we molecularly characterized 128 hav strains isolated during 20052009 in catalonia from patients with both sporadic ( n = 37 ) and outbreak ( n = 91 ) cases ( technical appendix figure 1 ) based on their viral protein 1 ( vp1 ) region ( 7 ) . deduced amino acid sequences were compared with those of hm-175 and gbm strains ( genbank accession nos . m14707 and x75215 , respectively ) and constituents of 2 of the commercial hav vaccines , havrix ( glaxosmithkline , rixensart , belgium ) and avaxim ( sanofi - pasteur , paris , france ) , respectively . six amino acid replacements , which have not been previously described , were detected ( table 1 ) . two were semiconservative replacements , v1171a and a1280v , and the other 4 were nonconservative , v1166 g , y1181s , r1189 t , and a1280e . the replaced amino acids were located in a refined 3-dimensional computer model of the hav protomer ( 11 ) , and their relative distances to residues 1102 , 1171 and 1176 , constituents of the immunodominant site ( 12 ) , and to residue 1221 , constituent of the glycophorin a binding site epitope ( 13 ) , were used as markers of the potential antibody - escaping phenotype . all replaced positions were located at ( 1171 ) or around ( 1166 , 1181 , 1189 , 1280 ) the viral immunodominant site near the 5-fold axis ( figure 1 ) , and thus strains bearing these replacements might be considered antigenic variants . in a previous study , several escape mutants to k34c8 monoclonal antibody ( mab ) , which recognizes the immunodominant site , were isolated ( 9 ) . among these mutants , 2 were defined by replacements w1170c ( c6 ) and a1187p ( p29 ) , which were located very close to the mutated residues detected in this study ( figure 1 ) . residue 1170 is located contiguous to residue 1171 and close to residues 1280 and 1181 . resistant ( mar ) mutants were used to mimic the behavior of the naturally isolated variants in neutralization assays with antivaccine serum specimens . results proved that mutant c6 is resistant to both antivaccine serum , as well to convalescent - phase serum , whereas mutant p29 is partially resistant to serum generated with avaxim vaccine ( table 2 ) . hepatis a virus protomer model ( 11 ; refined by ming luo , university of alabama , birmingham , al , usa ) , which includes the locations of all of the substituted residues in viral protein 1 detected in the isolated variants during 20052009 . c ) view of 2 adjacent protomers , showing the close contact of residues 1171 and 1280 . red , residues forming the immunodominant site ; yellow , residues substituted in monoclonal antibody  resistant mutants c6 ( w1170c ) and p29 ( a1187p ) ; green , residues substituted in the identified natural variants . the amino acid substitution v1171a detected in 1 variant is shown in red because this residue belongs to the immunodominant site . the first digit refers to the viral protein , i.e. , 1 for vp1 , and the following 3 digits refer to the amino acid position in the protein . * assays were performed by using vaccine and convalescent - phase serum samples as well as k34c8 mab . mab , monoclonal antibody ; havrix , havrix vaccine ( glaxosmithkline , rixenart , belgium ) ; avaxim , avaxim vaccine ( sanofi - pasteur , paris , france ) . following the model of the hepatitis a virus protomer of ming luo ( figure 1 ) . three neutralization assays were performed with each antivaccine serum sample , the convalescent - phase serum sample , and the mab k34c8 . as controls , neutralization of the d23 h7c27 mab escape variant ( 9 ) as well as that of the hm175/43c wild - type strain , was also measured . the highest dilution showing a log nt / n0 = 0.60 ( 75% neutralization ) of the wild - type strain was used to test the variants ; nt , the viral titer after neutralization ; n0 , the initial titer . neutralization limits were the following : log nt / n0>0.26 ( < 45% ) for resistant variants , 0.26>log nt / n0>0.60 ( 45%75% ) for partially resistant variants , and log nt / n0<0.60 ( > 75% ) for sensitive variants ( 9 ) . of the 6 antigenic variants isolated ( table 1 ) , 4 were obtained during an outbreak among msm in 20082009 . although the number of reported cases of this outbreak was of 186 , the number of molecularly analyzed samples , i.e. , 66 , was similar to that of the rest of analyzed samples , i.e. , 62 ( including other small outbreaks as well as sporadic cases ) . phylogenetic analysis of antigenic variants in the msm group suggested that they originated from a single patient ( technical appendix figures 1 , 2 ) , and thus 4 variants ( 6% of all isolated strains ) represent quite an important number for such an antigenically stable virus . in contrast , the multiple origins of the strains of the general group ( 42 phylogenetically distinguishable strains ; technical appendix figure 1 ) did not correlate with higher numbers of antigenic variants : only 2 were detected ( 3% of all isolated strains ) . an intriguing issue is why so many variants of the immunodominant 5-fold site arose during the msm 2008 - 2009 outbreak , considering the low fitness shown by mar mutants with replacements around this site . in particular , c6 and p29 mutants were rapidly outcompeted by a wild - type strain in the absence of antibodies , and although they were able to overcome the wild - type virus in the presence of the k34c8 mab , it was only after a slow process ( figure 2 ) , indeed indicating a very low fitness . this kind of mutant can only be selected throughout bottleneck events , such as the ingestion of minute amounts of viruses able to float some variants , unlikely in high - risk practices of hiv - positive patients whose viral load in stool may be as high as 10 genome copies / g , or the ingestion of a considerable amount of viruses by patients with low igg levels , who are unable to completely neutralize the infecting virus , thus allowing the viral population to replicate in the presence of antibodies . in fact , 4% ( 8/186 case - patients ) of the msm 20082009 outbreak patients had been vaccinated . however , in only 1 case was the vaccine administered during childhood following the complete dose schedule . in 5 cases , the patients had received only 1 dose of the vaccine during the 6-month period before any symptoms of infection , and the remaining 2 had received only 1 dose of the vaccine long before the infection . an incomplete vaccination schedule in an immunocompromised host could lead to a situation of only partial protection , providing suitable conditions for the emergence of an antigenic variant . unfortunately , samples from these vaccinated patients were not available ; however , it is obvious that the vaccinees likely contributed to the selection of such antigenic variants at a population scale . because in situations of no competition with the wild - type virus , the mar mutants replicate perfectly well , it may be inferred that the natural variants , once they are selected in an improperly vaccinated hiv - positive person , may spread to other , properly vaccinated persons . resistant ( mar ) mutants c6 ( w1170c ) and p29 ( a1187p ) were grown in competition with the hm175/43c ( wild - type virus ) in the presence ( a , b ) or in the absence ( c , d ) of the monoclonal antibody ( mab ) k34c8 . the mar / wild - type ratios were 1:100 ( 104 50% tissue culture infective dose [ tcid50 ] units of mar mutants mixed with 106 tcid50 units of the wild - type virus in the presence of the k34c8 mab ) and 1:1 ( 106 tcid50 units of mar mutants mixed with 106 tcid50 units of the wild - type virus in the absence of antibodies ) . in the competition experiments performed in the presence of antibodies , the initial viral mixtures as well as the viral progenies were neutralized with the mab prior each infection passage . the proportion of mutant and wild - type phenotypes at each passage was inferred from the chromatogram of the consensus sequences and using as marker mutations w1170c and a1187p in c6 and p29 mars , respectively ( 9 ) . isolation of so many variants in a single outbreak among the msm population , in a virus presenting such severe genomic and structural constraints , emphasizes the need to target this community with more effective information on risky sexual practices and vaccination programs . additionally , and particularly among hiv - positive msm , efforts should be made to completely accomplish the vaccination schedule , due to their lower level of immune response ( 14,15 ) . an additional concern is that this impaired response may contribute not only to a lower protection of the vaccinee but also to the emergence of antigenic variants . in the analyzed msm 20082009 outbreak , 4 variants were isolated that were located at or very close to the immunodominant site as well as to residues substituted in 2 mar mutants showing a phenotype of resistance to the protection offered by commercial vaccines . thus , a similar behavior of the natural variants can be postulated , and if this is the case , a new serotype could emerge .
six hepatitis a virus antigenic variants that likely escaped the protective effect of available vaccines were isolated , mostly from men who have sex with men . 
 the need to complete the proper vaccination schedules is critical , particularly in the immunocompromised population , to prevent the emergence of vaccine - escaping variants .