Development of a Virosomal RSV Vaccine Containing 3D-PHAD® Adjuvant: Formulation, Composition, and Long-Term Stability.

PURPOSE: Characterization of virosomes, in late stage preclinical development as vaccines for Respiratory Syncytial Virus (RSV), with a membrane-incorporated synthetic monophosphoryl lipid A, 3D-PHAD® adjuvant. METHODS: Virosomes were initially formed by contacting a lipid film containing 3D-PHAD® with viral membranes solubilized with the short chain phospholipid DCPC, followed by dialysis, later by adding solubilized 3D-PHAD to viral membranes, or to preformed virosomes from DMSO. RESULTS: Virosomes formed from lipid films contained the membrane glycoproteins G and F, at similar F to G ratios but lower concentrations than in virus, and the added lipids, but only a fraction of the 3D-PHAD®. By single particle tracking (SPT), the virosome size distribution resembled that seen by cryo-electron microscopy, but dynamic light scattering showed much larger particles. These differences were caused by small virosome aggregates. Measured by SPT, virosomes were stable for 300 days. 3DPHAD ® incorporation in virosomes could be enhanced by providing the adjuvant from DCPC solubilized stock, but also by adding DMSO dissolved adjuvant to pre-formed virosomes. Virosomes with 0.1 mg/mg of 3D-PHAD®/viral protein from DMSO induced antibody titers similar to those by virosomes containing 0.2 mg/mg of DCPC-solubilized 3D-PHAD®. CONCLUSIONS: Stable 3D-PHAD® adjuvanted RSV virosomes can be formulated.

The role of plasmodesma-located proteins in tubule-guided virus transport is limited to the plasmodesmata.

Intercellular spread of plant viruses involves passage of the viral genome or virion through a plasmodesma (PD). Some viruses severely modify the PD structure, as they assemble a virion carrying tubule composed of the viral movement protein (MP) inside the PD channel. Successful modulation of the host plant to allow infection requires an intimate interaction between viral proteins and both structural and regulatory host proteins. To date, however, very few host proteins are known to promote virus spread. Plasmodesmata-located proteins (PDLPs) localised in the PD have been shown to contribute to tubule formation in cauliflower mosaic virus and grapevine fanleaf virus infections. In this study, we have investigated the role of PDLPs in intercellular transport of another tubule-forming virus, cowpea mosaic virus. The MP of this virus was found to interact with PDLPs in the PD, as was shown for other tubule-forming viruses. Expression of PDLPs and MPs in protoplasts in the absence of a PD revealed that these proteins do not co-localise at the site of tubule initiation. Furthermore, we show that tubule assembly in protoplasts does not require an interaction with PDLPs at the base of the tubule, as has been observed in planta. These results suggest that a physical interaction between MPs and PDLPs is not required for assembly of the movement tubule and that the beneficial role of PDLPs in virus movement is confined to the structural context of the PD.

Contiguous gene syndrome due to a maternally inherited 8.41 Mb distal deletion of chromosome band Xp22.3 in a boy with short stature, ichthyosis, epilepsy, mental retardation, cerebral cortical heterotopias and Dandy-Walker malformation.

Microdeletions of Xp22.3 are associated with contiguous gene syndromes, the extent and nature of which depend on the genes encompassed by the deletion. Common symptoms include ichthyosis, mental retardation and hypogonadism. We report on a boy with short stature, ichthyosis, severe mental retardation, cortical heterotopias and Dandy-Walker malformation. The latter two abnormalities have so far not been reported in terminal Xp deletions. MLPA showed deletion of SHOX and subsequent analysis using FISH and SNP-arrays revealed that the patient had an 8.41 Mb distal deletion of chromosome region Xp22.31 --> Xpter. This interval contains several genes whose deletion can partly explain our patient's phenotype. His cortical heterotopias and DWM suggest that a gene involved in brain development may be in the deleted interval, but we found no immediately obvious candidates. Interestingly, further analysis of the family revealed that the patient had inherited his deletion from his mother, who has a mos 46,X,del(X)(p22)/45,X/46, XX karyotype.

Adaptation of positive-strand RNA viruses to plants.

The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.

Epidiaphyseal versus other intercalary allografts for tumors of the lower limb.

Epidiaphyseal intercalary reconstruction has become possible for bone tumors that extend into the epiphysis because advances in magnetic resonance imaging and chemotherapy allow close resection while sparing the juxtaarticular bone and joint. In a retrospective study, we questioned whether epidiaphyseal reconstructions around the knee had a clinical outcome (measured as long-term survival, complication rate, and functional score) comparable with metadiaphyseal and diaphyseal reconstructions. Between 1988 and 1999, 14 epidiaphyseal, nine metaphyseal, and 12 diaphyseal reconstructions were done, and the median followup was 7.2 years. Kaplan-Meier analysis showed a 10-year survival rate of 79% for epidiaphyseal reconstructions, which did not differ from an 89% rate for metadiaphyseal and a 75% rate for diaphyseal reconstructions. Epidiaphyseal complications included two infections, five fractures, and three nonunion treatments. Complications for all 35 grafts included three infections, 12 fractures, and nine nonunion treatments. Ultimately, six grafts failed, with infection and length of resection as predisposing factors. All epiphyseal osteotomies had tumor-free margins and no local recurrences. The mean Musculoskeletal Tumor Society score for each type of intercalary reconstruction was between 23 and 24. Because the epidiaphyseal reconstruction avoids complications associated with joint reconstruction and the results are comparable with those of other types of intercalary grafts, these reconstructions should be considered if at least 1 cm of tumor-free juxtaarticular bone can be maintained.

Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection.

Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast. Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds. Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined. At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added. With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively. In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca. 50% of the protoplasts became infected. No infection occurred in the absence of PEG. Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection. In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected. Increasing ionic strength, however, increased both binding and infectivity. The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV. In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding. In the presence of PEG the effects were similar, although larger amounts of virus were bound. The percentage of infection was reduced by all additives to 5-25%. Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity. These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions. Observations in the electron microscope support the latter mechanism.

The route of tomato spotted wilt virus inside the thrips body in relation to transmission efficiency.

The route of tomato spotted wilt virus (TSWV) in the body of its vectors, Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae) was studied during their development. First instar larvae were allowed, immediately upon hatching, to acquire virus from mechanically infected Datura stramonium plants for 24 h. The rate of transmission by adults was determined in inoculation access feeding test on Emilia sonchifolia leaf disks. Thrips tissues were analysed for infection at 24 h intervals after the acquisition-access feeding period, and assayed by the whole-mount immuno-fluorescent staining technique. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Occasionally the first infections of the salivary glands were already detected in one-day-old second stage larvae. The intensity of the infection in the various organs of the thrips of each species was positively related to the transmission efficiency. In both thrips populations good agreement was found between the percentage of second instar larvae and adults with at least one infected salivary gland lobe and the percentage of transmitting adults. These results support the contention that the virus must reach the salivary glands before thrips pupation in order to be transmitted by old second instar larvae and adults.

Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis.

Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.

Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts.

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the virus-containing tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75% of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 microns in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

Capsid proteins of cowpea mosaic virus transiently expressed in protoplasts form virus-like particles.

The coding regions for cowpea mosaic virus (CPMV) capsid proteins VP37 and VP23 were introduced separately into a transient plant expression vector containing an enhanced CaMV 358 promoter. Significant expression of either capsid protein was observed only in protoplasts transfected simultaneously with both constructs. Immunosorbent electron microscopy revealed the presence of virus-like particles in extracts of these protoplasts. An extract of protoplasts transfected with both constructs together with RNA-1 was able to initiate a new infection, showing that the two capsid proteins of CPMV can form functional particles containing RNA-1 and that the 60-kDa capsid precursor is not essential for this process.

The nonstructural NSm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells.

The expression and subcellular location of the 33.6-kDa nonstructural protein NSm of tomato spotted wilt virus (TSWV) was analyzed in Nicotiana rustica plants and protoplasts as a function of time. Immunofluorescent studies in protoplasts isolated from TSWV-infected N. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. In situ, these tubules appeared specifically in the plasmodesmata, suggesting their involvement in cell-to-cell movement of the virus during systemic infection. In protoplasts transfected with an expression vector containing the NSm gene, similar tubules were formed, indicating that NSm has the ability to form these structures in the absence of other virus-specific components. To test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells. Spodoptera frugiperda and Trichoplusia ni cells were infected with a recombinant baculovirus expressing the NSm-gene (AcNPV/NSm). The efficient formation of NSm-containing tubules emerging from the surface of both cell types indicate that no plant-specific cell structures or proteins are involved in their development.

In situ localisation of beet necrotic yellow vein virus (BNYVV) in rootlets of susceptible and resistant beet plants.

Mechanisms of resistance to beet necrotic yellow vein virus (BNYVV) were studied by comparing the multiplication and distribution of BNYVV in root tissue of some beet accessions. Seedlings were infected either by soil containing resting spores of Polymyxa betae with BNYVV, or by a viruliferous zoospore suspension. With both inoculation methods high virus concentrations were obtained in rootlets of the susceptible cultivar 'Regina'. Using infested soil, low virus concentrations were found in the partially resistant cultivar 'Rima' and in the resistant accessions Holly and WB42. When a zoospore suspension was used, similar virus concentrations occurred in 'Rima' and Holly as in 'Regina', while a low virus concentration was found in WB42. By in situ localisation studies, using immunogold-silver labelling, virus was detected in 'Regina' after infection by soil or a zoospore suspension, but it could only be detected in the resistant accessions after infection by a zoospore suspension. In rootlets of 'Regina', 'Rima' and Holly, virus was found in the epidermis, cortex parenchyma, endodermis, and interstitial parenchyma, but in general not inside the vascular tissue. In WB42 the virus, occurring in small aggregates, seemed to be restricted to the epidermis and some cortex parenchyma cells. Comparing both the multiplication and distribution of BNYVV in rootlets of the accessions studied, it is concluded that the virus resistance mechanism in 'Rima' and Holly is different from that in WB42.

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins.

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.

Expression and subcellular location of the NSM protein of tomato spotted wilt virus (TSWV), a putative viral movement protein.

The 33.6-kDa nonstructural (NSM) protein gene, located on the ambisense M RNA segment of tomato spotted wilt virus (TSWV), was cloned and expressed using the Escherichia coli pET-11t expression system. The protein thus produced was purified and used for the production of a polyclonal antiserum. Western immunoblot analyses of TSWV-infected Nicotiana rustica plants showed NSM synthesis only during a short period early in systemic infection. Although NSM was found associated with cytoplasmic nucleocapsid preparations, it was absent from purified virus particles. Analyses of subcellular fractions from young, systemically infected leaves showed the presence of NSM in fractions enriched for cell walls and cytoplasmic membranes, respectively. Furthermore, immunogold labeling of tissue sections of TSWV-infected N. rustica plants showed that this protein was found associated with nucleocapsid aggregates in the cytoplasm and in close association with plasmodesmata. The data obtained provide evidence that NSM represents the viral movement protein of TSWV, involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures.

Isolation and characterization of tubular structures of cowpea mosaic virus.

Tubular structures involved in the cell-to-cell movement of cowpea mosaic virus (CPMV) were partially purified from infected cowpea protoplasts to identify the structural components. A relatively pure fraction could be obtained by differential centrifugation and this was analysed by PAGE and immunoblotting. Besides the movement protein (MP) and capsid proteins (CP) of CPMV, no other major infection-specific proteins could be detected, suggesting that host proteins are not a major structural component of the movement tubule.

Phloem loading and unloading of Cowpea mosaic virus in Vigna unguiculata.

Within their host plants, viruses spread from the initially infected cell through plasmodesmata to neighbouring cells (cell-to-cell movement), until reaching the phloem for rapid invasion of the younger plant parts (long-distance or vascular movement). Cowpea mosaic virus (CPMV) moves from cell-to-cell as mature virions via tubules constructed of the viral movement protein (MP). The mechanism of vascular movement, however, is not well understood. The characteristics of vascular movement of CPMV in Vigna unguiculata (cowpea) were examined using GFP-expressing recombinant viruses. It was established that CPMV was loaded into both major and minor veins of the inoculated primary leaf, but was unloaded exclusively from major veins, preferably class III, in cowpea trifoliate leaves. Phloem loading and unloading of CPMV was scrutinized at the cellular level in sections of loading and unloading veins. At both loading and unloading sites it was shown that the virus established infection in all vascular cell types with the exception of companion cells (CC) and sieve elements (SE). Furthermore tubular structures, indicative of virion movement, were never found in plasmodesmata connecting phloem parenchyma cells and CC or CC and SE. In cowpea, SE are symplasmically connected only to the CC and these results therefore suggest that CPMV employs a mechanism for phloem loading and unloading that is different from the typical tubule-guided cell-to-cell movement in other cell types.

Multiplication of tomato spotted wilt virus in its insect vector, Frankliniella occidentalis.

The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.

Construction and analysis of an Autographa californica nuclear polyhedrosis virus mutant lacking the polyhedral envelope.

An Autographa californica nuclear polyhedrosis virus (AcMNPV)-specific protein of 34 kDa (pp34) is shown to be involved in the morphogenesis of the polyhderal envelope of baculoviruses. The region of the AcMNPV genome encompassing EcoRI-H and -S (map positions 82.6-85.8) contains five open reading frames (ORFs) forming one transcriptional unit. The bacterial beta-galactosidase (lacZ) gene was inserted in phase with the N-terminal 12 amino acids of ORF3, thereby intervening this gene. A recombinant (AcMNPV/p34DZ5) was selected by the expression of lacZ (blue plaques). Protein analysis by SDS-PAGE and immunoblotting indicated that Spodoptera frugiperda cells infected with the recombinant lacked pp34. Electron microscopy of recombinant-infected cells showed that the electron-dense "spacers", normally present in wild-type AcMNPV-infected cells, were also absent. These results indicated that pp34 is dispensable for nonoccluded virus replication and is involved in the morphogenesis of the polyhedral envelope. The recombinants were infectious to fourth instar larvae of Spodoptera exigua. Recombinant polyhedra were more sensitive to weak alkali. This supports the hypothesis that the absence of the polyhedral envelope and the efficient release of occluded virions are responsible for the increased virulence of polyhedral envelope negative mutants.

Studies on the movement of cowpea mosaic virus using the jellyfish green fluorescent protein.

The jellyfish green fluorescent protein (GFP) coding sequence was used to replace the coat protein (CP) genes in a full-length cDNA clone of CPMV RNA-2. Transcripts of this construct were replicated in the presence of RNA-1 in cowpea protoplasts, and GFP expression could be readily detected by fluorescent microscopy. It was not possible to infect cowpea plants with these transcripts, but combined with a mutant RNA-2, in which the 48-kDa movement protein (MP) gene has been deleted infection did occur. With this tripartite virus (CPMV-TRI) green fluorescent spots were visible under UV light on the inoculated leaf after 3 days and a few days later on the higher leaves. These results show that the polyproteins encoded by RNA-2 do not possess an essential function in the virus infection cycle and that there is, contrary to what we have found so far for the proteins encoded by RNA-1, no need for a tight regulation of the amounts of MP and CPs produced in a cell. Subsequently, the GFP gene was introduced between the MP and CP genes of RNA-2 utilizing artificial proteolytic processing sites for the viral proteinase. This CPMV-GFP was highly infectious on cowpea plants and the green fluorescent spots that developed on the inoculated leaves were larger and brighter than those produced by CPMV-TRI described above. When cowpea plants were inoculated with CPMV RNA-1 and RNA-2 mutants containing the GFP gene but lacking the CP or MP genes, only single fluorescent epidermal cells were detected between 2 and 6 days postinoculation. This experiment clearly shows that both the capsid proteins and the MP are absolutely required for cell-to-cell movement.

The movement protein and coat protein of alfalfa mosaic virus accumulate in structurally modified plasmodesmata.

In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are structurally modified in that the desmotubule is absent and the diameter has increased drastically (almost twofold) when compared to plasmodesmata in uninfected cells or cells in which AMV infection had been fully established. Previously reported observations on virion-containing tubule formation at the surface of AMV-infected protoplasts suggest that AMV employs a tubule-guided mechanism for intercellular movement. Although CP and MP localization to plasmodesmata is consistent with such a mechanism, no tubules were found in plasmodesmata of AMV-infected tissues. The significance of these observations is discussed.