S-540956, a CpG Oligonucleotide Annealed to a Complementary Strand With an Amphiphilic Chain Unit, Acts as a Potent Cancer Vaccine Adjuvant by Targeting Draining Lymph Nodes

Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.

A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

Complete genome sequence of a novel mitovirus isolated from Paris polyphylla var. yunnanensis

Paris mitovirus 1 (ParMV1) is a positive-sense RNA virus isolated from diseased Paris polyphylla var. yunnanensis plants in Wenshan, Yunnan. The complete genome sequence of ParMV1 consists of 2,751 nucleotides with a genome structure typical of the mitoviruses. ParMV1 genome has a single open reading frame (ORF: 358-2,637 nt) that encodes RNA-dependent RNA polymerase (RdRp) with a molecular mass of 86.42 kDa. ParMV1 contains six conserved motifs (Ι-VΙ) that are unique to mitoviruses. In addition, the 5′and 3′ terminals of the genome have a stable secondary structure, and the reverse complementary sequence forms a panhandle structure. Comparative genome analysis revealed that ParMV1 shares 23.1–40.6% amino acid (aa) and 32.3–45.7% nucleotide (nt) sequence identities with the RdRp of other mitoviruses. The phylogenetic tree inferred from RdRp aa sequence showed that ParMV1 clusters with mitoviruses, and hence should be considered as a new member of the genus Mitovirus in the family Motiviridae. This is the first report of a novel mitovirus infecting Paris polyphylla var. yunnanensis.

Employing CRISPR/Cas9 for de-repression of specific miRNA-target genes in rice

MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or translation repression, thereby regulating various development and physiological processes in eukaryotic orgasms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM), can block endogenous mature miRNAs activity and eliminate the inhibition to their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve de-repression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 and OsGRF8 carrying in-frame mutants with disrupting the miR396 recognition sites, escape from miR396-mediated post-transcriptional silence, resulting in enlarged grain size and increased the brown planthopper (BPH) resistance in their respective rice transgenic lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely de-repress particular target genes of functional importance for trait improvement in plants.

Electrochemical Resistive DNA Biosensor for the Detection of HPV Type 16

In this work, a low-cost and rapid electrochemical resistive DNA biosensor based on the current relaxation method is described. A DNA probe, complementary to the specific human papillomavirus type 16 (HPV-16) sequence, was immobilized onto a screen-printed gold electrode. DNA hybridization was detected by applying a potential step of 30 mV to the system, composed of an external capacitor and the modified electrode DNA/gold, for 750 µs and then relaxed back to the OCP, at which point the voltage and current discharging curves are registered for 25 ms. From the discharging curves, the potential and current relaxation were evaluated, and by using Ohm’s law, the charge transfer resistance through the DNA-modified electrode was calculated. The presence of a complementary sequence was detected by the change in resistance when the ssDNA is transformed in dsDNA due to the hybridization event. The target DNA concentration was detected in the range of 5 to 20 nM. The results showed a good fit to the regression equation ΔRtotal(Ω)=2.99 × [DNA]+81.55, and a detection limit of 2.39 nM was obtained. As the sensing approach uses a direct current, the electronic architecture of the biosensor is simple and allows for the separation of faradic and nonfaradaic contributions. The simple electrochemical resistive biosensor reported here is a good candidate for the point-of-care diagnosis of HPV at a low cost and in a short detection time.