Evidence that the amino acid region 124-203 of glycoprotein G from the respiratory syncytial virus (RSV) constitutes a major part of the polypeptide domain that is involved in the protection against RSV infection.

The first 230 residues of the 298-amino acid glycoprotein G of respiratory syncytial virus (RSV) are sufficient to confer complete resistance to challenge with live RSV, whereas the first 180 residues completely failed (Olmsted et al. (1989) J. Virol. 63, 411-420). The characterization of a protective epitope corresponding to the amino acid region 174-187 of the G protein (Trudel et al. (1991) Virology 185, 749-757) suggests that interruption of this region in the 180 residue truncated polypeptide may be responsible for its inability to confer protection and consequently that the 174-187 region may play a major role in the protection effected by the protein G. To support these hypotheses, we examined the ability of the amino acid region 124-203 of glycoprotein G to confer protection. The corresponding peptide was expressed as a non-fusion protein in a recombinant vaccinia virus designated VG27. Immunization of BALB/c mice with this recombinant efficiently induced the production of antibodies capable of recognizing both the parental glycoprotein G and peptide 174-187. Furthermore, upon challenge with RSV, a significant decrease of infectious particles was found in the lungs of mice immunized with VG27 as compared with non-immunized mice. Our results suggest that the 124-203 amino acid region of the RSV G protein constitutes a major part of the domain involved in protection.

Antibody response to rubella virus proteins in different physical forms.

The immunogenicity of different antigens, containing rubella virus hemagglutinating (HA) membrane protein, was studied using live virus, beta-propiolactone-inactivated virus, detergent and lipid-free octamers and virosomes. Whole virus particles, live or inactivated, induced hemagglutination inhibition (HAI) antibodies in rabbits after one subcutaneous injection of 0.16 micrograms of HA protein. Hemagglutinin rosettes or virosomes failed to induce antibodies even at a dose of 120 micrograms. Apparently, the extraction of viral membrane hemagglutinin, for the preparation of a rubella subunit vaccine, led to destruction of the antigenicity responsible for the induction of hemagglutination inhibiting antibodies. These results are discussed in the light of earlier studies on the preparation of a rubella subunit vaccine.

Rapid virus subunit visualization by direct sedimentation of samples on electron microscope grids.

Airfuge direct ultracentrifugation of viral samples on electron microscope grids offers a rapid way for concentrating viral particles or subunits to facilitate their detection and study. Using the A-100 fixed angle rotor (30 degrees) with a K factor of 19 at maximum speed (95,000 rpm), samples up to 240 microliters can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10- to 40-fold increase in sensitivity depending on the size of particles. Application of this technique to rubella virus permitted better visualization of viral membrane subunits on the particles. Rubella hemagglutinin immuno-stimulating complexes preparations were also better visualized and their morphology conserved after direct ultracentrifugation on the specimen grids. Similar observations are reported for respiratory syncytial virus associated subunits.

E1 glycoprotein of rubella virus carries an epitope that binds a neutralizing antibody.

We identified by immunoprecipitation and Western blot analysis, using a monoclonal antibody that neutralizes rubella virus, that E1 glycoprotein carries an epitope linked with neutralization. Glycosidase treatment of virus does not prevent blotting of this monoclonal antibody with the E1 glycoprotein, dissociating this epitope from the hemagglutination epitope which is linked with the oligosaccharide side chains. We also investigated by Western blot analysis human serum reactivity toward E1 glycoprotein and the two other structural proteins of rubella virus, E2 and C: all positive sera detected E1 and C, irrespective of their titers, indicating the importance of glycoprotein E1 in immunity. Frequent lack of reactivity against E2 might suggest that this glycoprotein is either less exposed or less immunogenic.

Identification of rubella virus structural proteins by immunoprecipitation.

Immunoprecipitation of [3H]amino acid labelled virus with monoclonal or human convalescent rubella sera and subsequent analysis by electrophoresis and fluorography, revealed three structural proteins of rubella virus: VP3: 59,000; VP2: 44,800; and VP1: 33,000.

Hemagglutination inhibition and virus neutralizing response of rabbits inoculated with bovine herpesvirus 1 subunit vaccine.

The immunogenicity of bovine herpesvirus type 1 (BHV-1) hemagglutinin has been investigated. Both live and nonionic detergent solubilized vaccines were prepared and 5000 hemagglutinating units (HAU) were injected subcutaneously into rabbits. Both types of vaccine induced a good antibody response but live virus was four times more efficient in inducing hemagglutination inhibiting and neutralizing antibodies than either Triton X-100- or octylglucoside-solubilized subunit vaccine. Blotting analysis revealed that five proteins, of 105,000, 90,000, 74,000, 64,000 and 54,000 mol. wt, were recognized by the serum of vaccinated animals. Triton X-100-solubilized vaccine did not induce antibodies against the 105,000 and 64,000 mol. wt proteins, indicating the important role of VP 90,000 and VP 74,000 in hemagglutination and neutralization. The order in which antibodies to the different viral proteins were induced was VP 90,000, (VP 105,000, VP 64,000, VP 54,000) and VP 74,000. Our data indicate that VP 90,000 is the hemagglutinin. Using convalescent serum from intranasally infected animals, we could identify nine structural proteins for BHV-1; VP 105,000, VP 90,000, VP 74,000, VP 64,000, VP 54,000, VP 50,000, VP 47,000, VP 40,000 and VP 31,000.

One-step primary reconstruction for complex craniofacial resection with PEEK custom-made implants.

PURPOSE: Reconstruction of large craniofacial defects has largely improved since custom-made implants have been developed in the past decade. For large lesions in fronto-orbital region (such as osteomeningioma), we applied a simple and reliable protocol to perform optimal primary reconstruction with PEEK (polyetheretherketone) specific implant at the same time of the resection. MATERIAL AND METHODS: Our protocol is based on virtual preoperative surgery with a planned bone resection that allows engineering of a specific implant to accurately fit to the defect during the surgery. Thus tumour removal and optimal immediate reconstruction are performed easily in a single-step procedure. The use of navigation is required to perform accurate resection according to the planning. We report our experience in five patients requiring complex orbito-frontal reconstruction. RESULTS: Planned resection was always achieved with accurate placement of the implant. Optimal orbital reconstruction is allowed and permits exophthalmos correction and orbital contour symmetry. No major complication was observed. CONCLUSION: We provide a simple one-step technique to reconstruct the orbit while achieving symmetric cosmetic and functional results, reducing operative time and avoiding donor site morbidity.

[Septic pseudarthrosis of the mandible and endodontic treatment]. / Pseudarthrose septique mandibulaire et traitement endodontique.

INTRODUCTION: Septic pseudarthrosis of the mandible is an uncommon complication with several etiologies. The presence of a tooth next to a fracture site is one of the etiological factors. Conservative management of a tooth in or near the mandibular fracture site is often the issue when treating fracture of a toothed part. OBSERVATION: A 49 year-old male patient was hospitalized in our department for a bifocal fracture of the mandible (right parasymphysis and left sub-condylar). An open reduction internal fixation with plates and screws was performed. The immediate postoperative period was uneventful except for persistent tooth pain in the parasymphyseal fracture site. At 1 postoperative month, the fracture site was stable and signs of consolidation were documented by the panoramic view. A dentist performed root canal treatment on tooth 42, 8 weeks after surgery, because of recurrent complaint by the patient. At 6 months, clinical and radiological examinations revealed mandibular pseudarthrosis. DISCUSSION: This type of pseudarthrosis case is relatively infrequent. It occurs after endodontic treatment of a healthy tooth close to the fracture site (performed after the physiological delay before bone healing). The bacterial colonization of the fracture site could be related to this endodontic treatment. This case raises questions on the need for endodontic treatment of a tooth near a mandibular fracture site.

Virosome preparation: differences between influenza and rubella hemagglutinin adsorption.

Rubella and influenza virosomes were prepared from preformed liposomes or dried lipid films with or without nonionic detergent beta-D-octylglucoside. The preformed liposomes and lipid films were prepared from lecithin and dicetyl phosphate (3.5:1). Viral hemagglutinin rosettes were prepared from purified viruses after solubilization with Triton X-100 (1%), centrifugation through a continuous sucrose gradient containing 30 mM octylglucoside, and dialysis. Analysis of virosomes by sucrose density gradient centrifugation, hemagglutination assay, and electron microscopy revealed that rubella hemagglutinin did not require the presence of detergent to form virosomes, whereas influenza hemagglutinin could absorb efficiently to liposomes only in the presence of detergent.

Neutralizing response of rabbits to an experimental rubella subunit vaccine made from immunostimulating complexes.

The purpose of this study was to evaluate experimentally the immunogenicity in rabbits of rubella subunits adsorbed to the adjuvant Quil A. The adsorbed viral proteins form structurally defined ImmunoStimulating COMplexes (ISCOMs). Rubella ISCOMs were tested for their capacity to induce neutralizing and hemagglutination-inhibiting antibodies, in comparison with a commercial live attenuated vaccine. Rubella ISCOMs were as efficient as the live vaccine in inducing neutralizing and hemagglutination inhibiting antibodies, suggesting the possibility of developing an ISCOMs subunit vaccine.

Protection of BALB/c mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein.

A synthetic peptide homologous to amino acids 174-187 of the G glycoprotein of the A2 strain of human respiratory syncytial (RS) virus (G/174-187) was shown to induce protection from live virus challenge of BALB/c mice after immunization with three doses of 50 micrograms of peptide coupled to keyhole limpet hemocyanin. Immunized mice showed high levels of circulating RS-specific antibodies as detected by ELISA assay; however, no neutralizing antibodies were found. Moreover, an important short-term cytotoxic T-cell response was observed with lymphocytes isolated from the lungs but not from the spleen of immunized mice. This response was lost 24 weeks after immunization; however, mice remained protected against challenge with live RS virus. In addition, a monoclonal antibody that specifically binds to peptide G/174-187 was found efficient in conferring passive protection from challenge: this data further supports our results on the importance of the 174-187 region in protection. Another peptide, spanning amino acids 144 to 159, was shown to induce neutralizing antibodies but did not confer protection.

Gene mapping of infectious bovine rhinotracheitis viral DNA genome.

A bovine herpesvirus I (BHV-I) HindIII genomic bank spanning 89% of the entire genome was constructed and individual fragments analyzed for their capacity to select specific mRNAs which were then expressed by in vitro translation assays. This procedure allowed the mapping of more than 20 viral polypeptides to discrete regions of the DNA genome. Some polypeptides map in neighboring HindIII fragments while most seem encoded in single fragments. In particular, the coding sequences for an abundant 94 kDa polypeptide, which is the potential unglycosylated precursor of gII glycoprotein, have been assigned to the small 3.6 kbp HindIII genomic fragment M. The localization of structural and non-structural gene-coding sequences will help to characterize viral polypeptides and eventually, a better understanding of BHV-I infection will be gained.

Immunovirological studies on human respiratory syncytial virus structural proteins.

Immunovirological studies suggest that human respiratory syncytial virus may well be composed of five structural proteins as are other members of the Paramyxoviridae family: the two external membrane glycoproteins H (90 000) and Fo (F1, 49 000; F2, 20 000; disulfide linked), the internal membrane protein M (34 000), the nucleoprotein N (42 000), and a protein (78 000) designated P that could be the equivalent of the polymerase of the morbillivirus and paramyxovirus genus. Neutralizing monoclonal antibodies showed, by immunoprecipitation and immunoblotting, that the fusion protein carries neutralizing epitopes. One monoclonal antibody, which shows a high neutralizing titer, immunoblotted directly with the F1 fragment (49 000) of the fusion protein. Analysis in mice of the immunogenicity of the structural proteins separated on sodium dodecyl sulphate gels indicated that, under our conditions, only the fusion protein dimer Fo and its F1 fragment were capable of inducing neutralizing antibodies.

Comparison of caprine, human and bovine strains of respiratory syncytial virus.

A new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.

Experimental polyvalent ISCOMs subunit vaccine induces antibodies that neutralize human and bovine respiratory syncytial virus.

The purpose of the present study was to evaluate experimentally, in guinea-pigs, the immunogenicity of respiratory syncytial (RS) virus subunit vaccines. Immunostimulating complexes (ISCOMs), made from the surface proteins of both human (Long) and bovine (A-51908) RS strains adsorbed to the adjuvant Quil A, were assayed for their capacity to induce neutralizing antibodies, in comparison to experimental live virus vaccines. Serums from animals vaccinated with either the human or bovine RS subunit vaccines were equally efficient in neutralizing human or bovine RS virus. ISCOMs prepared with bovine RS virus proteins were significantly (p less than 0.05%) more efficient than their human counterpart, in inducing neutralizing antibodies, suggesting their greater potential as a subunit vaccine.

Control of infectious bovine rhinotracheitis in calves with a BHV-1 subunit-ISCOM vaccine.

An ISCOM subunit vaccine was prepared by adsorption of purified viral membrane proteins of BHV-1 on the glycoside Quil A and assayed in six-month-old seronegative calves. Groups of five animals were given three intramuscular doses of 50 or 25 micrograms BHV-1/ISCOMs or a commercial attenuated vaccine. Both types of vaccine induced seroconversion: the ISCOM vaccine consistently gave a serological response superior to that of the attenuated vaccine with maximal titres of 1/608 in haemagglutination inhibition and 1/53 in neutralization for the 50 micrograms dose compared to 1/152 haemagglutination units and 1/16 neutralizing titres for the attenuated vaccine. Calves vaccinated with the ISCOM vaccine were protected upon challenge whereas control animals showed signs of respiratory distress, and calves vaccinated with the attenuated vaccine developed only mild respiratory tract infection and mild increased rectal temperature. Virus shedding was reduced 100-fold in the attenuated vaccine group compared to 10,000-fold reduction in the ISCOM vaccine groups. The high level of protection induced indicates the potential of BHV-1/ISCOM as a subunit vaccine.

Initiation of cytotoxic T-cell response and protection of Balb/c mice by vaccination with an experimental ISCOMs respiratory syncytial virus subunit vaccine.

Respiratory syncytial virus is an important human pathogen causing serious lower respiratory tract infections of children and elderly people. Previous studies on the development of experimental subunit vaccines either expressed by recombinant DNA technology or prepared from purified viral proteins absorbed on adjuvant (ISCOMs) have shown promise. The present work reports on the effectiveness of an experimental ISCOMs vaccine in initiating humoral and cell-mediated immune responses and in providing overall protection upon live virus challenge in Balb/c mice; results indicate that vaccination by the intramuscular route is more effective, even if vaccination by the intranasal route also significantly reduced virus shedding.

Vaccination of rabbits with a bovine herpesvirus type 1 subunit vaccine: adjuvant effect of ISCOMs.

Bovine herpesvirus type 1 has two major immunogenic surface glycoproteins: a 90 kDa haemagglutinin and the 130 (74 + 54) kDa glycoprotein. These proteins were purified by rate zonal sucrose density gradient centrifugation after extraction with nonionic detergent Triton X-100. For the preparation of ISCOMs, the glycoproteins were further adsorbed during a second rate zonal centrifugation, to micelles of Quil A glycoside, already added to the gradient. Haemagglutinating peaks were collected and used as subunit vaccine in rabbits: groups of three animals were injected with 50, 10 and 5 micrograms protein. Seroconversion was followed by ELISA, haemagglutination inhibition, neutralizing and plaque reduction assays. ISCOMs and unadsorbed subunits were efficient in inducing neutralizing as well as haemagglutination inhibiting antibodies: ISCOMs gave a higher level of response. The efficiency demonstrated by ISCOMs suggests their potential as a subunit vaccine.

Identification of a synthetic peptide as part of a major neutralization epitope of respiratory syncytial virus.

A 7000 Mr cleavage fragment of the F1 subunit that carries the major neutralization epitope has been identified by chemical and enzymatic cleavage of the fusion protein of respiratory syncytial (RS) virus (Long strain) with an efficient RS virus-neutralizing monoclonal antibody. Based on the published mRNA-deduced sequence of the A2 strain, coupled to the hydropathicity profile and prediction of protein conformation, the neutralization epitope has tentatively been localized on the first third of the F1 protein N-terminal, probably in the region of amino acids 215Ser to 236Glu. Analysis of three peptides covering different portions of the 212Cys to 236Glu region of the F1 fusion protein identified a peptide (Cys X 216Asn to 236Glu) that reacted strongly with the neutralizing monoclonal antibody and that was efficient in blocking neutralization and in plaque-reducing assays, confirming that the neutralization epitope was localized in that region. Further analysis with two other synthetic peptides (212Cys to 222Glu and Cys X 221Ile to 236Glu) indicated that the dodecapeptide Ile-Glu-Phe-Gln-Lys-Asn-Asn-Arg-Leu-Leu-Glu mimicked either the whole or a major part of the neutralization epitope. This opens a promising avenue for the simple design of a synthetic peptide vaccine to control RS virus infection.