Dear prof Racaniello and colleagues,
Since some time I’ve been listening to your immunology podcast (loved the episodes on checkpoint immunotherapy and CAR T) and since the Covid-19 crisis started I’ve been listening regularly to This week in virology as well. Have recommended the podcasts to many friends and colleagues.
Since some time I’ve heard about people who suffer a long course of Covid-19 disease with chronic fatigue syndrome like complaints. A colleague of mine manifests these symptoms (which really sucks) and a friend who is an occupational physician has told me about many patients with similar complaints as well. Recently Ed Yong, a great journalist writing for The Atlantic wrote about this phenomenon (https://www.theatlantic.com/health/archive/2020/06/covid-19-coronavirus-longterm-symptoms-months/612679/). Ed Yong compares the symptoms of this chronic Covid-19 course to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).
A quote from the article:
“It’s not clear why this happens. Akiko Iwasaki, an immunologist at Yale, offers three possibilities. Long-haulers might still harbor infectious virus in some reservoir organ, which is missed by tests that use nasal swabs. Or persistent fragments of viral genes, though not infectious, may still be triggering a violent immune overreaction, as if “you’re reacting to a ghost of a virus,” Iwasaki says. More likely, the virus is gone but the immune system, having been provoked by it, is stuck in a lingering overactive state.”
I was wondering about your thoughts on this.
Gerben Breimer, pathologist (in training) from the Netherlands
Greetings from Moscow!
I am a long time fan of both IMMUNE and TWIV, and it is very useful for me as a researcher (thanks a lot!).
On Immune 32 (june 5) you joked about infecting people with SARS-CoV-2 to study cross-reactive pre-existing memory activation. I think we found a way to do that without infecting anyone.
As you know, T cell receptor (TCR) diversity is extremely high, and because of this you could use TCR sequence as a barcode to quantitatively track individual T cell clones in time. Idea is to collect samples longitudinally, sequence TCR repertoire on each time point and look for TCR sequences which increase (and later decrease) their frequencies after the infection (or vaccination: works great with a live yellow fever vaccine!).
Two donors we sampled and sequenced TCR repertoires last year for a different project got mild COVID-19 this March. So we were able to sample them again after infection and use TCR repertoire sequencing to identify T cell clonal expansions and track them back to the pre-infection (and pre-pandemic) T cell repertoire. Surprisingly, a bunch of COVID-19 reactive CD4+ clones (but few CD8+) were found in central memory subpopulation from one year before, suggesting cross-reactive memory indeed participates in immune response to SARS-CoV-2. Here is our preprint (First author is Anastasia Minervina, also a big fan of yours!) describing this results:
Thank you again for your hard work!
Mikhail Pogorelyy, PhD
Department of genomics of adaptive immunity,
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Thank you for all these microbe.tv podcasts! I discovered Immune back in January when I was trying to find podcasts or lectures about mucosal immunity. From Immune, I discovered TWIV and then the whole world of microbe.tv and got distracted from my initial interest in mucosal immunity by all your coverage of COVID-19. Now I listen nearly all the time and usually several times to each episode so I can absorb as much as possible. But now I’ve come full circle and am back to mucosal immunity. Vincent and others on TWIX podcasts have noted that SARS-CoV-2 seems to replicate in the gut but that there is no pathway for the infection to get from the gut to the lungs or nasal cavity. Fine. But is there a way for that gut infection to educate the mucosa in the lung and nasal cavity such that a stomach infection by SARS-CoV-2 would confer at least some immunity against infection in the lung or nasal cavity? Does IGA/Tregs/etc. travel among the various mucosal sites or are they trapped in the gut mucosa? If a child contracts the gut version (through fecal-oral transmission) and then later contracts the respiratory version would they have any additional immunity? Also, can you recommend any Mucosal Immunity 101 type podcasts or lectures? (I’ve listened to Brianne’s lectures (which are great!) and another really good one from Albert Einstein School of Medicine, but I think I have only scratched the surface.) Thank you so much for these podcasts. I can’t overstate how much I enjoy them.
Dear Immunites/Immunos/Immunes and TWiVites (How should we refer to ourselves?)
–this is a long email, so for the sake of taking less time you can skip down to “Peptide MegaPools are Risky Business”
My name is Jesus Contreras Rodriguez, I am a rising 3rd year PhD candidate at the Johns Hopkins Graduate Program in Immunology under the mentorship of Dr. Nilabh Shastri. My lab studies mechanisms of immune surveillance (how the immune system can distinguish friend from foe, and cells with altered antigen processing), specifically, I study the generation of peptides derived from non-AUG reading frames destined for MHC class I presentation.
I have been an avid TWiV and Immune listener ever since I joined the Shastri Lab two years ago when we were still housed in UC Berkeley, and thanks to TWiV and Immune I’ve learned of many papers that have shaped my PhD project and future research interests, and I’ve grown to love Virology and systems immunology. Your podcast series was the only thing keeping my brain from turning into mush during the lockdown, and currently, they are much-needed motivation as I return to the lab and contemplate viral models for the study of nonAUG encoded MHC-I epitopes (JHops re-opened research labs 6/17).
On a side note, I want to perform a plaque assay in some way shape or form before I finish my doctoral studies! Vincent, how can I do this?! <==come to NYC when we can travel again! (vr)
Recently, I presented the Grifoni 2020 study discussed in Immune-32 and TWiV-620 to my lab and we both liked and cringed at this study. The purpose of this email is to raise a question about the methodology of this study and others like it, as well as share with the rest of the Immune and TWIV fan-base the Shastri-lens for reading papers relating to immunopeptidomics.
Immunopeptidomics has become a promising academic marriage between immunology and bioinformatics that has great potential for unlocking the peptidome presented by MHCI and MHCII, with recent advancements in epitope prediction and mass spectrometry paving the way for increasingly more sensitive peptide identification. Below, I hope to explain my reasoning for critiquing this study.
Peptide MegaPools are Risky Business
While this method of identifying the source of immunogenic peptides is high throughput, the generation of these peptide pools (and of all the peptides in this study) has many caveats. The main problem is that each individual peptide in the pool was not HPLC purified following synthesis (they used crude peptides), meaning that a plethora of reaction intermediates (think truncated, heterogenous, and side-chain reaction peptides/aggregates) are present in each individual peptide tube the authors used and eventually pooled. Crude peptide synthesis reactions can range from 50-70% target peptide and contain organic and inorganic salts from the synthesis reaction that can wreak havoc to cells in unthinkable ways, and affect peptide binding.
(Thinking in terms of how these peptides are synthesized) Even if the deprotection or addition of one amino acid to the growing peptide chain is 80-99% efficient, that leaves behind 1-19% of unreacted amino ends (protected or otherwise) that can then serve as substrates for the following amino-acid addition step (generally in traditional solid-phase peptide synthesis where synthesis occurs C->N). After the generation of a 9-mer or 10-mer peptide (say for the CD8 T cell studies in Grifoni 2020) the final crude peptide reaction will contain anywhere from ~40-20% (or even more!) peptides by mass that are not your target peptide. Back-of-the-envelope/rough calculations will place the total concentration of these non-target peptides (upon resuspension) to be ~100-10microM with individual off-target peptides ranging from 100-10nM. Now, take into account that a homogeneous population of T-cells (say, a reporter T-cell hybridoma or OT-I CD8’s) can respond to their cognate peptide-MHC when the [peptide] is as low as 1.0femtoM (T-cells are extremely sensitive!) and in some instances into the sub-femtoM range. In pooling crude peptides, the relative concentration of off-target products will be higher than that of one individual desired peptide.
Also, peptides can compete with each other for binding to surface MHC, since exogenous loading of peptides (peptide pulsing as was done in Grifoni 2020) to MHC relies on the affinity of a peptide for a given MHC molecule binding groove and displacement of a previously-bound peptide. Truncated Variants of a peptide can still bind to MHC and, if anything, compete for access to the binding pockets. The variability in T-cell responses presented by Grifoni et al 2020, while representing a trend, could be due to these off-target events especially since the authors do not indicate the severity of CoVID-19 suffered by each infected individual data-point (patient/donor) in the study.
Taking the above into account, I view the figures relating to CD8+ Tcell responses with a Tonka-Truck full of salt, let alone the CD4+ Tcell figures where the peptides are longer and therefore introduce much more variation on the off-target reaction products. An alternative would be to elute MHC class I peptides from cells that are infected with SARS-CoV-2 and then running the cleaned-up eluate and identifying the SARS-CoV-2 peptides by MS (no easy job by any means, requires a lot of sample by mass, and one will need patients with the same HLA alleles to remove variability in peptide sequence due to HLA-binding requirements).
Don’t get me wrong, I enjoyed and appreciate the Grifoni 2020 paper and the overall trends in antibody and pre-existing immunity it discovered. Generating an in silico list of and synthesizing peptides is no joke, and acquiring pure peptides (<99% pure) is an expensive and time-consuming process. The Sette and Crotty labs have contributed priceless tools for the identification of peptides and I would love nothing less than to shake the hands of each author and PI in the study.
However, as immunologists interested in identifying viable targeted vaccine targets for humoral and cell-mediated immunity against SARS-CoV-2 we must be wary of prioritizing expediency for the sake of information over the careful and deliberate approach that has advanced our respective fields in the past decades.
I appreciate every single podcast your group has published, and I look forward to the possibility of hearing my question in a future TWiV/Immune episode. A big shout-out to my life-long friend Teresa Lupone for introducing me to this podcast and helping nurture my love for research, Rafael Rivera and Valerie Zapata at the MCB grad program UC Berkeley, and the Shastri Lab and Immunology community at Johns Hopkins as well as all the future immunologists this pandemic has made/enlisted.
Vincent, please always keep it grumpy. Grumpy means you care.
Jesus Contreras Rodriguez
Ph.D. Candidate, Graduate Program in Immunology
Johns Hopkins School of Medicine
Good Morning Twiv Gang,
It is a comfortable temperature this morning in Boise Idaho. I do not check the actual temperature when I first get up in the morning but simply step out and decide whether I need to wear a jacket or not when I ride my bike into work. I tried to listen to twiv 301 on viruses in the ocean, but my iPhone gave out 5 minutes into the podcast. So I switched over to #544 since I already had that one downloaded. I had a question on immunodominance. Excuse me this is a new idea for me and my question will probably be somewhat imprecise. I understand that there is no dominant epitope in influenza viruses. Is there an immunodominant epitope in the Hepatitis A virus. I assume that for Hepatitis B the immunodominant epitope is the surface antigen. Please correct me if I am wrong. The reason I asked this question is that I heard on a YouTube lecture that any antibodies to hepatitis A virus are protective. I just want to make sure I understand this idea.
My next question is on cytokine storms. I have enclosed this paper on “cytokine storms” and wanted to get your viewpoint. I must admit that if you were to ask me what is a “cytokine storm” I would be at a loss for words.
Dr. Barker I have appreciated your lectures on the immune system. I used them last year to teach myself immunology. And I did use the Kuby to supplement the lectures. The innate immune system is super interesting. Studying your lectures put me a much better position to read and understand the flood of information on SARS-Co-2. I have been following both the virology and immune podcasts religiously. It is the only way I can hope to keep up or try to keep up with the flood of information. Now when I have time I try to backtrack and listen to older ones. It has helped me focus on learning. Under the current circumstances I cannot take a leisurely approach to my education.
I recently volunteered to be a PI for a serological study. Finally after many years of practicing medicine I get a chance to do some research. This has been a goal of mine for over 20 years, but due to life events it never materialized. So I grabbed this opportunity when it appeared. Some day I hope to be able to do laboratory work. Perhaps after this is finished. Thanks again for tickling my scientific curiosity.
He sent “Is a “Cytokine Storm” Relevant to COVID-19?” from JAMA
Hi Immune Crew,
I’m taking an anatomy and physiology class and this question came up when we were discussing blood typing and transfusions. It makes sense that someone who is Rh- will only produce anti-Rh antibodies if they are exposed to the antigen, but it makes less sense to me that someone with type O blood will produce anti-A and anti-B antibodies without being exposed to either antigen. The sources I’ve looked at don’t say how this happens, only that it does. How do these antibodies develop? Is there another source of these antigens besides erythrocytes? If there is, wouldn’t it have to be a transient source or risk constant agglutination?
Thanks for taking the time to make this podcast, the TWi-verse has been a great source of information over the last few months.
Dear team Immune,
Thank you for all of your work to help your listeners understand the immunology and virology of SARS-CoV-2. I discovered your podcast in early April and am greatly appreciative of your insights on this virus.
My question is on the detectable immune response. I’ve been exposed to Covid-19 repeatedly – I was in northern Italy at the end of February and in NYC the first two weeks of March, during which I had dinner with one friend who developed a fever 2 days later, and kissed someone who developed a fever 4 days later – both of whom later tested positive for Covid antibodies. I then flew to California and 10 days later my roommate developed Covid symptoms and later tested positive for antibodies. During the week she was sick with a 101 fever and bad cough, we still ate dinner together, I was around her while she was coughing, and we shared the same space. I never developed symptoms and, despite my friends accusing me of being patient 0, have twice tested negative for antibodies. What is your hypothesis on why that is? Could my innate immune system have fought off any virus I was exposed to? If I mounted an adaptive immune response could my neutralizing antibody levels not be over the threshold for a positive serology test? Would you speculate that I may have helper T cells that may recognize the virus? I acknowledge that without data it’s impossible to know, but I’d still be interested in your speculations. And, if you know of any researchers in the SF Bay Area looking for Covid study participants that fit my description I’d be happy to volunteer.
Thanks so much,
Hi Immune Team,
I saw this paper on UCSF Grand rounds last week on impaired type one interferon activity. The presenter spent 10 seconds on the paper!! This was a bit of an annoyance. I had several questions and thought it might be a good discussion for everyone. The paper on page 2 says that plasmacytoid dendritic cells are the main source of IFN-alpha. I think my problem is I do not understand how dendritic cells are classified. The paper appears to suggest that alpha and beta type I interferons are affected differently by SARS-CoV-2. I am perplexed. What are plasmacytoid dendritic cells good for? Kuby 8th edition page 95 says that plasmacytoid dendritic cells secrete type III interferons.
I have enclosed a link to a second paper, Longitudinal analyses reveal immunological misfiring in severe covid-19. https://www.medrxiv.org/content/10.1101/2020.06.23.20138289v3 I sent it along in case the first was not interesting enough. This paper was much harder for me to understand. I am still working my way through Dr. Racaniellos virology and Dr. Barker’s great immunology videos. They are pushing me into worlds not yet explored.
All of you rock!! You are helping me to expand my reaches. Heck I even started listening to the parasitology podcasts. They are a nice break from covid-19.
Thanks for all that you do to educate the public.