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To demonstrate that antibodies against amino acids (aa) 603–622 of the SIV gp41 transmembrane glycoprotein enhance infection of SIV in vivo.A synthetic peptide derived from aa 603–622 of SIVmac251 gp41 was synthesized and tested for immunogenicity in rabbits and SIV-infected rhesus macaques. Next, SIV-naive animals were immunized with either a recombinant vaccinia virus expressing the SIV gp160 envelope glycoprotein (Wrgp160) and boosted three times with aa 603–622 (group 1, four animals), wild-type vaccinia virus and boosted with aa 603–622 (group 2, two animals), or VVrgp160 followed by three doses of an irrelevant peptide (group 3, two animals). Animals were challenged with SIVmac251.Peptide aa 603–622 was immunogenic in rabbits. SIV-infected rhesus monkeys immunized with the peptide developed two-three log increases in antibodies to this peptide and antibodies that could enhance SIV infection in vitro. SIV-naive rhesus macaques in group 1 had higher levels of antibody to the peptide by enzyme-linked immunosorbent assay and higher levels of enhancing antibodies at the time of SIV challenge than the animals in groups 2 or 3. Following challenge with SIVmac251 the group 1 animals had detectable p27 antigen longer than animals in group 2 and 3 and died of simian AIDS before the respective animals in the two control groups (P<0.05 by log-rank test).aa 603–622 of SIV gp41, like aa 579–613 of HIV gp41, can stimulate production of antibodies that enhance SIV and HIV infection in vitro. Furthermore, immunization with this peptide suppressed beneficial effects of a gp160 vaccine and appeared to enhance SIV infection in vivo.