A major challenge in vaccine design is to identify antigen presentation and delivery systems capable of rapidly stimulating both the humoral and cellular components of the immune system to elicit a strong and sustained immunity against different viral isolates. Approaches to achieve this end involve live attenuated and inactivated virions, viral vectors, DNA, and protein subunits. This review reports the state of current antigen delivery, and focuses on two innovative systems recently established at our labs. These systems are the filamentous bacteriophage fd and an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

Historically, live attenuated virus vaccines were used to control many viral diseases, such as measles, smallpox, mumps and rubella. These vaccines elicit both humoral and cell-mediated immune memory responses[1-5], however, they are associated with genetic instability and residual virulence[6-7]. For these reasons, current human clinical trials using live attenuated viral vaccines are rare, and research has been limited to non-human primate studies[8] to understand the mechanisms about the protection elicited. The possible causes of pathogenicity may include: (1) reversion to a virulent form of the attenuated virus[9]; (2) recombination of the vaccine strain with wild type, pathogenic virus[10]; (3) ability of viral genomes to persist in tissues or the proviral genomes to integrate into the host genome[11]; and (4) the dysregulation of the immune system by viral proteins[12]. As alternatives to live vectors, a variety of other antigen delivery systems are available for vaccine research.

One of the most pursued strategies is based upon the use of a huge range of viral and bacterial vectors, which are defined as non-pathogenic vehicles containing inserted genes from pathogens for their expression and subsequent induction of specific immune responses. Viral vectors such as poxvirus, adenovirus, vesicular stomatitis virus, adenovirus-associated virus (AAV), alphavirus, cytomegalovirus and lentivirus, to name some but not all, have been explored[13]. In addition, vectors have been developed from bacteria such as Shigella[14] and Salmonella[15], by exploiting their ability to be delivered via the oral route, which is the natural route of infection for a variety of bacteria. Because so many of the factors governing the induction of optimal immunity are incompletely understood (such as the activation of the innate immune system by various components of vectors and their effects upon adaptive immunity), it is hard to know exactly what would make the best vector for any given vaccine. Given the expensive and long development process required for clinical testing, the choice of one vector over another for each vaccine has been largely based upon the key characteristics of the vector and results of preclinical studies rather than comparison of vectors and administration protocols, since regimens, doses, and matrices of prime-boost combinations could lead to innumerable variables. Some of these vectors, in particular adenovectors and poxvectors, including both canarypox and modified vaccinia Ankara, have been employed in clinical trials for an anti-HIV-1 vaccine[16-20], all obtaining unsatisfactory results. More recently, "The STEP trial", which utilized an adenovirus vector encoding HIV proteins, was performed as a proof of concept for prevention or control of HIV infection[21,22]. The trial was stopped when it was clear that the vaccine was not demonstrating efficacy.

One major concern regarding the use of vectors is the immune response against the vector itself,[23,24] especially in persons who have been previously exposed to the virus or a related virus. Other concerns are the disputed risk of integration[25], the limited gene-insert capacity (in AAV and alphavirus vectors[26,27]), the safety issue in neurotropism (such as in the vectors based on herpes virus or vesicular stomatitis virus[28]), and in bacterial vaccine vectors given orally, the transfer of heterologous genes to other bacteria[29].

In addition to the use of vectors, antigens can be delivered by injecting plasmid DNA encoding immunogenic proteins under the control of eukaryotic promoters. DNA vaccination was first described in 1993[30] and accepted as one of the most promising ways of immunization. Overall, available evidence suggests that DNA vaccination is generally well tolerated[31-33], and two veterinary vaccines have been already licensed[34]. The fate of the DNA after injection has been a matter of concern due to the risk of integration of the nucleic acids into the host genome. However, all available data suggest that frequency of integration is below the frequency of spontaneous mutation[35,36]. Notwithstanding these premises, failures and triggering of disease exacerbation instead of protection have also been reported on the use of DNA vaccines. One of the main criticisms for DNA vaccination is their relatively low efficacy, mainly because a large amount of DNA is needed to be injected in order to achieve a strong response[37]. Improvement of the immune response to DNA vaccines has been attempted by aiming at enhanced plasmid uptake by exploring different routes of administration (gene-gun, electroporation), delivery of the antigen in combination with various immunostimulatory cytokines[38], and by using DNA vaccines in combination with adjuvants or cytokines in order to locally recruit different subsets of professional antigen-presenting cells (APCs)[39].

Virus-like particles (VLPs) represent another recent important biotechnological advancement. They are composed of one or several recombinantly expressed viral proteins, which spontaneously assemble into supramolecular structures resembling infectious viruses or, in some cases, subviral particles.

VLPs, like viruses, are comprised of one or more proteins arranged geometrically into dense, repetitive arrays. These structures are characteristics of microbial antigens, and the mammalian immune system has evolved to respond vigorously to this arrangement of antigens. Thus, due to their highly repetitive surface, VLPs are able to induce strong B-cell responses by efficiently crosslinking the membrane-associated immunoglobulin molecules that constitute the B-cell receptor. In addition to their ability to stimulate B-cell-mediated immune responses, VLPs have been shown to be highly effective in stimulating CD4 proliferative responses and cytotoxic T lymphocyte (CTL) responses[40-44]. Currently, VLP-based vaccines are in various stages of development, spanning preclinical evaluation to market. Vaccines for hepatitis B (Recombivax and Energix) and human papillomavirus (Gardasil and Cervarix) have been licensed commercially[41]. Furthermore, VLPs may also be used as a platform for inducing immune responses against antigens of choice. This can be achieved by constructing chimeric VLPs that display heterologous epitopes[45,46]. However, generating chimeric VLPs is largely empirical; it is almost impossible to predict whether individual peptides will be compatible with VLP assembly or whether the insertion will be immunogenic. Another important limitation of this approach is the size and the nature of the epitopes that can be inserted into the VLPs, in particular into their immunodominant regions.

Other promising strategies for vaccine delivery include the use of virosomes (liposomes carrying viral envelope proteins)[47-51], or subunit vaccines expressed in plant[52-56] or insect cells[57-61].

In summary, a variety of vaccine carriers and delivery modalities have been developed in recent years. However, the failure of clinical trials, based on available formulations, indicates that novel vaccine modalities are still very much needed to complement the existing array of options. In this context, we propose two innovative concepts for vaccine design based on the filamentous bacteriophage fd and the E2 protein from the pyruvate dehydrogenase (PDH) complex of Bacillus stearothermophilus (B. stearothermophilus). Both of the delivery systems combine the advantages of safety with the capability to elicit both humoral and cellular antigen-specific immune responses.

Vaccine is one of the most powerful and cost-effective tools of modern medicine. Currently, there is a renaissance in vaccine research due to the fact that healthcare authorities are increasingly recognizing the public health benefit and cost-effectiveness of vaccines. Since vaccines are administered a few times at most, they are also more cost-effective than many other drugs in treating the subsequent diseases. In recent years, several economists have argued convincingly that the cost savings are even more impressive because they go beyond the cost of medical care, and should include income lost due to illness and its sequelae[82]. The revival of interest in vaccines has of course also been underpinned by a rapidly expanding body of knowledge in the fields of immunology and by the impressive biotechnological advancement in recent years. Today, purification of microbial elements, genetic engineering, and improved knowledge of immune protection allow direct creation of attenuated mutants, expression of vaccine proteins in a variety of vectors, purification and even synthesis of microbial antigens, and induction of immune responses through manipulation of DNA, RNA, and proteins. A major aim is to integrate information gathered from classical pre-clinical trials with that derived from different fields of investigation. In this context, several approaches are being pursued to improve rationally designed delivery systems. We have been exploring the advantages of two innovative antigen display systems derived from non-pathogenic prokaryotic organisms, based on the filamentous bacteriophages fd and the acyltransferase component (the E2 protein) from the PDH complex of B. stearothermophilus. The fd and E2 antigen delivery systems combine the safety with the capability to trigger both humoral and cellular antigen-specific immune response, even in the absence of adjuvants. Both of these vehicles gain access to APCs, and intersect the human leukocyte antigen class I and II intracellular compartments, inducing specific cytotoxic T-lymphocyte responses. Moreover, the E2DISP delivery system offers a unique opportunity to display up to 60 copies of individual or multiple heterologous polypeptides, by preserving their native structure on the surface of a single VLP. These antigen presentation systems could be combined with other well-studied vaccines in an attempt to elicit full-spectrum immune responses.