| Background and Objection:Influenza, a contagious, acute respiratory disease caused by an influenza virus, is a worldwide pandemic and hazardous infection. Influenza virus infection represents a serious threat to public health. Each year, it affects a significant portion of population worldwide. Vaccinaion is the most efficient method to control influenza pandemics. Inactivated and live attenuated influenza virus vaccines are currently available. Commercial inactivated vaccines suffer from limited efficacy in cross-protective immunity and routes of administration, given by intramuscular injection. In contrast, live attenuated influenza virus vaccines are intranasally administered and activate all components of the immune system, inducing both a balanced systemic and local immune response and a broad humoral and cellular response. Their protective efficacy, therefore, lasts for longer periods.Most live attenuated vaccines utilize the virus that possesses cold-adapted temperature-sensitive phenotype. Inducing such temperature-sensitive phenotype is time consuming. Virus can be attenuated by other mechanisms. For instance, microRNA (miRNA) regulates viral replication through repression of translation. We reasoned that such technology can be used for generating live attenuated influenza virus for vaccine. Many miRNAs have a distinctive cell- and tissue-specific pattern of expression. Lungs are the main target organ for influenza viral infection. Let-7b expression is abundant in pulmonary tissues. We inserted the miR-let-7b target sequence into a 2009 pandemic (H1N1) virus genome (A/Nanjing/NJU-108/2009) to engineer a recombinant virus miRT-H1N1. We demonstrated that this recombinant virus was unable to infect HBE cells (human bronchial epithelial cells) that express a high level of corresponding miRNA-let-7b. Upon production of miRNA-let-7b the viral replication was restricted and the virus became attenuated.In the present study, we wanted to determine whether miRT-H1N1 virus produced an attenuated phenotype in vivo. It is well known that mice, particularly the BALB/c strain, are currently intensively employed for studying pathogenesis and preliminary efficacy studies for influenza vaccines [31]. The BALB/c mice were selected for this study. We investigated viral replication efficiency, elicited immune responses and protective efficacy in a mouse model immunized with the microRNA response elements (MREs)-containing influenza A virus.Methods:1.Experimental infection of miceVirus stocks were propagated in specific-pathogen free (SPF) chicken Embryos. After 48 h, the allantoic fluid were collected and detected for rescued virus which the HA result was positive. The 50% tissue culture infectious dose (TCID50) for each virus was determined by serial titration of virus in Madin-Darby canine kidney (MDCK) cells and calculated by the method developed by Reed and Muench. Six-week-old female BALB/c mice under chloral hydrate anesthesia, were intranasally infected with 105 TCID50 of WT-H1N1, miRT-H1N1 or scbl-H1N1 viruses in 50 μl phosphate-buffered saline (PBS). Animals were weighed daily and their mortality was monitored for 14 days after infection. Four mice per group were euthanized on day 3 and 5 post-infection and lung tissues were collected for viral titration. The recombinant virus was sequenced and identified by RT-PCR.2. Evaluation of immunogenicity in miceSix-week-old female BALB/c mice were primed with 50μl of PBS,105 TCID50 of miRT-H1N1 virus intranasally on day 0, and boosted with the same dose on day 28. Blood samples were collected for antibody detection on day 28 and day 42 post-vaccination. Serum antibody titers were determined by hemagglutination inhibition (HI) and microneutralization assays. Specific seral IgG were detected by enzyme-linked immunosorbent assay (ELISA).Meanwhile, six mice per group were euthanized two weeks after the second vaccination. Trachea-lung and nasal washes were harvested for detection of mucosal sIgA by ELISA. Single spleen cell suspensions were prepared and stimulated with purified H1N1 virus on day 28 post-vaccination. Culture supernatants were harvested for detecting interleukin (IL)-4 and interferon (IFN)-γ with ELISA after 48h of culture.3. Analysis of efficacy against wt-H1N1 ChallengeMice were intranasally challenged with 100 LD50 of WT-H1N1 virus at 42 days post-vaccination (dpv). Mock-infected control mice only received 50 μl PBS. Challenged mice were monitored and their symptoms, body weight and survival were recorded for 14 days. Three mice per group were euthanatized and the lung tissues were harvested on day 5 post-challenge. The paraffin-embedded tissues were sliced for hematoxylin-and-eosin (H&E) staining. The histopathologic score (HPS) was determined. IL-6, IFNy, and TNF-a. mRNA level was detected by quantitative RT-PCR.Results:1. Experimental infection of mice(1) The hemagglutinin titer of miRT-H1N1, scbl-H1N1 and WT-H1N1 virus was 1:29,1:28 and 1:28 respectly. The miRT-H1N1 virus replicated as well as WT-H1N1 or scbl-HlNlvirus.(2) Symptoms included rough fur, quietness, and loss of appetite and weight in all of the WT-H1N1 virus-infected mice. All of the mice infected with the wild-type virus were euthanized on days 5 and 8 because of the severity of their symptoms (lost more than 25% of their body weight), whereas the survival rates for the mice inoculated with miRT-H1N1 virus were 100%. The MLD50 of WT-H1N1 virus was 105-3TCID50. The control virus scbl-H1N1 displayed similar MLD50 value to wild type, as expected (105.38 TCID50). In contrast, the miRT-H1N1 virus was not lethal against any mice at such dose, resulting in increased doses (MLD50>107TCID50). We also analyzed RNA sequences of miRT-H1N1 virus isolated from lungs of infected mice. Sequence anaylysis showed no mutations in the miR-let-7b target region.2. Antibody responses to miRT-H1N1 immunization in miceAll mice immunized with miRT-H1N1 virus showed appreciable neutralizing activity against the homologous WT-H1N1 virus. Similarly, the levels of HI antibodies were higher in mice immunized with miRT-H1N1 virus than those treated with the PBS control. The geometric mean titer (GMT) of HI antibody against the WT-H1N1 virus was 403, at 28 days after the first immunization, which was sharply increased to 2032, two weeks after the second immunization. Mice developed robust IgG antibodies (GMT 905; 28 days post-first immunization). A much stronger IgG antibody response (GMT 2873) appeared after the booster (42 days post-immunization). Furthermore, immunization with the miRT-H1N1 virus induced higher levels of sIgA in trachea-lung (GMT 64) and nasal washes (GMT 16) compared with the control group. Additionally, stronger IL-4 and IFN-g production was detected in spleen cells isolated from mice immunized with miRT-H1N1 virus compared with mock-immunized controls.3. Protective efficacy of miRT-H1N1 virus immunization in miceAll animals vaccinated with miRT-H1N1 were fully protected against the lethal HINlinfection with 100 MLD50of WT-H1N1 virus as demonstrated by the absence of signs of disease and limited weight loss after challenge compared with mock-vaccinated groups. In contrast, mock-vaccinated mice succumbed to challenge within 8 days post-challenge (dpc). The miRT-H1N1 virus-immunized mice group showed significantly lower or undetectable level of WT virus in the lungs at 3 and 5 dpc than the mock-vaccinated mice.We analyzed lung histopathology in mice challenged with WT-H1N1 virus, after 5 days post-challenge. Severe pneumonia pathology with alveolar collapse, inflammatory infiltrates, necrotizing bronchitis and extensive hemorrhagic lesions, was detected in the lungs of mock-vaccinated group. By contrast, only mild histopathological alterations were detected in the lungs of miRT-H1N1-vaccinated mice. The HPS was significantly lower in miRT-H1N1-vaccinated mice than in the control mice. Lower levels of the IFN-β,IL-6 and TNF-α were detected at days 3 and 5 following challenge in the miRT-H1N1 vaccinated group compared with PBS control groups.Conclusion:1. The miRT-H1N1 virus was attenuated and not lethal in all in mice, suggesting that the miRT-H1N1 virus was safe to mice. We demonstrated that the miRT-H1N1 virus replication is let-7b dependent. This feature allows efficient virus production in chicken embryos and directly used as live vaccine that can be attenuated once the infection was established invivo.2. The miRT-H1N1 elicited robust immunity in mice, and protected the immunized mice from infection with homologous H1N1 viruses. Our results proved the principle that MREs-containing influenza attenuated virus can be used as live-attenuated vaccine against pandemic viruses. |
PDF Full Text Request