Differential humoral and cellular immunity induced by vaccination using plasmid DNA and protein recombinant expressing the NS3 protein of dengue virus type 3 (original) (raw)
Related papers
Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines
PLoS ONE, 2011
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the Nterminal 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-c, thus suggesting the involvement of this branch of the immune system in the protection.
2020
Dengue virus represents the main arbovirus affecting humans, but there are no effective drugs or available worldwide licensed vaccine formulations capable of conferring full protection against the infection. Experimental studies and results generated after the release of the licensed anti-DENV vaccine demonstrated that induction of high-titer neutralizing antibodies does not represent the sole protection correlate and that, indeed, T cell-based immune responses plays a relevant role in the establishment of an immune protective state. In this context, this study aimed to further demonstrate protective features of immune responses elicited in immunocompetent C57BL/6 mice immunized with three plasmids encoding DENV2 nonstructural proteins (NS1, NS3, and NS5), which were subsequently challenged with a DENV2 strain naturally capable of inducing lethal encephalitis in immunocompetent mouse strains. The animals were immunized intramuscularly with the DNA vaccine mix and complete protection...
Archives of Virology, 2010
A DENV-2 plasmid named pEII*EIII/NS1*, containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST-E and GST-NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design of a recombinant subunit vaccine against dengue disease.
PLoS neglected tropical diseases, 2015
Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell response...
Archives of Virology, 2008
In an effort to develop a suitable DNA vaccine candidate for dengue, using dengue-3 virus (DENV-3) as a prototype, the genes coding for premembrane (prM) and envelope proteins (E) were inserted into an expression plasmid. After selecting recombinant clones containing prM/E genes, protein expression in the cell monolayer was detected by indirect immunofluorescence and immunoprecipitation assays. After selecting three vaccine candidates (pVAC1DEN3, pVAC2DEN3 and pVAC3DEN3), they were analyzed in vivo to determine their ability to induce a DENV-3-specific immune response. After three immunizations, the spleens of the immunized animals were isolated, and the cells were cultivated to measure cytokine levels by ELISA and used for lymphoproliferation assays. All of the animals inoculated with the recombinant clones induced neutralizing antibodies against DENV-3 and produced a T cell proliferation response after specific stimuli. Immunized and control mice were challenged with a lethal dose of DENV-3 and observed in order to assess their survival capability. The groups that presented the best survival rate after the challenge were the animals vaccinated with the pVAC3DEN3 clones, with an 80% survival rate. Thus, these data show that we have manufactured a vaccine candidate for DENV-3 that is able to induce a specific immune response and protects mice against a lethal challenge.
Journal of General Virology, 1990
Antibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the nonstructural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies pre
Virus Genes, 2014
The NS3 protein is a multifunctional nonstructural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a Histagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-a in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.
Virology, 2007
We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1-and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1-and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.
PLOS Neglected Tropical Diseases, 2015
Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a viruscapture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.