These new vaccines are obtained by identifying the protein or proteins of an infectious agent capable of inducing a protective immune response in the similar way as that induced by the complete infectious agent. This is either by the identification of those proteins which have neither immunological nor replicative interest, or those which could be related to virulence and are therefore not necessary. By means of genetic engineering techniques, corresponding genes can be selected, cloned and expressed in different vectors or removed by selective deletion. A variant of this system would be obtaining the protein or proteins by protein synthesis once a protein of immunological interest has been identified. 

Another important feature related to the strategy of obtaining these new vaccines is the possibility of incorporating sequences of other antigens that can increase the stimulation of B and T lymphocytes, and even the release of cytokines. Thus, antigen presentation to the immune system can be improved.

Based on the different methodologies used (recombination, gene deletion) or the type of product obtained (inactive proteins, deleted-attenuated and recombinant vaccines), the new generation vaccines can be classified in the following groups:

CLASSIFICATION OF NEW GENERATION PORCINE VACCINES

INACTIVATED PROTEINS:

Sub-unit vaccines: Foot-and-mouth disease Classical swine fever Porcine parvovirus (experimental)

Synthetic protein vaccines: Foot-and-mouth disease

DELETED LIVE VACCINES: Aujesky's disease

LIVE RECOMBINANTS Experimental only

DNA VACCINES Experimental only

INACTIVATED PROTEINS:

The most commonly used molecular techniques to obtain large amounts of antigenic proteins are:

The technique of recombinant DNA  

This technique is based on the production of a protein or proteins from a infectious agent without needing the microorganism itself. This occurs by means of genetic engineering techniques which fragment the corresponding DNA, and express it in various "in vitro" expression vectors. Thus, large amounts of a single protein (sub-unit) or of several proteins of an infectious agent are produced. These can be used as a sub-unit vaccine. The stages of this method are the following:

Once the protein/s of immunological interest of a certain causal agent and its sequence are identified, the DNA fragment that codes this protein can be isolated and inserted in a plasmid which works as a vector for transference and this is then inserted in the expression vector (the type of plasmid used depends on the type of expression vector). 

Some of these expression vectors accept the new gene and produce the protein that it codes. By means of labelling systems it is possible to identify the vector which expresses the new gene. 

INACTIVATED PROTEINS:

Electron microscopy of the structure of a VLP ("virus like particles") of several proteins of "blue tongue" virus. Courtesy of P. Roy.

The most commonly used expression vectors are bacteria, mainly E. coli, yeasts and above all baculovirus. Bacteria have problems with adequate glycosylation of polypeptides produced, meaning that normally the proteins obtained have lower immunogenic capacity.

The baculovirus is being used increasingly in the production of sub-unit vaccines, owing to its great capacity for expression. The baculovirus is an insect virus which can be replicated in stable insect cell lines and whose expression promoter is the polyhedrin gene. This gene represents approximately 60% of the total proteins of the baculovirus and can be substituted by foreign genes.

By means of this system, proteins have been produced and expressed in insect cells against animal viruses such as: blue tongue, porcine and canine parvovirus, classical swine fever and the African horse sickness virus. It has even been possible to express several proteins simultaneously with a similar form as the virion ("Virus like particles VLP"). This is the case with "blue tongue", and the resulting product has high immunogenic capacity.

The first sub-unit vaccine against foot-and-mouth virus (FMV) was achieved using the technique of recombinant DNA in the mid-1980s. The gene of the VP1 protein of FMV was cloned and expressed in E. Coli, producing a large amount of VP.

Unfortunately, the immune response obtained with this sub-unit vaccine was greatly inferior to that obtained with the conventional inactivated vaccine. In order to achieve an immune reaction similar to the conventional vaccine, approximately 1000 times more VP 1 is needed.

A sub-unit vaccine has recently been developed against the classical swine fever virus (CSFV), made exclusively of the E 2 glycoprotein. At an experimental level this protein induces immunity and protection against virulent CSFV. The gene of the E 2 protein has been cloned and expressed by means of a baculovirus system. The protein produced is inoculated in experimental pigs, producing neutralizing antibodies able to protect against infection by the virulent virus.

Production of synthetic proteins:

Synthetic vaccines. When epitopes or antigenic determinants of immunological interest are identified in the complex structure of a protein, as with the VP 1 protein of foot-and-mouth virus where we know that the epitope for the induction of neutralizing antibodies is located between amino acids 140 and 160, its sequence can be chemical synthesized and a synthetic peptide made which is identical to that of the virus. This is known as a synthetic vaccine. Unfortunately, in the case of foot-and-mouth disease the levels of protection achieved with the synthetic protein are found in less than 50% of the animals. 

The cause of this lack of protective immunity appears to be because the epitope situated between amino acids 140 and 160 is recognised effectively by the B lymphocytes, but not by the T lymphocytes. Work is currently being done on the identification of epitopes which can effectively stimulate the T lymphocytes for their inclusion in a future vaccine.