HA1 (Hemagglutinin) quantitation for influenza A H1N1 and H3N2 high yield reassortant vaccine candidate seed viruses by RP-UPLC.

The only effective measure to decrease morbidity and mortality caused by the influenza virus in the human population is worldwide vaccination. Vaccination produces neutralizing antibodies that target the HA1 subunit of the HA (hemagglutinin) protein and are strain specific. The effectiveness of new influenza vaccines are linked to two factors, the correct prediction of the circulating strains in the population in a particular season and the concentration of the HA1 protein in the vaccine formulation. With the advent of the licensing of quadrivalent vaccines, pharmaceutical manufacturers are under considerable pressure due to time constraints and dedicated resources to deliver 194-198 million doses (2020-2021 U.S. market) of vaccine. Considering the valuable resources needed to produce the influenza vaccine in a timely manner, the efficient quantitation of the HA1 protein (the main component in the influenza vaccine) is required. Currently the only method approved by regulatory agencies for quantitation of the HA antigen in vaccines is the single radial immunodiffusion assay (SRID), an antibody dependent assay that is not time efficient. Time efficient methods that are antibody independent e.g. reverse phase-high performance liquid chromatography (RP-HPLC) or size exclusion-HPLC (SE-HPLC) are available. An improved method implementing reverse phase-ultra performance liquid chromatography (RP-UPLC) has been developed to quantitate the HA1 protein antigen present in the high yield reassortant vaccine seed viruses from influenza A H1N1 and H3N2 subtypes harvested from inoculated embryonated chicken eggs. This method differentiates between high yield and lower yielding reassortants in order to select the best vaccine candidate seed virus with the highest growth 'in ovo'. This direct capability to monitor the HA1 concentration of potential reassortant seed viruses and to choose the best yielding HA influenza reassortant when faced with multiple viral seed candidates provides a major advantage on the industrial scale to the influenza vaccine process.

CC chemokine CCL1 receptor CCR8 mediates conversion of mesenchymal stem cells to embryoid bodies expressing FOXP3+CCR8+ regulatory T cells.

Embryoid bodies (EBs) are three dimensional aggregates of pluripotent stem cells primarily used to investigate morphogenesis and cell toxicity, are also attractive tools in regenerative medicine. While embryonic stem cells (ESCs) and induced pluripotent cells (IPSCs) have been shown to form EBs in mouse, primate and humans, EB formation have not been previously demonstrated in mesenchymal stem cells (MSCs). Here we show that rat MSCs form EBs; which express regulatory T cell (Treg) marker Foxp3 and CC chemokine CCL1 receptor CCR8. We show a novel method for formation of EBs from MSCs under stress and demonstrate that the induction of FoxP3+ CCR8+ EBs is dependent upon CCL1 gradients which mediate cell proliferation, migration and invasion of mTregs. The identification of EBs and novel FoxP3+ CCR8+ regulatory T cells (mTregs) for selective conversion and isolation of bone marrow derived MSCs offers novel avenues for research, diagnosis and treatment.