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A toxicity and immunogenicity study, evaluating the safety of a polyvalent DNA prime/protein boost HIV-1 vaccine (DP6-001), was examined in rabbits. Animals were primed with a cocktail of six different DNA plasmids expressing five HIV-1 env genes and one gag gene followed by boosting with five gp120 proteins homologous to the DNA vaccines. The vaccine was shown to be immunogenic as evident from the induction of high-titered anti-Env and anti-Gag antibodies. There was an absence of detectable adverse effects on key toxicology parameters. Although plasmids persisted in the injection sites following single administration for 64 days, no evidence of integration into the host genomic DNA was observed. These studies demonstrate that a novel polyvalent DNA prime/protein boost vaccine lacks signs of toxicity and DNA integration in a rabbit model, and immunogenicity and toxicology data support clinical testing of the vaccine in humans.

Objective: To analyze the mechanisms of release and the extracellular fate of the HIV-1 Tat protein and to determine the Tat domain binding to the extracellular matrix. Design and methods: Release of Tat was studied by pulse-chase experiments with Tat-transfected COS-1 cells in the presence or absence of different serum concentrations, temperatures and drugs inhibiting the classical secretion pathway or endo-exocytosis, such as brefeldin A and methylamine. The binding of extracellular Tat to heparan sulfate proteoglycans (HSPG) was determined by using trypin, heparin or heparinase in pulse-chase experiments, by get shift and competition assays with radiolabeled heparin, and by heparin-affinity chromatography. The mapping of the Tat binding site to heparin was defined by functional assays of rescue of Tat-defective HIV-1 proviruses. Results: Tat is released in the absence of cell death or permeability changes. Tat release is dependent upon the temperature and serum concentration, and it is not blocked by brefeldin A or methylamine. After release, a portion of the protein remains in a soluble form whereas the other binds to extracellular matrix (ECM)-associated HSPG. The HSPC-bound Tat can be retrieved into a soluble form by heparin, heparinase or trypsin. Binding to heparin is competed out by heparin-binding factors such as basic fibroblast growth factor (bFGF), and it is mediated by the Tat basic region which forms a specific complex with heparin which blocks HIV-1 rescue by exogenous Tat and allows purification of a highly biologically active protein. Conclusions: These results demonstrate that Tat exits from intact cells through a leaderless secretion pathway which shares several features with that of acid FGF or bFGF. The released Tat binds to HSPG through its basic region and this determines its storage into the ECM, as occurs for bFGF.

The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gp120 known to contain a neutralizing epitope, this epitope was localized between amino acids 307 and 320 (V3 loop) in the external glycoprotein molecule. The monoclonal antibody did not interfere with the binding of gp120 to CD4, or with the subsequent step of CD4-induced shedding of gp120 from the viral envelope. However, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.

The envelope glycoprotein (gp120) of HIV-1 was labeled with fluorescein by using 6-[4,6-dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4-positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein-labeled gp120 with CD4 was observed in a solid-phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV-1-infected cells on CEM cells was significantly inhibited in the presence of fluorescein-labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein-labeled gp120 was approximately 5 x 10(-10) cm2sec-1, with nearly 61% of the receptor molecules being mobile. Binding of anti-gp120 monoclonal antibody to the CD4-gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.

The immunogenicity of a polyvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1Ba-L, whereas the virus load was reduced in the remaining animals compared to naïve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-κB, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNFα). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-κB activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.

Kaposi’s sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130 cas . Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.

An enzyme immunoassay using the purified native gp160 for the detection of human immunodeficiency virus type 1 (HIV-1) antibody was developed. This assay was determined to be highly specific, since (i) 157 serum samples that were confirmed negative by Western blot (immunoblot) (WB) were negative, (ii) 41 serum samples from populations with medical conditions that might cause nonspecific assay reactivity were all negative, and (iii) all 15 serum samples that showed false-positive reactions in one or more commercial HIV-1 screening tests were negative. The assay gave 100% specificity with a randomly selected and unlinked panel of 1,000 serum samples from healthy blood donors. The sensitivity of the assay was assessed by testing 238 samples confirmed as HIV-1 antibody positive by a standardized WB assay. All 238 serum samples (100%) were reactive in the native gp160 assay. In a dilution panel of 14 weakly WB-positive serum samples, 7 samples reacted two-to fivefold more strongly in the gp160 assay than in a virus lysate-based assay; the remaining 7 samples gave comparable reactivities in the two tests. The reactivities of 13 of these 14 serum samples in the gp160 assay were higher than in a commercial enzyme immunoassay that uses a recombinant envelope protein as the antigen. The native gp160 assay was more sensitive to identify seroconversion. In a well-characterized panel of sequential blood samples from a seroconverter, the new assay detected antibodies at least one sample ahead of the other commercial assays tested.

Immunization of mice and rats with purified external glycoprotein gp120 from two divergent human immunodeficiency virus type 1 (HIV-1) isolates resulted in the development of seven hybridomas secreting monoclonal antibodies able to recognize regions of gp120 which are common among divergent strains of HIV-1. These monoclonal antibodies cross-reacted with env glycoproteins from one African (Rutz), one Haitian (RF), and three North American viral isolates, namely IIIB, MN, and 451 by either immunoblot or radioimmunoprecipitation assays. All recognized denatured gp120 in immunoblots with the exception of one which required a conformationally intact glycoprotein for reactivity. The gp120 epitopes identified by these antibodies were mapped by screening of an env gene library in the lambda gt11 expression system. Three out of four epitopes were found to reside in the amino-terminal half of gp120 (Cys9 to Cys35, Thr44 to Glu72 and Val108 to Met130), the other was located in the middle region (Thr221 to Ser255). By virtue of their extent of cross-reactivity these reagents might provide a unique resource for the detection of new viral isolates related to HIV-1.

In addition to their properties as sequence-specific inhibitors of gene expression, sequence nonspecific phosphorothioate oligodeoxynucleotides have been shown to protect against the cytopathic effects of HIV-1. Although these compounds are effective inhibitors of HIV-1 reverse transcriptase in vitro, it is not certain that they exert their cytoprotective effect only in this manner. Initial binding of the HIV-1 virion to cells involves the interaction of the viral envelope protein gp120 with CD4. In this report, we describe flow cytometric data and a solid-phase ELISA assay that document the ability of a phosphorothioate deoxycytidine 28-mer to interfere with this interaction by competing with gp120 binding to CD4. The biological importance of this interaction is demonstrated by the fact that phosphorothioate oligodeoxycytidine inhibits syncytium formation resulting from HIV-1-induced cell fusion. These data suggest that phosphorothioate oligodeoxynucleotides may exert their cytoprotective effects, perhaps at least in part, by interfering with the binding of HIV-1 to the target cells.

Lipopolysaccharide (LPS) has been purified from Bacteroides gingivalis. The purity of this LPS is evidenced on ultracentrifugation, immunoelectrophoresis, and chemical analyses. On comparison with LPS from aerobic enteric organisms, LPS from Bacteroides gingivalis exhibits minimal potency when tested for pyrogenicity in rabbits and mitogenicity in mouse spleen cells. On the other hand, purified LPS from Bacteroides gingivalis exhibits high potency in its ability to stimulate 45Ca release from prelabeled fetal rat bones and to inhibit Ca influx into osteoclast-like cells.

The bone resorptive factor and amphipathic antigen (AcA) previously identified by us in preparations fromActinomyces viscosus have been partially purified, characterized chemically, and compared. They elute at the same location on chromatography with Ac 22. The fatty acid composition of AcA and the bone resorptive factor is the same. Some differences in carbohydrate composition are observed. TheActinomyces factor does not affect calcium influx or cyclic AMP in isolated bone cells. Therefore it is concluded that AcA stimulates resorption either by gaining entrance into bone cells or by way of a yet undetermined second messenger.

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.

A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.

Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.

Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nM, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents PTH, isoproterenol, and 8-bromo-cAMP, which were previously shown to reduce alkaline phosphatase activity in osteoblast-like cells, antagonized the effects of dexamethasone. Moreover, in the presence of dexamethasone, lower concentrations of these agents were required for inhibitory effects on alkaline phosphatase.

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.