Extensive genomic recoding by codon-pair deoptimization selective for mammals is a flexible tool to generate attenuated vaccine candidates for dengue virus 2.

The four serotypes of dengue virus (DENV) are the leading etiologic agent of disease caused by arthropod-borne viruses (arboviruses) in the world, with billions at risk of DENV infection spread by infected mosquitoes. DENV causes illness ranging from dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV proliferates well in two different host systems, an invertebrate mosquito vector and vertebrate primate host, which have a distinct difference in their preference of codon pairs (CP) for translation (different "codon pair bias"). Consequently, arboviruses must delicately balance the use of codon pairs between mammals and arthropods, which presents an Achilles' heel that we have exploited by specifically shifting the codon pair preference in the E and NS3 ORFs away from mammals while keeping the CPB favorable for mosquito ORFs. Here we report that recoding of the ORFs has led to variants that were over-attenuated in rhesus macaques although induction of protective antibodies in animals vaccinated with the smallest recoded ORF (E) was observed. The flexibility of our synthetic vaccine design (by decreasing the number of unfavorable CPs in the E ORF), allowed us to construct two new vaccine candidates (EhminA and EhminB) with intermediate attenuation in cell culture and neonatal mice, a result demonstrating proof of concept. New DENV vaccine candidates are being developed based on selective attenuation by dramatic recoding, with flexibility in balancing the attenuation and immunogenicity by marrying rational design and empirical modification.

Dengue: a growing threat requiring vaccine development for disease prevention.

Dengue disease is the most prevalent mosquito-borne viral infection in humans. At least one half of the global population is estimated at risk of infection and an estimated 390 million people are infected each year. Over the past few years, dengue burden continued to increase, mainly impacting developing countries. Alarming changes in dengue epidemiology were observed highlighting a spread from tropical to subtropical regions as well as urban to rural areas. An increase in the co-infections with the four serotypes has also been noticed, involving a shift in the targeted population from pediatric to adult. Facing these global changes, authorities will have to reinforce preventive actions and adapt healthcare management. New prophylactic strategies are urgently needed to prevent severe forms of dengue disease. The lack of specific antiviral therapies available turns vaccine development into a socio-economic challenge. In this review, we propose an update on the dengue global trends and different vaccine strategies in development. A particular attention will be paid to up-to-date information on dengue transmission and the protective efficacy of newly commercialized tetravalent dengue vaccine Dengvaxia®, as well as the most advanced candidate vaccines in clinical development.

The Good, the Bad, and the Shocking: The Multiple Roles of Dengue Virus Nonstructural Protein 1 in Protection and Pathogenesis.

Dengue virus (DENV) is the most prevalent medically important mosquito-borne virus in the world. Upon DENV infection of a host cell, DENV nonstructural protein 1 (NS1) can be found intracellularly as a monomer, associated with the cell surface as a dimer, and secreted as a hexamer into the bloodstream. NS1 plays a variety of roles in the viral life cycle, particularly in RNA replication and immune evasion of the complement pathway. Over the past several years, key roles for NS1 in the pathogenesis of severe dengue disease have emerged, including direct action of the protein on the vascular endothelium and triggering release of vasoactive cytokines from immune cells, both of which result in endothelial hyperpermeability and vascular leak. Importantly, the adaptive immune response generates a robust response against NS1, and its potential contribution to dengue vaccines is also discussed.

Prevention and Control Strategies to Counter Dengue Virus Infection.

Dengue is currently the highest and rapidly spreading vector-borne viral disease, which can lead to mortality in its severe form. The globally endemic dengue poses as a public health and economic challenge that has been attempted to suppress though application of various prevention and control techniques. Therefore, broad spectrum techniques, that are efficient, cost-effective, and environmentally sustainable, are proposed and practiced in dengue-endemic regions. The development of vaccines and immunotherapies have introduced a new dimension for effective dengue control and prevention. Thus, the present study focuses on the preventive and control strategies that are currently employed to counter dengue. While traditional control strategies bring temporary sustainability alone, implementation of novel biotechnological interventions, such as sterile insect technique, paratransgenesis, and production of genetically modified vectors, has improved the efficacy of the traditional strategies. Although a large-scale vector control strategy can be limited, innovative vaccine candidates have provided evidence for promising dengue prevention measures. The use of tetravalent dengue vaccine (CYD-TDV) has been the most effective so far in treating dengue infections. Nonetheless, challenges and limitation hinder the progress of developing integrated intervention methods and vaccines; while the improvement in the latest techniques and vaccine formulation continues, one can hope for a future without the threat of dengue virus.

Achieving safe, effective, and durable Zika virus vaccines: lessons from dengue.

Newly proposed candidate Zika virus vaccines might or might not succeed in raising safe, effective, and durable protection against human Zika virus infections or syndromes. Analyses of a clinically tested and licensed dengue vaccine that failed to protect seronegative individuals from breakthrough or enhanced dengue infections suggest that poor T-cell immunity might have contributed to protection failure. Because of the similarity of Zika and dengue viruses, an analogous unwanted outcome might occur with some Zika virus vaccine designs. A successful Zika virus vaccine requires challenge experiments that are done at long intervals after immunisation and that identify protection as the absence of viraemia and the absence of an anamnestic antibody response. T-cell immunity might be an essential component of safe, efficacious, and durable Zika virus vaccines.

Paradoxical role of antibodies in dengue virus infections: considerations for prophylactic vaccine development.

Highly effective prophylactic vaccines for flaviviruses including yellow fever virus, tick-borne encephalitis virus and Japanese encephalitis virus are currently in use. However, the development of a dengue virus (DENV) vaccine has been hampered by the requirement of simultaneous protection against four distinct serotypes and the threat that DENV-specific antibodies might either mediate neutralization or, on the contrary, exacerbate disease through the phenomenon of antibody-dependent enhancement (ADE) of infection. Therefore, understanding the cellular, biochemical and molecular basis of antibody-mediated neutralization and ADE are fundamental for the development of a safe DENV vaccine. Here we summarize current structural and mechanistic knowledge underlying these phenomena. We also review recent results demonstrating that the humoral immune response triggered during natural DENV infection is able to generate neutralizing antibodies binding complex quaternary epitopes only present on the surface of intact virions.

T cell immunity to dengue virus and implications for vaccine design.

Dengue virus infections are increasing at an alarming rate in many tropical and subtropical countries and represent, in some of these areas, a leading cause of hospitalization and death among children. The lack of a clear definition of the correlates of protection from severe dengue disease represents a major hurdle for vaccine development. In particular, the role of T lymphocytes during dengue infection remains unclear and there is evidence suggesting that these cells may be important for both protective immunity and/or immunopathology. In this review we discuss the findings that support a protective role of T cells versus those supporting their involvement in pathogenesis. A better understanding of T cell immunity is urgently needed for the development of safe and efficacious vaccines.

Current issues in the economics of vaccination against dengue.

Dengue is a major public health concern in tropical and subtropical areas of the world. The prospects for dengue prevention have recently improved with the results of efficacy trials of a tetravalent dengue vaccine. Although partially effective, once licensed, its introduction can be a public health priority in heavily affected countries because of the perceived public health importance of dengue. This review explores the most immediate economic considerations of introducing a new dengue vaccine and evaluates the published economic analyses of dengue vaccination. Findings indicate that the current economic evidence base is of limited utility to support country-level decisions on dengue vaccine introduction. There are a handful of published cost-effectiveness studies and no country-specific costing studies to project the full resource requirements of dengue vaccine introduction. Country-level analytical expertise in economic analyses, another gap identified, needs to be strengthened to facilitate evidence-based decision-making on dengue vaccine introduction in endemic countries.

Key concepts, strategies, and challenges in dengue vaccine development: an opportunity for sub-unit candidates?

Despite 70 years of research that has intensified in the past decade, a safe and efficacious dengue vaccine has yet to be available. In addition to the expected challenges such as identifying immune correlates of protection, the dengue vaccine field has faced additional hurdles including the necessity to design a tetravalent formulation and the risk of antibody-mediated disease enhancement. Nevertheless, tetravalent live attenuated vaccine candidates have reached efficacy trials and demonstrated some benefit, despite imbalanced immunogenicity and incomplete protection against the four serotypes. Meanwhile, the development of sub-unit dengue vaccines has gained momentum. As the target of most of the neutralizing antibodies so far reported, the virus envelope E protein has been the focus of much effort and represents the leading dengue sub-unit vaccine candidate. However, its notorious poor immunogenicity has prompted the development of innovative approaches to make E-derived constructs part of the second generation dengue vaccines portfolio.

Points for Consideration for dengue vaccine introduction - recommendations by the Dengue Vaccine Initiative.

Dengue is a public health problem in the tropics and subtropics. There are several vaccine candidates in clinical development. However, there may be gaps in the new vaccine introduction after vaccine licensure before it becomes available in developing countries. In anticipation of the first dengue vaccine candidate to be licensed, Dengue Vaccine Initiative (DVI) and, its predecessor, Pediatric Dengue Vaccine Initiative (PDVI) have been working on points for consideration to accelerate evidence-based dengue vaccine introduction, once a vaccine becomes available. In this paper, we review the history of PDVI and its successor, the DVI, and elaborate on the points of consideration for dengue vaccine introduction.

Tackling dengue fever: Current status and challenges.

According to recent statistics, 96 million apparent dengue infections were estimated worldwide in 2010. This figure is by far greater than the WHO prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas.This article aims at bringing to light the current epidemiological and clinical status of the dengue fever. The relationship between genetic mutations, single nucleotide polymorphism (SNP) and the pathophysiology of disease progression will be put into perspective. It will also highlight the recent advances in dengue vaccine development.Thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. However, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk.

Development of a recombinant, chimeric tetravalent dengue vaccine candidate.

Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

A role for vector control in dengue vaccine programs.

Development and deployment of a successful dengue virus (DENV) vaccine has confounded research and pharmaceutical entities owing to the complex nature of DENV immunity and concerns over exacerbating the risk of DENV hemorrhagic fever (DHF) as a consequence of vaccination. Thus, consensus is growing that a combination of mitigation strategies will be needed for DENV to be successfully controlled, likely involving some form of vector control to enhance a vaccine program. We present here a deterministic compartmental model to illustrate that vector control may enhance vaccination campaigns with imperfect coverage and efficacy. Though we recognize the costs and challenges associated with continuous control programs, simultaneous application of vector control methods coincident with vaccine roll out can have a positive effect by further reducing the number of human cases. The success of such an integrative strategy is predicated on closing gaps in our understanding of the DENV transmission cycle in hyperedemic locations.

Mouse models of dengue virus infection for vaccine testing.

Dengue is a mosquito-borne disease caused by four serologically and genetically related viruses termed DENV-1 to DENV-4. With an annual global burden of approximately 390 million infections occurring in the tropics and subtropics worldwide, an effective vaccine to combat dengue is urgently needed. Historically, a major impediment to dengue research has been development of a suitable small animal infection model that mimics the features of human illness in the absence of neurologic disease that was the hallmark of earlier mouse models. Recent advances in immunocompromised murine infection models have resulted in development of lethal DENV-2, DENV-3 and DENV-4 models in AG129 mice that are deficient in both the interferon-α/ß receptor (IFN-α/ß R) and the interferon-γ receptor (IFN-γR). These models mimic many hallmark features of dengue disease in humans, such as viremia, thrombocytopenia, vascular leakage, and cytokine storm. Importantly AG129 mice develop lethal, acute, disseminated infection with systemic viral loads, which is characteristic of typical dengue illness. Infected AG129 mice generate an antibody response to DENV, and antibody-dependent enhancement (ADE) models have been established by both passive and maternal transfer of DENV-immune sera. Several steps have been taken to refine DENV mouse models. Viruses generated by peripheral in vivo passages incur substitutions that provide a virulent phenotype using smaller inocula. Because IFN signaling has a major role in immunity to DENV, mice that generate a cellular immune response are desired, but striking the balance between susceptibility to DENV and intact immunity is complicated. Great strides have been made using single-deficient IFN-α/ßR mice for DENV-2 infection, and conditional knockdowns may offer additional approaches to provide a panoramic view that includes viral virulence and host immunity. Ultimately, the DENV AG129 mouse models result in reproducible lethality and offer multiple disease parameters to evaluate protection by candidate vaccines.

Development of the Sanofi Pasteur tetravalent dengue vaccine: One more step forward.

Sanofi Pasteur has developed a recombinant, live-attenuated, tetravalent dengue vaccine (CYD-TDV) that is in late-stage development. The present review summarizes the different steps in the development of this dengue vaccine, with a particular focus on the clinical data from three efficacy trials, which includes one proof-of-concept phase IIb (NCT00842530) and two pivotal phase III efficacy trials (NCT01373281 and NCT01374516). Earlier studies showed that the CYD-TDV candidate had a satisfactory safety profile and was immunogenic across the four vaccine serotypes in both in vitro and in vivo preclinical tests, as well as in initial phase I to phase II clinical trials in both flavivirus-naïve and seropositive individuals. Data from the 25 months (after the first injection) active phase of the two pivotal phase III efficacy studies shows that CYD-TDV (administered at 0, 6, and 12 months) is efficacious against virologically-confirmed disease (primary endpoint) and has a good safety profile. Secondary analyses also showed efficacy against all four dengue serotypes and protection against severe disease and hospitalization. The end of the active phases in these studies completes more than a decade of development of CYD-TDV, but considerable activities and efforts remain to address outstanding scientific, clinical, and immunological questions, while preparing for the introduction and use of CYD-TDV. Additional safety observations were recently reported from the first complete year of hospital phase longer term surveillance for two phase 3 studies and the first and second completed years for one phase 2b study, demonstrating the optimal age for intervention from 9 years. Dengue is a complex disease, and both short-term and long-term safety and efficacy will continue to be addressed by ongoing long-term follow-up and future post-licensure studies.

Clinical evaluation strategies for a live attenuated tetravalent dengue vaccine.

Butantan Institute is a public Brazilian biomedical research-manufacturer center affiliated to the São Paulo State Secretary of Health. Currently, Butantan is one of the main public producers of vaccines, antivenoms, and antitoxins in Latin America. The partnership between Butantan and the National Institutes of Health (NIH) of the United Sates has been one of the longest and most successful partnerships in the development and manufacturing of new vaccines. Recently, Butantan Institute has developed and manufactured a lyophilized tetravalent live attenuated dengue vaccine with the four dengue viruses attenuated and licensed from the Laboratory of Infectious Diseases at The National Institutes of Allergy and Infectious Diseases (LID/NIAID/NIH). The objective of this paper is to describe the clinical evaluation strategies of a live attenuated tetravalent dengue vaccine (Butantan-DV) developed and manufactured by Butantan Institute. These clinical strategies will be used to evaluate the Butantan-DV Phase III trial to support the Butantan-DV licensure for protection against any symptomatic dengue caused by any serotype in people aged 2 to 59 years.

Preclinical and clinical development of a dengue recombinant subunit vaccine.

This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

Nucleic acid (DNA) immunization as a platform for dengue vaccine development.

Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.