Purification of flavivirus VLPs by a two-step chromatographic process

Flaviviruses are enveloped viruses with positive-sense, single-stranded RNA, which are most commonly transmitted by infected mosquitoes. Besides for example dengue viruses (DENV), which have been already for decades posing challenges to public health worldwide, zika virus (ZIKV) and yellow fever virus (YFV) are flaviviruses that have caused significant outbreaks in the last few years. Thus, based on our experience of expressing virus-like particles (VLPs) of several different flaviviruses in recombinant mammalian cells, this work focuses on the development of efficient chromatographic purification processes for zika and yellow-fever VLPs. ZIKV has been discovered in 1947, and since 2007 it has caused isolated outbreaks in Pacific Islands. However, in 2015 it was identified for the first time in Brazil and then quickly spread to over 60 countries between 2015 and 2016. Although most zika patients are asymptomatic, in a small proportion of adults ZIKV infection can cause Guillain-Barré syndrome, and in fetuses of infected women it frequently causes serious congenital malformations, especially in the central nervous system. Since it can be transmitted also by the sexual route and can persist for very long periods in body fluids (including sperm), the development of a vaccine is needed to prevent the spread of the virus to non-endemic countries and to prevent outbreaks to periodically occur in regions where the virus is already circulating. Yellow fever virus is a highly lethal virus, which causes death in about 6-10% of non-vaccinated individuals. In past centuries, before the introduction of the current live-attenuated vaccine, 10% of the population of cities like Philadelphia (USA) and Barcelona (Spain) died in YF outbreaks. The current vaccine is very safe and provides life-long protection from a single dose. However, it can also cause fatal adverse effects in a small proportion of vaccines, and the egg-based production is limited in capacity. This latter fact led to worldwide vaccine shortages during an outbreak in Africa in 2016 and in Brazil in 2017-2018. Although the WHO introduced during the African outbreak in 2016 the use of a fractional (1/5) dose as an emergency measure to control outbreaks, even if using fractional doses of the current vaccine, shortage would be an issue if YF outbreaks spread and especially if it gets to be locally transmitted in Asia, where the mosquito vector is widespread. In this work, a two-step chromatographic process was developed for the purification of zika and yellow fever VLPs from CHO- and HEK293-derived cell culture supernatant, building on previous experience acquired on the purification of yellow fever whole virus from Vero cell culture (Pato et al., 2014, doi: 10.1016/j.vaccine.2014.02.036). The initial clarification of the cell culture suspension was performed by centrifugation and/or filtration, followed by anion exchange chromatography and then a multimodal chromatographic step. The anion exchanger used was a Q membrane adsorber, due to its easy scalability, simplicity to handle, absence of diffusional limitations, and good performance at high flow rates for the capture of large molecules such as VLPs. This capture step allowed a high degree of concentration and an efficient DNA removal. In order to enhance HCP removal, a CaptoCore 700 multimodal column was used in a flow-through mode, allowing contaminants to be adsorbed while VLPs were excluded by size. Samples from all steps of the process were characterized by immunoassays, total protein determination, SDS-PAGE and Western blot. The promising results obtained for zika and yellow fever VLPs indicate that this process could be potentially applied also to other flavivirus VLPs that we have been expressing in our lab, such as DENV1-4, SLEV, CPCV and ILHV. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for the production of cell culture-derived recombinant flavivirus vaccines. Acknowledgements: T. P. Pato (Biomanguinhos, FIOCRUZ, Brazil) for fruitful discussions, and B. S. Graham (VRC, NIH, USA) for fruitful discussions and sharing reagents

Virus-like particles (VLPs) as a platform for the development of yellow fever and Zika virus vaccine candidate

Flaviviruses are arboviruses that have been posing serious challenges to global health since 2015. Zika virus (ZIKV) emerged in 2015 in Brazil and quickly spread to over 60 countries in Africa, Asia and the Americas, causing Guillain-Barré syndrome in adults and serious congenital malformations in fetuses of infected mothers. Besides mosquito-borne transmission, zika virus can persist for months in sexual fluids and thus poses risk also to non-endemic countries due to sexual transmission of returning travelers. Although the number of cases decreased significantly due to herd immunity in affected countries, development of a vaccine for ZIKV is of great importance to avoid future resurgence of the virus in endemic areas or future spread to currently non-endemic regions. Yellow fever (YF) is a “historically devastating disease” (Paules and Fauci, 2017), which in past centuries killed approximately 10% of the population of cities like Philadelphia and Barcelona. Although a very effective vaccine exists for YFV, it can cause fatal adverse effects in a small proportion of vaccinees, and recent outbreaks have shown that due to its limited production in embryonated eggs the risk of serious vaccine shortages is high. Fractionating the vaccine dosis (1/5) was the emergency solution introduced by the WHO in 2016 to stop an outbreak in Africa, and is currently being adopted for mass vaccination in Brazil to try to stop the serious outbreak ongoing since 2017. The potential risk of YFV spreading to highly populated areas with no vaccination coverage, where the mosquito vector is present, such as Asia, makes urgent the development of new YFV vaccines. In this context, virus-like particles (VLPs) can be a promising platform for developing safe and effective vaccines for YFV, ZIKV and other flaviviruses. In this work, we developed stable recombinant cell lines constitutively expressing the structural prM (pre-membrane) and E (envelope) proteins of ZIKV and YFV. Sucrose cushion ultracentrifugation and TEM images have confirmed that VLPs resembling in both size and shape the respective native viruses are formed. In order to optimize expression, cell transfection protocol was optimized using different transfection reagents, media and host cell lines, including CHO, HEK293, BHK, MDCK and Vero cells. Stable cell lines derived from CHO-K1 and HEK293-3F6 gave the most promising results and were followed for up to 20 weeks post-transfection in the presence and absence of the selection marker, showing that cells grow to high densities with high viabilities and keeping the expression of VLPs. The use of FACS to sort for high producer cells allowed obtaining enriched cell pools producing significantly higher amounts of VLPs and confirmed the hypothesis that secreted VLPs can be transiently detected on the cell membrane surface. Kinetic studies to evaluate different culture media and cultivation conditions under batch, pseudoperfusion and perfusion mode were carried out with the final aim of increasing productivity and reducing production costs. Ongoing studies are focusing, on one hand, to purify the ZIKV and YFV VLPs for immunogenicity studies and, on the other hand, to express VLPs of other flaviviruses circulating in the Americas, as preparedness measure for future threats. VLPs of DENV 1, 2, 3 and 4, as well as of Saint Louis encephalitis (SLEV), Ilheus (ILHV) and Cacipacore (CPCV) viruses have already been successfully expressed by transient transfection. Acknowledgements: The authors wish to gratefully acknowledge the Vaccine Research Center of NIAID/NIH (USA) for transferring the DENV, SLEV, ILHV and CPCV gene constructs developed during a sabbatical work of L. R. Castilho and used in this work. References: Paules CI, Fauci AS (2017), Yellow Fever - Once Again on the Radar Screen in the Americas, N Engl J Med 376:1397-1399, doi: 10.1056/NEJMp1702172

Production of Zika virus-like particles (VLPs) by perfusion processes

Zika virus (ZIKV) emerged as a major international public health concern in 2015 and rapidly spread to more than 80 countries in Africa, Asia and the Americas. ZIKV infection has been shown to cause Guillain-Barré syndrome in adults, as well as severe congenital malformations in fetuses from as much as 42% of infected mothers (Brasil et al., 2016, doi:10.1056/NEJMoa1602412). While no ZIKV vaccine becomes approved for human use, periodic outbreaks will continue to occur in endemic regions and the risk of spreading to non-endemic regions will continue to exist, especially because ZIKV persists in body fluids for very long time after infection and can be transmitted via the sexual route. Among many different vaccine platforms currently under study, virus-like particles (VLPs) are a promising alternative for the development of vaccines, since three-dimensional structures, constituted by recombinant structural proteins of the virus but lacking the viral genome, are able to display the antigen in a repetitive pattern, triggering a robust immune response. In this work, we investigated the production of Zika virus-like particles by both intermittent and continuous perfusion processes, using a recombinant HEK293 cell pool previously generated in our laboratory, which constitutively expresses the VLPs. In order to improve production levels, we first enriched the recombinant cell pool for high producers by means of fluorescence-activated cell sorting (FACS). Using this FACS-enriched cell pool, small-scale shake flask studies showed that intermittent perfusion (also known as pseudoperfusion) with daily medium exchange enhanced viable cell density by 3.5 fold and VLP titer by 4 fold when compared to batch cultures. Continuous perfusion in a controlled stirred-tank bioreactor was carried out using an ATF-2 unit as cell retention device. A steady-state viable cell concentration of 25-30 × 106 cells/mL was maintained at a cell-specific perfusion rate (CSPR) of 50-60 pL/cell/day. VLP titers inside the bioreactor were higher than in the harvest, evidencing product retention by the ATF hollow fiber, especially from day 14 of cultivation on. Our results show that the use of cell lines constitutively expressing zika VLPs, cultured in stirred-tank perfusion bioreactors, represents a promising system for the production of a VLP-based Zika vaccine candidate. This process could potentially be more cost-effective than traditional viral vaccine platforms based on batch production of whole viruses, especially considering that VLPs can be produced in lower biosafety level plants, and that perfusion systems are characterized by higher volumetric productivities, reduced bioreactor sizes, smaller plant footprint and lower investment costs when compared to batch processes

Backreaction and order reduction in initially contracting models of the universe

The semiclassical backreaction equations are solved in closed Robertson-Walker spacetimes containing a positive cosmological constant and a conformally coupled massive scalar field. Renormalization of the stress-energy tensor results in higher derivative terms that can lead to solutions that vary on much shorter time scales than the solutions that would occur if the higher derivative terms were not present. These extra solutions can be eliminated through the use of order reduction. Four different methods of order reduction are investigated. These are first applied to the case when only conformally invariant fields, with and without classical radiation, are present. Then they are applied to the massive conformally coupled scalar field. The effects of different adiabatic vacuum states for the massive field are considered. It is found that if enough particles are produced, then the Universe collapses to a final singularity. Otherwise it undergoes a bounce, but at a smaller value of the scale factor (for the models considered) than occurs for the classical de Sitter solution. The stress-energy tensor incorporates both particle production and vacuum polarization effects. An analysis of the energy density of the massive field is done to determine when the contribution from the particles dominates.Comment: 58 pages, 19 figure

Use of a biphasic perfusion process based on mild hypothermia for recombinant glucocerebrosidase (GBA) production

The main goal of this study was to develop an innovative CHO-based process for the production of glucocerebrosidase (GBA), an enzyme used for the replacement therapy of Type 1 Gaucher disease. The focus of the present study was on the development of a perfusion process, combining strategies that are commonly used for process optimization: temperature reduction, and supplementation of the culture medium with productivity enhancers, such as short chain fatty acids. The effects of mild hypothermic conditions combined with valeric acid supplementation were first studied in batch shake flasks for two clones (CHO-GBA-36K and CHO-GBA-65P), developed previously using as host the cell lines CHO.K1 (ATCC CCL-61) and CHO.PRO5 (a glycosylation mutant developed by Stanley et al. Cell 6:121, 1975), respectively. A DOE approach was used (Table 1) to select the most promising cultivation conditions to be further applied to a perfusion process. The best performance regarding both cell growth and GBA production was obtained for the CHO-GBA-65P clone under condition [1], at 31ºC with no valeric acid (Table 1). Under this condition, CHO-GBA-65P achieved a maximum qP of 58.4 mU/106 cells/d, which is 4.2 fold higher than qP at the control condition [2] and 2.7 fold higher than the maximum qP obtained for the CHO-GBA-36K clone, which was achieved at 31ºC with 2 mM valeric acid supplementation (condition [3]). Please click Additional Files below to see the full abstract

Multimodal chromatography combining steric exclusion and cation exchange as an intermediate downstream step to purify yellow fever virus-like particles

Yellow fever (YF) is an hemorrhagic viral disease transmitted by infected mosquitoes, which is endemic in many African and Central/South American countries. The severe symptoms and the high mortality rate of the disease can have devastating effects in case an outbreak occurs in an area where the population is non-vaccinated. Before the current YF vaccine became available, outbreaks in cities like Barcelona (Spain) and Philadelphia (USA) led to the death of approximately 10% of the population. Recent outbreaks have shown that YF continues to be a major public health threat due to production capability issues and shortage of vaccine stockpiles, which even led to the use of an emergency fractional (1/5) dose in Africa in 2016 and in Brazil in 2018. Yellow fever virus-like particles (VLPs) represent an interesting alternative to develop a new YF vaccine. With the aim of developing an efficient and affordable process to purifiy yellow fever VLPs, in this work we developed a multimodal strategy combining cation exchange (CEX) and steric exclusion chromatography (SXC) under conditions where the product of interest does not bind to the CEX adsorber, whereas many contaminants do. In this way, the product of interest is retained just due to steric exclusion by the polyethylene glycol (PEG) added to the mobile phase. Product desorption can be achieved by decreasing PEG concentration, while contaminants remain bound to the adsorber and are eluted in the regeneration step. To the best of our knowledge, the application of such a multimodal strategy has not been published before. Please click Download on the upper right corner to see the full abstract

PRAKIRAAN KEBUTUHAN ENERGI LISTRIK DI PROVINSI NTT TAHUN 2019-2029 MENGGUNAKAN METODE GABUNGAN DAN LEAP

ABSTRAKPenelitian ini bertujuan untuk memprakirakan Kebutuhan Energi Listrik per pelanggan tarif Rumah Tangga, Bisnis, Sosial dan Pemerintah, dan Industri di Provinsi Nusa Tenggara Timur (NTT) Tahun 2019 – 2029 menggunakan Perangkat Lunak LEAP (Long-range Energy Alternative Planning System) dengan skenario pertumbuhan penduduk dan pertumbuhan ekonomi. Dalam perencanaan prakiraan energi listrik ini juga digunakan skenario metode gabungan yakni gabungan beberapa metode seperti metode ekonometri, kecenderungan dan analitis untuk mendapatkan elastisitas.Hasil prakiraan menggunakan metode gabungan menunjukkan rata-rata kenaikan jumlah pelanggan sebesar 0,95% pertahun. Konsumsi energi listrik pada tahun 2029 meningkat sebesar 988,2275 GWh dengan rata-rata kenaikan 1,15% pertahun. Daya tersambung pada tahun 2029 juga meningkat sebesar 1651,73 MVA dengan rata-rata kenaikan sebesar 6,08% pertahun. Hasil prakiraan menggunakan perangkat lunak LEAP menunjukkan rata-rata kenaikan sebesar 0,97% pertahun. Konsumsi energi listrik meningkat 995,0894 GWh dengan rata-rata kenaikan sebesar 1,20% pertahun. Daya tersambung juga meningkat sebesar 1114,52 MVA dengan rata-rata kenaikan sebesar 2,77%. ABSTRACT The forecast utilize the Long-Range Energy Alternative Planning System (LEAP) software to estimate the population, the number of customers, the power connected to the customer rate sector, the energy consumption of the customer rate sector, and the total energy consumption. In planning electricity forecasting in NTT Province for the years of 2019-2029, a combined method scenario is used combining different methods such as econometrics, trends and analytical.The results using the combined method show an average increase in customer numbers of 0.95% per year. Electricity consumption increased by 988.2275 GWh in 2029, with an average increase of 1.15% per year. The connected load also increased by 1651.73 MVA in 2029, with an average increase of 6.08% per annum. Estimates using LEAP software show an average increase of 0.97% per year. Electricity consumption increases by 995.0894 GWh with an average of 1.20% per year. The connected load also increased by 1114.52 MVA with an average of 2.77%

Evaluation of IRES-mediated expression and different signal peptides for the development of CHO clones producing an anti-PCSK9 monoclonal antibody

High levels of low-density lipoprotein (LDL) in the blood are associated to an increased risk of cardiovascular diseases, which are a leading cause of death worldwide. Despite this scenario, only minor progress was made since the introduction of statins as lipid-lowering synthetic drugs in the 1980s. Among patients suffering from high cholesterol levels, about 20% present strong adverse effects to statins or don’t manage to decrease their LDL levels to normality. Since 2015, a new class of monoclonal antibodies (mAbs) was approved by FDA and EMA. These mAbs target the enzyme PCSK9 that is involved in the regulation of LDL cholesterol homeostasis, allow a higher amount of LDL receptors to be available on the surface of hepatic cells and thus enable a significant reduction of the circulating cholesterol levels. In this study, we selected signal peptides and IRES elements from the literature to use for the construction of different tricistronic expression vectors. For the light chain, two different signal peptides were evaluated (SP1 and SP2), whereas for the heavy chain just one signal peptide was used (SP3). An EMCV IRES element was placed between the light chain (LC) gene and SP3. Downstream of the heavy chain (HC) gene, an attenuated IRES (att-IRES) element was inserted, followed by the antibiotics resistance gene for selection. In order to enable the evaluation of a stringent double selection (two different antibiotics) upon co-transfection with two plasmids, two sets of vectors were constructed, having either geneticin (neo) or hygromycin B as selection marker. Thus, a total of 4 vectors were constructed, having the following tricistronic cassette structure: CMV-IE promoter, SP1 or SP2, LC gene, EMCV IRES, SP3, HC gene, att-IRES, neo or hygro. CHO.K1 (ATCC, USA) previously adapted to suspension culture in TC-LECC medium (Xell AG, Germany) were transfected (or co-transfected) using Lipofectamine 3000 (Gibco, USA), using either circular or linearized plasmids. Cell culture supernatants harvested 48h post transfection showed a similar expression level for all constructs. After approximately 2 months under selection pressure with the respective antibiotics, the stable cell pool that had been transfected with circular SP1-LC-SP3-HC-NEO was chosen for further studies. Cells were cultivated in shake flasks, attaining 13.9E6 cells/mL on day 7 and showing an specific growth rate of 1.2 d-1 during the exponential growth phase. The mAb was purified from cell culture supernatant using protein A affinity chromatography, showing high purity and homogeneity. Affinity of the purified mAb to PCSK9 was tested, confirming the success of the approach adopted in this work

Perfusion process for the production of a new, VLP-based yellow fever vaccine candidate

Yellow fever (YF) is an acute viral hemorrhagic disease endemic in tropical areas of Africa, Central and South America, which is transmitted by the bite of infected mosquitoes. It is a “historically devastating disease” (Paules and Fauci, 2017) that killed during outbreaks in past centuries, before the introduction of the current vaccine, approximately 10% of the population of cities like Philadelphia (USA) and Barcelona (Spain). According to Garske et al. (2014), YF caused in 2013 78,000 deaths worldwide, which is a disease burden comparable to influenza. In the past few years, outbreaks in Angola (2016) and in Brazil (2017-2018) led to the depletion of the WHO vaccine stockpile and to the introduction of the emergency use of a fractional dose (1/5). Furthermore, the Angola outbreak in 2016 caused the first cases of YF ever to occur in Asia (11 imported cases to China), rising the concern about approximately 2 billion immunologically naïve people who would be at high risk in Asia in case local transmission of the virus starts to occur (Wilder-Smith et al., 2019). The urgent need for a new YF vaccine becomes evident from two major issues concerning the current vaccine, which consists of a live-attenuated virus propagated in chicken embryos: (i) vaccine shortage due to limitations in the manufacturing technology; (ii) rare, but fatal adverse effects. Therefore, this work focuses on the development of a safe, non-replicating YF vaccine, produced by a high-productivity perfusion process. Stable recombinant HEK293 cell lines constitutively expressing the structural proteins prM (pre-membrane) and E (envelope) of YFV were generated, enabling long-term production and secretion of recombinant virus-like particles (VLPs). FACS (fluorescence activated cell sorting) was used to sort the transfected population for high producer cells and allowed obtaining an enriched cell pool producing significantly higher amounts of VLPs. Small scale kinetic studies under intermittent perfusion (pseudoperfusion) were performed in order to investigate possible feeding strategies and to evaluate the use of short-chain fatty acids as productivity enhancers. Subsequently, perfusion runs were carried out in stirred-tank bioreactors in order to investigate optimal conditions for VLP production, as well as to evaluate different cell retention devices (e.g. inclined lamella settler and ATF-2). Partial retention of the VLPs in the perfusion bioreactor system occurred when the ATF-2 was used. VLPs produced by perfusion were purified by a two-step chromatographic process, and transmission electron microscopy (TEM) images confirmed the expected size and morphology of the VLPs, enabling their use in mouse immunogenicity studies. References: Garske T, Van Kerkhove MD, Yactayo S, Ronveaux O, Lewis RF, Staples JE, Perea W, Ferguson NM, Yellow Fever Expert Committee (2014). Yellow fever in Africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data. PLoS Medicine 11:e1001638. Paules CI, Fauci AS (2017), Yellow fever - once again on the radar screen in the Americas, N Engl J Med 376: 1397-1399. Wilder-Smith A, Lee V, Gubler DJ (2019), Yellow fever: is Asia prepared for an epidemic? The Lancet 19:241-242