Characterization of antigen presentation capability for neoantigen-based products using targeted LC-MS/MS method.

The generation of an immune response in neoantigen-based products relies on antigen presentation, which is closely analyzed by bioassays for T-cell functions such as tetramer or cytokine release. Mass spectrometry (MS) has the potential to directly assess the antigen-presenting capability of antigen-presenting cells (APCs), offering advantages such as speed, multi-target analysis, robustness, and ease of transferability. However, it has not been used for quality control of these products due to challenges in sensitivity, including the number of cells and peptide diversity. In this study, we describe the development and validation of an improved targeted LC-MS/MS method with high sensitivity for characterizing antigen presentation, which could be applied in the quality control of neoantigen-based products. The parameters for the extraction were carefully optimized by different short peptides. Highly sensitive targeted triple quadrupole mass spectrometry combined with ultra-high performance liquid chromatography (UHPLC) was employed using a selective ion monitoring mode (Multiple Reaction Monitoring, MRM). Besides, we successfully implemented robust quality control peptides to ensure the reliability and consistency of this method, which proved invaluable for different APCs. With reference to the guidelines from ICH Q2 (R2), M10, as well as considering the specific attributes of the product itself, we validated the method for selectivity, specificity, sensitivity, limit of detection (LOD), recovery rate, matrix effect, repeatability, and application in dendritic cells (DCs) associated with neoantigen-based products. The validation process yields satisfactory results. Combining this approach with T cell assays will comprehensively assess cell product quality attributes from physicochemical and biological perspectives.

GMP-grade microcarrier and automated closed industrial scale cell production platform for culture of MSCs.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix® and ACISCP system, we inoculated 1.5 × 107 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 1010 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix® microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

Requirments for primary human hepatocyte.

'Requirements for Primary Human Hepatocyte' is the first set of guidelines on Primary Human Hepatocyte in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for Primary Human Hepatocyte, which is applicable to the quality control for Primary Human Hepatocyte. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of Primary Human Hepatocyte for applications.

COVID-19 vaccines: progress and understanding on quality control and evaluation.

The outbreak of COVID-19 has posed a huge threat to global health and economy. Countermeasures have revolutionized norms for working, socializing, learning, and travel. Importantly, vaccines have been considered as most effective tools to combat with COVID-19. As of the beginning of 2021, >200 COVID-19 vaccine candidates, covering nearly all existing technologies and platforms, are being research and development (R&D) by multiple manufacturers worldwide. This has posed a huge obstacle to the quality control and evaluation of those candidate vaccines, especially in China, where five vaccine platforms are deployed in parallel. To accelerate the R&D progress of COVID-19 vaccines, the guidances on R&D of COVID-19 vaccine have been issued by National Regulatory Authorities or organizations worldwide. The Center for Drug Evaluation and national quality control laboratory in China have played a leading role in launching the research on quality control and evaluation in collaboration with relevant laboratories involved in the vaccine R&D, which greatly supported the progression of vaccines R&D, and accelerated the approval for emergency use and conditional marketing of currently vaccine candidates. In this paper, the progress and experience gained in quality control and evaluation of COVID-19 vaccines developed in China are summarized, which might provide references for the R&D of current and next generation of COVID-19 vaccines worldwide.

AP-3-dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells.

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.

Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials.

BACKGROUND: Although several COVID-19 vaccines have been developed so far, they will not be sufficient to meet the global demand. Development of a wider range of vaccines, with different mechanisms of action, could help control the spread of SARS-CoV-2 globally. We developed a protein subunit vaccine against COVID-19 using a dimeric form of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. We aimed to assess the safety and immunogenicity of this vaccine, ZF2001, and determine the appropriate dose and schedule for an efficacy study. METHODS: We did two randomised, double-blind, placebo-controlled, phase 1 and phase 2 trials. Phase 1 was done at two university hospitals in Chongqing and Beijing, China, and phase 2 was done at the Hunan Provincial Center for Disease Control and Prevention in Xiangtan, China. Healthy adults aged 18-59 years, without a history of SARS-CoV or SARS-CoV-2 infection, an RT-PCR-positive test result for SARS-CoV-2, a history of contact with confirmed or suspected COVID-19 cases, and severe allergies to any component of the vaccine were eligible for enrolment. In phase 1, participants were randomly assigned (2:2:1) to receive three doses of the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart, in either a two-dose schedule or a three-dose schedule. Investigators, participants, and the laboratory team were masked to group allocation. For phase 1, the primary outcome was safety, measured by the occurrence of adverse events and serious adverse events. For phase 2, the primary outcome was safety and immunogenicity (the seroconversion rate and the magnitude, in geometric mean titres [GMTs], of SARS-CoV-2-neutralising antibodies). Analyses were done on an intention-to-treat and per-protocol basis. These trials are registered with ClinicalTrials.gov (NCT04445194 and NCT04466085) and participant follow-up is ongoing. FINDINGS: Between June 22 and July 3, 2020, 50 participants were enrolled into the phase 1 trial and randomly assigned to receive three doses of placebo (n=10), the 25 µg vaccine (n=20), or the 50 µg vaccine (n=20). The mean age of participants was 32·6 (SD 9·4) years. Between July 12 and July 17, 2020, 900 participants were enrolled into the phase 2 trial and randomly assigned to receive two doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150), or three doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150). The mean age of participants was 43·5 (SD 9·2) years. In both phase 1 and phase 2, adverse events reported within 30 days after vaccination were mild or moderate (grade 1 or 2) in most cases (phase 1: six [60%] of ten participants in the placebo group, 14 [70%] of 20 in the 25 µg group, and 18 [90%] of 20 in the 50 µg group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 µg group, 50 [33%] of 150 in the two-dose 50 µg group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 µg group, and 65 [43%] of 150 in the three-dose 50 µg group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 µg group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 µg vaccine group, two [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 µg vaccine group, and six [4%] in the three-dose 50 µg vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 µg vaccine group, and five [3%] in the three-dose 50 µg vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 µg vaccine group, and two [1%] in the three-dose 50 µg vaccine group), but none was considered vaccine related. In phase 2, on the two-dose schedule, seroconversion rates of neutralising antibodies 14 days after the second dose were 76% (114 of 150 participants) in the 25 µg group and 72% (108 of 150) in the 50 µg group; on the three-dose schedule, seroconversion rates of neutralising antibodies 14 days after the third dose were 97% (143 of 148 participants) in the 25 µg group and 93% (138 of 148) in the 50 µg group. In the two-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the second dose were 17·7 (95% CI 13·6-23·1) in the 25 µg group and 14·1 (10·8-18·3) in the 50 µg group. In the three-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the third dose were 102·5 (95% CI 81·8-128·5) in the 25 µg group and 69·1 (53·0-90·0) in the 50 µg group. INTERPRETATION: The protein subunit vaccine ZF2001 appears to be well tolerated and immunogenic. The safety and immunogenicity data from the phase 1 and 2 trials support the use of the 25 µg dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy. FUNDING: National Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Mesenchymal stem cells modified by FGF21 and GLP1 ameliorate lipid metabolism while reducing blood glucose in type 2 diabetic mice.

OBJECTIVE: The purpose of this study was to investigate the therapeutic effects of genetically modified mesenchymal stem cells (MSCs) in the treatment of type 2 diabetes mellitus (T2DM) in order to identify a new method for treating diabetes that differs from traditional medicine and to provide a new means by which to fundamentally improve or treat diabetes. METHODS: MSCs derived from adipose tissue were modified to overexpress FGF21 and GLP1, which was achieved through lentiviral particle transduction. The cells were transplanted into BKS.Cg-Dock7m+/+Leprdb/Nju mice (T2DM mouse model). Injections of physiological saline (0.1 mL) and liraglutide (0.5 mg/kg) were used as negative and positive controls, respectively. ELISA or Western blotting was used for protein analysis, and quantitative real-time PCR was used for gene expression analysis. RESULTS: Genetic modification had no effects on the morphology, differentiation ability, or immunophenotype of MSCs. Moreover, MSC-FGF21+GLP1 cells exhibited significantly increased secretion of FGF21 and GLP1. In the T2DM mouse model, the transplantation of MSC-FGF21+GLP1 cells ameliorated the changes in blood glucose and weight, promoted the secretion of insulin, enhanced the recovery of liver structures, and improved the profiles of lipids. Moreover, FGF21 and GLP1 exerted synergistic effects in the regulation of glucolipid metabolism by controlling the expression of insulin, srebp1, and srebp2. CONCLUSION: Stem cell treatment based on MSCs modified to overexpress the FGF21 and GLP1 genes is an effective approach for the treatment of T2DM.

Deconstructing transcriptional variations and their effects on immunomodulatory function among human mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. METHODS: We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. RESULTS: MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. CONCLUSION: This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.

The potential markers of NK-92 associated to cytotoxicity against K562 cells.

Markers associated to NK cytolytic activity are in a great need to regulate NK cell immunotherapy products. We assume that biomarkers which response to cytolysis will change their transcription, expression or secretion. To find NK-92 indicator to cytolytic activity, we have evaluated the potential markers by quantifying the expression of well-known cytotoxicity functional molecules (cytokine IFN-γ, Granzyme B, perforin, CD69 and CD107a), and explored candidate markers by a sweeping transcription picture of NK-92 using a direct cytolysis model (incubation with K562). We found that IFN-γ secretion was highly correlated to cytotoxicity of NK-92, neither Granzyme B, perforin secretion, nor CD69, CD107a positive population were upregulated by K562 stimulation. RNAseq revealed 432 genes expression changed during cytolysis, several genes (BIRC3, CSF2, VCAM1 and TNFRSF9) mRNA expression were validated by real time RT-PCR under K562 being killed or protected from being killed conditions. Results suggested IFN-γ secretion, BIRC3 and TNFRSF9 transcription in NK-92 were responsive to K562 cytolysis. In a word, our results confirmed one marker and reveal an array of novel candidate markers associated with NK-92 cytotoxicity. Further studies are greatly needed to determine the roles these new makers play in NK-92 cytolysis process.

Distinct mucosal microbial communities in infants with surgical necrotizing enterocolitis correlate with age and antibiotic exposure.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most common surgical emergency in preterm infants, and pathogenesis associates with changes in the fecal microbiome. As fecal samples incompletely represent microbial communities in intestinal mucosa, we sought to determine the NEC tissue-specific microbiome and assess its contribution to pathogenesis. DESIGN: We amplified and sequenced the V1-V3 hypervariable region of the bacterial 16S rRNA gene extracted from intestinal tissue and corresponding fecal samples from 12 surgical patients with NEC and 14 surgical patients without NEC. Low quality and non-bacterial sequences were removed, and taxonomic assignment was made with the Ribosomal Database Project. Operational taxonomic units were clustered at 97%. We tested for differences between NEC and non-NEC samples in microbiome alpha- and beta-diversity and differential abundance of specific taxa between NEC and non-NEC samples. Additional analyses were performed to assess the contribution of other demographic and environmental confounding factors on the infant tissue and fecal microbiome. RESULTS: The fecal and tissue microbial communities were different. NEC was associated with a distinct microbiome, which was characterized by low diversity, higher abundances of Staphylococcus and Clostridium_sensu_stricto, and lower abundances of Actinomyces and Corynebacterium. Infant age and vancomycin exposure correlated with shifts in the tissue microbiome. CONCLUSION: The observed low diversity in NEC tissues suggests that NEC is associated with a bacterial bloom and a distinct mucosal bacterial community. The exact bacterial species that constitute the bloom varied by infant and were strongly influenced by age and exposure to vancomycin.

Bacterial DNA is present in the fetal intestine and overlaps with that in the placenta in mice.

Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.

Gene-edited MLE-15 Cells as a Model for the Hermansky-Pudlak Syndromes.

Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body-like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3ß1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.

Review of the current use and evaluation of cell substrates for producing biologicals in selected countries.

In 2010, the WHO guidance document for the evaluation of cell substrates for producing biologicals was replaced with updated recommendations and in May 2013 an implementation workshop on the new recommendations was held in Beijing, China. As part of this workshop, a survey of the use and evaluation of cell substrates for producing biologicals was undertaken and the information obtained was updated in June 2014. The purpose of survey was to capture the status of national requirements related to cell substrates in various countries with particular emphasis on whether or not the updated WHO recommendations had been, or were to be, incorporated into national requirements. This paper reports the outcome of the survey and is based on information provided by regulators in eleven countries. Since the publication of the updated WHO recommendations, several activities such as the implementation workshop and publications have been undertaken by the WHO. The aim of these activities, including the publication of this article, is to contribute to the implementation of WHO recommendations so as to reduce regulatory gaps between national requirements and globally agreed expectations.

Early life establishment of site-specific microbial communities in the gut.

Fecal sampling is widely utilized to define small intestinal tissue-level microbial communities in healthy and diseased newborns. However, this approach may lead to inaccurate assessments of disease or therapeutics in newborns because of the assumption that the taxa in the fecal microbiota are representative of the taxa present throughout the gastrointestinal tract. To assess the stratification of microbes in the newborn gut and to evaluate the probable shortcoming of fecal sampling in place of tissue sampling, we simultaneously compared intestinal mucosa and fecal microbial communities in 15 neonates undergoing intestinal resections. We report three key results. First, when the site of fecal and mucosal samples are further apart, their microbial communities are more distinct, as indicated by low mean Sørensen similarity indices for each patient's fecal and tissue microbiota. Second, two distinct niches (intestinal mucosa and fecal microbiota) are evident by principal component analyses, demonstrating the critical role of sample source in defining microbial composition. Finally, in contrast to adult studies, intestinal bacterial diversity was higher in tissue than in fecal samples. This study represents an unprecedented map of the infant microbiota from intestinal mucosa and establishes discernable biogeography throughout the neonatal gastrointestinal tract. Our results question the reliance on fecal microbiota as a proxy for the developing intestinal microbiota. Additionally, the robust intestinal tissue-level bacterial diversity we detected at these early ages may contribute to the maturation of mucosal immunity.

The prevalence of neutralizing antibodies against AAV serotype 1 in healthy subjects in China: implications for gene therapy and vaccines using AAV1 vector.

Recombinant adeno-associated virus serotype 1 (AAV1) has attracted tremendous interest as a promising vector for gene therapy and vaccine applications. However, the presence of AAV1 neutralizing antibodies as a consequence of exposure to wild type AAV1 can limit significantly effective gene transfer for biologics based AAV1 vector. Prior studies have reported that a prevalence of AAV1 neutralizing antibodies ranged from 10% to 50% in different countries around the world, and up to 79% in Dutch subjects. However, few studies have reported on the AAV1 neutralizing antibody prevalence in Chinese subjects. In this study, a high-throughput luciferase-based virus neutralization assay was established and standardized for critical parameters, including the appropriate cell line, and the optimal viral infection dose, and the infection time with homologous AAV1 vaccinated mice and guinea pig sera. Then, a total of 500 healthy individual serum samples from two separate regions of China were screened for the AAV1 neutralizing antibodies by conducting a non-randomized, cross-sectional analysis. Interestingly, a high prevalence of AAV1 neutralizing antibody (69.8%) was found in all individuals. There was significant difference observed for prevalence by gender (P = 0.042), age range (P = 0.011) and geographic origin (P < 0.001). The percentage of positive AAV1 neutralizing antibodies (NT50 > 10) in teenagers (year <18, as of 2012) was significant lower than that of adults (19-56, as of 2012) (P = 0.011), indicating the optimal vaccination period of childhood. The current study provides a useful insight for the future development of AAV1-based vaccination and gene therapy strategies in Beijing and Anhui provinces of China.

Mycobacterium smegmatis in skin biopsy specimens from patients with suppurative granulomatous inflammation.

Formalin-fixed, paraffin-embedded skin biopsy specimens, including 72 suppurative granulomatous inflammation (SGI) and 47 non-SGI controls, were tested for mycobacteria by using a broad-range PCR and a suspension array identification system. Mycobacterium smegmatis was detected in 13 (18.1%) of the SGI skin biopsy specimens, which was significantly more than 2 (4.3%) in the controls (odds ratio, 5.73; 95% confidence interval, 1.21 to 27.06; P = 0.028).

Detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus PCR coupled with electrospray ionization/mass spectrometry.

As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.

Comparison on virulence and immunogenicity of two recombinant vaccinia vaccines, Tian Tan and Guang9 strains, expressing the HIV-1 envelope gene.

BACKGROUND: The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. METHODOLOGY/PRINCIPAL FINDINGS: To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. CONCLUSIONS/SIGNIFICANCE: Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.

Comparison of two high-throughput assays for quantification of adenovirus type 5 neutralizing antibodies in a population of donors in China.

BACKGROUND: The presence of various levels of Adenovirus serotype 5 neutralizing antibodies (Ad5NAb) is thought to contribute to the inconsistent clinical results obtained from vaccination and gene therapy studies. Currently, two platforms based on high-throughput technology are available for Ad5NAb quantification, chemiluminescence- and fluorescence-based assays. The aim of this study was to compare the results of two assays in the seroepidemiology of Ad5NAb in a local population of donors. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based neutralizing antibody detection test (FRNT) using recombinant Ad5-EGFP virus and the chemiluminescence-based neutralizing antibody test (CLNT) using Ad5-Fluc were developed and standardized for detecting the presence of Ad5NAb in serum samples from the population of donors in Beijing and Anhui provinces, China. First, the overall percentage of people positive for Ad5NAb performed by CLNT was higher than that obtained by FRNT (85.4 vs 69.9%, p<0.001). There was an 84.5% concordance between the two assays for the 206 samples tested (144 positive in both assays and 30 negative in both assays). All 32 discordant sera were CLNT-positive/FRNT-negative and were confirmed positive by western blot. Secondly, for all 144 sera positive by both assays, the two assays showed high correlation (r = 0.94, p<0.001) and close agreement (mean difference: 0.395 log(10), 95% CI: -0.054 log(10) to 0.845 log(10)). Finally, it was found by both assays that there was no significant difference observed for titer or prevalence by gender (p = 0.503 vs 0.818, for two assays); however, age range (p = 0.049 vs 0.010) and geographic origin (p = 0.007 vs 0.011) were correlated with Ad5NAb prevalence in northern regions of China. CONCLUSION: The CLNT assay was relatively more simple and had higher sensitivity than the FRNT assay for determining Ad5NAb titers. It is strongly suggested that the CLNT assay be used for future epidemiological studies of Ad5NAb in other localities.