Research Article Discordant Correlation between Serological Assays Observed When Measuring Heterosubtypic Responses against Avian Influenza H5 and H7 Viruses in Unexposed Individuals Eleonora Molesti, 1 Francesca Ferrara, 1 Giulia Lapini, 2 Emanuele Montomoli, 2 and Nigel Temperton 1 1 Viral Pseudotype Unit, Medway School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime, Chatham ME4 4TB, UK 2 Department of Molecular and Developmental Medicine, University of Siena, Via Aldo Moro 3, 53100 Siena, Italy Correspondence should be addressed to Nigel Temperton; n.temperton@kent.ac.uk Received 28 February 2014; Revised 11 May 2014; Accepted 11 May 2014; Published 11 June 2014 Academic Editor: Yanjin Zhang Copyright © 2014 Eleonora Molesti et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. he human population is constantly exposed to multiple inluenza A subtypes due to zoonotic spillover and rapid viral evolution driven by intrinsic error-prone replication and immunological pressure. In this context, antibody responses directed against the HA protein are of importance since they have been shown to correlate with protective immunity. Serological techniques, detecting these responses, play a critical role for inluenza surveillance, vaccine development, and assessment. As the recent human pandemics and avian inluenza outbreaks have demonstrated, there is an urgent need to be better prepared to assess the contribution of the antibody response to protection against newly emerged viruses and to evaluate the extent of preexisting heterosubtypic immunity in populations. In this study, 68 serum samples collected from the Italian population between 1992 and 2007 were found to be positive for antibodies against H5N1 as determined by single radial hemolysis (SRH), but most were negative when evaluated using haemagglutination inhibition (HI) and microneutralisation (MN) assays. As a result of these discordant serological indings, the increased sensitivity of lentiviral pseudotypes was exploited in pseudotype-based neutralisation (pp-NT) assays and the results obtained provide further insight into the complex nature of humoral immunity against inluenza A viruses. 1. Introduction he constant rapid evolution of HPAI H5 and H7 viruses driven by intrinsic error-prone replication and increased by immune pressure signiicantly inluences the sensitivity of available serological assays. Moreover, the antigenic variation of inluenza viruses can also limit the eicacy of prepandemic human vaccines, vaccine strain selection, and results in the necessity to update inluenza vaccines to include contempo- rary viruses and to monitor those that are distinct from the current vaccine strains [1]. he human population is con- stantly exposed, during a lifetime (by natural infection and/or vaccination) to diferent inluenza A subtypes with an associ- ated increase in the memory B cell repertoire. his antibody repertoire may also be cross-reactive to closely related variant viruses making it more diicult to develop sensitive and speciic serological assays [2–4]. he adaptive homosubtypic antibody responses to the antigenic sites of the HA and NA of individual inluenza strains can discriminate between inlu- enza subtypes and current seasonal inluenza vaccines can boost strain-speciic responses with little protection against antigenically drited or shited strains. However, it has been shown that exposure to one subtype of inluenza A can also induce immunity that is cross-protective against other inluenza subtypes. Such adaptive immune response, called “heterosubtypic” immunity, elicits an antibody response to epitopes that are highly conserved amongst strains [5–7]. hese more conserved epitopes, which are less accessible than those on the HA globular head, are predominantly localized in the membrane-proximal stalk region of HA [8]. From Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 231365, 12 pages http://dx.doi.org/10.1155/2014/231365