Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from challenge.

Despite the existence for some time of effective prophylactic vaccines, hepatitis B virus (HBV) infection remains an important global concern. Improvements on existing vaccines could be beneficial, especially in situations where it is desirable or necessary to induce protective immunity more rapidly or with fewer doses. We have compared, in chimpanzees, a current HBV vaccine that contains recombinant hepatitis B surface antigen HBsAg) adsorbed to alum, with two novel vaccine strategies that have proven superior to the current vaccine in mice.

Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation.

Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll-like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood.

Structure based design of a series of potent and selective non peptidic PTP-1B inhibitors.

A series of benzotriazole phenyldifluoromethylphosphonic acids were found to be potent PTP-1B inhibitors. Molecular modeling on the X-ray crystal structure of the lead structure led to the design of potent PTP-1B inhibitors that show moderate selectivity against TC-PTP, a very closely related protein tyrosine phosphatase.

Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities.

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll-like receptor 9 (TLR9). CpG ODN of the B-Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN-alpha secretion by plasmacytoid DC were found to occur only with A-Class ODN.

History of vaccines and positioning of current trends.

The history of vaccine development spans a relatively short period of time in comparison to the history of human civilization. However, monumental advances in the field of vaccines have been made in effort to combat infectious disease. These advances have led to a reduction, and in one case the complete eradication, of the burden of some infectious diseases of the world.

DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice.

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers.

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA.

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN.

Structure of protein tyrosine phosphatase 1B in complex with inhibitors bearing two phosphotyrosine mimetics.

Protein tyrosine phosphatases (PTPases) are signal-transducing enzymes that dephosphorylate intracellular proteins that have phosphorylated tyrosine residues. It has been demonstrated that protein tyrosine phosphatase 1B (PTP1B) is an attractive therapeutic target because of its involvement in regulating insulin sensitivity (Elcheby et al. Science 1999, 283, 1544-1548).

The potential of CpG oligodeoxynucleotides as mucosal adjuvants.

The development of mucosal vaccines for humans has been hindered by the lack of safe yet effective mucosal adjuvants. Bacterial toxins are commonly used as adjuvants in animal models, but they are too toxic for use in humans. A novel class of adjuvant is CpG DNA, which contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs).

Immune-mediated destruction of transfected myocytes following DNA vaccination occurs via multiple mechanisms.

The delivery of antigenic proteins in the context of a DNA vaccine leads to the intracellular synthesis of antigen and the induction of both humoral and cellular immune responses. Subsequent to immune activation, any transfected cell expressing the immunogenic protein should, by the rules of immunology, become a legitimate target for removal by immune-mediated mechanisms. Herein, we have used an indirect assay of myocyte integrity following intra-muscular (i.

The use of CpG DNA as a mucosal vaccine adjuvant.

CpG DNA has been shown to be a potent adjuvant in many disease models. Most studies using CpG DNA as adjuvant have used parenteral delivery, but more effective protection against mucosal pathogens could be achieved with effective mucosal immunization. Recently, mucosal immunization with CpG DNA as an adjuvant has been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses.

CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage.

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP.

Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV.

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2.

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported.

Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments.

Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.

Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests.

Refinement of an in vitro cell model for cytochrome P450 induction.

Induction of cytochromes P450 (P450s) by drugs can lead to drug-drug interactions. Primary hepatocytes have been reported to retain inducible P450s. To optimize the use of primary hepatocytes for predicting induction of P450 (CYP 3A and 2B) expression in vivo, both culture conditions and expression of induction potentials were investigated.

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C.

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus.

Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition.

Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2.

Ser-228 has been shown to be essential for the catalytic activity of the human cytosolic phospholipase A2 (cPLA2). However, its involvement in catalysis has not yet been demonstrated. Using site-directed mutagenesis, active-site directed irreversible inhibitors, and the novel fluorogenic substrate 7-hydroxycoumarinyl gamma-linolenate, evidence is presented to show that the hydroxyl group of Ser-228 is the catalytic nucleophile of cPLA2.

A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains.

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3.

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus.

Selective inhibitors of COX-2.

The main target of non-steroidal anti-inflammatory drugs (NSAIDs) is prostaglandin G/H synthase (PGHS), also known as cyclooxygenase (COX), which exists as two isoforms. In order to evaluate the contributions of PGHS isoforms to physiological and pathological conditions and their sensitivity to inhibition by non-steroidal anti-inflammatory drugs, we have established high level expression systems of recombinant human PGHS isoforms. The inducible form of PGHS, termed PGHS-2, has been purified and characterized with respect to substrate specificity, product formation, enzymatic activity, glycosylation, heme content, quaternary structure, and modification by aspirin.

High-level expression of active human cyclooxygenase-2 in insect cells.

Active human cyclooxygenase-2 (Cox-2) was expressed at high levels in insect cells using a recombinant baculovirus. The specific activity of Cox-2 in the microsomes of infected cells was 0.51 mumol O2/min/mg and was comparable to that obtained for partially purified Cox-2 from ovine placenta (0.