A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice.

A gene fragment encoding for the amino acids (aa) 286-426 from the dengue Envelope (E) protein was expressed in Escherichia coli as two forms of fusion proteins. In one case, the E fragment was fused to the first 45 aa of the P64k protein from Neisseria meningitidis (PD2) while, in the other, it was inserted within the lipoil-binding domain of the aforementioned bacterial protein (PD3). PD2 was obtained as insoluble form within the cytoplasm of the bacteria while PD3 was distributed equally as soluble and insoluble forms. The insoluble forms of each protein as well as the soluble fraction of PD3 were semipurified to test the antigenicity and the immunogenicity in mice. The forms containing the entire P64k protein exhibited the highest recognition with different polyclonal and monoclonal antibodies. Consequently, the neutralizing antibodies elicited by the recombinant proteins were higher in the case of PD3 forms than with PD2, independently of the solubility status. In addition, mice inoculated with the semipurified insoluble form of PD3 were partially protected against lethal challenge with dengue-2 virus, administered by intracerebral inoculation. The results suggested the folding and carrier capacity of the P64k protein over the E fragment, converting PD3 as an attractive vaccine candidate against dengue-2 virus.

A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococcal P64k protein carrier, induces a functional immune response in mice.

Previously we have reported the capacity of the fusion protein PD3, composed of the P64k protein and the envelope (E) fragment from amino acids (aa) 286-426 of dengue-2 virus (DEN-2), to induce a functional immune response in mice against the homologous virus. In that case, the E fragment was inserted within the lipoyl-binding domain of the meningococcal P64k protein. In the present study, to test the functionality of the same E region from dengue-1 (DEN-1), a similar construct was made. Furthermore, another alternative of fusion protein was also constructed where the same E fragment from DEN-1 was fused to the C-terminus of the P64k protein. The recombinant proteins obtained (PD11 and PD10) were semi-purified and analysed for their antigenicity, immunogenicity and the ability to protect mice against lethal challenge. Both molecules exhibited the same recognition patterns against anti-DEN-1 polyclonal antibodies. In addition, when administered to mice, they elicited high levels of neutralizing antibodies and induced significant protection against lethal challenge with DEN-1 after intracerebral inoculation. These results reveal the availability of two sites within the P64k for the further insertion of DEN fragments, enabling a construct carrying two fragments from heterologous serotypes within the same molecule of this protein carrier.

Induction of neutralizing antibodies and partial protection from viral challenge in Macaca fascicularis immunized with recombinant dengue 4 virus envelope glycoprotein expressed in Pichia pastoris.

A recombinant vaccine that expresses the envelope (E) gene of dengue virus type 4 was tested for immunogenicity and protection in Macaca fascicularis. One hundred micrograms of semipurified recombinant E protein (E4rec) expressed in Pichia pastoris was used to immunize three animals. Neutralizing antibodies to dengue 4 virus with a titer of 1:30 were detected in all immunized monkeys prior to challenge. Animals were challenged with 10(5) plaque-forming units of dengue 4 virus. One vaccine-immunized monkey was protected from viremia, while the other two were partially protected. Monkeys immunized with E4rec elicited the highest neutralizing antibody titers (P < 0.05) ranging from 1:85 to 1:640 at day 30. In both immunized and control animals, the longest duration of viremia correlated with earliest and highest level of IgM antibody to dengue virus. The vaccinated animals showed anamnestic antibody responses upon virus challenge, indicating successful priming by the recombinant vaccine. Our results suggest that E4rec expressed in P. pastoris can provide partial protection against viremia. However, the results were not effective enough to use it as a vaccine candidate. Further work is required to improve the quality of the immunogen.

The fusion site of envelope fragments from each serotype of Dengue virus in the P64k protein, influence some parameters of the resulting chimeric constructs.

To characterize the effect of the envelope fragment fusion site in the P64k protein from Neisseria meningitidis several chimeric constructs were obtained. One variant consisted in the insertion of the E fragment from each Dengue serotype within the lipoil binding domain of the P64k, whereas the other was based on the fusion of the envelope fragment at the C-terminus of the same meningoccocal protein. The results of the expression study revealed the majoritary levels with the C-terminus fusion variants of each serotype. In contrast, the highest proportion of soluble protein was reached with the insertion variants independently of the viral serotype. On the other hand, a significant level of degradation was detected for the semipurified forms of the insertion variants being remarkable in the Dengue 2 construct. Finally, the recognition by Dengue murine antibodies was similar independently of the fusion site. Regarding these results, we can affirm the suitability of the C-terminus fusion variants for further vaccine development as well as for a diagnostic system.

Long-term memory cellular immune response to dengue virus after a natural primary infection.

OBJECTIVES: This study was conducted to examine the memory T-cell response to dengue virus 20 years after a primary infection. We took advantage of the exceptional epidemiologic situation in Cuba, where the population initially suffered two large successive epidemics due to dengue virus 1 and 2 respectively over a 4-year period. Thereafter, no dengue virus circulation was subsequently observed, except for the Santiago de Cuba municipality. DESIGN: T-cell response was evaluated in peripheral blood mononuclear cells (PBMCs) from 20 individuals with history of a primary infection by dengue virus 1 or 2. Methods previously shown to induce lymphoproliferation of CD4+ memory T-cell subpopulations were used. We evaluated the proliferative responses generated in those PBMCs after stimulation with dengue virus 1, 2, 3 and 4 antigens in a serotype-specific and serotype-crossreactive way. RESULTS: Serotype-specific and serotype-crossreactive lymphoproliferative responses in all PBMCs donated by dengue immune donors were observed. The serotype-crossreactive response for dengue 2 was stronger than for the rest of the serotypes. CONCLUSIONS: This is the first report of cellular memory lymphocyte response specific for dengue virus detected 20 years after a primary infection by dengue.