NEW GENERATION VACCINES

The advances in the last few years in the knowledge about the immune response and about molecular biology have allowed the identification of a large number of infectious agents and proteins of immunological interest and their expression in different vectors of amplification. The elimination of those proteins that are not of immunological interest or are not related to the virulence of the agent is now possible. Thus, new vaccines have been created which do not contain the whole infectious agent and that, among other advantages, allows the serological discrimination between sick and vaccinated animals.

Thanks to the knowledge of the immune response against infectious agents and to the development of different genetic techniques, new vaccines are being created that will allow some of the problems caused by the use of conventional vaccines to be solved. 

What are these new vaccines?

The basis of these new vaccines is, in the first place, the identification of the protein/s of the infectious agent that are able to induce an immune response in a similar way to that produced by the whole agent. Secondly, the identification of those proteins that are not immunogenic, do not have a role in replication, or that are related to virulence; thus, these proteins are not necessary. Using genetic engineering, the genes coded for these proteins can be selected, cloned and  expressed using different vectors; they can also be eliminated by selective deletion. A variation of this system is the chemical production of the selected proteins once they have been identified. 

New generation vaccines can be classified in the following groups based on the different technologies used (inactivated proteins, deleted-attenuated vaccines, recombinant vaccines) 

INACTIVATED PROTEINS.

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

Recombinant DNA technique.  

This technique is based on the production of proteins from an infectious agent without using the microorganism. Using genetic engineering techniques, DNA is fragmented expressed in vitro in different vectors . Thus, large quantities of a protein (subunit) are produced (sometimes more than one protein is produced). This can be used as a subunit vaccine. The different steps of this method are:

Once the fragment of DNA and its sequence are known, (1) this fragment is isolated and (2) inserted into a plasmid. (3) This plasmid is then introduced into an expression vector (E. coli, Baculovirus). (4) Some will accept the gene and thus produce the recombinant protein. (5) Most of them will not.. 

Once the relevant protein/s  from an etiologic agent has been  identified and sequenced, the DNA fragment codifying these proteins is isolated and then inserted into a plasmid that acts as the vector for the transference. Later, this is inserted in the expression vector (the type of plasmid will depend on the vector for expression) 

Some of these expression vectors will accept the new gene and then produce the protein. Using different  labeling techniques the identification of those vectors expressing the new gene will be possible.  

The most frequently used vectors for expression are bacteria, especially E. coli, yeasts and baculovirus. Bacteria present some problems for adequately glycosilating the produced polypeptides, that's why usually, the obtained proteins have lower immunogenic properties. 

Baculovirus are being increasingly used for the production of subunit vaccines due to their great capability of expression. Baculovirus is an insect virus able to replicate in established insect cell lines and whose expression promoter is the gene of polyhydrine. This gene produces up to 60% of the total protein of the virus and can be replaced by foreign genes. Using this system, different proteins against several animal viruses have been produced in insect cells: blue tongue disease, porcine parvovirus, and African equine fever. In some cases, the production of several proteins at the same time simulating the virion particle ("Virus like particles" of VLP), has been possible. This is the case with blue-tongue disease; the obtained product has a high immunogenic capability.    

 VLP ("virus like particles"). Microscopic estructure of blue-tongue disease. Courtesy from P. Roy.

Foot-and-mouth disease lesions in a pig.

The  first sub-unity vaccine against foot-and-mouth disease virus (FMDV) was obtained in the mid-eighties using recombinant DNA techniques. The gene for VP-1 protein was cloned and expressed in E. coli, and large quantities of VP were produced.  

However, the immune response induced with this vaccine turned out to be much smaller than that produced by the conventional inactivated vaccine. In order to obtain a response similar to that of the conventional vaccine, 1000 times more of VP-1 was necessary . 

A new sub-unity vaccine against Classical Swine Fever Virus (CSFV) has recently been developed. This vaccine consists of just glycoprotein gp 55, which has been shown to induce immunity and protection against virulent CSFV. The gene of gp 55 has been cloned and expressed using baculovirus. The protein obtained is then inoculated into the pigs which then produce neutralizing antibodies that are able to protect against the infection by the virulent virus. 

Production of synthetic proteins. 

Synthetic vaccines. When epitopes or antigenic determinants are identified in the complex structure of a protein, as happens with VP-1 of FMD, which is known to be located between aminoacids 140 and 160, it is then possible to chemically synthesize them and then produce a synthetic peptide identical to that of the virus; this is known as a synthetic vaccine. However, the number of protected animals (in the case of foot-and-mouth disease) is less than 50% of the total.  

The cause of this lack of protective immunity seems to be due to the fact that the epitope located between aminoacids 140 and 160 is effectively recognized by B lymphocytes, but not by T lymphocytes. The identification of epitopes able to stimulate T lymphocytes is nowadays underway in order to include them in a future vaccine.