Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

Abstract

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LT(G33D)), originally produced by some enterotoxigenic Escherichia coli (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LT(G33D). Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LT(G33D) elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LT(G33D.) Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LT(G33D). Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine.

A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

“…Neutralization assay showed that the titer of neutralization antibody against JEV was relatively low and may not account for the in vivo protection. The contribution of the NS1-induced immune response should not be ruled out, since flavivirus NS1 protein has been shown to induce protective immune responses in animals (67)(68)(69)(70)(71)(72)(73). Notably, immunization with ChinDENV elicited significantly increased JEV antigen-specific T cell responses (IFN-␥ and IL-2), suggesting a possible role for cellular immunity in the defense against JEV challenge in ChinDENV-immunized mice.…”

DENV-2 subunit proteins fused to CR2 receptor-binding domain (P28)-induces specific and neutralizing antibodies to the Dengue virus in mice

“…[11][12][13][14] Conversely the non-structural protein 1 (NS1) is both expressed on the surface of the infected cells and secreted. NS1 elicits an immune protective response in natural infections and in experimental models, [15][16][17] without the adverse effects that involve antibody-dependent enhancement.…”

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 …”

“…In a fi rst approach, they evaluated the safety and immunogenicity in mice of a recombinant DV2 NS1 protein expressed in E. coli and formulated with three diff erent adjuvants: alum, Freund's adjuvant or a non-toxic variant of E. coli heat-labile enterotoxin LTG33D (Amorim et al, 2012). Mice receiving four doses of the NS1-LTG33D combination (10 μg NS1 plus 1 μg LTG33D) by the s.c. route elicited the strongest antibody response, with a balanced IgG1/IgG2a ratio and enhanced avidity to the NS1 protein.…”