INSERM U3701 and Liver Unit2, CHU Necker, Faculté de Médecine Necker Enfants-Malades, 156 rue de Vaugirard, 75015 Paris, France
Author for correspondence: Dina Kremsdorf. Fax +33 1 40 61 55 81. e-mail kremsdor{at}necker.fr
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It is well established that HBV surface antigen (HBsAg) is a major target of the humoral and cellular immune response against HBV. Within HBsAg, the a determinant is an important target of the humoral immune response (Brown et al., 1984 ). The antigenic a determinant (residues 124147) has a highly conformational structure that consists of two loops held by disulphide bridges (cysteines 124137 and 139147) projecting from the virus surface (Brown et al., 1984
). In recent years, envelope mutants have been detected following vaccination or anti-HBs immunoglobulin therapy. Vaccine-associated HBsAg mutations have principally been identified in the a determinant (Carman et al., 1993
). These mutants, possibly selected under vaccine pressure, may escape neutralization by vaccine-induced anti-HBs (Hsu et al., 1999
). However, such HBV mutants are also present in chronic, asymptomatic HBV carriers. The most frequently identified mutation is the glycine-to-arginine change at position 145 of HBsAg (Carman et al., 1990
, 1993
). This mutation has, in particular, been identified in HBV-infected children vaccinated against HBV and living in regions of virus endemicity (Oon et al., 1995
). Amino acid changes have been also identified in the a determinant in liver transplant recipients treated with monoclonal or polyclonal anti-HBs immunoglobulin (Carman et al., 1996
; McMahon et al., 1992
; Protzer-Knolle et al., 1998
).
HBV vaccination of chronic HBV carriers is a promising therapeutic strategy. However, an important issue when designing future trials is to assess whether vaccine therapy in HBV chronic carriers may induce the selection of escape mutants. In this context, the aim of this study was to determine whether the vaccination of chronic HBV carriers would induce the selection of a mutant in the antigenic a determinant and whether HBV envelope genetic variability might affect the response of chronic carriers to vaccination.
The 19 patients investigated have participated in a pilot clinical study concerning the vaccination of 32 chronic HBV carriers with active HBV replication (Pol et al., 1994 ). Vaccination consisted of three intramuscular injections of GenHevac B (HBsAg and pre-S2 protein, Pasteur-Mérieux) at 1-month intervals. The response to vaccinotherapy was defined by a reduction of at least 50% of serum HBV DNA 3 months after the last injection (tested by using the Quantiplex bDNA branch or Murex kit).
HBV serological markers, virus load and sequence analysis of the antigenic a determinant were studied in 12 patients (five responders and seven non-responders) before and 6 and/or 12 months after vaccinotherapy (Table 1; Fig. 1
). The HBV serological pattern of patients was assessed by using a standard enzyme immunoassay (Abbott). All patients were HBsAg- and hepatitis B e antigen (HBeAg)-positive before vaccinotherapy. Anti-HBe was detected in four of five responders (Table 1
). For the last responder patient (no. 21), anti-HBe was detected 21 months after initiation of treatment (data not shown). HBV sequences of the antigenic a determinant were compared first with HBV subtypes (Fig. 1
). Eight of the sequences were related to the adw2 subtype (cases 3, 12, 17, 21, 22, 24, 29 and 30) and four to the ayw2 subtype (cases 5, 10, 16 and 50). A response to vaccination was observed in patients infected with the ayw (2/4) and adw (3/8) subtypes. Vaccine therapy was undertaken with the GenHevac B vaccine (Pasteur-Mérieux) containing HBs and preS2 proteins from the ayw subtype. The sequence data thus demonstrated that a response to vaccination was independent of the infecting strain. This agreed with previous findings, demonstrating that the epitopes recognized after vaccinotherapy are present in conserved regions of the proteins (Couillin et al., 1999
). No differences were observed in the sequences obtained before and after treatment from any of the patients, whether or not they responded to vaccination (Fig. 1
). This result suggests that vaccinotherapy does not induce the emergence of escape mutants in the a antigenic determinant.
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In order to investigate the potential role of HBV genetic variability outside the antigenic a determinant in the response to vaccination, the complete sequences of the envelope proteins of five non-responders and five responders were obtained before and 6 months after vaccination. Table 2 summarizes the genetic variations observed when the pre- and post-treatment sequences were compared. A total of ten amino acid changes were observed in six patients. These changes were detected regardless of the response to treatment. However, the same amino acid change was never seen in more than one patient. Thus, we could not define a specific domain implicated in the response to vaccinotherapy. In one patient, the mutation at position 216 in the S domain introduced a stop codon, deleting the last 11 amino acids of the envelope protein. Two substitutions (S115T in preS1 and T54P in preS2) corresponded to subtype variations. Amino acid changes G16D, T55A in preS1 and Q122K in the S domain corresponded to a reversion to a wild-type sequence. The variation at position 122 in the S domain has been described previously as being important for the recognition of antibodies against the d/y subtype epitope (Okamoto et al., 1989
). This type of reversion from mutated to wild-type HBsAg had been reported between a mother and her vaccinated child (Chong-Jin et al., 1999
). The last four mutations (H60P, Q89R and P96T in preS1 and E164G in S) did not correspond to subtype variations. Two of these substitutions (Q89R and P96T), identified in two responder patients, were located in preS1 domains involved in the B cell response. The E164G mutation located in the S domain was observed in a non-responder patient. The implications of this amino acid change as an escape mutant require further investigation.
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References |
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Received 31 May 2000;
accepted 30 October 2000.
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