IN-VIVO SELECTION OF DUCK HEPATITIS-B VIRUS PRE-S VARIANTS WHICH ESCAPE FROM NEUTRALIZATION

To better understand the role of specific residues within the duck hep atitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutraliz ation. A highly neutralizing monoclonal antibody (Mab 900) which recog nizes an epitope (83)IPQPQWTP(90) localized previously on the DHBV pre -S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only two in vivo neutralization rounds with Mab 900, five different pre-S mutant genom es were identified, which harbored point mutations affecting only prol ine residues located at position 90 within this epitope ((83)IPQPQWTP( 90)) and/or at a distance at position 5. We have shown that a single ( P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity nor in vivo infectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P9 0H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapid in vivo selection and identification of DHBV pre-S variants and provid e evidence that residues within and at a distance from the neutralizat ion epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity. (C) 1997 Academic Press.