Hepatitis A Virus Vaccine Escape Variants and Potential New Serotype Emergence

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.

recognized, which suggests that severe structural constraints occur in the capsid that prevent the more extensive substitutions necessary for the emergence of a new serotype. Indeed, negative selection of replacements affecting residues encoded by rare codons of the capsid surface has been documented, indicating a critical role played by such rare codons (9). Since these residues are located quite near or even at the epitope regions, the need to maintain such rare codons might prevent the emergence of new serotypes (9). We have recently noted that fine-tuning translation kinetics selection, or the right combination of preferred and rare codons in the capsid coding region, is necessary to get regulated ribosome traffic to guarantee the proper capsid folding (10).
In this context, it seems quite unlikely that a new serotype will emerge, although the emergence of new variants is not impossible if the virus population is forced through bottleneck conditions such as immune selective pressures. We investigated the presence of antigenic variants among sporadic and outbreak cases of hepatitis A.

The Study
We molecularly characterized 128 HAV strains isolated during 2005-2009 in Catalonia from patients with both sporadic (n = 37) and outbreak (n = 91) cases (Technical Appendix, www.cdc.gov/EID/content/17/4/pdfs/10-1169-Techapp.pdf) 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, V1166G, Y1181S, R1189T, 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.
Additionally, residue 1187 is in close contact with residue 1189. Since HAV natural isolates cannot be grown in vitro, C6 and P29 monoclonal antibody-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).
Of the 6 antigenic variants isolated (Table 1)

Conclusions
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 2008-2009 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. A → E 1280 *Position strain HM175 (GenBank accession no. M14707). 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.  -0.08 ± 0.14 -0.08 ± 0.14 -0.02 ± 0.04 -0.08 ± 0.14 P29 ( (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 N t /N 0 = -0.60 (75% neutralization) of the wild-type strain was used to test the variants; N t , the viral titer after neutralization; N 0 , the initial titer. Neutralization limits were the following: log Nt/N 0 >-0.26 (<45%) for resistant variants, -0.26>log Nt/N 0 >-0.60 (45%-75%) for partially resistant variants, and log Nt/N 0 <-0.60 (>75%) for sensitive variants (9).