Prophylactic vaccination is one of the most effective interventions in medicine and is responsible for substantial decreases in morbidity and mortality by many pathogens worldwide [1,2]. Although the concept of vaccination has existed since the early 1000s, it was not until the 1700s that the first experimental proof for vaccination emerged. Edward Jenner demonstrated that exposure to cowpox virus protected individuals from infection by the related, but much more virulent, smallpox virus [3]. Today, even as our understanding of the immune system rapidly expands, the basic idea behind early strategies of vaccination still holds: protection is best achieved by activating the immune system before an exposure to an infectious pathogen.

Prophylactic vaccines are usually derived from inactivated or attenuated strains of a pathogen or from macromolecules such as proteins, glycoproteins, and polysaccharides that are synthesized by the pathogen [4-6]. These vaccines prevent or limit infection because they induce robust, specific B cell mediated antibody response and a variety of T cell mediated cellular immune responses, including development of cytotoxic cells. Cytokines generated during the early and late phase of the immune response allows communication between the immune cells as well as fine tunes the global immune response for effective elimination of the pathogen. The induction of such B and T cell mediated responses, and the subsequent generation of B and T cell memory, requires cooperation and activation of the innate immune system. Vaccines often include adjuvants to trigger the host innate immune responses by activating dendritic cells, macrophages, and other innate immune cells during early phases of the response. The importance of adjuvants in vaccination has long been recognized but how adjuvants worked remained unclear until a proposal by Charles Janeway in 1989 [7]. The focus on adjuvants and their actions quickly became an area of intense research, especially after the discovery of the Toll-like receptors in 1997 [8]. Prophylactic vaccines derived from inactivated or attenuated strains of a pathogen have natural adjuvants present in the pathogen itself. Vaccines derived from macromolecules must first be mixed with synthetic adjuvants before administration in order to boost immune responses. Currently, only a limited number of adjuvants are approved for use in humans in the United States. These include alum and a few lipid based emulsions [9]. Other adjuvants have the capability to enhance immune responses but are not currently licensed for use in vaccines, in part, due to potential toxic side effects [9]. Natural components of pathogens (i.e. LPS, muramyl di-peptide, flagellin) and nucleic acid mimics (i.e. CpG, PolyIC) are on this list [9,10]. Exposure to wild-type pathogen after vaccination recalls pathogen specific memory cells that mount a robust and rapid response, often preventing infection completely or significantly limiting its severity, duration, and spread. In the absence of vaccination, the adaptive immune response to a wild-type pathogen is slow to develop allowing the pathogen to replicate and spread quickly in the absence of pathogen-specific memory response.

Traditional vaccines are remarkably effective, but there are limitations to their production and distribution. The need for developing alternative safe and effective vaccine platforms is urgent. First, the primary measure of the immunogenicity of a vaccine has traditionally been antibody titers [11]. Generating neutralizing antibody against the pathogen to prevent it from establishing an infection has proved to be a successful strategy for vaccination for many pathogens. However, it is becoming increasingly clear that T cell responses are critical at mediating clearance of many infections as well as preventing the establishment of chronic infections [12-14]. As such, the most effective vaccines should induce rapid and robust CD4 and CD8 T cell mediated immune responses. Second, traditional vaccines can be time consuming to produce. For example, the traditional seasonal influenza virus vaccine takes roughly five to six month to manufacture [15] and any pitfalls in the workflow could lengthen this time. Finally, the majority of traditional vaccines rely on cold storage, a limiting factor to distribution of vaccines across the world particularly in less or under developed nations [16].

Research in developing new vaccine platforms with the potential to elicit robust but broad B and T cell responses and mitigate other limitations of traditional vaccines is ongoing. A number of such vaccine platforms include virus like particles, liposomes, polymeric nanoparticles, and metal-based nanoparticles [17-19]. Recently, gold nanoparticle (Au-NP) based vaccines have been receiving substantial attention due to the potential advantages over traditional vaccine platforms. First, gold nanoparticles are customizable, able to be synthesized in various shapes and sizes by relatively straightforward chemical synthesis [17,20]. From an immunological perspective, this customizability has functional significance. After injection, nanoparticles smaller than 100 nm readily enter lymphatic tissues [21,22] and those less than 50 nm accumulate in multiple immune cell subsets in the secondary lymphoid compartment [23]. Localization of AuNPs to lymphatic tissues and cells is notable as it mimics what occurs during a natural infection. Pathogens and their components gain access to the lymphatic network and migrate to lymph nodes via lymphatic flow or after being internalized by resident antigen presenting cells. In the secondary lymphoid compartments, antigenic determinants (called epitopes) recognized by B cells induce antibody responses while those degraded into small peptide epitopes can be presented to and activate T cell responses. Secondly, a number of macromolecules capable of inducing broad and robust immune responses can be attached to Au-NPs by either covalent or non-covalent mechanisms [17,24]. These include antibodies [17] and other antigenic proteins [25], T cell activating peptides derived from pathogens [26,27], and nucleic acids, including siRNA [28]. Importantly, these antigenic determinants can be attached in the presence of adjuvants, such as CpG [29], which enhances innate immune responses induced during vaccination. Third, Au-NPs are relatively stable [20,30]. Data from Tao et al. [30] demonstrated that Au-NPs conjugated with the influenza specific peptide M2e and the adjuvant CpG could be dried into a powder and stored at room temperature, potentially breaking the cold storage chain for distribution of vaccines. Importantly, these dried Au- NPs could be easily reconstituted with water without any observable aggregation. Finally, and perhaps most importantly, Au-NPs and have a good safety profile [6,31], which is essential for the development and use of any vaccine. There is some concern that smaller Au-NPs or Au- NPs coated with certain peptides or proteins may cross the blood brain barrier when administered intravenously [32,33]; however, Au-NPs can, like traditional vaccines, be administered by injecting through subcutaneous and intradermal routes [22] and therefore mitigate this concern.

Data from numerous laboratories indicate that the shape of the particle can have a significant impact on immunogenicity [17,20]. The malleability of gold particles allow synthesis into Au-NPs of various shapes and gives added advantages to this vaccine approach. When mice were administered Au-NPs intranasally, rod shaped particles had a more prolonged effect on microglia cell activation than spheres or urchin shaped particles [34]. When microglial cells were stimulated in vitro, rod shaped particles induced GM-CSF and IL-1α secretion, while sphere and urchin shaped particles induced only GM-CSF and IL-1α secretion respectively. A recent study by Nikkura et al. [25] demonstrated that when Au-NPs ~40nm in size were coated with the West Nile Virus E protein, spherical particles induced higher levels of antibody and induced pro-inflammatory cytokine secretion while rod shaped particles were more efficiently internalized by professional antigen presenting cells. Finally, using a mouse model of breast cancer, Black et al. demonstrated that rod shaped Au-NPs were taken up more efficiently by tumor cells resulting in the particles being distributed throughout the tumor [35]. In contrast, sphere shaped Au-NPs were only located around the periphery of the tumor. This later finding may be useful for developing therapeutic vaccines, particularly for diseases such as multi-drug resistant tuberculosis where granulomas pose particular challenge for the immune system. Taken as a whole, the data on size and shape of Au-NPs suggest that efficient Au-NP based vaccines should be a mixture of particles of varying sizes and shapes designed to induce more broad and robust immune responses.

The most challenging aspect in developing nanoparticle based vaccines will be choosing the appropriate epitopes and/or antigens (i.e. longer peptide fragments or whole proteins) for coating Au-NPs. A large number of B and T cell epitopes have been well characterized and submitted to the Immune Epitope Database (IEDB) [36]. However, these are largely based on peptide screens and it is unclear whether these induce T cell responses during infection. Improvements on T cell epitope selection is possible by using immunoproteomic techniques to identify those epitopes that are naturally processed and presented by infected cells [37,38]. These techniques have identified novel epitopes not present in IEDB but that are presented on infected cells [37,38] and to which seropositive patients have circulating T cells [38]. Regardless of the epitope identification method used, formulating Au- NPs containing multiple epitopes/antigens to induce antibody and CD4 and CD8 T cell responses will be essential. Indeed, numerous studies have demonstrated that a broad immune response is critical for clearance of many infections, including hepatitis B [13], hepatitis C [12], and influenza [14].

In addition to choosing the appropriate targets to include in the AuNP based vaccines, ensuring the proper processing of these targets will be essential in generating a protective immune response. In order to generate robust and specific CD4 and CD8 T cell responses, internalization of pathogens and/or antigens by antigen presenting cells (APCs) is critical. A number of APC subsets, including dendritic cells and macrophages are important in this process. These cells internalize extracellular antigen and shuttle it into the endosomal compartment where it can be degraded into smaller peptide epitopes. These peptide epitopes are then loaded onto MHC class II molecules in a special vesicular compartment followed by their export and display on the cell surface. Presentation of peptide/MHC-II complexes to CD4 T cells, each bearing a specific receptor, ensures cell activation. Because Au-NPs are efficiently internalized by professional antigen presenting cells [23,25], loading Au-NPs with multiple epitope/proteins, would be sufficient to generate CD4 T cell responses but may not be as efficient at generating CD8 T cell responses since, for the majority of these antigens, targeting to the cytosolic compartment is pivotal. The proteolytic environment of the late endosome is harsh and it is possible that MHC class I epitopes loaded onto Au-NPs would be degraded before encountering MHC class I molecules and, therefore, impair CD8 T cell responses. Therefore, in order to induce the most broad and robust CD4 and CD8 T cell responses, Au-NPs and/or their associated epitopes/antigens must be targeted to the appropriate antigen presenting cell or cellular compartment to ensure optimal processing and presentation to T cells. To this end, CD8 T cell responses may be enhanced by targeting Au-NPs and associated epitopes/antigens to antigen presenting cells capable of cross presentation [39]. A number of methods can be used to promote cross presentation and thereby trigger CD8 T cell responses. First, Au-NPs coated with epitopes/ antigens can be targeted to cross presenting dendritic cells by also including antibodies specific to cell surface proteins on dendritic cells that are involved in cross presentation of antigens, namely DEC-205 [40,41] or the mannose receptor [42,43]. Targeting DEC-205 may be the more attractive method as this pathway also allows for efficient MHC class II presentation [44] whereas cross presentation involving the mannose receptor seems to be specific for soluble antigen [42,43]. A second potential mechanism to target Au-NPs attached epitopes to the class I pathway is to include cell penetrating peptides (CPPs), small peptides that allow efficient delivery of molecules into the cells. Despite internalization into endocytic compartments, certain cell penetrating peptides allow escape of cargo into the cytosol [45], thereby allowing longer peptides or proteins attached to the Au-NP to be naturally processed by the classical proteasome-class I machinery. Similar to targeting epitopes to cross presenting DCs, targeting Au-NPs to the cytosolic compartment via CPPs may enhance class II processing and CD4 T cell activation as data indicate that 30-40% of the class II restricted response during a viral infection is proteasome mediated [46,47]. Like class I restricted epitopes, class II epitopes dependent on the proteasome for processing do not survive the harsh proteolytic environment of the endosome [48]. As is the case for size and shape, AuNP based vaccines should include a number of antigens targeted to the appropriate cellular compartment to ensure robust T cell activation. One can envision a vaccine formulation in which spherical Au-NPs are coated with antibody epitopes and rod shaped particles are coated with T cell epitopes that are targeted to the classical endosomal pathway (CD4 T activation), the intracellular proteasomal pathway (both CD4 and CD8 T activation), and/or cross presenting cell subsets (both CD4 and CD8 T activation). Both spherical and rod shaped Au-NPs can also be conjugated to any number of adjuvants to boost innate and adaptive responses and lead to more efficient memory B and T cell generation, thus offering enhanced protection to vaccinated individuals.

The new and improved Au-NP vaccine formulations, with different shapes and sizes of the nanoparticle, designed to direct antigens and their epitopes to different internal compartments in antigen presenting cells provides a multifaceted approach. While such an approach is likely to generate a broad and robust immune response, its potential to generate robust memory B and T cells during these early responses remains to be tested. With the number of travelers moving across the world by plane on a daily basis (estimated at 815 million in domestic and international travel in 2012; http://www.rita.dot.gov/bts/press_ releases/bts016_13), the spread of infectious agents across national and international borders can occur quickly. This is illustrated by the recent emergence of Dengue virus [49] and Chikungunya virus [50,51] in the United States and the entry of Ebola virus infected individuals into the US and other countries [52,53]. In addition, the threat of new emerging diseases is ever present with the emergence of Severe Acute Respiratory Syndrome (SARS) [54-56], Middle East Respiratory Syndrome (MERS) [57,58] and a number of new influenza virus strains [59,60]. With these known and unknown dangers to human and animal health, the importance of developing a new vaccine platform cannot be overstated.