Using Multiomics to Study the Immune Response to SARS-CoV-2

Multiomics approaches combining protein and gene expression analyses are used in various applications, including analyes of human immune response to the SARS-CoV-2 virus. In this interview, Dr. Barbara Molon and Prof. Antonella Viola’s team at the Instituto di Ricerca Pediatrica Città della Speranza (IRP) in Padova, Italy, describe how they use a single cell multiomics approach to study the SARS-CoV-2 immune response. Installation of the BD Rhapsody^™ Express Single-Cell Analysis System and remote training of the team happened during the height of the COVID-19 lockdown. Dr. Molon and the team members talked to us about their work, their expectations from the multiomics technology and its future in the field of immunology.

What impact has the COVID-19 pandemic had on your research?

Barbara Molon: We are an immunology lab and the study of the immune system in both physiological and pathological contexts is our main focus. From the very beginning, we have been interested not only in basic biology but also in a translational approach in our research. In particular, we aim at investigating leading mechanisms underpinning pathological situations and use the knowledge gained to develop new therapeutic strategies to finally improve clinical outcomes for patients. In this way, we have been involved in studying multiple pathologies. For many years, I have been studying cancer and inflammation and would say that inflammation is one major topic of our research. The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has dramatically affected all of us and also our research.

From the very beginning, it was clear that the host immune response to the virus makes a difference in the clinical outcomes of patients. As you know, there are several studies currently investigating the role of the immune system in the control of the spreading of the SARS-CoV-2 virus, which also represents a challenging opportunity to investigate and understand host-pathogen interactions in infections. So, we jumped into this project.

Why are you using multiomics to study the SARS-CoV-2 immune response?

Barbara Molon: Of course, we moved to use the single-cell approach as cell heterogeneity represents a hallmark of both physiological and pathological conditions, and importantly,  biologists and clinicians believe that this cell heterogeneity can have important therapeutic implications. As the use of single-cell analysis represents an important requirement to study disease pathogenesis, we got in touch with BD specialists and asked about the combination of classical single-cell transcriptomics with the multiomics approach. This was very interesting for us as we believed that the use of oligo-conjugated antibodies with the transcriptional profile could dramatically improve the resolution and the clustering of immune cell populations. Besides, this approach could also provide information about what happens after translation. We believed that this kind of approach could give us more information about the disease and improve general knowledge about the cells in a reliable way.

How are you studying the SARS-CoV-2 immune response?

Barbara Molon: We are running this project in collaboration with the Infectious Diseases Unit of the University of Padova Hospital and the Human Technopole in Milan. The idea was to create a kind of a task force to study patients infected by the SARS-CoV-2 virus, at different time points, both during and after the infection, using the multiomics approach. In general, we wanted to have a sort of a dynamic picture of the immune response in a patient over a period to better characterize the immune response to the virus, to elucidate SARS-CoV-2 pathogenesis, and to identify new targets that can help in therapy.

How did your BD Rhapsody™ System installation go?

Fabio Munari: The installation of the BD Rhapsody^™ Express Single-Cell Analysis System occurred during the lockdown in Italy. We were here in the lab since we were working with COVID-19 samples. A few days after installation, we had a very complete and precise remote training by BD, we were the first in Europe! Although the BD Field Application Specialist, Serge Scherrer, and the Technical Sales Specialist, Werner Rodriguez, were always available for any questions and technical problems, we were able to manage very well the BD Rhapsody^™ workflow by our own, right after the training. Then we started working by ourselves and were able to generate more than 100 libraries in a couple of weeks. It was tough, but we made it. It was very helpful to have two BD Rhapsody^™ Express Single-Cell Analysis Systems since we could run two cartridges at once. This improved a lot our workflow. Of course, in the beginning, it was very difficult for us since our experience is mainly in cell biology and flow cytometry and not genomics, but we soon improved our skills, mainly due to the support we constantly received from BD. Just to give you an example, when we began this project, even if we had clear in mind what we wanted to do, we had no idea about the type and the amount of BD panel kit to order, together with all the reagents and instrument necessary for library preparation. We were running against the clock, but the support from BD was crucial to start the project as soon as the samples arrived. For me, it was a good experience because it was a time of great teamwork and collaboration and we have learned a lot in a very short time.

Roberta Angioni: We are very satisfied with the BD technical support. We started only with an idea of the type of analysis to perform on COVID-19 patient samples. With the support of  BD, we were able to design the experiments, perform the single-cell multiomics approach and collect good data.

Ricardo Sanchez Rodriguez: As my colleagues said the training was an entirely different experience due to the lockdown but thanks to the professionalism of BD specialists, we were able to generate the libraries in a short time.

Do you think multiomics will be used more and more with flow cytometry?

Barbara Molon: I think that having the combination of a cell sorter with a BD Rhapsody™ System would be effective from a technical point of view. To enrich cell populations that are very rare and to study them directly ex vivo from patients for example, without any manipulation, would be the ideal way to study patient specimens.

I think that, especially for personalized medicine research, it would be very important to improve this kind of approach and to make it less expensive. It is necessary to improve the technical process and reduce costs. This is my idea from a translational point of view.

On the other hand, we are also basic immunologists, and technologies like this will allow us to understand mechanisms that have not been discovered yet. In my view, it is one of the best approaches for immunology.

Fabio Munari: I think that right now, the main bottleneck for this technique is the cost of sequencing. It is a very good approach but is still quite unaffordable for many labs.

Roberta Angioni: Single-cell multiomics approaches should be used alongside flow cytometry. In my opinion, for too long scientists have focused their research on specific cell types, often underestimating what surrounded them. With this kind of analysis, we can take into account the modifications that concomitantly occur in different cellular subsets and at different levels, including transcriptional and proteomic differences.

Is the return on investment good?

Fabio Munari: Yes, of course. We still must sequence the library and do all the analysis, but we are confident that it will be worth the money.

Barbara Molon: For the moment it is just hope, but maybe in the future, we could get something out of it.

Ricardo Sanchez Rodriguez: Absolutely. All our quality controls showed high reproducibility to generate different libraries for whole transcriptome, immune panel and VDJ.

How complementary is multiomics with flow cytometry? 

Barbara Molon: The two approaches are complementary, but we don’t yet have the data from the multiomics approach. You can use a panel of up to 400 antibodies for the multiomics approach and we have used only about 52. We can improve the numbers and get a lot of information from transcriptomics.

Roberta Angioni:  In my opinion,  there are advantages in using the multiomics approach together with flow cytometry. Indeed, with the flow cytometry, we can have a good general picture of our sample, acquiring millions of events in a very short time. This could be a cost-effective and winning starting point to identify a cellular subset of interest. This could be studied deeper with the multiomics approach later. Conversely, the BD Rhapsody™ System could be exploited to have an untargeted analysis of the sample, thus avoiding the restricted vision on a single cellular compartment. In this way, flow cytometry could help with the validation of the obtained results. Thus, they could be used with several pipelines and with different objectives. However, they are perfectly complementary and I think it could be a successful strategy for high-impact discoveries.

What is the future of immunology?

Antonella Viola: The study of the recognition and elimination of pathogens by our immune system will always be strategic, even in the years to come. On the other hand, discoveries tell us more and more that a fundamental role of the immune system is the maintenance of homeostasis. The constant interaction of the immune system with microbes to not to be attacked, but rather to be exploited for specific functions useful to our body, is based on mechanisms that are only partially understood. The cross-talk between microbes and the immune system, how the two worlds talk and shape each other, and the consequences of this dialogue or its interruption on our health are fascinating topics to explore. However, besides basic and general events, there is a crucial need to understand the mechanisms underlying the individual variability in the immune response. This is essential not only when facing a pandemic but also when treating patients with immune-related disorders, such as autoimmune diseases, or in cancer immunotherapy. The identification of individual responses and molecular signatures is crucial for the effective development of therapeutic approaches designed for the patient.  In this respect, I think that the multiomics approach in the research phase will be tremendously useful.

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