mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

BACKGROUND: Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. METHODS: Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. RESULTS: miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. CONCLUSIONS: miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates.

Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5' to 3') a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

Evaluation of the effects of applying the ventricular care bundle (VCB) method for reducing ventilator-associated pneumonia (VAP) in the intensive care unit of a general Chinese tertiary hospital.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a severe complication that occurs within patients who must use ventilators in the intensive care unit (ICU). Ventilator care bundles (VCB) have been applied across many developed regions and have produced positive results in controlling VAP. In this study, we report on the implementation and effects of using VCBs to manage VAP in a general tertiary hospital in the Inner Mongolia Autonomous Region of China. METHODS: A targeted surveillance method was used to survey all the patients (n=4,716) in the ICU from June 1, 2017 to May 31, 2019. Patients from June 1, 2017 to May 31, 2018, and June 1, 2018, to May 31, 2019, were respectively divided into 2 groups: the control group (2,029 patients) and intervention group (2,687 patients). These dates were selected because VCB was implemented from June 1, 2018, in our institution. The variables that were associated with VCB and observed were the head-of-bed elevation, oral care, maintenance of the pressure for the cuff of the endotracheal tube, aspiration of subglottic secretion, daily sedation vacation protocol, daily extubation assessment results, and hand hygiene. After collecting the data, the compliance of VCB, ventilator use ratio, and the incidence rate of VAP in these 2 groups were compared. RESULTS: We observed that compliance with all of the intervention measures for VCB improved results in the intervention group compared to the control. Furthermore, the compliance rate of hand hygiene increased from 71.99% to 91.97%, and the head-of-bed elevation of 30°-45° increased from 62.02% to 85.96%. All differences between these two groups were statistically significant, according to the χ 2 -test. The ventilator use ratio was statistically and significantly lower in the intervention group (34.86%) compared to the control group (40.29%) (χ 2 =95.513, P<0.001). The incidence rate of VAP was statistically and significantly lower in the intervention group (13.70‰) compared to the control group (18.85‰) (χ 2 =5.471, P=0.019). CONCLUSIONS: Our results show that VCB prevents VAP. Therefore, personnel training, clinical supervision, and surveillance feedback could promote a reduction in intervention measures.

Drug resistance of healthcare-associated pathogenic bacteria and carbapenem-resistant Acinetobacter baumannii homology in the general intensive care unit.

BACKGROUND: The objective of this study was to understand the distribution and drug resistance of healthcare-associated infection (HAI) pathogens in an intensive care unit (ICU) of a general tertiary hospital in Inner Mongolia, and to classify carbapenem-resistant Acinetobacter baumannii (CR-AB) in ICU patients and environmental samples. Additionally, this study aimed to provide scientific evidence for the use of clinical antibiotics and effective prevention and control measures for CR-AB outbreak. METHODS: The distribution and drug resistance of pathogens isolated from patient's samples in the ICU of 12 Hospitals from January to May 2019 were retrospectively analyzed. Meanwhile, CR-AB isolated from patients and environmental samples were collected and classified by pulsed-field gel electrophoresis (PFGE). RESULTS: The pathogens isolated from ICU samples, mainly Gram-negative bacteria (63.07%), were CR-AB, Klebsiella pneumoniae, and Pseudomonas aeruginosa; the main Gram-positive bacteria (22.13%) were Enterococcus faecium and Staphylococcus aureus; and fungi accounted for the remaining (14.80%). The samples mainly came from sputum (41.09%). Among non-fermenting bacteria, the resistance rates of CRAB to piperacillin, piperacillin/tazobactam, and other treatments were higher than those of Pseudomonas aeruginosa (P<0.05). Meanwhile, the resistance rates to ampicillin/sulbactam and compound sulfamethoxazole were lower than those of Pseudomonas aeruginosa (P<0.05). The resistance rates of Klebsiella pneumoniae to piperacillin/tazobactam, ceftazidime, and others were higher than those of Escherichia coli (P<0.05). Among Gram-positive bacteria, the resistance rates of Enterococcus faecium to erythromycin, clindamycin, and other treatment were higher than those of Staphylococcus aureus (P<0.05). A total of 62 bands were obtained from 63 strains of CR-AB by electrophoresis. Also, 16 clusters (A-P) were obtained with a 74% similarity coefficient, among which K, L, and N types (more than 9 strains) were more common. CONCLUSIONS: Gram-negative bacteria were the primary pathogens of HAI in the ICU, and their drug resistance was serious. There is homology in the PFGE typing of CR-AB. Therefore, hospitals should strengthen the surveillance of drug-resistant pathogenic bacteria. Additionally, further cleaning and disinfection measures are needed to improve environmental hygiene and prevent outbreaks of HAI.

Healthcare-associated infection prevention and control management in a tertiary hospital and an overall evaluation.

BACKGROUND: Healthcare-associated infection (HAI) is a crucial factor influencing medical quality. Studies about HAI management situations are rare, especially for the Inner Mongolia region of China. Therefore, this study aimed to investigate management procedures and the overall evaluation of HAI in order to inform HAI management improvement more scientifically. METHODS: A questionnaire was used to investigate HAI-related prevention and control indicators in tertiary hospitals in the Inner Mongolia region from July 2018 to June 2019. RESULTS: The survey showed that the mean incidence rate of HAI was 3.79%. The mean rate of hand hygiene compliance of healthcare workers (HCWs), inpatient's antibiotics-use rate, and the detection of the antibiotic ratio before therapy was 54.34%, 34.33%, and 25.40%, respectively. The mean of the surgical site infection (SSI) rate of the level I incision and the preventive antibiotics-use ratio of the level I incision was 1.31% and 28.89%, respectively. The mean of the multi-drug resistant organism (MDRO) infection rate was 0.40% and the mean of the MDRO detection rate was 18.55%. The mean of the central line-associated bloodstream infection rate was 2.24%, the ventilator-associated pneumonia (VAP) rate was 11.17%, and the catheter-associated urinary tract infection (CAUTI) rate was 1.95‰. As for the overall evaluation, 19 (35.85%) hospitals had a bad grade, 18 (33.96%) hospitals had a medium grade, and 16 (30.19%) hospitals had a good grade. CONCLUSIONS: The incidence rate of HAI in tertiary hospitals in the Inner Mongolia region is higher than the national level. Also, the overall evaluation of bad-grade hospitals and their deficiencies should be used as an example to improve the HAI management level.

Homeobox B8 Targets Sterile Alpha Motif Domain-Containing Protein 9 and Drives Glioma Progression.

Gliomas are the most commonly occurring tumors of the central nervous system. Glioblastoma multiforme (GBM) is the most malignant and aggressive brain cancer in adults. Further understanding of the mechanisms underlying the aggressive nature of GBM is urgently needed. Here we identified homeobox B8 (HOXB8), a member of the homeobox family, as a crucial contributor to the aggressiveness of GBM. Data mining of publicly accessible RNA sequence datasets and our patient cohorts confirmed a higher expression of HOXB8 in the tumor tissue of GBM patients, and a strong positive correlation between the expression level and pathological grading of tumors and a negative correlation between the expression level and the overall survival rate. We next showed that HOXB8 promotes the proliferation and migration of glioblastoma cells and is crucial for the activation of the PI3K/AKT pathway and expression of epithelial-mesenchymal transition-related genes, possibly through direct binding to the promoter of SAMD9 (Sterile Alpha Motif Domain-Containing Protein 9) and activating its transcription. Collectively, we identified HOXB8 as a critical contributor to the aggressiveness of GBM, which provides insights into a potential therapeutic target for GBM and opens new avenues for improving its treatment outcome.

High-throughput screening of active compounds against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 ±â€¯1.41 and 3.13 ±â€¯0.98 µM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.

Active surveillance of ventilator-associated pneumonia in the intensive care unit and establishment of the risk grading system and effect evaluation.

BACKGROUND: To discuss ventilator-associated pneumonia (VAP) patient's clinical characteristic and related factors in the intensive care unit (ICU), and to establish a risk grading system for VAP patients in the ICU in order to provide a reference for VAP prevention. METHODS: A total of 1,513 patients in eight ICUs who received mechanical ventilation between June 2015 and June 2018 were randomized and into two groups, with 908 patients in the model group and 605 patients in the verification group. The model group was used to analyze the influencing factors of VAP and establish a risk grading system, while the verification group was used to verify the risk grading system. A receiver operating characteristic (ROC) curve was used to evaluate the predictive effect of the grading system. RESULTS: During the 3-year study period, of the 1,513 total patients, 188 patients were infected with VAP, leading to an incidence rate of 12.43% (188/1,513) and an infection rate of 15.23‰ (188/12,347). ICU length of stay, mechanical ventilation days, frequency of oral care, unused subglottic secretion drainage, tracheotomy, APACHE II score, and combined antibiotics use were risk factors of VAP infection for patients who received mechanical ventilation in the modeling group (P<0.05). In a VAP risk-grading system established based on risk factors, the high, medium and low-grade patients had a statistically significantly different VAP infection rate in the model group, and patients with a high grade had a higher risk of VAP infection. Patients' data in the model and verification groups were used to draw a ROC curve which showed a good predictive effect. CONCLUSIONS: This study establishes and verifies the VAP risk grading system for patients who receive mechanical ventilation. It is helpful in high-risk patient surveillance and in reducing and preventing VAP infection.

Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay.

Human respiratory syncytial virus (RSV) is the single most important cause of lower respiratory tract disease in infants and young children and a major viral agent responsible for respiratory tract disease in immunosuppressed individuals and the elderly, but no vaccines and antiviral drugs are available. Herein the recombinant RSV (rRSV) encoding enhanced green fluorescence protein (EGFP, rRSV-EGFP) was constructed and the potential for screening anti-RSV drugs was investigated. The recombinant plasmid of pBRATm-rRSV-EGFP, containing T7 transcription cassette composed of T7 promoter, RSV antigenomic cDNA with EGFP gene, HDV ribozyme (δ), and T7 terminator in the order of 5' to 3', was constructed and cotransfected into BHK/T7-9 cells together with helper plasmids encoding N, P, L, and M2-1 gene, respectively. The rescued rRSV-EGFP was confirmed by increasing expression of EGFP over blind passages and by RT-PCR. rRSV-EGFP was comparable to the other two recombinant RSVs encoding red fluorescent protein (RFP, rRSV-RFP) or luciferase (Luc, rRSV-Luc) in the growth kinetic, and there was a difference in sensitivity between them for screening anti-RSV agents based on infection of HEp-2 cells. The EGFP-encoding rRSV has been constructed and rescued successfully and has the potential for high-throughput anti-RSV drug screening in vitro.

A Built-In CpG Adjuvant in RSV F Protein DNA Vaccine Drives a Th1 Polarized and Enhanced Protective Immune Response.

Human respiratory syncytial virus (RSV) is the most significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection.

A single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8+ T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production.

Enhanced humoral and CD8+ T cell immunity in mice vaccinated by DNA vaccine against human respiratory syncytial virus through targeting the encoded F protein to dendritic cells.

Human respiratory syncytial virus (RSV) is the most important cause of serious lower respiratory tract infection in infants, the elderly, and the immunocompromised population. There is no licensed vaccine against RSV until now. It has been reported that targeting antigen to DEC205, a phagocytosis receptor on dendritic cells (DCs), could induce enhanced CD4+ and CD8+ T cell responses in mice. To develop RSV DNA vaccine and target the encoded antigen protein to DCs, the ectodomain of fusion glycoprotein (sF, amino acids: 23-524) of RSV was fused with anti-DEC205 single-chain Fv fragment (scDEC) and designated scDECF. Following successful expression from the recombinant plasmid of pVAX1/scDECF, the recombinant protein of scDECF was found capable of specifically binding to DEC205 receptor on CHOmDEC205 cells, and facilitating uptake of RSV F by DC2.4 cells in vitro. Furthermore, the higher levels of RSV-specific IgG antibody responses and neutralization antibody titers, as well as RSV F-specific CD8+ T cell responses were induced in mice immunized intramuscularly by pVAX1/scDECF than by the control plasmid of pVAX1/scISOF encoding sF protein fused with isotype matched control single-chain Fv fragment (scISO). Compared with pVAX1/scISOF, both the ratio of IgG2a/IgG1, >1, and the enhanced IFN-γ cytokine were induced in mice following pVAX1/scDECF immunization, which exhibited a Th1 dominant response in pVAX1/scDECF vaccinated mice. Notably, the elevated efficiency of RSV F protein bound by DCs in vivo could also be observed in mice inoculated by pVAX1/scDECF. Collectively, these results demonstrate the enhanced IgG and CD8+ T cell immune responses have been induced successfully by DNA vaccine against RSV by targeting F antigen to DCs via the DEC205 receptor, and this DC-targeting vaccine strategy merits further investigation.

DNA vaccine encoding central conserved region of G protein induces Th1 predominant immune response and protection from RSV infection in mice.

Human respiratory syncytial virus (RSV) can cause serious infection in the lower respiratory tract, especially in infants, young children, the elderly and the immunocompromised population worldwide. Previous study demonstrated the polypeptide (amino acids 148-198) of RSV attachment (G) glycoprotein, corresponding to the central conserved region and encompassing CX3C chemokine motif, could induce antibodies and protection from RSV challenge in mice [1,2]. In this study, we evaluated the immune efficacy of the recombinant DNA vaccine of pVAX1/3G148-198 encoding RSV G protein polypeptide. RSV specific serum IgG antibodies with neutralizing activity were stimulated following prime-boost immunization of pVAX1/3G148-198 intramuscularly, and the ratio of IgG2a/IgG1 was 4.93, indicating a Th1 biased immune response. After challenged intranasally with RSV Long, the vaccinated mice showed both decreased lung RSV titers, pulmonary inflammation and body weight loss. The results suggest that pVAX1/3G148-198 DNA vaccine may be an effective RSV vaccine candidate, and deserves further exploration.

Kinesin-1 inhibits the aggregation of amyloid-ß peptide as detected by fluorescence cross-correlation spectroscopy.

Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-ß (Aß) generated by the proteolytic processing of amyloid-ß precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Aß generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Aß with kinesin-1 at the single-molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Aß over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

[Construction and rescue of the minigenome of bovine parainfluenza virus type 3 based on T7 promoter expression system].

Objective: To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3). Methods: We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy. Results: We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3. Conclusion: This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

Single chain variable fragment against aß expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-ß (Aß) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aß N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aß fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aß aggregation or disaggregation in vitro. Here, a model of cell-free Aß "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and Aß fibril maturation. The effect of A8 scFv on Aß fibrillogenesis was markedly more significant when administered at the start of the Aß folding reaction. Furthermore, the results also showed that pre-formed Aß fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aß aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" Aß aggregation and will assist in the development of therapeutic strategies for AD.

Sublingual administration of a helper-dependent adenoviral vector expressing the codon-optimized soluble fusion glycoprotein of human respiratory syncytial virus elicits protective immunity in mice.

Sublingual (s.l.) immunization has been described as a convenient and safe way to induce mucosal immune responses in the respiratory and genital tracts. We constructed a helper-dependent adenoviral (HDAd) vector expressing a condon-optimized soluble fusion glycoprotein (sFsyn) of respiratory syncytial virus (HDAd-sFsyn) and explored the potential of s.l. immunization with HDAd-sFsyn to stimulate immune responses in the respiratory mucosa. The RSV specific systemic and mucosal immune responses were generated in BALB/c mice, and the serum IgG with neutralizing activity was significantly elevated after homologous boost with s.l. application of HDAd-sFsyn. Humoral immune responses could be measured even 14weeks after a single immunization. Upon challenge, s.l. immunization with HDAd-sFsyn displayed an effective protection against RSV infection. These findings suggest that s.l. administration of HDAd-sFsyn acts as an effective and safe mucosal vaccine against RSV infection, and may be a useful tool in the prevention of RSV infection.

Intranasal immunization with a helper-dependent adenoviral vector expressing the codon-optimized fusion glycoprotein of human respiratory syncytial virus elicits protective immunity in BALB/c mice.

BACKGROUND: Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract. Currently, there is no clinically approved vaccine against RSV infection. Recent studies have shown that helper-dependent adenoviral (HDAd) vectors may represent effective and safe vaccine vectors. However, viral challenge has not been investigated following mucosal vaccination with HDAd vector vaccines. METHODS: To explore the role played by HDAd as an intranasally administered RSV vaccine vector, we constructed a HDAd vector encoding the codon optimized fusion glycoprotein (Fsyn) of RSV, designated HDAd-Fsyn, and delivered intranasally HDAd-Fsyn to mice. RESULTS: RSV-specific humoral and cellular immune responses were generated in BALB/c mice, and serum IgG with neutralizing activity was significantly elevated after a homologous boost with intranasal (i.n.) application of HDAd-Fsyn. Humoral immune responses could be measured even 14 weeks after a single immunization. Immunization with i.n. HDAd-Fsyn led to effective protection against RSV infection on challenge. CONCLUSION: The results indicate that HDAd-Fsyn can induce powerful systemic immunity against subsequent i.n. RSV challenge in a mouse model and is a promising candidate vaccine against RSV infection.

[Antibody responses induced by mucosal DNA vaccine encoding the codon-optimized F protein of human respiratory syncytial virus (RSV) delivered with attenuated Salmonella typhimurium].

OBJECTIVE: In order to investigate antibody responses by musosal DNA vaccines encoding the codon-optimized F protein of human respiratory syncytial virus (RSV) delivered with attenuated Salmonella typhimurium aroA strain SL7207 (SL7207) via intranasal or intragastric routes. METHODS: After the codon-optimized F gene was synthesized, we constructed eukaryotic expression plasmid pcDNA3.1/Fsyn and transformed it into SL7207 to get recombinant SL7207 of SL7207/pcDNA3.1/Fsyn. Then, SL7207/pcDNA3.1/Fsyn was exploited to evaluate its immunogenicity in BALB/c mice administered intranasally or intragastrically. The resultant serum and mucosal antibody responses were analyzed by indirect ELISA. RESULTS: Compared with intragastric immunization group, intranasal immunization group achieved higher level of RSV specific serum IgG and secretion IgA (SIgA) (P < 0.05). Moreover, F protein expressed by the codon-optimized F gene of Fsyn was of elevated immunogenicity, compared with wild type F (Fwt) (P < 0.05). CONCLUSION: Intranasal immunization and codon optimization approaches can improve antibody responses of RSV DNA vaccine delivered by attenuated Salmonella typhimurium aroA strain SL7207.