J-LEAPS vaccines initiate murine Th1 responses by activating dendritic cells.

The Ligand Epitope Antigen Presentation System (LEAPS) converts a peptide containing a T cell epitope as small as 8 amino acids into an immunogen and directs the nature of the subsequent response. Tandem synthesis of the J peptide (a peptide from the beta-2-microglobulin) with peptides of 15 or 30 amino acids from HSV-1 or HIV made them immunogenic and promoted Th1 immune responses. Immunization of A/J or C57BL/6 mice with J-LEAPS heteroconjugates containing an epitope from the HSV-1 glycoprotein D (JgD) or an epitope from the HIV gag protein (JH) emulsified with Seppic ISA51 induced increased levels of IL-12p70 by day 3 and increased levels of interferon gamma (IFN-gamma) on days 10 and 24. Interestingly, levels of IL-10, TNF-alpha, and IL-6 did not change. Neither the H nor the gD peptides alone elicited responses and only weak responses followed immunization with the J peptide. Bone marrow (BM) cells became CD86 and CD11c positive within 48 h of treatment with JgD or JH. JH or JgD treatment promoted IL-12p70 production and expression of CD8 denoting the maturation and activation of a subclass of myeloid DCs. Pure cultures of immature myeloid DCs also responded to JgD treatment, forming clusters, developing dendrites, and producing IL-12p70 within 24 h. The JH or JgD treated bone marrow cells (JgD-DC) were necessary and sufficient to activate splenic T cells to produce IFN-gamma and the JgD-DC provided an antigen specific booster response to T cells from JgD immunized mice. Adoptive transfer of JgD-DC was also sufficient to initiate protective antigen specific immunity from lethal challenge with HSV-1. The J-LEAPS vaccines appear to act as an adjuvant and immunogen on DC precursors in a unique manner to promote activation and maturation into IL-12p70 producing DCs which then can initiate sufficient Th1 immune responses to elicit protection without production of acute phase cytokines.

A L.E.A.P.S. heteroconjugate vaccine containing a T cell epitope from HSV-1 glycoprotein D elicits Th1 responses and protection.

The L.E.A.P.S. heteroconjugate vaccine antigen (JgD), composed of a T cell epitope from glycoprotein D (gD(8-23)) of herpes simplex virus (HSV) linked with a peptide sequence from beta-2-microglobulin (aa38-50), elicited protection against lethal intraperitoneal (IP) challenge and prevented disease signs in most, and limited disease progression, for the rest of BALB/c mice challenged in the epidermal abrasion-zosteriform spread mouse infection model. JgD elicited a Th1 response in vaccinated mice as indicated by delayed type hypersensitivity (DTH) responses to HSV antigen, and gD and virion specific antibodies with an IgG2a/IgG1 >1. Vaccination with the JgD peptide delayed the onset of disease signs, reduced severity of the disease and reduced mortality rates in mice with different MHC backgrounds as compared to their respective control mice. CD8 cells were demonstrated as important for initiation of the immune response to JgD and CD4 cells and interferon gamma (IFN-gamma) for delivering immune protection in BALB/c mice, as indicated in monoclonal antibody ablation studies. JgD, and other J-L.E.A.P.S. vaccine antigens, appear to prime T cells to initiate a Th1 response, which is subsequently boosted upon viral challenge to result in protection.

Characterization and performance of membranes designed for macroencapsulation/implantation of pancreatic islet cells.

Amphiphilic polymer membranes were synthesized for macroencapsulation of cells and characterized by select chemical and biological techniques. The membranes were prepared by crosslinking hydrophilic poly(N,N-dimethyl acrylamide) (PDMAAm) main chains with hydrophobic di-, tri-, and octa-methacrylate telechelic polyisobutylene (PIB) stars. The hydrophilic/hydrophobic composition and the molecular weights between crosslink sites (both M(c,hydrophilic) and M(c,hydrophobic)) were controlled by synthesis conditions. Small tubular membranes were made by in situ rotational copolymerization/crosslinking and filled with pancreatic rat islets. The water-swelling behavior, mechanical properties, and oxygen and insulin diffusion were studied. Macroencapsulatory performance of these membranes was investigated in vitro by macroencapsulation of pancreatic rat islets within tubular membranes for up to 1.5 months, and studying the insulin secreting ability of encapsulated islets in culture. The membranes are robust and maintain their integrity for the period of encapsulation. They allow oxygen and insulin diffusion. Macroencapsulated islets maintained their viability and insulin secretion over an extended period (i.e., 45 days).

Synthesis, permeability and biocompatibility of tricomponent membranes containing polyethylene glycol, polydimethylsiloxane and polypentamethylcyclopentasiloxane domains.

The synthesis of "smart" tricomponent amphiphilic membranes containing poly(ethylene glycol) (PEG), polydimethylsiloxane (PDMS) and polypentamethylcyclopentasiloxane (PD(5)) domains is described. Contact angle hysteresis indicates that in air, the surfaces of such PEG/PD(5)/PDMS membranes are enriched by the hydrophobic components, PDMS and PD(5), while in water, the surfaces are rich in the hydrophilic PEG. The oxygen permeability of a series of membranes with varying M(c,hydrophilic) (M(n,PEG)=4600, 10,000 and 20,000 g/mol) and varying PEG/PD(5)/PDMS compositions was studied. Oxygen permeability increased with the amount of PDMS in the membrane. The molecular weight cut-off (MWCO) ranges and permeability coefficients of insulin through a series of PEG/PD(5)/PDMS(=29/14/57) membranes with varying M(c,hydrophilic) were determined. Insulin permeability is directly related to M(c,hydrophilic) of the membrane. MWCO studies show that the membranes are semipermeable to, i.e., allow the transport of smaller proteins such as insulin (M(n)=5733 g/mol, R(s)=1.34 nm) and cytochrome c (M(n)=12,400 g/mol, R(s)=1.63 nm), but are barriers to larger proteins such as albumin (M(n)=66,000 g/mol, R(s)=3.62 nm). Implantation of representative membranes in rats showed them to be biocompatible. According to these studies, PEG/PD(5)/PDMS membranes may be suitable for biological applications, e.g., immunoisolation of cells.

Multiple mechanisms for HSV-1 induction of interferon alpha production by peripheral blood mononuclear cells.

UV-inactivated, infectious, and other forms of herpes simplex virus 1 (HSV-1) induced interferon (IFN) production by different routes in myeloid origin mononuclear cells (MOMC) (consisting predominantly of monocytes). GM-CSF activated the MOMC (G-MOMC) to produce greater amounts of interferon while differentiation to DC, by the addition of granulocyte macrophage colony stimulating factor (GM-CSF) and calcium ionophore (GA-MOMC), reduced the levels of interferon production upon challenge with some HSV strains. UV-inactivated virus induced more interferon than infectious virus. L-fucose, an antagonist of the mannose receptor, inhibited the induction of IFN-alpha by UV-inactivated virus and gB(-) virus (defective in penetration) in MOMC and GA-MOMC but not G-MOMC. L-fucose had little effect on interferon induction by infectious HSV-1. The insensitivity of the G-MOMC to fucose inhibition distinguishes these interferon producing cells from the pDC2 cells previously described as natural interferon producing cells. The mannose receptor appears to be involved in the response to non-infectious forms of HSV but infectious virus appears to use a different pathway. These studies suggest that non-infectious virions and HSV infected cell debris effectively stimulate monocytes and pre-dendritic cells to produce IFN-alpha to initiate host protection against HSV infection.

The ability of an HSV strain to initiate zosteriform spread correlates with its neuroinvasive disease potential.

The requirements for disease development in the mouse epidermal scarification-zosteriform model of HSV infection are likely to parallel those required for primary HSV disease of humans. HSV-1 strains, which are neuroinvasive in the mouse footpad model of HSV encephalitis, caused local site lesions within 3 days and secondary zosteriform lesions along the dermatome within approximately 5 days. HSV-1 strains, which are not neuroinvasive, failed to progress to zosteriform lesion development and local site lesions were mild or absent. Relative differences in the rate and extent of zosteriform lesion development paralleled the behavior of the viruses in the mouse footpad model of neuroinvasion. In conclusion, the viral properties which are important for neuroinvasiveness appear to also determine the ability of an HSV strain to cause zosteriform disease.

Changes in BiP (GRP78) levels upon HSV-1 infection are strain dependent.

BiP (grp78) is a chaperone protein which can also regulate the unfolded protein response of the cell. Levels of BiP increased in cells infected by the small plaque producing, cell associated, neuroinvasive strains of HSV-1 (SP7, 490) but decreased in cells infected with KOS, a large plaque, attenuated strain. BiP protein synthesis continued early in infection and BiP was sequestered and its degradation was limited during SP7 infection. BiP protein synthesis stopped and the protein was degraded in KOS infected cells. These viral strain dependent differences in BiP concentration may influence other aspects of the viral interaction with the target cell and its host.

Intrastrain variants of herpes simplex virus type 1 isolated from a neonate with fatal disseminated infection differ in the ICP34.5 gene, glycoprotein processing, and neuroinvasiveness.

Two intrastrain variants of herpes simplex virus type 1 (HSV-1) were isolated from a newborn with fatal disseminated infection. A small-plaque-producing variant (SP7) was the predominant virus (>99%) in the brain, and a large-plaque-producing variant (LP5) was the predominant virus (>99%) in the lung and gastrointestinal tract. EcoRI and BamHI restriction fragment patterns indicated that SP7 and LP5 are related strains. The large-plaque variants produced plaques similar in size to those produced by HSV-1 KOS. Unlike LP5 or KOS, SP7 was highly cell associated and processing of glycoprotein C and glycoprotein D was limited to precursor forms in infected Vero cells. The large-plaque phenotype from KOS could be transferred into SP7 by cotransfection of plasmids containing the EK or JK EcoRI fragment or a 3-kb plasmid with the UL34.5 gene of HSV-1 KOS together with SP7 DNA. PCR analysis using primers from within the ICP34.5 gene indicated differences for SP7, LP5, and KOS. Sequencing data indicated two sets of deletions in the UL34.5 gene that distinguish SP7 from LP5. Both SP7 and LP5 variants were neurovirulent (lethal following intracranial inoculation of young BALB/c mice); however, the LP5 variant was much less able to cause lethal neuroinvasive disease (footpad inoculation) whereas KOS caused no disease. Passage of SP7 selected for viruses (SLP-5 and SLP-10) which were attenuated for lethal neuroinvasive disease, were not cell-associated, and differed in the UL34.5 gene. UL34.5 from SLP-5 or SLP-10 resembled that of KOS. These findings support a role for UL34.5 in promoting virus egress and for neuroinvasive disease.

Immunization with a LEAPS heteroconjugate containing a CTL epitope and a peptide from beta-2-microglobulin elicits a protective and DTH response to herpes simplex virus type 1.

A ligand epitope antigen presentation system (LEAPS) heteroconjugate vaccine containing a CTL epitope (H1) from the HSV-1 immediate early protein ICP27 (322-332) and a peptide sequence (J) from beta-2-microglobulin (35-50) elicited protection from intraperitoneal viral challenge and promoted DTH responses. The H1 peptide and other H1 containing heteroconjugates did not elicit protection or DTH responses. Antibody to the H1 peptide could not be detected by ELISA following vaccination with peptide, heteroconjugate or natural infection. The LEAPS heteroconjugate appears to prime a Thl-like response which is subsequently boosted by infection. These studies show that attachment of the J peptide can make a CTL epitope into a vaccine which is immunogenic and promotes a protective Th1 type of response.

The antiviral xanthate compound D609 inhibits herpes simplex virus type 1 replication and protein phosphorylation.

The mechanism of antiviral action of tricyclodecan-9-yl-xanthogenate (D609) was investigated in vitro. D609 inhibited herpes simplex virus type 1 (HSV-1) replication without apparent cytotoxicity. It reduced phosphorylation of virus-infected cell polypeptides and inhibited the HSV-1 encoded protein kinase (US3 PK) and, to a lesser extent, cellular protein kinase C in vitro. Virus production was reduced by D609 at concentrations greater than 3.8 microM, with complete inhibition at 75.2 microM at an MOI of 1 PFU/cell or less. Addition of D609 could be delayed until 7 h post-infection and still inhibit virus replication. Phosphorylation of infected cell viral polypeptides of 34 (similar molecular weight to the substrate of the viral US3 protein kinase) and 69 kDa was inhibited at 18.4 microM. Treatment of infected or uninfected cells with 37.6 microM D609 reduced protein phosphorylation to background levels. A concentration of 1.9 microM D609 in vitro inhibited the viral US3-encoded PK, which had been purified from infected cell lysates by affinity chromatography and identified by specific antibody. Purified cellular protein kinase C was inhibited at 75.2 microM D609 whereas other cellular kinases including casein kinase 1 and cAMP dependent kinase were not inhibited at concentrations as high as 188 microM D609. Collectively these data indicate that the mechanism of antiviral action of D609 is by inhibition of protein kinases and protein phosphorylation affecting a late step in HSV replication.

Inhibition of the G2/M transition of the cell cycle by methyl-2,5-dihydroxycinnamate in human lymphoid cells.

Immortalized human lymphoid cells treated with Methyl-2,5-dihydroxycinnamate (MDHC), a stable analog of erbstatin, inhibited the G2/M transition of the cell cycle. The MDHC inhibition of the cell cycle was observed at concentrations well below the IC50 for the inhibition of the EGF receptor and sufficiently below that reported to induce protein cross-linking. The effect of MDHC upon the cell cycle is relatively stable, since unlike erbstatin, inhibition of the G2/M transition was observed 32 hours following removal of the drug. PHA stimulated human peripheral blood mononuclear cells (PBMC) were much less sensitive to MDHC. This study shows that MDHC acts on cells lacking an EGF receptor and the target of MDHC is involved in promoting progression of the cell cycle.

A block in glycoprotein processing correlates with small plaque morphology and virion targetting to cell-cell junctions for an oral and an anal strain of herpes simplex virus type-1.

The characteristics of two clinical isolates of HSV-1 obtained from an oral (424) and an anal (490) lesion were compared with the highly passaged KOS strain. In contrast to KOS, the clinical isolates produced small plaques, were more cell-associated and the predominant viral glycoprotein species for gC and gD in infected cell lysates was the precursor, high mannose glycoform. Total virus production in Vero cells was equivalent for the three virus strains in one-step growths. Pulse-chase studies of glycoprotein C processing showed a reduction in rate at 7.5 h post infection and a significant block in processing at 10.5 h post infection for 424 and 490 but not KOS. Similar results were obtained for gD. The significant reduction in glycoprotein processing for 424 and 490 suggests a block in transport of viral glycoproteins or virions to and through the Golgi apparatus. Extracellular virions and the cell surface, prior to cell lysis, contained the processed gC glycoform suggesting a competent cellular glycan processing system. Upon co-infection of 424 or 490 with KOS or a gC- KOS strain, gC was processed to levels equivalent to KOS indicating that 424 and 490 are not inhibitory but that an activity(s) encoded by KOS facilitates maturation of gC from 424 and 490. Unlike KOS infected Vero cells, virion-containing vacuoles were observed in the cytoplasm at 12 h p.i. and extracellular virions were concentrated at cell-cell junctions of 424 or 490 infected cells but not in the perinuclear region. These results suggest that intracellular transport of viral glycoproteins and virions in 424 and 490 infected cells is different from KOS infected cells. The reduced level of viral glycoprotein maturation, virus release, cell surface presence and presence of virions at cell-cell junctions are consistent with small plaque production in tissue culture cells.

Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines.

Amantadine and related compounds stabilized the structure of purified pig brain clathrin coated vesicles (CCV) at biologically relevant concentrations. Incubation of purified CCV for 30 min at 25 degrees C or 37 degrees C caused the release of clathrin, as determined by a centrifugation assay, and a reduction in the number of coated vesicles, by electron microscopy. Amantadine (10 mM), tromantadine (1 mM), amidine D295 (cyclohexylcarboximidamide-(N-benzyl)hydrochloride (10 mM), chloroquine (0.1 mM) and monodansylcadaverine (10 mM) significantly reduced the extent of dissociation.

Autologous antibody is protective against HSV-1 infection of the immunocompromised mouse.

The protective capability of autologous anti-herpes simplex virus type 1 (anti-HSV-1) antibody was analyzed in immunosuppressed mice. Immunologically naive, immunosuppressed mice infected with a low-passage clinical HSV-1 isolate developed local site lesions, monoplegia, paraplegia, and died within 8 days. Mice that had recovered from a previous HSV-1 infection and were immunosuppressed with cyclophosphamide and then rechallenged with live virus showed no symptoms and survived. Untreated mice that had recovered from infection (primed mice) had high titers of anti-HSV-1 antibody and a delayed type hypersensitivity (DTH) response to virus challenge. Cyclophosphamide treatment, but not lethal irradiation, could ablate the DTH response, resulting in a lack of antivirus cell-mediated immunity. Antibody was the only demonstrable protective immune function in the cyclophosphamide-treated animals. This indicates that cell-mediated immunity is not required for protection against HSV-1 challenge in individuals with virus-specific antibody.

Tromantadine inhibits a late step in herpes simplex virus type 1 replication and syncytium formation.

Addition of tromantadine after virus penetration inhibited HSV-1 induced syncytium formation and virus production in HEp-2 and VERO cells and acted additively with neutralizing antibody in blocking virus spread and cytopathology. Inhibition of syncytium formation in VERO cells infected with 0.01 pfu/cell of HSV-1 GC+ was observed at a concentration greater than 25 micrograms/ml. The extent of inhibition was dependent upon the multiplicity of infection and cell type. Tromantadine inhibited a late event in HSV-1 replication which appeared to be sensitive to cycloheximide. Reversal of the inhibitory effect of tromantadine on syncytium formation required new protein synthesis. HSV-1 gB, gC, and gD were synthesized in the presence of tromantadine and could be detected on the cell surface by immunofluorescence. Tromantadine most likely inhibits a cellular process that is required for syncytium formation, such as glycoprotein processing, which occurs after the synthesis of the fusion protein but before its expression on the cell surface.

Mild acidic pH inhibition of the major pathway of herpes simplex virus entry into HEp-2 cells.

Penetration of the KOS strain of herpes simplex virus type 1 (HSV-1) and the MS and 333 strains of herpes simplex virus type 2 (HSV-2) into HEp-2 cells at pH 6.3 was at least 100-fold less efficient than at pH 7.4. Penetration of two low passage clinical isolates was completely blocked at pH 6.3. The syncytium-forming HSV-1 strains GC and MP were less sensitive than KOS to the mild acidic conditions. The inhibition was completely reversed upon neutralization of the medium. Penetration was assayed by plaque production following protection from acid inactivation upon virus entry. Penetration of HSV-1 KOS into Vero and HEL diploid fibroblast cells was similarly inhibited. HSV-1 KOS grown in 2-deoxy-D-glucose and monensin was also extensively inhibited at pH 6.3 but virus grown in 2-deoxy-D-glucose penetrated more slowly than normal virus at pH 7.4. Electron microscopy of HSV-1 KOS infection indicated that fusion and endocytosis occur at both pH 7.4 and 6.3 but that fusion predominates at pH 7.4 and endocytosis predominates at pH 6.3. These results indicate that fusion at the plasma membrane is the major route of productive entry for HSV, that strains of HSV can differ in their pH dependence for penetration and this may determine whether virus infection can occur following endocytic uptake.

Herpes simplex virus type 1 penetration initiates mobilization of cell surface proteins.

Changes in membrane structure resulting from herpes simplex virus 1 (HSV-1) penetration were detected using fluorescence photobleaching recovery methods. The effect could be blocked by inhibitors of viral and cellular processes involved in virus penetration. A rapid mode of HSV-1 strain KOS penetration into VERO cells at 37 degrees C normally occurs after a 5 min lag period and is 90-95% complete within 20-30 min. Rates of cell surface protein diffusion increase 2-3-fold after 5 min and return to normal after 25-30 min, this return correlating temporally with the penetration of the virus. At pH 6.3 the lag period preceeding penetration of HSV is increased to 20 min and penetration proceeds much more slowly than at pH 7.4. Inhibition of virus penetration with cytochalasin B or with the antiherpes drug tromantadine also prevents the HSV-1-induced increase in cell surface protein mobility. Colchicine, which does not block HSV-1 penetration, prevents the recovery of the membrane following virus penetration. Therefore, the changes in membrane structure characterized by increased cell surface protein mobility seem to be caused by virus penetration. Cytoskeletal function and integrity are required for the initiation of, and cell recovery from, virus penetration. A pH-sensitive activity, likely to be a virion fusion glycoprotein, is also required.

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid.

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.

Flow cytometric evaluation of anti-herpes drugs.

A rapid means of screening drugs for toxicity and anti-herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV-1 (multiplicity of infection 1-10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4-n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.