| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 103 === Human respiratory syncytial virus (RSV) causes lower respiratory tract infection such as bronchiolitis and pneumonia and is the leading cause of hospitalization of infants and young children. The peak incidence of RSV-related hospitalization is between the second and the sixth month of age. In countries with temperate climates that have well defined RSV seasons, approximately 50% of RSV-related hospitalization occurs in infants younger than 6 months and almost all children are infected by RSV at least once by 2 years of age. Predisposing conditions for the development of serious RSV disease include young age, prematurity, chronic lung disease (CLD), congenital heart disease (CHD), neuromuscular impairment, immunodeficiency, and Down syndrome. RSV epidemics occur depending on geographic location; they cluster during the autumn and last until spring in temperate climate and generally coincide with the rainy season in tropical areas. Treatment of RSV respiratory illness is mainly supportive. Monthly palivizumab is currently recommended to high-risk children as a prophylaxis for RSV infection during winter season. The so-called “RSV season” for RSV immunoprophylaxis in high-risk infants in Taiwan might be different from other country with defined seasonal peaks of RSV infections such as United States. The nationwide epidemiologic data are limited and the total burden of RSV infection remains undefined in Taiwan Therefore, the aims of my study include: 1. Perform a population-based study to determine severity, risk factors, seasonality, and medical cost of RSV-associated hospitalization in Taiwan. 2. To investigate the phylogenetic relationship, evolutionary variability, CTL epitopes and population dynamics of the RSV F protein gene of clinical isolates in northern Taiwan over ten consecutive seasons. 3. To determine the clinical effectiveness and safety of the novel six consecutive monthly doses of palivizumab prophylaxis protocol for the prevention of RSV infection in high-risk preterm infants. 4. To estabilish the RSV DNA vaccine platform.
Firstly, we analyzed the annual population-based incidence, underlying diseases and characteristics of hospitalizations due to RSV in Taiwanese children under 5 years of age from 2004 to 2007 by using Taiwan’s National Health Insurance database. A total of 11081 children with RSV-associated hospitalization were studied. Average annual population-based hospitalization incidences were 1077 and 232 per 100,000 children-year in children under 6 months and under 5 years of age, respectively. The peak incidence was between one and two month of age. The male-to-female incidence risk ratio was 1.4:1 (P<0.001). There was a significant seasonal distribution with consistent peaks in the spring and autumn every year (P <0.001). A total of 373 (3.3%) had repeated RSV infection. The 943 children (8.5%) with underlying diseases were older (P =0.001), required longer intensive care unit (ICU) stays (P <0.001), higher rate of endotracheal intubation (P<0.05), and higher medical cost (P <0.001). A total of 888 (8%) patients required ICU care. The younger age (P < 0.001) and underlying diseases (P < 0.001) were independent predictors of requiring ICU care. RSV infection occurred biennially with peaks in spring and fall in Taiwan. Patients with underlying diseases needed longer hospital stay, ICU stay and higher medical cost. Younger age, prematurity, congenital heart disease and cerebral palsy are predictors of ICU care.
Secondly, we studied the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000–2010. RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2010 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ) and maximum likelihood (ML) methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. From a total of 325 clinical RSV strains, phylogenetic analysis showed that 83 subgroup A strains (RSV-A) could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B) had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114) in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little changed before 2000, then slowed down between 2000 and 2005, and significantly evolved faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. We found that before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were clearly different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence in RSV has significantly increased in recent 5 years. These observations may be valuable for vaccine design and epidemiological studies.
Thirdly, a novel six consecutive monthly doses of palivizumab for RSV prevention protocol has been approved for high risk preterm infants since December 2010. We conducted an observational prospective study at Department of Pediatrics of Mackay Memorial Hospital from April 2011 to March 2013 to determine the clinical effectiveness and safety of this novel protocol for the prevention of RSV infection. We enrolled high-risk infants who received palivizumab prophylaxis as study group and followed up for 12 months. Historic control, those who were born and followed up in the same hospital between July 2000 and June 2008, were enrolled for propensity score matching. Primary endpoint was RSV-related hospitalization, and secondary endpoints included the length of hospital stay and intensive care unit (ICU) care. During the study period, we enrolled 127 infants (108 infants born at ≤ 28 weeks and 19 infants born at 29–35 weeks with CLD). They completed 6-dose palivizumab as scheduled. Among the study group, the RSV-related hospitalizations were 2 (1.6%) within 6 months and 5 (3.9%) within 12 months after discharge. We matched 127 infants in the control group with 127 infants in the study group by propensity score matching. The reductions of RSV-related hospitalization rates were 86% (10.2% vs 1.6%, P =0.002) within 6 months after discharge and 78% (15.7% vs 3.9%, P =0.004) within 12 months after discharge. Compared to the control group, the rate of ICU care significantly decreased from 7.1% to 0.8% (P =0.024) within 6 months after discharge and from 7.9 % to 0.8% (P =0.014) within 12 months after discharge. Adverse events were recorded in 6.4% injections. This six monthly intramuscular administration of palivizumab is effective for prevention of RSV hospitalization in regions with no single seasonal peak of RSV infection such as Taiwan.
Fourthly, in order to protect most of the healthy baby with no underlying disease from respiratory syncytial virus infection, the establishment of respiratory syncytial virus DNA vaccines research platform is needed. We chose the mouse strain C57 / BL6 as experimental subjects: DNA vaccine by intramuscular vaccination to bilateral thigh with a total 100 μg (each leg 50 μg), for a total of three times of immunizations with an interval of 10 days. To measure HRSV-specific serum IgG, sera will be obtained from blood samples collected from the tail prior to each immunization. Ten days after the third immunization, mice were sacrificed for immunological analysis of spleen cells. Mice was challenged with 106 PFUs RSV and then recorded for weight change daily. We used the mouse serum to assay for antibody response. The experiments showed that mice can not generate F protein antibody after three F protein DNA vaccines. We also analyzed cellular immune response to evaluate the effectiveness of the vaccine. Experimental results showed that when injected with the F protein DNA and recombinant GM-CSF, the dendritic cells and macrophages cell surface MHC class II were upregulated. That CD4 T cells display a certain degree of activation also echoes the increases of MHC class II expression. However, further analysis of interferon-γ by T cells falied to demonstrate the increase of interferon-γ performance and CD4 and CD8 T cell proliferative ability were not significant increased through the anti-CD3 antibody stimulation. From above experimental results, when F protein DNA vaccine and simultaneous injection of recombinant GM-CSF DNA, the spleen CD4 T cells and antigen presenting cells partially activated, but the degree might not be so high as to promote cell proliferation. There were no increase in the amount of interferon-γexpressed by CD4 T cells; this implied there might not be good Th1 immune response. In order to get a more significant effect of the vaccine, we need to adjust the dose by DNA, the adjuvant, or the protocol of vaccination. We also need to search more literature and conducted further experiments.
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