A mild maculopapular rash developed in three volunteers (two vaccinees and one placebo)

A mild maculopapular rash developed in three volunteers (two vaccinees and one placebo). 10% of the rDEN430-4995 vaccinees. None of the rDEN430-4995 vaccinees became viremic, yet 95% developed a four-fold or greater increase in neutralizing antibody titers. Thus, rDEN430-4995 was demonstrated to be safe, highly attenuated, and immunogenic. However, an asymptomatic localized erythematous rash at the injection site was seen in 17/20 rDEN430-4995 vaccinees. Therefore, alternative DENV4 vaccine strains were selected for further clinical development. == INTRODUCTION == Dengue has emerged as the worlds most important mosquito-borne viral infection. The World Health Organization estimates that there are 50100 million cases of dengue annually in the 2 2.5 billion people at risk for infection.1These infections result in hundreds of thousands of hospitalizations and approximately 20, 000 deaths each year. Children bear the brunt of the dengue-associated burden of disease, which is estimated to be as high as 616,000 disability-adjusted life years.2 There are four antigenically distinct serotypes of dengue virus (DENV), namely DENV1, DENV2, DENV3, and DENV4. All of them are capable of causing the full spectrum of dengue disease, which ranges from an undifferentiated febrile illness to classic dengue fever to life-threatening dengue hemorrhagic fever/dengue shock syndrome.3Although long-term homotypic immunity is generated after infection with a single DENV serotype,4heterotypic protection is less durable.57Pre-existing immunity to one DENV serotype has been identified as a risk factor for more severe disease upon a secondary, heterotypic infection.810For these reasons, an effective dengue vaccine should induce long-lived protective immunity against all four DENV serotypes simultaneously, be attenuated and immunogenic in both DENV-negative persons and in persons with various levels of immunity to one or more DENVs, and exhibit an acceptable safety profile. Encouraged by the success of the live attenuated yellow fever vaccine, several live attenuated dengue vaccine candidates are being evaluated in clinical trials.1113However, formulating a live attenuated tetravalent dengue vaccine that is sufficiently attenuated for each of the monovalent components and immunogenic for all four DENV serotypes has been challenging. Components of several live attenuated vaccines that were attenuated and immunogenic in animal models as monovalent candidates exhibited either insufficient attenuation or insufficient immunogenicity in a tetravalent formulation.1418Attenuation phenotypes that have been identified in preclinical studies such as temperature sensitivity, small-plaque phenotype, or reduced level or frequency of viremia in non-human primates, have not consistently predicted a satisfactory level of infectivity, attenuation, or immunogenicity in humans.12,13,1922Therefore, it seems prudent to develop a panel of attenuated vaccine candidates for each DENV serotype and to identify in phase 1 studies the best four monovalent candidates for inclusion in a tetravalent vaccine. The dengue vaccine development strategy pursued by the Laboratory of Infectious Diseases is based on the introduction of an attenuating 30-nucleotide deletion mutation (30) into the 3 untranslated region (UTR) of DENV1 and DENV4, and on chimerization of DENV2 and DENV3 with rDEN430.23Additional approaches for developing an appropriately attenuated DENV3 vaccine are based on the use of more extensive deletions in the 3-UTR and on replacement of the 3-UTR of DEN3 with that derived from DEN430.24 We have previously reported the safety and immunogenicity of rDEN430, a highly immunogenic DEN4 vaccine that was safe and well tolerated by volunteers, with minimal reactogenicity. rDEN430 was tested at doses ranging from 105to 101plaque-forming units (PFU).25,26At the highest dose, 25% of vaccinees developed a transient increase in alanine aminotransferase (ALT) levels, and 50% developed an asymptomatic maculopapular rash.25In response to the observed elevation in serum ALT levels in some vaccinees at the 105PFU dose, it was decided to further attenuate rDEN430 by the introduction of previously identified mutations that reduced replication in human liver cells. Two additional vaccine candidates were created: rDEN430-200,201, which encodes two charge-to-alanine substitutions at amino acid residues 200 and 201 of the nonstructural 5 (NS5) protein27and rDEN430-4995, which encodes a single residue change at Methasulfocarb amino acid 158 of NS3. This NS3 mutation was originally identified as one that promoted efficient virus replication in Vero cellsin vitroand was attenuatingin vivo.28In preclinical studies, rDEN430-200,201 and rDEN430-4995 were more attenuated than rDEN430 in Methasulfocarb severe combined immunodeficiency (SCID) mice implanted with a human tumor cell line (SCID-HuH-7 mice) and in rhesus macaques.29,30Although both viruses were Methasulfocarb highly attenuated in rhesus monkeys, both induced moderate levels of Mouse Monoclonal to Rabbit IgG neutralizing antibodies and prevented replication of challenge DENV4. We previously reported that compared with rDEN430, rDEN430-200,201 exhibited increased attenuation, as shown by a reduced frequency of rash, ablation of elevation of ALT levels and viremia in dengue-negative humans, and retention of sufficient Methasulfocarb immunogenicity.31rDEN430 and rDEN430-200,201 were passaged and manufactured in Vero cells, and both viruses acquired Vero cell adaptation mutations during passagein vitro; virus titers greater than 107PFU/mL were achieved during manufacture Methasulfocarb of the clinical trials material. Because the 4995 mutation promoted efficient virus replication in Vero cells, thereby decreasing the likelihood of additional Vero cell adaptation mutations, and because it decreased replication in human.