Enhancement of DNA vaccine efficacy by targeting the xenogeneic human chorionic gonadotropin, survivin and vascular endothelial growth factor receptor 2 combined tumor antigen to the major histocompatibility complex class II pathway.

BACKGROUND: A number of strategies have been used to improve the efficacy of the DNA vaccine for the treatment of tumors. These strategies, ranging from activating CD4+ T cell, manipulating antigen presentation and/or processing to anti-angiogenesis, focus on one certain aspect in the functioning of the vaccine. Therefore, their combination is necessary for rational DNA vaccines design by synergizing different regimens and overcoming the limitations of each strategy. METHODS: A DNA fragment (HSV) encoding the C terminal 37 amino acids of human chorionic gonadotropin ß chain (hCGß), 5 different HLA-restricted cytotoxic T lymphocyte epitopes from human survivin and the third and fourth extracellular domains of vascular endothelial growth factor receptor 2 (VEGFR2) was inserted into the sequence between the luminal and transmembrane domain of human lysosome-associated membrane protein-1 cDNA for the construction of a novel DNA vaccine. RESULTS: This novel vaccine, named p-L/HSV, has a potent antitumor effect on the LL/2 lung carcinoma model in syngeneic C57BL/6 mice. The immunologic mechanism involved in the antitumor effect referred to the activation of both cellular and humoral immune response. In addition, the tumor vasculature was abrogated as observed by immunohistochemistry in p-L/HSV immunized mice. Furthermore, the immunized mice received an additional boost with p-L/HSV 6 months later and showed a strong immune recall response. CONCLUSIONS: The present study indicates that the strategies of combining antitumor with antiangiogenesis and targeting the tumor antigen to the major histocompatibility complex class II pathway cooperate well. Such a study may shed new light on designing vaccine for cancer in the future.

Immune responses to T-cell epitopes of SARS CoV-N protein are enhanced by N immunization with a chimera of lysosome-associated membrane protein.

In our previous study by Gupta et al, dominant T-cell epitopes of SARS CoV-N(N) protein were predicted by software. The spectrum of interferon (IFN)-gamma responses of Balb/c mice immunized against two different forms of SARS CoV-N plasmid was then analyzed. A cluster of dominant T-cell epitopes of SARS CoV-N protein was found in the N-terminus (amino acids 76-114). On the basis of this study, four different plasmids were constructed: (i) DNA encoding the unmodified N (p-N) or N(70-122) (p-N(70-122)) as an endogenous cytoplasmic protein or (ii) DNA encoding a lysosome-associated membrane protein (LAMP) chimera with N (p-LAMP/N) or N(70-122) (p-LAMP/N(70-122)). The immune responses of mice to these four constructs were evaluated. The results showed marked differences in the responses of the immunized mice. A single priming immunization with the p-LAMP/N construct was sufficient to elicit an antibody response. Enzyme-linked immunospot (ELISpot) assay indicated that p-LAMP/N(70-122) and p-LAMP/N plasmids both elicited a greater IFN-gamma response than p-N. p-N and p-N(70-122) constructs induced low or undetectable levels of cytokine secretion. We also found that the p-LAMP/N(70-122) construct promoted a long-lasting T-cell memory response without an additional boost 6 months after three immunizations. These findings show that DNA vaccines, even epitope-based DNA vaccines using LAMP as chimera, can elicit both humoral and cellular immune responses.

Prediction of class I T-cell epitopes: evidence of presence of immunological hot spots inside antigens.

MOTIVATION: Processing and presentation of major histocompatibility complex class I antigens to cytotoxic T-lymphocytes is crucial for immune surveillance against intracellular bacteria, parasites, viruses and tumors. Identification of antigenic regions on pathogen proteins will play a pivotal role in designer vaccine immunotherapy. We have developed a system that not only identifies high binding T-cell antigenic epitopes, but also class I T-cell antigenic clusters termed immunological hot spots. METHODS: MULTIPRED, a computational system for promiscuous prediction of HLA class I binders, uses artificial neural networks (ANN) and hidden Markov models (HMM) as predictive engines. The models were rigorously trained, tested and validated using experimentally identified HLA class I T-cell epitopes from human melanoma related proteins and human papillomavirus proteins E6 and E7. We have developed a scoring scheme for identification of immunological hot spots for HLA class I molecules, which is the sum of the highest four predictions within a window of 30 amino acids. RESULTS: Our predictions against experimental data from four melanoma-related proteins showed that MULTIPRED ANN and HMM models could predict T-cell epitopes with high accuracy. The analysis of proteins E6 and E7 showed that ANN models appear to be more accurate for prediction of HLA-A3 hot spots and HMM models for HLA-A2 predictions. For illustration of its utility we applied MULTIPRED for prediction of promiscuous T-cell epitopes in all four SARS coronavirus structural proteins. MULTIPRED predicted HLA-A2 and HLA-A3 hot spots in each of these proteins.

Expression of lysosome-associated membrane proteins in human colorectal neoplasms and inflammatory diseases.

The lysosome-associated membrane proteins (LAMPs)-1 and -2 are major constituents of the lysosomal membrane. These molecules are known to be among the most glycosylated proteins of several types of cells and cancer cells, and their expression in cancer cells is marked by a distinct difference in the structures of the oligosaccharides as compared to nonmalignant cells. We analyzed by immunohistochemistry the intensity and distribution of LAMP-1 and LAMP-2 in 9 human colorectal cancer cases and in 16 control cases, including inflammatory diseases (diverticulitis, ulcerative colitis, and Crohn's disease). LAMP proteins were expressed more intensely in the epithelium of colorectal neoplasms than in normal mucosa (P < 0.05), and no significant differences were found between adenoma and cancer cells (P > 0.05) in the same tissue section. Further, in sites of inactive inflammatory diseases and nonneoplastic areas in cancer specimens, no significant increases in epithelial LAMP proteins were observed, even in the proliferative zone of the lower crypt epithelium. Northern blot analysis showed increased expression of LAMP-1 and LAMP-2A in two of three colorectal cancers examined and increased LAMP-2B in all three cancers. Our findings suggest that LAMPs are related to neoplastic progression, but there is no direct association between the expression of LAMP molecules and cell proliferation.

Fucosyltransferases in Schistosoma mansoni development.

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.

Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency.

Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 microg/ml, the size of particles was optimized to approximately 100--250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINE-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.

Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease.

We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.

Antigen-specific induction of peripheral T cell tolerance in vivo by codelivery of DNA vectors encoding antigen and Fas ligand.

Fas ligand (FasL, CD95L) induces apoptosis in activated T cells with upregulated Fas (CD95) expression through the process termed activation-induced cell death (AICD). We postulated that coexpression of antigen and FasL within individual antigen-presenting cells would lead to antigen-specific activation of T cells and to their consequent deletion by FasL-mediated AICD. A DNA-gelatin coacervate containing transferrin cell ligand, calcium, and the lysosomatropic agent chloroquine, a formulation previously shown to achieve high-level transfection of immune and muscle cells in vivo, was used to codeliver plasmids encoding FasL and antigen. Mice developed a strong cytolytic T cell response to beta-Gal when injected with DNA encoding beta-galactosidase (LacZ) model antigen, either as naked DNA or DNA nanoparticles, but failed to respond when there was concomitant injection of nanoparticles containing both the LacZ and murine FasL DNA vectors. This loss of T cell response was systemic, specific for beta-Gal, complete when nanoparticles were administered before antigen challenge, and decreased the T cell response from prior immunization with LacZ DNA. In effect, this "tolerization" injection induced antigen-specific peripheral tolerance in study mice, and represents a possible approach to the treatment of autoimmune diseases and transplantation rejection.

Differential expression of the lysosome-associated membrane proteins in normal human tissues.

The lysosome-associated membrane proteins LAMP-1 and LAMP-2 have closely related structures, with 37% sequence homology, and are major constituents of the lysosomal membrane. Their roles are unknown, but they are thought to be structural or functional components of the lysosomal membrane. Recent reports suggest that despite their similar structure and common localization, LAMP-1 and LAMP-2 may have different functions. In our further study of these two molecules, the presence of LAMP-1 and LAMP-2 in a variety of human tissues was analyzed by immunohistochemistry, and their localization was compared to that of cathepsin D, a lysosomal hydrolase. the tissue content of LAMP-1 and LAMP-2 and their respective mRNAs were also analyzed by Northern and Western blotting. The LAMP molecules were detected by immunohistochemistry primarily in metabolically active cells, with a cytoplasmic distribution similar to that of cathepsin D and consistent with their predominant localization in lysosomes. However, there were marked differences in the intensity of staining and, in some cases, the localization of the three proteins. For example, there was much stronger staining for LAMP-2 than LAMP-1 in brain tissue and prostate ductal cells. These differences in localization were consistent with the results obtained in Western blotting of protein extracted from the tissues. The pattern of mRNA expression was similar in all of the examined tissues, with a single mRNA identified for LAMP-1 and two splice variant forms seen for LAMP-2. Our studies of these molecules in human tissues support the conclusion that the expression of the molecules is independently controlled in some tissues, suggesting that the molecules may have independent as well as similar functions.

Gene transfer by DNA-gelatin nanospheres.

A DNA and gelatin nanoparticle coacervate containing chloroquine and calcium, and with the cell ligand transferrin covalently bound to the gelatin, has been developed as a gene delivery vehicle. In this study, the coacervation conditions which resulted in the formation of distinct nanoparticles are defined. Nanospheres formed within a narrow range of DNA concentrations and achieved incorporation of more than 98% of the DNA in the reaction. Crosslinking of gelatin to stabilize the particles does not effect the electrophoretic mobility of the DNA. DNA in the nanosphere is partially resistant to digestion with concentrations of DNase I that result in extensive degradation of free DNA but is completely degraded by high concentrations of DNase. Optimum cell transfection by nanosphere DNA required the presence of calcium and nanospheres containing transferrin. The biological integrity of the nanosphere DNA was demonstrated with a model system utilizing DNA encoding the cystic fibrosis transport regulator (CFTR). Transfection of cultured human tracheal epithelial cells (9HTEo) with nanospheres containing this plasmid resulted in CFTR expression in over 50% of the cells. Moreover, human bronchial epithelial cells (IB-3-1) defective in CFTR-mediated chloride transport were complemented with effective transport activity when transfected with nanospheres containing the CFTR transgene.

DNA-polycation nanospheres as non-viral gene delivery vehicles.

Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such as gelatin and chitosan were evaluated as gene delivery vehicles. DNA-nanospheres in the size range of 200-750 nm could transfect a variety of cell lines. Although the transfection efficiency of the nanospheres was typically lower than that of lipofectamine and calcium phosphate controls in cell culture, the beta-gal expression in muscle of BALB/c mice was higher and more sustained than that achieved by naked DNA and lipofectamine complexes. This gene delivery system has several attractive features: (1) ligands can be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; (2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; (3) other bioactive agents or multiple plasmids can be co-encapsulated; (4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; (5) the nanosphere can be lyophilized for storage without loss of bioactivity.

Controlled gene delivery by DNA-gelatin nanospheres.

A novel system for gene delivery, based on the use of DNA-gelatin nanoparticles (nanospheres) formed by salt-induced complex coacervation of gelatin and plasmid DNA, has been developed. These particles were spherical, with a size range of 200-700 nm, contained 25-30% (w/w) DNA, and were stabilized by cross-linking of gelatin. As a consequence of being controlled by the cross-linking density of the gelatin matrix, the average release rate of DNA from nanospheres synthesized under standard conditions was 2.2%/day in serum. Nanosphere DNA incubated in bovine serum was more resistant to nuclease digestion than was naked DNA. Various bioactive agents could be encapsulated in the nanospheres by ionic interaction with the matrix components, physical entrapment, or covalent conjugation. Transfection of cultured cells with a luciferase plasmid was enhanced by conjugating human transferrin onto the nanosphere and coencapsulating the endolysolytic agent chloroquine. Under our experimental conditions, gene expression in mice subsequent to intramuscular injection of nanospheres containing 1 microg of a beta-galactosidase plasmid was greater and more prolonged than was observed after injection of an equal amount of naked DNA or DNA complexed with Lipofectamine.

CD44 expression in the stromal matrix of colorectal cancer: association with prognosis.

CD44, a cell hyaluronate receptor, is implicated in the metastatic behavior of some cancer cells. This study analyzed CD44 expression in topographic tissue sites of colorectal cancers to determine its association with patient survival and clinicopathological characteristics. Immunohistochemical localization of the core CD44 and the v6 splice variant domains was examined by use of paraffin-fixed sections from 133 stage II or III colorectal cancers that previously had been evaluated for other diagnostic markers. Expression in malignant epithelium, stromal matrix, and stromal cells was compared to patient survival by univariate, multivariate, and bootstrap (reproducibility) analysis. Core CD44 staining was present in the malignant epithelium of 85% of tumors, the stromal matrix of 90%, and the stromal cells of 98%. The v6 splice variant domain was present in the epithelium of 77% of tumors but was less frequent in the stromal matrix (12%; P < 0.001) and stromal cells (17%; P < 0.001). Absence of core CD44 immunoreactivity in the stromal matrix was associated with increased death rate (hazard ratio, 2.4; 95% confidence interval, 1.2-4.8; P = 0.02), making this one of the most significant adverse prognostic variables, along with an age of 60 years or older, poor differentiation of the cancer, extramural venous invasion, chromosome 18q allelic loss, and nonwhite race. This study shows that core CD44 and v6 splice variant antigens are differentially expressed in the epithelium and stroma of colorectal cancers. A model that includes core CD44 immunoreactivity in stromal matrix along with other prognostic factors may improve identification of high-risk and low-risk patients.

Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition marker.

During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, i.e., a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical "dense bodies" or mature lysosomes described in other cells are not present in resting neutrophils.

The enhanced immune response to the HIV gp160/LAMP chimeric gene product targeted to the lysosome membrane protein trafficking pathway.

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 carboxyl-terminal 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.

Relationship between CD44 expression and cell proliferation in epithelium and stroma of colorectal neoplasms.

The cell surface glycoprotein CD44 is expressed primarily in the region of cell replication in the lower crypt epithelium of colorectal mucosa, and its expression is markedly increased in colorectal neoplasms, suggesting that expression is linked to proliferation. The association between CD44 expression and replication in individual cells was therefore analyzed by double-label immunohistochemistry for CD44 and the cell-cycle-dependent protein proliferating cell nuclear antigen (PCNA). Enhanced expression of CD44 in colorectal neoplasms occurred not only in epithelial cells but also in stromal cells, including lymphocytes and macrophages. On a topographical basis, the cellular localization of CD44 and PCNA were commonly different. Quantitatively, in all cell types studied (epithelial cells and stroma of colorectal mucosa, adenomas, and carcinomas) PCNA was present most frequently in cells lacking CD44. Statistical analysis by logistic regression models indicated that cells negative for CD44 had a higher probability of being positive for PCNA than did cells positive for CD44 (P < 0.001). These data suggest that the enhanced level of CD44 in colorectal neoplasms is asynchronous with cell replication and reflects mechanisms that act on nonproliferative stromal lymphocytes and other mononuclear cells as well as the epithelial cells.

Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen.

Presentation of antigenic peptides by MHC class II molecules to CD4+ T cells is critical to the generation of antitumor immunity. In an attempt to enhance MHC class II antigen processing, we linked the sorting signals of the lysosome-associated membrane protein (LAMP-1) to the cytoplasmic/nuclear human papilloma virus (HPV-16) E7 antigen, creating a chimera (Sig/E7/LAMP-1). Previously, we found that expression of this chimera in vitro and in vivo with a recombinant vaccinia vector targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells compared to vaccinia expressing wild-type E7. In the current study, we tested these recombinant vaccinia for in vivo protection against an E7+ tumor, TC-1, which was derived from primary epithelial cells of C57BL/6 mice cotransformed with HPV-16 E6 and E7 and c-Ha-ras oncogenes. All mice vaccinated with 1 x 10(7) plaque-forming units of wild-type E7-vaccinia showed progressive tumor growth when challenged with a tumorigenic dose of TC-1 tumor cells; in contrast, 80% of mice vaccinated with the chimeric Sig/E7/LAMP1 vaccinia remained tumor free 3 months after tumor injection. Furthermore, treatment with the Sig/E7/LAMP-1 vaccinia vaccine cured mice with small established TC-1 tumors, whereas the wild-type E7-vaccinia showed no effect on this established tumor burden. These findings point out the therapeutic limitations of recombinant vaccinia expressing unmodified tumor antigens. Further, they demonstrate that modifications that reroute a cytosolic tumor antigen to the endosomal/lysosomal compartment can profoundly improve the in vivo therapeutic potency of recombinant vaccines.

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency.

Lysosome-associated membrane protein-1-mediated targeting of the HIV-1 envelope protein to an endosomal/lysosomal compartment enhances its presentation to MHC class II-restricted T cells.

A subset of endogenously synthesized Ags can be processed for class II-restricted presentation, probably through multiple mechanisms. Processing of exogenous Ags for class II-restricted presentation appears to occur in unique endosomal processing compartments with lysosomal characteristics including the presence of the lysosomal membrane protein LAMP-1. Therefore, we attempted to enhance the efficiency of class II-restricted presentation of an endogenous Ag, the HIV-1 envelope (env) protein, by specifically targeting the Ag to class II processing compartments through the pathway followed by LAMP-1. Because the env protein associates tightly with CD4 shortly after synthesis, we first targeted the env protein using a chimeric CD4 protein consisting of the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of LAMP-1. When co-expressed with this chimeric protein, the env protein was efficiently localized to lysosome-like compartments. Enhanced stimulation of env-specific CD4+ T cell clones by APC expressing the env protein and the CD4-LAMP-1 chimera was readily demonstrated in both cytotoxicity assays and proliferation assays. We also targeted the env protein directly as a chimeric protein consisting of the extracellular domain of the env protein and the transmembrane and cytoplasmic domains of LAMP-1. The proliferative response of env-specific CD4+ T cell clones to the env-LAMP-1 chimera was greatly enhanced compared with wild-type env protein, especially when limiting numbers of stimulator cells were used. The enhanced stimulatory capacity of APC expressing LAMP-1-targeted Ags has important implications for vaccine design.

Internalization of type 1 complement receptors and de novo multivesicular body formation during chemoattractant-induced endocytosis in human neutrophils.

Upon activation of human neutrophils by chemoattractants, functionally important proteins are rapidly transported from intracellular granules and storage vesicles to the plasma membrane. This is accompanied by a marked increase in the rate of endocytosis and by ligand-independent internalization of type 1 complement receptors (CR1). To define the pathway of endocytosis, we used gold-conjugated BSA in a pulse-chase protocol. This tracer was initially internalized into small endocytic vesicles which rapidly traversed the cytoplasm and coalesced to form large, conspicuous multivesicular bodies. Within 5 min after addition of the chemoattractant, multivesicular bodies contained > 60% of the cell-associated BSA-gold. CR1 colocalized with the endocytic tracer in both the early endosomes and multivesicular bodies. In unstimulated cells, there was much less uptake of BSA-gold and multivesicular bodies were rarely seen. Using the acidotropic amine, DAMP, and anti-DNP antibodies, we found that the multivesicular bodies were acidified but the early endosomes did not concentrate DAMP. Neither the early endosomes nor the multivesicular bodies initially contained the lysosomal membrane antigens hLAMP 1 or 2, but hLAMP-positive structures subsequently joined the multivesicular bodies. The rapid activation of the endocytic pathway upon stimulation of neutrophils allowed us to visualize the de novo formation and maturation of multivesicular bodies. Our observations suggest that vesicles containing ion pumps and acid hydrolases fuse with multivesicular bodies, giving them characteristics of lysosomes, and that these are the probable sites of degradation of CR1. The observations do not support models which would require transport of CR1 from multivesicular bodies to defined, pre-existing lysosomes for degradation.