was supported by The German National Academic Foundation. effective vaccine being of paramount importance (1C4). Rapid progress in this area has been hindered in part by lack of a widely available small animal model for HIV entry. Currently available animal models include nonhuman primates and immunodeficient humanized mice, neither of which is readily available or amenable to genetic modifications (5, 6). Some viral pathogens exhibit a narrow host range, one of those being HIV. HIVs entry into target cells is mediated by binding of its trimeric envelope spike (gp160) to human CD4 (hCD4) (7) and subsequently to a coreceptor such as human CXCR4 (8) or human CCR5 (hCCR5) (9C11). hCCR5 is of particular interest because CCT245737 it CCT245737 seems to be the primary coreceptor used for transmission (12, 13), as evidenced by the finding that homozygous deletion in the CCR5 allele confers resistance against HIV-1 acquisition (14, 15) and can also lead to long-term control of HIV after stem cell transplantation (16). Finally, HeLa cells and other HIV-resistant cells, including mouse cells, support viral entry when they are engineered to express hCD4/hCCR5/hCXCR4 (17C19). Here, we describe a hCCR5- and hCD4-expressing luciferase reporter mouse that can be used to measure HIV pseudovirus entry and antibody-mediated protection against initial infection in vivo. Results hCCR5-2A-hCD4 Construct. To overcome HIVs host-restriction at the CCT245737 level of viral entry, we coexpressed hCCR5 and hCD4 on a single poly-protein transcript separated by a ribosomal skip 2A peptide sequence (hCCR5-2A-hCD4) (Fig. 1and 0.0001; Fig. 3and at day 4 in and = 6 infected mice (filled circles) vs. controls that did not receive the AdV-hCCR5-2A-hCD4 virus, = 3 mice (open circles). ( 0.0001. Neutralizing Human Anti-HIV Monoclonal Antibodies Mediate Protection in HIV-LUCAdV Mice. Antibodies are key components of most protective vaccines (29, 30) and thus are thought to be essential for protection against HIV infection. In support of this idea, passive administration of potent broadly neutralizing monoclonal antibodies RaLP can provide sterilizing immunity against simian/HIV (SHIV) infection in macaques (31C35), and they seem to delay HIV rebound in humans (36, 37). In addition, plasma concentration of anti-HIV IgG antibodies specific for the V1V2 loop region was inversely correlated with infection risk in the recent RV144 vaccine trial (38, 39). However, the mechanisms that mediate the protective effects in RV144 are poorly understood, as exemplified CCT245737 by the finding that the vaccine assessed in the RV144 trial did not elicit broadly neutralizing antibodies (38C40). We selected six potent, broadly neutralizing anti-HIV antibodies to examine their effects on HIV entry in vivo. NIH45-46G54W (41), 3BNC117 (42), 3BNC60 (42), VRC01 (43), and b12 (44) all target the CD4 binding site, whereas PG16 (45) targets the V1/V2 loop region. These antibodies have varying levels of neutralizing activity (IC50) in the TZM-bl cell assay against HIVYU-2 in vitro, ranging CCT245737 from 0.01 to 2.30 g/mL (Fig. 4and and 0.05, ** 0.01) or doseCresponse curves (and Tables S1 and S2) (52). DoseCresponse experiments were performed to compare the in vivo activity of 3BNC60GR/LR, 3BNC117GR/LR, and VRC01GR/LR to WT controls. GR/LR mutant and WT forms of the individual antibodies were present at similar serum concentrations at the time of pseudovirus injection (Fig. S3= 0.0027 and = 0.0044, respectively; Fig. 4= 0.1848 and = 0.0830, respectively; Fig. 4 em F /em ). When testing 1-79 and 1-74 for binding to the HIVYU-2 trimer expressed on the surface of 293T cells, we found that their binding is relatively weak compared with the neutralizing antibodies examined above (Fig. 4 em G /em ). Thus, antibodies that bind weakly to the HIV trimer.