Monitoring and external control of pH in microfluidic droplets during microbial culturing.

BACKGROUND: Cell-based experimentation in microfluidic droplets is becoming increasingly popular among biotechnologists and microbiologists, since inherent characteristics of droplets allow high throughput at low cost and space investment. The range of applications for droplet assays is expanding from single cell analysis toward complex cell-cell incubation and interaction studies. As a result of cellular metabolism in these setups, relevant physicochemical alterations frequently occur before functional assays are conducted. However, to use droplets as truly miniaturized bioreactors, parameters like pH and oxygen availability should be controlled similar to large-scale fermentation to ensure reliable research. RESULTS: Here, we introduce a comprehensive strategy to monitor and control pH for large droplet populations during long-term incubation. We show the correlation of fluorescence intensity of 6-carboxyfluorescein and pH in single droplets and entire droplet populations. By taking advantage of inter-droplet transport of pH-mediating molecules, the average pH value of several million droplets is simultaneously adjusted in an a priori defined direction. To demonstrate the need of pH control in practice, we compared the fermentation profiles of two E. coli strains, a K12-strain and a B-strain, in unbuffered medium with 5 g/L glucose for standard 1 L bioreactors and 180 pL droplets. In both fermentation formats, the commonly used B-strain E. coli BL21 is able to consume glucose until depletion and prevent a pH drop, while the growth of the K12-strain E. coli MG1655 is soon inhibited by a low pH caused by its own high acetate production. By regulating the pH during fermentation in droplets with our suggested strategy, we were able to prevent the growth arrest of E. coli MG1655 and obtained an equally high biomass yield as with E. coli BL21. CONCLUSION: We demonstrated a comparable success of pH monitoring and regulation for fermentations in 1 L scale and 180 pL scale for two E. coli strains. This strategy has the potential to improve cell-based experiments for various microbial systems in microfluidic droplets and opens the possibility for new functional assay designs.

The prevalence of CRF55_01B among HIV-1 strain and its connection with traffic development in China.

CRF55_01B is a relatively "young" HIV strain. At present, we do not know much about its transmission characteristics in China. So, to describe the transmission characteristics of CRF55_01B strain among provinces and HIV infected people, and to analyze the reasons for its rapid epidemic in China, a total of 1237 subjects infected with CRF55_01B from 31 provinces spanning a period of 12 years from 2007 to 2018 were enrolled in this study. By constructing a molecular network and Bayesian correlation analysis, we found that CRF55_01B increased exponentially from 2005 to 2009 after its origin in Shenzhen, and increased rapidly after 2010. CRF55_01B began to spread to other provinces in 2007. After 2010, the strain showed a trend of rapid spread and epidemic from Guangdong-Shenzhen to other provinces in China. Guangdong, Shenzhen, Hunan, Beijing, Guangxi, Hubei, Jiangxi, Guizhou, Hebei, Anhui, Shanghai, Shandong, Henan, and Yunnan were the key provinces of CRF55_01B transmission. CRF55_01B, although originating from men who sex with men (MSM), was transmitted among heterosexuals in 2010. Males in heterosexuals played a crucial role in the transmission and diffusion of this strain. We also revealed that CRF55_01B might spread rapidly along with the rapid development of the Beijing-Guangzhou and Beijing-Kowloon railways. This study suggests that if we detect the spread of MSMs in time through molecular monitoring in the early stage of the epidemic, it can help us control the epidemic early and prevent its spread, which is of great significance to China's national prevention and control of HIV-1.

Immunogenicity and Efficacy of Live Infectious Bronchitis 793/B.08IR Vaccine in SPF Chickens.

Infectious bronchitis virus (IBV) has a variety of serotypes with relatively limited cross-protection leading the disease to be a major problem in the poultry industry. The IBV 793/B strain has identified to circulate in Iran; therefore, the development of a specific vaccine to protect against the virulent virus has received attention. In this regard, the live IB 793/B vaccine (793/B.08IR) was developed in the Razi Vaccine and Serum Research Institute. In this study, the immunogenicity of 793/B.08IR vaccine via different routes of vaccination and efficacy of the vaccine were determined in specific-pathogen-free (SPF) chickens. Three treatment groups of 10 SPF chickens received the vaccine via eye drops, spray, and drinking water. The sera were collected from the chicks at 3 and 6 weeks after the vaccination, and IBV specific antibody was measured using enzyme-linked immunosorbent assay (ELISA) and serum neutralization (SN) test. To evaluate 793/B.08IR vaccine efficacy, 10 SPF chickens were vaccinated using eye drops. Moreover, 10 unvaccinated chickens were separately retained as negative controls. The birds were challenged with the virulent virus 3 weeks following the vaccination. Five days after the challenge, the tracheal swab was taken for virus reisolation. In the immunogenicity test, the ELISA titers of three vaccinated groups were significantly higher than the background values obtained in the control group (p<0.0001). The mean value of ELISA titer in the spray vaccinated group was higher than the spray and drinking water vaccinated groups 3 weeks following the vaccination; however, the difference was not statistically significant. No differences were observed in antibody titers among the three vaccinated groups 6 weeks after the vaccination. The results of the SN test confirmed the data obtained from the ELISA. The results of antibody titer and its increasing trend in chickens showed that 793/B.08IR vaccine induce proper immunity against the virus. In the efficacy test, IBV was isolated from 90% of the unvaccinated controls and 10% of vaccinated groups. The results of the recovery of the virus after the challenge showed that 793/B.08IR vaccine can provide a significantly improved protection against the pathogen in SPF vaccinated chickens.

Awareness and acceptance of H1N1 vaccination among physicians: Experience of 2017 vaccination campaign.

CONTEXT: India experienced pandemic phase of H1N1 in May 2009 to December 2010. The postpandemic phase went on from January 2011 to December 2014. As per the WHO, all countries should immunize their health-care workers as a first priority to protect the essential health infrastructure. AIMS: The aim of the study is to assess the level of awareness and acceptance of influenza vaccine among physicians and also the perception of physicians regarding H1N1 infection. This study also examined time of vaccine administration in relation with efficacy concerns based on literature. SETTINGS AND DESIGN: A vaccination campaign was conducted for all health-care workers of Seth GSMC and KEM Hospital, Mumbai, in the month of July 2017 based on which a cross-sectional observational study was conducted among the physicians of the same institute. METHODS: After ethical clearance, a prevalidated pretested survey based on a pilot survey of 20 physicians was distributed among physicians, which was based on the awareness and acceptance of H1N1 vaccination among physicians and perception of H1N1 infection. Effective sample size was 272. STATISTICAL ANALYSIS USED: Descriptive statistics and Chi-square test were generated for the survey responses. All the continuous variables were reported as mean, median, and range. Categorical variables were reported as tables and pie charts. P < 0.05 was taken as significant. Data analysis was done with SPSS version 21. RESULTS: The overall vaccine compliance was 29.8%. This study has found that area of work, deficiency in knowledge about adverse effect of vaccine, misconceptions regarding vaccine, and concerns about efficacy and duration of vaccine are the important factors which lead to decreased vaccine compliance. Furthermore, it is found during the study that timing of vaccination was not given due importance as considering the epidemiological pattern. CONCLUSIONS: More emphasis should be given to education sessions and counseling of physicians regarding H1N1 vaccination and oseltamivir therapy. At administrative level, more focus should be given on timing of vaccination and other logistics. Vaccine campaigns should be conducted ideally 1 month before expected rise in cases. Quadrivalent vaccine would be more appropriate over trivalent based on epidemiology of infection in India.

Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by reversed-phase high-performance liquid chromatography method.

Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

Immunogenicity and safety of a cell culture-based quadrivalent influenza vaccine in adults: A Phase III, double-blind, multicenter, randomized, non-inferiority study.

Quadrivalent influenza vaccines (QIVs), which include both B lineage strains, are expected to provide broader protection than trivalent influenza vaccines (TIVs). The non-inferiority, immunogenicity, and safety of a cell culture-based investigational QIVc and 2 TIVs (TIV1c, TIV2c), in adults (≥18 y), were evaluated in this Phase III, double-blind, multicenter study. A total of 2680 age-stratified subjects were randomized (2:1:1) to receive 1 dose of QIVc (n = 1335), TIV1c (n = 676), or TIV2c (n = 669). TIV1c (B/Yamagata) and TIV2c (B/Victoria) differed only in B strain lineage. The primary objective was to demonstrate non-inferiority of the hemagglutinin-inhibition antibody responses of QIVc against TIVc, 22 d post-vaccination. Secondary objectives included the evaluation of immunogenicity of QIVc and TIVc in younger (≥18 - <65 y) and older (≥65 y) adults. Hemagglutinin inhibition assays were performed at days 1 and 22. Solicited local and systemic adverse events (AEs) were monitored for 7 d post-vaccination, and unsolicited AEs and serious AEs until day 181. QIVc met the non-inferiority criteria for all 4 vaccine strains and demonstrated superiority for both influenza B strains over the unmatched B strain included in the TIV1c and TIV2c, when geometric mean titers and seroconversion rates with TIVc were compared at day 22. Between 48%-52% of subjects experienced ≥1 solicited AE, the most common being injection-site pain and headache. Serious AEs were reported by ≤1% of subjects, none were vaccine-related. The results indicate that QIVc is immunogenic and well tolerated in both younger and older adults. The immunogenicity and safety profiles of QIVc and TIVc were comparable at all ages evaluated.

IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells.

Regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16%±1.8 vs. 5.53%±1.2, p<0.05). Despite observing a higher total number of dead CD4(+) cells in the IDO group, there was a more abundant live CD4(+)CD25(+) subpopulation in this group. Further analysis reveales that these CD4(+) CD25(+) cells have the capacity to expand in the presence of IDO expressing DFs. Greater number of CTLA-4(+) cells and high expression of TGF-ß and IL-10 were found in CD4(+) cells of the IDO group compared to those of the controls. This finding confirmed a suppressive functionality of the expanded Tregs. Furthermore, CD4(+) CD25(+) cells isolated from the IDO group showed an alloantigen specific suppressive effect in a mixed lymphocyte reaction assay. These results confirm that IDO expressing dermal fibroblasts can expand a population of suppressive antigen specific Tregs. In conclusion, IDO expressing dermal fibroblasts have the capacity to stimulate the expansion of a subset of Tregs which can be used to generate antigen-specific immune tolerance.