Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

Abstract

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Differential humoral and cellular immunity induced by vaccination using plasmid DNA and protein recombinant expressing the NS3 protein of dengue virus type 3

“…Finally, in other studies, it has been observed that the DNA vaccines encoding the full-length NS3 induced a cellular immune response against this epitope with the production of IFN-γ. Therefore, results suggest the participation of a cellular immune response in the mechanism of protection induced by the DNA vaccines based on the NS3 protein [41]. …”

Polyclonal antibodies against recombinant dengue virus NS3 protein

“…El ADN complementario obtenido se usó como plantilla para amplificar mediante PCR dos regiones del extremo C-terminal de NS3, dominio en el cual se encuentran los epítopos más antigénicos de la proteína (10). Para ello, se usó el genoma de la cepa de referencia 16681 de DENV2 (NC_001474.2), del cual se extrajo la secuencia de nucleótidos que correspondía a la proteína NS3.…”

Clonagem e expressão das proteínas recombinantes NS1 e NS3 do vírus da dengue tipo 3

“…PREUGSCHAT; STRAUSS, 1993;COSTA et al, 2011;FALGOUT;LAI, 1993;FALGOUT et al, 1991;LEUNG, D. et al, 2001;LI, H. et al, 1999;LI, J. et al, 2005;YUSOF et al, 2000). Quando utilizado o sistema eucariótico para expressão da proteína NS3, também são observadas proteínas de tamanhos menores que aquela correspondente à proteína inteira, mas com perfil diferente daquelas observadas em sistemas procarióticos (COSTA et al, 2011).…”

“…Quando utilizado o sistema eucariótico para expressão da proteína NS3, também são observadas proteínas de tamanhos menores que aquela correspondente à proteína inteira, mas com perfil diferente daquelas observadas em sistemas procarióticos (COSTA et al, 2011).…”

“…A proteína NS3 é uma proteína conservada entre os quatro sorotipos do vírus da dengue e possui epítopos de células T em seu interior, alvo principal para células T CD4+ e CD8+ de resposta de células T (APPANNA et al, 2007;MATHEW;ROTHMAN, 2008) além de induzir uma resposta protetora em camundongos quando utilizada como uma vacina de DNA (COSTA et al, 2011) esses dados demonstram a potencial utilização da proteína NS3 como um complemento para uma vacina contra a doença, por exemplo, em conjunto com o domínio 3 da proteína E do envelope viral ou a NS1, os quais são capazes de gerar forte resposta humoral.…”