This study was designed to test whether the immune responses induced by the concomitant administration of PCV13 + TIV to antigens A/HIN1, A/H3N2 MK0683 and B are noninferior to those induced by TIV alone (TIV + Placebo), and that the immune responses to the PCV13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) induced by PCV13 + TIV are noninferior to those induced by PCV13 administered 1 month after TIV. The safety profile of PCV13 + TIV compared with that

of each agent alone was also assessed. The immune responses induced by PCV13 + TIV were compared with those of TIV alone (Placebo + TIV), as measured by the standard hemagglutination inhibition (HAIs) assays for the TIV strains (A/H1N1, A/H3N2, and B) 1 month after TIV vaccination, and with PCV13 alone in a subset of 605 participants, as measured by a standardized enzyme-linked immunosorbent assay for serotype-specific immunoglobulin G (IgG) 1 month after PCV13 vaccination [13]. For TIV antigens (A/H1N1, A/H3N2, and B), a responder was defined as a participant achieving a ≥4-fold increase in HAI titres from prevaccination to 1 month postvaccination. A comparison between the two treatment groups (PCV13 + TIV relative to Placebo + TIV) was based on the difference in proportions of responders. Noninferiority was declared if the lower limit of the

2-sided 95% confidence interval (CI) for the difference in the proportion of responders between groups ([PCV13 + TIV] − [Placebo + TIV]) was greater than −0.10 consistent with existing literature [14]. Serotype-specific anticapsular polysaccharide IgG geometric mean concentrations (GMCs) were

calculated for each of the click here 13 pneumococcal serotypes. A comparison between the two treatment groups (PCV13 + TIV relative to PCV13) was based on the ratio of GMCs for each of the pneumococcal serotypes. Noninferiority was declared if the lower limit of the 2-sided 95% CI for the GMC ratio ([PCV13 + TIV]:PCV13) was >0.5 (2-fold criterion) calculated 1-month after PCV13 vaccination. PCV13 efficacy data in the adult populations are not yet available. For the purpose of comparing groups administered PCV13 with and without TIV, a 0.5 margin was applied. This definition Levetiracetam was considered to be reasonable on the basis of GMC ratios of 2- to 3-fold seen among serotypes, and across several of the infant PCV7 or PCV9 efficacy trials [15]. These differences are not manifested as differences in efficacy among the serotypes. Therefore, geometric mean immune response values that are within a 2–3-fold range are unlikely to manifest as a clinically significant change in the effectiveness of the vaccine. This noninferiority margin was consistent with relevant publications at the time of study design [14]. Additionally, the immune response of PCV13 + TIV was assessed based on the European Medicines Agency (EMA) “Note for Guidance on Harmonisation of Requirements for Influenza Vaccines” [16].

Samples were normalized using median of all samples baseline transformation and quantile normalization algorithms. Pathway and Gene Ontology (GO) analysis were performed with the novel informatics I-BET-762 chemical structure package InnateDB (www.innatedb.ca). Microarray data has been deposited at ArrayExpress, a MIAME compliant public archive at EMBL-EBI (accession number E-TABM-853). Seven subjects (5 male and 2 female, ages 22–39, median 27 years) were recruited to receive three sequential oral BCG Moreau Rio de Janeiro (approximately 107 viable bacilli) challenges (see Section 2). All subjects completed all visits. Scoring results of symptoms after each vaccination dose are shown in Fig. 1. One subject reported moderate

symptoms (abdominal discomfort and loose stool), and one reported more severe symptoms (loose stools on 2 days). Other symptoms were mild and non-specific. Five subjects reported upper

respiratory tract symptoms after the first challenge, none after the second, and one after the third. After each challenge four (different) subjects recorded gastrointestinal symptoms. Interestingly, the frequency and persistence of symptoms was highest after the first challenge (see Fig. 1, total 28-day aggregate score: 60). After the second challenge there were fewer symptoms confined mainly to the first 4 days, with a 28-day aggregate score of 26. After the third challenge there was the lowest number of symptoms, present as a low-level this website background with an aggregate score of 24. All subjects had received parenteral immunization with BCG in the past, and therefore IFNγ secretion in response to antigen stimulation could be detected

at baseline, as expected (Fig. 2). There was little increase in the frequency of cells responding to PPD or Ag85 stimulation detected by ELISPOT until 6 months after the first challenge (3 months after the third—Fig. 2A). This late onset elevated response to PPD persisted until 12 months, whereas that to Ag85 declined from Resveratrol a peak at 6 months, possibly a result of the larger variety of antigens present in PPD. The detection of IFNγ secretion into supernatant after 7 days in vitro stimulation was generally less sensitive than ELISPOT ( Fig. 2B), although there was a trend to a response to PPD and Ag85, peaking at 12 and 6 months, respectively, with no response detected to MPB70. Microarray analysis of whole blood from vaccinated individuals showed remarkably limited statistically relevant change in gene expression following each of the vaccine challenges. Out of >48,000 probes, only 6 and 9 genes were significantly differentially expressed at both days 4 and 7, respectively, after the first challenge, compared to day 0 and all these genes were down-regulated (Table 2). Importantly, further challenges did not detectably change gene expression. No pathway or GO term was over-represented on day 4. However, at day 7, an over-representation of GO terms related to cytoskeleton (p-value 0.

09 M Tris borate, 2 mM EDTA, pH 7.8) at 90 mV for 60 min. cDNA was prepared from 2 μg of total RNA using the Superscript™ First-Strand Synthesis System (Invitrogen, Paisley, UK) with random hexamer primers according to the manufacturer’s protocol. Samples were incubated at 65 °C for 5 min then held

on ice for 1 min before the addition of Superscript III reverse transcriptase. Samples were then incubated at 25 °C for 10 min followed by reverse transcription at 50 °C for 50 min. The reaction was terminated by heating to 85 °C for 5 min to inactivate the enzyme. Quantitative PCR was carried out on a 7500 Real Time PCR Sequence Detection System (Applied Biosystems, Foster City, CA). TaqMan analysis was performed in a 25 μl reaction mixture containing 30 ng BMS-777607 molecular weight cDNA, TaqMan Universal PCR Master Mix (comprising AmpliTaq Gold DNA polymerase, dNTPs with dUTP, passive reference and optimised selleck chemical buffer) and Assay-on-demand™ gene expression assay mixes containing specific primers and probes (all from Applied Biosystems). The PCR conditions comprised a 2 min incubation at 50 °C followed by a 10 min polymerase activation at 95 °C. This was followed by 40 cycles alternating between 95 °C for 15 sections and 60 °C for 1 min each.

Amplification curves were analysed using the SDS version 3.2 software (Applied Biosystems, Foster City, CA). The baseline and threshold values were set and the Ct values extracted for each gene of interest. Relative quantification was calculated using the geometric mean of two selected house-keeping genes, gapdh and mvp. Relative gene expression

levels were calculated using the equation 2−ΔCt. An arbitrary classification system was applied to the data quantifying relative expression levels Rolziracetam as ‘high’ >0.5, ‘moderate’ between 0.02 and 0.5, ‘low’ between 0.001–0.02 and ‘negligible’ <0.001. All transport experiments were conducted in standard buffer solution (SBS) comprising Hank’s Balanced Salt Solution (HBSS) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Cell layers were allowed to equilibrate in SBS for 60 min at 37 °C before TEER measurements were taken. Each condition was carried out in quadruplicate and only layers with a resistance >250 Ω cm2 were accepted for experimentation. For transport studies with radiolabelled markers, donor compartments were filled with 0.51 ml (apical to basolateral (AB) transport) or 1.51 ml (basolateral to apical (BA) transport) of SBS containing 25 nM 3H-digoxin and/or 6.55 μM 14C-mannitol. Receiver compartments were filled with 1.5 ml (AB transport) or 0.5 ml (BA transport) of SBS. At the start and end of the experiment, 10 μl samples were taken from the donor compartments for determination of the initial and final concentration. Every 30 min over a 2 h period, 300 μl samples (AB transport) and 100 μl samples (BA transport) were taken from the respective receiver chambers and replaced with the same volume of SBS.

, 1990). While an extensive body of empirical evidence supports gender as a strong determinant of health

(Krieger, 2003 and Sen and Östlin, 2008), other determinants of obesity risk contribute to a more complex picture; the effects of these determinants are difficult to disentangle (Verbrugge, 1985). In health disparities research, obesity risk is often attributed to racial and ethnic differences (Cossrow and Falkner, 2004 and Wang and Beydoun, 2007). However, socioeconomic factors and population density (rural, urban) also play important roles (Wang and Beydoun, 2007 and Zhang and Wang, 2004). In the literature, unique differences in community resiliency, culture, and geography have been found to be associated with attenuated obesity risk, especially among particular subpopulations Dasatinib mouse (Wang and Beydoun, 2007). Although studying complex causal pathways to disease development is of significant value to obesity Vorinostat research, public health practice often necessitates more applied science, requiring data that can enumerate specific subpopulation needs. At this more granular level, subpopulation health data can aid

program planning and fieldwork by tailoring interventions to specifically address key geo-social factors that influence obesity risk (Frieden, 2010). Information on key attributes of targeted populations (e.g., subgroup obesity prevalence, health profiles and/or health behaviors) can be used to plan programs that address group- or culturally-specific covariates including food preparation style, social norms surrounding eating, etc. Such data provides validation of agency decisions to invest federal funds in obesity prevention. Unfortunately, for most communities, access to subpopulation health data is sparse. In this article, we contribute to public health practice by presenting two case studies of CPPW communities that collected subpopulation health data to document community needs. We specifically described the prevalence of overweight and obesity, and the health risk profiles of low-income women in a clinic setting in rural West Virginia

(WV, Case-Community PD184352 (CI-1040) No. 1)2 and urban Los Angeles County (LA County, Case-Community No. 2).3 We chose these two specific communities because surveillance of obesity by population density (rural and urban) were key focus areas in the national CPPW program during 2010–2012. We analyzed cross-sectional data from health assessments conducted during the first 15 months of the national CPPW program in rural WV and urban LA County. Both communities participated in several local CPPW interventions and enhanced evaluation activities, including interval assessments of body mass index (BMI) and self-reported dietary behaviors of low-income community-dwelling adults. In WV, CPPW funded interventions in a six-county area. This region is largely rural (U.S.

All animal procedures were approved by local Animal Care Committee and are in accordance with the NIH Guide for the care and use of laboratory animals. Organotypic hippocampal slice cultures were prepared according to the method of Stoppini et al. (1991), with modifications (Valentim et al., 2003, Cimarosti et al., 2005, Horn et al., 2005 and Frozza et al., 2009). Briefly, 400-μm-thick hippocampal slices were prepared from 6 to 8-day-old male Wistar rats using a McIlwain tissue chopper and separated in ice-cold Hank’s balanced salt solution (HBSS) www.selleckchem.com/TGF-beta.html composed of (mM): glucose

36, CaCl2 1.26, KCl 5.36, NaCl 136.89, KH2PO4 0.44, Na2HPO4 0.34, MgCl2 0.49, MgSO4 0.44, HEPES 25; fungizone 1% and gentamicin 0.1 mg/mL, pH 7.2. The slices were placed on Millicell culture insert and the inserts were transferred to a 6-well culture plate. Each well contained 1 mL of tissue culture medium consisting of 50% minimum essential medium, 25% HBSS, 25% heat inactivated horse serum supplemented

with (mM, final concentration): glucose 36, HEPES 25 and NaHCO3 4; fungizone 1% and gentamicin 0.1 mg/mL, pH 7.3. Organotypic cultures were maintained in a humidified incubator gasified with 5% CO2 atmosphere at 37 °C for 30 days. Culture medium was changed three times a week. Aβ25–35 and Aβ35–25 (reverse peptide) stock solutions (675 μM) were prepared in sterile distilled water and stored at −20 °C. To obtain the fibrillar form of Aβ25−35 peptide, an aliquot of the stock solution was incubated under 37 °C during the 4 days preceding its use in culture (Casal et al., 2004). The so-called non-fibrillar Aβ corresponds to the peptide that was not subjected to the TSA HDAC mouse aforementioned activation process and was therefore added to the culture directly from stock solution. On the 28th in vitro day, the medium was replaced by a serum reduced medium (2.5%) into which 25 μM of fibrillar/non-fibrillar Aβ25–35 or Aβ35–25 was added or not (control slices). Previous experiments showed that this concentration (25 μM) of Aβ25–35 had the most toxic effect (data not shown), at least for the fibrillar peptide form. Cellular damage was assessed by fluorescent image analysis of propidium iodide (PI)

uptake (Noraberg et al., 1999). One hour before the end of the treatments, which means after 47 h of Aβ25–35 or Aβ35–25 exposure, 7.5 μM of PI was either added to the medium and incubated for 1 h. PI uptake is indicative of significant membrane injury (Macklis and Madison, 1990). Cultures were observed with an inverted microscope (Nikon Eclipse TE 300) using a standard rhodamine filter set. Images were captured and then analyzed using Scion Image software (http://www.scioncorp.com). After capture of images, the area where PI fluorescence (transformed in pixels) was detectable above the background was analyzed using the “density slice” option of Scioncorp Software through the division of PI fluorescence by the total area of the slice (Valentim et al.

CR formulations provide certain advantages when compared to their IR counterparts. CR formulations can reduce peak to trough fluctuations in the plasma concentration–time profile (compared to multiple-dose administration of an IR product), hence reducing fluctuation-related side effects and/or sub-therapeutic concentrations. CR formulations can increase the exposure over time of drugs with a short elimination half-life, and can be used to target delivery into distal regions

Akt inhibitor of the intestine (e.g. colon), or where there is a need for targeted delivery for the treatment of a specific disease, such has Crohn’s disease (Langer, 1990, Rubinstein, 2005 and Thombre, 2005). This can lead to an increased patient compliance. Furthermore, CR formulations can be of use in drug find more development when the standard IR formulation is not an alternative due to unfavourable pharmacokinetic properties of the drug candidate (Langer, 1990, Rubinstein, 2005 and Thombre, 2005). One of the main goals when developing a CR formulation of a marketed drug is

to achieve, at least, the same exposure as the equivalent dose of their IR counterpart. In general however the relative bioavailability of a CR formulation compared to its IR counterpart is expected to be less than 100% (European Medicines Agency, 2013). Several physiological factors can influence the observed whatever differences in systemic exposure between IR and CR. A CR formulation is intended to release its drug content within 12–24 h, in contrast the small intestinal transit time is around 2–5 h (Davis et al., 1986, Fallingborg et al., 1989 and Yu et al., 1996). Therefore a majority of the dose should be released into distal regions of the small intestine and the colon, where the residence time in the colon is about 12–24 h (Coupe et al., 1992, Davis et al., 1986 and Fallingborg et al., 1989). The extended release may limit the absorption potential for a drug formulated as CR as, in

general, the distal regions of the intestine provide a less favourable environment for drug absorption. For instance, the reduced surface area available for absorption in the distal region of the GI tract may limit the absorption for poorly permeable compounds (Tannergren et al., 2009 and Watts and Lllum, 1997), the intestinal pH increases towards the distal portion of the intestine consequently limiting the aqueous solubility of basic compounds (Fallingborg et al., 1989). Finally, the lack of bile salts, less fluid volume in the colon, differences in the regional permeability and possible degradation by colonic microflora can also have a negative impact on the drug absorption of CR formulations (Lennernas, 2014a, Schiller et al., 2005, Sutton, 2009 and Tannergren et al., 2009).

p.i., but none of the animals showed rise in body temperature (data not shown). At both 3 and 14 d.p.i. there was no virus replication in the brains, spleen and intestine (data not shown). This study confirmed the attenuated phenotype of a A/17/California/2009/38 pandemic LAIV candidate in a ferret model. The results of immunogenicity study showed that a single dose of pandemic LAIV was sufficient to induce adequate immune responses against the wild type strain. Moreover, vaccinated animals

proved to be protected against challenge with a virulent wild type pandemic H1N1 virus (Table 2). The monovalent LAIV contained 7.0 log EID50 selleckchem per 0.5 ml dose for adults and 6.5 log EID50 for children. Following successful preclinical studies, a Phase I/II randomized, controlled, double-blind clinical study was carried out in 120 adults aged 18–60 years randomly divided into groups to receive

either the vaccine (100) or the placebo (20) administered intranasally in two doses given at 21 days apart. Standard haemagglutinin inhibition (HAI) assays were performed and influenza virus-specific serum IgG and IgA antibodies in nasal swabs were tested by enzyme-linked immunosorbent assays (ELISA) using whole purified virus at 16 HAU per 0.05 ml for absorption. No clinically significant solicited adverse events attributable to the LAIV VE-822 solubility dmso were detected seven days after vaccination (Table 3). The few reactions reported were of short duration and without sequelae. HAI and ELISA tests were also used to determine the serological

response in 66 adult subjects (Table 4). Although post-vaccination serum HA antibody titres were low, cumulative data from both assays resulted in 42.5% and 70.2% conversion after the first and second inoculation, respectively. Peripheral blood mononuclear cells were obtained for analysis by cytokine tests at various times following the first and second vaccination from a limited number of volunteers (16 vaccinees and 9 placebo recipients, respectively). Fig. 1 represents post-vaccination nearly changes (n-fold) of cellular immune response mediated with virus-specific CD3+CD4+IFNγ+ and CD3+CD8+IFNγ+ memory T cells in volunteers who received LAIV and placebo. After revaccination, the mean increases of both CD4+ and CD8+ memory cells were significantly higher in vaccinated volunteers compared with the placebo group. Interestingly, the same effect of vaccination was also observed in vaccinees without reliable conversions of HAI antibody titres. Even after a single vaccination, the rate of volunteers with a significant increase of these cells in the blood (i.e. results exceeding 2 standard deviations of placebo mean value) was 37.5% (CD8+) and 75.0% (CD4+). After revaccination, the percentage of individuals with significant rises in CD8+ and in CD4+ cells was 68.8%. HAI test results in children were much higher, i.e. 41.4% and 83.

Contributors: Study concept and design: Drs. Ambrose and Wu. Acquisition of data: Drs. Ambrose and Wu. Analysis and interpretation of data: all authors. Drafting of the manuscript and critical revision of the manuscript for important intellectual ABT-199 manufacturer content: all authors. Statistical analysis: Dr. Wu. All authors approved the final manuscript for submission. Financial disclosures: Drs. Ambrose, Wu, Jones, and Mallory are employees

of MedImmune, LLC, Gaithersburg, MD. Funding/support: This research was funded by MedImmune, LLC. Role of the sponsor: All authors are employees of MedImmune, LLC who worked collaboratively in the design of the analysis and interpretation of the data, and reviewed and approved the manuscript. Additional contributions: Editorial assistance was provided by Susan E. DeRocco, PhD, and Gerard P. Johnson, PhD, of Complete Healthcare Communications, Inc. (Chadds Ford, PA) and funded by MedImmune, LLC. “
“The tick Rhipicephalus (Boophilus) microplus has a significant economic impact on cattle breeding industry worldwide, estimated at billions of dollars

annually [1] and [2]. This parasite causes a variety of deleterious effects in cattle, mainly as result of bodyweight reduction, blood loss and the transmission of disease-causing agents [1] and [2]. The intensive use of acaricides in order to control tick infestation raises concerns as to the potential presence of pesticide this website residues in milk, meat, and the environment [3]. For these reasons, a tick vaccine, as an alternative control method, is a major economic issue [4] and [5]. It has been repeatedly demonstrated that the

stimulation of bovine immune system by tick proteins vaccination induces a protective immune response against R. microplus [6]. In 1986, a protective protein from R. microplus old named Bm86 was discovered, when this antigen became the first tick antigen to compose a commercial vaccine against an ectoparasite [7]. Although vaccine formulations based on Bm86 in most cases elicit protective immune responses against R. microplus, they vary considerably in terms of protection level depending, among other things, on the genetic variability of tick and bovine populations [8], [9], [10], [11], [12] and [13]. Therefore, the discovery of new tick antigens focusing on those displaying minimal genetic variability among R. microplus populations could improve vaccination efficacy and reduce variation in the protection level afforded by the Bm86-based vaccines. However, except for a few studies [14], data regarding cross-reactivity between tick proteins are scarce, although some tick antigens have been shown to induce cross-protective immunity against some tick species [14] and [15]. Another strategy to enhance anti-tick vaccine efficacy is to combine two or more antigens [16].

Due to an ageing population, the number of the most common upper limb fractures – proximal humeral fractures and distal radius fractures – are expected to increase by about 10% every five years to 2036 (Sanders et al 1999). Following an upper limb fracture, patients are often referred to physiotherapy for rehabilitation to reduce pain, improve range of movement and strength, and to regain function (AIHW 2008). Even though the aims of physiotherapy are clear, the interventions used during the rehabilitation phase can vary greatly. These interventions can include thermal modalities, ultrasound,

electrical stimulation, continuous passive movement, electromyographic biofeedback, soft tissue mobilisation, mobilising and strengthening exercises, application of resting or dynamic splints, advice, and education selleck kinase inhibitor (Bertoft et al 1984, Clifford, 1980, Lundberg et al 1979, Michlovitz et al 2001). Exercise is a common intervention after upper limb fracture. For example, Michlovitz et al (2001) found that exercise was prescribed to at least 90% of patients receiving rehabilitation after distal radius fracture. The application click here of exercise is also consistent with the third key principle of fracture management – movement (Adams and Hamblen, 1995).

Previous research has identified that therapeutic exercise is beneficial across a broad range of health conditions (Taylor et al 2007). However, previous systematic reviews of trials of upper limb fracture management have not focused on the effect of exercise (Handoll et al 2003, Handoll et al 2006). In addition, clinical practice

guidelines for the treatment of distal radius fractures concluded that there was weak evidence to support the use of a home exercise program (Lichtman et al 2010). New trials of physiotherapy rehabilitation have been published since the two reviews were completed in 2003 and 2006. Physiotherapists need current evidence about the effectiveness of treatment techniques to help them make clinical decisions about patient care and to allocate limited therapy resources for people with upper limb fractures. Therefore, the specific research question for this systematic review was: What is the effect of exercise on reducing enough impairment and increasing activity in the rehabilitation of people with upper limb fractures? Relevant randomised and quasi-randomised controlled trials were identified using a search strategy (See Appendix 1 on the eAddenda for full search strategy) from the earliest date possible until January 2011 in the following electronic databases: CINAHL, MEDLINE, Embase, AMED, SPORT Discus, PubMed, PEDro and the Cochrane Central Register of Controlled Trials. To ensure all relevant studies were captured, manual reference list checks and citation tracking of included studies using Web of Science were performed. One reviewer examined the study titles and abstracts to determine if they satisfied the inclusion criteria.

In this study, we developed HPV 16/18/58 trivalent L1 VLP vaccines and compared the type specific neutralizing antibody levels induced by the trivalent vaccine with those by corresponding monovalent vaccines. We found that the HPV 58 containing trivalent vaccine could induce high titers of HPV specific antibodies against all component types, and that the type specific neutralizing antibody levels were interfered by co-immunized antigens. HPV 16, 18, 58 L1 genes were codon optimized according to the codon usage bias of Sf9 cells. All modification was made according to Table 1. Optimized genes were synthesized by Sangon Corp. (Shanghai, China) and constructed into

pFastBac I (Invitrogen). Optimized genes were uploaded to Genbank, and the accession numbers are GU556964 (HPV 16 L1), GU556965 (HPV 18 L1) and GU556966 (HPV 58 L1), respectively. HPV 6 and 11 L1 genes were obtained by our lab previously Veliparib datasheet [30], [31] and [32]. L1 genes were expressed in baculovirus expression system and purified by CsCl ultracentrifugation

as described previously [33]. The purity of L1 was evaluated by SDS-PAGE with Coomassie blue staining. VLPs were further verified by transmission electron microscopy (TEM) [31]. We formulated pentavalent, trivalent, bivalent and monovalent vaccines with high and low dose of antigens, with or without Aluminium adjuvant according to Table 2. High dose vaccines contained 5 μg VLPs of each type, while low dose vaccines contained 0.1 μg VLPs of each type. Protease Inhibitor Library price The adjuvant we used here is Aluminium hydroxide (Sigma–Aldrich). All the vaccines were formulated in a total volume of 100 μl in PBS. The control vaccine

contained 100 μl PBS only. Balb/c mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and kept in the animal facility of the Institute of Basic Medical Science, Chinese Academy of Medical Sciences. All experimental protocols were approved by the Institutional Animal Care and Use Committee. Experiment groups immunized with different vaccine formulations were listed in Table 2. Briefly, for the long-term experiments, mice (n = 4 per group) were immunized intramuscularly with Trivalent-1 vaccine, Mono 16, 18, 58 vaccines or PBS, respectively at week 0, 2, 4, and were given an extra boost at week 52. Serum samples were collected at 2 week’s interval for first 12 Parvulin weeks and then at 10 week’s interval until week 52. Samples were also collected 2 weeks after the extra boost. All samples were analyzed by ELISA for type specific antibody responses [30]. Serum samples collected at week 4 and 6 were analyzed for neutralizing antibody level (pseudo-neutralization assay). For dose adjustment experiments, mice (n = 4 per group) were immunized intramuscularly with Trivalent-1, Trivalent-2, Mono 16, Mono 18 and Mono 58 vaccines, respectively at week 0, 2, 4. Serum samples collected at week 4 and 6 were analyzed by pseudo-neutralization assay.