[Update on smallpox vaccines]. / Actualités sur la vaccination antivariolique.

Smallpox is among the most dangerous pathogens that could be used by bioterrorists. The former vaccines produced by scarification on the flanks of calves or sheep could be used to protect the whole French population when used with bifurcated needles. They should be replaced by a second-generation vaccine grown in cell culture and, eventually later by new and safer third-generation vaccines using non-replicative viral strains.

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein.

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells.

Immunocytochemical reactions on biological specimens depend on many factors, the most crucial one being the maintenance of antigenicity. Antigens are vulnerable at each stage during preparation for electron microscopy. One of the least traumatic methods of preparing biological tissues for post-embedding immunolabelling includes the following steps: (1) physical stabilization of the native biological material by rapid freezing (cryofixation) and keeping the immobilized biological sample at low temperature, thereby avoiding any movements of water, ions and macromolecules; (2) dehydrating the frozen biological material by freeze-drying at low temperature; (3) embedding of the dehydrated specimen. Here we show that embedding of chemically unfixed dendritic cells in Spurr's resin after cryofixation and freeze-drying enables the conservation of fine ultrastructure without cell distortion or shrinkage. Furthermore, we demonstrate the feasibility of protein localization in ultrathin sections by immunolabelling of the major histocompatibility class II molecules.

Enveloped virus is the major virus form produced during productive infection with the modified vaccinia virus Ankara strain.

Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain that may be useful as a vaccine vector. Ultrastructural examination of purified MVA showed that most of the viral particles are enveloped in contrast to the Copenhagen strain (COP). In CsCl gradients, the majority of the MVA particles displayed a light buoyant density characteristic of the enveloped form. Consistent with these results, MVA particles were poorly labeled with antibodies against the surface of intracellular mature virus but strongly labeled with antibodies against an envelope antigen. Furthermore, MVA was more resistant than the COP strain to neutralization by mouse anti-COP antibodies. These results suggest that the MVA strain may be particularly suitable for the engineering of envelope proteins and that MVA may be able to resist the humoral immunity displayed by previously vaccinated individuals.

Schnitzler syndrome: heterogeneous immunopathological findings involving IgM-skin interactions.

The Schnitzler syndrome is the association of chronic urticaria, intermittent fever, osteosclerotic bone lesions and a monoclonal IgM gammopathy. It is not yet firmly established whether the monoclonal immunoglobulin component plays a part in the pathophysiology of the urticarial lesions. Immunoblotting on epidermal and dermal human skin extracts as well as immunoelectron microscopic (IEM) studies on Lowicryl K4M-embedded skin sections were performed in three patients with the Schnitzler syndrome. Western blotting on epidermal extracts showed the presence of IgM-kappa antiskin autoantibodies in two patients. These antibodies displayed the same isotype as the monoclonal components and recognized a 280-290-kDa antigen in one patient and a 200-kDa antigen in the other patient. IEM studies showed sparse IgM deposits in the epidermis, around the keratinocytes, near the desmosomes in one patient and dense deposits below the lamina densa, in the region of the anchoring fibrils, in another patient. Antiskin IgM autoantibodies of the same isotype as their monoclonal gammopathies can be present in the serum of some patients with the Schnitzler syndrome. These IgM antibodies seem to deposit in vivo in the epidermis and at the dermal-epidermal junction, in the region of the anchoring fibrils. These findings suggest that the monoclonal gammopathy plays a part in the pathophysiology of the skin rash. They also suggest patient heterogeneity both in the skin antigens that are recognized as well as in their localization.

Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes.

The in vitro interactions between vaccinia virus (VV) and monocyte-derived human dendritic cells (DC) have been studied to gain a better understanding of the mechanisms involved in the induction of an immune response by VV. This work showed that VV binds to DC less efficiently than to HeLa cells (HeLa). Capping of viral antigens on the DC surface and electron microscopic examinations suggested that VV enters into DC mainly by endocytosis instead of fusion as for HeLa. Early viral-encoded proteins were expressed in DC but late viral proteins and viral DNA synthesis did not occur. Nevertheless, when successfully infected, DC expressed a similar amount of a foreign, viral-encoded protein, as HeLa, if the early component of the p7.5 promoter was used. VV infection did not lead to DC maturation as determined by following the level of several cell surface markers associated with maturation, but an inhibition of the expression of the costimulatory molecule CD80 was noticed. The proliferation of allogeneic peripheral blood lymphocytes (PBL) was stimulated by VV-infected DC or inhibited depending on the particular donor lymphocytes employed. PBL from VV-vaccinated individuals with good memory responses to VV antigens proliferated in the presence of infected autologous DC. PBL from individuals with poor memory responses to VV and one unvaccinated individual also proliferated, albeit to a lower level, in the presence of infected autologous DC. These results suggest that VV-infected DC could both stimulate memory cells and prime naive cells in vitro.

Inefficient measles virus budding in murine L.CD46 fibroblasts.

Infection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L. CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L. CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation.

A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome.

Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes. The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase. Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini. Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression. Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase. The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies.

A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain.

Measles viruses (MV) can be isolated from the brains of deceased subacute sclerosing panencephalitis patients only in a cell-associated form. These viruses are often defective in the matrix (M) protein and always seem to have an altered fusion protein cytoplasmic tail. We reconstituted a cell-free, infectious M-less MV (MV-DeltaM) from cDNA. In comparison with standard MV, MV-DeltaM was considerably more efficient at inducing cell-to-cell fusion but virus titres were reduced approximately 250-fold. In MV-DeltaM-induced syncytia the ribonucleocapsids and glycoproteins largely lost co-localization, confirming the role of M protein as the virus assembly organizer. Genetically modified mice were inoculated with MV-DeltaM or with another highly fusogenic virus bearing glycoproteins with shortened cytoplasmic tails (MV-Delta(tails)). MV-DeltaM and MV-Delta(tails) lost acute pathogenicity but penetrated more deeply into the brain parenchyma than standard MV. We suggest that enhanced cell fusion may also favour the propagation of mutated, assembly-defective MV in human brains.

The assembly of the measles virus nucleoprotein into nucleocapsid-like particles is modulated by the phosphoprotein.

Measles virus nucleoprotein encoded from the vaccinia virus genome assembles into nucleocapsids similar in many respects to those observed during a natural measles virus infection. The influence of the measles virus phosphoprotein on nucleocapsid assembly has been studied using a vaccinia virus recombinant encoding both the nucleoprotein and the phosphoprotein. Infection of cells with the virus recombinant resulted in the formation of cytoplasmic inclusions in which the nucleoprotein and the phosphoprotein colocalized. Electron microscopic examination suggested that these inclusions contained characteristic nucleocapsid filaments. The buoyant density of nucleocapsids assembled in the presence of the phosphoprotein was found to be slightly higher than that of nucleocapsids assembled in its absence. Furthermore, the phosphoprotein partially inhibited the formation of nucleocapsids, a process which was extremely efficient when the nucleoprotein was expressed alone. Analysis of the nucleic acid content of nucleocapsids showed that they packaged heterologous RNA into a micrococcal nuclease-resistant form. These experiments demonstrate that the measles virus phosphoprotein regulates the efficiency with which the nucleoprotein assembles into nucleocapsids and the structural conformation they acquire.

Location, DNA sequence and transcriptional analysis of the DNA polymerase gene of orf virus.

Degenerate oligonucleotides representing conserved regions of various DNA polymerases hybridized to a region located 26 kb from the left end of the orf virus (OV) strain NZ-2 genome. DNA sequence analysis of this region revealed a 3024 bp open reading frame able to encode a protein with 56 percent amino acid identity to the DNA polymerase of vaccinia virus (VAC) and with significant homology to other DNA polymerases. Early transcripts derived from the open reading frame were detected in RNA purified from OV-infected cells, and 5' ends were mapped to a region 8-19 nt downstream from an A/T-rich sequence that resembles VAC early promoters. Unlike the VAC gene, the OV DNA polymerase makes almost exclusive use of G/C coding options. Attempts to substitute the activity of the OV DNA polymerase for its VAC counterpart were unsuccessful. This may indicate that the OV DNA polymerase is incompatible with VAC accessory proteins.

A vaccinia virus transfer vector using a GUS reporter gene inserted into the I4L locus.

A vaccinia virus (VV) transfer vector is described which enables integration of heterologous sequences into the I4L locus (ribonucleotide reductase-encoding gene) through co-insertion of a GUS selection marker. I4L- VV recombinants formed blue plaques when an agarose overlay containing XGluc (5-bromo-4-chloro-3-indolyl-beta-glucuronide) was added to the infected cell monolayer. Viruses already containing a lacZ reporter gene were also suitable recipients for the selection procedure since infection with a VV lacZ recombinant did not produce any blue plaques with XGluc. The addition of a synthetic early promoter downstream from the GUS cassette initiated the predicted-size transcript during an infection. Insertion of genes with VV p7.5-promoters into the I4L, J2R and K1L loci of the same virus produced viable virus recombinants even though recombination between these loci could be demonstrated. These techniques should be valuable for the further development of VV as a polyvalent vector.

Biological properties of recombinant HIV envelope synthesized in CHO glycosylation-mutant cell lines.

N-glycosylation of the human immunodeficiency virus type-1 envelope (Env) glycoprotein precursor (gp160) occurs by transfer of Glc3Man9GlcNAc2 onto the nascent protein. Maturation then occurs via cleavage of the three Glc residues, which starts during translation. These events are considered necessary to create Env functional conformation: treatment with "alpha"-glucosidase inhibitors, but not alpha-mannosidase inhibitors (i) impairs gp160 cleavage into gp120 and gp41, (ii) diminishes the accessibility of gp120 V3 region, (iii) prevents gp120 binding to its CD4 receptor, and (iv) prevents gp41-mediated membrane fusion. These inhibitors are of therapeutic interest. Here, using a collection of parent and mutant CHO cells that possess mutations in different steps of glycosylation, we reassessed the role of glycans in both the processing and the properties of recombinant gp160 expressed from a vaccinia virus vector. Mutant cells were as follows: Lec23 (which lacks alpha-glucosidase I activity) produces a collection of triglucosylated structures (Glc3Man7-9GlcNAc2); LEC10 (which has increased GlcNAc transferase III activity) produces complex glycans with a bisected GlcNAc residue; Lec1 (which lacks GlcNAc transferase I) and Lec3.2.8.1 (which lacks GlcNAc transferase I and has decreased activity of CMP-NeuNAc and UDP-Gal translocases) produce Man5GlcNAc2 glycans at complex or hybrid sites. As expected, glycosylation of Env produced from mutants was affected but, irrespective of the glycosylation phenotype, (i) similar quantities of Env were synthesized, (ii) the immunoreactivity of V3 was similar, (iii) gp160 was efficiently cleaved into gp120 and gp41, (vi) Env was exposed at the cell membrane, (v) secreted gp120 bound CD4, and (vi) membrane gp41 was able to induce membrane fusion with CD4+ cells. Thus, the glycosylation alterations examined are dispensable for Env processing and biological activity in CHO cells. In particular, removal of the three outer Glc residues was not required per se for Env folding in this system because functional Env is obtained from Lec23 cells: it appears therefore that lack of modification is not equivalent to drug inhibition of modification. These data are discussed in the light of previous reports describing the use of glycosidase inhibitors to alter glycosylation.

The vaccinia virus J5L open reading frame encodes a polypeptide expressed late during infection and required for viral multiplication.

A number of open reading frames (ORFs) are found in the vaccinia virus (VV) genome whose activities in the viral life cycle have not yet been determined. This report examines one such ORF, designated J5L, which was demonstrated to be essential for viral multiplication. Stable inactivation of the J5L ORF by insertion of a lacZ ORF was impossible unless another copy of the J5L ORF was present in the VV genome. Fusion genes between the J5L ORF and either the lacZ gene or the VV K1L gene were employed to study its temporal expression as well as its protein product. These experiments showed that J5L is transcribed late in infection and gives rise to a protein product which migrates by SDS-PAGE with the expected molecular weight (16 kDa). Numerous unsuccessful attempts to establish a stable cell line expressing J5L suggest that the J5L gene product could be cytotoxic.

Recombinant MUC 1 vaccinia virus: a potential vector for immunotherapy of breast cancer.

Breast cancer is considered as the major cause of mortality by cancer for women. Even if chemotherapy, radiotherapy and surgery have improved the life expectancy of patients bearing tumours, breast cancer is responsible for the death of 42,000 women per year in USA and 25,000 women in France. In this context, cancer vaccines may add an attractive alternative therapeutic strategy to the current existing treatments. We describe here the construction of recombinant vaccinia viruses co-expressing a tumour associated antigen (MUC 1) and an "adjuvant" cytokine, which have potential applications in the active immunotherapy of breast cancer. Indeed, recombinant vaccinia viruses have been extensively used during the past decade to induce a protective response against a whole variety of pathogens, and has proven to be of great value in the elicitation of a cellular immune response leading to the rejection of tumour grafts in mouse models.

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.

In vitro and in vivo study of the ectromelia virus homolog of the vaccinia virus K1L host range gene.

Ectromelia virus encodes a protein which is homologous to the product of the vaccinia virus host range gene, K1L, except for eight conservative and two non-conservative substitutions and an additional threonine residue at the carboxyl terminus. Unlike the vaccinia virus gene, the ectromelia virus homolog failed to support optimal virus replication in RK-13 cells and appeared to be expressed 20-fold less efficiently. This lower level of expression was not due to the genetic background of the virus, K1L RNA transcription, sequence of the K1L RNA leader, or stability of K1L RNA or protein. Infections of RK-13 cells with ectromelia or vaccinia virus mutants lacking an intact K1L gene resulted in transient expression of early genes followed by a rapid and irreversible cessation of both virus and host protein synthesis. Infections of the disease-susceptible ANCR or -resistant C57BL/6 mice with the K1L-lacking ectromelia virus yielded a pathogenesis pattern indistinguishable from wild-type, suggesting that the ectromelia virus homolog of vaccinia virus K1L is not important for ectromelia virus in vivo replication and spread.

Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines.

Vaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybrid protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.