Dengue virus vaccine development.

CHAPTER SEVEN

Dengue Virus Vaccine Development Lauren E. Yauch, Sujan Shresta1 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5.

Virology and Epidemiology of DENV Infection Adaptive Immune Response to DENV Dengue Vaccine Objectives and Challenges Animal Models for Testing Dengue Vaccine Candidates Dengue Vaccine Approaches 5.1 Recombinant subunit protein vaccines/subviral particles 6. DNA Vaccines 7. Viral Vectored Vaccines 7.1 Vaccinia 7.2 Adenovirus vectors 7.3 Alphavirus replicon particles 8. Inactivated Whole Virus 9. Live Attenuated 9.1 University of Hawaii/WRAIR 9.2 Mahidol University 9.3 CDC/Inviragen 9.4 NIAID/NIH 9.5 DENV Chimeras 9.6 Acambis/Sanofi Pasteur (ChimeriVax) 10. Moving Forward References

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Abstract Dengue virus (DENV) is a significant cause of morbidity and mortality in tropical and subtropical regions, causing hundreds of millions of infections each year. Infections range from asymptomatic to a self-limited febrile illness, dengue fever (DF), to the life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The expanding of the habitat of DENV-transmitting mosquitoes has resulted in dramatic increases in the number of cases over the past 50 years, and recent outbreaks have occurred in the United States. Developing a dengue vaccine is a global health priority. DENV vaccine development is challenging due to the existence of four serotypes of the

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virus (DENV1–4), which a vaccine must protect against. Additionally, the adaptive immune response to DENV may be both protective and pathogenic upon subsequent infection, and the precise features of protective versus pathogenic immune responses to DENV are unknown, complicating vaccine development. Numerous vaccine candidates, including live attenuated, inactivated, recombinant subunit, DNA, and viral vectored vaccines, are in various stages of clinical development, from preclinical to phase 3. This review will discuss the adaptive immune response to DENV, dengue vaccine challenges, animal models used to test dengue vaccine candidates, and historical and current dengue vaccine approaches.

1. VIROLOGY AND EPIDEMIOLOGY OF DENV INFECTION Dengue virus (DENV) is the etiologic agent of dengue fever (DF), the most prevalent arthropod-borne viral illness in humans. DENV belongs to the Flaviviridae family and is related yellow fever virus (YFV), hepatitis C virus, West Nile virus, Japanese encephalitis virus (JEV), and St. Louis encephalitis virus. DENV is an enveloped virus with a single-stranded, positive-sense RNA genome. The DENV genome is 10.7 kb and contains a 50 methyl guanosine cap, 50 untranslated region (UTR), single open reading frame, and a 30 UTR (Clyde, Kyle, & Harris, 2006). The RNA genome is translated as a single polyprotein that is then cleaved into three structural proteins (capsid (C), premembrane (prM), and envelope (E)) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by both viral and host proteases. prM likely functions as a chaperone for E during virion assembly (Mukhopadhyay, Kuhn, & Rossmann, 2005). prM is cleaved by furin to M in the trans-Golgi resulting in the formation of mature virions containing E and M (Murphy & Whitehead, 2011). However, this cleavage is incomplete (especially in mosquito cells), so many immature virions that contain prM are released (van der Schaar et al., 2007). The E protein is structurally conserved among flaviviruses and consists of three domains (EDI, EDII, and EDIII) (Kuhn et al., 2002; Rey, Heinz, Mandl, Kunz, & Harrison, 1995). The E protein interacts with a cellular receptor(s) and viral uptake occurs via receptor-mediated endocytosis followed by fusion of the viral and endosomal membranes and release of the nucleocapsid into the cytoplasm (Heinz et al., 1994; Mukhopadhyay et al., 2005). Translation and replication of the viral genome occurs in the cytoplasm in association with intracellular membranous structures. Virus assembly takes place at intracellular membranes, and viral particles pass through the Golgi and are exocytosed via secretory vesicles (Heinz et al., 1994).

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The four serotypes of DENV (DENV1–4) are transmitted to humans primarily by the mosquitoes Aedes aegypti and Aedes albopictus. The habitat of DENV-transmitting mosquitoes has expanded, and in the last 50 years, the incidence of infections has increased 30-fold (WHO, 2009). Infections with DENV can be asymptomatic or cause a spectrum of clinical disease ranging from an acute, debilitating, febrile illness (DF) to the more lifethreatening dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS). Typical symptoms of DF consist of fever, retro-orbital headache, myalgia, rash, nausea, and vomiting. DHF is characterized by increased vascular permeability, hemorrhagic manifestations, thrombocytopenia, and, in the case of DSS, shock (WHO, 2009). Epidemiological observations have revealed that secondary infection with a different dengue serotype is the single greatest risk factor for manifestations of severe disease. In addition to the individual’s immune status, genetic host factors and viral virulence have also been postulated to affect disease severity (Halstead, 2007; Rico-Hesse, 2007). Thus, the development of dengue disease likely depends on complex interplays between host and viral factors. DENV is endemic in Southeast Asia, the Western Pacific, Central and South America, the Caribbean, and Africa. Recent outbreaks have occurred in the United States in Hawaii (2001), Texas (2005), and Florida (2009–2011) (Adalja, Sell, Bouri, & Franco, 2012). Based on a recent publication reporting new, evidence-based estimates of the global burden of dengue, 3.6 billion people live in dengue-endemic areas and the virus causes approximately 400 million infections and 100 million symptomatic cases annually (Bhatt et al., 2013). Over 2 million cases of severe dengue disease and over 20,000 deaths are estimated to occur each year (Gubler, 2012). Despite these high numbers of global morbidity and mortality associated with DENV infection, no effective antiviral therapy or vaccine exists at present and treatment is largely supportive in nature.

2. ADAPTIVE IMMUNE RESPONSE TO DENV The adaptive immune response presumably affords a lifelong immunity against challenge with the same DENV serotype, but only transient cross-protection against a heterologous DENV serotype, after which the memory response may play a pathological role during a secondary infection (Kyle & Harris, 2008). An early study in human volunteers found homologous immunity lasted as long as 18 months, and heterologous immunity for 2–3 months (Sabin, 1952). Epidemiological studies in Thailand and Cuba support a role for the immune system in disease enhancement, as

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most cases of DHF/DSS occur during secondary infections with a heterologous DENV serotype (Burke, Nisalak, Johnson, & Scott, 1988; Guzman et al., 1987; Guzman et al., 2000; Halstead, Nimmannitya, & Cohen, 1970; Sangkawibha et al., 1984; Vaughn et al., 2000). Infants born to dengue-immune mothers are also at greater risk for DHF/DSS, during the period of time (between 6 and 9 months of age) when circulating maternal antibodies levels wane to subprotective levels (Halstead, 1988; Kliks, Nimmanitya, Nisalak, & Burke, 1988). Thus, both actively and passively acquired DENV-specific antibodies are associated with severe dengue disease. Consequently, the immunologic investigation of DENV infection has been dominated by studies examining the role of adaptive immunity in DENV pathogenesis. Subneutralizing concentrations of DENV-specific antibodies may contribute to viral replication and disease severity via a phenomenon known as “antibody-dependent-enhancement” (ADE). According to the ADE hypothesis, DENV-antibody complexes are formed and bind to Fc receptors (FcR) on cells such as macrophages, facilitating viral entry and replication. Increased viral loads resulting from ADE then drive the production of inflammatory mediators that increase vascular permeability. Supporting the ADE hypothesis, nonneutralizing DENV-specific antibodies increased viral replication in peripheral blood leukocytes in vitro (Halstead & O’Rourke, 1977; Halstead, O’Rourke, & Allison, 1977), and studies using a variety of monoclonal antibodies have since shown that neutralizing antibodies can promote ADE in vitro when present at subneutralizing concentrations (Morens, Halstead, & Marchette, 1987; Pierson et al., 2007). Studies with monkeys have confirmed ADE of DENV replication in vivo. Specifically, monkeys receiving passive transfer of DENV-immune human sera (Halstead, 1979) or a humanized DENV-specific IgG1 monoclonal antibody (Goncalvez, Engle, St Claire, Purcell, & Lai, 2007) had higher viral loads than control monkeys. ADE resulting in disease enhancement was recently demonstrated using a mouse model of DENV infection: infection in the presence of DENV-reactive monoclonal antibodies or immune sera resulted in increased disease severity and turned a nonlethal illness into a lethal disease resembling human DHF/DSS (Balsitis et al., 2010; Zellweger, Prestwood, & Shresta, 2010). In addition to a pathogenic role for antibodies in severe dengue disease, altered T-cell responses during secondary infections with heterologous serotypes have been postulated to contribute to cytokine storm and immunopathogenesis of DHF/DSS. Studies with human samples have shown that

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serotype cross-reactive T cells are preferentially activated during secondary infection, and these cross-reactive T cells exhibit suboptimal degranulation and enhanced TNF and IFN-g production (Mangada & Rothman, 2005; Mongkolsapaya et al., 2003, 2006). TNF is suspected to cause endothelial cell dysfunction or damage, leading to plasma leakage, a hallmark of DHF/DSS. At present, despite several decades of research investigating the role of T cells in the context of DENV pathogenesis, direct evidence demonstrating a pathogenic role for DENV-specific T cells is not yet available. In fact, one study of DENV-infected adults found the breadth and magnitude of the T-cell response during secondary DENV infection was not significantly associated with disease severity (Simmons et al., 2005), and a recent study of T-cell responses in donors in a DENV hyperendemic area supports an HLA-linked protective role for CD8þ T cells (Weiskopf et al., 2013). An important protective role for CD8 þ T cells during primary DENV2 infection was also identified using a mouse model (Yauch et al., 2009). These recent studies are beginning to examine the role of T cells in the context of protection, and are starting to implicate a key role for T cells, in particular CD8 þ T cells, in anti-DENV immunity. In addition to T cells, virus-specific antibodies are likely to play a protective role against DENV infection in humans. Sera from infected individuals or anti-DENV monoclonal antibodies can neutralize epitopes that are required for viral entry (Crill & Roehrig, 2001) and can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) (Garcia et al., 2006; Laoprasopwattana et al., 2007). In addition, the amounts of preexisting, heterologous neutralizing antibodies and ADCC activity in presecondary infection plasma samples negatively correlate with plasma viremia levels and disease severity (Endy et al., 2004; Laoprasopwattana et al., 2007). Studies with mouse models have shown that passive transfer of neutralizing monoclonal antibodies can confer protection from lethal challenge (Kaufman et al., 1989; Kaufman, Summers, Dubois, & Eckels, 1987) and antibody-mediated control of flavivirus infection in vivo correlates with neutralizing activity in vitro (Diamond, Shrestha, Marri, Mahan, & Engle, 2003; Kaufman et al., 1987; Oliphant et al., 2005). The majority of neutralizing antibodies against DENV are directed against the E protein, and the most potently neutralizing bind EDIII (Crill & Roehrig, 2001; Megret et al., 1992; Roehrig, 2003; Shrestha et al., 2010; Sukupolvi-Petty et al., 2010, 2007; Wahala et al., 2010). Although not part of the virion, NS1 is also a target of the host antibody response, as the protein is expressed on the surface of infected cells and is also secreted (Muller & Young, 2013).

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NS1 is a complement-fixing antigen, and NS1-specific antibodies can protect via complement-dependent killing of infected cells. Recent studies examining the human DENV-specific antibody response have identified neutralizing antibodies that bind EDIII (Beltramello et al., 2010; de Alwis et al., 2011), as well as neutralizing antibodies that recognize a complex epitope present on the virion but not on soluble E protein (de Alwis et al., 2012). prM/M is also a dominant target of the human DENV-specific antibody response, however prM/M-specific antibodies were shown to be broadly cross-reactive and weakly or nonneutralizing (Beltramello et al., 2010; de Alwis et al., 2011; Dejnirattisai et al., 2010). These studies have begun to decipher features of a protective anti-DENV antibody response in humans. Collectively, studies to date demonstrate that DENV-specific antibodies can both protect against infection and, under certain conditions, enhance infection and disease severity, whereas the role of T cells remains to be fully elucidated. Thus, the adaptive immune response to dengue can be both protective and pathogenic, which complicates vaccine development, as discussed in the succeeding text.

3. DENGUE VACCINE OBJECTIVES AND CHALLENGES Several DENV vaccines are currently under development, including some in phase 3 safety and efficacy testing (Table 7.1). These include inactivated, live attenuated, recombinant subunit, viral vectored, and DNA vaccines. Dengue vaccine development has focused on eliciting a neutralizing antibody response, as T cells are assumed to play a minor or secondary role in dengue vaccine-mediated protection. The WHO has published guidelines on the clinical evaluation of dengue vaccines in endemic areas (WHO Initiative for Vaccine Research, & World Health Organization. Dept. of Immunization Vaccines and Biologicals, 2008) and on the quality, safety, and efficacy of live attenuated dengue vaccines (WHO, 2011). The successful development of live attenuated vaccines for the flaviviruses YFV and JEV suggest a DENV vaccine is feasible. However, DENV vaccine development is more complicated due to the existence of four serotypes of DENV that a vaccine must induce protection against. Viral interference, which is when one or more serotype(s) replicates better than the others and the immune response against that serotype dominates, has been an issue in tetravalent DENV vaccine development. Another significant challenge to dengue vaccine development is the potential for

Table 7.1 Vaccines in development Type Approach

Developer

Status

Recombinant Affinity-purified E protein subunit

Hawaii Biotec/ Merck

Phase 1

Recombinant EDIII protein fused to carrier subunit protein

Preclinical

DNA monovalent

prM and E of DENV1

NMRC

Phase 1

DNA tetravalent

prM and E of DENV1–4

NMRC

Phase 1

DNA tetravalent

EDIII from DENV1–4, synthetic Inovio consensus (SynCon™) human codon optimized

DNA shuffle

DNA shuffling of codonoptimized DENV1–4 E to generate single chimeric antigen

NMRC/Maxygen Preclinical

DNA

NS1

Various

Adenoviral vector

Recombinant adenoviral vector NMRC/GenPhar Preclinical expressing DENV1–4 prM and E

Alphavirus replicon particles

VRP expressing prM and E or Global Vaccines soluble E dimers from DENV1–4

Preclinical

Inactivated monovalent

Purified, inactivated DENV1

WRAIR

Phase 1

Inactivated tetravalent

Purified, inactivated DENV1–4

WRAIR

Phase 1

Live attenuated tetravalent

Tissue culture-passaged

WRAIR/GSK

Phase 2

Live attenuated tetravalent chimeric

Tissue culture-passaged DENV2 CDC/Inviragen backbone and prM/E from DENV1–4

Phase 2


Gene deletion (D30 30 UTR deletion mutations)

NIAID/NIH

Phase 1


YFV/DENV chimera

Acambis/Sanofi Pasteur

Phase 3

Preclinical

Preclinical

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nonneutralizing antibody responses to enhance DENV infection and disease. A dengue vaccine must induce antibody responses to all four serotypes simultaneously, and must provide long-lasting immunity to avoid the risk of ADE. Long-term studies are needed to evaluate the duration of vaccineinduced immunity, as epidemiological studies of sequential outbreaks in Cuba (DENV1 followed by DENV2 and DENV3) revealed that 20 years or more between DENV infections resulted in DHF/DSS, and the risk of severe disease was actually greater at longer intervals (Alvarez et al., 2006; Guzman et al., 2000, 2002). Another challenge of DENV vaccine development is that the correlates of protection, that is, the immune functions responsible for protection, are presently unknown. Therefore, vaccine efficacy must be measured as protection from infection in human vaccinees. Neutralizing antibodies are thought to be best surrogate for vaccine-induced protection, and high DENV neutralizing antibody titers (measured by plaque-reduction neutralization tests (PRNT)) in monkeys have been correlated with protection (Clements et al., 2010; Guirakhoo et al., 2004). However, there is no proof that neutralizing antibodies are absolutely required to protect. In fact, numerous studies in monkeys found a lack of correlation between neutralizing antibody titers and protection (Blaney, Matro, Murphy, & Whitehead, 2005; Raviprakash, Porter, et al., 2000; Robert Putnak et al., 2005; Scott et al., 1980; Simmons, Porter, Hayes, Vaughn, & Putnak, 2006; White et al., 2013). Similarly, studies with mouse models have revealed a lack of correlation between neutralizing antibody titers and protection (Brien et al., 2010; Zellweger, Miller, Eddy et al., 2013). Additionally, a live attenuated vaccine candidate recently tested in a phase 2b trial induced high titers of neutralizing antibodies against DENV2 but was ineffective at preventing DENV2 infection (Sabchareon et al., 2012). Thus, different features of the anti-DENV antibody response, such as ADCC and complement-fixation, or PRNT assays using cell types other than the standard epithelial cell lines for measurement of neutralization activity may correlate with antibodymediated protection against DENV in vivo. Based on recent studies implicating a role for CD8þ T cells in protection against DENV in humans and mouse models, certain T-cell-mediated functions may also correlate with protection in vivo. As DENV is a significant public health problem in many resource-poor countries, a dengue vaccine must be manufactured economically, which is difficult, as the vaccine needs to include viruses or antigens from all four serotypes. The vaccine must be safe and not cause DF-like disease. Both

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safety and efficacy must be tested in different ethnicities, and the vaccines must be safe and immunogenic in children and adults. DENV cocirculates in areas with other flaviviruses, including YFV and JEV, and therefore a dengue vaccine needs to be effective in inducing an immune response in flavivirus-immune individuals. Studies have found preexisting immunity to YFV resulted in enhanced DENV-specific antibody responses following DENV vaccination or infection (Bancroft et al., 1984; Carey, Myers, & Rodrigues, 1965; Dorrance et al., 1956; Guirakhoo et al., 2006; Poo et al., 2010; Scott et al., 1983). Thus, it appears dengue vaccination in flavivirus-immune individuals is feasible, albeit the precise features of the anti-DENV antibody response (in terms of specificity, isotype, avidity, and in vivo protective capacity) in flavivirus-immune versus flavivirus-naive individuals are as yet unknown, and none of these published studies examined the anti-DENV T-cell responses. Finally, live attenuated vaccines must be evaluated for neurovirulence in nonhuman primates (NHP) although testing a rodent model may be sufficient in the future (Monath et al., 2005; WHO, 2011). Neurovirulence testing is particularly important for vaccines created using the YF 17D backbone, as that vaccine has been associated with neurotropic disease (Khromava et al., 2005).

4. ANIMAL MODELS FOR TESTING DENGUE VACCINE CANDIDATES Although the natural hosts for DENV are humans and mosquitoes, a sylvatic cycle involving NHP has been observed in Africa and Southeast Asia (Diallo et al., 2003; Wolfe et al., 2001). NHP used in dengue vaccine research include rhesus monkeys (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and owl monkeys (Aotus nancymaae). NHP develop viremia and an antibody response upon DENV infection but show very few clinical signs of disease observed in humans (Halstead, Casals, Shotwell, & Palumbo, 1973; Scherer, Russell, Rosen, Casals, & Dickerman, 1978). Rhesus monkeys infected with DENV develop transient viremia lasting 3–6 days (Blaney et al., 2005, 2007; Guirakhoo et al., 2001). After subcutaneous (s.c.) infection, the virus quickly spreads to the regional lymph nodes and can be isolated from the skin and distant lymph nodes, and rarely from the spleen, thymus, liver, lungs, and bone marrow (Marchette, Halstead, Falkler, Stenhouse, & Nash, 1973). Some hallmarks of human clinical disease have been observed in NHP after s.c. infection, including leukopenia and

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thrombocytopenia (Halstead & Palumbo, 1973; Marchette et al., 1973). In one study, infection of rhesus monkeys with DENV via the intravenous (i.v.) route resulted in hemorrhage and petechiae (Onlamoon et al., 2010). In NHP, vaccine efficacy and safety have typically been measured by changes in the duration of viremia, peak viral titer, and magnitude of the antibody response. Important differences have been noted between vaccination of humans and NHP; in particular, shorter immunization protocols are effective in NHP. Two months between doses of a live attenuated vaccine protected rhesus monkeys (Simmons, Burgess, Lynch, & Putnak, 2010), whereas in humans, 3 months between doses of a live attenuated vaccine did not significantly enhance immunity (Sun et al., 2003). In addition, a live attenuated tetravalent vaccine protected monkeys from DENV challenge but was not protective in a human phase 2b trial, although the reasons for the lack of efficacy remain to be determined (Guirakhoo et al., 2004; Sabchareon et al., 2012). Wild-type mice are highly resistant to infection with DENV clinical isolates. Mouse models that have been developed for studying dengue pathogenesis and testing vaccine and antiviral candidates include intracerebral (i.c.) inoculation with mouse brain-adapted virus, infection of immunocompromised mice (including mice lacking components of the interferon (IFN) response), and mouse–human chimeras (Zompi & Harris, 2012). DENV infection of suckling mice via the i.c. route causes encephalitis and death and has been used to test the efficacy of DENV vaccines (Blaney et al., 2001; Bray et al., 1989; Eckels et al., 1984; Falgout, Bray, Schlesinger, & Lai, 1990; van Der Most, Murali-Krishna, Ahmed, & Strauss, 2000). However, both the route of infection and outcome are not relevant to human dengue disease. The WHO guidelines suggest the suckling mouse/encephalitis model is not useful for testing the safety and efficacy of dengue vaccine candidates but could be used to test vaccine lot consistency (WHO, 2011). Some of the immunocompromised mice and mouse–human chimeras develop signs of dengue disease observed in humans, including fever, increased vascular permeability, and thrombocytopenia after DENV infection. Severe combined immunodeficiency (SCID) mice transplanted with human liver cells (SCID-HuH-7) have been used to test the attenuation of live attenuated dengue vaccines by measuring viral titers (Blaney, Hanson, Hanley, Murphy, & Whitehead, 2004; Blaney et al., 2005). Mice lacking both type I and type II IFN receptors (AG129) are highly susceptible to DENV and were developed to test dengue vaccine candidates

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(Johnson & Roehrig, 1999). The AG129 mice develop paralysis even when inoculated via a peripheral route, although infection with certain DENV strains or infection in the presence of DENV-specific antibodies leads to a severe disease mimicking DHF/DSS (Balsitis et al., 2010; Jelinek et al., 2002; Prestwood, Prigozhin, Sharar, Zellweger, & Shresta, 2008; Tan et al., 2010; Zellweger et al., 2010). A live attenuated vaccine candidate (DENVax) has been tested in AG129 mice (Brewoo et al., 2012; Huang et al., 2003); however, results assessing dengue vaccine-induced immune responses in these mice with compromised or altered immune system should be interpreted with caution. Due to the limitations of the animal models and the lack of known correlates of protection, protection mediated by DENV vaccine candidates, in particular live attenuated vaccines that replicate poorly in animal models, will ultimately be defined by the ability to protect humans from DF and DHF/DSS (WHO, 2011).

5. DENGUE VACCINE APPROACHES 5.1. Recombinant subunit protein vaccines/subviral particles Recombinant subunit vaccines have several advantages for DENV vaccination compared with live attenuated vaccines. Protein vaccines are safe, inducing a balanced immune response to the four serotypes should be feasible, and the immunization schedule can be accelerated, which reduces the risk of incomplete immunity and the potential for ADE. The disadvantages of these vaccines include the requirement for adjuvant and multiple doses to achieve optimal immunogenicity, and they may not be as efficient at inducing long-lasting immunity as live attenuated vaccines. The target of subunit vaccine development for dengue has been the E glycoprotein, as the majority of neutralizing epitopes on the DENV virion are in the E protein. Recombinant E protein has been produced using Escherichia coli, baculovirus/insect cells, yeast, and mammalian cells. E. coli has been used to express truncated versions of E that are fused to other carrier proteins. EDIII, which is believed to be the receptor-binding domain, has been the focus of these E. coli-expressed fusion proteins. EDIIIs from DENV1–4 fused to the E. coli trpE protein and expressed in E. coli were recognized by immune ascites fluid from mice infected with the homologous, but not heterologous, serotypes (Fonseca, Khoshnood, Shope, & Mason, 1991; Mason, Zugel, Semproni, Fournier, & Mason, 1990). EDIII from DENV2 fused to the maltose-binding protein (MBP) from E. coli

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induced neutralizing antibodies in immunized mice and partially protected against lethal DENV2 i.c. challenge (Simmons, Nelson, Wu, & Hayes, 1998). Tetravalent immunization of mice (intramuscular (i.m.), in alum) with EDIII-MBP fusion proteins from each of the four serotypes resulted in neutralizing antibody responses against all four serotypes (Simmons, Murphy, & Hayes, 2001). Immunization of mice with the recombinant E protein together with a DENV2 DNA vaccine encoding prM and E induced high titer antibody and neutralizing antibody responses, as measured by enzyme-linked immunosorbent assay (ELISA) and PRNT50, respectively (Simmons, Murphy, Kochel, Raviprakash, & Hayes, 2001). The DENV2 EDIII-MBP fusion protein, along with the prM/E DNA vaccine and a purified inactivated virus (PIV), was tested in rhesus monkeys in various combinations of prime–boost vaccination (Simmons et al., 2006). The highest neutralizing antibody titers were observed following combination DNA and recombinant protein vaccination; however, only PIV vaccination protected monkeys from viremia after challenge with DENV2. Immunization of mice with DENV2 EDIII fused to the meningococcal P64k protein induced neutralizing antibodies and partial protection from lethal i.c. DENV2 challenge (Hermida et al., 2004). Vaccination of cynomolgus monkeys with this recombinant protein in Freund’s adjuvant protected from DENV2 challenge (Hermida et al., 2006), and green monkeys vaccinated with the fusion protein formulated with serogroup A capsular polysaccharide from Neisseria meningitidis (adsorbed on alum) developed neutralizing antibody titers against DENV2 and were partially protected from DENV2 challenge (Valdes, Hermida, et al., 2009). Finally, an EDIII-C chimeric protein expressed in E. coli induced neutralizing antibodies in mice (Valdes, Bernardo, et al., 2009). When aggregated with oligodeoxynucleotides, the protein also induced a stronger cell-mediated immune (CMI) response and protected 70% of mice from i.c. DENV2 challenge. Thus, a wide variety of E. coli-expressed EDIII containing fusion proteins have been generated and tested in both mouse and NHP models. The yeast Pichia pastoris has been used to generate recombinant E protein from DENV4 (Guzman et al., 2003). To improve secretion, the E protein was truncated at the C-terminus to remove the hydrophobic membrane anchor. Cynomolgus monkeys immunized with recombinant E plus alum developed neutralizing antibodies but were only partially protected against DENV4 challenge. Advantages of the baculovirus and insect cell expression system include high yields and proper processing and glycosylation of the expressed protein.

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DENV E produced by baculovirus has been shown to be in its native conformation and immunogenic. Recombinant baculovirus encoding DENV4 C-M-E-NS1-NS2A was expressed in Spodoptera frugiperda (Sf )-derived Sf9 cells (Zhang et al., 1988). Rabbits immunized with infected Sf9 cell lysate developed a low titer antibody response against prM, E, and NS1, and immunized mice did not develop virus-neutralizing antibodies but were protected from i.c. lethal challenge. Rhesus monkeys were then immunized with the lysate, which induced low levels of antivirion antibodies, but vaccination did not significantly protect monkeys from DENV4 challenge (Eckels et al., 1994). C-terminally truncated E and part of the M protein from DENV1 were expressed in Sf cells (Putnak et al., 1991). Immunization of BALB/c mice with the recombinant protein in complete and incomplete Freund’s adjuvant induced neutralizing antibodies and protected some mice from DENV1 i.c. challenge. Similarly, C-terminally truncated DENV2 and DENV3 E proteins expressed in Sf9 cells induced neutralizing antibodies in mice (Delenda, Staropoli, Frenkiel, Cabanie, & Deubel, 1994). Recombinant DENV2 E protein protected against lethal DENV2 i.c. challenge, and immunization with DENV3 E protein was partially protective against heterologous DENV2 infection. Vaccination of cynomolgus monkeys with the recombinant E protein only partially protected from viral challenge (Velzing et al., 1999). A hybrid E protein containing 36 amino acids from M, EDI and EDII from DENV2 E, and EDIII from DENV3 was constructed and expressed in Sf21 cells (Bielefeldt-Ohmann, Beasley, Fitzpatrick, & Aaskov, 1997). The recombinant protein was recognized by a panel of DENV-reactive monoclonal antibodies and inhibited binding of DENV2 and DENV3 to human cells. Immunization of mice induced DENV2and DENV3-specific antibody and cross-reactive T-cell responses. Expression of E along with prM allows for the secretion of E from cells, and the integrity of the neutralizing epitopes on E are maintained (Fonseca, Pincus, Shope, Paoletti, & Mason, 1994). Expression of prM and E DENV proteins in cells can generate virus-like particles (VLP), which contain the glycosylated viral proteins in a lipid membrane. DENV VLP have been generated from E and prM constructs expressed in yeast (Sugrue, Fu, Howe, & Chan, 1997), insect (Kelly, Greene, King, & Innis, 2000; Kuwahara & Konishi, 2010), and mammalian (Konishi & Fujii, 2002; Zhang et al., 2011) cells. The VLP are similar to infectious virions in terms of structure but are safer as they are noninfectious. The E contained in the VLP was shown to be equivalent to E produced in infected cells (Konishi & Fujii, 2002; Kuwahara & Konishi, 2010), and immunization of rabbits and mice

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with the VLP induced neutralizing antibodies (Kelly et al., 2000; Konishi & Fujii, 2002; Sugrue et al., 1997; Zhang et al., 1988). To avoid the drawbacks of expressing the E protein in E. coli, yeast, and baculovirus/insect cell systems, including expression of the protein in a nonnative conformation, low yields, and modest immunogenicity, the Drosophila melanogaster Schneider 2 (S2) cell expression system has been utilized by Hawaii Biotech to express the E protein (Coller, Clements, Bett, Sagar, & Ter Meulen, 2011). S2 cells were stably transformed with constructs expressing full-length prM and 80% of the E protein (C-terminally truncated; 80E) from the four DENV serotypes (strains DENV1 258848, DENV2 PR159/S1, DENV3 CH53489, and DENV4 H241) (Clements et al., 2010; Robert Putnak et al., 2005). Glycosylated recombinant 80E proteins were produced at high levels (10–40 mg/L) in native-like conformation. Immunogenicity of the DENV2-80E recombinant protein was tested in rhesus monkeys (Robert Putnak et al., 2005). DENV2-80E was given with five different adjuvant formulations, including AS04-OH, AS04-PO, AS05, AS08 (all produced by GlaxoSmithKline (GSK)), and alum. Monkeys were immunized at 0 and 3 months, and all animals seroconverted after the second dose. The highest neutralizing antibody titers were observed when DENV2-80E was given with AS04, AS05, or AS08. The booster immunization increased neutralizing antibody titers, which then dropped before challenge. DENV2-80E partially protected monkeys from wild-type DENV2 challenge; most vaccinated monkeys had no detectable live virus but some had DENV RNA in the sera as measured by realtime RT-PCR. Immunization of BALB/c mice with 80E subunits from the four serotypes in ISCOMATRIX® adjuvant induced long-lasting neutralizing antibody titers against all serotypes (Clements et al., 2010). The neutralizing antibody titers were similar when the antigens were given as tetravalent or monovalent immunization, implying no antigenic interference with the tetravalent formulation. Rhesus monkeys immunized with low doses (1 or 5 mg) of each of the four DENV-80E proteins (along with the DENV2 NS1 protein to enhance immunogenicity) induced neutralizing antibodies against all four serotypes, and monkeys were protected against challenge with DENV2 or DENV4. These vaccine candidates were recently transferred from Hawaii Biotech to Merck. A phase 1 trial of the DENV180E vaccine candidate (three doses of 10 or 50 mg in alum) has been completed (Coller et al., 2011), and a phase 1 trial of a tetravalent formulation (V180) with ISCOMATRIX® began in 2012 (Clinicaltrials.gov NCT01477580).

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6. DNA VACCINES DNA vaccination involves cloning the gene(s) of interest into a plasmid backbone and delivering the DNA intradermally (i.d.), s.c., or i.m. The DNA is taken up by cells, the protein of interest is expressed, and antigenpresenting cells take the antigen to the draining lymph nodes (Gurunathan, Klinman, & Seder, 2000). DNA vaccination results in antigen expressed by both MHC class I and class II, leading to activation of CD8 þ and CD4þ T cells, as well as antibody responses. Other advantages include low cost, ease of production, and temperature stability. DNA vaccines are nonreplicating, are therefore safer than live attenuated vaccines, and have low reactogenicity. However, DNA vaccines are not highly immunogenic, and require multiple doses and coimmunization with adjuvants. Research done at the Naval Medical Research Center (NMRC) has led to the first dengue DNA vaccine tested in a clinical trial. In initial studies, the prM protein and 92% of the E protein from DENV2 (strain New Guinea C, NGC) (C-terminally truncated) were cloned into eukaryotic expression vectors (Kochel et al., 1997). E protein was expressed by transfected cells in vitro, and immunization of mice (i.d.) resulted in DENV2 neutralizing antibodies. Coimmunization with a plasmid expressing immunostimulatory CpG motifs improved the neutralizing antibody response, and mice vaccinated with the DENV2 prM/E vaccine and CpG-containing plasmid were significantly protected from lethal i.c. DENV2 challenge (Porter et al., 1998). The DENV2 prM/E DNA vaccine (D) was tested in mice along with the recombinant fusion protein containing DENV2 EDIII and MBP (R) as part of various prime–boost strategies (Simmons, Murphy, Kochel, et al., 2001). Mice received three doses of the vaccines alone or together: R/R/R, D/R/R, D/D/D, R/D/D, or RD/RD/RD. Modest levels of neutralizing antibody were induced by the DNA vaccine alone, whereas immunization with the DNA vaccine together with the recombinant protein induced high titer antibody and neutralizing antibody responses. The highest antibody titers (measured by ELISA) were observed following D/D/D or RD/RD/RD vaccination, whereas the highest neutralizing antibody responses (measured by PRNT) were induced by RD/RD/RD and R/R/R, and the lowest were induced by D/D/D and R/D/D. The DNA and protein vaccines were then tested in rhesus monkeys, along with a PIV (P) (Simmons et al., 2006). After the third dose, all monkeys had equivalent antibody titers by ELISA; the highest neutralizing antibody titers

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were observed following DR/DR/DR, P/P/P, and DP/DP/DP vaccination, and the lowest neutralizing antibody titers were observed in D/D/Dimmunized monkeys. Monkeys were challenged with DENV2 (strain S16803) 5 months after the last dose, and protection from DENV2 viremia was only seen with PIV alone. DNA vaccination alone or in combination with recombinant protein or PIV did not significantly reduce viremia. To increase the immunogenicity of the DENV2 prM/E DNA vaccine, antigen was targeted to lysosomes in an attempt to increase antigen presentation on MHC class II, thereby enhancing CD4þ T-cell and antibody responses (Raviprakash et al., 2001). The transmembrane and cytoplasmic regions of E were replaced with carboxy-terminal sequence of lysosomeassociated membrane protein (LAMP), which contains the endosomal/ lysosomal targeting sequences of LAMP. The modification resulted in DENV antigens colocalized with endogenous LAMP in transfected cells and significantly increased neutralizing antibody titers in mice (Lu et al., 2003; Raviprakash et al., 2001). DENV1 DNA vaccine candidates were created using truncated or fulllength E with or without prM from strain Western Pacific 74 (West Pac 74) (Raviprakash, Kochel, et al., 2000). Cells transfected with prM and fulllength E formed VLP in transfected cells and induced long-lasting neutralizing antibody responses in mice; therefore, this construct was selected for further study. Rhesus monkeys were vaccinated i.d. or i.m. with three or four doses of the DENV1 DNA vaccine (D1ME100) (Raviprakash, Porter, et al., 2000). I.m. immunization resulted in higher antibody levels than i.d., and protection from DENV1 challenge 4 months after the last immunization. Four of eight monkeys vaccinated i.m. were completely protected and four partially protected, despite very low neutralizing antibody titers. In contrast, i.d. vaccination did not protect. The D1ME100 vaccine was also tested in Aotus monkeys (Kochel et al., 2000). The monkeys received three doses i.d. or i.m., and all developed neutralizing antibodies and were partially or completely protected from viremia after DENV1 challenge 6 months after the third dose. To enhance the neutralizing antibody response, Aotus monkeys were coimmunized with the D1ME100 vaccine and plasmids expressing human immunostimulatory sequences (ISS) and/or Aotus GM-CSF (Raviprakash et al., 2003). In addition, delivery of the vaccine using the needle-free Biojector® was tested. Coimmunization with ISS or GM-CSF did not increase neutralizing antibody titers; however, Biojector® vaccination resulted in significantly higher neutralizing antibody titers for immunization with D1ME100 plus ISS and GM-CSF than needle

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injection (i.d.). D1ME100 given with the GM-CSF gene and ISS (whether via Biojector® or needle) induced stable neutralizing antibody responses that protected 87% of monkeys challenged with DENV1 6 months after a third vaccination. D1ME100 was compared with a candidate vaccine (D1MEVRP) expressing DENV1 prM and E in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) (Chen et al., 2007). Cynomolgus monkeys were vaccinated with three doses of the DNA vaccine (DDD) or the VRP (VVV) or given two doses of the DNA vaccine followed by a dose of the VRP (DDV). All regimens were immunogenic and protective, but the heterologous prime–boost of DDV induced the highest DENV1-specific IgG and neutralizing antibody titers and complete protection from DENV1 challenge. A tetravalent DNA (TDNA) vaccine was made and tested in rhesus monkeys as part of a prime–boost vaccination strategy with a tetravalent live attenuated vaccine (TLAV) boost (Simmons et al., 2010). The DNA constructs contained prM and full-length E from West Pac 74 (DENV1) and near wild-type Philippine strains from DENV2, 3, and 4. The DENV2 construct contained the LAMP sequences. Monkeys were primed with TDNA (1.25 mg of each serotype i.m. using Biojector®) or tetravalent PIV (TPIV) in alum, boosted 2 months later with TLAV, and challenged with DENV3 (strain CH53489) 8 months later. Monkeys immunized with TDNA/ TDNA/TLAV were partially protected, whereas TPIV/TLAV monkeys were completely protected from viremia. A phase 1 study of the monovalent D1ME100 has been completed (Beckett et al., 2011). Twenty-two flavivirus-naive adults received a high or low dose (5 or 1 mg) of the DNA vaccine using the Biojector® needle-free system at 0, 1.5, and 5 months. The vaccine was safe and well tolerated; the most commonly reported side effect was mild pain or tenderness at the injection site. However, the vaccine was poorly immunogenic. Of those receiving the high dose, only 41.6% (5/12) developed DENV1 neutralizing antibodies, and no neutralizing antibody responses were detected in the low dose group. E protein-specific T-cell IFN-g responses were detected in 50% and 83.3% of individuals in the low and high dose groups, respectively. Various approaches are being explored to enhance the immunogenicity of the DENV DNA vaccine, including alternative delivery strategies, plasmid modifications, testing as part of prime–boost strategies, and coimmunization with adjuvants (Danko, Beckett, & Porter, 2011). Danko et al. found formulation with the adjuvant Vaxfectin® enhanced the neutralizing antibody response in monkeys immunized with a

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tetravalent DNA vaccine (Danko, Beckett, & Porter, 2011), and a phase 1 study of the tetravalent DNA vaccine (TVDV) given with Vaxfectin® began in 2011 (Clinicaltrials.gov NCT01502358). In parallel, DNA shuffling and screening technologies were utilized to develop a single recombinant antigen containing epitopes from all four DENV serotypes (Apt et al., 2006). Three chimeric clones (one containing truncated E and two expressing full-length prM/E) induced neutralizing antibodies against all four serotypes and protected mice from lethal i.c. DENV2 challenge. The three clones were then used to immunize rhesus monkeys; some monkeys vaccinated with the constructs expressing prM/E developed neutralizing antibodies against all four serotypes, but only partial protection against DENV1 challenge and no protection against DENV2 was observed (Raviprakash et al., 2006). Konishi et al. developed a tetravalent DENV DNA vaccine containing constructs expressing prM and E from DENV1–4 (Konishi, Kosugi, & Imoto, 2006; Konishi, Terazawa, & Fujii, 2003; Konishi, Yamaoka, Kurane, & Mason, 2000). Mice immunized with 25 mg of each of the four constructs using a needle-free jet injector developed neutralizing antibodies against all four serotypes (Konishi et al., 2006). Simultaneous immunization with protein, in the form of DENV2 extraviral particles or inactivated JEV vaccine, enhanced the immunogenicity of the DNA vaccine (Imoto & Konishi, 2007). A synthetic consensus (SynCon™) human codon optimized DNA vaccine has been developed by Inovio Pharmaceuticals. A single plasmid was constructed containing consensus EDIII sequences from DENV1–4 (Ramanathan et al., 2009). In vivo electroporation of mice with the DNA vaccine induced neutralizing antibodies against the four serotypes. DNA vaccines based on the NS1 protein have also been created and tested in mice (Costa et al., 2007, 2006; Timofeev, Butenko, & Stephenson, 2004; Wu et al., 2003). As mentioned earlier, anti-NS1 antibodies can mediate complement-dependent killing of infected cells, and as the protein is not expressed on the virion, antibodies against NS1 cannot mediate ADE. A DNA vaccine expressing DENV2 NS1 induced moderate antibody responses and T-cell responses in mice and provided partial protection against i.v. DENV2 challenge (Wu et al., 2003). Coimmunization with a plasmid expressing IL-12 enhanced the protective efficacy. Vaccination with a plasmid containing the DENV2 NS1 gene fused to the secretory signal sequence of human tissue plasminogen activator (t-PA) was also found to be immunogenic and protective in mice challenged with DENV2 i.c.

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(Costa et al., 2007, 2006). A DNA vaccine expressing the DENV1 prM-ENS1 proteins induced greater ADCC and cytotoxic T-lymphocyte activity and better protection from lethal DENV1 i.c. challenge than a DNA vaccine expressing prM and E without NS1 (Zheng et al., 2011). Altogether, most of the DNA vaccine-based approaches for development of dengue vaccines have focused on eliciting immune responses to the prM and E proteins, and similarly to the recombinant E protein-based vaccines, these vaccine-induced immune responses are mainly evaluated for induction of anti-DENV antibodies. Results of the phase 1 trial of TVDV given with Vaxfectin® will be informative. A few candidates generate NS1specific B-cell and T-cell responses. Further advances in DNA vaccination technology that overcome the poor immunogenicity may lead to a successful DENV DNA vaccine in the future.

7. VIRAL VECTORED VACCINES Several viral vector platforms have been explored as delivery vehicles for DENV antigens, including vaccinia virus, adenovirus, and alphavirus vectors.

7.1. Vaccinia Advantages of poxviruses, including vaccinia virus, as vaccine vectors include the ability to insert large pieces of DNA, high levels of gene expression, lack of persistence or viral integration into the host genome, high immunogenicity, and relative ease of vaccine production (Drexler, Staib, & Sutter, 2004). However, early attempts using vaccinia virus as a vaccine vector for DENV antigens were disappointing. The vaccinia Western Reserve (WR) strain was used to express prM, E, NS1, and NS2A from DENV4 (Zhao et al., 1987). CV-1 monkey kidney cells infected with the recombinant virus expressed the structural proteins and NS1; however, infection of cotton rats did not result in an antibody response to prM or E, and only 1/11 animals had an antibody response to NS1, likely due to low level of gene expression. Mice immunized with recombinant viruses containing the structural proteins (with or without NS1 and NS2A) were protected from lethal DENV4 i.c. challenge despite a low titer antibody response to E (Bray et al., 1989). Immunization with recombinant viruses expressing DENV4 NS1 completely protected mice from i.c. DENV4 challenge, whereas vaccination with DENV2 NS1 resulted in only partial protection from DENV2 challenge (Falgout et al., 1990). To improve the

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immunogenicity of recombinant DNA-expressed E, various recombinant vaccinia virus strains were constructed that expressed full-length or C-terminally truncated E from DENV4 (Men, Bray, & Lai, 1991). Fulllength E was not secreted from recombinant virus-infected CV-1 cells, but several C-terminally truncated mutants were secreted extracellularly or expressed on the cell surface. Immunization of mice with vaccinia virus recombinants expressing the truncated proteins that were recognized by dengue hyperimmune ascitic fluid (i.e., were expressed in native conformation) protected from lethal encephalitis. Passive transfer of immune sera suggested anti-E antibodies mediated the protection. Due to safety concerns for the nonattenuated WR strain, the highly attenuated, replication-deficient modified vaccinia Ankara (MVA) was selected as a vector to express C-terminally truncated E proteins (80%) from DENV2 and DENV4 (Men et al., 2000). The MVA-DENV2 80%E, but not MVA-DENV4 80%E, induced neutralizing antibodies in mice after i. m. inoculation. Two doses of MVA-DENV2 80%E in rhesus monkeys induced a low antibody response and partial protection against DENV2 challenge, and three doses was completely protective.

7.2. Adenovirus vectors Adenovirus vectors have a number of advantages as vaccine vectors, including the adenovirus genome is well characterized and easy to manipulate, they can be rendered replication-defective to increase safety, they have broad tropism that allows for high levels of antigen expression in numerous cell types, and they are easy to produce and store (Tatsis & Ertl, 2004). Adenoviral vectors have been used for gene replacement therapy and as vaccine vectors and have been shown to induce robust CD8 þ T-cell and antibody responses against the transgene. Preexisting immunity to adenoviruses can affect immunization; however, this can be overcome by using adenoviruses from different species, such as chimpanzees. A recombinant, replication-deficient adenovirus (rAd) was constructed expressing the ectodomain of the DENV2 E protein and part of prM (Jaiswal, Khanna, & Swaminathan, 2003). Immunization of BALB/c mice (intraperitoneally (i.p.)) elicited DENV2-specific T-cell responses and neutralizing antibodies. A replication-deficient Ad vector was also used to express a chimeric antigen consisting of the EDIIIs of DENV2 and DENV4 (Khanam, Rajendra, Khanna, & Swaminathan, 2007). The vector was used as part of a heterologous prime–boost strategy: mice were immunized with

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the rAd (i.p.), followed by an i.d. boost with a plasmid vector encoding the EDIIIs. The vaccinations induced neutralizing antibodies and T-cell responses against DENV2 and DENV4. A tetravalent vaccine expressing the EDIII sequences from the four DENV serotypes was then created using the rAdV5 vector (Khanam, Pilankatta, Khanna, & Swaminathan, 2009). Prime–boost immunization of mice (rAd i.p. followed by plasmid i.d.) induced neutralizing antibody responses and T-cell responses against the four serotypes. A homologous prime–boost with the rAd vector encoding the DENV EDIIIs revealed anti-AdV5 Ab did not interfere with boosting the anti-DENV antibody response. The complex rAd-based vaccine platform (cAdVax), developed by GenPhar Inc., was used to construct a pair of adenoviral vectors that each express prM and E from two DENV serotypes: cAdVaxD(1-2) and cAdVaxD(3-4) (Holman et al., 2007; Raja et al., 2007). Vaccination of mice (i.p.) induced neutralizing antibody titers against all four serotypes and a broadly reactive T-cell response. Tetravalent vaccination was studied in rhesus monkeys by mixing the two bivalent vectors (Raviprakash et al., 2008). Two doses of the vaccines (i.m., 8 weeks apart) resulted in high titer neutralizing antibodies against all four serotypes and significantly protected against live DENV challenge 4 or 24 weeks after the second immunization. Complete protection against DENV1 and DENV3 viremia was observed; however, for DENV4, the duration of viremia after challenge at 24 weeks was reduced but the viral titers were increased compared with control vaccinated animals. Despite the induction of anti-Ad antibodies induced by the first dose, the second immunization was able to boost anti-DENV antibody titers.

7.3. Alphavirus replicon particles Alphavirus-derived replicon vaccines have shown promise as a platform for dengue vaccination. VEE VRP are nonreplicating VLP containing a modified genome expressing a protein of interest. Vaccination with VRP induces high levels of antigen expression in a single round of infection, and antigen presentation is robust due to the adjuvant activity of VRP and the targeting of the VRP to dendritic cells (DC) in the lymph nodes (MacDonald & Johnston, 2000; Thompson et al., 2006). A VRP expressing DENV1 prM and E (D1ME-VRP) was shown to be immunogenic and protective when given in three doses or as part of a heterologous prime–boost with a DENV1 DNA vaccine to cynomolgus monkeys (Chen et al., 2007). DENV2 prM and E have also been cloned into a VEE replicon vector

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and packaged into VRP (White et al., 2007). Immunization of mice (s.c.) resulted in DENV2-specific IgG and neutralizing antibodies, and a second immunization at 12 weeks resulted in increased neutralizing antibody titers that lasted for 30 weeks. Vaccination was protective: two doses of 1E6 infectious units (IU) in young mice completely protected against lethal i.c. DENV2 challenge, and lower doses induced partial protection. VRP expressing two configurations of the E protein (subviral particles (prM/ E), or soluble E dimers (E85)) were compared (White et al., 2013). Immunization of rhesus macaques with E85-VRP resulted in serotype-specific antibody responses targeting EDIII that developed more rapidly and to a higher titer than the prM-E-VRP response. Monkeys were then vaccinated with a tetravalent vaccine containing E85-VRP from the four serotypes. After 2 doses, all animals had robust neutralizing antibody responses against all four serotypes, and were partially protected from challenge with DENV1 and DENV2, and completely protected from DENV3 and DENV4. Importantly, antivector immunity from the first dose did not seem to reduce the effectiveness of second dose. The authors believe clinical trials with the tetravalent E85-VRP vaccine candidates are warranted. Overall, similarly to recombinant protein- and DNA-based vaccine approaches, viral vectored dengue vaccine candidates are focused on eliciting and evaluating E proteinspecific antibody responses. In contrast with recombinant E protein- and DNA-based vaccine approaches, no viral vectored vaccine has advanced to clinical phase 1 testing.

8. INACTIVATED WHOLE VIRUS Vaccination with inactivated DENV vaccines ideally should induce a balanced immune response without the viral interference that can occur with live attenuated vaccines. In addition, with inactivated vaccines, there is no risk of viral replication or reversion to wild-type virus that could occur with a live virus vaccine. However, inactivated DENV vaccines contain only the C, M, E, and NS1 proteins (Putnak, Barvir, et al., 1996; Putnak, Cassidy, et al., 1996) and therefore the immune response is directed only against these proteins, and there is no response to the other nonstructural proteins. Inactivated vaccines are less effective than live attenuated vaccines in inducing long-lasting immunity, and as with other nonliving vaccines, multiple doses and adjuvants will likely be necessary for optimal immunogenicity in unprimed individuals. In addition, inactivated vaccines may not be as efficient at inducing CMI as live vaccines. However, an

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inactivated vaccine for dengue may be useful as part of heterologous prime– boost vaccine regimen, for example, with a DNA vaccine. The Walter Reed Army Institute of Research (WRAIR) has developed PIV vaccine candidates. The DENV2 strain S16803 was grown in Vero (African green monkey kidney epithelial) cells, purified on sucrose gradients, and inactivated with formalin (Putnak, Barvir, et al., 1996). Immunization of mice and rhesus monkeys with PIV (absorbed on alum) induced a high titer neutralizing antibody response. Immunization was also protective; two doses protected mice from DENV2 i.c. challenge, and three doses in monkeys led to reduced or absent viremia after DENV2 challenge. A PIV was also made with the DENV2 strain 16681 grown in fetal rhesus lung (FRhL) cells and inactivated with formalin (Putnak, Cassidy, et al., 1996). This PIV was also immunogenic, and doses of 100 or 1000 ng (but not 10 ng) adjuvanted with alum significantly protected mice from lethal i.c. challenge. The DENV2 strain S16803 PIV was compared with a live attenuated vaccine (DENV2 PDK-50) and recombinant subunit protein vaccine (r80E) in rhesus monkeys (Robert Putnak et al., 2005). Monkeys were immunized at 0 and 3 months, and five different adjuvants (alum, or AS04-OH, AS04-PO, AS05, and AS08 from GSK) were tested with the PIV and r80E vaccines. All monkeys seroconverted after the second dose, and the highest neutralizing antibody titers were observed after vaccination with 5 mg of PIV adjuvanted with AS05 or AS08 or 5 mg r80E in AS05 or AS08. Unlike the live attenuated vaccine, the PIV and r80E vaccines did not induce stable antibody titers; the titers increased after the boost but declined before DENV2 challenge 2 months later. In addition, whereas vaccination with the live attenuated virus resulted in no viremia after challenge, some PIV-vaccinated monkeys had viremia. A subsequent study compared vaccination of rhesus monkeys with combinations of three nonreplicating DENV2 vaccine candidates: DNA vaccine expressing prM and E, EDIIIMBP fusion protein, and PIV (Simmons et al., 2006). After the third dose, all monkeys had high antibody titers (measured by ELISA) and neutralizing antibodies (measured by PRNT50). The highest neutralizing antibody titers were observed after vaccination with the DNA vaccine and fusion protein together; however, significant protection from DENV2 challenge 5 months after the last immunization was observed only with PIV vaccination. Protection correlated with total antibody levels (including antibodies against NS1) as measured by ELISA and antibody avidity, but not with neutralizing antibody titers.

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A TPIV vaccine was made from wild-type DENV1–4 strains grown in Vero cells and inactivated with formalin (Simmons et al., 2010). The TPIV was tested as part of a heterologous prime–boost strategy. Rhesus monkeys were primed with one dose of TPIV in alum and boosted 2 months later with a TLAV. TPIV immunization resulted in a low titer neutralizing antibody response, but boosting with TLAV increased titers. The highest neutralizing antibody titers were against DENV2, and the lowest were against DENV3. TPIV/TLAV vaccinated monkeys were completely protected from challenge with DENV1, 2, 3, or 4 at 8 months, and anamnestic neutralizing antibody responses were detected after the live viral challenge. A phase 1 clinical trial of the WRAIR DENV1-PIV began in 2011, and two phase 1 trials of the tetravalent TDENV-PIV candidate began in 2012 in a dengue-primed population (Clinicaltirals.gov NCT01702857) and in a nonendemic area (NCT01666652). The tetravalent vaccine candidates will be tested with three different adjuvants: alum, AS01E, and AS03B. As an alternative to formalin inactivation, psoralen-inactivation has been used to inactivate DENV. Psoralens intercalate between nucleic acids and covalently cross-link pyrimidines following UVA exposure. This method inactivates viruses while leaving immunogenic surface epitopes intact (Groene & Shaw, 1992). A psoralen-inactivated DENV1 vaccine has been tested in mice (Maves, Castillo Ore, Porter, & Kochel, 2010) and monkeys (Maves, Ore, Porter, & Kochel, 2011). Aotus monkeys immunized i.d. with three doses (10 ng each) of the inactivated DENV1 virus in alum developed DENV1-specific IgG and neutralizing antibodies and were moderately protected from DENV1 challenge. The authors suggest alternate routes of administration, higher or greater number of doses, or different adjuvants may enhance the immunogenicity. Thus, similarly to recombinant protein-, DNA-, and viral vector-based dengue vaccine candidates, studies with inactivated whole virus vaccines have primarily assessed vaccine-induced antibody responses in terms of the duration and levels of ELISA-binding and PRNT titers and the capacity to protect against lethal i.c. challenge of mice and viremia in monkeys. Unlike recombinant protein-, DNA-, and viral vector-based dengue vaccines that induce E (or NS1-)-specific antibody responses, vaccination with whole virus vaccines induces antibody responses against E, prM, and NS1.

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9. LIVE ATTENUATED Most dengue vaccine efforts have focused on developing live attenuated vaccines, and these are the furthest along in development and clinical testing. Live attenuated vaccines have a number of advantages including their ability to induce immune responses that mimic the response to natural infection, the induction of robust B- and T-cell responses, and the ability to confer lifelong immune memory (Pulendran & Ahmed, 2011). The most successful vaccines developed to date, including the smallpox vaccine, are live attenuated vaccines. Live attenuated vaccines can be produced at relatively low cost and may be effective after one dose. It has been estimated that a live attenuated DENV vaccine could be produced at an affordable cost in developing countries (Mahoney et al., 2012). Live attenuated DENV vaccine candidates must be attenuated for mosquitoes as well as humans, to prevent transmission after vaccination. The vaccine strains must be genetically stable to avoid reversion to wild-type viruses, and genetic stability must be monitored throughout manufacture. The major challenges of developing a live attenuated vaccine for DENV include the need for the vaccine to induce balanced immune responses to all four serotypes, and be sufficiently attenuated to not cause symptoms of DF. Viral interference is a key issue in tetravalent live attenuated dengue vaccine development and has been observed with live attenuated vaccine candidates in monkeys and human volunteers (Guy et al., 2009; Kanesathasan et al., 2001; Kitchener et al., 2006; Osorio, Brewoo, et al., 2011). Booster immunizations will likely be required to overcome the interference and induce immune responses against all four serotypes. If more than one dose is required, the time between vaccinations must be optimized to allow replication of all four strains in subsequent immunizations; that is, booster immunizations must be given after sterilizing immunity has waned. A study in rhesus monkeys found a second immunization with a live attenuated tetravalent vaccine at 4 months, but not 1 month, boosted neutralizing antibody titers (Blaney et al., 2005). Similarly, a study in humans found a second immunization 1 or 3 months after the first dose did not significantly increase neutralizing antibody titers (Sun et al., 2003) so subsequent studies boosted at 6 months (Simasathien et al., 2008; Sun et al., 2009). Prolonged immunization schedules seem to be necessary but may be difficult to implement or track in DENV endemic areas.

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Early dengue vaccine research attempted to attenuate the virus by serial passaging through mice. Passaging DENV through the brain of suckling mice via i.c. inoculation resulted in increased neurovirulence in mice (Cole & Wisseman, 1969; Sabin & Schlesinger, 1945) and attenuation in humans (Hotta, 1952; Sabin, 1952). After 7–10 passages through mice, the virus was deemed attenuated enough to test as a vaccine (Sabin, 1952). The fifteenth mouse-passaged virus was given to 16 human volunteers. The vaccine was safe; all volunteers developed a maculopapular rash, but systemic symptoms were absent or mild. The vaccine induced protective immunity, as the vaccinees were immune to exposure to DENV-infected mosquitoes 21–38 days after vaccination. A mouse-passaged DENV1 vaccine was found to protect adults and adolescents in Puerto Rico during a heterologous DENV outbreak (Bellanti et al., 1966). The heterologous protection developed in three weeks and lasted for at least 85 days. In 1971, the US Armed Forces Epidemiological Board initiated efforts to develop live attenuated DENV vaccines with the strategy of attenuation by serial tissue culture passage, and passaging began at the University of Hawaii in 1971 (Halstead & Marchette, 2003). Initial efforts focused on passaging wild-type DENV strains through various types of primary cells or cell lines, including primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Some wild-type and some attenuated strains were sent to Mahidol University in Thailand. Passaging of DENV in vitro was done simultaneously in Hawaii, Thailand, and at WRAIR.

9.1. University of Hawaii/WRAIR PR-159/S-1 is a vaccine strain that was derived at WRAIR by passaging a DENV2 clinical isolate, PR-159, through primary GMK cells and FRhL cells (Eckels, Brandt, Harrison, McCown, & Russell, 1976; Eckels, Harrison, Summers, & Russell, 1980; Harrison, Eckels, Sagartz, & Russell, 1977). PR-159/S-1 has in vitro and in vivo attenuation characteristics including temperature sensitivity, small plaque size (on rhesus monkey kidney epithelial LLC-MK2 cells), and reduced virulence for suckling mice and rhesus monkeys. The DENV2 vaccine strain was tested in six YFV-immune human volunteers (Bancroft et al., 1981). Five of six had viremia and seroconverted, including one who had symptoms of mild DF including fever, headache, and myalgia. A subsequent study tested the vaccine in 98 volunteers (Bancroft et al., 1984). Seroconversion was higher in YFV-immune individuals compared with naive volunteers

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(90% vs. 61%), and peak neutralizing antibody titers were higher in YFVimmune volunteers as well. A DENV1 vaccine candidate, 45AZ5, was derived by passaging a clinical isolate through FRhL cells followed by chemical mutagenesis with 5-azacytidine (McKee et al., 1987). Despite having markers of attenuation including temperature sensitivity, small plaque size, and reduced virulence in mice and monkeys, 45AZ5 was genetically unstable and caused DF in two volunteers. Similarly, a DENV3 vaccine candidate caused DF in recipients (Innis et al., 1988). The DENV4 strain H241 was passaged through PDK cells and FRhL cells to derive the H241, PDK35-TD3 FRhL p3 vaccine strain (Halstead, Eckels, Putvatana, Larsen, & Marchette, 1984). This strain was attenuated in vitro and in suckling mice and had low virulence in rhesus monkeys. It was next tested in five YFV-immune volunteers (Eckels et al., 1984). Only two subjects seroconverted, and those individuals developed mild clinical disease. Phenotypically changed virus was isolated from the volunteers with viremia, indicating the virus was genetically unstable. A DENV4 vaccine candidate was also developed at WRAIR. The DENV4 strain 341750 Carib was passaged in PDK cells 20 times and in FRhL-2 cells 4 times to derive 341750 Carib PDK-20/FRhL-4 (Marchette et al., 1990). The vaccine strain was less virulent than the parental strain in rhesus monkeys, yet the vaccine strain induced the development of neutralizing antibodies and hemagglutination inhibition (HAI) antibodies against DENV4. Monkeys immunized with the vaccine strain were protected from parental DENV4 challenge. Three doses (103, 104, or 105 plaqueforming units (PFU)) of the 341750 Carib PDK-20/FRhL-4 vaccine strain were then tested in human volunteers (Hoke et al., 1990). Five of 8 volunteers receiving 105 PFU developed viremia and antibody responses (neutralizing, HAI, and IgM) against DENV4. The viremic subjects also developed rash and slight temperature elevations. The vaccine was deemed safe and reasonably immunogenic and selected for further study as part of a tetravalent vaccine. The other strains selected were DENV1 45AZ5 PDK-20 FRhL3, DENV2 S16803 PDK-50 FRhL3, and DENV3 CH53489 PDK-20 FRhL3. These vaccine strains were tested in flavivirus-naive adults as monovalent or tetravalent vaccination (Sun et al., 2003). Monovalent recipients were given one or two doses 1 or 3 months apart, and the tetravalent vaccine was given in two or three doses at 1–4 month intervals. The doses of DENV1 and DENV2 were 10-fold higher than DENV3 and DENV4. The highest reactogenicity was observed with DENV1, and myalgia, rash,

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and fever were the most common symptoms. Viremia was detected in some of the volunteers, most often in DENV3 or tetravalent recipients. Seroconversion after one monovalent dose was 100% for DENV1, 92% for DENV2, 46% for DENV3, and 58% for DENV4; for tetravalent vaccination, seroconversion ranged from 30% to 70%. Seroconversion did not significantly differ between monovalent and tetravalent recipients, suggesting a lack of viral interference. The second dose of monovalent vaccination 30 or 90 days later was less reactogenic than the first dose, but did not boost antibody titers except to DENV3. Second and third doses of the tetravalent vaccine increased the number of seroconversions and neutralizing antibody titers. In collaboration with GSK, a subsequent phase 1 trial in flavivirus-naive adults tested 16 formulations of the tetravalent vaccine: DENV1 (45AZ5) PDK-20, DENV2 (S16803) PDK-50, DENV3 (CH53489) PDK-20, and DENV4 (341750) PDK-20 (Edelman et al., 2003). The formulations were variably reactogenic, and reactogenicity correlated with immunogenicity. Viremia was detected in 47% of recipients overall, primarily after the first dose. Overall, seroconversion to DENV1, 2, 3, and 4 were 69%, 78%, 69%, and 38%, respectively, and the highest neutralizing antibody titers were against DENV1. There was no consistent effect of a second immunization at day 28 on neutralizing antibody responses, and no formulation induced a tetravalent neutralizing antibody response after two doses. The poor response to the boost was likely due to the presence of heterotypic immunity, which prevented replication of the second dose. A new formulation, containing a higher passage DENV1 and lower passage DENV4 than the previous formulations (DENV1 (45AZ5) PDK-27, DENV2 (S16803) PDK-50, DENV3 (CH53489) PDK-20, and DENV4 (341750) PDK-6), was tested in cynomolgus macaques and found to induce a balanced tetravalent neutralizing antibody response (Koraka, Benton, van Amerongen, Stittelaar, & Osterhaus, 2007). It was then studied in seven DENV- and JEV-naive Thai children who were given two doses 6 months apart (Simasathien et al., 2008). The vaccine was safe, with no severe adverse events (SAE) observed. Symptoms were more frequently reported after the first vaccination and included fever, fatigue, headache, myalgia, and arthralgia. DENV4 viremia was detected in three volunteers. The vaccine was also immunogenic: 50% of the children seroconverted to DENV2 and DENV4 after the first dose, and after the second dose, six of seven recipients seroconverted to all four serotypes. The new tetravalent formulation was tested side by side with two older formulations in a double-blind, randomized phase 2 trial in

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71 flavivirus-naive adults (Sun et al., 2009). Volunteers were given two doses at 0 and 6 months. The new formulation was immunogenic; 63% of recipients developed a tetravalent neutralizing antibody response after two doses. Compared with the older formulations, the new formulation was less reactogenic and more immunogenic, and was therefore selected for future studies, including a phase 1/2 trial in infants (Watanaveeradej et al., 2011). Thirty-four infants (12–15 months of age) received two doses of the tetravalent DENV vaccine 6 months apart (PDK 27/50/20/6), and 17 infants received a control vaccine. The vaccine was safe; no vaccinerelated SAE were observed, although one subject had transiently elevated AST/ALT levels. The vaccine was also moderately immunogenic: after the second dose, 85.7% of recipients had trivalent neutralizing antibody responses and 53.6% had tetravalent responses. Two formulations of a new vaccine (TDEN) were produced using rederived master seeds from the PDK 27/50/20/6 precursor vaccine and were studied in a placebo-controlled phase 2 trial in 86 adults (Thomas et al., 2013). The two new formulations (F17 and F19) were compared with the precursor vaccine (F17/Pre: PDK 27/50/20/6). F19 had fourfold less DENV4 than F17 and F17/pre). No vaccine-related SAE were observed in the vaccinees, and symptoms were transient and mild to moderate in severity. Rash was the only symptom observed more often in DENV vaccine recipients versus placebo. DENV4 viremia was detected in some of the F17/Pre vaccinees and one F17 vaccinated subject; no viremia was detected for the other serotypes or in the F19 recipients. A second dose at 6 months increased antibody titers and broadened the response. Tetravalent seroconversion rates in DENV-unprimed subjects were 60% for F17 and 66.7% for F19 one month after the second dose. A third dose given 5–12 months later was ineffective at boosting neutralizing antibody titers. Altogether, the new formulations were safe and moderately effective, and the authors state studies in a larger number of adults and then children are warranted.

9.2. Mahidol University The DENV2 strain, 16681, was serially passaged through PDK cells 53 times to obtain 16681-PDK-53, which was tested in a phase 1 trial in Thailand (Bhamarapravati, Yoksan, Chayaniyayothin, Angsubphakorn, & Bunyaratvej, 1987). Five JEV- and DENV-naive volunteers and five JEV-immune volunteers were vaccinated. One patient became viremic, and all developed neutralizing antibodies that lasted for 1.5 years.

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DENV2-specific CD4 þ and CD8þ T-cell responses were detected in all vaccinees (Dharakul et al., 1994). When given in a bivalent formulation with a DENV4 vaccine strain, 1036 PDK 48, all subjects developed neutralizing antibodies against DENV2 and DENV4 (Bhamarapravati & Yoksan, 1989). The 16681-PDK-53 vaccine was also found to be safe and immunogenic in 10 flavivirus-naive American volunteers, who developed a DENV2 neutralizing antibody response that lasted for 2 years (Vaughn et al., 1996). Vaccine strains from each serotype obtained by passage through PDK cells or primary GMK cells were selected and tested in monovalent, bivalent, trivalent, and tetravalent vaccinations in Thai adults (Bhamarapravati & Sutee, 2000). The strains used were DENV1 PDK-13, DENV2 PDK-53, DENV3 PGMK-30/F3, and DENV4 PDK-48. The vaccine was safe and did not induce clinically significant symptoms. Of the volunteers that seroconverted, most had neutralizing antibodies 2 years after monovalent vaccination. All bivalent and trivalent vaccine recipients seroconverted to all serotypes in the vaccine, and of the tetravalent recipients, four of six developed neutralizing antibodies to all four serotypes, whereas two seroconverted to DENV1, 2, and 3 but not DENV4. The vaccine strains were produced by Aventis Pasteur and tested in a phase 1 trial in the United States in 40 flavivirus-naive adults (Kanesathasan et al., 2001). Subjects received a single dose of a monovalent vaccine or the tetravalent vaccine (containing 3.47–3.9 log10 PFU of each serotype). Mild symptoms including fever, headache, malaise, rash, and transient neutropenia were observed in the monovalent recipients. Tetravalent vaccination was more reactogenic than monovalent vaccination, and one volunteer developed a dengue-like syndrome. Viremia was detected in DENV3 and DENV4 monovalent recipients, and DENV3 was detected in the tetravalent vaccine recipients. All of the of DENV2, 3, and 4 monovalent recipients but only 60% of the DENV1 recipients seroconverted. Of the tetravalent recipients, only one of ten seroconverted to all four serotypes, and neutralizing antibody responses were directed primarily to DENV3. The vaccine induced DENV-specific T-cell responses (as measured by in vitro proliferation, IFN-g production, and cytotoxicity) in the tetravalent vaccine recipients; however, the responses to the four serotypes were not equivalent (Rothman et al., 2001). In an attempt to achieve a more balanced antibody response, seven tetravalent vaccine formulations were tested that differed in overall viral dose and the dose of each serotype (Sabchareon et al., 2002). Fifty-nine flavivirusnaive Thai adults received two vaccine doses 6 months apart. Five volunteers

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developed a DF-like illness, with headache, fever, and myalgia the most common symptoms. Some hematologic abnormalities were also observed including decreases in platelets, neutrophils, and lymphocytes, and some subjects had increased AST and ALT levels. The second dose was less reactogenic, but viremia was detected after both doses. After the second dose, 76% of subjects seroconverted to three serotypes and 71% seroconverted to all four. The DENV3 component was dominant; viremia detected after the first dose was mainly DENV3, all subjects seroconverted to DENV3 after one dose, and neutralizing antibody titers were highest against DENV3. Two formulations of a tetravalent vaccine that contained less DENV3 than previous formulations were then tested in Thai children (Sabchareon et al., 2004). Children 5–12 years of age received three immunizations— the second was given 3–5 months after first, and the third was given 8–12 months after the second. The vaccines were moderately reactogenic and induced symptoms including fever, myalgia, and rash. There were five severe reactions including one DF-like illness. After three doses, 89% and 100% of the recipients seroconverted to all four serotypes. DENV3 was still dominant, as indicated by a high prevalence of DENV3 viremia and high neutralizing antibody titers against DENV3. A planned phase 1b trial to test two formulations of the vaccine in adult Caucasians in Australia was halted after 10 recipients received one dose and developed a mild DF-like syndrome due to the DENV3 component (Kitchener et al., 2006). In an attempt to attenuate DENV3, the vaccine strain was plaque-purified and adapted to Vero cells (Sanchez et al., 2006). The Vero-adapted dengue serotype 3 vaccine, VDV3, was attenuated in vitro and in monkeys and was next tested in 15 volunteers in Hong Kong. All subjects had adverse reactions and the trial was halted. As a balanced immune response was not achieved with these vaccine candidates, they were not pursued further.

9.3. CDC/Inviragen Another live attenuated candidate was developed at the CDC and has been licensed by Inviragen. Chimeric viruses were cloned with the DENV2 PDK-53 vaccine strain developed at Mahidol University as a backbone, and the DENV2 structural proteins were replaced with the structural proteins from DENV1, 3, or 4 to create the tetravalent vaccine, DENVax. Attenuating mutations in PDK-53 are outside of the structural genes

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(Butrapet et al., 2000); therefore, all four chimeric strains should retain the DENV2 PDK-53 attenuation markers. DENV2/DENV1 chimeras were created using the C, M, and E proteins of the Mahidol DENV1 PDK-13 vaccine virus or wild-type DENV1 16007 and were found to be attenuated in vitro and in mice (Huang et al., 2000). DENV2/1, DENV2/3, and DENV2/4 chimeras were created by cloning prM and E from wild-type DENV1 (strain 16007), DENV3 (strain 16562), and DENV4 (strain 1036) into two genetic variants of the DENV2 PDK-53 vaccine virus, or the parental strain, 16681 (Huang et al., 2003). The chimeras retained the DENV2 PDK-53 attenuation markers, including temperature sensitivity, small plaque size in LLC-MK2 cells, lack of neurovirulence in newborn mice, and reduced replication in C6/36 mosquito cells. Monovalent and tetravalent chimeric vaccine (DENVax) formulations were tested in AG129 mice (Brewoo et al., 2012; Huang et al., 2003). Monovalent DENVax-1, 2, or 3 significantly protected against lethal DENV1 or DENV2 challenge. Tetravalent vaccination induced neutralizing antibody responses against all four serotypes and protected against challenge with DENV1 or DENV2. Three different formulations, differing in the dose of each serotype, of the tetravalent chimeric DENVax vaccine were tested in cynomolgus macaques (Osorio, Brewoo, et al., 2011). Monkeys were given two vaccinations 60 days apart. Low-level DENV2 viremia was detected, yet all monkeys developed neutralizing antibodies against all four serotypes after one or two doses. Monkeys also developed a DENV2-specific T-cell response. The most balanced antibody response was observed with the formulation containing 103 PFU of DENV1 and DENV2 and 105 PFU of DENV3 and DENV4. All monkeys were completely protected against challenge with DENV3 or DENV4 30 days after the second immunization, and the high-dose formulation (105 PFU of each serotype) completely protected against DENV1 and DENV2 as well. Based on these results, tetravalent DENVax is being tested in phase 1 clinical trials (Osorio, Huang, Kinney, & Stinchcomb, 2011), and a phase 2 study in healthy volunteers between 1.5 and 45 years of age began in 2011 (Clinicaltrials.gov NCT01511250).

9.4. NIAID/NIH A genetics approach was undertaken by researchers in the Laboratory of Infectious Diseases at the National Institute of Allergy and Infectious

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Diseases (NIAID) with the goal of attenuating the virus without significantly reducing immunogenicity. Reverse genetics was used to introduce deletions, from 30 to 262 nucleotides (nt), into the 30 UTR of DENV4 cDNA (Men, Bray, Clark, Chanock, & Lai, 1996). Mutants that were attenuated in LLC-MK2 cells were selected and tested in rhesus monkeys. Some mutants were attenuated in vivo, in terms of reduced viremia and neutralizing antibody titers, compared with the parental wild-type DENV4 virus. A DENV4 30 nt 30 UTR deletion mutant (rDENV4D30) that was attenuated in monkeys was selected and tested in 20 healthy adults in a phase 1 trial (Durbin et al., 2001). Volunteers received 105 PFU s.c. Low titer viremia was detected in 14 volunteers, and 100% developed neutralizing antibody responses against DENV4. The vaccine was well tolerated: Asymptomatic rash was observed in subjects with viremia, and 5 volunteers had a transient increase in serum ALT levels. The vaccine was attenuated for mosquitoes as well. Compared with the wild-type parental virus, the vaccine strain was restricted in infecting A. aegypti midgut and in disseminating from the midgut to the salivary gland. In addition, vaccine recipients did not transmit the virus to A. albopictus mosquitoes (Troyer et al., 2001). The rDENV4D30 vaccine was further evaluated in phase 2 placebocontrolled trial (Durbin et al., 2005). A dose deescalation was done, and vaccinees (20 per group) received 103, 102, or 101 PFU. All doses were well tolerated and immunogenic. Some recipients developed a mild rash and neutropenia, but only 1/60 had an elevated serum ALT level. Almost all recipients (97%) seroconverted (defined as a fourfold increase in neutralizing antibody titers) to DENV4 after a single inoculation. These results supported the inclusion of this vaccine strain in a tetravalent formulation. In parallel, DENV4 mutants were generated in an attempt to derive a vaccine candidate that would not induce the hepatotoxicity observed in volunteers receiving 105 PFU of the rDENV4D30 vaccine (Hanley, Lee, Blaney, Murphy, & Whitehead, 2002). Five attenuating mutations were introduced into rDENV4D30 and were tested in SCID-HuH-7 mice and rhesus monkeys (Hanley et al., 2004). One mutant (rDENV4D30200,201) that was significantly attenuated in rhesus monkeys compared with wild-type DENV4 and rDENV4D30 was selected and tested in a phase 1 trial (McArthur et al., 2008). Volunteers received 105 PFU of rDENV4D30-200,201, which was well tolerated; no ALT elevations or viremia were detected, and all 20 volunteers seroconverted after one dose. Toward the goal of creating a tetravalent vaccine, the group introduced the 30 nt 30 UTR deletion into a full-length DENV1 cDNA clone to create

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rDENV1D30 (Whitehead, Falgout, et al., 2003). This virus was attenuated similarly to rDENV4D30 in rhesus monkeys and completely protected against DENV1 challenge, with no viremia detected in vaccinated monkeys. A phase 1 study of the rDENV1D30 DENV1 vaccine was conducted in adult volunteers (Durbin et al., 2006a). Twenty vaccinees received 103 PFU, which was well tolerated. The most common adverse events were an asymptomatic rash and neutropenia, which were observed in 40% and 45% of the recipients, respectively. Viremia was detected in 9/20 subjects and was slightly higher titer than rDENV4D30-induced viremia. The vaccine was highly immunogenic, as 95% of the recipients seroconverted and had neutralizing antibodies against DENV1 that lasted for the 6 months of the study. A subsequent study found a second immunization with rDENV1D30 4 or 6 months after the first dose was safe; however, it was not infectious and it did not boost antibody titers, indicating the first vaccination induced sterilizing immunity that lasted for at least 6 months (Durbin, Whitehead, et al., 2011). For DENV3, unlike DENV1 and DENV4, the D30 mutation was not sufficiently attenuating. rDENV3D30 was not attenuated in mosquitoes, SCID-HuH-7 mice, or monkeys (Blaney, Hanson, Firestone, et al., 2004). As an alternate attenuating strategy, the DENV3 M and E proteins were cloned into the rDENV4 backbone to create rDENV3/4(ME) and rDENV3/4D30(ME) chimeras, which were attenuated in mice, mosquitoes, and rhesus monkeys. The two chimeras were comparably attenuated, indicating the D30 mutation did not confer additional attenuation. No viremia was detected in immunized monkeys yet all seroconverted, and they were protected against challenge with the parental DENV3. Additional DENV3 vaccine candidates were created, including rDENV3D30/31, which contains an additional 31 nt deletion in the 30 UTR, and rDENV3-30 D4D30, which was created by replacing the entire 30 UTR of rDENV3 with the 30 UTR of rDENV4D30 (Blaney et al., 2008). Both viruses were attenuated in SCID-HuH-7 mice and rhesus monkeys; immunization of monkeys resulted in neutralizing antibody responses and protection from wild-type DENV3 challenge. rDENV3D30/31 was also attenuated for mosquitoes. Similarly, the D30 mutation in DENV2 did not sufficiently attenuate the virus to be considered for a human vaccine. rDENV2D30 was attenuated in SCID-HuH-7 mice and not infectious for A. aegypti mosquitoes, but was only slightly attenuated in rhesus monkeys compared with rDENV2 and wild-type DENV2 (Blaney, Hanson, Hanley, et al., 2004). To further

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attenuate rDENV2D30, a point mutation in NS3 that had been previously demonstrated to attenuate rDENV4D30 (Hanley et al., 2004) was made. rDENV2D30-4995 was found to be further attenuated in SCID-HuH-7 mice compared with rDENV2D30. In other approaches to create DENV2 vaccine candidates, the structural genes (CME or ME) of DENV2 were cloned into rDENV4 or rDENV4D30 (Whitehead, Hanley, et al., 2003). Chimeras (without the D30 deletion) were attenuated in SCID-HuH-7 mice, mosquitoes, and rhesus monkeys. rDENV2/4D30(CME) was more attenuated than rDENV2/4(CME) and did not replicate in monkeys; rDENV2/4(ME) was similarly attenuated when cloned with or without the D30 deletion. Due to its attenuation and immunogenicity, rDENV2/4D30(ME) was deemed a promising vaccine candidate and was tested in 20 DENV-naive adults (Durbin et al., 2006b). The volunteers received 103 PFU, which was safe and immunogenic. A mild asymptomatic rash and mild neutropenia were observed in some subjects. All volunteers seroconverted to DENV2 and neutralizing antibodies were maintained for the 6 months of the study. Low magnitude viremia was detected in 11 volunteers, and the D30 mutation was unchanged in the viremic volunteers, confirming that the mutation was stable. Three tetravalent vaccine formulations were tested in animals (Blaney et al., 2005). TV-1 was composed of 105 PFU of the four D30 viruses; TV-2 contained 105 PFU of rDENV1D30, rDENV4D30, rDENV2/ 4D30, and rDENV3/4D30; and TV-3 contained 105 PFU of rDENV1D30, rDENV2D30, and rDENV4D30, and 106 PFU of rDENV3/4D30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice, and all three formulations were attenuated in rhesus monkeys. TV-1- and TV-3-immunized monkeys all seroconverted after one dose, whereas TV-2 required a booster immunization to achieve high titers against DENV2 and DENV3. Boosting at 4 months, but not 1 month, increased neutralizing antibody titers. A single dose of TV-2 protected against challenge with DENV1, 3, and 4, and two doses protected from challenge with DENV2. Two doses of TV-3 also completely protected against DENV2 challenge. These results supported testing TV-2 and TV-3 in clinical trials. A phase 1 trial investigated a single dose of four different formulations of a live tetravalent vaccine in 113 flavivirus-naive volunteers (Durbin et al., 2013). The vaccines were well tolerated, with no SAE or fever induced in any subject. The only side effect that occurred with a significantly higher incidence in vaccinees compared with placebo recipients was an

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asymptomatic rash observed in 64.2% of vaccinees. Low-level viremia was detected in most (73%) recipients, and in the majority (64%) of viremic subjects, one serotype of virus was detected. One dose of each formulation induced a trivalent or better neutralizing antibody response in 75–90% of the volunteers. Black race correlated with lower seropositivity and a reduced incidence of viremia, which was interesting as the black race is associated with resistance to DENV infection (Blanton et al., 2008; Halstead et al., 2001). Formulation TV003, containing 103 PFU each of rDENV1D30, rDENV2/4D30, rDENV3D30/31, and rDENV4D30, induced the most balanced neutralizing antibody response and a trivalent or better response in 90% of recipients after a single dose. However, only 50% of recipients seroconverted to DENV2. Phase 1 trials testing two different formulations (TV003 and TV005, which contains a higher dose of rDENV2/4D30 than TV003) of the tetravalent vaccine (TetraVax-DV) began in 2011 in flavivirus-naive adults (Clinicaltrials.gov NCT01436422) and flavivirusimmune adults (NCT01506570). A phase 2 trial in Brazil is planned. The safety and immunogenicity of vaccination of DENV-immune individuals was investigated (Durbin, Schmidt, et al., 2011). Individuals who had received a monovalent DENV vaccine were given a second immunization with a heterotypic monovalent attenuated vaccine 0.6–7.4 years later. Replication and safety were comparable in immunized and naive volunteers. In contrast to naive individuals, most volunteers who received a second DENV vaccination developed a broad, heterotypic neutralizing antibody response. However, in one cohort, preexisting DENV2 immunity impaired seroconversion to a DENV1 vaccine. The D30 vaccines have a number of advantages. Attenuation is due to deletions in 30 UTR, so both T-cell and antibody responses can be induced against wild-type DENV structural and nonstructural proteins. Deletion mutants are more stable than point mutations and therefore these strains are unlikely to revert to wild-type viruses. In addition, as the four vaccine strains contain the same deletion, potential recombination between the four viruses will not lead to reversion of wild-type virus.

9.5. DENV Chimeras Chimeric viruses were constructed using recombinant DNA technology (Bray & Lai, 1991). Using the cDNA of DENV4, the C, prM, and E genes were replaced with structural genes from DENV1 or DENV2. The DENV2/DENV4 chimera was attenuated, providing a proof of

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concept for producing attenuated, chimeric dengue vaccine strains. The chimeras were attenuated in rhesus monkeys (Bray, Men, & Lai, 1996). Monkeys vaccinated with DENV1/DENV4 or DENV2/DENV4 chimeras developed neutralizing antibodies against DENV1 and DENV2, respectively, and were protected against challenge with DENV1 or DENV2. Monkeys immunized with an equal mixture of DENV1/DENV4 and DENV2/DENV4 chimeras were protected from challenge with DENV1 or DENV2.

9.6. Acambis/Sanofi Pasteur (ChimeriVax) Research begun at the NIH and St. Louis University (Bray & Lai, 1991; Chambers, Nestorowicz, Mason, & Rice, 1999) and continued at Acambis (now part of Sanofi Pasteur) resulted in the creation of chimeric viruses containing the DENV structural proteins on the YF 17D backbone. The YF 17D vaccine backbone was selected because of the safety, long duration of immunity, and rapid onset of immunity induced by the YFV 17D vaccine, which has been used for over 60 years. To create a DENV2 chimeric strain, ChimeriVax-DENV2, the prM and E genes from the DENV2 PUO218 strain were cloned into a cDNA infectious clone of 17D (Guirakhoo et al., 2000). ChimeriVax-DENV2 was nonneurovirulent for 4-week-old mice and was genetically stable. Inoculation of rhesus monkeys resulted in brief viremia, a neutralizing antibody response, and complete protection from challenge with wild-type DENV2. DENV1, DENV3, and DENV4 chimeras were then constructed using the prM/E sequences from DENV clinical isolates (Guirakhoo et al., 2001). The chimeras replicated to high titers in Vero cells, were nonneurovirulent in 4-week-old mice, and were immunogenic in rhesus monkeys. Monkeys immunized with a tetravalent vaccine (ChimeriVax-DENV1–4) seroconverted to all four viruses after one dose (except 1 of 6 did not seroconvert to DENV4). Preexisting immunity from YF 17D vaccination (YF-VAX) did not significantly affect the neutralizing antibody response. A phase 1 trial found the safety profiles of YF-VAX and ChimeriVaxDENV2 were similar, and no SEA were observed (Guirakhoo et al., 2006). All recipients seroconverted to DENV2 after vaccination with 5 log10 PFU of the vaccine, and preexisting immunity to YFV did not interfere with DENV2 seroconversion. In fact, all YFV-immune subjects also seroconverted to the other DENV serotypes, whereas seroconversion to the other serotypes was low in YFV-naive subjects.

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Vaccine lot viruses of ChimeriVax-DENV1–4 were made using current good manufacturing practice (cGMP) (Guirakhoo et al., 2004). Neurovirulence was tested in cynomolgus monkeys after i.c. inoculation with the tetravalent vaccine and was found to be reduced compared with YF-VAX vaccination. Vaccine induced-protection was also tested in cynomolgus monkeys. Monkeys received a single immunization s.c. with a high or low dose (3 or 5 log10 PFU of each vaccine strain) of the tetravalent vaccine and were challenged with wild-type DENV strains 6 months later. All monkeys seroconverted to all four serotypes, and 22/24 were protected from challenge. Viral interference was studied in cynomolgus monkeys vaccinated with the chimeric vaccine strains (Guy et al., 2009). Interference was observed in monkeys given equivalent doses of each chimeric vaccine strain, with DENV4 dominating, and several approaches were investigated to overcome the interference. Immunization with bivalent vaccines at separate sites with different draining lymph nodes, preexisting flavivirus immunity, decreasing the dose of the dominant serotype, and boosting at 1 year all improved the development of a balanced antibody response. The ChimeriVax strains were highly attenuated for A. albopictus and A. aegypti mosquitoes in terms of infection and dissemination (Higgs et al., 2006; Johnson et al., 2004). Growth of the vaccine strains was also studied in human myeloid DC and hepatic cell lines in vitro (Brandler et al., 2005). The vaccine strains were not attenuated for replication in DC compared with wild-type DENV or YF 17D but replicated to lower titers than YF 17D in HepG2 and THLE-3 cells (but not HuH-7 cells), suggesting the vaccine strains may be less hepatotropic than YF 17D and therefore have less risk of inducing the hepatic failure that has been occasionally been observed after YF 17D vaccination. Importantly, the chimeric viruses were found to be genetically and phenotypically stable throughout the manufacturing process (Mantel et al., 2011; Monath et al., 2005). A tetravalent vaccine (TDV), containing 5 log10 tissue culture infective doses (TCID50) of each recombinant serotype, was tested in flavivirus-naive adults (Morrison et al., 2010). Two groups of 33 volunteers received the vaccine at 0, 4, and 12–15 months or saline for first injection followed by two doses of the TDV. The vaccine was safe, with no vaccine-related SAE. Low-level viremia was observed primarily after the first dose and was mainly DENV4. Each dose of the vaccine increased neutralizing antibody titers, and all volunteers receiving three doses seroconverted to all four serotypes. The TDV was tested in children and adolescents (2–5, 6–11, or

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12–17 years of age) and adults in a nondengue endemic area (Mexico City) (Poo et al., 2010). Subjects received three doses at 0, 3.5, and 12 months or YF-VAX followed by two doses of TDV. The vaccine was safe, with no vaccine-related SAE reported, and immunogenic. Seropositivity against each serotype after three doses of TDV ranged from 77% to 92% and from 85% to 94% in the YF/TDV recipients. A phase 1 trial was then conducted in the Philippines, a dengue-endemic country (Capeding et al., 2011). Children, adolescents, and adults received three doses of the TDV vaccine at 0, 3.5, and 12 months. Reactogenicity was similar in adults and children, with headache, injection site pain, fever, and myalgia most frequently reported. A low level of viremia (primarily DENV4) was detected in some recipients, most frequently after the first dose. After three doses, 100% of adults seroconverted to all four serotypes, and seroconversion ranged from 83% to 100% in children/adolescents. CD8þ T-cell responses against YF 17D NS3 and DENV-specific CD4þ T-cell responses were detected in volunteers vaccinated with the tetravalent chimeric vaccine (Guy et al., 2008). IFN-g dominated over TNF for both CD4þ and CD8 þ T-cell responses. After one vaccine dose, responses were serotype-specific and dominated by DENV4 but broadened after a booster immunization. A phase 2a study was designed to examine the safety and efficacy of TDV vaccination in flavivirus-immune individuals (Qiao, Shaw, Forrat, WartelTram, & Lang, 2011). One dose of the TDV was given to persons who had been vaccinated with monovalent live attenuated DENV1 or DENV2 vaccines, or YF-VAX 1 year prior, or flavivirus-naive adult volunteers. Prior flavivirus immunity did not increase reactogenicity or the incidence of viremia, but it did increase immunogenicity. In flavivirus-naive recipients, the neutralizing antibody response after one dose of TDV was directed predominantly to DENV3 and DENV4, whereas in DENV1-, DENV2-, and YF-primed recipients a more balanced neutralizing antibody response was observed. A phase 2 study was conducted in 199 children (2–11 years of age) in Peru who had varying levels of preexisting flavivirus immunity from YF vaccination (Lanata et al., 2012). Children received 3 doses of TDV at 0, 6, and 12 months. The reactogenicity observed was similar to previous studies; injection site pain, headache, malaise, fever were most commonly reported and decreased with subsequent vaccinations. No vaccine-related SAE were reported. Viremia was detected in 44% of the 97 individuals tested and was mainly DENV4. Vaccination was immunogenic as well and resulted in 94% of recipients seroconverting to all four DENV serotypes with comparable neutralizing antibody titers to the four serotypes.

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Results of a phase 2b study of TDV were reported in 2012. The CYDTDV vaccine was given to children 4–11 years of age in dengue-endemic Thailand (Sabchareon et al., 2012). The primary analysis included data from 2452 vaccine recipients and 1221 controls. More than 90% of the children had preexisting antibodies against DENV or JEV, and 70% were seropositive against at least one DENV serotype. Three injections of the vaccine were given at 0, 6, and 12 months, and the subjects were followed for 13 months after the last dose. The vaccine was safe with no vaccinerelated SAE and immunogenic. Neutralizing antibody titers increased after one dose and increased further after the second and third doses and then decreased 1 year later. However, the overall protective efficacy in preventing symptomatic dengue infection was only 30.2%. The efficacy for the individual serotypes was 55.6% for DENV1, 9.2% for DENV2, 75.3% for DENV3, and 100% for DENV4. DENV2 was the most common infecting serotype, which skewed the overall efficacy. The antibody neutralization data did not correlate with protection, as neutralizing antibody titers (measured by PRNT50) increased after each dose and were highest against DENV2 and DENV3, yet the subjects were not protected against DENV2 infection. The authors suggest in the future performing neutralization studies on cells that express FcR, which are targets of DENV in vivo. The PRNT also does not distinguish between balanced neutralizing antibody responses to the four serotypes, or less protective cross-reactive responses. In addition, antibodies have other functions besides neutralization, including ADCC, which may be important for protection. Another potential reason for the low efficacy includes an antigenic mismatch between the DENV2 vaccine strain and the DENV2 strain that resulted in infections. Finally, the lack of a DENV-specific T-cell response may have contributed to the poor efficacy, as these chimeric vaccines consist of YFV, not DENV, nonstructural proteins, which are the dominant targets of the anti-DENV T-cell response in humans and mouse models (Weiskopf et al., 2013, 2011; Yauch et al., 2010). Despite the disappointing protection observed, the study results were informative and may spur investigations that lead to the identification of correlates of protection. Importantly, the vaccine was safe, with no vaccinerelated SAE induced, and there was no disease enhancement observed in the presence of nonprotective immunity during the short duration of the study. Phase 3 studies involving 30,000 individuals in Latin America and Asia started in 2011 and will provide more data on the efficacy of this vaccine (Clinicaltrials.gov NCT01374516 and NCT01373281).

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10. MOVING FORWARD Years of dengue vaccine research have brought us close to the point of having a licensed vaccine. Although the results of the CYD-TDV phase 2b trial were disappointing, the findings were important in directing future vaccine development and will hopefully lead to the identification of immune correlates of protection. The trial results highlighted the need to study pre- and postvaccination immune responses in both flavivirus-naive and flavivirus-immune individuals in more detail. The lack of efficacy against DENV2 despite neutralizing antibodies measured by PRNT using Vero cells suggests neutralization assays on cell types that express FcR may be more relevant. In addition to examining neutralization, other antibody functions can be studied as well. The titer, class, subclass, and avidity of antibodies specific for E, prM, and NS1 can be determined. The ability of vaccine-induced antibodies to mediate ADCC and fix complement can also be analyzed. The magnitude, breadth, and functionality, including cytokine production and cytotoxicity, of both CD4þ and CD8 þ T-cell responses should also be investigated. As mentioned earlier, recent studies point to an important protective role for CD8 þ T cells in the immune response to DENV. Vaccines that induce robust T- and B-cell responses may prove to be superior to those vaccines that induce robust antibody responses but weak T-cell responses. Overall, the vaccines currently in clinical trials are safe, and no disease enhancement has been observed in vaccinated humans to date. However, long-term studies, both in NHP and humans, are required to ensure waning immunity does not predispose vaccinees to severe dengue disease. The WHO recommends following subjects for approximately 3–5 years after the last vaccination (WHO, 2011). Although no disease enhancement following DENV vaccination has been reported, recent studies of the human antibody response to DENV found prM/M-specific antibodies are broadly cross-reactive and weakly or nonneutralizing (Beltramello et al., 2010; de Alwis et al., 2011; Dejnirattisai et al., 2010), suggesting it may be prudent to minimize the anti-prM antibody response to avoid ADE. Animal models provide the necessary tools for dissecting the mechanisms of vaccine-mediated protection. As some of the vaccine studies discussed earlier suggest that vaccine-induced immune responses differ in flavivirusnaive versus flavivirus-immune individuals, animal models provide the tools to evaluate vaccine-induced immune responses under well-defined naive

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versus immune infection settings. Thus, vaccine-induced immune responses in animal models of dengue disease should be studied in more detail, including analyzing the magnitude and quality of the T-cell responses. The existing murine and NHP animal models can also be improved, and/or new models developed. Manipulating the virus or mouse immune system may lead to more relevant models (Zompi & Harris, 2012). For instance, passaging of DENV though monkeys may result in the isolation of a strain more virulent for monkeys. Mice lacking only the type I IFN receptor may prove to be a more relevant model than AG129 mice. In addition, adoptive transfer studies may be useful for studying subunit and inactivated vaccines. Wild-type mice can be immunized with these nonreplicating vaccines, followed by transfer of immune components from the vaccinated wild-type mice into IFN receptor-deficient mice. The IFN receptor-deficient mouse models serve as a stringent challenge assay, and the adoptive transfer system allows for thorough analysis of vaccine-induced humoral versus cellular response in normal mice. The lack of an adequate animal model for evaluating live attenuated dengue vaccine-induced immune responses has prompted the development of a dengue human challenge model (DHCM). In a recent study, subjects previously vaccinated with the WRAIR/GSK live attenuated tetravalent vaccine (TDV) were challenged with underattenuated DENV strains to evaluate the safety of challenge with the underattenuated strains and to evaluate the relationship between vaccine-induced neutralizing antibody titers and protection (Sun et al., 2013). Subjects who had received the TDV 12–42 months previously, or naive controls, were challenged with underattenuated DENV1 or DENV3. All 5 vaccinated subjects challenged with DENV1 were protected, and 2 of 5 challenged with DENV3 were protected. The 4 naive control recipients developed DF upon challenge. Neutralizing antibody titers correlated with protection in all but 1 subject who was protected from DENV1 challenge despite no detectable neutralizing antibodies. The DENV3 challenge was associated with significant elevations in AST/ALT. This study demonstrated the feasibility of human challenge to evaluate DENV vaccine candidates. A DHCM workshop, sponsored by the WRAIR and the NIH, was held in 2011, and the consensus was that a DHCM could be developed safely, if appropriate challenge strains can be identified and produced under cGMP (Durbin & Whitehead, 2013). Safety is a major concern for a DHCM, as challenge of vaccine recipients with underattenuated strains could put the subjects at risk for developing severe disease. Additionally, there is no approved therapeutic that could be used to

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treat recipients who develop DF or DHF/DSS. However, a DHCM could provide valuable information on the immune response to DENV and potentially lead to the identification of immune correlates of protection. A DHCM could also be useful for selecting vaccine candidates for field studies.

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