of 6 million cells per mL. Critical parameters and operating conditions impacting the yield and quality of the Ad vaccine will be identified and elevated in a rational process operating strategy that will lead to high-cell density productive infection in bioreactors. Process development and scale-up will be followed by a downstream processing, evaluation of immunogenicity, formulation and stability assays, and protective capacity assessments after viral challenge in the target animals. Recombinant adenoviruses have been generated carrying the NDV coding sequences for the fusion (F) or the hemagglutinin-neuraminidase (HN) proteins, and also for co-expression of both genes in a bicistronic construction. Phylogenetic analyses were primarily conducted to ensure a high degree of sequence identity of the genes cloned with the genoype of locally circulating strains. Recombinant protein expression was also designed and analyzed under different regulatory sequences aiming for selection of the most immunogenic variant. Following the initial phases of project execution, the subsequent steps will define the final parameters for high-cell density infection and rAd5 production for the animal studies. Here we discuss in detail the completed and upcoming project steps as well as the different strategies implemented to achieve the set objectives supporting the main goal of sustainable technology transfer and capacity building of the NVI in Ethiopia

Multivalent Influenza vaccine production in HEK-293 cells in response to pandemic threats

Influenza virus infects millions of people every year worldwide, with elderly and very young people among the most critically affected. Strains that constitute a pandemic threat are characterized by the severity of the clinical manifestations and mortality rates and tend to require the urgent production of hundreds of millions of vaccine doses in very short periods of time. There is an evident need to develop new generations of influenza vaccines based on robust production systems such as mammalian or insect cell cultures. These systems may allow, in contrast to production in embryonated chicken eggs, a faster response capacity, a superior manufacturing process control and a more reliable and better characterized product. Please click Download on the upper right corner to see the full abstract

Impact of in vitro HIV infection on human thymic regulatory T cell differentiation

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment

Transient Expression in HEK-293 Cells in Suspension Culture as a Rapid and Powerful Tool: SARS-CoV-2 N and Chimeric SARS-CoV-2N-CD154 Proteins as a Case Study

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein

Image_1_Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.tiff

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment.</p

Data_Sheet_1_Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.docx

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment.</p

Membrane chromatography-based downstream processing for cell-culture produced influenza vaccines

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3–62.5% viral recovery was achieved, with the removal of 86.3–96.5% host cell DNA and 95.5–99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies

Impact of extended-release niacin on immune activation in HIV-infected immunological non-responders on effective antiretroviral therapy

Background Background: Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) is involved in immune dysregulation during HIV infection. Niacin (vitamin B3) could control the excess of tryptophan depletion and represents a potential strategy to improve immune functions and CD4 count recovery in immunological non-responder HIV-infected individuals on antiretroviral therapy (ART). Methods Methods: In the CTN PT006 phase 2 pilot randomized trial, 20 adults on ART with CD4 ≤ 350 cells/µl, despite an undetectable viral load (VL) for at least 3 months, received 2000 mg of extended-release (ER)-niacin orally once daily for 24 weeks. Side effects, VL, CD4/CD8 counts, lipid profile, T-cell activation and senescence, Tregs and Th17 cell frequencies, Kyn/Trp ratio, and levels of IL-6, IP-10, sST2, I-FABP, and LBP were assessed following ER-niacin treatment. Results Results: Thirteen participants completed the study. Treatment was interrupted in 4 patients due to loss of follow-up or personal reasons and 3 patients were discontinued due to comorbidity risks. All participants maintained a VL < 40 copies/ml, while ER-niacin did not affect CD4 and CD8 cell counts. Plasma levels of triglycerides, total, and LDL cholesterol significantly decreased, following ER-niacin treatment. ER-niacin also diminished Kyn plasma levels and slightly decreased CD4 T-cell activation. However, no improvement in CD8 subsets, Kyn/Trp ratio, Th17/Treg balance, and plasma inflammatory markers was observed. Conclusions Conclusions: Although ER-niacin combined with ART was well-tolerated among immune non-responders and decreased plasma lipids, it did not improve systemic inflammation, Kyn/Trp ratio, and CD4 cell recovery. Overall, ER-niacin was not effective to overcome chronic inflammation in PLWH