Immunogenicity and Efficacy of Flagellin-Envelope Fusion Dengue Vaccines in Mice and Monkeys

Abstract

The envelope (E) protein of flaviviruses includes three domains, EI, EII, and EIII, and is the major protective antigen. Because EIII is rich in type-specific and subcomplex-specific neutralizing epitopes and is easy to express, it is particularly attractive as a recombinant vaccine antigen. VaxInnate has developed a vaccine platform that genetically links vaccine antigens to bacterial flagellin, a Toll-like receptor 5 ligand. Here we report that tetravalent dengue vaccines (TDVs) consisting of four constructs, each containing two copies of EIII fused to flagellin (R3.2x format), elicited robust and long-lived neutralizing antibodies (geometric mean titers of 200 to 3,000), as measured with a 50% focus reduction neutralization test (FRNT 50 ). In an immunogenicity study, rhesus macaques (n ‫؍‬ 2) immunized subcutaneously with 10 g or 90 g of TDV three or four times, at 4-to 6-week intervals, developed neutralizing antibodies to four dengue virus (DENV) serotypes (mean post-dose 3 FRNT 50 titers of 102 to 601). In an efficacy study, rhesus macaques (n ‫؍‬ 4) were immunized intramuscularly with 16 g or 48 g of TDV or a placebo control three times, at 1-month intervals. The animals that received 48-g doses of TDV developed neutralizing antibodies against the four serotypes (geometric mean titers of 49 to 258) and exhibited reduced viremia after DENV-2 challenge, with a group mean viremia duration of 1.25 days and 2 of 4 animals being completely protected, compared to the placebo-treated animals, which all developed viremia, with a mean duration of 4 days. In conclusion, flagellin-EIII fusion vaccines are immunogenic and partially protective in a nonhuman primate model. Dengue disease poses a significant health threat to almost onehalf of the world's population residing in the Asian-Pacific region and Latin America, where the disease is endemic, as well as to travelers and military personnel. Currently, no licensed dengue vaccine is available. Several vaccine candidates based on live attenuated dengue viruses (DENVs) or dengue virus-flavivirus chimeras are currently being evaluated in phase II or III clinical trials (for reviews, see references 1-3). However, some of these vaccines require lengthy immunization regimens of up to 1 year for the development of immunity to all four serotypes, which seems to make them less well suited for travelers and military personnel. In a series of recently published phase IIb/III trials conducted in five Asian countries in which dengue is endemic, the Sanofi-Aventis chimeric yellow fever (YF) 17D-DENV-1 to -4 CYD tetravalent dengue vaccine (TDV) showed an acceptable safety profile and 56% overall efficacy (4); however, the vaccine failed to confer significant protection against DENV-2 (5). These findings underscore the need for the development of alternative vaccine platforms, such as those based on purified inactivated virus (6), DNA (7), and recombinant subunit truncated envelope (E) proteins (e.g., 80% envelope protein [80E]) (8). Some of these alternative vaccine candidates hav...

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

“…Another promising study reported improved antibody titers (∼60% increase), improved viral neutralization titers (∼3-fold increase), and improved Th1 cytokine profile (∼2-fold increase in IFNγ and TNFα expression) when comparing the effectiveness of flagellin-modified and wildtype porcine circovirus type 2 Cap protein (64). Additionally, many other studies evaluating the effectiveness of recombinantly fusing flagellin to PBV have shown promising results, with the majority reporting improved overall immunogenicity and/or survival when compared to antigen administered alone (65)(66)(67)(68)(69)(70)(71). Unsurprisingly, results like these have propelled multiple flagellin-fused PBVs targeting influenza to clinical trials (72)(73)(74)(75).…”

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

“…When expressed as a recombinant polypeptide, EDIII preferentially folds into its native conformation [14] and has been shown to elicit potently neutralizing antibodies that target tertiary epitopes displayed on wild-type virus [15,16]. While DENV EDIII recombinant protein vaccines have been described in the past 10 years, few have undergone immunogenicity trials in non-human primates [17–25] with even fewer evaluating protection [18,21,23–25]. Those that have been evaluated in protection studies are all EDIII fusion proteins, with EDIII fused to DENV2 capsid protein [18], the P64K protein from N. meningitidis [21,24,25], and Salmonella enterica flagellin [23].…”

“…While DENV EDIII recombinant protein vaccines have been described in the past 10 years, few have undergone immunogenicity trials in non-human primates [17–25] with even fewer evaluating protection [18,21,23–25]. Those that have been evaluated in protection studies are all EDIII fusion proteins, with EDIII fused to DENV2 capsid protein [18], the P64K protein from N. meningitidis [21,24,25], and Salmonella enterica flagellin [23]. Fusion proteins were employed as EDIII carriers that are also immunostimulatory via innate immune pathways.…”

Dengue vaccine: an update on recombinant subunit strategies

“…Tetravalent candidate evaluated in mice; monovalent DV1 and DV2 candidates evaluated in NHPs Hermida et al, 2006;Lazo et al, 2014;Valdés et al, 2009a] EDIII-capsid fusion protein (DIIIC) expressed in E. coli Monovalent DV2 candidate evaluated in NHPs [Gil et al, 2015;Suzarte et al, 2014;Valdés et al, 2009b;Zuest et al, 2015] Tetravalent EDIII-STF2 fusion proteins expressed in E. coli Tetravalent candidate evaluated in mice and NHPs [Liu et al, 2015] EDIII-HBcAg fusion protein expressed in P. pastoris Monovalent DV2 candidate evaluated in mice Recombinant EDII-EDIII-NS1 fusion protein expressed in Drosophila S2 Tetravalent lipidated consensus EDIII (LcED III) expressed in E. coli Tetravalent candidate evaluated in mice [Chen et al, 2013;Chiang et al, 2011;Leng et al, 2009] Lipidated EDIII (LED III) expressed in E. coli Monovalent candidates (DV1,2,4) evaluated in mice [Chiang et al, 2011[Chiang et al, , 2013a MixBiEDIII: bivalent EDIIIs (DV1-2/ DV3-4) expressed in E. coli Tetravalent candidate evaluated in mice [Zhao et al, 2014] Bivalent EDIII (DV1-2) expressed in E. coli Bivalent candidate (DV1-2) evaluated in mice [Zhang et al, 2015] Tetravalent chimeric EDIII expressed in P. pastoris Tetravalent candidate evaluated in mice [Etemad et al, 2008] Non-structural proteins Recombinant DV2 NS1 protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Amorim et al, 2012] Recombinant DV2 NS1-DEC205 fusion protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Henriques et al, 2013] Full-length recombinant DV2 NS3 protein expressed in E. coli Monovalent DV2 candidate evaluated in mice [Ramírez et al, 2014] Capsid protein Recombinant capsid protein expressed in E. coli Monovalent DV2 candidate evaluated in mice and NHPs …”

“…Other vaccine candidates produced with this platform were well tolerated and immunogenic in humans (Treanor et al, 2010;Taylor et al, 2011) and a fl agellin-based West Nile Virus vaccine protected mice against experimental infection with this virus (McDonald et al, 2007). A tetravalent dengue vaccine candidate was created by replacing the D3 domain of STF2 with the E protein domain III from each DV serotype and fusing an additional copy of the domain III fragment to the C-terminus of STF2 (Liu et al, 2015). Proteins were produced in E. coli.…”