CCR5, CXCR4, and CD4 are clustered and closely apposed on microvilli of human macrophages and T cells.

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

A critical site in the core of the CCR5 chemokine receptor required for binding and infectivity of human immunodeficiency virus type 1.

Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (approximately 98-99% identical) CCR5 of African green monkeys (AGMs) avidly binds beta-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.

Immunogenic characterization of a tissue culture-derived vaccine that affords partial protection against avian coccidiosis.

The immunogenicity of a tissue culture-derived vaccine generated from an Eimeria tenella-infected cell line in a serologically defined bird line, and the ability to confer protection against homologous challenge in young chicks was examined. The cell line, SB-CEV-1/F7, was infected with E. tenella sporozoites and the resulting 72-h postinfection cell-free supernatants were adjuvanted and used to immunize Leghorn chicks homozygous for the B19 haplotype. Peripheral blood and splenic lymphocytes from these immunized birds proliferated in vitro in response to both sporozoite and SB-CEV-1/F7 tissue culture-derived parasite antigens. In addition, splenic immune lymphocytes obtained from birds previously exposed to E. tenella in vivo responded to these tissue culture-derived parasite antigens in vitro. To evaluate the efficacy of the vaccine, B19B19 chicks were vaccinated s.c. with adjuvanted 72-h postinfection cell-free supernatants or an ammonium sulfate precipitate derivative thereof, orally boosted, and then subjected to homologous parasite challenge at 10 d of age. The level of protection (body weight gain, cecal lesions) was assessed 6 d after challenge. Performance results from four battery trials demonstrated that vaccinated birds were significantly protected against weight loss compared to unimmunized, challenged controls. In addition, in two of the four trials, vaccinated birds were significantly protected against lesions. These results provide strong evidence that tissue culture-derived parasite antigens obtained from the E. tenella-infected SB-CEV-1/F7 cell line are immunogenic in birds and can provide partial protection against E. tenella clinical coccidiosis.