The Enhanced Immune Response to the HIV gp160/LAMP Chimeric Gene Product Targeted to the Lysosome Membrane Protein Trafficking Pathway

Abstract

The lysosome-associated membrane proteins (LAMP), found in the outer membrane of lysosomes and also in a multilaminar compartment that contains major histocompatibility complex class II (MHC II) proteins, are directed to their localization by a cytoplasmic carboxylterminal sequence. Our studies of the immune response to LAMP-targeted proteins has led to the application of a HIV-1 gp160/LAMP chimeric gene as a novel means to enhance the MHC II presentation of gp160. Immunofluorescence microscopy confirmed that the gp160/LAMP protein had a cellular localization corresponding to that of lysosomes. Pulse-chase analysis confirmed that the rates of synthesis of gp160/LAMP and wild type gp160 were comparable and that both proteins were processed to gp120 at similar rates. However, the gp160/LAMP was degraded more rapidly than the wild type gp160. MHC II-mediated T cell proliferation assays performed with cloned human cell lines showed that gp160/LAMP stimulated greater responses than did the wild type gp160. Moreover, mice vaccinated with recombinant vaccinia expressing gp160/LAMP had greater gp160-specific lymphoproliferation responses and higher titers of anti-V3 loop antibodies than mice vaccinated with recombinant vaccinia expressing wild type gp160.Much of the effort in developing an effective vaccine for HIV-1 has focused on the envelope protein. In addition to protein vaccines, some vaccine strategies have featured recombinant viruses that express the envelope protein (1-11), and other more recent studies have employed DNA immunization (12-17). Envelope-specific humoral and cell-mediated responses have been demonstrated by use of these approaches. However, one problem with DNA vaccines in general is that the vaccine antigen made in cells taking up the vector may not enter the major histocompatibility complex class II (MHC II) 1 antigen processing and presentation pathway, which conventionally operates only in professional antigen presenting cells (APC) that express MHC II molecules. This pathway is believed to be accessed primarily by the endocytosis or phagocytosis of extracellular proteins, with transport of the protein to endosomal/lysosomal compartments where it is proteolytically degraded, and the antigenic peptides loaded onto MHC II molecules for transport to the cell surface and presentation to CD4 ϩ T cells (18 -20). Some endogenously synthesized membrane proteins, including the influenza hemagglutinin and HIV envelope protein, are also presented by MHC II molecules, presumably by endocytosis from the cell surface or other suggested pathways (21-24).We have hypothesized that the targeting of recombinant antigens to the lysosome-associated membrane protein (LAMP) trafficking pathway will enhance the loading of endogenously synthesized antigen onto MHC II molecules and consequently elicit an enhanced CD4 ϩ helper T cell response with the resulting increase in both humoral and cell-mediated immunity. The basis of this approach is to create a chimeric antigen containing the endosomal/lysosomal localization ...

Effect of the V3 Loop Deletion of Envelope Glycoprotein on Cellular Responses and Protection against Challenge with Recombinant Vaccinia Virus Expressing gp160 of Primary Human Immunodeficiency Virus Type 1 Isolates

“…Processing of the proteins to yield the WT and ⌬V3 gp120 was first apparent at 60 min and more so at 120 min. The gp160 protein bands were present at each time point throughout the experiment, consistent with previous results that only a fraction of the gp160 protein is processed to the constituent gp120 and gp41 subunits (48). Beginning at 60 min, the intensity of the ⌬V3 mutant gp160 band was decreased by 71% relative to the value observed at 15 min, and at 240 min, the intensity decreased by over 90% as determined by densitometry.…”

Targeting Antigen in Mature Dendritic Cells for Simultaneous Stimulation of CD4+ and CD8+ T Cells

“…Therefore, we pursued alternative methods to establish a single rVV vector capable of infecting a single DC population capable of stimulating both CD4 ϩ and CD8 ϩ T cells. Using a molecular approach that directly targeted pp65 into the lysosomal compartment to facilitate presentation in the context of MHC class II (17)(18)(19)(20)(21)(22), we observed that the intracellular processing machinery for the class II pathway in DC remains intact for at least 16 h after the delivery of a maturation signal, and that such mature infected DC can effectively induce CD4 ϩ T cell responses. Moreover, the hybrid pp65 protein fused to both the LAMP1 transmembrane-cytoplasmic tail and an ER sorting signal did not interfere with concurrent processing of the protein via the class I pathway.…”

“…Recently, Wu and colleagues showed that linking the sorting signals of LAMP1 to the cytoplasmic/nuclear human papilloma virus E7 Ag routed this endogenously synthesized Ag into the MHC class II pathway, resulting in enhanced presentation to CD4 ϩ cells both in vitro and in vivo (17,19). The use of LAMP1-targeting sequences to improve MHC class II Ag presentation has also been shown with HIV gp160 (18,20,21), EBV, and influenza virus (22).…”

“…Molecular approaches directly targeting intracellular Ags into the appropriate subcellular compartment for MHC class II processing have been recently described (17)(18)(19)(20)(21)(22). The lysosomal-MHC class II processing compartment is characterized by several specific membrane proteins that function to resist degradation by hydrolases, maintain an acidic environment, and promote fusion with other membrane organelles, such as endosomes and phagosomes (23).…”

HIV-1 p55Gag Encoded in the Lysosome-associated Membrane Protein-1 as a DNA Plasmid Vaccine Chimera Is Highly Expressed, Traffics to the Major Histocompatibility Class II Compartment, and Elicits Enhanced Immune Responses

“…With a variety of plasmids, antigens, and gene delivery systems, LAMP/antigen chimeras were targeted to the lysosomal membrane and found to elicit enhanced immune responses as compared with vaccines encoding unmodified, native antigens. Antigen/LAMP chimeras encoded in vaccinia virus vectors included HIV-1 gp160/LAMP (33,34), human papilloma virus E7/LAMP (35,36), and cytomegalovirus pp65/LAMP (37). Naked DNA plasmid antigen/LAMP chimera vaccines include human papilloma virus E7/LAMP (38) and dengue virus 2 premembrane/envelope/LAMP (39,40).…”