Safety, Tolerability, and Immunogenicity of Booster Dose with MVC-COV1901 or MVC-COV1901-Beta SARS-CoV-2 Vaccine in Adults: A Phase I, Prospective, Randomized, Open-Labeled Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines based on variant strains have been in use as booster doses to update immunity against circulating variants. Here we present the results of a phase one prospective, randomized, and open-labeled trial to study the safety and immunogenicity of a booster dose consisting of a subunit vaccine based on the stabilized prefusion SARS-CoV-2 spike protein, MVC-COV1901, or its Beta version, MVC-COV1901-Beta. Participants aged ≥18 and <55 years who received two or three prior doses of MVC-COV1901 vaccines were enrolled and were to receive a booster dose of either 15 mcg of MVC-COV1901, 15 mcg, or 25 mcg of MVC-COV1901-Beta in a 1:1:1 ratio. Adverse reactions after either MVC-COV1901 or MVC-COV1901-Beta booster doses after two or three doses of MVC-COV1901 were comparable and mostly mild and transient. At four weeks after the booster dose, participants with two prior doses of MVC-COV1901 had higher levels of neutralizing antibodies against ancestral SARS-CoV-2, Beta, and Omicron variants than participants with three prior doses of MVC-COV1901, regardless of the type of booster used. MVC-COV1901 and MVC-COV1901-Beta can both be effectively used as booster doses against SARS-CoV-2, including the BA.4/BA.5 Omicron variants.

Evaluating the Neutralizing Ability of a CpG-Adjuvanted S-2P Subunit Vaccine Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants of Concern.

BACKGROUND: Variants of concern (VoCs) have the potential to diminish the neutralizing capacity of antibodies elicited by vaccines. MVC-COV1901 is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine consisting of recombinant prefusion stabilized spike protein S-2P adjuvanted with CpG 1018 and aluminum hydroxide. We explored the effectiveness of MVC-COV1901 against the VoCs. METHODS: Serum samples were taken from rats and phase 1 clinical trial human subjects immunized with a low, medium, or high dose of MVC-COV1901. The neutralizing titers of serum antibodies were assayed with pseudoviruses coated with the SARS-CoV-2 spike protein of the wild-type (WT), D614G, Alpha, or Beta variants. RESULTS: Rats vaccinated twice with vaccine containing high doses of antigen retained high levels of neutralization activity against the Beta variant, albeit with a slight reduction compared to WT. After the third dose, neutralizing titers against the Beta variant were noticeably enhanced regardless of the amount of antigen used for immunization. In humans, vaccinated phase 1 subjects still showed appreciable neutralization abilities against the D614G, Alpha, and Beta variants, although neutralizing titers were significantly reduced against the Beta variant. CONCLUSIONS: Two doses of MVC-COV1901 were able to elicit neutralizing antibodies against SARS-CoV-2 variants with an overall tendency of inducing higher immune response at a higher dose level. The neutralizing titers to the Beta variant in rats and humans were lower than those for WT and the Alpha variant. An additional third dose in rats was able to partially compensate for the reduction in neutralization against the Beta variant. We have demonstrated that immunization with MVC-COV1901 was effective against VoCs.

Adding Pay-for-Performance Program to Routine Care Was Related to a Lower Risk of Depression Among Type 2 Diabetes Patients in Taiwan.

Background: Patients with type 2 diabetes (T2DM) often experience depression during treatment, negatively influencing their treatment compliance and clinical outcomes. Recently, the pay-for-performance (P4P) program for chronic diseases, with high-cost and high-risk feature, such as T2DM, has been implemented and has been operational for several years. Nevertheless, its effect on the risk of developing depression among T2DM cases is unknown. This study aims to explore the association of P4P use with the subsequent risk of developing depression among these patients. Methods: This cohort study used a nationwide health insurance database to identify patients 20-70 years of age newly diagnosed with T2DM who enrolled in the P4P program between 2001 and 2010. From this group, we enrolled 17,022 P4P users and then 17,022 non-P4P users who were randomly selected using propensity-score-matching. Enrolled patients were followed until the end of 2012 to record the occurrence of depression. The Cox proportional hazards regression was utilized to obtain the adjusted hazard ratio (aHR) for P4P use. Results: During the study period, a total of 588 P4P users and 1,075 non-P4P users developed depression at incidence rates of 5.89 and 8.41 per 1,000 person-years, respectively. P4P users had a lower depression risk than did non-P4P users (aHR, 0.73; 95% Confidence Interval, 0.65-0.80). This positive effect was particularly prominent in those receiving high-intensity use of the P4P program. Conclusion: Integrating P4P into routine care for patients with T2DM may have beneficial effects on curtailing the subsequent risk of depression.

Safety and immunogenicity of a Recombinant Stabilized Prefusion SARS-CoV-2 Spike Protein Vaccine (MVC-COV1901) Adjuvanted with CpG 1018 and Aluminum Hydroxide in healthy adults: A Phase 1, dose-escalation study.

BACKGROUND: This was a phase 1, dose-escalation open-label trial to evaluate the safety and immunogenicity of MVC-COV1901, a SARS-CoV-2 S-2P protein vaccine adjuvanted with aluminum hydroxide and CpG 1018. METHODS: Between September 28 and November 13 2020, 77 participants were screened. Of these, 45 healthy adults from 20 to 49 years of age were to be administered two doses of MVC-COV1901 in doses of 5 µg, 15 µg, or 25 µg of spike protein at 28 days apart. There were 15 participants in each dose group; all were followed for 28 days after the second dose at the time of the interim analysis. Adverse events and laboratory data were recorded for the safety evaluation. Blood samples were collected for humoral, and cellular immune response at various time points. Trial Registration: ClinicalTrials.gov NCT04487210. FINDINGS: Solicited adverse events were mostly mild and similar. No subject experienced fever. After the second dose, the geometric mean titers (GMTs) for SARS-CoV-2 spike-specific immunoglobulin G were 7178.2, 7746.1, 11,220.6 in the 5 µg, 15 µg, and 25 µg dose groups, respectively. The neutralizing activity were detected in both methods. (Day 43 GMTs, 538.5, 993.1, and 1905.8 for pseudovirus; and 33.3, 76.3, and 167.4 for wild-type virus). The cellular immune response induced by MVC-COV1901 demonstrated substantially higher numbers of IFN-γ- producing cells, suggesting a Th1-skewed immune response. INTERPRETATION: The MVC-COV1901 vaccine was well tolerated and elicited robust immune responses and is suitable for further development. FUNDING: Medigen Vaccine Biologics Corporation.

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 µg or 5 µg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.

Identification of Impaired Second-Phase Insulin Secretion in Various Degrees of Glucose Tolerance in a Chinese Population.

AIM: Impaired insulin sensitivity and insulin secretion (ISEC) are major pathophysiologies of type 2 diabetes (T2DM). ISEC has two phases: the first and second phases (second ISEC). In this study, we derived equations to identify patients with second ISEC deficiency (ISEC-D). METHODS: Data from 96 patients, namely 19 with a normal fasting plasma glucose (FPG) level, 21 with prediabetes, and 56 with T2DM, were enrolled. They underwent a modified low-dose graded glucose infusion test, which was originally proposed by Polonsky et al. The test results were interpreted as the slopes of the changes of plasma insulin against the glucose levels, which were considered second ISEC. Patients with the lowest quartile of the slopes were defined as having ISEC-D. We built three models: Model 0: FPG, Model 1: FPG + waist circumference, and Model 2: Model 1 + fasting plasma insulin. The area under the receiver operating characteristic (aROC) curve was used to determine the predictive power of these models. RESULTS: Among the metabolic syndrome components, FPG had the largest aROC curve (78.2%). Although aROC curves of Models 1 and 2 (85.2% and 91.5%, respectively) were higher than the aROC curve of FPG, no difference was observed between Models 1 and 0. By contrast, the aROC curve of Model 2 was higher compared with Model 1. CONCLUSIONS: FPG showed the largest aROC curve. Model 2 had the highest predictive power, which could identify patients with ISEC-D with a sensitivity and specificity of 94.3% and 82.6%, respectively. These two models could be conveniently used in daily practice.

The estimation of first-phase insulin secretion by using components of the metabolic syndrome in a chinese population.

Aims. There are two phases of insulin secretion, the first (FPIS) and second phase (SPIS). In this study, we built equations to predict FPIS with metabolic syndrome (MetS) components and fasting plasma insulin (FPI). Methods. Totally, 186 participants were enrolled. 75% of participants were randomly selected as the study group to build equations. The remaining 25% of participants were selected as the external validation group. All participants received a frequently sampled intravenous glucose tolerance test, and acute insulin response after the glucose load (AIRg) was obtained. The AIRg was considered as FPIS. Results. When MetS components were only used, the following equation was built: log (FPIS) = 1.477 - 0.119 × fasting plasma glucose (FPG) + 0.079 × body mass index (BMI) - 0.523 × high-density lipoprotein cholesterol (HDL-C). After FPI was added, the second equation was formulated: log (FPIS) = 1.532 - 0.127 × FPG + 0.059 × BMI - 0.511 × HDL-C + 0.375 × log (FPI), which provided a better accuracy than the first one. Conclusions. Using MetS components, the FPIS could be estimated accurately. After adding FPI into the equation, the predictive power increased further. We hope that these equations could be widely used in daily practice.

Estimation of the disposition index based on components of metabolic syndrome.

Decreased insulin sensitivity (IS) and impaired insulin secretion are major pathological features of type 2 diabetes (T2DM). The product of these factors is the disposition index (DI). We aimed to develop an equation for predicting DI. We enrolled 167 participants in our study. We randomly assigned 126 (75%) of the participants to the study group, whose data would be used to build the equation for estimating the DI. The remaining 41 participants comprised the external validation group. A frequently sampled intravenous glucose-tolerance test was performed for all participants, and the IS, the glucose sensitivity, the acute insulin response to the glucose load, and the DI were determined. Three factors were selected from multiple linear regression analysis, and we constructed the equation log (DI) = 2.449 - 0.113 × fasting plasma glucose + 0.046 × body mass index - 0.612 × high-density lipoprotein cholesterol. Using this equation, the calculated log (DI) significantly correlated with the measured log (DI) in the external validation group (r = 0.428, p = 0.007). By using the equation based on the demographic data and measurements of metabolic syndrome components, the DI could be predicted with acceptable accuracy (r = 0.428). Because of the relationships between the MetS and demographic parameters, this method of predicting DI may help further clinicians' understanding of the underlying pathological mechanisms in T2DM.

Increased risk of ischemic stroke after hyperosmolar hyperglycemic state: a population-based follow-up study.

BACKGROUND: Although much attention has been focused on the association between chronic hyperglycemia and cerebrovascular diseases in type 2 diabetes mellitus (DM) patients, there is no data regarding the risk of ischemic stroke after a hyperosmolar hyperglycemic state (HHS) attack. The objective of this study was to investigate the risk of ischemic stroke in type 2 DM patients after an HHS attack. METHODS: From 2004 to 2008, this retrospective observational study was conducted on a large cohort of Taiwanese using Taiwan's National Health Insurance Research Database (NHIRD). We identified 19,031 type 2 DM patients who were discharged with a diagnosis of HHS and 521,229 type 2 DM patients without an HHS diagnosis. Using the propensity score generated from logistic regression models, conditional on baseline covariates, we matched 19,031 type 2 DM patients with an HHS diagnosis with the same number from the comparison cohort. The one-year cumulative rate for ischemic stroke was estimated using the Kaplan-Meier method. After adjusting covariates, Cox proportional hazard regression was used to compute the adjusted one-year rate of ischemic stroke. RESULTS: Of the patients sampled, 1,810 (9.5%) of the type 2 DM patients with HHS and 996 (5.2%) of the comparison cohort developed ischemic stroke during the one-year follow-up period. After adjusting for covariates, the adjusted HR for developing ischemic stroke during the one-year follow-up period was 1.8 (95% C.I., 1.67 to 1.95, P<0.001) for type 2 DM patients with HHS compared with those without HHS. CONCLUSION: Although DM is a well-recognized risk factor for atherosclerosis, type 2 DM patients that have suffered a HHS attacks are at an increased risk of developing ischemic stroke compared with those without HHS.

Predicting metabolic syndrome by using hematogram models in elderly women.

BACKGROUND: Low-grade inflammatory status was thought to be a major underlying mechanism in MetS. White blood cell (WBC) count was one of the inflammatory markers identified to be associated with MetS. Moreover, not only WBC but also hemoglobin (Hb) and platelet (PLT) were all associated with MetS. OBJECTIVE: In this study, we tried to build models by the hematogram components. In this way, we can not only predict the occurrence of MetS with a relatively low-cost and routine lab test, but also can understand more about the relationships between low grade inflammation and MetS. METHODS: We randomly collected subjects over 65 years old from MJ Health Screening Center's database between 1999 and 2008. After excluding subjects with medications for hypertension, hyperlipidemia and/or diabetes, 13,132 female were eligible for analysis. RESULTS: All the MetS components, hematogram parameters and age were higher in group with MetS. In the correlation matrix, all these three hematogram parameters (WBC, Hb and PLT) were correlated with MetS components except for the correlation between Hb and HDL-C. The ROC curves showed that the model 3 (PLT + Hb + WBC) had greatest area under the curve of 0.631 with the sensitivity of 58.1% and specificity of 61.4%. CONCLUSIONS: Our findings have shown that all the three hematogram parameters are related to MetS. The results not only shed light on the complex relationships, but also demonstrate a common and easy model to aid clinicians to be more aware of the occurrence of MetS.

Evaluation of second phase insulin secretion with simple surrogates derived from the mixed meal tolerance test in patients with type 2 diabetes.

The major contributors to the pathogenesis of type 2 diabetes are impaired insulin action and insulin secretion, including second phase insulin secretion (2nd ISEC). This study aimed to compare surrogates derived from the mixed meal tolerance test (MTT) with 2nd ISEC derived from modified low-dose graded glucose infusion (M-LDGGI) in patients with type 2 diabetes. We were subsequently able to decide which surrogate would be performed easily and accurately. Twenty type 2 diabetes patients were enrolled. They received both MTT and M-LDGGI. The standardized MTT meals were provided at 8:00 A.M. and 12:00 P.M. The M-LDGGI was a simplified version of the Polonsky method; only two 80-min stages of glucose infusion (2 and 6 mg/kg/min) were given. The slopes of the insulin to glucose curve during the test were regarded as the 2nd ISEC. First, we used the area under the insulin curve (AUC(IN)) during MTT to quantify the 2nd ISEC. The best correlated AUC(IN) was from 60-240 min. Second, the slopes between any two time points of the plasma insulin to glucose level (SLOPE(I/G)) were also assessed. The time period best correlated with 2nd ISEC was from 0-120 min (SLOPE0₋120). Finally, the insulin-to-glucose ratio (IGr) of each time point was used to estimate the 2nd ISEC, and the best correlation was observed at 180 min. In conclusion, estimating 2nd ISEC surrogates derived from MTT proved to be possible. The most accurate surrogate is the SLOPE0₋120, while IG(r180) is another less precise but more convenient method.

The combined effect of individual and neighborhood socioeconomic status on cancer survival rates.

BACKGROUND: This population-based study investigated the relationship between individual and neighborhood socioeconomic status (SES) and mortality rates for major cancers in Taiwan. METHODS: A population-based follow-up study was conducted with 20,488 cancer patients diagnosed in 2002. Each patient was traced to death or for 5 years. The individual income-related insurance payment amount was used as a proxy measure of individual SES for patients. Neighborhood SES was defined by income, and neighborhoods were grouped as living in advantaged or disadvantaged areas. The Cox proportional hazards model was used to compare the death-free survival rates between the different SES groups after adjusting for possible confounding and risk factors. RESULTS: After adjusting for patient characteristics (age, gender, Charlson Comorbidity Index Score, urbanization, and area of residence), tumor extent, treatment modalities (operation and adjuvant therapy), and hospital characteristics (ownership and teaching level), colorectal cancer, and head and neck cancer patients under 65 years old with low individual SES in disadvantaged neighborhoods conferred a 1.5 to 2-fold higher risk of mortality, compared with patients with high individual SES in advantaged neighborhoods. A cross-level interaction effect was found in lung cancer and breast cancer. Lung cancer and breast cancer patients less than 65 years old with low SES in advantaged neighborhoods carried the highest risk of mortality. Prostate cancer patients aged 65 and above with low SES in disadvantaged neighborhoods incurred the highest risk of mortality. There was no association between SES and mortality for cervical cancer and pancreatic cancer. CONCLUSIONS: Our findings indicate that cancer patients with low individual SES have the highest risk of mortality even under a universal health-care system. Public health strategies and welfare policies must continue to focus on this vulnerable group.

Selection and characterization of vaccine strain for Enterovirus 71 vaccine development.

Enterovirus 71 (EV71) has recently emerged as an important neurotropic virus in Asia because effective medications and prophylactic vaccine against EV71 infection are not available. Based on the success of inactivated poliovirus vaccine, the Vero cell-based chemically inactivated EV71 vaccine candidate could be developed. Identification of EV71 vaccine strain which can grow to high titer in Vero cell and induce cross-genotype virus neutralizing antibody responses represents the first step in vaccine development. In this report we describe the characterization and validation of a clinical isolate E59 belonging to B4 sub-genotype based on VP1 genetic analysis. Before selected as the vaccine strain, the genetic stability of E59 in passage had been analyzed based on the nucleotide sequences obtained from the Master Virus Seed, Working Seed banks and the virus harvested from the production lots, and found to be identical to those found in the original isolate. These results indicate that E59 vaccine strain has strong genetic stability in passage. Using this vaccine strain the prototype EV71 vaccine candidate was produced from 20L of Vero cell grown in serum-containing medium. The production processes were investigated, characterized and quantified to establish the potential vaccine manufacturing process including the time for virus harvest, the membrane for diafiltration and concentration, the gel-filtration chromatography for the down-stream virus purification, and the methods for viral inactivation. Finally, the inactivated virion vaccine candidate containing sub-microgram of viral proteins formulated with alum adjuvant was found to induce strong virus neutralizing antibody responses in mice and rabbits. Therefore, these results provide valuable information for cell-based EV71 vaccine development.

Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus.

The neutralization titer of a hemagglutinin (HA)-specific neutralizing antibody against new isolates reflect both the antigenic drift and the conformation status of HA protein in these new influenza viruses. Since most antigenic sites are in the HA1 domain of HA, using HA1 domain of influenza virus as antigen is of great importance in vaccine development. In this study, we investigate different purification processes for optimizing the immunological properties of an Escherichia coli-expressed HA1 domain (rH5HA1) of influenza H5N1 virus. rH5HA1 was expressed as inclusion bodies and extracted with 6M guanidine hydrochloride (GnHCl)/PBS buffer. The best condition for generating HA1-specific neutralization determinants is on-column oxidative refolding procedures with GSH/GSSG and l-arginine buffer. Others refolding procedures such as using high-pH buffer and/or different detergent solubilizations were found to be ineffective producing neutralization epitope recognized by a HA1-specific neutralizing monoclonal antibody that was raised against H5N1 virus.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design.

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.

High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture.

Developing an effective vaccine against enterovirus 71 (EV71) infection provides the best means to control the disease. We have previously reported that large-scale preparation of a low immunogenic EV71 strain can be achieved using serum free microcarrier Vero cell culture in a 2-l bioreactor [Wu SC, Liu CC, Lian WC. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 2004;22:3858-64]. This present work further investigated the virus growth and the immunogenicity of two high immunogenic strains (EV71-075 and EV71-117) prepared in serum-free microcarrier cell cultures. Our results showed that serum free culture increased cell death rate after infection, reduced the virus specific productivity, but resulted in elicitation of higher neutralizing titers in immunized mice as compared to that parallel obtained in serum-containing cultures. Therefore, serum-free microcarrier culture is a valuable process for developing inactivated EV71 vaccines.

A novel maternally transcribed homeobox gene, Eso-1, is preferentially expressed in oocytes and regulated by cytoplasmic polyadenylation.

The homeobox gene families play important roles in the transcriptional regulation of gene expression prior to and during embryo development. To identify novel homeobox genes expressed in early embryonic development, we conducted a degenerated oligonucleotide polymerase chain reaction (PCR) to screen a mouse embryonic stem (ES) cell cDNA library. A novel homeobox-containing gene, Eso-1, which is preferentially expressed in ES cells and ovaries, was identified. The full-length Eso-1 cDNA was found to be 1,710 bp with a predicted homeodomain that has no significant homology to previously reported homeodomain proteins. Eso-1 was mapped to chromosome 14A3. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that Eso-1 was expressed through oogenesis and continuing to be expressed through to the blastocyst stage. De novo expression of Eso-1 started at 13.5 days postcoitum in the ovaries, which coincides with the initiation of oogenesis. Northern blot analyses demonstrated that Eso-1 is preferentially expressed in both ovaries and ES cells as a 1.7-kb transcript. Results from whole mount in situ hybridization revealed that Eso-1 in oocytes showed increased expression from primordial to antral follicles. The 3'-untranslated region of Eso-1 transcripts contained cytoplasmic polyadenylation sequences while the length of poly (A) tails changed during oocyte maturation, indicating that Eso-1 expression is controlled by time-dependent translational activation. We suggest that the novel homeodomain protein, Eso-1, plays a role during oocyte maturation and early embryonic development.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates.

The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni(2+)-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166-180, 356-375, and 396-410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156-175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156-175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1-30, 86-100, 306-320, and 351-365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2K(d) CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-gamma secretion cell assay and analysis by flow cytometry, peptides containing residues 81-95 were found to be capable of stimulating both CD4(+) and CD8(+) cell proliferation in vitro. We also only observed that peptides corresponding to residues 336-350 were capable of stimulating IFN-gamma production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

The effect of testosterone supplement on insulin sensitivity, glucose effectiveness, and acute insulin response after glucose load in male type 2 diabetics.

Our understanding of the effect of androgens on insulin action and glucose metabolism is incomplete. Several different models and methods have been used to study androgen effects, with some studies indicating that higher testosterone levels are associated with increased insulin resistance. In polycystic ovary syndrome, where high testosterone levels are frequently found, affected patients have a higher risk of diabetes. In contrast, increased insulin resistance was found in both hypergonadotropic and hypogonadotropic men with hypoandrogenism, patients with Klinefelter's syndrome and men with idiopathic gonadotropin deficiency. Insulin resistance is considered to be one of the cornerstones in the state that ultimately leads to clinically established type 2 diabetes mellitus. In addition, men with type 2 diabetes have relative hypogonadism. Therefore, supplementation with testosterone might play a role in improving both insulin resistance and hypogonadism. The study population consisted of 11 male patients with type 2 diabetes. Their mean age was 57.7 +/- 3.41 years, the body mass index (BMI) was 24.4 +/- 1.02 kg/m2, and the waist-to-hip ratio (W/H) was 0.91 +/- 0.05. The patients were all treated with oral hypoglycemic agents. The men received androgen injections every 3 weeks intramuscularly for 12 weeks. The injections were testosterone depot 100 mg/3 weeks. Insulin sensitivity, glucose effectiveness and area under acute insulin response were calculated from "minimal model" algorithms. There were no significant differences in the value of BMI, W/H ratios, plasma lipid concentrations, testosterone, homeostasis model assessment (HOMA) of insulin sensitivity, and beta-cell function, before and after supplementation of testosterone. Furthermore, the insulin sensitivity (SI) (1.04 +/- 0.25, 1.11 +/- 0.36 x 10(-5) min(-1/)pM; p = 0.43), glucose effectiveness (EG) (0.018 +/- 0.003, 0.017 +/- 0.002 min(-1); p = 0.29), and acute insulin response (AIR) after a glucose load (45.7 +/- 24.3, 50.1 +/- 32.5 pM; p = 0.45) did not change significantly after supplmentation with testosterone. In our study, there was no improvement of SI, EG, and AIR after 3 months of Testosterone Depot treatment in type 2 diabetes, but we believe that duration and dosage of the androgen therapy might play an important role in improving insulin sensitivity. The mechanisms by which testosterone causes insulin resistance is unknown, and larger studies on androgen treatment in type 2 diabetic patients are necessary.

Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development.

Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.