Different chimeric antibody-like molecules consisting of the four human CD4 extracellular domains (amino acids 1-369) fused to different parts of human IgG1 and IgM heavy-chain constant regions have been created and expressed in mammalian cells. For both IgG1 and IgM fusion proteins, the best expression in COS cells was observed for molecules lacking the CH1 domain of the heavy-chain constant region. The chimeric molecules are potent inhibitors of human immunodeficiency virus (HIV) infection and HIV-mediated cytotoxicity. A CD4:IgG1 hinge fusion protein, which was analyzed in more detail, binds efficiently to HIV gp160 and human Fc receptors and shows complement-assisted inhibition of viral propagation in culture. Half-life studies after intravenous application of the latter human fusion protein into mice and monkeys showed significant prolongation of serum survival compared to soluble CD4. An IgG2b murine homolog of the human CD4:IgG1 hinge fusion protein was prepared and evaluated in mice, where it was found to be nontoxic and to have no detectable effect on the humoral response to soluble antigen.
After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This problem is compounded by the fact that the pandemic-driven situation in 2009 has roughly doubled the overall vaccine demand. Modern cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the increasing worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumors have fomented uncertainty among the general population and physicians, and also discredit officially accepted test results and product licensing. This article provides an overview of the safety profile of the cell culture technology, of the cells and of the final vaccine product.
A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU), Novartis Vaccines and Diagnostics) is produced using Madin-Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10(-6) to 10(-16) infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.
Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10(34). Residual MDCK-DNA is < or =10 ng per dose and the ss-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.
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