Generation of candidate human influenza vaccine strains in cell culture: rehearsing the European response to an H7N1 pandemic threat

Background: Although H5N1 avian influenza viruses pose the most obvious imminent pandemic threat, there have been several recent zoonotic incidents involving transmission of H7 viruses to humans. Vaccines are the primary public health defense against pandemics, but reliance on embryonated chickens eggs to propagate vaccine and logistic problems posed by the use of new technology may slow our ability to respond rapidly in a pandemic situation. Objectives: We sought to generate an H7 candidate vaccine virus suitable for administration to humans whose generation and amplification avoided the use of eggs. Methods: We generated a suitable H7 vaccine virus by reverse genetics. This virus, known as RD3, comprises the internal genes of A/Puerto Rico/8/34 with surface antigens of the highly pathogenic avian strain A/Chicken/Italy/13474/99 (H7N1). The multi-basic amino acid site in the HA gene, associated with high pathogenicity in chickens, was removed. Results: The HA modification did not alter the antigenicity of the virus and the resultant single basic motif was stably retained following several passages in Vero and PER. C6 cells. RD3 was attenuated for growth in embryonated eggs, chickens, and ferrets. RD3 induced an antibody response in infected animals reactive against both the homologous virus and other H7 influenza viruses associated with recent infection by H7 viruses in humans. Conclusions: This is the first report of a candidate H7 vaccine virus for use in humans generated by reverse genetics and propagated entirely in mammalian tissue culture. The vaccine has potential use against a wide range of H7 strains.

Vaccine glycoproteins tagged with the human Fc domain as second generation vaccine candidates

Traditional vaccines such as inactivated or live attenuated vaccines, are gradually giving way to more biochemically defined vaccines that are most often based on a recombinant antigen known to possess neutralizing epitopes. Such vaccines can offer improvements in speed, safety and manufacturing process but an inevitable consequence of their high degree of purification is that immunogenicity is reduced through the lack of the innate triggering molecules present in more complex preparations. Targeting recombinant vaccines to antigen presenting cells (APCs) such as dendritic cells however can improve immunogenicity by ensuring that antigen processing is as efficient as possible. Immune complexes, one of a number of routes of APC targeting, are mimicked by a recombinant approach, crystallizable fragment (Fc) fusion proteins, in which the target immunogen is linked directly to an antibody effector domain capable of interaction with receptors, FcR, on the APC cell surface. A number of virus Fc fusion proteins have been expressed in insect cells using the baculovirus expression system and shown to be efficiently produced and purified. Their use for immunization next to non-Fc tagged equivalents shows that they are powerfully immunogenic in the absence of added adjuvant and that immune stimulation is the result of the Fc-FcR interaction.

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Aluminium and the human breast

The human population is exposed to aluminium (Al) from diet, antacids and vaccine adjuvants, but frequent application of Al-based salts to the underarm as antiperspirant adds a high additional exposure directly to the local area of the human breast. Coincidentally the upper outer quadrant of the breast is where there is also a disproportionately high incidence of breast cysts and breast cancer. Al has been measured in human breast tissues/fluids at higher levels than in blood, and experimental evidence suggests that at physiologically relevant concentrations, Al can adversely impact on human breast epithelial cell biology. Gross cystic breast disease is the most common benign disorder of the breast and evidence is presented that Al may be a causative factor in formation of breast cysts. Evidence is also reviewed that Al can enable the development of multiple hallmarks associated with cancer in breast cells, in particular that it can cause genomic instability and inappropriate proliferation in human breast epithelial cells, and can increase migration and invasion of human breast cancer cells. In addition, Al is a metalloestrogen and oestrogen is a risk factor for breast cancer known to influence multiple hallmarks. The microenvironment is established as another determinant of breast cancer development and Al has been shown to cause adverse alterations to the breast microenvironment. If current useage patterns of Al-based antiperspirant salts contribute to causation of breast cysts and breast cancer, then reduction in exposure would offer a strategy for prevention, and regulatory review is now justified.