This is the accepted manuscript of a paper published in the Journal of Virology (Koel BF, M?gling R, Chutinimitkul S, Fraaij PL, Burke DF, van der Vliet S, de Wit E, Bestebroer TM, Rimmelzwaan GF, Osterhaus ADME, Smith DJ, Fouchier RAM, de Graaf M, Journal of Virology 2015, 89, 3763?3775, doi:10.1128/JVI.02962-14). The final version is available at http://dx.doi.org/10.1128/JVI.02962-14 ; The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. ; This work was supported by a ZonMW VICI grant and NIH contracts HHSN266200700010C and HHSN272201400008C, NIH Director's Pioneer Award DP1-OD000490-01, European Union FP7 program EMPERIE (223498), European Union FP7 program ANTIGONE (278976), and program grant P0050/2008 from the Human Frontier Science Program. P.L.F. receives funding from the EU FP7 project PREPARE (602525). M.D.G. was funded by a Marie Curie fellowship under contract PIEF-GA-2009-237505. E.D.W. is supported by the Intramural Research Program of NIAID, NIH.