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This paper describes an international collaboration to carry out studies that contributed to the understanding of pathogenesis, diagnosis, treatment, and prevention of several diseases of public health importance for Thailand and the United States. In Kamphaeng Phet Province, Thailand, febrile syndromes, including encephalitis, hepatitis, hemorrhagic fever, and influenza-like illnesses, occurred commonly and were clinically diagnosed, but the etiology was rarely confirmed. Since 1982, the Kamphaeng Phet Provincial Hospital, the Thai Ministry of Public Health, and the US Army Component of the Armed Forces Research Institute of Medical Sciences, along with vaccine manufacturers and universities, have collaborated on studies that evaluated and capitalized on improved diagnostic capabilities for infections caused by Japanese encephalitis, hepatitis A, dengue, and influenza viruses. The collaboration clarified clinical and epidemiological features of these infections and, in large clinical trials, demonstrated that vaccines against Japanese encephalitis and hepatitis A viruses were over 90% efficacious, supporting licensure of both vaccines. With the introduction of Japanese encephalitis vaccines in Thailand's Expanded Program on Immunization, reported encephalitis rates dropped substantially. Similarly, in the US, particularly in the military populations, rates of hepatitis A disease have dropped with the use of hepatitis A vaccine. Studies of the pathogenesis of dengue infections have increased understanding of the role of cellular immunity in responding to these infections, and epidemiological studies have prepared the province for studies of dengue vaccines. Approximately 80 publications resulted from this collaboration. Studies conducted in Kamphaeng Phet provided experience that contributed to clinical trials of hepatitis E and HIV vaccines, conducted elsewhere. To provide a base for continuing studies, The Kamphaeng Phet-AFRIMS Virology Research Unit (KAVRU) was established. This paper reviews the origins of the collaboration and the scientific observations made between 1982 and 2012.

Coccidioides immitis is endemic in the soil of the desert Southwest. It causes a respiratory infection that is usually mild, but can last months and may disseminate beyond the lung. Disseminated infections can be fatal or require life-long therapy. Development of an effective vaccine may be a successful method of preventing serious disease. In this paper, we show that immunostimulatory-oligodeoxynucleotides (ISS-ODN) are an effective adjuvant for a recombinant coccidioidal protein known as antigen 2/proline rich antigen. Protective immunity induced by this ISS-ODN-based vaccine requires IL-12, interferon-gamma and MHC Class II-restricted T-cells. Cytotoxic CD8 T-cells are not required. This study elucidates the mechanisms needed to elicit successful immunity against coccidioidomycosis, and holds promise for development of an effective coccidioidal vaccine against coccidioidomycosis.

Effective vaccination-based control of intracellular pathogens or parasites and various tumours is dependent upon induction of cytotoxic lymphocytes and other mechanisms of cellular immunity. Such responses are usually described as being antagonistic to an antibody-based immune response. This paper elaborates on previous studies that have demonstrated that conjugation of a fusion protein (FP, incorporating copies of the variable number of tandem repeat sequence of human mucin-1 (MUC1)) to oxidized mannan results in a significant shift from a type-2 response towards a type-1 response. This response induces complete protection upon challenge of immunized mice with MUC1 expressing tumour cells. This report details experiments in which the balance between type-1 and type-2 anti-MUC1 responses is manipulated by altering the dose of mannan-FP (M-FP) delivered. It is also shown that type-1 and type-2 responses may be induced simultaneously by administration of both forms of the antigen (FP/M-FP). Further, when a type-2 response is induced after FP immunization, a type-1 response can also be established by subsequent immunization with M-FP without adversely affecting the initial response. The converse also applies when M-FP is used for the initial immunizations, followed by FP administration. Delivery of interleukin-1 beta as a cytokine adjuvant with M-FP immunizations also enhanced antibody responses to levels fourfold that induced by M-FP alone without adversely affecting the cytotoxic activity induced by M-FP immunization. Contrary to the type-1/type-2 paradigm, cellular and antibody responses to MUC1 were not antagonistic. These results have important implications for the development of vaccination strategies against pathogens for which both the cellular and humoral compartments of the immune response contribute to protection.

This paper describes a field trial involving 13 calves raised on a farm known to be endemic for Trichophyton verrucosum infection. Seven calves were immunized by subcutaneous inoculations with a crude ribosomal fraction (CRF) of T. verrucosum suspended in aluminium hydroxide as adjuvant. Six animals were sham-immunized with buffer suspended in the adjuvant and served as controls. Two injections were given: one at the age of 2 weeks and a second 2 weeks later. CRF was prepared from T. verrucosum cultures grown in a vitamin-enriched liquid medium. The fungal mat was disrupted mechanically and CRF was separated from the cell-free extract by differential ultracentrifugation. The CRF was characterized biochemically (RNA and protein content) and physically (electron microscopy). The protection induced by vaccination was assessed through a clinical follow-up of the animals to determine the presence and duration of dermatophyte infection following their exposure to a T. verrucosum -contaminated environment. Vaccination with the CRF resulted in a statistically significant decrease in the period during which clinical signs of dermatophytosis were observed (from a mean of 9.5 weeks to 3.7 weeks). To assess the humoral response, serum samples were taken before each vaccination and 2 weeks after the second inoculation. For the cell-mediated immune (CMI) response assessment, whole-blood samples were taken 2 weeks after the second vaccination. The presence of anti- T. verrucosum antibodies in the sera was determined by ELISA and the CMI response was assessed in vitro by the lymphocyte stimulation test. The results indicate that both the cell-mediated and humoral components of the immune system were stimulated by vaccination. The overall results of the present research indicate that the T. verrucosum CRF shows potential as a basis for the development of a vaccine against bovine tricophytosis.

Using conalbumin as a model antigen, we demonstrate in this paper that liposomal antigen differently influences the activation of the immune system depending on the mode of association of the antigen with the liposomal vehicle whether it is by encapsulation or surface linkage. This conclusion is based on in vivo data showing that encapsulated antigen induces a short-lasting response dominated by IgG1 production while surface-linked antigen has a longer-lasting effect characterized by increased production of IgM, IgG2a, IgG3 as well as of IgG1. The in vivo data were complemented by in vitro proliferation studies carried out on spleen cells or macrophage-depleted spleen cells obtained from mice sensitized in vivo and rechallenged in vitro on day 4 following sensitization. Rechallenge was carried out in the absence or presence of anti-IL1. The data indicate that, in contrast to what is generally observed in vivo, liposomes alone potentiate spleen cell proliferative response in a dose-dependent manner. This liposomal effect totally obscures the antigen-specific proliferation that was expected with encapsulated antigen without masking that induced by surface-linked antigen. The mode of antigen association also influences anti-cytokine responsiveness as demonstrated by the insensitivity of the surface-linked antigen response to the presence of anti-IL1 and the significantly decreased response observed with encapsulated antigen under identical conditions. The response to both liposomal antigenic formulations was almost totally abolished in adherent cell-depleted cultures. The overall results therefore suggest that encapsulated and surface-linked antigens activated different immune pathways.

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950's, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of beta-chemokines. We describe that the beta-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

The initial reports, more than a decade and a half ago. that genetically engineered DNA can be directly delivered into animals resulting in the induction of immune responses served to create the field of DNA vaccines. Since that time, there has been a great deal of progress in our understanding of the basic biology of this important platform. Interest in the platform for clinical applications has been strong. Many infectious disease and cancer targets have been studied pre-clinically and have progressed into clinical trials. Interestingly, DNA vectors make up about 19% (246 out of 1311 total trials) of all gene therapy vector platforms studied in phase I to III trials in 2007. These data support the overall preclinical success and phase I safety of the platform, When we look at all open enrolling DNA vaccine/therapy trials, 68% are trials testing DNA therapies for cancer, while 11% are for treatment of cardiovascular diseases. Surprisingly, while some of the more attention getting DNA trials are in the area of infectious diseases, infectious disease trials make up roughly just 5% of the current open human clinical trials testing this platform. These include vaccines to treat or prevent HIV-1, cytomegalovirus, and Influenza, malaria and HCV among others. The majority of open clinical trials for the DNA platform are being tested in the United States (68%) with the United Kingdom (15%) and Germany following (8%) as additional important areas for clinical evaluation. It is clear that the clinical acceptance of this platform is growing rapidly as regulatory agency’s become more and more comfortable with the enviable safety profile of this platform. In theory, this conceptually safe vaccine approach represents an important non-live platform for the induction of both cellular and humoral immune responses. The DNA vaccine platform is unique in that its straight forward use of molecular design seems particularly amenable to scale up and manufacturing. Furthermore, the ability to drive cellular immune responses in a non live platform is an extremely compelling aspect of DNA vaccine technology, suggesting that a critical shift has occurred in vaccine development. Before these studies, live infection was believed to be required to induce strong cellular immunity and in particular, the elusive induction of killer Cytotoxic T Cells (CTL). The use of the DNA approach promised to overcome the safety concerns associated with live vaccines. Furthermore through manipulation of the plasmid encoded antigens, a DNA vaccine could obviate any potential side effects of vaccination, yet in theory produce a similar immune response resulting in protective immunity. Unfortunately, this early optimism did not last. Based on immune responses observed in the first clinical trials of DNA vaccines in the areas of HIV, Influenza, Malaria, and Cancer among others, concerns were raised about the low levels of immunogenicity induced by this platform in humans. Furthermore, the immune responses in large animals and important primate model systems were much lower than expected based on the responses observed to be induced in small animal models. For more than the last 7 years DNA vaccines became relegated to a supporting role in vaccine development, as they were primarily being examined clinically for their ability to prime the immune response in prime boost vaccine trials. Even more worrisome was that in this more limited arena, concerns about the immunogenicity of the platform in the clinic and in larger animals lingered. It was under this cloud of controversy that last summer leading researchers in the DNA vaccine field gathered in Malaga, Spain to debate and discuss the latest results in advancing the DNA vaccine platform. The mood at the opening of the conference was sober. In the past decade and a half, the DNA vaccine concept has been tested and applied against a variety of pathogens and tumor antigens. Many of the vaccines developed in through these studies are among the most creative ever designed for any vaccine application. There was a great deal of interest in improving DNA vaccine potency in a safe manner. Many technical improvements were discussed at the meeting including strategies for gene optimization, improved RNA structural design, novel formulations, immune adjuvants and improved delivery devices, regulatory T cell manipulations and cytokine modulations among others. Combination studies were also described. Consensus antigens based vaccine strategies were presented that suggest that such designs may allow specific vaccine prototypes to drive cross reactive immune responses to changing pathogens to some extent. Studies were presented using novel delivery devices to improve transfection efficiencies including the Gene Gun and electroporation technologies. Novel formulations or adjuvants were also presented including results using vaxfectin, or with cytokine adjuvants showing improved effects on the resulting immune responses in different models of host induced immunity. As the conference progressed the results presented using improved DNA vaccine combination approaches that were able to drive improved levels of cellular immune responses in many important animal model systems started to lift the mood of the participants. In human studies, prime boost approaches appeared to generate improved and consistent levels of IFNγ responses than had previously been reported with other platforms. This data suggested that through continued modifications of the technology, further improved responses can be generated. Exciting data was generated using the electroporation platforms where T cell responses in non human primates were driven to levels previously not observed with other DNA vaccine approachs. In some of these studies the levels of T cell immunity induced were similar to or superior to those induced by live viral vectors. All of these results were very encouraging for investigators in the field. The safety record of DNA vaccines in humans remained with out blemish, which was also encouraging. However, the field has been craving a true clinical success. Unfortunately, for this meeting this year a true clinical success was still elusive. However, it was progress in the veterinary market that served to further improve the energy and optimism of the conference. It was reported at the meeting that four different veterinary use plasmid encoded products were licensed over the past few years. One approval was for a vaccine for protecting Salmon against Hematopoietic Necrosis Virus. This interesting vaccine is directly injected into hatchlings and results in the generation of protective immunity in farm-raised fish. A second impressive story was the report of a DNA vaccine that targets West Nile Virus infection by driving vaccine-induced anti-WNV antibodies and protects horses from this emerging pathogen. This interesting result was in the large animal arena which is a surprising but encouraging story. An exciting development was the presentation of a DNA vaccine used to treat canine melanoma that was conditionally licensed and shows extreme promise to alter the outcome of this fatal disease in dogs. This exciting development represents the first license for any immune therapy approach in this arena. A fourth product was described that delivers plasmid encoding GHRH to treat fetal loss in pigs using electroporation as its delivery platform for a plasmid product was also presented, shortly after the conference this product was also approved for animal health use. This represents the first such delivery platform success. These commercial successes for several groups including Merial, Novartis, Vical, Wyeth and VGX, have helped to demonstrate the commercial potential of this technology. These developments together with the clinical responses, helped to energize the conference attendees. This issue brings together many important research studies that were presented at the meeting. The many articles from diverse investigators from around the globe serve to underscore the important progress in this area and the continued vitality of the field. Advances in basic research, formulations, manufacturing advances, plasmid design, delivery schemes, clinical investigation into human and animal health are reported. The Malaga meeting appears to have been a true turning point for the field, both commercially and clinically. As this collection of papers underscores, field appears to be more determined, more focused and more productive than ever. The positive energy gathered from this meeting is providing significant momentum for further growth and collaboration with in the field. The next few years will be critical for DNA vaccine technology and the collaborative nature of the field will be central to the field’s success. We are actively looking forward to the next meeting and seeing the continued expansion and progress in the DNA vaccine arena and crossing the next threshold of progress together.