Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: Preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant.

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness.

Therapeutic vaccination against relevant high virulence clinical isolates of Mycobacterium tuberculosis.

The purpose of this study was to attempt to develop therapeutic or post-exposure vaccines that could slow progressive disease in guinea pigs infected by low dose aerosol with very high virulence Beijing isolates of Mycobacterium tuberculosis, currently classified as Category C biodefense pathogens by the NIH and CDC. After screening several candidates we focused on the use of three candidates; these were a pool of bacterial iron acquisition proteins, a pool of antigens recognized by T cells from chronically infected mice thought to represent proteins made by the bacillus in response to decreases in local oxygen tension, and a bacterial lipoprotein that is a potent TLR2 agonist. When delivered in a potent GLA-based adjuvant [targeting TLR4 and TLR9], in most cases we were unable to reduce the bacterial load in the lungs. However, the pathologic appearance of lungs from these animals showed that, while primary lesions were most unaffected and had become necrotic, the development of large, lung consolidating secondary lesions seemed to have been mostly prevented. In animals given both a priming vaccination and a boost the effects were prominent, and almost certainly contributed to significantly prolonged survival in these animals. In a biodefense situation, this prolonged survival would be potentially long enough to allow for the organism to be identified and a drug susceptibility profile determined.

Immunomodulatory and physical effects of phospholipid composition in vaccine adjuvant emulsions.

Egg phosphatidylcholine is commonly used as an emulsifier in formulations administered parenterally. However, synthetic phosphatidylcholine (PC) emulsifiers are now widely available and may be desirable substitutes for egg-derived phospholipids due to stability, purity, and material source considerations. In earlier work, we demonstrated that a squalene-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) emulsion provided equivalent physical stability compared to a squalene-egg PC emulsion. In the present manuscript, we evaluate the physical stability of vaccine adjuvant emulsions containing a range of other synthetic phosphatidylcholine emulsifiers. Besides the POPC emulsion, the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) emulsion showed good particle size and visual stability compared to emulsions made with other synthetic phospholipids. Moreover, comparable immune responses were elicited by squalene emulsions employing various synthetic PC or egg PC emulsifiers in combination with an inactivated influenza vaccine or a recombinant malaria antigen, and these responses were generally enhanced compared to antigen without adjuvant. Therefore, we show that (1) some synthetic PCs (DMPC, POPC, and to a lesser extent 1,2-dioleoyl-sn-glycero-3-phosphocholine) are effective stabilizers of squalene emulsion over a range of storage temperatures while others are not (1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine) and (2) the immunogenicity of stable squalene emulsions is similar regardless of PC source.

Immunomodulatory and physical effects of oil composition in vaccine adjuvant emulsions.

Squalene-based oil-in-water emulsions have been used for years in some seasonal and pandemic influenza vaccines. However, concerns have been expressed regarding squalene source and potential biological activities. Little information is available regarding the immunomodulatory activity of squalene in comparison with other metabolizable oils in the context of oil-in-water emulsions formulated with vaccines. The present work describes the manufacture and physical characterization of emulsions composed of different classes of oils, including squalene, long chain triglycerides, a medium chain triglyceride, and a perfluorocarbon, all emulsified with egg phosphatidylcholine. Some differences were apparent among the non-squalene oils in terms of emulsion stability, including higher size polydispersity in the perfluorocarbon emulsion, more rapid visual instability at 60°C for the long-chain triglyceride and perfluorocarbon emulsions, and an increased creaming rate in the medium-chain triglyceride emulsion at 60°C as detected by laser scattering optical profiling. The biological activity of each of these emulsions was compared when formulated with either a recombinant malaria antigen or a split-virus inactivated influenza vaccine. Overall, vaccines containing the squalene emulsion elicited higher antibody titers and more abundant long-lived plasma cells than vaccines containing emulsions based on other oils. Since squalene-based emulsions show higher adjuvant potency compared to the other oils tested, non-squalene oils may be more suitable as carriers of amphiphilic or hydrophobic immunostimulatory molecules (such as TLR agonists) rather than as stand-alone adjuvants.