Antibodies to a Single, Conserved Epitope in Anopheles APN1 Inhibit Universal Transmission of Plasmodium falciparum and Plasmodium vivax Malaria

International audienc

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates

<div><p>Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia^® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. <i>In vitro</i> transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, <i>in vitro</i> ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, <i>in vitro</i> ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.</p></div

Immunogenicity of CYD viruses in rhesus monkeys.

<p>Longitudinal geometric mean virus neutralization titers in rhesus monkeys immunized with a tetravalent formulation of recombinant CYD viruses. A group of 4 monkeys was immunized with a tetravalent formulation containing 10⁵ PFU of each of rYFD1-PUO359, rYFD2-PUO218, rYFD3-PaH881/88 and rYFD4-1228 (TVP-980) viruses via the subcutaneous route. A negative control group of 4 monkeys was included that received saline via intramuscular route. Both groups were immunized at 0, and 6 months. Virus neutralizing antibody titers were measured over a period of 9 months using a LiCor-based neutralization assay. The geometric mean virus neutralization titers at each time point are presented representing the virus neutralization results for DENV1, DENV2, DENV3, and DENV4.</p

Recombinant chimeric Yellow fever dengue constructs.

<p>Recombinant chimeric Yellow fever dengue constructs.</p

Summary of recombinant viruses recovered.

<p>Summary of recombinant viruses recovered.</p

Confirmation of recombinant virus recovery.

<p><b>A</b>. Infectivity of RNA transcripts was determined in Vero cells. <b>B</b>. Virus recovery was also confirmed by immunostaining using pan-flavivirus antibody 4G2. <b>C</b>. Virus recovery and identity was confirmed by RT-PCR using primers specific to YF (1), D4-MY01 (2), D4-MY01/YF junction (3), 5’ YF/D3 junction (4) and 3’ YF/D3 junction (5).</p

Outline of recombinant YF-17D-dengue chimeric virus recovery.

<p>The chimeric constructs were designed such that recombinant viruses can be recovered either by the single-plasmid (linearized single plasmid) or the two plasmid approach (<i>in-vitro</i> ligation of DNA fragments). The full-length chimeric cDNA clone or <i>in-vitro</i>-ligated chimeric DNA was linearized with XhoI to allow for run-off transcription. A LONG-PCR step was also performed in some cases to generate templates for <i>in vitro</i> transcription. Chimeric full-length viral RNA transcripts thus generated were transfected into Vero cells for virus recovery.</p