Targeting Genetic Vaccines

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There are three primary ways to target an antigen to a particular cell or organ: (1) targeting the delivery system for uptake by a specific cell, (2) linking the antigen to a targeting ligand, or (3) using DNA that is transcriptionally regulated to only become active in the target cell. For a specific cell type, modifications can be made to the antigen to direct it to different pathways of antigen processing and presentation.

4.3.1. Targeting Uptake

There are a variety of surface receptors that are potential targets for APC specific DNA or post-transcriptional antigen delivery. Fc receptors are thought to bind immune complexes and facilitate the opsonization of particulates. This binding activates DCs by up-regulation of costimulatory molecules (86). CTLA-4 is another ligand that has been used to target DCs, and is thought to bind B7.1/B7.2 at a high affinity. Some chemokines act through binding to DC cell surface receptors and can be employed as well. Certain proteins such as CD36 and a^5 integrins are involved with receptor mediated phagocytosis (87) and others such as DEC205 (or the human homolog LY75) and DC-SIGN, which are DC markers mediate receptor mediated endocytosis (88). All of these are potential targets for use in directing DNA or antigen specifically to APCs (Table 2). A good example is the addition of mannose or mannan to a delivery system to target the mannose receptor on the DC surface. Targeting this receptor has led to 2-fold increase in phagocytosis of particle formulations by APC in vitro (89) and has also been used to increase transfection of cultured DCs (96). The addition of mannan to the surface has also been associated with an increase in antibody and cell mediated immune responses in vivo (65).

4.3.2. Intracellular Targeting

Targeting of antigen to different cellular compartments may influence the way that antigen is processed and presented. Conceivably, an antigen normally processed by the MHC class II pathway that is instead presented on the MHC class I pathway could lead to immune responses that primarily elicit CTL activity instead of antibody secretion (isotype switching), and possibly a more relevant therapy for cancer.

One of the most common cellular localization sequences used for targeting an antigen fusion partner to the MHC class I pathway is ubiquitin. Ubiquitin marks proteins for degradation by the proteosome into small peptides which are then transported to the endoplasmic reticulum for loading onto MHC class I molecules. Addition of ubiquitin to plasmid fusion constructs usually increases CTL responses at the cost of humoral responses (91-95). However, in one study, a ubiquitin fusion construct demonstrated a decrease in humoral response whereas CTL response remained unchanged (96). Further examination of ubiquitin fusion constructs will be required for generalization of this strategy. Calreticulin (CRT) is a particularly interesting candidate for cancer vaccines because it has both MHC class I targeting capacity and antiangiogenesis properties (the ability to inhibit blood vessel growth to the site of a tumor) (97-166). Addition of CRT to fusion constructs has shown to exhibit notable antitumor activity when given as a DNA vaccine for HPV-16 E7 antigen. It is believed that the antiangiogenesis properties of CRT are involved in this observed response (161).

Targeting the MHC class II pathway may also be a logical strategy if a humoral response is desired. This pathway can be targeted through fusion with lysosomal associated proteins such as LAMP-1 (162-164) or LIMP II (91). An antigen can also be targeted to the cell surface (165). Another apparent mechanism for increasing MHC class II processing is targeting antigen for secretion. This antigen could then be taken up by a DC and associated with class II molecules in the lysosome. Interestingly, both humoral and/or cell mediated immunity are increased by using this strategy (166,167). This phenomenon may involve a cross-priming mechanism to allow antigen to enter the cytoplasm.

4.3.3. Transcriptional Targeting

One of the most commonly used mammalian promoters in genetic vaccines is the cytomegalovirus promoter (pCMV). This is an extremely strong viral promoter that is capable of mediating high levels of antigen expression in many cell types. However, some of the expression products in a genetic vaccine, such as the immunomodulating

Table 2

Some Examples of the Genetic Vaccine Targeting Strategies^

Table 2

Some Examples of the Genetic Vaccine Targeting Strategies^

Ligand

Target

Ref.

IgG Fc Fragment

DC Fc receptor

(141)

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