New Approaches to Vaccine Development​

The rapid development of molecular biological techniques coupled with major advances in genomics and proteomics has formed the bedrock of new vaccine development strategies.. These techniques have been applied to the identification of potential antigenic targets for vaccine development and facilitated new methods for the expression and delivery of candidate genes. The common strategies arising from this include the development of recombinant live vector vaccines expressing heterologous genes, as delivery systems, and DNA vaccines (eukaryotic antigen expression vectors).
1. Recombinant Live Vectors
Recombinant live vectors expressing heterologous proteins have the potential benefits of live vaccines without the need to isolate and attenuate the actual pathogen. Several attenuated bacterial and viral vectors have been constructed with varying levels of complexity to deliver proteins from a range of pathogens. In principle, the introduction of these living vaccines should stimulate strong cellular responses as the transgene products will be expressed and processed within the host leading to MHC presentation. The stimulation of cytotoxic T cells via
MHC class I presentation is of particular importance in immunity to viruses and other intracellular pathogens.
2. Recombinant BCG Vectors
BCG is the most commonly used vaccine in the world. Several recombinant BCG (rBCG) candidate vaccines containing genes from several different pathogens have been developed and tested, and in general these proved effective at stimulating both B and T cell responses. Protective immunity in animal models has been demonstrated for rBCG vaccines expressing the outer surface protein (OspA) of Borrelia burgdorferi,  the merozoite surface protein 1 (MSP-1)7 and the Leishmania protective antigen (LRC1). Other studies have shown immunogenicity for rBCG vaccines but no protection was demonstrated. Likewise, varying levels of heterologous protein have been reported. The variability in protective responses and protein production may be due to differences in the shuttle vectors used, stability of the rBCG vaccines used and the route of administration. Despite the variability in responses, the immunogenicity of rBCG vector vaccines shows promise.
3. Recombinant Salmonella Vectors
Salmonella spp. invade the gut and as such are good candidates for oral delivery of vaccines. In common with other Gram-negative bacteria, Salmonella possess type 3 secretion secretions (T3SS), which promote their invasiveness. One system is used to infect non-phagocytic cells while the second is required for intracellular replication and spread within the host. Salmonella Pathogenicity Island 2 (SPI2-T3SS) creates a needle-like complex through which bacterial effector proteins are delivered into the cell. It has been shown that the SPI2-T3SS can be used
to deliver heterologous antigenic proteins.13 The introduction of proteins through this route should result in intracellular processing of proteins for presentation to T cells. Significantly, the SPI2-TSS has been shown to be active in dendritic cells, the major professional antigen presenting cells of the immune system. In one study,15 using S.typhimurium strain, translational fusions between Salmonella effector proteins (SseF or SseJ) and ovalbumin and L. monocytogenes proteins listeriolysin O (Llo) and p60. The last two are protective antigens in various models of listerial infection.16 Mice orally immunised with the recombinant S. typhimurium carrying the fusion proteins showed reduced Listeria burden. As found in other studies, this protection was significantly greater than that observed with constitutively expressed proteins. In clinical studies,18 mucosal delivery of an attenuated S.typhimurium carrying an SopE-HIV Gag fusion protein from a lethal plasmid showed some level of Il-2 response to the Gag protein as determined by ELISPOT. However, the response was low compared with that to Salmonella antigen alone. Only one shot of vaccine was used, so it may be possible to enhance the responsiveness. Importantly, the vaccine was well tolerated in the trial subjects, suggesting good safety prospects.
A recombinant S. typhimurium strain which secretes the M. tuberculosis virulence protein ESAT-6 was tested in mice and demonstrated significant protection to subsequent challenge with M. tuberculosis as measured by a reduction in the number of tubercle bacilli in the lungs.
Co-immunisation with a DNA vaccine using ESAT-6 ligated to mammalian expression vectors showed no enhancement, although priming with the S. typhimurium vaccine followed by mixed immunisation showed a synergistic effect with some enhanced protection in the spleens of the M. tuberculosis-infected mice.
4 .Recombinant Adenovirus Vectors
Adenoviruses, the primary cause of the common cold, show some promise as delivery systems for heterologous antigenic proteins. Attenuated human adenoviruses (HAd7) have been used for many years to immunise US military personnel and their families with no significant sideeffects.
 More recently, trials of attenuated human adenovirus 5 (HAd5) in non-human primates have shown protective immunity against Ebola virus. Protective immunity has also been shown against HIV-1 in non-human primates using a HAd5 vector expressing the HIV-1 gag gene. Adenovirus vectors are stable and generally safe vectors with good efficacy. Furthermore, adenovirus vectors have been shown to be reliable and induce strong expression of transgenes. As they are replication-defective, horizontal transmission is very unlikely. The major disadvantages of using HAd vectors in humans is the presence of pre-existing immunity to adenoviruses themselves. This arises from routine exposure to the wild-type viruses. HAd vectors are highly immunogenic in their own right and memory responses coupled with prior exposure to the wild types is likely to be the cause of reduced responses to heterologous proteins encoded in the vectors by neutralising antibodies to the vector itself. There may also be destruction of transduced cells expressing vector antigens. This type of memory response is the probable reason for the recent withdrawal from clinical trials of an adenovirus vector vaccine against HIV-1.25 Different approaches have been undertaken to bypass this problem, such as prior boosting with the recombinant DNA, microencapsulation of the vector and the use of rarer types of adenoviruses. A further strategy to overcome the preimmune interference is the use of non-human adenoviruses as vectors in humans. Pre-existing antibodies to bovine
adenovirus 3 (BAd3) are not prevalent in the human population. Replication-defective BAd3 vectors have been shown to express heterologous proteins in various human cell types in vitro. The use of non-human adenoviruses shows promise in the development of new vector vaccine delivery systems in humans.

5.Recombinant Vaccinia Vectors
Vaccinia has been used in humans since the pioneering work of Edward Jenner at the end of the 18th century. As the prototype vaccine, it is perhaps unsurprising that recombinant vaccinia vectors expressing a multitude of genes from across the microbial world have been described.
 However, vaccinia is not recommended for use in individuals with compromised immune systems. In view of the ever-increasing population of HIV-positive individuals and ageing populations coupled with the immunogenicity of the vaccine and its ability to replicate in humans, the use of vaccinia as a vector vaccine in humans is limited without significant attenuation as seen in the modified vaccinia virus Ankara (MVA). MVA has been demonstrated to be safe in immune-suppressed non-human primates, suggesting a possible future role in vaccine development.
 One alternative is to use other members of the pox family of viruses such as the avipoxviruses. These are replication-deficient in mammalian cells and offer some possibility for use in humans.

6. DNA Vaccines
The basis of this concept is the introduction of plasmid DNA encoding antigenic transgenes into the host cells in which the transgenes are expressed. In practice, although they do elicit specific immune responses, such vaccines have shown low immunogenicity, probably through reduced ability of the proteins to enter antigen processing pathways or loss of the plasmid prior to sufficient concentrations of protein being expressed. Immunogenicity can be augmented by including cytokine genes in the plasmid. Another approach is to target the vaccine to the antigen processing pathways using a gene encoding a targeting protein for the processing pathways. This has been demonstrated using the gene encoding the lysosome-associated membrane protein (LAMP). The use of an LAMP/gag chimera has been shown to induce long-term immunological memory in mice. Furthermore, a single dose of the plasmid was sufficient to induce a secondary CD8+ T cell response without CD4+ T-helper cell assistance. The inclusion of genes encoding immunomodulatory molecules, in particular cytokines, would not just enhance the immunogencity of the vaccine but also allow manipulation of the immune response by altering the T-helper cell polarisation into the subset, i.e. TH₁ or TH₂ appropriate to the pathogen. Additionally, the inclusion of other molecules that influence the maturation of dendritic cells or molecules, such as chemokines that promote recruitment of other antigen-presenting cells to the immunisation site, open up possibilities not available in more conventional vaccine preparations. Such considerations may also apply to recombinant vector vaccines.