Alteration of glycosylation renders HIV sensitive to inactivation by normal human serum.

Retroviruses from various mammalian species, excluding humans, are effectively inactivated in normal human serum (NHS). Recent studies have shown that NHS inactivation of retroviruses occurs through natural Ab recognition of a terminal glycosidic moiety on the viral envelope that is acquired during replication in the host cell. This carbohydrate structure (the alpha-galactosyl epitope) is expressed on the cells of most mammals, with the exception of humans and other Old World primates. In this study, NHS sensitivity of HIV was assessed following viral propagation in human cells that were manipulated to express the alpha-galactosyl epitope. HUT-78 cells were transduced with an exogenous alpha1-3-galactosyl transferase gene, which codes for the terminal glycosyl transferase responsible for generation of the alpha-galactosyl epitope. The transduced HUT-78 cells expressed high levels of the alpha-galactosyl epitope on their membrane surface, rendering them sensitive to killing in NHS. Similarly, HIV passaged through these cells acquired the alpha-galactosyl epitope in association with the envelope glycoprotein gp120 and was also effectively inactivated in NHS. Viral inactivation was abolished by the addition of a synthetic disaccharide that contains the alpha-galactosyl epitope, indicating that virolysis is mediated by anti-alpha-galactosyl natural Ab. These results demonstrate that, like other retroviruses bearing the alpha-galactosyl epitope, HIV modified to express this epitope is inactivated in NHS. Furthermore, these data suggest that expression of the alpha-galactosyl epitope on the surface of viruses may have implications in the interspecies transmission of such viruses to humans.

Retroviral vector producer cell killing in human serum is mediated by natural antibody and complement: strategies for evading the humoral immune response.

The introduction of retroviral vector producer cells (VPC) into tumors as a means of increasing transduction efficiency has recently been employed in human gene therapy trials. However, the fate of these xenogeneic cells in humans is not well understood. In the present study, we used an in vitro model to examine the survival of commonly used VPC lines in serum from humans and various other species. VPC derived from the murine NIH-3T3 cell line, including PA317, Psi CRIP, and GP + E-86, were effectively killed in sera from Old World primates, including human and baboon. Conversely, the same murine cell lines survived exposure to sera from dog, rabbit, rat, and mouse. This pattern of serum killing parallels the occurrence of the anti-alpha-galactosyl natural antibody (Ab) found exclusively in Old World primates. The anti-alpha-galactosyl Ab targets the terminal glycosidic structure Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl epitope) found on the surface of mammalian cells, excluding Old World primates. All murine-derived VPC tested expressed high levels of the alpha-galactosyl epitope as determined by FACS analysis. VPC killing was complement-mediated, because preincubation of human serum with a functionally blocking anti-C5 mAb completely abolished cell lysis. Furthermore, addition of soluble galactose(alpha 1-3)galactose (Gal alpha 1-3Gal) to human serum or down-regulation of the alpha-galactosyl epitope on the surface of VPC effectively reduced VPC killing, indicating that complement activation by these cells is primarily initiated by natural antibody recognition of the alpha-galactosyl epitope. Finally, VPC incubated with human serum for 8 hr in the presence of complement inhibition continued to produce viable retroviral particles, thus demonstrating a correlation between VPC and particle survival. Taken together, these data suggest that elimination of the alpha-galactosyl epitope or complement blockade may provide a strategy to prolong the survival of VPC and the particles that they produce in vivo.

A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody.

Type C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C1q to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of "natural antibody" [Ab] (anti-alpha-galactosyl Ab) to the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence of anti-alpha-galactosyl natural Ab. Blockade or depletion of anti-alpha-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti-alpha-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the alpha-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-alpha-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the alpha-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.

Importance of the amino terminus of the interleukin-8 receptor in ligand interactions.

Interleukin-8 (IL-8) and growth regulatory gene/melanoma growth stimulatory activity (GRO/MGSA) are small polypeptide molecules involved in the chemotactic response of certain cell types. Two receptors have been described which interact with IL-8, designated type 1 and type 2. IL-8 binds with high affinity to both receptors, whereas GRO/MGSA and neutrophil-activating peptide-2 demonstrate a high degree of binding only to the type 2 receptor. The two forms of IL-8 receptor are members of the rhodopsin seven-helix membrane-spanning superfamily, and share a high degree of overall homology, although the amino termini are very divergent. By using conserved restriction enzyme sites, a series of chimeric IL-8 receptor molecules were constructed between the type 1 and type 2 receptors and transfected into human 293 kidney epithelial cells. These chimeric molecules altered regions of the receptor presented to the ligand. The ability of the chimeric receptors to bind IL-8 was determined, as well as the ability of IL-8 and GRO/MGSA to inhibit radiolabeled IL-8 binding. The amino terminus of the IL-8 receptors was found to be important for differential binding of GRO/MGSA and IL-8. In addition, a series of peptides was also constructed to further investigate which residues of IL-8 receptor interact with IL-8. These peptides also identified the amino-terminal sequence of the IL-8 receptors as being important in interacting with IL-8.