Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. A total of ~350 mL of blood, 2 lymph node samples and 12 microbiome samples were obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches – NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design.
Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.
Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.
The efficacy of two strains of Lactobacillus probiotics (Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) immobilized in microcapsules composed of pea protein isolate (PPI) and alginate microcapsules was assessed using a mouse model of Citrobacter rodentium-induced colitis. Accordingly, 4-week-old mice were fed diets supplemented with freeze-dried probiotics (group P), probiotic-containing microcapsules (group PE) (lyophilized PPI–alginate microcapsules containing probiotics), or PPI–alginate microcapsules containing no probiotics (group E). Half of the mice (controls, groups P, PE, and E) received C. rodentium by gavage 2 weeks after initiation of feeding. Daily monitoring of disease symptoms (abnormal behavior, diarrhea, etc.) and body weights was undertaken. Histopathological changes in colonic and cecal tissues, cytokine expression levels, and pathogen and probiotic densities in feces were examined, and the microbial communities of the distal colon mucosa were characterized by 16S rRNA sequencing. Infection with C. rodentium led to marked progression of infectious colitis, as revealed by symptomatic and histopathological data, changes in cytokine expression, and alteration of composition of mucosal communities. Probiotics led to changes in most of the disease markers but did not have a significant impact on cytokine profiles in infected animals. On the basis of cytokine expression analyses and histopathological data, it was evident that encapsulation materials (pea protein and calcium alginate) contributed to inflammation and worsened a set of symptoms in the cecum. These results suggest that even though food ingredients may be generally recognized as safe, they may in fact contribute to the development of an inflammatory response in certain animal disease models.
Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.
While meeting the pandemic demand of SARS-CoV-2 testing, clinical laboratories worldwide tend to adopt new test systems offering cost-effective and faster test outcomes. However, the reliability of SARS-CoV-2 test results has paramount importance in the management of such a health crisis. Therefore, this study sought to determine the accuracy of the test results from a novel duplex Microchip RT-PCR test system using patient saliva samples and nasal swabs stabilized in Viral Transport Medium (VTM) with reference threshold Cycle Values (Ct). The VTM used to stabilize these samples during transport was found to be inhibitory to the RT-PCR. Therefore, all the samples were subjected to spin column purification of total RNA to remove the influence of VTM. A total of 70 patient samples, including 24 positive- and 31 negative-saliva in VTM samples and 15 positive nasal swab samples, were tested. Results obtained from both the sample types were compared to their reference values and no false positive or false negatives were observed. From this data, accuracy, specificity, and sensitivity were determined to be 100% applying the corresponding formulae. The limit of detection with 95% confidence probability was determined to be 2.5 copies/μl in the original sample.
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