Plant-derived vaccine protects target animals against a viral disease.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

Iscom immunization with synthetic peptides representing measles virus hemagglutinin.

Synthetic peptides representing the measles virus (MV) hemagglutinin (MVH) were incorporated into immunostimulating complexes (iscoms) and used for immunization of rabbits. Nine regions of MVH were selected on the basis of hydropathy and antigenicity profiles, by use of the known primary structure of MVH. Six linear and three branched types of peptides were synthesized and conjugated to palmitic acid before incorporation into the iscom structure. Five of the anti-peptide sera reacted by ELISA with the homologous peptide but did not react with MV in the native state, indicating that either the selected sites are not represented on the surface of MV, or they could be a conformational epitope. Human-anti MV and rabbit anti-MV did not react with the peptides.

Mapping the antigenic structure of porcine parvovirus at the level of peptides.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Isolation and characterization of a soluble antigen complex of Plasmodium falciparum with pyrogenic properties.

A soluble antigen complex, previously designated antigen no. 7 (Ag7) on the basis of the pattern obtained by crossed immunoelectrophoresis of culture supernatants of P. falciparum, was isolated by affinity chromatography. It was shown to be synthesized at the schizont stage of the parasite growth cycle and to be located on the surface of the schizonts. Antibodies to Ag7 did not inhibit the growth of the parasite in vitro. Ag7 is recognized by immune human sera from many parts of the world and it stimulated the production of specific antibody in mice when incorporated into immune-stimulating complex (ISCOM) structures. It also specifically stimulated in vitro proliferation of lymphocytes from clinically immune adults. That it induced the secretion of interleukin 1 by human monocytes and was pyrogenic in rabbits was of particular interest. Thus Ag7 has endotoxin-like properties which make it a possible candidate for an antitoxic malaria vaccine.

Immunological reactivity of bovine viral diarrhea virus proteins after proteolytic treatment.

The immunological reactivity of bovine viral diarrhea virus proteins after proteolytic treatment is described. The results indicate that the epitopes detected are very dependent on conformation of the protein. A partially protease resistant 22 kD fragment of the biotype-specific p80 is identified.

Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid.

The pestiviruses are small enveloped RNA viruses and are causative agents of economically important animal diseases in cattle, swine, sheep and goats worldwide. We used the polymerase chain reaction to amplify one common fragment of several different strains of both hog cholera virus and bovine virus diarrhea virus (BVDV). The fragment is located at the 5'-end of the genome immediately upstream of the open reading frame. This is a highly conserved region among the different published pestivirus sequences. An internal restriction digest of the amplified fragment with XhoI and PstI was performed in order to confirm specificity of the amplified fragment. The fragment was sequenced for a number of different BVDV strains, and the sequences obtained were compared to those published and used to deduce genetic relationships between strains. Apart from this common fragment we have amplified several other fragments of the Danish BVDV strain Ug59 and obtained specific amplification fragments of the expected size.

Adjuvants.

A number of adjuvants of importance in veterinary vaccines are reviewed. These include mineral salt adjuvants, oil adjuvants, hydrophilic and hydrophobic bloc polymers, hydrocarbons and surface active agents. The current and future applications of these substances and their possible modes of action are discussed. Creation of an adjuvant register by the newly formed European Adjuvant Group should facilitate accumulation of information on adjuvants.

A novel subunit ISCOM vaccine against bovine virus diarrhoea virus.

The preparation and preliminary testing of a subunit ISCOM (immunostimulating complex) vaccine against bovine virus diarrhoea virus (BVDV) is described. Vaccination of calves with this vaccine yields high neutralising titres against a panel of Danish BVDV field isolates. The serological difference between virus isolates and vaccine strain selection is discussed.

Edible vaccines.

The ultimate vaccine is an oral vaccine which given once protects against a multitude of diseases. Furthermore this ultimate vaccine needs to be very stable and inexpensive to produce. Probably this latter condition can be met only if the vaccines are produced in plants. Such vaccines are called 'edible vaccines'. Edible vaccines can be produced in plants in many ways. Using recombinant plantvirus, CPMV, it was shown that plants can produce massive amounts of chimaeric virus particles which protect after a single injection the target animal against disease. The final step, oral administration, is being addressed at present. Preliminary experiments by others suggest that this step may be solved sooner than expected.

Epidemiological studies of piglet diarrhoea in intensively managed Danish sow herds. III. Rotavirus infection.

The prevalence of rotavirus infection was studied in 1090 litters from 26 sow herds. Samples of normal, semifluid and watery stools were examined for rotavirus by an ELISA-test on faeces. Rotavirus was detected in 77% of the herds and in 30.5% of the litters (prevalence rates). The highest prevalence rate was seen in piglets between 21 and 41 days of age. Gilts' litters had a very high prevalence during the first week of life. Apart from this, no difference was found between litters from gilts and older sows. Rotavirus was detected more frequently in semiliquid, loose stools than in normal or watery stools, and an association between virus detection and diarrhoea could not be demonstrated. However, litters which shedded rotavirus during the suckling period had lower weight gains and higher incidence rates of respiratory diseases than virus-free litters. Litters weaned at 2 weeks in battery cages had slightly increased risk of shedding rotavirus compared to litters weaned in more traditional systems. The study revealed that rotavirus is widespread in Danish swine herds. The findings give evidence to suggest that the type of mild diarrhoea in 3-week-old piglets known as steatorrhoea or "white scours" may be associated with rotavirus infection, possibly in combination with E. coli and other agents. The high prevalence in piglets weaned at 2 weeks plus the higher morbidity and mortality among such piglets sustain the conclusion that piglets should not be weaned before 3 weeks of age or below a body weight of 6-7 kg.

Assay of detergents by rocket electrophoresis in agarose gels containing red blood cells: "rocket hemolysis".

A method is described for quantitation of charged detergents using their hemolytic property in an electrophoresis assay in agarose gels containing red blood cells. After electrophoresis the zone of hemolysis is directly proportional to the concentration of detergent in the sample. Using this technique we have determined the smallest detectable concentration for the negatively charged detergents, sodium dodecyl sulfate (SDS) and Quil A to about 10 micrograms/ml and 25 micrograms/ml, respectively and the positively charged cetyltrimethylammonium bromide (CTAB) to about 10 micrograms/ml.