The protein DIIIC‐2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV‐2

Abstract

Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum. Dengue virus (DENV) is a mosquito-borne flavivirus and the causative agent of dengue and severe dengue. It is estimated that nearly half of the world's population is at risk of infection, with up to 50 million people infected each year and frequent epidemic activity in Southeast Asia, South America and Western Pacific regions. 1,2 Infection with one serotype confers immunity to infection with the same dengue serotype, but does not prevent infection with the others. Viral and host factors have been proposed to have a role in the development of the severe disease, but epidemiological evidence has led to the realization that the majority of severe cases occur in individuals who suffer secondary or sequential DENV infections. 3,4 This implies that immunity to a heterotypic virus is not only ineffective in preventing secondary infection, but may also enhance the disease.Primary prevention through dengue vaccines is considered a research priority in the dengue agenda. Although a vaccine is feasible, its development faces several challenges: (a) a dengue vaccine must be able to protect against the four serotypes; (b) long-term protection is needed; (c) no suitable animal model exists for dengue; and (d) although the protective role of neutralizing antibodies is widely held, correlates of protection need to be defined.There are several dengue vaccine candidates at advanced preclinical and clinical stages, although no vaccine is licensed. The most advanced strategies (phase II-III) are based on live attenuated viruses and are led by the Sanofi Pasteur's ChimeriVax-dengue vaccine candidate (Paris, France). Despite the balanced reactogenicity and immunogenicity profile of the tetravalent ChimeriVax-dengue vaccine candidate, three doses are required during 1 year to induce high neutralizing antibody seroconversion...

Dengue vaccine: an update on recombinant subunit strategies

“…Monkeys immunized subcutaneously with 100 μg of EDIIIC2 (plus 10 μg of ODN 39M) in alum developed a functional immune response that conferred partial protection from viremia when challenged thirty days aft er the fourth dose (Gil et al, 2015). A tetravalent vaccine candidate containing the DV2 EDIIIC combined with the EDIII-P64k fusion proteins from DV1, DV3 and DV4 was immunogenic and protected mice against challenge with lethal (DV1, DV2, DV4) or encephalitis-inducing (DV3) virus .…”

“…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 …”

Tetravalent dengue DIIIC protein together with alum and ODN elicits a Th1 response and neutralizing antibodies in mice

“…To combine B cell-and T cell epitopes in a single recombinant DENV construct, a fusion protein of the dengue virus E domain III (DIII) and the capsid (C) protein (DIIIC) has been developed as a potential vaccine candidate. DIIIC of dengue serotype 2 (DIIIC-2) induces a protective immune response in mice and in monkeys when injected as aggregates, which form spontaneously when DIIIC is mixed with oligodeoxynucleotide (ODN) [14,19,20]. Immunization with DIIIC induces IFN-␥-and TNF-␣ producing CD4 + and CD8 + T cells, whereas CD4 + T cells contribute the majority of cytokines as shown by CD4-or CD8-depletion before in vitro stimulation of splenocytes from immunized mice [14].…”

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.…”