Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines.

BACKGROUND: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. AIMS: In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? RESULTS: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-ß4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. CONCLUSION: Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

A novel hepatitis B vaccine containing Advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing.

Although current HBV vaccines have an outstanding record of safety and efficacy, reduced immunogenicity is a problem in those of older age, or having renal impairment or diabetes mellitus. In this study, we tested the ability of Advax™ adjuvant, a novel polysaccharide adjuvant based on delta inulin, to enhance the immunogenicity of hepatitis B surface antigen (HBs) in mice and guinea pigs by comparison to the traditional alum adjuvant. Advax™ provided antigen-sparing, significantly enhanced both anti-HBs antibody titers, and anti-HBs CD4 and CD8 T-cells, with increases in Th1, Th2 and Th17 cytokine responses. Unlike alum, the adjuvant effect of Advax™ was seen even when injected 24h before the HBs antigen. Advax™ adjuvant similarly enhanced humoral and cellular immune responses in guinea pigs to a third generation preS-HBs antigen. Advax™ adjuvant when combined with HBs antigen could provide enhanced protection over current generation HBV vaccines for immunization of low responder populations.

Pushing the frontiers of T-cell vaccines: accurate measurement of human T-cell responses.

There is a need for novel approaches to tackle major vaccine challenges such as malaria, tuberculosis and HIV, among others. Success will require vaccines able to induce a cytotoxic T-cell response--a deficiency of most current vaccine approaches. The successful development of T-cell vaccines faces many hurdles, not least being the lack of consensus on a standardized T-cell assay format able to be used as a correlate of vaccine efficacy. Hence, there remains a need for reproducible measures of T-cell immunity proven in human clinical trials to correlate with vaccine protection. The T-cell equivalent of a neutralizing antibody assay would greatly accelerate the development and commercialization of T-cell vaccines. Recent advances have seen a plethora of new T-cell assays become available, including some like cytometry by time-of-flight with extreme multiparameter T-cell phenotyping capability. However, whether it is historic thymidine-based proliferation assays or sophisticated new cytometry assays, each assay has its relative advantages and disadvantages, and relatively few of these assays have yet to be validated in large-scale human vaccine trials. This review examines the current range of T-cell assays and assesses their suitability for use in human vaccine trials. Should one or more of these assays be accepted as an agreed surrogate of T-cell protection by a regulatory agency, this would significantly accelerate the development of T-cell vaccines.

The future of human DNA vaccines.

DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans.

Advax™, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses.

Advax™ adjuvant is derived from inulin, a natural plant-derived polysaccharide that when crystallized in the delta polymorphic form, becomes immunologically active. This study was performed to assess the ability of Advax™ adjuvant to enhance influenza vaccine immunogenicity and protection. Mice were immunized with influenza vaccine alone or combined with Advax™ adjuvant. Immuno-phenotyping of the anti-influenza response was performed including antibody isotypes, B-cell ELISPOT, CD4 and CD8 T-cell proliferation, influenza-stimulated cytokine secretion, DTH skin tests and challenge with live influenza virus. Advax™ adjuvant increased neutralizing antibody and memory B-cell responses to influenza. It similarly enhanced CD4 and CD8 T-cell proliferation and increased influenza-stimulated IL-2, IFN-γ, IL-5, IL-6, and GM-CSF responses. This translated into enhanced protection against mortality and morbidity in mice. Advax™ adjuvant provided significant antigen dose-sparing compared to influenza antigen alone. Protection could be transferred from mice that had received Advax™-adjuvanted vaccine to naïve mice by immune serum. Enhanced humoral and T-cell responses induced by Advax™-formulated vaccine were sustained 12months post-immunization. Advax™ adjuvant had low reactogenicity and no adverse events were identified. This suggests Advax™ adjuvant could be a useful influenza vaccine adjuvant.

Technologies for enhanced efficacy of DNA vaccines.

Despite many years of research, human DNA vaccines have yet to fulfill their early promise. Over the past 15 years, multiple generations of DNA vaccines have been developed and tested in preclinical models for prophylactic and therapeutic applications in the areas of infectious disease and cancer, but have failed in the clinic. Thus, while DNA vaccines have achieved successful licensure for veterinary applications, their poor immunogenicity in humans when compared with traditional protein-based vaccines has hindered their progress. Many strategies have been attempted to improve DNA vaccine potency including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime-boost strategies. This review summarizes these advances in DNA vaccine technologies and attempts to answer the question of when DNA vaccines might eventually be licensed for human use.

Enhancement of neutralizing humoral response of DNA vaccine against duck hepatitis B virus envelope protein by co-delivery of cytokine genes.

We explored in the duck hepatitis B virus (DHBV) model the impact of duck interferon gamma (Du-IFNgamma) or interleukin 2 (Du-IL2) co-delivery on humoral neutralizing response induced by DNA-based vaccine encoding DHBV preS/S large envelope protein. Co-delivery of either Du-IL2 or Du-IFNgamma encoding plasmids considerably increased the magnitude of anti-preS humoral response. Moreover, co-administration of cytokine genes led to a significant (p<0.001) enhancement of neutralizing anti-DHBV antibody response, which was more pronounced for Du-IFNgamma. Our data suggest that co-delivery of cytokine and envelope protein encoding plasmids will be a valuable approach for the development of a potent therapeutic DNA vaccine against chronic hepatitis B.

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable.

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized [13C]glutamine at high rates and accumulated 13C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of [13C]glucose initially synthesized from [13C]glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72 h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.