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Expert Review of Vaccines

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A recombinant, chimeric tetravalent denguevaccine candidate based on a dengue virusserotype 2 backbone

Jorge E. Osorio, Derek Wallace & Dan T. Stinchcomb

To cite this article: Jorge E. Osorio, Derek Wallace & Dan T. Stinchcomb (2016) A recombinant,chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone,Expert Review of Vaccines, 15:4, 497-508, DOI: 10.1586/14760584.2016.1128328

To link to this article: http://dx.doi.org/10.1586/14760584.2016.1128328

Accepted author version posted online: 04Dec 2015.Published online: 22 Feb 2016.

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REVIEW

A recombinant, chimeric tetravalent dengue vaccine candidate based on adengue virus serotype 2 backboneJorge E. Osorioa, Derek Wallaceb and Dan T. Stinchcombc

aDepartment of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, US; bClinicalDevelopment, Takeda Vaccines Pte. Ltd, Singapore, Singapore; cScientific Affairs and Policy, Takeda Vaccines, Inc., Deerfield, IL, US

ABSTRACTDengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1–4),necessitating tetravalent dengue vaccines that can induce protection against all four DENV.Takeda’s live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuatedDENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 clonedinto the attenuated DENV-2 ‘backbone’. In Phase 1 and 2 studies, TDV was well tolerated bychildren and adults aged 1.5–45 years, irrespective of prior dengue exposure; mild injection-sitesymptoms were the most common adverse events. TDV induced neutralizing antibody responsesand seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that maybe necessary for broad protection against dengue fever.

ARTICLE HISTORYReceived 16 September 2015Accepted 2 December 2015Published online22 February 2016

KEYWORDSDengue; live attenuatedtetravalent vaccine; TDVdevelopment; safety; clinicalimmunology; dengue-seropositive participants;dengue-endemic

Dengue is the most significant mosquito-borne viraldisease in humans, causing an estimated 390 millioninfections annually [1]. In the past 50 years, the recog-nized incidence of dengue fever cases has increased30-fold [2]. Subclinical infections and dengue fever arethe most common manifestations of dengue infection,with either no clinical symptoms or symptoms includ-ing fever, headache, arthralgia, myalgia, retro-orbitalpain, rash, bleeding, thrombocytopenia, or leucopenia[3,4]. A small proportion of patients go on to developsevere life-threatening dengue hemorrhagic fever ordengue shock syndrome [5,6].

Dengue circulates mainly in tropical and subtropicalregions as one of four serotypes (DENV-1–4). Patterns ofserotype predominance and pathogenesis varyannually, even within the same regions [7–17]. Distinctviral genotypes and lineages within these genotypesadd to the complexity of these patterns. Where geno-typing is done, lineage replacement can be observed,with certain genotypes demonstrating greater epidemicpotential [18,19]. Nevertheless, naturally acquiredimmunity to dengue appears to be serotype-specificand lifelong [20,21].

At present, treatment for dengue fever is limited tosupportive care, so safe and effective dengue vac-cines are urgently needed. Tetravalent vaccines arerequired because of the annual and geographic var-iations in circulation and relative prevalence of thefour dengue serotypes and because of concerns

about antibody-dependent enhancement of denguedisease [22]. The risk of severe dengue is increasedduring secondary infections with a heterologous den-gue serotype, prompting the need for a vaccine thatcan induce simultaneous and durable immunity to allfour existing DENV serotypes. Several vaccines are indevelopment; this review concerns the clinical safetyand immunogenicity of Takeda’s live attenuated tet-ravalent dengue vaccine candidate (TDV) (previouslyreferred to as ‘DENVax’ in some earlier publications).

TDV design and development

The construction of TDV has been reviewed previously[23,24]. Briefly, the basis of TDV comprises an attenu-ated DENV-2 virus strain (TDV-2) termed PDK-53,derived by 53 serial passages in primary dog kidneycells of a DENV-2 strain that was isolated from a patientin Thailand [23]. This is accompanied by three chimericviruses containing the pre-membrane (prM) and envel-ope (E) protein genes of DENV-1, -3, and -4 geneticallyengineered into the attenuated TDV-2 genome back-bone (TDV-1, TDV-3, and TDV-4, respectively; Figure 1)[23,25].

Three key mutations in the TDV-2 backbone contri-bute synergistically and individually to result inimpaired replication of all four TDV viruses in vitro, inmice, in nonhuman primates [23,25–27], in carrier Aedesaegypti mosquitoes [26,28], and in humans [29–34]. The

CONTACT Jorge E. Osorio osorio@svm.vetmed.wisc.edu

EXPERT REVIEW OF VACCINES, 2016VOL. 15, NO. 4, 497–508http://dx.doi.org/10.1586/14760584.2016.1128328

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low replicative capacity in humans and mosquitoesensures that the vaccine is highly unlikely to be trans-missible from a vaccinated individual via mosquitoes innature [26]. Since all four TDV components share thethree identical attenuating mutations, recombinationbetween TDV viruses cannot generate more pathogenicviruses, and reversion of all three mutations andconsequent loss of attenuation is unlikely. The TDVcomponents are genetically stable during the manufac-turing process, ensuring retention of the attenuatingmutations [26]. Even if one mutation were to revert towild-type, the other two are sufficient to maintain theattenuated phenotype [35].

Despite the mutations that attenuate both its repli-cation and its neurovirulence, TDV remains immuno-genic and protective against wild-type strains in mice[25]. Mouse studies have shown that neutralizing anti-body titers elicited by the tetravalent formulation weresimilar to the homologous titers elicited by each mono-valent virus component [25]. TDV has been shown toprotect against challenge with wild-type dengueviruses in both mouse [36] and nonhuman primate[27,37] animal models. These studies illustrated TDV’sprotective role via two very different models of dengueinfection: infection by certain strains in interferon (IFN)receptor-deficient (AG129) mice is pathogenic and mayreflect severe disease that manifests in the absence ofinnate immunity [38], while the nonhuman primatemodel measures dengue virus replication and viremiain an intact primate immune background but in theabsence of clinical signs. Finally, in a mouse model,neutralizing antibodies induced by TDV exhibitedbroad cross-neutralizing activity against a number ofcontemporary DENV-4 isolates from different geogra-phical locations, suggesting potential effectiveness ofthe vaccine to protect against different DENV-4 geno-types circulating in endemic areas [36]. TDV and

monovalent TDV-4 or TDV-2 vaccines provided statisti-cally significant protection against mortality and anysigns of morbidity following DENV-4 challenge inAG129 mice [36].

TDV immunogenicity in dengue-naïve andseropositive individuals

TDV was designed to induce humoral and cellularimmune responses to protect against all four dengueserotypes, to allay concerns regarding antibody-dependent enhancement, and to ensure the vaccine iseffective, irrespective of age, prior exposure to dengue,or predominant circulating serotype. The premembraneand envelope proteins unique to each of the four den-gue serotypes are needed to induce the neutralizingantibodies to DENV-1–4 [39]. The conserved nonstruc-tural (NS) proteins within the dengue backbone arerequired to generate T cell-mediated responses to den-gue infection [37,40,41], and antibodies against NS1 areassociated with cross-protective humoral immuneresponses [36,42,43].

Humoral immune responses elicited by TDV

Immunogenicity data from two Phase 1 studies of TDVin healthy, dengue-naïve adults conducted in Colombia[32] and the United States [30] have been previouslyreviewed [24]. Both were randomized, double-blind,placebo-controlled studies in which TDV was adminis-tered twice, 90 days apart, either subcutaneously orintradermally. ‘Low-dose’ and ‘high-dose’ formulationswere evaluated; the high-dose formulation containedapproximately 2.5-fold more TDV-1, 10-fold moreTDV-2, 10-fold more TDV-3, and approximately equalamounts of TDV-4 per dose than the low-dose formula-tion. Both TDV formulations and both routes of delivery

5’- C E NS1 NS3 NS52AprM 2B 4A 4BDENV-2 backbone(TDV-2)

5’- C –3’

–3’

–3’

–3’

E NS1 NS3 NS52AprM 2B 4A 4B TDV-4

5’- C E NS1 NS3 NS52AprM 2B 4A 4B TDV-3

5’- C E NS1 NS3 NS52AprM 2B 4A 4B TDV-1

Figure 1. Genetic structure and design of TDV. Shown are the gene arrangements of the parental DENV-2 PDK-53 backbone(TDV-2), into which the prM and E genes of DENV-1, -3, and -4 were cloned to create the three chimera viruses: TDV-1, -3 or -4.Arrowheads indicate the three pivotal mutations in DENV-2 PDK-53 that resulted in the attenuated phenotype of the vaccineviruses. Different colors indicate the different origins of the prM and E genes in the chimeras.

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induced neutralizing antibodies to all four dengue virusserotypes in both studies [30,32]. TDV consistentlyinduced the highest geometric mean titers (GMTs) toDENV-2, followed by DENV-1, DENV-3, and then DENV-4. Seroconversion rates to the individual serotypes fol-lowed the GMT trends. The second dose marginallyimproved seroconversion for DENV-4, as well as multi-valent seroconversion rates.

In a third Phase I study (termed DEN 104), severaldifferent formulations and dosing schedules were evalu-ated in flavivirus-naïve healthy adults [34]. Two doses ofTDV were given in opposite arms on the same daybecause in preclinical studies, multiple administrationsat the same time rapidly generated protective, multi-valent immune responses [37]. It was hoped that givingtwo doses, one in each arm at the same visit, mightincrease compliance by reducing the number of visitsrequired to achieve the same level of immunogenicity. Adifferent formulation, in which the concentration of TDV-4 was three times higher than in the ‘high-dose’ TDVformulation used in previous studies, was also given todetermine whether increasing the amount of TDV-4would increase the vaccine’s immunogenicity againstthis serotype [34]. Finally, a one-tenth TDV dose wasalso evaluated to assess the impact of any reduction inpotency that might occur during vaccine storage.

The participants were injected subcutaneously onDays 0 and 90 at three centers in the United States.The dosing schedules and the composition of the dif-ferent formulations are described in Tables 1 and 2 andthe notes below them.

As in the previous Phase I studies [30,32], the stron-gest neutralizing antibody responses (Table 1) and thehighest serotype-specific seroconversion rates (Table 2)were elicited by the backbone vaccine virus, TDV-2,followed by TDV-1 and -3; TDV-4 generated the lowestresponses. The booster dose at Day 90 did not statisti-cally significantly increase neutralizing antibody GMTsto higher levels than those observed at Day 30 for anyof the DENV in any of the groups (Table 1).

Double administration on the same day did notstatistically significantly increase DEN-4 GMTs or ratesof seroconversion to multiple serotypes compared withthe two-dose (Day 0, Day 90) schedule (compare datafor Groups 1 and 2 and Groups 4 and 5 in Tables 1 and2). Increasing the concentration of TDV-4 slightlyincreased TDV’s DENV-4 immunogenicity in terms ofseroconversion rates and GMTs, but at the expense ofDEN-1 seroconversion rates and neutralizing antibodies(compare data for Groups 1 and 4 in Tables 1 and 2).Diluting TDV 10-fold had little impact on levels of GMTsto any serotype (see Group 6 in Table 1), which may

Table 1. Geometric mean titers (GMTs) observed 30 days after each dose with different dosing schedules and formulations in TDVPhase 1 study DEN 104l [34].

Dosing schedule and formulation(No. of patients)

DEN-1 DEN-2 DEN-3 DEN-4

Day 0 Day 30 Day 120 Day 0 Day 30 Day 120 Day 0 Day 30 Day 120 Day 0 Day 30 Day 120

Group 1D01 TDV† + PD90 TDV(n = 25)

6 429 223 5 6412 1598 5 62 51 5 8 12

Group 2D0 2TDVD90 P(n = 25)

7 474 137 5 4843 1479 5 151 42 5 19 9

Group 3D0 2TDVD90 TDV(n = 24)

5 370 216 5 6274 1894 5 70 57 5 12 12

Group 4D0 TDVH4 + PD90 TDVH4 + P(n = 21)

6 46 39 5 4954 1402 5 47 30 5 46 23

Group 5D0 2TDVH4D90 2TDVH4(n = 21)

5 69 65 5 5408 1254 5 62 51 5 22 26

Group 6D0 0.1TDVD90 0.1TDV(n = 24)

7 386 180 5 7035 1356 5 37 26 5 17 13

†TDV formulation formerly described as the ‘high-dose’ formulation, i.e. TDV-1: 2×104 plaque-forming units (PFU); TDV-2: 5×104 PFU; TDV-3: 1×105 PFU; TDV-4: 3×105 PFU.

D0, first study injection(s) on Day 0; D90, second study injection(s) on Day 90; TDV, tetravalent dengue vaccine formulation; 2TDV, injection of TDV dose inboth arms on the same visit; TDVH4, TDV formulation containing three-fold higher TDV-4 (i.e. TDV-1: 2×104 PFU; TDV-2: 5×104 PFU; TDV-3: 1×105 PFU;TDV-4: 1×106 PFU); 2TDVH4, injection of TDVH2 dose in both arms on the same visit; 0.1TDV, one-tenth dose TDV (i.e. TDV-1: 2×103 PFU; TDV-2: 5×103

PFU; TDV-3: 1×104 PFU; TDV-4: 3×104 PFU); P, placebo (phosphate-buffered saline).

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suggest that the reductions in overall titer of the liveattenuated vaccine that might occur during its naturalshelf life should not have any meaningful impact onclinical immunogenicity.

The randomized, double-blind, placebo-controlledPhase 2 study, DEN 203, was the first evaluation ofTDV in dengue-exposed populations that includedadults and children (as young as 1.5 years old) livingin four geographically dispersed dengue-endemiccountries: Puerto Rico, Colombia, Singapore, andThailand [33]. The first stage of the study designinvolved an age-descending phase during whichhealthy participants aged from 45 down to 1.5 yearswere sequentially enrolled and randomized to receiveTDV or phosphate-buffered saline placebo in four age-descending groups, once safety had been establishedin the previous, older group. The second stage was anexpansion phase, in which a single group of childrenaged 1.5–11 years were enrolled and randomized toreceive TDV or placebo once safety had been estab-lished in all the age groups from Stage I. All participantswere injected subcutaneously on Days 0 and 90 withthe TDV formulation formerly described as the ‘high-dose’ formulation, that is TDV-1: 2×104 PFU; TDV-2:5×104 PFU; TDV-3: 1×105 PFU; TDV-4: 3×105 PFU, orPBS placebo. All participants are being followed for

3 years for severe adverse events and passive surveil-lance for dengue disease; long-term immunogenicity isbeing assessed in Stage 1 participants. The study popu-lation included participants who were seropositive forone or multiple DENV at enrollment, with baseline ser-opositivity to one or more serotypes being more pre-valent among adults (84% overall) than children (39%)in these endemic countries, as expected.

TDV administration induced robust neutralizing anti-body responses (Table 3) and seropositivity increased inall age groups after TDV vaccination. As in all the PhaseI studies, after one or two doses of TDV, GMTs ofneutralizing antibodies were highest for DEN-2, fol-lowed by DEN-1, DEN-3, and DEN-4 (Table 3), in bothseronegative and seropositive participants [33]. TDVinduced higher levels of neutralizing antibodies to allfour DENV in the participants who were initially sero-positive for dengue [33], consistent with findings thatthe preimmunity to flaviviruses (including dengue) hasa priming effect on both the humoral and cellularresponse to subsequent dengue vaccination [44–46].

At Day 120, that is 30 days after the second TDVdose, the seropositivity rates for DENV-1–3 were >95%in each of the five study groups [33]. The DENV-4seropositivity rate was slightly lower, varying from73% to 100% in all age groups among the Stage I

Table 2. Seroconversion rates to DENV-1–4 observed after each dose with different dosing schedules and formulations in TDV Phase1 DEN 104 study [34].

Dosing schedule and formulation (No. of patients)

DENV-1 DENV-2 DENV-3 DENV-4

Day 30 Day 120 Day 30 Day 120 Day 30 Day 120 Day 30 Day 120

Group 1D01 TDV† + PD90 TDV(n = 25)

100 100 96 100 84 100 36 60

Group 2D0 2TDVD90 P(n = 25)

95.8 95.8 100 100 100 95.8 58.3 33.3

Group 3D0 2TDVD90 TDV(n = 24)

100 95.7 100 100 87.5 91.3 33.3 52.2

Group 4D0 TDVH4 + PD90 TDVH4 + P(n = 21)

81 84.2 100 100 85.7 94.7 81 73.7

Group 5D0 2TDVH4D90 2TDVH4(n = 21)

89.5 94.1 100 100 94.7 94.1 57.9 76.5

Group 6D0 0.1TDVD90 0.1TDV(n = 24)

100 95.8 95.8 95.8 66.7 83.3 29.2 41.7

†TDV formulation formerly described as ‘high-dose’ formulation, i.e. TDV-1: 2×104 plaque-forming units (PFU); TDV-2: 5×104 PFU; TDV-3: 1×105 PFU; TDV-4:3×105 PFU.

D0, first study injection(s) on Day 0; D90, second study injection(s) on Day 90; TDV, tetravalent dengue vaccine formulation; 2TDV, injection of TDV dose inboth arms on the same visit; TDVH4, TDV formulation containing three-fold higher TDV-4 (i.e. TDV-1: 2×104 PFU; TDV-2: 5×104 PFU; TDV-3: 1×105 PFU;TDV-4: 1×106 PFU); 2TDVH4, injection of TDVH2 dose in both arms on the same visit; 0.1TDV, one-tenth dose TDV (i.e. TDV-1: 2×103 PFU; TDV-2: 5×103

PFU; TDV-3: 1×104 PFU; TDV-4: 3×104 PFU); P, placebo (phosphate-buffered saline).

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participants and 94.3% in the Stage II expansion groupof 1.5–11 year olds. The first TDV dose had little impacton the number of adults who became seropositive tomultiple viruses (see Day 28 data for Group 1 inTable 4), most likely because 79% of the TDV-vacci-nated adults were already seropositive for all fourDENV at enrollment. However, the vaccine demon-strated a much greater immunogenic impact amongthe younger age groups, with almost three-quarters ofthe 1.5–11-year-old expansion group becoming seropo-sitive to all four serotypes after a single dose (Group5 Day 28 data in Table 4). This proportion increased to93% after two doses (versus 30% in the placebo group).

In summary, TDV induces neutralizing antibodyresponses to all four DENV in adults and children asyoung as 1.5 years, even after a single dose, irrespectiveof prior dengue exposure. These humoral immunogeni-city findings with different formulations and dosingstrategies were used to inform ongoing and subse-quent clinical studies of TDV.

Cell-mediated immune responses elicited by TDV

Data are emerging to suggest that neutralizing anti-body titers, previously thought to be a correlate ofprotection in dengue infection, may not be sufficientto predict or induce complete vaccine efficacy [41,47–49]. CD8+ T cells are thought to play a key role incontrolling viral infections. They recognize viral epi-topes in the context of human leukocyte antigen classI molecules on the surface of infected cells, and follow-ing activation, they kill cells directly or via secretion ofcytokines such as IFN-γ and TNF-α [49]. MultifunctionalCD8+ T cell responses are thus an important compo-nent of protection following dengue infection.

With regard to cell-mediated immunity, differentDENV serotypes are associated with distinct immuno-dominance patterns. DENV-1, DENV-2, and DENV-4 allelicit a CD8+ T cell response that is mainly focused onnonstructural proteins (primarily NS3, NS4b, and NS5).By contrast, DENV-3 elicits CD8+ T cell responses that

Table 4. Percentage of seropositivity to multiple serotypes observed at baseline and 30 days after each dose in the different agegroups in TDV Phase 2 DEN 203 study [33].

Studyday

Number ofserotypes

Stage I Stage II

Group 1 (21–45 years) Group 2 (12–20 years) Group 3 (6–11 years) Group 4 (1.5–5 years) Group 5 (1.5–11 years)

TDV(n = 24)

Placebo(n = 14)

TDV(n = 14)

Placebo(n = 14)

TDV(n = 14)

Placebo(n = 14)

TDV(n = 14)

Placebo(n = 14)

TDV(n = 159)

Placebo(n = 53)

Day 0 Naïve 21 7 32 7 57 82 70 92 58 45Monovalent 0 7 5 7 14 0 13 8 9 15Bivalent 0 0 5 0 0 0 4 0 5 4Trivalent 0 14 5 0 10 12 0 0 5 6Tetravalent 79 71 55 86 19 6 13 0 22 30

Day 28 Naïve 0 7 9 7 0 71 0 92 0 51Monovalent 0 0 0 7 0 12 0 8 1 9Bivalent 4 7 9 0 0 0 9 0 5 6Trivalent 17 7 23 7 14 6 26 0 21 6Tetravalent 79 79 59 79 86 12 65 0 72 28

Day 120 Naïve 0 0 0 14 0 59 0 85 0 51Monovalent 0 7 0 0 0 18 0 0 0 9Bivalent 0 7 5 0 0 0 0 0 1 8Trivalent 8 21 23 14 14 6 0 0 4 2Tetravalent 88 64 73 71 86 18 96 15 93 30

TDV, tetravalent dengue vaccine formulation containing TDV-1: 2×104 PFU; TDV-2: 5×104 PFU; TDV-3: 1×105 PFU; TDV-4: 3×105 PFU.

Table 3. Geometric mean titers (GMTs) observed 30 days after each dose in the different age groups in TDV Phase 2 DEN 203study [33].

Study group (Age)Study treat-ment(N at baseline)

DEN-1 DEN-2 DEN-3 DEN-4

Day 0 Day 28 Day 120 Day 0 Day 28 Day 120 Day 0 Day 28 Day 120 Day 0 Day 28 Day 120

Stage I: Group 1 (21–45 years) TDV (n = 24) 334 1157 1187 13 2314 2410 228 538 630 77 131 210P (n = 14) 487 431 390 12 195 290 276 238 290 59 66 42

Stage I: Group 2 (12–20 years) TDV (n = 22) 103 905 1300 16 1727 1754 75 325 341 21 57 83P (n = 14) 200 205 164 9 431 410 283 305 290 57 46 69

Stage I: Group 3 (6–11 years) TDV (n = 21) 13 377 552 7 2357 983 13 315 377 8 61 65P (n = 17) 11 11 14 546 14 17 8 9 14 6 7 14

Stage I: Group 4 (1.5–5 years) TDV (n = 23) 14 395 582 5 1135 460 9 130 196 7 27 41P (n = 13) 5 5 5 1 5 5 5 5 5 5 5 5

Stage II: Group 5 (1.5–11 years) TDV (n = 159) 20 522 701 14 1497 593 17 207 324 10 45 75P (n = 53) 29 29 30 18 16 16 24 19 19 11 11 11

TDV, tetravalent dengue vaccine formulation containing TDV-1: 2×104 PFU; TDV-2: 5×104 PFU; TDV-3: 1×105 PFU; TDV-4: 3×105 PFU; P, placebo (phosphate-buffered saline).

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target both structural (C, M, and E) and NS proteins [41].Furthermore, it has been suggested that protectionagainst DENV-2 may be more T cell dependent thanthe other serotypes [41].

The T cell responses elicited by TDV were investi-gated in flavivirus-naïve adults who took part in a US-based Phase 1 study [30] 3 months after the primaryimmunization and 14 and 180 days after the secondimmunization given on Day 90 [50]. TDV elicited CD8+ Tcells that recognized mainly the NS3, as well as NS5 andNS1 proteins of DENV-2. These T cells were predomi-nantly multifunctional, producing IFN-γ and TNF-α.Importantly, these multifunctional DENV-2 reactive Tcells were cross-reactive in that they could also recog-nize NS1/3/5 peptides from DENV-1,-3, and -4. ThisCD8+ T cell response to TDV was evident after primaryvaccination, was not increased by the booster vaccina-tion, and was detectable 6 months after the lastimmunization.

In summary, the use of DENV-2 as a backbone givesTDV the capacity to elicit multifunctional, cross-reactive,and durable CD 8+ T cell-mediated immune responses[50] that may contribute to protection against dengue[41,49,51,52].

Ongoing and planned Phase 2 and 3 clinical studieshave incorporated analyses of cell-mediated immunityto enable more complete characterization of TDV’simmunogenicity and its potential for efficacy.

TDV safety in dengue-naïve and previouslyexposed individuals

In the Phase 1 study, DEN 104 conducted in a US-basedcohort of healthy, flavivirus-naive adults [34], all doses,dosing schedules, and formulations of TDV were gen-erally well tolerated. Neither simultaneous administra-tion of two doses of TDV in each arm nor theformulation that contained three-fold more of theTDV-4 component led to higher incidences of treat-ment-related unsolicited AEs. Mild injection-site pain,headache, fatigue, and myalgia were the most commonAEs after both doses, consistent with the earlier Phase Istudies [30,32]. Interestingly, the frequency of AEs wasnot substantially lower in participants who received theone-tenth dose formulation. The incidence of AEs waslower after the second dose in all formulation anddosage groups.

Three patients reported symptoms after the primaryimmunization that led to nonadministration of the sec-ond dose. One participant who had received TDV inboth arms (Group 4) had severe muscle pain and jointpain on Day 9 and reported feeling feverish and havingchills on that day; the AEs resolved within 2 days after

treatment with paracetamol. Another participant whoreceived the one-tenth TDV dose had nonsevere jointswelling and joint pain on Day 16; the participantreceived a 6-day course of ibuprofen, and these AEsresolved within 8 days. A third participant, also in theone-tenth dosing group, had nonsevere photophobiaon Day 15, which resolved within 4 days. However, theycontinued with protocol safety evaluations and immu-nogenicity assays until the end of the study with nofurther incident.

Because the Phase 2 DEN 203 study of TDV involvedthe first use of TDV in children and adolescents, and itsfirst use in volunteers previously exposed naturally todengue, a strict safety-based age-descending protocolwas followed when first administering the vaccine tosuccessively younger cohorts of patients. Only after theData Safety Monitoring Board had reviewed 28-daysafety data from all the older age groups and at least12 vaccinees aged 1.5–5 years was TDV administered inthe expansion group of 212 children aged 1.5–11 years [33].

Seropositivity for dengue at baseline varied widelywithin the study groups, with 84% of 21–45-year-oldsbeing seropositive for any dengue serotype, comparedwith 39% of 1.5–11-year-olds. Nevertheless, no newsafety signals were detected in this dengue-exposedpopulation from the primary immunization until30 days after the second dose (Day 120). No partici-pants withdrew from this study due to adverse events,nor did any deaths occur. Although SAEs were reported;none was vaccine-related and all resolved withoutsequelae. No hematology or chemistry-related AEswere reported, nor were any clinically significant meanchanges from baseline observed for any hematology orchemistry laboratory parameters.

Unsolicited treatment-related AEs were observed inthe same proportion of TDV- and placebo-treated par-ticipants (7.2% of each study group). As in the Phase Istudies, the most frequent unsolicited AEs were head-ache (3.2% of TDV-vaccinees and 2.7% of placebo-trea-ted participants), injection-site pain (2.4% of TDV- and0% of placebo-treated participants), and pyrexia (1.6%of TDV- and 2.7% of placebo-treated participants). Rashwas observed infrequently – by 0.6–4.5% of TDV-vacci-nated participants among study groups aged <20 yearsand by 1.8–7.7% of placebo-injected participants in thesame age groups.

Solicited (i.e. self-reported) injection-site reactionswere more common in the TDV (37.3%) than in theplacebo groups (9.9%). The incidences showed no rela-tion to age; mild injection-site pain was reported by46% of TDV-treated participants aged 12–45 years and35% of children aged 1.5–11 years. The frequency and

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severity of systemic self-reported AEs, however, werecomparable between TDV and placebo groups, andwere mostly mild. The most common were headache(27.3% with TDV versus 29.7% with placebo), fever(18.5% with TDV versus 19.8% with placebo), and mus-cle pain (17.5% with TDV versus 17.1% with placebo).

In summary, TDV has been generally well-toleratedfollowing at least one administration in healthy children(N = 225) and adults (N = 291) who were either sero-negative (N = 400) or seropositive (N = 116) for dengue[30,32–34]. The most common AEs associated with TDVare mild and transient local injection site reactions.Despite careful observation, rashes were not observedfollowing TDV administration at any higher frequencythan with placebo administration in either adults orchildren. No significant changes in neutrophil, platelet,or white blood cell counts were observed in any of thePhase 1 or 2 clinical studies of TDV to date.

TDV replication

TDV replication findings in Phase 1 study DEN 104 weresimilar to those in other Phase 1 studies [24]. Transientviral RNA indicative of TDV-2 replication was the mostcommonly detected and occurred with all formulationsand dose schedules, mostly within the first 2 weeksafter the first dose [34]. TDV-3 and TDV-4 RNA weredetected in some participants during the same period.No replication was detected in any participant for anyvaccine virus after the second dose, and no associationwas observed between TDV virus replication and AEincidence.

In the Phase 2 study 203, transient TDV-2 replicationwas the only type detected; predominantly within thefirst couple of weeks after the first dose and mainly inseronegative individuals [33]. At Day 7, TDV-2 viral RNAwas detected in 8–39% of the TDV-vaccinated partici-pants in all age groups; on Day 14, the range was15–28% of TDV-vaccinated study groups aged<20 years. During the 2 weeks following the seconddose, only one participant in the 12–20-year age groupand one from the 21–45-year age group were TDV-2RNA-positive. Again, TDV-2 replication did not causeany detectable clinical signs: no association wasobserved between the presence of viral RNA and unso-licited or solicited local or systemic AEs. TDV replicationwas not increased in seropositive individuals. Thus, pre-exposure to wild-type DENV did not enhance the repli-cative capability of these attenuated DENV.

These data suggest that the immune responseinduced by the first dose of TDV is usually sufficientto inhibit TDV viral replication following second expo-sure to the live, attenuated dengue viruses [33].

Viremia is generally of short duration and below thethreshold required to infect mosquitoes that may bitea TDV-vaccinated individual. The 50% mosquito infec-tious dose for DENV-1–4 has been found to range from6.29 to 7.52 log10 RNA copies/mL of plasma [53], andour Phase 1 studies have shown individual titers ofTDV-2, -3, and -4 ranging from 3.6 to 4.9 log10 gen-ome equivalents (ge)/mL among subjects with detect-able TDV viral RNA [24]. Furthermore, the mutationsthat attenuate TDV in mammals also reduce transmis-sion and trafficking of the vaccine viruses into the A.aegypti salivary gland [28]. Thus, transmission of TDVfrom a vaccinated individual to mosquitos is highlyunlikely.

Summary

The live attenuated TDV induces robust humoral andcellular immune responses in adults and children, irre-spective of prior exposure to dengue. TDV inducesneutralizing antibody responses to all four DENV sero-types and protects against challenge with wild-typedengue viruses in both mouse and nonhuman primateanimal models. Humoral immunogenicity is highestagainst DENV-2, followed by DENV-1, DENV-3, andthen DENV-4. This vaccine’s TDV-2-based backbonedesign may be responsible for the cell-mediatedimmune responses it induces to dengue NS proteins,which may further contribute to protection againstdengue infection.

TDV has demonstrated an acceptable tolerability andsafety profile in adults and children, irrespective of priorexposure to dengue.

Ongoing Phase 2 studies are being carried out toexplore the potential for a one-dose vaccination regi-men, and approximately 3,000 adults and children havebeen enrolled to date. Data from these studies will beimportant in confirming safety and immunogenicityprior to progression to a large-scale efficacy study thatis expected to start in 2016.

Expert commentary

Substantial resources are being devoted to developingdengue vaccines as researchers strive to address theincreasing burden of dengue worldwide. The candi-dates currently in development are significantly differ-ent from one another in approach and design, in thehopes of identifying a vaccine technology that showshigh levels of protection against all four serotypes in allage groups.

Sanofi Pasteur’s vaccine candidate, CYD, is a liveattenuated tetravalent chimeric vaccine that

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incorporates the prM and E genes from DENV-1–4 into ayellow fever vaccine backbone [54]. CYD is the mostadvanced in its clinical development, with a Phase 2b[48] and two Phase 3 efficacy trials completed inSoutheast Asia and Latin America [47,55]. Three dosesof the tetravalent CYD vaccine provided protectionfrom all-type dengue fever in 56.5% and 60.8% of vac-cinated subjects in the Asian and Latin American stu-dies, respectively [47,55]. However, the serotype-specific protection varied considerably, with protectionfrom dengue fever caused by DENV-2 the lowest at 35%and 42.3% in the two studies, followed by DENV-1 at50% and 50.3%, and then DENV-3 (78.4% and 74%protection) and DENV-4 (75.3 and 77.7% protection)[47,55]. Protection also varied with dengue immunestatus at enrollment: protection was lower in initiallyseronegative individuals (35.5% and 43.2%, respectively,in the Asian and Latin American studies) and higher ininitially seropositive individuals (74.3% and 83.7%,respectively) [47,55]. Analysis of immunogenicity sub-sets within each trial demonstrated that the vaccineinduced balanced neutralizing antibody responses toall four DENV after the three doses, but these responseswere insufficient to generate balanced protection[47,55]. It may be necessary to induce higher levels ofneutralizing antibodies to DENV-1 and DENV-2 than toDENV-3 and DENV-4 to provide broader protectionagainst all four serotypes. In one cluster study, protec-tion from dengue infection correlated with higher neu-tralizing antibody responses to DENV-2 than DENV-1 orDENV-4 [56]. Alternatively, more multitypic or epitope-specific humoral responses and/or cellular immuneresponses to dengue NS proteins may be necessary toimprove protection against DENV-1 and DENV-2 and inseronegative individuals [57]. Furthermore, a recentanalysis of long-term follow-up data and a post hocanalysis of efficacy by age from the Phase 2b andAsian Phase 3 CYD efficacy studies showed lower effi-cacy and increased risks of hospitalization in CYD-vac-cinated children who were younger than 9 years(particularly in 2–5-year-olds) [58].

In addition to TDV and CYD, other vaccine candi-dates in clinical trials include recombinant dengueviruses developed by researchers at the NationalInstitute of Allergy and Infectious Diseases (NIAID) atthe National Institutes of Health. The NIAID vaccinescomprise live wild-type dengue strains that containattenuating mutations in the 3ʹ untranslated region.Data from Phase 1 studies of two different admixturesof monovalent vaccines, TV003 and TV005, have beenpublished, and Phase 2 studies are ongoing [59–61].The Butantan Institute is developing a lyophilized tetra-valent formulation of the NIAID candidates that is in

Phase 1/2 testing; no data are available to date [62–65].GSK has a tetravalent purified formalin-inactivated vac-cine candidate, which is undergoing Phase 1 testingusing different adjuvants in dengue-naïve and seropo-sitive individuals [61]. Merck’s recombinant subunit vac-cine comprising recombinant envelope proteinsrepresenting each serotype expressed in a DrosophilaS2 cell expression system [66] is also in Phase 1 devel-opment [61]. The Naval Medical Research Center(NMRC)’s monovalent DNA vaccine, comprising DENV-1 prM and E expressed from a plasmid vector undercontrol of the human cytomegalovirus promoter/enhancer has completed Phase 1 clinical testing [61];a tetravalent dengue DNA vaccine was assessed in aPhase 1 study [67], but the data have not beenpublished.

Although guidelines exist for the harmonization ofthe assessment of human antibodies to dengue viruses,there is significant inter-laboratory variability in metho-dology details for the plaque reduction neutralizationtest (PRNT50) assay [68]. This limits the possibility fordirect comparisons of seroconversion data or GMT titersfrom trials of different vaccines. In addition, methods ofpresenting GMTs and seropositivity rates vary betweendengue vaccine researchers. Some, including Takeda,present GMTs and seropositivity rates at a given timepoint, such as 30 days after vaccination (i.e. the datapresented refer to the specific measurements obtainedat that time point). Others provide peak GMTs, where atiter referenced at a given time point represents thehighest titer measured in the interval since vaccination;and cumulative seropositivity rates, where an individualwho was previously seropositive is considered seropo-sitive at a future time point regardless of actual seros-tatus at that time. These factors highlight theimportance of obtaining clinical efficacy data on eachof the vaccine candidates in order to improve ourunderstanding of how immune responses to vaccina-tion translate to protection against symptomatic den-gue infection.

Five-year view

This is an unprecedented time in the history of denguevaccine development. National and international healthagencies face the challenge of reversing the trends ofincreased epidemic dengue activity and the increasingincidence of dengue hemorrhagic fever [2]. The WHOhas a goal of reducing mortality and morbidity due todengue virus by 50% and 25%, respectively, by the year2020 [69]. In addition, dengue is a healthcare priority inmany Latin American and Asian countries, where epi-demics occur regularly. More effective control of the

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mosquito vectors through more efficient breeding sitecontrol and insecticide deployment, Wolbachia infec-tion, or genetic modification of mosquitoes may helpto reduce transmission, but implementation may bemany years away [61]. It is hoped that dengue vaccinesmay provide a quicker, more direct solution for peopleliving with the threat of dengue.

Over the next 5 years, the chimeric CYD is likely tobe the first vaccine to be licensed, followed by thelive attenuated vaccine developed by Takeda and theNIAID vaccine candidate. CYD will be introduced firstin Latin America and Asian regions [70]. Takeda iscontinuing its development of TDV to meet growingunmet medical needs in endemic countries throughongoing Phase 2 studies and a Phase 3 efficacy studythat will commence in 2016. Butantan is progressingwith clinical studies of a tetravalent formulation ofthe NIAID vaccine candidates in Brazil, with theintention of initiating a Phase 3 efficacy study [71].Given the outcomes of long-term safety studiesand efficacy analyses [58]. it is unlikely that CYDwill be widely adopted for use in younger seronega-tive children.

Nevertheless, even partially effective vaccines will bebeneficial in reducing dengue epidemics, reducing theprobability of infection, clinical symptoms, and severedisease within each vaccinated individual and poten-tially reducing dengue transmission by mosquitoes [61].For example, modeling studies showed that vaccinesthat have 70–90% efficacy against all dengue serotypescould have the potential to reduce the frequency andmagnitude of epidemics over the short to medium term[72,73]. However, a clear understanding of the epide-miology and seroprevalence of dengue in a particular

region will be required to model the impact of vaccinesthat offer low efficacy against one or more serotypes[74]. Whether the vaccine candidates from Takeda,NIAID, GSK, or others are able to provide robust protec-tion against all dengue serotypes, including in serone-gative individuals, will become evident in the next5 years.

Additional efficacy studies, together with analysesof the humoral and cellular immunogenicity of vaccinecandidates, may increase our understanding of howthe immune response to dengue vaccines translatesinto lower rates of dengue infection, transmission, andoverall disease burden. Human dengue challenge stu-dies with attenuated dengue viruses may provideadditional information on potential correlates of pro-tection [75,76]. The authors believe that in the next5 years, we will witness both a reduction in incidenceof this devastating disease and a better understandingof how vaccines may protect people of all ages fromdengue.

Financial & competing interests disclosure

The studies described in this review were funded by Inviragenand Takeda Vaccines, Inc. J Osorio and D Stinchcomb wereemployed by Inviragen at the time the reviewed studies werecarried out, and are currently Senior Scientific Advisors toTakeda Vaccines, Inc. D Wallace is an employee of TakedaVaccines, Inc. The authors have no other relevant affiliationsor financial involvement with any organization or entity with afinancial interest in or financial conflict with the subject mat-ter or materials discussed in the manuscript apart from thosedisclosed. Editorial assistance with preparation of the manu-script was provided by Samantha Santangelo, PhD, funded byTakeda Vaccines Inc.

Key issues

● Dengue is caused by infection with one of four mosquito-borne serotypes (DENV-1–4), and tetravalent dengue vaccines (TDV) that can inducesimultaneous immunity to all four DENV over a prolonged period are urgently needed.

● Patterns of DENV predominance and pathogenesis vary, but DENV-2 is often associated with secondary infection and severe disease.● Takeda’s TDV comprises an attenuated DENV-2 strain (TDV-2) plus three chimeric viruses containing the pre-membrane (prM) and envelope (E)

protein genes of DENV-1, -3, and -4 cloned into the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4).● PrM and E are needed to induce the neutralizing antibodies to DENV-1–4; the nonstructural (NS) proteins expressed from the dengue backbone

are required to generate T cell-mediated responses to dengue infection, and antibodies against NS1 are associated with cross-protective humoralimmune responses.

● TDV protected against challenge with wild-type DENV in both mouse and non-human primate animal models.● In Phase 1 and 2 studies, TDV induced strong neutralizing antibody responses and seroconversion to all four DENV in adults and children as

young as 1.5 years, even after a single dose, irrespective of prior dengue exposure. Humoral immunogenicity was always highest against DENV-2,followed by DENV-1, DENV-3, and then DENV-4.

● TDV also elicited cross-reactive T cell-mediated responses, which may contribute to protection against dengue, to NS proteins of all four DENV.● Low levels of vaccine viral replication may occur after the first dose; this is mainly TDV-2-specific, of short duration, and below the threshold

required to infect mosquitoes that may bite a TDV-vaccinated individual.● In Phase 1 and 2 studies conducted in the United States, South America, and Southeast Asia that included endemic regions, TDV was generally

well tolerated by adults and children, irrespective of prior dengue exposure, with mild injection-site symptoms being the most commonlyreported.

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