Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by 85%. Specificity of the assay was tested using two different subtypes of viruses H1N1 (A/Puerto Rico/8/1934 and A/Brisbane/59/2007), and H3N2 virus (A/Aichi/68) with their respective subtype specific stem specific monoclonal antibodies: M145, Aca-1, Aca-2 (H1N1 specific) and Aca-3 (H3N2 specific). Subtype specific mAbs blocked membrane fusion, while a mismatch in virus subtype and the mAb resulted in lack of blocking. We also studied the effect of H1N1 head specific mAb Aca-4, which not only blocked attachment of the virus, but also demonstrated blocking of membrane fusion. Results were validated by testing pre- and post- sera from 2009 seasonal Influenza vaccination and to show that at higher Ab concentration the majority of virus (85%) was blocked from attaching cells, but at lower Ab concentration, where attachment could not be prevented, blocking of membrane fusion was still in effect - up to 50%. Sera screening experiments showed that sera antibodies work beyond just blocking attachment. They also may neutralize the already attached virus by blocking fusion of the viral membrane in the endosomes. The assay has the capacity to monitor blocking of attachment and fusion in a single run. Therefore, it is more representative regarding the natural process of infection and the corresponding neutralization pathways. The assay is unique in terms of assessing stem specific antibodies; stem specific response and its measurement are relevant for the advancement of a universal influenza vaccine.

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