Immunogenicity and protective efficacy of a recombinant fusion protein containing the domain III of the dengue 1 envelope protein in non-human primates.

Recombinant fusion proteins containing the aa 286-426 of the dengue envelope protein fused to P64k protein from Neisseria meningitidis have been previously reported. Particularly, the immunogenicity and protective capacity of the dengue 2 recombinant protein was demonstrated in Macaca fascicularis monkeys. Here we evaluate the recombinant fusion protein containing the domain III of the dengue 1 envelope protein (PD10) in non-human primates (M. fascicularis and rhesus monkeys) and compare the effect of aluminum hydroxide and Freund adjuvant on the immunity induced. The PD10 protein emulsified in Freund adjuvant was highly immunogenic in M. fascicularis and rhesus monkeys. Following dengue 1 virus challenge, animals immunized with PD10 in Freund adjuvant were protected from viremia. However, monkeys receiving PD10 in aluminum hydroxide developed a poor antibody response and were not protected from viral challenge. These preliminary experiments are encouraging. Other formulations or vaccine schedules are being studied in an attempt to find regimens that enhance immunological protection.

Primary and secondary infections of Macaca fascicularis monkeys with Asian and American genotypes of dengue virus 2

The goal of this study was to compare the immune response and the protection capacity induced by the dengue virus 2 (DENV-2) American and Asian genotypes in Macaca fascicularis monkeys. Animals were infected with American or Asian DENV-2 strains and challenged 1 year later with a DENV-2 Asian genotype strain. The viremia and monkey antibody levels were similar for the different strains after primary and secondary infection; however, the functionality of the antibody response was different. A limited viral replication was demonstrated after the secondary infection in all the monkeys. No virus was isolated in tissue culture, while reverse transcription-PCR showed a late positive reaction in four of five challenged monkeys. The immunoglobulin M response pattern and the detection of antibodies to specific proteins by Western blotting supported the protection data. Despite the demonstration of the protective effect after homologous challenge, a strong anamnestic antibody response was observed(AU)

Anamnestic antibody response after viral challenge in monkeys immunized with dengue 2 recombinant fusion proteins.

The suitability of dengue 2 envelope domain III recombinant fusion proteins [(fusion (PD5) and insertion (PD3) variants)] for inducing functional antibodies and a protective immune response in nonhuman primates has been reported. However, the evaluation of the antibody response after immunization did not correlate with the protection data as measured by viremia detection. Here, we characterized the anamnestic immune response after viral challenge in monkeys immunized with the dengue 2 recombinant proteins in an attempt to define correlates of protection useful for vaccine studies. Monkeys immunized with PD5 (most protected group) exhibited an earlier increase in the anti-DENV-2 IgM response after challenge compared to control animals. Hemagglutination-inhibiting (HAI) antibodies were increased significantly earlier in PD5-immunized animals compared to those immunized with PD3. The fully protected monkeys showed the earliest HAI antibody response. These results underline the usefulness of the anamnestic antibody response for supporting protection data. The induction of an early HAI and IgM antibody response after challenge suggest a protective role against dengue virus (DENV) infection in monkeys, supporting their use as correlates of protection in vaccine studies.

Primary and secondary infections of Macaca fascicularis monkeys with Asian and American genotypes of dengue virus 2.

The goal of this study was to compare the immune response and the protection capacity induced by the dengue virus 2 (DENV-2) American and Asian genotypes in Macaca fascicularis monkeys. Animals were infected with American or Asian DENV-2 strains and challenged 1 year later with a DENV-2 Asian genotype strain. The viremia and monkey antibody levels were similar for the different strains after primary and secondary infection; however, the functionality of the antibody response was different. A limited viral replication was demonstrated after the secondary infection in all the monkeys. No virus was isolated in tissue culture, while reverse transcription-PCR showed a late positive reaction in four of five challenged monkeys. The immunoglobulin M response pattern and the detection of antibodies to specific proteins by Western blotting supported the protection data. Despite the demonstration of the protective effect after homologous challenge, a strong anamnestic antibody response was observed.

Biological and Molecular Properties of Dengue 2 Strains Isolated during the DHF/DSS Cuban Epidemic, 1981.

To study some biological and molecular properties of nine DENV-2 strains isolated during the 1981 Cuban epidemic, temperature sensitivity, viral plaque size, the kinetic of virus replication in newborn mice inoculated by intracerebral route, the influence of pH medium on virus-cell attachment phase and the restriction enzyme pattern were studied. Strains were classified in two patterns according to temperature sensitivity, plaque size, and virus replication in mouse brain and cell culture and restriction enzymatic pattern the changes observed differentiate clearly the strains isolated at the beginning and at the end of the epidemic suggesting that viruses with different characteristics circulated.

Amino acid changes in the recombinant Dengue 3 Envelope domain III determine its antigenicity and immunogenicity in mice.

The immunogenicity of the Envelope fragment from amino acid 284 to 426 of Dengue viruses, obtained as fusion proteins with P64k in Escherichia coli, has been previously tested by our group. Here, we studied two fusion proteins with P64k carrying the Envelope fragment from two strains of Dengue 3: H87 prototype strain (PD9) and an isolate from the Nicaragua 1994 outbreak (PD18). Sequence comparison of the Dengue Envelope fragments showed four amino acid differences. Only PD18 reacted with human antisera and induced a higher functional immune response in mice than PD9. Moreover, mice immunized with PD18 were less susceptible to Dengue 3 administered intracerebrally than those immunized with PD9. The results reveal that not all sequences of the Dengue Envelope fragment, at least in the context of P64k, are antigenic and generate a functional immune response against the native virus. This finding has direct implications for the design of vaccines based on fragments of the Envelope protein.

A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates.

We have previously reported the construction and evaluation in mice of recombinant fusion proteins formed by a fragment (aa 286-426) of the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macaca fascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Four doses of the proteins adjuvated in Freund's adjuvant were administered and the kinetics of antibody induction was monitored by ELISA and neutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challenge with 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results constitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model.

PCR detection of dengue virus using dried whole blood spotted on filter paper.

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.

Dengue 3 epidemic, Havana, 2001.

In June 2001, dengue transmission was detected in Havana, Cuba; 12,889 cases were reported. Dengue 3, the etiologic agent of the epidemic, caused the dengue hemorrhagic fever only in adults, with 78 cases and 3 deaths. After intensive vector control efforts, no new cases have been detected.