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We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine.

A dengue-2 (DEN-2) DNA vaccine coding for the premembrane and envelope (E) proteins and a recombinant fusion protein containing the B domain of the DEN-2 E protein fused to the maltose-binding protein (MBP) of Escherichia coli both elicited neutralizing antibody in mice. In order to achieve more rapid protective immunity as well as to increase the persistence of neutralizing antibody, we primed mice with the DNA vaccine (D), the recombinant MBP protein (R), or both (RD) given simultaneously, and then boosted twice with either the R (R/R/R or D/R/R) or D (D/D/D or R/D/D) constructs alone or the RD (RD/RD/RD) combination. All of the recombinant protein vaccines were given with alum as an adjuvant. The serum antibody response measured by enzyme-linked immunosorbent assay was highest in D/D/D mice and RD/RD/RD mice. The D/R/R mice showed an intermediate response, and the R/D/D and R/R/R showed the lowest response. The geometric mean (GM) 50% neutralizationtiter (50% plaque reduction neutralization, or PRNT50) was marginally higher for RD/RD/RD mice (891) at 9 months after priming than that for R/R/R mice (724). T he lowest GM PRNT50 titers were seen in the D/D/D mice (33) and R/D/D mice (40), and the D/R/R group had a slightly higher titer (156) than these 2 groups. The predominant antibody subclass for the D/D/D mice was immunoglobulin (Ig) G2a, similar to mice infected with live virus. The R/R/R mice showed an exclusive IgGI antibody response, and the RD/RD/RD response also was predominantly IgGI. The antibody subclass pattern of the R/D/D and D/R/R mice showed a more balanced distribution of both IgG1 and IgG2a. Investigating the neutralizing capacity of antibody subclasses suggested that both IgG1 and IgG2a could neutralize DEN-2 virus. Our observations indicate that the combination RD prime-boost regimen warrants further investigation as a vaccine strategy to prevent dengue infection.

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the naïve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.