Manipulation of the immune systems of humans and animals is a recognised manner of avoiding or treating certain diseases or conditions.
The mechanisms by which the immune system controls disease include the induction of neutralising antibodies (a humoral immune response), and the generation of cellular or T-cell responses. The latter include T-helper cells (TH) and cytotoxic T-lymphocytes (CTL). In instances of viral infection e.g. polio or hepatitis, antibodies provide protection by preventing the virus from infecting cells. Antibodies can also protect against bacteria e.g. pneumococci and staphylococci, by use of bactericidal mechanisms and by neutralising bacterial toxins.
T-cells can be stimulated when peptide fragments from an antigen are bound to molecules known as MHC I or MHC II (major histocompatibility complex, class I or class II) and are displayed on the surface of professional APCs (antigen presenting cells) such as DCs (dendritic cells) or macrophages. The T-cells contain antigen receptors which they employ to monitor the surface of cells for the presence of the peptide fragments from the antigen. The antigen receptors on TH cells recognise antigenic peptides bound to MHC II molecules. By contrast, the receptors on CTLs react with antigens displayed on class I molecules.
The stimulated T-cells amplify the immune response in that when a T-cell recognises a target cell which is infected with the pathogen, or that contain an epitope which it recognises, a chain of events is triggered and these eventually result in death of the infected cells. In addition, some T-cells can stimulate secretion of cytokines or lymphokines, which in turn can exert effects that ultimately lead to inactivation or eradication of pathogens.
Although there are many vaccines on the market there is a need to produce more effective and broad ranging vaccines for a number of diseases or conditions. There also remains a need for protection against infective agents or pathogens against which vaccines are currently unavailable or ineffective. In addition, there is a need for effective, single-dose vaccines, which are particularly desirable for economic reasons, for ease of delivery, and for patient or subject compliance.
Most vaccines suffer from the disadvantage that they are not able to induce an optimal combination of the various types of humoral and cellular responses so as to be immunologically effective. For instance, some vaccines only stimulate antibody responses when both antibody and cellular responses would be more efficacious. In other instances, multiple doses of the vaccines eg booster shots are required in order to attain protection against the relevant infective agent.
In some other instances, IgE production is induced along with other desired immunoglobulins such as IgA, IgG and IgM. Vaccines that induce IgE are not desirable, as the immunoglobulin is involved in allergic responses.
Stimulation of IgA production is a “first line” defense for pathogens that infect via entry through a mucosal site or surface. Thus, vaccines that can generate a high IgA secretory immune response without enhancing IgE production would also be valuable.
In yet other instances, although a vaccine results in stimulation of APCs, the degree of immune stimulation is sub-optimal. For example, dendritic cells or DCs are characterised by a series of subset of cells that can be distinguished from each other by surface molecules some of which are specific ligands that bind receptors on T cells. Accordingly, it would be desirable to produce a vaccine which would selectively target a subset of DCs, eg a subset capable of efficient CD8 T-cell priming since these T-cells play a vital role in protective immunity against many intracellular pathogens and cancer, but are notoriously difficult to induce.
Further, with regard to vaccines extracellular antigens traditionally do not enter the MHC-I processing pathway in most cells. In general, the production of CTL immunity using nonliving vaccines is unlikely although alternative routes of processing and presentation for class 1 have been proposed in APC through the uptake of apoptotic cells, immune complexes and particles [1]. Non-infective viral like particles (VLP) composed of the surface Hepatitis B protein or yeast retro-transposon protein particles have been shown to be efficiently processed for MHC I presentation by macrophages to induce CD8 CTL responses in vitro and in vivo [2, 3]. VLPs are multimeric, lipid-containing protein particles the lipid content of which comprises more than 50% of the dry weight.
However, since Hepatitis B core protein particles fail to be immunogenic, and have a lower lipid content, it has been proposed that VLP are immunogenic not by virtue of size, but by biochemical composition. This would be consistent with the proposal that when antigen is presented in formulations containing lipid or detergent, they are able to fuse with the APC, possibly by damaging the cell membrane, and thus gain entry into the cytoplasm.
The use of microspheres within which are entrapped antigens have been explored as a possible vaccine composition. The microspheres are made from biodegradable polyesters of lactic and glycolic acids (PLA and PLGA). The microspheres are constructed such that their size and polymer composition control the rate at which they degrade. As the microspheres degrade, the entrapped antigen is released therefrom, and provides for a controlled release of antigen for stimulating the immune response. It is unlikely that these molecules would interface and react with immune cells in the same way as protein particles the make-up of which are biologically compatible with cellular membranes.
However, the difficulties with this form of vaccine composition include antigen stability, the size of the spheres and the antigen-release kinetics, all of which still need to be resolved so as to produce a vaccine with good antigenicity and lasting immunogenicity, and to produce a vaccine that can be manufactured and administered economically [4].
In U.S. Pat. No. 4,225,581, a composition comprising a mixture of heterogenous particles ranging in size is described as being useful for delivering antigens that are adsorbed onto the surface of the polymeric particles to the body. However, the successful delivery, antigenicity and immunogenicity of such a vaccine was not illustrated or shown. Specifically, there was no reference to the induction of CD8 T cell responses, or even processing into the MHC class I presentation pathway. The polymeric material of the particles would be expected to have similar characteristics as PLA or PLGA microparticles discussed above.
Thus, it was not known prior to the present invention if the size per se of particles administered as part of vaccines could induce immunogenic responses.
In work leading up to the present invention, the inventor has surprisingly found that microparticles about the same size as viruses associated with an antigen provide strong cellular and humoral antibody responses in subjects.