Latest advancements in process intensification to support global demand for affordable vaccines

Vaccines have a profound impact on global health, preventing illness, death, and improving the quality of life across the globe. However, the current costs of vaccine manufacturing and distribution often prevent the poorest segments of the world’s population from accessing these critical medicines. Vaccine manufacturing for global distribution typically requires large and expensive manufacturing facilities that result in high vaccine Cost of Goods and impede developing countries from initiating or expanding in-country manufacturing capabilities. One of the strategies to address this is to intensify vaccine production processes. This presentation will give an overview of a project funded by the Bill & Melinda Gates foundation that focusses on applying the latest process intensification technologies to develop a platform that dramatically increases process efficiency, decreases production scale and reduces vaccine COGs to a price of $0.15 per dose or less. This vaccine manufacturing platform combines Vero cell lines optimized for virus propagation and media capable of supporting high cell density cell growth, high cell density single-use bioreactors and high efficiency and single step purification technologies. Together, these technologies enable vaccine yields to be significantly increased, which in turn allows commercial manufacturing in a small-footprint, low cost “micro-facility” capable of delivering \u3e40M doses of vaccine per year for a CAPEX of not more than$10M and low operational costs. Such “micro-facilities” can be rapidly and inexpensively commissioned, drastically reducing vaccine COGs, facilitating rapid response and resulting in commercial manufacturing at lab scale. The platform is currently being established using Sabin IPV as the target vaccine. Current status is that Vero cell lines expressing 2-4 fold higher cell specific virus titers have been selected. These have been successfully cultured in high cell density, single-use bioreactors up to 40 million cells per mL. Following infection with Sabin poliovirus vaccine strains, a single chromatographic step using a novel membrane has resulted in 90% recoveries at 95% purity. For IPV, these yield intensifications mean that the entire commercial scale process can be operated in isolators in a footprint of ±30 m2. Combination of four of these manufacturing units in a single “micro-facility” would be capable of delivering \u3e40 million doses of trivalent sIPV per year. Performance of the manufacturing process in isolators also allows the manufacture of viral vaccines that currently require high biological safety containment in an inexpensive facility design. This presentation will discuss the technologies used in the vaccine manufacturing platform and data obtained to date on Sabin IPV in more detail

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration

everal lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4β1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells

Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells

Interactions in the tumour microenvironment can promote chronic lymphocytic leukaemia (CLL) cell survival, proliferation and drug resistance. A detailed comparison of three co-culture systems designed to mimic the CLL lymph node and vascular microenvironments were performed; two were mouse fibroblast cell lines transfected with human CD40LG or CD31 and the third was a human microvascular endothelial cell line, HMEC-1. All three co-culture systems markedly enhanced CLL cell survival and induced a consistent change in CLL cell phenotype, characterized by increased expression of CD38, CD69, CD44 and ITGA4 (CD49d); this phenotype was absent following co-culture on untransfected mouse fibroblasts. In contrast to HMEC-1 cells, the CD40LG and CD31-expressing fibroblasts also induced ZAP70 expression and marked CLL cell proliferation as evidenced by carboxyfluorescein succinimidyl ester labelling and increased Ki- 67 expression. Taken together, our data show that co-culture on different stroma induced a remarkably similar activation phenotype in CLL cells but only the CD40LG and CD31-expressing fibroblasts increased ZAP70 expression and CLL cell proliferation, indicating that ZAP70 may play a critical role in this process. This comparative study reveals a number of striking similarities between the co-culture systems tested but also highlights important differences that should be considered when selecting which system to use for in-vitro investigations

Interaction with Vascular Endothelium Enhances Survival in Primary Chronic Lymphocytic Leukemia Cells via NF-kB Activation and De novo Gene Transcription

Chronic lymphocytic leukemia (CLL) cells rapidly undergo apoptosis in vitro, suggesting that the in vivo microenvironment provides crucial antiapoptotic signals. Overexpression of the antiapoptotic proteins Bcl-2 and Mcl-1 is a hallmark of CLL, and their expression is further enhanced in the lymphoid tissues. However, the high levels of Mcl-1 found in peripheral blood samples, coupled with its short half-life, led us to hypothesize that it must be actively maintained in the peripheral circulation. Coculture of CLL cells with human vascular endothelial cells significantly enhanced tumor cell survival, an effect that was not observed with normal B cells. This was associated with elevated levels of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-XL and marked increased expression of CD38 and CD49d, both of which are associated with clinically aggressive disease. Because CD38, CD49d, and some Bcl-2 family genes are transcriptional targets for NF-κB, we assessed NF-κB activation following coculture with endothelial cells. DNA binding of the NF-κB subunit Rel A was significantly increased and strongly correlated with changes in transcription of CD38, CD49d, BCL2, MCL1, and BCLXL, effects that were reversed by a peptide inhibitor of Rel A. These effects were not observed following coculture with nonendothelial cell lines. Therefore, CLL cells receive specific survival signals following interaction with endothelial cells mediated through the activation of NF-κB and the induction of downstream target genes. This type of interaction in the peripheral vasculature may explain the constitutive NF-κB activation and the overexpression of Bcl-2 family proteins commonly seen in this disease

The Arctic in the 21st century: changing biogeochemical linkages across a paraglacial landscape of Greenland

The Kangerlussuaq area of southwest Greenland encompasses diverse ecological, geomorphic and climate gradients that function over a range of spatial and temporal scales. Ecosystems range from the microbial communities on the ice sheet, through moisture stressed terrestrial vegetation (and their associated herbivores) to freshwater and oligosaline lakes. These ecosystems are linked by a dynamic glacio-fluvial-aeolian geomorphic system that transports water, geological material, organic carbon and nutrients from the glacier surface to adjacent terrestrial and aquatic systems. This paraglacial system is now subject to substantial change due to rapid regional warming since 2000. Here we describe changes in the eco- and geomorphic systems at a range of timescales, and explore rapid future change in the links that integrate these systems. We highlight the importance of cross-system subsidies at the landscape scale and importantly, how these might change in the near future as the Arctic is expected to continue to warm

Radiocarbon dating of fluvial organic matter reveals land-use impacts in boreal peatlands

This study measured the effects of land use on organic matter released to surface waters in a boreal peat catchment using radiocarbon dating of particulate and dissolved organic carbon (POC and DOC), DOC concentration, stable carbon and nitrogen isotope composition, and optical measurements. Undisturbed sites invariably released modern DOC and POC (<20 years old), and seasonal forcing had little impact on the age distribution. Release of pre-1950 carbon was detected at peat extraction, agricultural and drained sites, and was consistently observed at agricultural and peat extraction areas throughout the seasons. Conventional mean DOC ages reached 3,100 (±122) years before collection. On average, DOC concentrations were up to 38% higher at impacted sites compared to natural areas, but there was no significant effect of land use on surface water DOC concentrations. The study indicates that the true extent of land use impacts is not necessarily detectible through changes in DOC concentration alone: Radiocarbon dating was essential to show that leaching of old soil organic matter at modified sites had replaced, rather than supplemented, the modern DOM that is usually released from pristine peatlands. Relationships between the specific fluorescence intensity of DOM and its radiocarbon age were identified, indicating that optical techniques may provide a method for the detection of changes in DOM age