Enhancing Pandemic Laboratory Preparedness through Rapid Biopsy Sample Handling

PI(s)/Head responsible for the resource:

Annika Karlsson

Host organisation(s):

Department of Laboratory Medicine, Karolinska Institutet

Resource description:

“In this proposal we aim to extend our ongoing RAPID-SEQ project on pandemic laboratory preparedness for SARS-CoV-2 and metagenomics to include research and surveillance on enteroviruses, which have the capacity to cause large worldwide outbreaks. Historically, poliomyelitis is the most well-known and feared enterovirus disease, but many other disease outbreaks have been caused enteroviruses. This includes recent reports on neonatal cases with fulminant and fatal hepatitis due to echovirus 11, polio-like acute flaccid myelitis due to enterovirus D68 (EV-D68), myocarditis due to coxsackie B3 and B4 and neonatal sepsis and meningoencephalitis. Despite the serious consequences and threats from enterovirus infections, Sweden lacks a comprehensive program for surveillance of enteroviruses, apart from the WHO mandated surveillance for poliovirus.

This proposal aims to build capacity and preparedness for comprehensive laboratory surveillance of enteroviruses by:

1. Implementing NGS-based methods for partial and whole genome sequencing (WGS) of enteroviruses and rhinoviruses.
2. Performing enterovirus genotyping on respiratory specimens.
3. Analysing (i) genotypes from patients sampled at an intensive care unit vs other unit, (ii) circulating genotypes over time, and (iii) genotypes across age groups.

Our laboratory is excellently suited to take on the above tasks as being the largest clinical microbiology laboratory in Sweden with continuous access to large numbers of patient samples and results from routine diagnostics. In addition, we are the national reference laboratory for non-polio enteroviruses and a partner in the European non-polio enterovirus network (ENPEN). Within ENPEN we have contributed to several studies on EV-D68. In this proposal we will extend this work to other species and types of enteroviruses and rhinoviruses. We will build on the capacity in our current PLP project (RAPID-SEQ) by adapting the workflow for SARS-CoV-2 to another pathogen.”

Research findings:

To maintain the readiness for infectious diseases ANA Futura BSL3 Core Facility, has expanded current projects on SARS-CoV-2, Flavivirus (TBEV, Dengue, Yellow Fever, Japanese Encephalitis),HIV, hantavirus, Hep B, Hep B/D, and Hep C to include Rift Valley fever phlebovirus, Monkeypox virus (MPXV), and the following bacterial pathogens: Mycobacterium tuberculosis (including GMM),Burkholderia pseudomallei/Melioidos Burkholderia mallei, Brucella species, Francisella tularensis, Yersinia pestis. Protocols, SOPs, have been developed to handle propagation of the pathogens and handling of various clinical samples including blood, sputum, wound secretions, urine and tissue samples (biopsies) from the lung, liver, lymph nodes, bone marrow, and brain in accordance with safety regulations and risk assessment. Through the process we have refined logistics for sample preparation, inactivation, and transportation. Through our work we enable high-end research on patient´s immune responses but also promote research on sequencing and genotyping of isolated pathogens, antimicrobial resistance, diagnostic and prognostic biomarkers as well as drug discovery (REF).

Sorting of single cells from patient infected by a highly pathogenetic virus. SOPs for single cell sorting from HIV-infected cell samples obtained from children living with HIV (CLWH) and uninfected control children within the BSL3 lab has been validated. The sorting and 10X genomics were optimized using identical equipment in the BSL2 environment at CIM, MedH. Risk assessment was conducted before sorting of the cells in the BSL3. Single-cell RNA (scRNA) and TCR sequencing was performed on bulk and CMV-specific T cells. After sorting of T cells, cell viability and cell count was evaluated, and samples were pooled. GEM generation was conducted using 10X Genomics. Through this process, HIV is inactivated, and the cDNA generated can be transported to the BSL2 lab for downstream analysis. The library preparations were subjected to extensive round of QC on the Bioanalyzer. Sequencing was conducted by NGI, SciLifeLab. Using antibody barcodes, cell populations were mapped using PCA and UMAP analysis. Transcriptome data revealed reduced TCR diversity in the CMV-specific T cells from CLWH. The results are included in a manuscript entitled “Altered antiviral T cell immunity against common viruses in pediatric HIV” by Anna Olofsson, et.al. Usage of organoid models for the study of cell-pathogen interactions. Similarly, SOPs for handling of brain organoids has been generated in collaboration with Dr Neogi, with whom Dr Karlsson has a joint EU grant (see excel). This approach will be available to researchers in the future and be expanded to the use of organoid models for the evaluation of virus-cell/tissue interactions.

PhenoPCM proof of concept study on material collection and compatibility for multi-omics. The PhenoPCM project demonstrated the feasibility of collecting biopsy samples and preparing them for multi-omics analysis. By testing different sample handling methods—including fresh freezing, embedding for tissue sections, and standard formalin fixed paraffin embedded (FFPE) tissue preservation—we confirmed that each approach provided suitable material for comprehensive protein, DNA, and RNA analysis. The study involved multiple biopsy types collected from routine clinical procedures, highlighting practical integration with healthcare settings and showing promising results in terms of quality and quantity of extracted biomolecules. This proof-of-concept establishes a reliable framework to quickly and effectively collect and analyze clinical biopsy samples. Such capability is essential for rapid molecular investigation, significantly enhancing preparedness for future health crises and supporting timely diagnostics and treatments. Manuscript is under preparation describing part. Based on these learnings, we have now initiated a process to replicate these workflows and test proteome analysis in Lund and Gothenburg connecting respective university hospitals and SciLifeLab local platforms.

Rapid protein level analysis development To assess how the new clinical sample collection workflows and material can be used for rapid development of new tests and assays, we have selected an use-case to analyse immune-phenotypes of tumor tissue. This is relevant area in infection biology but applicable also on cancer immunotherapy field to improve precision medicine; hence the capability can be maintained via precision medicine development between pandemics. Immune checkpoint inhibitors (ICIs) have substantially improved outcomes for cancer patients, including some non-small-cell lung cancer (NSCLC) patients. However, the currently used assays for treatment selection based on PD-L1 expression or tumor mutational burden have low predictive accuracy. Proteomics has been shown to offer phenotypic insights into tumors immune phenotype not explained by genomics or immunohistochemistry; but no clinical proteomics tool has reached the clinic, yet. In this proof-of-concept application for rapid assay development, we develop PIONeer (Proteomics-based Immuno-Oncology Response Panel) assay for clinically relevant patient stratification using mass spectrometry (MS)-based targeted proteomics. In this study, we focused on characterizing tumor immune infiltration and general histological and sample quality features, covering a total of 98 protein markers. We showed the relevance of the designed panel for predicting response to immune checkpoint inhibitors using a publicly available dataset. We then designed a parallel reaction monitoring (PRM) method measuring 187 peptides. In a cohort of resected lung tumor samples, we showed highly accurate quantification of 129 peptides from 83 proteins using spiked-in heavy isotope-labeled internal standards. We further validated the method’s performance inan independent cohort of biopsy samples. Manuscript is under preparation describing this research part.

In summary, our project has enabled the establishment of a comprehensive and secure process for handling various clinical samples from subjects with highly pathogenic infectious disease and analysing immune responses and pathogenic interactions (1-6). Through a multidisciplinary approach and collaboration with leading experts and institutions, we are poised to make significant strides in the field of molecular, microbiological, and immunological biosciences (7-10). Knowledge generated through the SARS-CoV-2 pandemic imply that early pathogenic event may cause long-standing impact on health, e.g., post-acute sequelae of COVID-19 (11, 12). To grasp some of the underlying mechanisms early access to biopsied from different anatomical locations do enhance our ability to conduct molecular and immunological biosciences analysis. The work has created the framework, and the long-term aim is to create a biobank of tissue samples and clinical microbial isolates originating from different sanctuary sites creating a unique resource for research, testing vaccines and drugs. Moreover, we have connected the developed hospital sample flow for advanced research and precision medicine sampling with SciLifeLab platforms to demonstrate rapid assay development for immune phenotyping of tissues. Altogether, this project has so far connected hospital sampling to SciLifeLab analysis capabilities and formed a template to speed up such process not only at Karolinska Univ. Hospital but also elsewhere.

Impact on prepardness for future pandemics:

One promising avenue to bridge this knowledge gap regarding host immune defence mechanisms against infectious agents is through the implementation of rapid sample handling using established SOPs for a variety of highly pathogenetic microorganisms. Our framework has potential to be a powerful tool to supply researchers with valuable tissue specimens and streamlined and safe propagation of the pathogens. The central principle guiding these programs is the swift processing of samples, enabling accurate downstream applications, including direct ex vivo immune cell analysis and viral or bacterial propagation. Additionally, the developed SOPs ensure the proper inactivation and secure transfer of biological samples, such as fixed cells, RNA, DNA, and proteins, to other research infrastructures for further analysis. As a testbed for this development, we have used the BSL3 laboratory at Huddinge connected with infections disease clinical and immunology research centre. This can be a dedicated competence centre in future pandemics.

The Solna sample central has become a laboratory with expertise for advanced sample preparation, improving synergy among routine, research and pandemic preparedness needs. The lab is well connected to the hospital and during the project period the accessibility to SciLifeLab infrastructure for faster processes for specific advanced analysis have increased, and thus strengthening the link between research, infrastructure and clinical routines. The lab has been aligned with good data practices to integrate different data types into a clinical decision support system, molecular tumour board portal. This is an important aspect to transfer knowledge back to healthcare if experimental information needs to be incorporated to clinical decision-making during health crisis. We foresee that our project with all our formed national collaborations, our European connections, and our proof-of-concept project, have and will continue to improve Sweden’s possibilities to join and capitalize on preparedness and rapidly respond to viral threats, by providing a sample and data handling centre to bridge the gap between healthcare and research. Moreover, the continues use of this system for precision medicine development provides sample and data generation for monitoring emerging health threats as well as maintains the operative system intact between pandemics.

Contact information:

Annika Karlsson
Researcher
Email: annika.karlsson@ki.se