LATEST GENERATION AUTOVACCINES

1.- BACTERIAL CAPSULAR EXOPOLYSACCHARIDES

2.- BACTERIAL BIOFILMS: THE ROLE OF EXOPOLYSACCHARIDES IN CHRONIC PROCESSES

3.- LIPOSOMES: ANTIGEN VEHICLES

4.- LATEST GENERATION AUTOVACCINES

4.- LATEST GENERATION AUTOVACCINES

The optimization of efficacy is the objective of preparing autovaccines with purified antigens (exopolysaccharides) and liposomes. This article explains the process by which exopolysaccharide-producing bacterial variants are selected, and their advantages with respect to classical autovaccines.

Autovaccines are prepared specifically for a given farm and for a limited number of animals, based on the bacterium or bacteria isolated on the farm in question. The rationale for using such vaccines is the existence of different bacterial strains and serotypes that while causing the same disease, may differ from one farm to another.

Autovaccines provide an adapted and rapid solution to minor problems or new pathologies. Their use in turn limits the need for antibiotics, thereby reducing the cost of treatments, avoiding the development of bacterial resistance, and preventing the appearance of residues in the meat or milk destined for human consumption.

Autovaccines offer an adapted and quick solution for minor problems and new pathologies; they are used when no other authorized products are available, and are always produced under veterinary prescription.

They are used when no other authorized products are available, and are always produced under veterinary prescription. The preparation of autovaccines requires a prior diagnosis, for which reason they adapt perfectly to the pathological process involved. In some countries such as the United States, the Food and Drug Administration (FDA) authorizes the preparation of autovaccines against viruses, though in the European Union manufacture is only authorized against bacteria.

Classical autovaccines: bacterins. Most commercial bacterial vaccines, and in general all autovaccines available on the market fundamentally consist of a large number of inactivated bacteria which are resuspended in an adjuvant. In order to prepare such vaccines it suffices to multiply the bacteria in an adequate culture medium, inactivate them (via heat, irradiation, etc.), separate the bacterial bodies, and combine them with an oily adjuvant or with aluminum hydroxide. These autovaccines have demonstrated their efficacy in certain processes. However, in many cases the humoral response of the host animal to a simple bacterin is inadequate, since it occurs in a nonspecific manner against the bacterial body as a whole. Moreover, the presence of whole bacterial bodies and of adjuvants may sometimes induce secondary reactions at the injection site.

Phase variation. Bacteria do not express the same antigens in vitro as in vivo — a mechanism known as phase variation (Fig. 1).

Fig. 1: Bacteria do not always express the same antigens in vitro as in vivo. Photograph of Salmonella spp. colonies.

When bacteria grow in vivo, they must cope with a hostile environment in which certain nutrients are not abundant, and where the host attempts to eliminate them in different ways. In such situations the bacteria use certain survival strategies that are entirely useless when growing in vitro, in a particularly "favorable" medium. For example, under in vivo conditions iron and certain oligoelements are scarce, and the bacteria must produce proteins that bind or capture such elements. In vivo, bacteria must express adhesion factors (fimbria, pili, adhesins, etc.) to avoid being eliminated with body fluid or organic movements. Moreover, in vivo, bacteria produce a thick exopolysaccharide capsule that protects them from phagocytosis.

These structures are lost in vitro because in energy terms the production of exopolysaccharides, adhesins, iron-sequestering proteins, etc., is too costly and unnecessary. In fact, the bacteria sometimes lose the genetic information needed to produce these structures, and are never again able to express them. The way in which phase variation occurs is simple: in vitro, bacterial variants appear among the bacterial population that no longer express these antigens, and since these cells do not waste energy on other activities, their growth rate increases considerably. In only a few generations, the bacteria that no longer express the antigens required in vivo become predominant and eliminate the bacteria that still express such antigens. Under in vivo conditions the opposite applies, however, i.e., the variants that produce such antigens are able to proliferate within the host, while the bacteria that do not are eliminated.

In the classical manufacture of vaccines and autovaccines, laboratories often work with bacteria that no longer express such antigens, since the capacity has been lost through successive replications in vitro.

Limitations of the classical autovaccines

Veterinary autovaccines typically use whole-body bacteria, and only some commercial vaccines add purified antigens such as for example bacterial toxins. In general, a bacterin is not sufficient to secure humoral protection, because the bacteria do not express the necessary antigens. In these cases it is necessary to work with special media and/or growth protocols that stimulate antigen production. For example, if the iron contents in the growth medium are deliberately reduced, the bacteria will express iron-sequestering proteins.

Liposomal autovaccines: selection of variants

The manufacture of liposomal autovaccines has two purposes: to select exopolysaccharide-producing bacterial variants and to purify them for use as antigens.

The latest generation veterinary autovaccines are prepared from purified antigens following the model developed in human medicine.

The first criterion for the selection of bacteria is their growth phase. When bacteria multiply in vitro, they consume the nutrients found in the medium and modify the characteristics of the latter — as a result of which their initially exponential growth pattern becomes stationary. Under these conditions the bacteria no longer multiply and even begin to die; however, at this point they produce certain antigens that were not previously expressed, since the bacteria are obliged to activate new genes in the modified medium.

Accordingly, capsular exopolysaccharide production is maximum when the stationary phase of growth is reached. The objective of good liposome autovaccine manufacturing practice is to secure maximum exopolysaccharide production while avoiding the appearance of dead bacterial remains.

The photograph shows collagen pellets on which a biofilm has developed (below), and others (above) on which no such film has formed. The bacteria were grown under the same environmental conditions, though in one case exopolysaccharide (and thus biofilm) production was favored, and not in the other. In this way, in the laboratory it is possible to select and maintain an exopolysaccharide-producing population despite the existence of phase variation phenomena.

In addition to modification of the growth media as a way to select bacterial populations, other mechanical procedures can be used (Fig. 2). By using microspheres in the culture medium, it is possible to ensure that most exopolysaccharide-producing bacteria, bound together within a biofilm (see April article), adhere to the surface of the spheres, while the non-producing of phase varying bacteria remain free in the medium.

Liposome autovaccines: antigen purification

The use of vaccines exclusively prepared with purified antigen is highly developed in human medicine. In fact, each year millions of children throughout the world are vaccinated against meningitis caused by Streptococcus pneumoniae and Haemophilus influenzae, using only capsular exopolysaccharide as antigen, and transport protein. Specifically against S. pneumoniae, vaccines are used that contain 25 mg of each of the 3 different serotypes included in the formulation. These 23 serotypes are in turn implicated in over 85% of all pneumococcal infections.

In veterinary practice, the high costs involved limit the preparation of vaccines with so many serotypes. This is the case of Streptococcus suis in Spain. In theory, a vaccine against porcine meningitis including serotypes 2, 1, _, 8, 4, 7 and 9 would afford protection against 87% of the observed clinical problems - though the associated production costs would make their use prohibitive.

The alternative at the present time is to develop autovaccines containing the purified antigen or serotypes found in one given farm, and in which liposomes substitute the corresponding transport proteins.