Antibodies generated by candidate HIV-1 vaccines in a phase I clinical trial were assessed for neutralizing activity with a panel of eight well-characterized, genetically diverse clade B primary isolates having an R5 phenotype. The vaccines consisted of one of three different recombinant canarypox vectors expressing membrane-anchored HIV-1(MN)gp120 (ALVAC vCP205, vCP1433, and vCP1452) followed by boosting with a soluble gp160 hybrid consisting of MNgp120 and the majority of gp41 from strain IIIB. Serum samples from a subset of volunteers in each arm of the trial, containing moderate to high titers of neutralizing antibodies to HIV-1 MN, were analyzed. Competition assays with peptides revealed that the majority of neutralizing activity was specific for the MN-V3 loop. Despite MN-specific neutralization titers that sometimes exceeded 1:500, no neutralization of primary isolates was detected and, in some cases, mild infection enhancement was observed. In addition, little or no neutralization of the HIV-1 IIIB heterologous T cell line-adapted strain of virus was detected. These results reinforce the notion that monovalent HIV-1 ENV is a poor immunogen for generating cross-reactive neutralizing antibodies.

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Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Prom7?+ Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-speciffc RNA probe. (13). The two strains employed, a transgenic mouse line hemizygous for a Pro347 -_+Ser mutation in the pig rhodopsin gene and a normal strain, shared the same genetic background, C57BL/6. The transgenic strain was constructed by using C57BL/6 mice and maintained by crossing hemizygous individuals with C57BL/6 mice. Potentially transgenic embryos were generated by mating hemizygous transgenic male mice to C57BL/6 female mice. As a result, half of the embryos should be transgenic. The normal mice, C57BL/6J -c2J/c2J (The Jackson Laboratory), were homozygous for albino, allowing identification of chimeras by coat color mosaicism and by observation of mosaic fundi with indirect ophthalmoscopy. Chimeras expressing the transgene were identified by standard tail-DNA analysis.Histology. Three chimeras expressing the transgene were sacrificed at 7 weeks of age, and the one lacking the transgene, at 14 weeks. Enucleated eyes were immersed in 0.1 M cacodylate buffer, pH 7.3/3% glutaraldehyde. Following overnight fixation at 4°C, eyes were bisected through the optic nerve head along either a superior-inferior or nasaltemporal axis. The eyes were postfixed in 2% osmium tetroxide and embedded in low-viscosity Spurr resin (14). Sections included the entire hemisphere that passed near the optic nerve head. Sections 0.5-1 ,um thick were taken and counterstained with toluidine blue dye.Construction and Synthesis of Probes for in Situ Hybridization. Two RNA probes were used for in situ hybridization; one specific for mouse rhodopsin and the other, pig rhodopsin. These probes were derived from DNA sequences in the 3' untranslated portions of the respective rhodopsin genes. The DNA fragments were cloned in the Novagen T-vector