Cold-adapted influenza vaccine carrying three repeats of a respiratory syncytial virus (RSV) fusion glycoprotein epitope site protects BALB/c mice and cotton rats against RSV infection.

Respiratory syncytial virus is the major cause of respiratory viral infections, particularly in infants, immunocompromised populations, and the elderly (over 65 years old), the prevention of RSV infection has become a priority. In this study, we generated a chimeric influenza virus, termed LAIV/RSV/HA-3F, using reverse genetics technology which contained three repeats of the RSV fusion protein neutralizing epitope site II to the N terminal in the background of the hemagglutinin (HA) gene of cold adapted influenza vaccine A/California/7/2009 ca. LAIV/RSV/HA-3F exhibited cold-adapted (ca) and attenuated (att) phenotype. BALB/c mice immunized intranasally with LAIV/RSV/HA-3F showed robust immunogenicity, inducing viral-specific antibody responses against both influenza and RSV, eliciting RSV-specific humoral, cellular and mucosal immune responses. LAIV/RSV/HA-3F also conferred protection as indicated by reduced viral titers and improved lung histopathological alterations against live RSV virus challenge. Mechanismly, single-cell RNA sequencing (scRNA-seq) and single-cell T cell antigen receptor (TCR) sequencing were employed to characterize the immune responses triggered by chimeric RSV vaccine, displaying that LAIV/RSV/HA-3F provided protection mainly via interferon-γ (IFN-γ). Moreover, we found that LAIV/RSV/HA-3F significantly inhibited viral replication in the challenged lung and protected against subsequent RSV challenge in cotton rats without causing lung disease. Taken together, our findings demonstrated that LAIV/RSV/HA-3F has potential as a promising bivalent vaccine with dual purpose candidate for the prevention of influenza and RSV, and preclinical and clinical studies warrant further investigations.

A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge.

Influenza viruses present a significant threat to global health. The production of a universal vaccine is considered essential due to the ineffectiveness of current seasonal influenza vaccines against mutant strains. mRNA technology offers new prospects in vaccinology, with various candidates for different infectious diseases currently in development and testing phases. In this study, we encapsulated a universal influenza mRNA vaccine. The vaccine encoded influenza hemagglutinin (HA), nucleoprotein (NP), and three tandem repeats of matrix protein 2 (3M2e). Twice-vaccinated mice exhibited strong humoral and cell-mediated immune responses in vivo. Notably, these immune responses led to a significant reduction in viral load of the lungs in challenged mice, and also conferred protection against future wild-type H1N1, H3N2, or H5N1 influenza virus challenges. Our findings suggest that this mRNA-universal vaccine strategy for influenza virus may be instrumental in mitigating the impact of future influenza pandemics.

Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor.

Rolling circle amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogens without the need for sequencing. In this study, a surface plasmon resonance DNA biosensor based on RCA with a gold (Au) nanoparticle surface was established for isothermal identification of DNA. The probes included a specific padlock probe, a capture probe (CP), which is bound to biotin, and an Au nanoparticle-modified probe, which hybridizes with the RCA products. The CP was assembled on gold nanoparticles to increase its ability to bind and hybridize. The linear padlock probe, which was designed to circularize by ligation upon recognition of the bacterial pathogen-specific sequence in 16S rDNA, hybridizes to fully complementary sequences within the CP. Upon recognition, each target gene DNA is distinguished by localization onto the corresponding channel on the chip surface. Then, the immobilized CPs act as primers to begin the in situ solid-phase RCA reaction, which produces long single-stranded DNA. The RCA products fixed on the chip surface cause significant surface plasmon resonance angle changes. We demonstrated that six different bacterial pathogens can be identified simultaneously and that 0.5 pM of synthetic oligonucleotides and 0.5 pg µl(-1) of genomic DNA from clinical samples can be detected by this method with low background signals. Therefore, the multiplex diagnostic method provides a highly sensitive and specific approach for the rapid identification of positive samples.

BRAF mutations in patients with non-small cell lung cancer: a systematic review and meta-analysis.

BACKGROUND: BRAF mutations have been well described in non-small cell lung cancer (NSCLC) for several years, but the clinical features of patients harboring BRAF mutations are still not well described. We performed a meta-analysis to identify common clinical features in NSCLC patients carrying BRAF mutations. METHODS: We identified clinical studies that examined the association between BRAF mutations and features of NSCLC within PubMed, Embase and ISI Science Citation Index database up to October 2013. The effect size of clinical features was estimated by odds ratios (ORs) with 95% confidence interval (CI) for each study, using a fixed-effects or random-effects model. RESULTS: Ten studies with a total of 5599 NSCLC patients were included. There was a 3% (170/5599) BRAF mutation rate. BRAF mutations in NSCLC were significantly associated with adenocarcinomas (ADCs) (compared with non-ADCs, OR = 4.96, 95%CI = 2.29-10.75). There were no significant differences in gender, smoking and stage in patients with and without BRAF mutations. The BRAFV600E mutation was more frequent in women than non-BRAFV600E mutations (OR = 0.27, 95%CI = 0.12-0.59), and was closely related to never smokers (OR = 0.14, 95%CI = 0.05-0.42). CONCLUSIONS: These findings have important implications for the prediction of the NSCLC sub-types more accurately combined with other genetic changes.

Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.

BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC. METHODS: We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model. RESULTS: 25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation. CONCLUSIONS: This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

High-resolution melting analysis for accurate detection of BRAF mutations: a systematic review and meta-analysis.

The high-resolution melting curve analysis (HRMA) might be a good alternative method for rapid detection of BRAF mutations. However, the accuracy of HRMA in detection of BRAF mutations has not been systematically evaluated. We performed a systematic review and meta-analysis involving 1324 samples from 14 separate studies. The overall sensitivity of HRMA was 0.99 (95% confidence interval (CI) = 0.75-0.82), and the overall specificity was very high at 0.99 (95% CI = 0.94-0.98). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 68.01 (95% CI = 25.33-182.64), 0.06 (95% CI = 0.03-0.11), and 1263.76 (95% CI = 393.91-4064.39), respectively. The summary receiver operating characteristic curve for the same data shows an area of 1.00 and a Q* value of 0.97. The high sensitivity and specificity, simplicity, low cost, less labor or time and rapid turnaround make HRMA a good alternative method for rapid detection of BRAF mutations in the clinical practice.

High-sensitivity PCR method for detecting BRAF V600E mutations in metastatic colorectal cancer using LNA/DNA chimeras to block wild-type alleles.

The response to epidermal growth factor receptor (EGFR)-targeted therapy in metastatic colorectal cancer (mCRC) is variable because of intra-tumor heterogeneity at the genetic level, and consequently, it is important to develop sensitive and selective assays to predict patient responses to therapy. Low-abundance BRAF V600E mutations are associated with poor response to treatment with EGFR inhibitors. We developed a method for the detection of BRAF V600E mutations in mCRC using real-time wild-type blocking PCR (WTB-PCR), in which a chimera composed of locked nucleic acids and DNA is incorporated to amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. Mixing experiments showed that this method is exquisitely sensitive, with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000. To demonstrate the applicability of this approach for mCRC patients, we assessed the V600E mutations in 50 clinical cases of mCRC by real-time WTB-PCR. The percentage of patients with V600E mutation as determined by WTB-PCR (16%, 8/50) was higher than by traditional PCR (10%, 5/50), suggesting an increased sensitivity for WTB-PCR. By calculating the ΔC q for real-time traditional PCR, which amplifies all BRAF alleles, versus WTB-PCR, which selectively amplifies mutant BRAF, we demonstrated that among the V600E-positive mCRC patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 0.05 to 52.32%. In conclusion, WTB-PCR provides a rapid, simple, and low-cost method to detect trace amounts of mutated BRAF V600E gene.