Dengue serotype 1 vaccine development: The live attenuated DEN1 vaccine candidate virus rDEN1del30 has been evaluated in Phase I clinical trials and was found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies have been fully enrolled to determine the number of doses and the timing of the two doses needed to achieve optimal immunization. The studies involve two doses of vaccine given at an interval of 4 or 6 months. The results will be fully reported pending complete analysis, but preliminary analysis indicates that the first dose is highly immunogenic but the second dose is poorly infectious at 6 months. Trials to determine the human infectious dose 50 are planned. Dengue serotype 2 vaccine development: rDEN2/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine, rDEN4del30, have been replaced by those of the prototypic DEN2 NGC virus. The live attenuated DEN2 vaccine candidate virus rDEN2/4del30(ME) has been evaluated in Phase I clinical trials and found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies are in progress to address the number and timing of two doses of this vaccine candidate. Enrollment is not complete, and results of the trial will be reported pending completion. Trials to determine the human infectious dose 50 are planned. Dengue serotype 3 vaccine development;rDEN3/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine rDEN430 have been replaced by those of a DEN3 wild type virus. rDEN3/4del30(ME) or placebo was evaluated at a dose of 1,000 or 100,000 PFU in 28 healthy dengue-naive adult volunteers per dose. Less than 40% of vaccinees were infected with either dose indicating this vaccine candidate is not sufficiently infectious to be used as a component of a tetravalent dengue vaccine formulation. Studies have been initiated with an additional DEN3 vaccine candidate in which the DEN3-UTR (untranslated region) has been replaced with that of DEN4del30, and initial trials indicate the virus is safe and more infectious than rDEN3/4del30(ME) and that it can be used as the DEN3 component of a tetravalent vaccine. Dengue serotype 4 vaccine development: rDEN4∆30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4∆30 parent virus. In a previous study, 5 of 20 vaccinees who received rDEN4∆30 at 100,000 PFU developed moderately elevated levels of serum alanine aminotransferase (ALT). Mutational analysis of DENV-4 revealed that the 200,201 mutation of NS5 greatly restricted virus replication in human hepatocarcinoma HuH-7 cells. In pre-clinical animal studies, the vaccine candidate rDEN4∆30-200,201 was shown to be more attenuated than rDEN4∆30 in both SCID-HuH-7 mice and rhesus macaques. In the present study, twenty-eight healthy adult volunteers were randomized to receive either 100,000 PFU of vaccine (20) or placebo (8) as a single subcutaneous injection. The vaccine infected all vaccinees and was well tolerated without inducing ALT elevations. Although virus was not recovered from the serum of any vaccinee, moderate levels of neutralizing antibody were induced. The restricted replication of rDEN430-200,201 in SCID HuH-7 mice and rhesus monkeys was also observed in humans, thus validating the pre-clinical approach of selecting a mutant virus with a decreased level of replication in human liver cells. rDEN4∆30-200,201 is a promising candidate and can be considered for inclusion in a tetravalent DEN virus vaccine. We currently consider the rDEN4del30 vaccine our DEN4 leading candidate for inclusion in the tetravalent vaccine for two reasons. At a proposed dose of 1000 PFU, it is economical to produce and has an acceptable safety profile;and, rDEN4del30 appears slightly more immunogenic in humans than rDEN4del30-200,201;this greater immunogenicity likely would translate into a more durable immunity. A second candidate is rDEN4del30-4995. rDEN4∆30-4995 is a live attenuated dengue virus type 4 (DEN4) vaccine candidate specifically designed as a further attenuated derivative of the rDEN4∆30 parent virus. In a previous study, 5/20 vaccinees who received 100,000 plaque forming units (PFU) of rDEN4∆30 developed a transient serum ALT elevation and 10/20 developed an asymptomatic maculopapular rash. In the current study, 28 healthy adult volunteers were randomized to receive 100,000 PFU of rDEN4∆30-4995 (20) or placebo (8) as a single subcutaneous injection. The vaccine was safe, well-tolerated, and immunogenic. An asymptomatic generalized maculopapular rash and elevations in ALT levels were observed in 10% of the rDEN4∆30-4995 vaccinees. None of the rDEN4∆30-4995 volunteers became viremic, yet 95% developed a four-fold rise in neutralizing antibody titers. Thus rDEN4∆30-4995 was demonstrated to be safe, highly attenuated, and immunogenic. However, an asymptomatic localized erythematous rash at the injection site was seen in 17/20 rDEN4∆30-4995 vaccinees, and, therefore, alternative DEN4 vaccine strains were selected for further clinical development. Two tetravalent formulations will be evaluated in humans in the next FY. One will consist of rDEN1del30, rDEN2/4del30(ME), rDEN3-3Ddel30, and DEN4-del30 and the second will have rDEN4∆30-200,201 as the DEN4 component. An additional study designed to evaluate the safety of our DEN vaccine viruses in subjects immune to a heterologous dengue virus has been completed, and the data is being analyzed. Preliminary analysis of this trial, in which four combinations of heterologous first and second vaccines were studied, indicated that the vaccine viruses were infectious, safe, and immunogenic in the presence of preexisting heterologous immunity. Only one of the four combinations showed increased replication of vaccine virus, an expected finding in which preexisting immunity enhances virus replication, but this small increase in viremia was not accompanied by an increase in signs or symptoms of dengue virus disease. West Nile virus vaccine development trials in humans: Phase I trials are ongoing with a live attenuated West Nile virus vaccine candidate, WNVDEN4del30. Studies at 1000 and 100,000 infectious units per vaccinee indicated the vaccine was safe with low viremia, but was immunogenic in only 80% of vaccinees. To achieve a higher take rate, a study is ongoing in which two doses of 100,000 infectious units are given six months apart. The study is fully enrolled, and preliminary analysis indicates that the vaccine given as two doses is safe and immunogenic with up to 100% of vaccinees developing a rise in neutralizing antibody titer.
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