**1. Introduction**

The classic approach for the development of vaccines for infectious diseases has comprised of immunization with live attenuated or inactivated agents [1]. The introduction of genetic engineering expanded the approaches of vaccine development to the application of recombinantly expressed antigens and immunogens as immunization agents [2]. Both viral and non-viral vectors expressing surface proteins and antigens have been used for immunization, first in animal models followed by human clinical trials [3]. Taking this approach has elicited strong humoral and cellular immune responses and has provided protection against challenges with lethal doses of infectious agents [4]. Similarly, recombinantly expressed tumor antigens and tumor cell proteins have elicited immune responses in vaccinated animals and provided protection against challenges with tumor cells [5].

The standard procedure for non-viral vector-based immunization involves the application of conventional DNA plasmids for the expression of the antigen in question [6]. Various approaches to improve the efficacy of delivery and the expression of antigens include polymer and liposome-based coating of plasmid vectors [7,8]. DNA delivery based on both microparticles and nanoparticles has provided promising strategies for vaccine development. Microparticle systems promote the passive targeting of antigen presenting cells (APCs) through size exclusion and supports sustained DNA presentation to cells through the degradation and release of encapsulated vaccines [7]. On the other hand, nanoparticle encapsulation provides increased internalization, enhanced transfection efficiency, and improved uptake across mucosal surfaces. Appropriate biomaterial selection can enhance immune stimulation and activation through triggering innate immune response receptors [7]. Moreover, nanoparticle-based delivery can target DNA to professional APCs. Encapsulation also adds flexibility to administration routes generating systemic and mucosal immunity resulting in more effective humoral and cellular protective immune responses.

One alternative has been to apply alphavirus-based vectors, which due to the presence of the alphavirus replicon provides a self-amplifying mechanism generating substantial gene amplification and thereby enhanced expression of the gene of interest. The increased expression levels relate to improved immune responses, but also allows the potential use of reduced quantities of plasmid DNA for vaccinations. Although the focus in this review concerns DNA-based genetic antigen preparations, a short presentation of application of alphavirus RNA replicons and alphavirus replicon particles is included. The basics of the self-amplifying replicon function is briefly described below.
