Wesley, R. given birth to to DENV2-immune or DENV2-na?ve dams were immunized with either DENV2 VRP or Rabbit Polyclonal to DP-1 live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the power of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic. Dengue viruses (DENV) are members of the family and one of the most important groups of emerging viruses of global significance today (36, 66). There are four distinct antigenic serotypes (DENV1, DENV2, DENV3, and DENV4), all of which are capable of causing a spectrum of diseases in humans ranging from asymptomatic infections to debilitating classical dengue fever and severe and often fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (36, 68). DENV is usually transmitted to humans primarily by the mosquito for 5 h through a 5-ml cushion of 20% (wt/vol) sucrose. The sedimented computer virus was further purified by density gradient centrifugation in a 10 to 40% iodixanol gradient at 163,700 for 120 min. Virus-containing fractions were pooled, and purified computer virus was concentrated by centrifugation at 72,000 for 5 h. The computer virus was resuspended in phosphate-buffered saline (PBS)-1% fetal bovine serum and stored at ?80C. Cloning the DENV2 prM/E cassette into the VEE replicon plasmid. cDNA of DENV2 prM/E genes was obtained from the mouse-neuroadapted DENV2 strain NGC RNA genome by reverse transcription-PCR. We designed a start codon and a stop codon flanking the sequence encoding (5 to 3) the C-terminal domain name of the capsid gene made up of the prM signal sequence (20 amino acids), the prM gene, and the E gene. The sequences of the primers used to amplify this gene cassette are as follows: forward primer, 5 AGTCTAGTCCGCCAAGATGTTGAACAGGAGACGCAGAACTGCAGG; reverse Serotonin Hydrochloride primer, 5 GGCGCGCCTTAGGTCTGCACCATAACTCCCAAATACAGCGT. The amplified regions were initially cloned into PCR cloning plasmids, and their sequences were confirmed. The prM/E gene cassette was cloned into the multicloning site of the VEE replicon vector pVR21 (2) using ApaI and AscI sites upstream and downstream of the 26S subgenomic-RNA transcription start site, respectively, by overlapping extension Serotonin Hydrochloride PCR to generate pVRDENV2prM/E. The clone was linearized at a unique NotI site downstream of the VEE 3 untranslated region and poly(A) tract, and full-length T7 transcripts were generated in vitro using an mMessage mMachine kit (Ambion) as previously described (14). To package the recombinant replicon genome into VRP for delivery in vitro and in vivo, the replicon RNA was mixed with two helper RNAs also transcribed in vitro using T7 polymerase. One helper encoded only the capsid gene, and the other encoded only Serotonin Hydrochloride the glycoproteins from a cDNA clone of VEE, V3000. The helper RNAs had the replicase genes and the for 30 min, and the VRP were partially purified and concentrated by sedimentation at 72,000 for 3 h through a 5-ml cushion of 20% (wt/vol) sucrose dissolved in PBS. The pelleted VRP were resuspended overnight in endotoxin-free PBS with 1% donor calf serum at 4C, followed by storage at ?80C. Each VRP preparation was safety tested to ensure the absence of replication-competent computer virus that could have arisen by nonhomologous recombination. Ten percent of the preparation was used to inoculate BHK cells, and the presence of cytopathic effect was monitored during two sequential passages as an indication of the presence of replication-competent computer virus. Preparations that resulted in cytopathic effect failed the safety Serotonin Hydrochloride test and were discarded. VRP were titrated in BHK cells by indirect immunofluorescence assays (IFA). Cells seeded in eight-well chamber slides were infected with serial dilutions of the concentrated VRP preparation for 18 h at 37C, fixed in methanol for 10 min at 4C, and incubated sequentially with mouse polyclonal anti-DENV antibody, biotinylated anti-mouse immunoglobulin G (IgG), and avidin conjugated to fluorescein isothiocyanate. Replicon-infected fluorescent cells were.