Dengue is a mosquito-borne disease of global public health significance that is caused by four serologically and genetically related viruses (DENV-1 to DENV-4). Most human DENV infections are asymptomatic, but clinical cases can range in severity from a relatively mild self-limiting illness to a severe life-threatening disease. Infection with one serotype of DENV results in life-long homotypic immunity but only short term heterotypic protection. There are no licensed vaccines or antivirals for dengue due in part to difficulty in developing small animal models that mimic the systemic disease seen in humans. Consequently, an important advance was the description of models of DENV-2 infection in AG129 mice (deficient in interferon alpha/beta and gamma receptor signaling) that resemble human disease. However, the need for well characterized models of disease due to DENV-1, -3, and -4 still remains. Here we describe a new AG129 mouse model utilizing a non-mouse-adapted Thai human DENV-4 strain 703-4. Following intraperitoneal challenge, animals experience a rapidly progressive lethal infection without developing neurologic clinical signs of disease. High virus titers are seen in multiple visceral tissues including the liver, spleen and large intestine, and the infected animals develop vascular leakage and thrombocytopenia, hallmarks of human dengue. Taken together, our studies demonstrate that this model is an important addition to the field of dengue research particularly in understanding similarities and differences in the pathologic basis of the disease caused by different DENV serotypes and in determining comparative efficacy of putative vaccines and antivirals.

Subunit antigens are attractive candidates for vaccine development as they are safe, cost-effective, and rapidly produced. Nevertheless, subunit antigens often need to be adjuvanted and/or formulated to produce products with acceptable potency and efficacy. Here we describe a simple method for improving the potency and efficacy of a recombinant subunit antigen by its immobilization on nickel-chelating nanolipoprotein particles (NiNLPs). NiNLPs are membrane mimetic nanoparticles that provide a delivery and presentation platform amenable to binding any recombinant subunit immunogens featuring a poly-histidine tag. A His-tagged, soluble truncated form of the West Nile virus (WNV) envelope protein (trE-His) was immobilized on NiNLPs. Single inoculations of the NiNLP-trE-His produced superior anti-WNV immune responses and provided significantly improved protection against a live WNV challenge compared to mice inoculated with trE-His alone. These results have broad implications in vaccine development and optimization, as NiNLP technology is well suited to many types of vaccines, providing a universal platform for enhancing the potency and efficacy of recombinant subunit immunogens.Rational design of vaccines, made possible by an enhanced understanding of the mechanisms that trigger an effective immune response, includes the optimization of immunogen generation, addition of adjuvants, and improvement of delivery. Numerous vaccine formulations have been very successful, but no single technology covers the key requirements of the ideal vaccine, namely impeccable safety, low cost, rapid preparation, and high potency (1). The three former characteristics are found with subunit antigens (i.e. purified protein components from pathogen candidates) (2), though these immunogens suffer from poor potency and thus often are formulated with adjuvants to improved their immunogenicity (3,4). However, adjuvants themselves can have unwanted side-effects, and few are approved for use in humans (4). The development of an adjuvant-free technology to hoeprich2@llnl.gov, peter.mason@novartis.com. ‖ Current address: Department of International Health, Kobe University Graduate School of Health Sciences, Kobe, 654-0142 Japan ⊥ Current Address: Novartis Vaccines and Diagnostics, Cambridge, Massachusetts 02139 Supporting Information Available: Experimental details, Figure S1 showing NiNLP purification, Figure S2 showing AFM characterization of NiNLP, Figure S3 showing AFM analyses of trE-His binding, Figure S4 showing individual serological responses. This material is available free of charge via the Internet at http://pubs.acs.org. Multivalency and shape are likely important determinants of vaccine potency and efficacy since valency may increase affinity and/or aggregate receptors, leading to activation (5), and the immune system has likely evolved to react more strongly to particles of the size of invading microorganisms. Based on these concepts multiple investigators have developed multimeric, particulate antigens as vaccine candidate...

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Dengue is a mosquito-borne disease caused by four related but distinct dengue viruses, DENV-1 to DENV-4. Dengue is endemic in most tropical countries, and over a third of the world's population is at risk of being infected. Although the global burden is high, no vaccine or antiviral is licensed to combat this disease. An obstacle complicating dengue research is the lack of animal challenge models that mimic human disease. Advances in immunocompromised murine infection models resulted in development of lethal DENV-2, DENV-3 and DENV-4 models in AG129 mice, which are deficient in both the IFN-a/b receptor (IFN-a/bR) and the IFN-c receptor (IFN-cR). These models mimic features of dengue disease in humans. Here, we characterized lethal infection of AG129 mice by DENV-4 strain TVP-376 and found that AG129 mice developed clinical signs of illness and high viral loads in multiple tissues and succumbed 5 days after infection. Moreover, the splenic and hepatic histopathology of TVP-376-infected mice demonstrated the presence of cell activation and destruction of tissue architecture. Furthermore, infected mice had heightened levels of circulating cytokines. Comparison of the virulence phenotypes of DENV-4 strain TVP-376 and DENV-2 strain D2S10 revealed that TVP-376-induced mortality occurred in the absence of both IFN-a/bR and IFN-cR signalling, but not with intact signalling from the IFN-cR, whereas D2S10 required the absence of IFN-a/bR signalling only, indicating that it is more virulent than TVP-376. In conclusion, TVP-376 is lethal in AG129 mice, and this model provides a useful platform to investigate vaccine candidates and antivirals against DENV-4.

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