Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases.

Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation.

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77- to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9- to 7.1-fold or 22.5- to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3- to 8-fold or 91- to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

Cold chain and virus-free chloroplast-made booster vaccine to confer immunity against different poliovirus serotypes.

The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high-cost, limited supply of IPV, persistent circulating vaccine-derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low-cost cold chain-free plant-made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50-fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant-derived adjuvants after single priming with IPV significantly increased VP1-IgG1 and VP1-IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17-10.17 log2 titre) and seropositivity (70-90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant-cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA-approved antigens and adjuvants are discussed.

Plant-made oral vaccines against human infectious diseases-Are we there yet?

Although the plant-made vaccine field started three decades ago with the promise of developing low-cost vaccines to prevent infectious disease outbreaks and epidemics around the globe, this goal has not yet been achieved. Plants offer several major advantages in vaccine generation, including low-cost production by eliminating expensive fermentation and purification systems, sterile delivery and cold storage/transportation. Most importantly, oral vaccination using plant-made antigens confers both mucosal (IgA) and systemic (IgG) immunity. Studies in the past 5 years have made significant progress in expressing vaccine antigens in edible leaves (especially lettuce), processing leaves or seeds through lyophilization and achieving antigen stability and efficacy after prolonged storage at ambient temperatures. Bioencapsulation of antigens in plant cells protects them from the digestive system; the fusion of antigens to transmucosal carriers enhances efficiency of their delivery to the immune system and facilitates successful development of plant vaccines as oral boosters. However, the lack of oral priming approaches diminishes these advantages because purified antigens, cold storage/transportation and limited shelf life are still major challenges for priming with adjuvants and for antigen delivery by injection. Yet another challenge is the risk of inducing tolerance without priming the host immune system. Therefore, mechanistic aspects of these two opposing processes (antibody production or suppression) are discussed in this review. In addition, we summarize recent progress made in oral delivery of vaccine antigens expressed in plant cells via the chloroplast or nuclear genomes and potential challenges in achieving immunity against infectious diseases using cold-chain-free vaccine delivery approaches.

Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.

Minimum amount of physical activity for reduced mortality and extended life expectancy: a prospective cohort study.

BACKGROUND: The health benefits of leisure-time physical activity are well known, but whether less exercise than the recommended 150 min a week can have life expectancy benefits is unclear. We assessed the health benefits of a range of volumes of physical activity in a Taiwanese population. METHODS: In this prospective cohort study, 416,175 individuals (199,265 men and 216,910 women) participated in a standard medical screening programme in Taiwan between 1996 and 2008, with an average follow-up of 8·05 years (SD 4·21). On the basis of the amount of weekly exercise indicated in a self-administered questionnaire, participants were placed into one of five categories of exercise volumes: inactive, or low, medium, high, or very high activity. We calculated hazard ratios (HR) for mortality risks for every group compared with the inactive group, and calculated life expectancy for every group. FINDINGS: Compared with individuals in the inactive group, those in the low-volume activity group, who exercised for an average of 92 min per week (95% CI 71-112) or 15 min a day (SD 1·8), had a 14% reduced risk of all-cause mortality (0·86, 0·81-0·91), and had a 3 year longer life expectancy. Every additional 15 min of daily exercise beyond the minimum amount of 15 min a day further reduced all-cause mortality by 4% (95% CI 2·5-7·0) and all-cancer mortality by 1% (0·3-4·5). These benefits were applicable to all age groups and both sexes, and to those with cardiovascular disease risks. Individuals who were inactive had a 17% (HR 1·17, 95% CI 1·10-1·24) increased risk of mortality compared with individuals in the low-volume group. INTERPRETATION: 15 min a day or 90 min a week of moderate-intensity exercise might be of benefit, even for individuals at risk of cardiovascular disease. FUNDING: Taiwan Department of Health Clinical Trial and Research Center of Excellence and National Health Research Institutes.

Attributable mortality burden of metabolic syndrome: comparison with its individual components.

AIM: To estimate the national prevalence, mortality risk and population mortality burden of metabolic syndrome, and compare the values with those of its individual components. METHODS AND RESULTS: A total of 486,341 apparently healthy adults who went through a screening programme in Taiwan were recruited from 1994 onwards. As of 2007, 15,268 deaths had occurred at least one year after the examination. Six definitions of metabolic syndrome were used. Components of metabolic syndrome include obesity, hypertension, hyperglycaemia, dyslipidaemia and albuminuria. Hazard ratios (HRs) were calculated using the Cox proportional hazard model. The population mortality burden considered both national prevalence and HRs. The national prevalence of metabolic syndrome defined by the Adult Treatment Panel (ATP) III was 16.3%, the HR for all causes was 1.36 (95%, CI 1.31-1.41) and the HR for cardiovascular disease (CVD) was 1.63 (95%, CI 1.51-1.77). The population mortality burden of metabolic syndrome was 5.5% for all causes, in contrast to 9.0% for hypertension, 8.9% for albuminuria, 6.6% for diabetes, 3.5% for dyslipidaemia and 1.5% for obesity. For CVD it was 9.4%, lower than 10.7% for albuminuria and 25.0% for hypertension. CONCLUSION: The mortality burden of metabolic syndrome was relatively small at national level. Three of the five components of metabolic syndrome alone, namely hypertension, diabetes and albuminuria, contributed more than metabolic syndrome to all-cause mortality. Successful management of any of these three components would have achieved a greater impact on mortality than management of metabolic syndrome.

Cancer risks from betel quid chewing beyond oral cancer: a multiple-site carcinogen when acting with smoking.

OBJECTIVES: This cohort study is to assess the extent of cancer risks of betel quid chewing (without tobacco added) beyond oral cancer, as such information was limited from case-control studies. METHODS: The cohort, selected from participants in a medical screening program since 1994, consisted of 177,271 adult men with 19.2% chewers of betel quid. As of 2006, out of 4,840 deaths, 1,901 cancer deaths were identified. Mortality hazard ratios (HR) were estimated by Cox proportional hazard model. Life expectancy was calculated by life table method. RESULTS: One-third of smokers chewed (33%) but most of chewers smoked (90%). Risk for all cancer doubled among chewers (HR = 2.00). Risks of at least six cancer sites were increased among chewers: oral cavity (HR = 12.52), esophagus (HR = 5.64), liver (HR = 2.27), pancreas (HR = 2.67), larynx (HR = 6.24), and lung (HR = 2.43) with risks increased with increasing betel quid amount consumed. All-cancer age-adjusted mortality rates in Taiwan increased 25%, including 223% increase in oral cancer, during the last 20 years when chewing rate increased five- to tenfolds. Chewing on top of smoking increased the risks synergistically, and these two were responsible for at least half (50%) of all cancer deaths among 2 million chewers in Taiwan. Life expectancy of chewers was shorter than non-chewers by 5.93 years at age 20 and 5.55 years at age 40. CONCLUSION: In addition to oral cancer, significant increases were seen among chewers for cancer of the esophagus, liver, pancreas, larynx, lung, and all cancer. Chewing and smoking, as combined by most chewers, interacted synergistically and was responsible for half of all cancer deaths in this group. They were responsible for the recent increases in oral, esophageal, pancreatic, and liver cancer in Taiwan. Chewing and smoking shortened their life span by nearly 6 years.

Is high serum uric acid a risk marker or a target for treatment? Examination of its independent effect in a large cohort with low cardiovascular risk.

BACKGROUND: Cohort studies evaluating increased uric acid level as a cardiovascular disease (CVD) risk factor have shown variable results; studies are particularly lacking in lower risk populations. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 484,568 adults participating in a medical screening program in Taiwan since 1994 were followed up for a median of 8.5 years. Two subgroups were constructed: the first (n = 246,697; 51%) excluded participants with either overt CVD or overt CVD risk factors (including hypertension, diabetes, obesity, and hypertriglyceridemia) and the second (n = 157,238; 32%) further excluded individuals with early-stage CVD risk factors (including prehypertension, prediabetes, overweight, and borderline hypertriglyceridemia). PREDICTOR: Serum uric acid. OUTCOMES & MEASUREMENTS: All-cause and CVD mortality risk assessed using Cox proportional hazards models for categorical and continuous serum uric acid levels. As applicable, models adjusted for 14 variables. Population-attributable fraction was applied to compare contributions to mortality between high uric acid level and other CVD risk factors. RESULTS: In the total cohort, mean age was 41.4 +/- 14.0 years and 26.2% had serum uric acid levels >or=7 mg/dL. Through 2007, there were 16,246 deaths (3.4% of all participants), with 35.2% of deaths occurring in individuals with hyperuricemia. Adjusted HRs associated with serum uric acid levels >or=7 mg/dL for all-cause and CVD mortality were 1.10 (95% CI, 1.04-1.17) and 1.38 (95% CI, 1.20-1.58), respectively. In individuals with hyperuricemia, 64.3% had overt CVD risk factors and 82.5% had either overt or early-stage CVD risk factors. Individuals with serum uric acid levels >or=8 mg/dL without overt CVD risk factors constituted 13.5% of the total study population with hyperuricemia; in analyses excluding those with overt CVD risk factors, serum uric acid level >or=8 mg/dL was significantly associated with all-cause and CVD mortality, with HRs of 1.37 (95% CI, 1.18-1.60) and 2.30 (95% CI, 1.51-3.49), respectively. In the subgroup of those with serum uric acid levels >or=8 mg/dL but who lacked both overt and early-stage CVD risk factors, the HRs for all-cause and CVD mortality were also significant and were 1.39 (95% CI, 1.08-1.78) and 2.38 (95% CI, 1.24-4.54), respectively. HRs for individuals with the same risk profiles but with serum uric acid of 7.0-7.9 mg/dL were not significant. In all groups, inclusion of proteinuria and glomerular filtration rate in models substantially attenuated the association between uric acid level and outcomes. High uric acid levels contributed a relatively insignificant portion to mortality (1.2%) and CVD deaths (4.5%) in this population. LIMITATIONS: A single measurement of uric acid was used. CONCLUSION: Increased serum uric acid level is a minor, but significant, risk factor for all-cause and CVD mortality. However, except for a small proportion (13.5%), increased serum uric acid level is more a risk marker than a target for treatment and is not an independent risk. Determining appropriate groups to target in clinical trials for uric acid-lowering therapy is critical.

The reduction of tuberculosis risks by smoking cessation.

BACKGROUND: Smoking is known to aggravate tuberculosis (TB), but such information has been ignored in clinical practice, as it was not thought to be relevant. The aim of this study is to assess the benefits of smoking cessation on TB mortality reduction. METHODS: The study attempts to quantify smokers' risks on subsequent TB mortality and the change in such risks after smokers quit smoking. In this prospective cohort study, the TB mortality risks of smokers, never smokers and former smokers were compared, by using the Cox proportional model to estimate the hazard ratio (HR) of TB.The cohort, consisting of 486,341 adults, participated in standard medical screening programs since 1994, including 5,036 with self-reported TB history. Of 15,268 deaths identified as of 2007, 77 were coded as TB. RESULTS: Smokers with self-reported TB history (1.2%) had very high TB mortality (HR = 44.02). Among those without self-reported TB history, smoking increased TB mortality by nine-fold (HR = 8.56), but when they quit smoking, the risk was reduced by more than half (65%), to a level not different from those who had never smoked. The overwhelming majority of TB deaths (83%) occurred among those without self-reported TB history. Given the high smoking prevalence and the high HR, smoking accounted for more than one-third (37.7%) of TB mortality in Taiwan. Smokers reported less TB history but died more from TB than those who had never smoked. CONCLUSIONS: Smokers had very high TB mortality, as much as nine times those who had never smoked, but once they quit, the risk reduced substantially and was similar to those who never smoked. Smoking cessation has benefits to the smokers far beyond reducing TB risk, but successful tobacco control could favorably impact the TB mortality rate and reduce this public health burden, which has long haunted the Taiwanese population. Smoking cessation could reduce nearly one-third of tuberculosis deaths.

All-cause mortality attributable to chronic kidney disease: a prospective cohort study based on 462 293 adults in Taiwan.

BACKGROUND: Both end-stage renal disease and chronic kidney disease are increasing worldwide; however, the full effect of chronic kidney disease is unknown because mortality risks for all five stages are unavailable. We assessed prevalence and mortality risks for all stages of chronic kidney disease and quantified its attributable mortality in Taiwan. METHODS: The cohort consisted of 462 293 individuals aged older than 20 years who participated in a standard medical screening programme since 1994. As of Dec 31, 2006, we identified 14 436 deaths. Chronic kidney disease was determined by glomerular filtration rate and urinary protein. We estimated national prevalence in Taiwan from the cohort by adjusting age and educational levels. Hazard ratios (HRs) were calculated with Cox proportionate hazards model. We calculated mortality attributable to chronic kidney disease for national population and for low socioeconomic status. FINDINGS: The national prevalence of chronic kidney disease was 11.93% (95% CI 11.66-12.28), but only 3.54% (3.37-3.68) of participants in the cohort were aware of their disorder. Prevalence was substantially higher in the group with low socioeconomic status than in the high status group (19.87% [19.84-19.91] vs 7.33% [7.31-7.35]). 56 977 (12%) of cohort participants had chronic kidney disease; those with disease had 83% higher mortality for all cause (HR 1.83 [1.73-1.93]) and 100% higher for cardiovascular diseases (2.00 [1.78-2.25]), in a cohort that was observed for 13 years with median follow-up of 7.5 years (IQR 4.0-10.1). 10.3% (95% CI 9.57-11.03) of deaths in the entire population were attributable to chronic kidney disease, but 17.5% (16.27-18.67) of deaths in the low socioeconomic status population. 2350 (39%) deaths occurred before 65 years of age in those with chronic kidney disease. Regular users of Chinese herbal medicines had a 20% (odds ratio 1.20 [1.16-1.24]) increased risk of developing chronic kidney disease. INTERPRETATION: The high prevalence of chronic kidney disease and its associated all-cause mortality, especially in people with low socioeconomic status, make reduction of this disorder a public-health priority. Promotion of its recognition through the general public knowing their glomerular filtration rate and testing their urine is crucial to reduce premature deaths from all causes and to attenuate this global epidemic.

Making hypertensive smokers motivated in quitting: developing 'blood pressure equivalence of smoking'.

OBJECTIVE: To express the increased risk from smoking in terms of 'blood pressure' so that hypertensive smokers are motivated into quitting. METHODS: Mortality risks of smokers were compared with nonsmokers in a large worker cohort in Taiwan (n = 23755 with a 17-year follow-up) for all-cause and for cardiovascular diseases. The blood pressure equivalence of smoking was then identified by the difference in mortality risks between smokers and nonsmokers. RESULTS: Some interaction between hypertension and smoking was found to be synergistic. When hypertension and smoking co-existed, the all-cause mortality outcome [relative risk (RR) = 4.25] was larger than the sum or product of each individual risk for hypertension (RR = 2.16) or for smoking (RR = 1.97). The excess mortality risks of smoking for smokers were converted into a 'blood pressure equivalence'. The results demonstrate that the addition of smoking was similar to an increase of mortality risk approximately equivalent to an increase in blood pressure of 40 mmHg. CONCLUSIONS: Smoking cessation in hypertensive patients could provide a reduction of mortality risks similar to a permanent reduction of 40 mmHg in blood pressure, over and above any antihypertensive medications. Appreciating this relationship enables physicians to bridge the clinical disconnection and motivates hypertensive smokers to seek smoking cessation. The use of a 'blood pressure equivalence of smoking' can link the two separate risk factors and may lead to a paradigm shift in overcoming an existing clinical challenge.

Exploring the relationships between diabetes and smoking: with the development of glucose equivalent concept for diabetes management.

The extent of interaction between smoking and diabetes has been under-appreciated. Smokers had more diabetes, and when diabetes patients smoke, the combined mortality effect was greater than either the addition or multiplication of these two medical problems. Patients seen in the office are usually more interested in reducing blood glucose than in quitting smoking, and yet, smoking caused mortality risks, at a magnitude similar to or more than diabetes. The concept of "glucose equivalent of smoking" was developed to direct more attention to smoking in clinical management. Based on the follow-up observations from a large Asian cohort, the risk of an individual who smokes, from all-cause mortality, was found to be equivalent to an elevation of blood glucose by an average of 41mg/dl for the cohort in general and 68mg/dl for the diabetes in particular. By relating the message of smoking hazards in terms of "glucose equivalent", clinicians will be more alerted to counsel and patients will be more likely to quit. Appreciating this concept has a potential to change the paradigm of diabetes management, to bridge the clinical disconnect between the two, and to provide new ammunition for the diabetes epidemic in Taiwan.

Comparison of total hip and knee joint replacement in patients with rheumatoid arthritis and osteoarthritis: a nationwide, population-based study.

INTRODUCTION: Patients with rheumatoid arthritis (RA) and osteoarthritis (OA) may require total hip replacement (THR) or total knee replacement (TKR). The present study aimed to compare the demographic characteristics and medical costs of RA and OA patients from Taiwan who underwent either THR or TKR. METHODS: The medical records of patients who had undergone THR or TKR from 1 January 1996 to 31 December 2010 were obtained from the Taiwan National Health Insurance Research Database (NHIRD). In all, we found 49 and 146 RA patients who received THR and TKR, respectively, and 1,191 and 6,574 OA patients who received THR and TKR, respectively. The gender, age, Charlson comorbidity index (CCI), hospital grade, age at registration in the catastrophic illness dataset, and medical utilisation costs of the different groups were compared. RESULTS: There were statistically significant differences in age, CCI score, drug costs and surgery costs between RA and OA patients. Joint replacement incidence was lower in RA patients than in OA patients, and among patients who underwent THR, total medical costs incurred were higher for RA patients than OA patients. RA patients who underwent THR incurred a significantly greater total medical utilisation cost in the outpatient department (3 months before surgery and 12 months after surgery) than OA patients who underwent THR. CONCLUSION: Analysis of Taiwan NHIRD with regard to patients who had undergone either THR or TKR indicated that RA patients were younger than OA patients, and that significantly more medical resources were used for RA patients before, during and after hospitalisation for these procedures.

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs.

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.

The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs.

The heterodimer of glycoprotein 5 (GP5) and non-glycosylated matrix protein (M) is the leading target for the development of new generation of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) infection. It has been demonstrated that DNA vaccine co-expressing GP5 and M proteins as a fusion protein aroused better immunogenicity than that expressing GP5 or M alone, but it was no better than the DNA vaccine co-expressing GP5 and M proteins with two different promoters. Altered natural conformation of the co-expressed GP5 and M fusion protein was considered as the major cause. Glycine-proline-glycine-proline (GPGP) linker can minimize the conformational changes in tertiary structure and provide flexibility of the peptide chain. The objective of this study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in pigs. Three recombinant DNA constructs expressing GP5/M fusion protein without GPGP linker (pcDNA-56), GP5/M fusion protein conjugated by GPGP linker (pcDNA-5L6), and M/GP5 fusion protein conjugated by GPGP linker (pcDNA-6L5) were established. Sixteen PRRSV-free pigs were randomly assigned to four groups and inoculated intramuscularly with 3 consecutive doses of 500 µg of empty vector pcDNA3.1, pcDNA-56, pcDNA-5L6 or pcDNA-6L5 each at a 2-week interval followed by challenge with 5 × 10(5) TCID(50) PRRSV at 3 weeks after the final inoculation. All pcDNA-56-, pcDNA-5L6-, and pcDNA-6L5- but not pcDNA-3.1-inoculated pigs developed neutralizing antibodies (NAs) 3 weeks after the final inoculation and a gradual increase in NA titers after PRRSV challenge, indicating that pigs inoculated with these DNA constructs could establish a sufficient immune memory. The pcDNA-5L6- and pcDNA-6L5-inoculated pigs displayed lower level and shorter period of viremia and lower tissue viral load following PRRSV challenge than did the pcDNA-56-inoculated pigs. The strategy of co-expressing GPGP-linked GP5 and M fusion protein may be a promising approach for future PRRSV vaccine development, possibly via the improvement of natural conformation of the target fusion protein.

Immunogenicity of recombinant GP5 protein of porcine reproductive and respiratory syndrome virus expressed in tobacco plant.

The aim of the study was to evaluate the immunogenicity of the ORF5-encoded major envelop glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) expressed in tobacco plant as a potential pig oral vaccine in protection against PRRSV infection. Six-week-old PRRSV-free pigs were fed four times orally with 50g of chopped fresh GP5 transgenic tobacco leaves (GP5-T) (GP5 reaching 0.011% of total soluble protein) or wild-type tobacco leaves (W-T) each on days 0, 14, 28, and 42. Samples of serum, saliva, and peripheral blood mononuclear cells (PBMCs) were collected on days -1, 6, 13, 20, 27, 34, 41, and 48 after the initial oral vaccination. A similar vaccination-dependent gradual increase in the responses of serum and saliva anti-PRRSV total IgG and IgA, respectively, and in the levels of PRRSV-specific blastogenic response of PBMCs was seen in GP5-T-treated pigs; all statistically significant elevations occurred after the 2nd vaccination and were revealed after 20 days post-initial oral vaccination (DPIOV). Pigs fed on GP5-T also developed serum neutralizing antibodies to PRRSV at a titer of 1:4-1:8 after the 4th vaccination by 48 DPIOV. No detectable anti-PRRSV antibody responses and PRRSV-specific blastogenic response were seen in W-T-treated pigs. The present study has demonstrated that pigs fed on GP5-T could develop specific mucosal as well as systemic humoral and cellular immune responses against PRRSV. The results also support that transgenic plant as GP5-T can be an effective system for oral delivery of recombinant subunit vaccines in pigs.

Are Asians at greater mortality risks for being overweight than Caucasians? Redefining obesity for Asians.

OBJECTIVES: To assess whether overweight Asians, assessed on the basis of WHO criteria, are at greater mortality risk than overweight Caucasians, and to determine whether alternative cut-off points (BMI = 23.0-24.9 kg/m2 for overweight and BMI >or= 25.0 kg/m2 for obesity) suggested by the WHO Western Pacific Regional Office are appropriate. DESIGN: The cohort was followed prospectively until the end of 2001. All-cause and CVD mortality risks of the overweight and obese group, relative to the reference group (BMI = 18.5-24.9 or 18.5-22.9 kg/m2), were assessed using Cox regression analysis, adjusting for age, smoking and gender. Excess deaths were estimated with a method proposed by the US Centers for Disease Control and Prevention. SETTING: National Health Interview Survey (NHIS 2001) and a middle-aged perspective cohort in Taiwan. SUBJECTS: Subjects comprised 36 386 civil servants and school teachers, aged 40 years and older, who underwent a medical examination during 1989-1992. RESULTS: In the WHO-defined overweight group, Asians showed a significant increase in all-cause mortality risk compared with Caucasians. Asians showed risks equivalent to Caucasians' at lower BMI (around 5 units). Every unit of BMI increase, at 25.0 kg/m2 or above, was associated with a 9 % increase in relative mortality risk from all causes. Applying a cut-off point of 25.0 kg/m2 for obesity would result a prevalence of 27.1 %, while the traditional WHO cut-off point of 30.0 kg/m2 yielded obesity prevalence of 4.1 %. Excess deaths due to obesity accounted for 8.6 % of all deaths and 21.1 % of CVD deaths, based on the alternative cut-offs. CONCLUSIONS: In this Asian population, significant mortality risks started at BMI >or= 25.0 kg/m2, rather than at BMI >or= 30.0 kg/m2. The study supports the use of BMI >or= 25.0 kg/m2 as a new cut-off point for obesity and BMI = 23.0-24.9 kg/m2 for overweight. The magnitude of obesity-attributable deaths has been hitherto under-appreciated among Asians.

The effects of pre-disease risk factors within metabolic syndrome on all-cause and cardiovascular disease mortality.

The metabolic syndrome has been criticized for being "polluted with the inclusion of frank "diseases" with "pre-diseases". We assessed the effect of a single and a combination of "pre-disease" risk factors of metabolic syndrome on the overall and cardiovascular disease (CVD) mortality. These pre-disease risk factors included pre-diabetes, pre-hypertension, overweight and borderline hypertriglycerdemia and were defined as: fasting glucose at 110-125 mg/dL, systolic blood pressure at 120-139 mmHg, body mass index at 25-29.9 kg/m(2) and serum triglyceride at 150-199 mg/dL, respectively. The metabolic syndrome in this paper was based on the version defined by the ATP III. The cohort consisted of 35,259 adults (>==40 years) with a medium follow-up of 15 years. Relative risks (RRs) for all-causes, CVD and "CVD plus diabetes" mortality were calculated with the Cox proportional hazards model. Prevalence of the pre-disease risk factors (40.2%) was nearly four times larger than the metabolic syndrome (10.6%). Individual pre-disease risk factor was associated with significant increases of 13% and 67% (pre-diabetes), 22% and 62% (pre-hypertension), 23% and 32% (overweight) and 17% and 46% (borderline hypertriglyceridemia) on all-cause and "CVD plus diabetes" mortality, respectively. Smoking had comparable risks as "pre-diseases", and, as such, should also be considered as the fifth "pre-disease". Like metabolic syndrome, each "Pre-disease" is a major and significant risk factor for all cause and cardiovascular mortality, but unlike metabolic syndrome, the definition or clinical follow up of "Pre-disease" is simple and straightforward. Recognizing each of the four "pre-disease" as a clinical entity, a hitherto sub-clinical status but involving significantly increased mortality, can alert and justify early intervention through changing lifestyle and modifying biologic risk factors.

Assessing physical activity in an Asian country: low energy expenditure and exercise frequency among adults in Taiwan.

Leisure-time physical activity (LTPA) has been closely related to health improvement. The under-appreciation for energy output by nutritionists stems in part from limited data expressed in caloric equivalent. We converted the frequency, duration, and intensity of LTPA, reported from 15,390 adults in the Taiwan National Health Interview Survey 2001, into kilocalories (kcal). Half of Taiwanese adults admit to no LTPA. Women, lower education or income, younger age, smokers and chewers of betel quid; exercised significantly less than their counterparts. Less than 1/5 (18.9%) of the population in Taiwan was physically active at >or=750 kcal/week, and only 1/7 (13.9%) reached a more desirable goal of >or=1,000 kcal/week, compared with 1/3 in the U.S. The most disconcerting finding was the Taiwan unique U-shaped prevalence for males, with the 25-44 age group being the least active, >or=65 age group being the most active; and S-shaped for females, lowest at age 18-24 years and highest at the two older groups (45-64 and >or=65 years). LTPA was under-appreciated, particularly among the most productive work force (25-44-year group), who exercised with a prevalence only 1/4 of their U.S. counterparts. Expressing LTPA in kcal makes direct comparison easier. Invoking a goal of >or=750 kcal/week for Asians, attainable by exercising 4 hours/week, can facilitate nutritionists in assessing LTPA adequacy. Currently, 4/5 of adults in Taiwan failed to reach this goal. Recognizing the concept of cumulative energy expenditure, in contrast to disciplined daily work for 5 or more days, will encourage the infrequent exercisers such as "weekend warriors" to continue with their activities.