I’ve often won­dered about the Chi­nese for­eign min­is­ter, Wan Yi, declar­ing back in Decem­ber of 2019 that ‘the Unit­ed States is the trou­ble mak­er of the world.” At the time it was thought he was refer­ring to the ‘black hands’ he saw at work in Hong Kong and the right­eous com­ments about Xin­jiang province. I sus­pect that he was refer­ring to the deep state activ­i­ty which was only just being felt inside Chi­na at that time.

It was a night­mare sce­nario that shut­down the world. And yet, as we’ve seen as the COVID pan­dem­ic has played out, not all night­mare sce­nar­ios are equal. Some can be far more night­mar­ish than oth­ers. And that brings us to the fol­low­ing pair of arti­cles about the long-term night­mare impli­ca­tions of the SARS-CoV­‑2 virus that we have yet to real­ly under­stand. There’s the “known unknown” long-term impli­ca­tions of the impact that “long COVID” will ulti­mate­ly have on afflict­ed patients. But as the first arti­cle — a Nature Cell Biol­o­gy com­mu­ni­ca­tion — reminds us, we’re steadi­ly learn­ing more about those known unknowns of long COVID and the more we’re learn­ing the more dis­turb­ing it looks.

Specif­i­cal­ly, it appears that DNA dam­age is one of the long-term impacts of a SARS-CoV­‑2 infec­tion. As researchers dis­cov­ered, the SARS-CoV­‑2 virus appears to dis­rupt the DNA dam­age response (DDR) cel­lu­lar path­ways in mul­ti­ple ways, lead­ing to cel­lu­lar senes­cence. It’s a night­mare. Senes­cent cells are basi­cal­ly aged dys­func­tion­al cells noto­ri­ous for chron­i­cal­ly send­ing out inflam­ma­to­ry sig­nals, with a long-term risk of can­cer. Yep, it’s look­ing like SARS-CoV­‑2 might be dri­ving both aging and, per­haps even­tu­al­ly, can­cer.

It’s the kind of find­ings that should be rais­ing major alarm bells. The kind of alarm bells that trig­ger­ing a new “Oper­a­tion Warp Speed” inves­ti­ga­tion into whether or not a high­ly infec­tious air­borne aging+cancer virus was just unleashed across the globe. And that brings us to the sec­ond arti­cle below about an inves­ti­ga­tion recent­ly con­duct­ed by STAT News and Muck­rak­er into the US fed­er­al gov­ern­men­t’s $1.2 bil­lion RECOVER pro­gram set up to study both the long COVID dis­ease as well as treat­ments. As we’re going to see, not only has basi­cal­ly noth­ing come from that invest­ment but it’s not even clear who is ulti­mate­ly in charge and account­able for the pro­gram. Beyond that, these find­ings aren’t new. STAT News issues a sim­i­lar warn­ing about RECOV­ER’s lack of progress last year, prompt­ing con­gres­sion­al inquiries and pledges to pick up the pace. Instead, the delays have only com­pound­ed.

At this point, only a sin­gle tri­al to study a drug treat­ment for long COVID has been approved — for Pfiz­er’s rel­a­tive­ly expen­sive new drug Paxlovid — but even that tri­al is well behind sched­ule. Tri­als for oth­er exist­ing much cheap­er drugs like met­formin and nal­trex­one have been inex­plic­a­bly ignored. And that brings us to a very inter­est­ing update on the Oya­Gen Oya‑1 mys­tery. Recall the intrigu­ing sto­ry of Oya­Gen, the Rochester, NY-based com­pa­ny that claimed to have found a well-estab­lished safe old drug was effec­tive­ly ster­il­iz­ing SARS-CoV­‑2. These find­ings were derived in part­ner­ship with US gov­ern­ment researchers as Fort Det­rick. The founder of Oya­Gen even went to the media call­ing for some sort of atten­tion to be drawn to these find­ings and per­mis­sion for emer­gency clin­i­cal tri­als. Noth­ing came of that. Instead, as we lat­er saw, Oya­Gen basi­cal­ly sold the rights to Oya‑1 to Tonix Phar­ma­ceu­ti­cals. We are now informed by Tonix that the fed­er­al gov­ern­ment has refused to help spon­sor tri­als of the drug. Again, we’re talk­ing about a drug that had its effi­ca­cy against SARS-CoV­‑2 estab­lished by US researchers at Fort Det­rick. A very cheap drug with known safe dosages for humans. For some rea­son, the US gov­ern­ment just has no inter­est in this drug. or any oth­er drug that does­n’t come from a major phar­ma­ceu­ti­cal com­pa­ny and can’t fetch an expen­sive “new drug” price, it seems.

Oh, and it appears that long COVID’s symp­toms are alarm­ing­ly sim­i­lar to chron­ic-fatigue syn­drome (CSF). And yet, as we’re going to see, one of the only clin­i­cal tri­als the RECOVER pro­gram has actu­al­ly announced at this point for a pos­si­ble treat­ment for long COVID focus­es on exer­cise as a ther­a­py, that’s despite the fact that exer­cise ther­a­py has been shown to be poten­tial­ly dan­ger­ous for CFS patients. As a result, the CFS com­mu­ni­ty is also now alarmed by the RECOVER pro­gram’s deci­sion-mak­ing. This is a good time to recall the sor­did his­to­ry tying the emer­gence of CFS to Lyme dis­ease, its direct ties US biowar­fare research, and the decades of appar­ent coverup of that research in rela­tion to Lyme dis­ease and CFS.

So at the same time we’re learn­ing that SARS-CoV­‑2 could ulti­mate­ly be far more dam­ag­ing to the long-term health of the human species than pre­vi­ous­ly rec­og­nized, we’re also learn­ing that the NIH — an enti­ty that has been thor­ough­ly cap­tured by pri­vate inter­ests at this point — seems to have an inter­est in NOT learn­ing about this dis­ease. It’s all part of the night­mare.

Ok, first, here’s a Nature Cell Biol­o­gy com­mu­ni­ca­tion from the team that dis­cov­ered that high­ly alarm­ing evi­dence for long-term DNA dam­age induced by SARS-CoV-2’s attack on the cells own DNA dam­age response path­way. It’s the kind of dam­age with major long-term impli­ca­tions, rang­ing from accel­er­at­ed aging to poten­tial­ly can­cer:

Nature Cell Biol­o­gy
vol­ume 25, pages 526–527 (2023)

SARS-CoV­‑2 caus­es DNA dam­age, cel­lu­lar senes­cence and inflam­ma­tion

Fab­rizio d’Adda di Fagagna
Pub­lished: 09 March 2023

We dis­cov­ered that SARS-CoV­‑2 infec­tion caus­es DNA dam­age both in cul­tured cells and in vivo. Mech­a­nis­ti­cal­ly, SARS-CoV­‑2 degrades the enzyme CHK1, which leads to a reduc­tion in dNTPs and impaired DNA repli­ca­tion. More­over, inhi­bi­tion of the for­ma­tion of bind­ing pro­tein 53BP1 foci by the SARS-CoV­‑2 nucle­o­cap­sid pro­tein hin­ders the repair of dam­aged DNA. The ensu­ing accu­mu­la­tion of DNA dam­age caus­es cel­lu­lar senes­cence and inflam­ma­tion.

The ques­tions

SARS-CoV­‑2 — the virus respon­si­ble for the COVID-19 pan­dem­ic — has a greater impact on human health than oth­er res­pi­ra­to­ry virus­es have, although the mech­a­nisms of this are not ful­ly under­stood. Fur­ther ques­tions are the mol­e­c­u­lar basis of the long-term con­se­quences of COVID-19, known as ‘long COVID’, and why SARS-CoV­‑2 seems to accel­er­ate human aging. One poten­tial expla­na­tion for these obser­va­tions is that SARS-CoV­‑2 caus­es dam­age to nuclear DNA — the only irre­place­able cell com­po­nent. We there­fore aimed to test the hypoth­e­sis that dam­age to nuclear DNA might be the under­ly­ing cause of the many severe con­se­quences of SARS-CoV­‑2 infec­tion.

The dis­cov­ery

To estab­lish whether SARS-CoV­‑2 infec­tion results in acti­va­tion of the DNA dam­age response (DDR), we infect­ed human cell lines with SARS-CoV­‑2 and per­formed immunoblot analy­sis of DDR mark­ers. We showed that SARS-CoV­‑2 infec­tion acti­vat­ed the DDR, and we con­firmed the pres­ence of DNA frag­men­ta­tion through the use of comet assays. These events were accom­pa­nied by pro-inflam­ma­to­ry sig­nal­ing and the estab­lish­ment of cel­lu­lar senes­cence (a form of cel­lu­lar aging).

We next probed the mol­e­c­u­lar mech­a­nisms that caused the DNA dam­age. We dis­cov­ered that SARS-CoV­‑2 express­es pro­teins that, by dis­tinct mech­a­nisms, hijack cell nucleotide metab­o­lism. Specif­i­cal­ly, the viral fac­tors ORF6 and NSP13 pro­mot­ed the degra­da­tion of check­point kinase 1 (CHK1), an enzyme involved in coor­di­nat­ing the DDR. A reduc­tion in CHK1 lev­els is thought to result in the accu­mu­la­tion of rNTPs, which we pro­pose is need­ed to fuel viral repli­ca­tion (SARS-CoV­‑2 being a RNA virus). How­ev­er, the accu­mu­la­tion of rNTPs seemed to occur at the expense of dNTPs, which we detect­ed at low­er lev­els after SARS-CoV­‑2 infec­tion and which result­ed in impaired DNA repli­ca­tion and DNA dam­age.

In addi­tion, we found evi­dence that DNA breaks accu­mu­lat­ed because they were not effi­cient­ly repaired. Indeed, we dis­cov­ered that the SARS-Cov­‑2 nucle­o­cap­sid pro­tein impaired focal recruit­ment of the bind­ing pro­tein 53BP1 and decreased DNA repair by com­pet­ing with 53BP1 for asso­ci­a­tion with dam­age-induced long non-cod­ing RNAs. Over­all, these find­ings sug­gest that SARS-CoV­‑2 both induces DNA dam­age and impairs its repair, ulti­mate­ly caus­ing cells to age and spread inflam­ma­tion (Fig. 1). Final­ly, we demon­strat­ed that these events hap­pened in vivo in SARS-CoV-2-infect­ed mice and in patients with COVID-19.

The impli­ca­tions

Our find­ings reveal the pro­found impact that SARS-CoV­‑2 infec­tion has on cel­lu­lar biol­o­gy, threat­en­ing the most impor­tant cel­lu­lar con­stituent: nuclear DNA. The accu­mu­la­tion of DNA dam­age is known to be asso­ci­at­ed with can­cer and aging1. Although the long-term con­se­quences of severe COVID-19 on lung can­cer inci­dence are unknown at present, accel­er­at­ed aging phe­no­types have been report­ed2,3. Our results may pro­vide a mech­a­nis­tic expla­na­tion for post-COVID-19 syn­dromes with has­tened aging fea­tures, to which the estab­lish­ment of cel­lu­lar senes­cence and the trig­ger­ing of inflam­ma­to­ry process­es might be a cru­cial con­tribut­ing fac­tor. Indeed, chron­ic inflam­ma­tion is thought to be the under­ly­ing cause of lung fibro­sis4, brain degen­er­a­tion5 and over­all frailty. Thus, local events ini­tial­ly restrict­ed to the res­pi­ra­to­ry sys­tem may have sys­temic con­se­quences.

...
———–

“SARS-CoV­‑2 caus­es DNA dam­age, cel­lu­lar senes­cence and inflam­ma­tion” by Fab­rizio d’Adda di Fagagna; Nature Cell Biol­o­gy, vol­ume 25, pages 526–527 (2023); 03/09/2023

“Our find­ings reveal the pro­found impact that SARS-CoV­‑2 infec­tion has on cel­lu­lar biol­o­gy, threat­en­ing the most impor­tant cel­lu­lar con­stituent: nuclear DNA. The accu­mu­la­tion of DNA dam­age is known to be asso­ci­at­ed with can­cer and aging1. Although the long-term con­se­quences of severe COVID-19 on lung can­cer inci­dence are unknown at present, accel­er­at­ed aging phe­no­types have been report­ed2,3. Our results may pro­vide a mech­a­nis­tic expla­na­tion for post-COVID-19 syn­dromes with has­tened aging fea­tures, to which the estab­lish­ment of cel­lu­lar senes­cence and the trig­ger­ing of inflam­ma­to­ry process­es might be a cru­cial con­tribut­ing fac­tor. Indeed, chron­ic inflam­ma­tion is thought to be the under­ly­ing cause of lung fibro­sis4, brain degen­er­a­tion5 and over­all frailty. Thus, local events ini­tial­ly restrict­ed to the res­pi­ra­to­ry sys­tem may have sys­temic con­se­quences.”

It’s one of the most omi­nous con­se­quences we could dis­cov­er from SAR-CoV­‑2: DNA dam­age accu­mu­la­tion. It’s like an air­borne can­cer + aging virus. Maybe. We don’t real­ly know yet. More stud­ies are obvi­ous­ly nec­es­sary. But accord­ing to this study, SAR-CoV­‑2 appears to induce DNA dam­age through mul­ti­ple mech­a­nisms that dis­rupt­ed the cel­lu­lar DNA dam­age response (DDR) path­ways. First, it appears at least two viral pro­teins pro­mote the degra­da­tion of check­point kinase 1 (CHK1), an enzyme involved in coor­di­nat­ing the DDR. In addi­tion, the SARS-Cov­‑2 nucle­o­cap­sid pro­tein impaired focal recruit­ment of the bind­ing pro­tein 53BP1 and decreased DNA repair by com­pet­ing with 53BP1 for asso­ci­a­tion with dam­age-induced long non-cod­ing RNAs. In oth­er words, one of the virus­es pro­teins acts like a sponge sop­ping up the DNA dam­age sig­nals, fur­ther dis­rupt­ing the DDR. Final­ly, and per­haps most impor­tant­ly, they dis­cov­ered this not just in cell cul­tures but in infect­ed mice and human patients. This isn’t just hypo­thet­i­cal dam­age:

...
To estab­lish whether SARS-CoV­‑2 infec­tion results in acti­va­tion of the DNA dam­age response (DDR), we infect­ed human cell lines with SARS-CoV­‑2 and per­formed immunoblot analy­sis of DDR mark­ers. We showed that SARS-CoV­‑2 infec­tion acti­vat­ed the DDR, and we con­firmed the pres­ence of DNA frag­men­ta­tion through the use of comet assays. These events were accom­pa­nied by pro-inflam­ma­to­ry sig­nal­ing and the estab­lish­ment of cel­lu­lar senes­cence (a form of cel­lu­lar aging).

We next probed the mol­e­c­u­lar mech­a­nisms that caused the DNA dam­age. We dis­cov­ered that SARS-CoV­‑2 express­es pro­teins that, by dis­tinct mech­a­nisms, hijack cell nucleotide metab­o­lism. Specif­i­cal­ly, the viral fac­tors ORF6 and NSP13 pro­mot­ed the degra­da­tion of check­point kinase 1 (CHK1), an enzyme involved in coor­di­nat­ing the DDR. A reduc­tion in CHK1 lev­els is thought to result in the accu­mu­la­tion of rNTPs, which we pro­pose is need­ed to fuel viral repli­ca­tion (SARS-CoV­‑2 being a RNA virus). How­ev­er, the accu­mu­la­tion of rNTPs seemed to occur at the expense of dNTPs, which we detect­ed at low­er lev­els after SARS-CoV­‑2 infec­tion and which result­ed in impaired DNA repli­ca­tion and DNA dam­age.

In addi­tion, we found evi­dence that DNA breaks accu­mu­lat­ed because they were not effi­cient­ly repaired. Indeed, we dis­cov­ered that the SARS-Cov­‑2 nucle­o­cap­sid pro­tein impaired focal recruit­ment of the bind­ing pro­tein 53BP1 and decreased DNA repair by com­pet­ing with 53BP1 for asso­ci­a­tion with dam­age-induced long non-cod­ing RNAs. Over­all, these find­ings sug­gest that SARS-CoV­‑2 both induces DNA dam­age and impairs its repair, ulti­mate­ly caus­ing cells to age and spread inflam­ma­tion (Fig. 1). Final­ly, we demon­strat­ed that these events hap­pened in vivo in SARS-CoV-2-infect­ed mice and in patients with COVID-19.
...

It’s more or less a night­mare sce­nario. The kind of night­mare sce­nario that becomes more and more night­mar­ish the longer it does, espe­cial­ly for long COVID patients. Or at least it’s ear­ly evi­dence sug­gest­ing we could be look­ing at that long-term night­mare sce­nario. It sure would be nice if there was a robust research pro­gram into the nature long COVID. And that brings us to anoth­er night­mare sce­nario also play­ing out: the NIH’s $1.2 bil­lion RECOVER pro­gram ded­i­cat­ed to the study of long COVID has appar­ent­ly been a com­plete bust. An ongo­ing bust, with mon­ey still be spent but no one appar­ent­ly able to answer where it’s going, why there are so many delays, or who is ulti­mate­ly account­able. And while Con­gress explic­it­ly ear­marked that $1.2 bil­lion for the study of both long COVID and treat­ments, the RECOVER project appears to have neglect­ed the treat­ments almost entire­ly. The only drug tri­al announced is for Pfiz­er’s Paxlovid and even that is run­ning way behind sched­ule. Also, none of this is new. Alarm bells were raised about these delays last year and yet the delays have only com­pound­ed:

Stat News

The NIH has poured $1 bil­lion into long Covid research — with lit­tle to show for it

By Rachel Cohrs and Bet­sy Ladyzhets
April 20, 2023

WASHINGTON — The fed­er­al gov­ern­ment has burned through more than $1 bil­lion to study long Covid, an effort to help the mil­lions of Amer­i­cans who expe­ri­ence brain fog, fatigue, and oth­er symp­toms after recov­er­ing from a coro­n­avirus infec­tion.

There’s basi­cal­ly noth­ing to show for it.

The Nation­al Insti­tutes of Health hasn’t signed up a sin­gle patient to test any poten­tial treat­ments — despite a clear man­date from Con­gress to study them. And the few tri­als it is plan­ning have already drawn a firestorm of crit­i­cism, espe­cial­ly one inter­ven­tion that experts and advo­cates say may actu­al­ly make some patients’ long Covid symp­toms worse.

Instead, the NIH spent the major­i­ty of its mon­ey on broad­er, obser­va­tion­al research that won’t direct­ly bring relief to patients. But it still hasn’t pub­lished any find­ings from the patients who joined that study, almost two years after it start­ed.

There’s no sense of urgency to do more or to speed things up, either. The agency isn’t ask­ing Con­gress for any more fund­ing for long Covid research, and STAT and Muck­Rock obtained doc­u­ments show­ing the NIH refus­es to use its own mon­ey to change course.

“So far, I don’t think we’ve got­ten any­thing for a bil­lion dol­lars,” said Ezekiel Emanuel, a physi­cian, vice provost for glob­al ini­tia­tives, and co-direc­tor of the Health­care Trans­for­ma­tion Insti­tute at the Uni­ver­si­ty of Penn­syl­va­nia. “That is just unac­cept­able, and it’s a seri­ous dys­func­tion.”

Eric Topol, the founder and direc­tor of the Scripps Research Trans­la­tion­al Insti­tute, said he expect­ed the NIH would have launched many large-scale tri­als by now, and that test­ing treat­ments should have been an urgent pri­or­i­ty when Con­gress first gave the agency mon­ey in late 2020.

“I don’t know that they’ve con­tributed any­thing except more con­fu­sion,” Topol said.

Patients and researchers have already raised alarms about the glacial pace of the NIH’s ear­ly long Covid efforts. But a new inves­ti­ga­tion from STAT and the non­prof­it news orga­ni­za­tion Muck­Rock, based on inter­views with near­ly two dozen gov­ern­ment offi­cials, experts, patients, and advo­cates, and inter­nal NIH cor­re­spon­dence, let­ters, and pub­lic doc­u­ments, under­scores that the NIH hasn’t picked up the pace — instead, the delays have com­pound­ed.

It’s dif­fi­cult to pin­point exact­ly why progress is so stalled, experts and patients involved in the project empha­sized, because the NIH has obscured both who is in charge of the long Covid efforts and how it spent the mon­ey. The broad­er Biden admin­is­tra­tion has also missed oppor­tu­ni­ties for over­sight and account­abil­i­ty of the effort — despite the president’s lofty promis­es to focus on the dis­ease.

The NIH’s blun­ders have mas­sive ram­i­fi­ca­tions for the more than 16 mil­lion Amer­i­cans suf­fer­ing from long Covid, in addi­tion to those with oth­er, sim­i­lar chron­ic dis­eases. As the biggest gov­ern­ment-fund­ed study on this top­ic, the NIH ini­tia­tive, dubbed RECOVER, sets prece­dents for future research and clin­i­cal guide­lines. It will dic­tate how doc­tors across the coun­try treat their patients — and, in turn, impact people’s abil­i­ty to access work accom­mo­da­tions, dis­abil­i­ty ben­e­fits, and more.

...

In response to STAT and MuckRock’s ques­tions, the NIH and an insti­tute at Duke Uni­ver­si­ty man­ag­ing the clin­i­cal tri­als defend­ed the ini­tia­tive, with­out pro­vid­ing a clear expla­na­tion for the delays.

The NIH said it chose to fund a large-scale research pro­gram instead of small-scale stud­ies to make sure data and process­es could be shared across dif­fer­ent groups of patients, adding that clin­i­cal tri­als will be launch­ing soon. In these tri­als, stan­dard­ized study designs will allow the agency to test mul­ti­ple treat­ments across mul­ti­ple sites. If there are sig­nals a drug works, the agency said it can piv­ot to devote more resources there.

A Depart­ment of Health and Human Ser­vices spokesper­son said the agency has made progress over the last year in respond­ing to long Covid, and that there are research efforts under­way in addi­tion to the RECOVER pro­gram.

...

In 2020, Con­gress made an invest­ment of $1.2 bil­lion to learn more about the mys­te­ri­ous ongo­ing symp­toms that were afflict­ing some peo­ple infect­ed with Covid-19. That sort of mon­ey to fund research into a chron­ic con­di­tion like long Covid was vir­tu­al­ly unheard of.

The mon­ey was explic­it­ly ear­marked to fund both research to under­stand the dis­ease and clin­i­cal tri­als to test treat­ments that could bring patients relief. But more than two years in, the agency hasn’t start­ed test­ing a sin­gle treat­ment. Nor is it plan­ning to test many in the future. Instead, it’s focused on obser­va­tion­al research — and that, too, has pro­duced few insights.

The NIH is plan­ning five clin­i­cal tri­als, each of which will test treat­ments that may help with a major cat­e­go­ry of long Covid symp­toms. Some of these treat­ments will be drugs, while oth­ers will be behav­ioral ther­a­pies, such as cog­ni­tive retrain­ing. Each tri­al will include 300 to 900 patients, select­ed based on their symp­toms, accord­ing to details shared dur­ing a webi­nar in mid-April.

The only tri­al to be for­mal­ly announced so far will focus on Paxlovid, test­ing whether the drug alle­vi­ates symp­toms by mit­i­gat­ing any ongo­ing viral infec­tion in patients’ bod­ies. The study was sup­posed to start recruit­ing in Jan­u­ary.

But as of April, RECOVER hasn’t signed up a sin­gle patient for any of those clin­i­cal tri­als. And the time­line has slipped over and over again.

Ini­tial­ly, in a let­ter to mem­bers of Con­gress prompt­ed by STAT’s March 2022 report­ing on the initiative’s slow start, the NIH told law­mak­ers that the agency expect­ed to launch clin­i­cal tri­als by that fall. But by August, the esti­mat­ed launch had slipped to “by the end of 2022.” Then, anoth­er delay became pub­lic in Decem­ber, when one of the NIH offi­cials lead­ing RECOVER told advis­ers that clin­i­cal tri­als would begin by the first quar­ter of 2023. Now, Duke Uni­ver­si­ty, which is over­see­ing the clin­i­cal tri­al infra­struc­ture, told STAT and Muck­Rock it expects the first patients to sign up for tri­als this sum­mer.

[Click to read the entire doc­u­ment.]

Emanuel said the pace of tri­als shows lit­tle urgency on the part of NIH.

“If you don’t have the patho­bi­ol­o­gy fig­ured out, you try things. You don’t just slow, slow, slow, walk it,” he said.

All five clin­i­cal tri­al pro­to­cols are going through safe­ty reviews, and the Food and Drug Admin­is­tra­tion is review­ing the tri­als that will test Paxlovid and oth­er drugs, the Duke Clin­i­cal Research Insti­tute said. The insti­tute plans to share these pro­to­cols pub­licly when reviews are com­plete, but did not pro­vide an esti­mate for when that will hap­pen.

Faster progress is pos­si­ble. A sim­i­lar study at Stan­ford, which received fund­ing direct­ly from Pfiz­er, was also announced in Octo­ber 2022 but has already begun recruit­ing patients. This tri­al was “able to be more flex­i­ble and get the study start­ed faster” in com­par­i­son to RECOVER because it’s small­er, said Upin­der Singh, the study’s prin­ci­pal inves­ti­ga­tor. Singh and her col­leagues are only test­ing Paxlovid and doing so at only one loca­tion, rather than com­par­ing dif­fer­ent treat­ments.

Duke was also sup­posed to cre­ate a patient reg­istry to col­lect infor­ma­tion about long Covid patients, but that ini­tia­tive hasn’t been launched, either.

...

Rather than pri­or­i­tiz­ing treat­ments from the start, the NIH used much of its long Covid fund­ing on a large-scale study to track long Covid symp­toms and learn how the dis­ease works. This choice has frus­trat­ed patients because thou­sands of oth­er stud­ies have already answered many major ques­tions about the con­di­tion.

“We didn’t need to recre­ate” exist­ing stud­ies that already answered these ques­tions, said Cole, the long Covid patient. Researchers have been com­pil­ing lists of com­mon symp­toms since sum­mer 2020, she said. For Cole, fatigue and brain fog are the most debil­i­tat­ing aspects of the con­di­tion.

And even the symp­tom study is mov­ing slow­ly, in part because the ini­tia­tive has failed to bring in healthy peo­ple who could be com­pared against long Covid patients. RECOVER quick­ly filled its slots for peo­ple who had Covid more than 30 days pri­or to their recruit­ment, but is still look­ing for peo­ple who were infect­ed recent­ly, study lead Leo­ra Hor­witz said in a state­ment. Most study sites closed enroll­ment for long Covid patients at the end of August 2022.

The major­i­ty of the sci­en­tif­ic find­ings to emerge from RECOVER so far have been based on small groups of patients or on elec­tron­ic health records, rather than on the thou­sands of peo­ple who signed up to par­tic­i­pate.

The crawl­ing pace of the government’s long Covid efforts stand in stark con­trast with the government’s wild­ly suc­cess­ful part­ner­ship with the phar­ma­ceu­ti­cal indus­try to get Covid-19 vac­cines to mar­ket in less than 12 months. There are no ongo­ing efforts to sup­port inde­pen­dent pri­vate-sec­tor com­pa­nies or researchers try­ing to study treat­ments for long Covid through the NIH, even though some have proved promis­ing. Just this month, the White House left long Covid out of a $5 bil­lion effort to research next-gen­er­a­tion Covid-19 treat­ments and vac­cines.

Long Covid researchers feel there needs to be greater urgency. Singh com­pared the pres­sure that she’s cur­rent­ly under to the pres­sure many sci­en­tists faced ear­li­er in the pan­dem­ic when study­ing vac­cines and treat­ments. “We as a sci­en­tif­ic com­mu­ni­ty need to focus on long Covid and find solu­tions for long Covid,” she said.

Topol echoed this sen­ti­ment, cit­ing a recent opin­ion piece in Sci­en­tif­ic Amer­i­can that called for an Oper­a­tion Warp Speed for long Covid treat­ments. “That’s what should have hap­pened,” he said.

It’s almost impos­si­ble to tell where the NIH’s $1.2 bil­lion pot of long Covid mon­ey has gone.

There is no sin­gle NIH offi­cial respon­si­ble for lead­ing RECOVER, and the ini­tia­tive has failed to share basic infor­ma­tion that would typ­i­cal­ly be avail­able for a gov­ern­ment research project of this scale.

Unlike Oper­a­tion Warp Speed and oth­er Covid efforts, the NIH has out­sourced much of the work of run­ning RECOVER to out­side orga­ni­za­tions. New York Uni­ver­si­ty, RTI Inter­na­tion­al, Mayo Clin­ic, Mass­a­chu­setts Gen­er­al Hos­pi­tal, and Duke Uni­ver­si­ty are respon­si­ble for var­i­ous parts of the ini­tia­tive.

Many of the research projects asso­ci­at­ed with RECOVER have been fund­ed through these orga­ni­za­tions rather than direct­ly from the NIH. This process makes it hard to track how deci­sions are made or how mon­ey is spent through pub­lic data­bas­es, said Michael Siev­erts, a mem­ber of the long Covid Patient-Led Research Col­lab­o­ra­tive who has a back­ground in fed­er­al bud­get­ing for sci­en­tif­ic research.

Pub­lic records requests that Muck­Rock filed to the agency in late 2022, intend­ed to answer ques­tions about RECOVER’s fund­ing, are still incom­plete as of mid-April. Siev­erts has sim­i­lar­ly asked ques­tions to NIH offi­cials and received no respons­es.

The orga­ni­za­tion of RECOVER itself is con­vo­lut­ed, and dif­fi­cult to fig­ure out even for patient advo­cates who are direct­ly involved, they said. It’s advised by a com­plex series of com­mit­tees, some of which aren’t even post­ed on the initiative’s web­site. There’s no one per­son ulti­mate­ly respon­si­ble for coor­di­nat­ing among the dif­fer­ent insti­tutes — and requests for infor­ma­tion about the lead­er­ship hier­ar­chy have been ignored.

“They don’t have an org chart for the entire thing that exists, after two-plus years,” said Diana Güthe, the founder of Sur­vivor Corps and a RECOVER advis­er who has asked at near­ly every meet­ing she’s attend­ed.

Lau­ren Stiles, a patient advo­cate and Pres­i­dent and CEO of Dysau­tono­mia Inter­na­tion­al who serves on sev­er­al RECOVER com­mit­tees, shared sim­i­lar con­cerns.

“There’s a com­plete lack of trans­paren­cy. When we ask who made this deci­sion … they won’t tell us,” Stiles said.

As a result, when RECOVER says it’s run­ning out of funds, it’s hard to iden­ti­fy who is respon­si­ble for major deci­sions.

In response to ques­tions about the initiative’s bud­get, the NIH said it has no mon­ey avail­able for addi­tion­al pro­gram­ming. The agency said $811 mil­lion has been legal­ly com­mit­ted to var­i­ous activ­i­ties, and the rest is ear­marked to sup­port future research activ­i­ties.

The bud­get restric­tions are hav­ing prac­ti­cal impacts already.

A RECOVER advi­so­ry com­mit­tee respon­si­ble for rank­ing and eval­u­at­ing poten­tial treat­ment options was put on hia­tus “due to a lack of funds,” the committee’s leader told mem­bers in late Jan­u­ary, per an email exchange shared with STAT and Muck­Rock that has not been pre­vi­ous­ly report­ed.

The NIH told STAT and Muck­Rock that the com­mit­tee was paused because the clin­i­cal tri­al med­i­cines, devices, and treat­ment pro­grams have been cho­sen. How­ev­er, the agency said that the RECOVER clin­i­cal tri­als are “adap­tive plat­form tri­als,” which means they are designed with the inten­tion of remov­ing and adding treat­ments as new infor­ma­tion becomes avail­able.

This cur­rent bud­get squeeze didn’t come with­out warn­ing: The NIH was well-aware last sum­mer that the agency wouldn’t have enough mon­ey to run clin­i­cal tri­als that matched the initiative’s goals of reach­ing patients with diverse symp­toms.

One of RECOVER’s co-chairs wrote to Con­gress in June that “addi­tion­al resources are nec­es­sary” to test the full range of treat­ments need­ed.

But the Biden admin­is­tra­tion isn’t tak­ing any action to get more fund­ing with­in the agency, or from law­mak­ers.

NIH act­ing Direc­tor Lawrence Tabak told patient advo­cates that the agency isn’t plan­ning on direct­ing any fur­ther fund­ing for RECOVER with­in the agency. The agency said that such a request would poten­tial­ly under­cut a failed request for sup­ple­men­tal fund­ing that Con­gress ignored last year.

[Click to read the entire doc­u­ment.]

The Biden admin­is­tra­tion didn’t request any new funds for RECOVER in its 2024 bud­get, a large­ly aspi­ra­tional doc­u­ment that reflects the administration’s finan­cial pri­or­i­ties.

The bud­get did include $130 mil­lion in long Covid-relat­ed asks for oth­er agen­cies, includ­ing for the Health Resources and Ser­vices Admin­is­tra­tion to sup­port care for long Covid patients with com­plex needs and to edu­cate pri­ma­ry care providers, and for the Agency for Health­care Research and Qual­i­ty to research the deliv­ery of long Covid care and to estab­lish long Covid care hubs.

There’s also lit­tle account­abil­i­ty for NIH lead­ers to dis­close how funds are spent or respond to oth­er con­cerns with RECOVER because an enti­ty intend­ed to over­see long Covid research across the fed­er­al gov­ern­ment hasn’t been cre­at­ed.

In April 2022, Pres­i­dent Biden issued a pres­i­den­tial mem­o­ran­dum call­ing on fed­er­al agen­cies to “har­ness the full poten­tial” of the gov­ern­ment, in part­ner­ship with pri­vate sec­tor part­ners, to respond to long Covid.

The fol­low-through has been lack­ing on the initiative’s high­est-pro­file goal.

In August, in a con­gres­sion­al­ly man­dat­ed nation­al long Covid research plan, the Biden admin­is­tra­tion said it would cre­ate an Office of Long Covid Research and Prac­tice at HHS. This month, HHS put out a fact sheet tout­ing the administration’s progress in reach­ing its goals — and omit­ted any men­tion of the office.

An HHS spokesper­son said that the depart­ment is work­ing to devel­op the office, and request­ed fund­ing in next year’s White House’s bud­get for the Office of the Assis­tant Sec­re­tary for Health to coor­di­nate response efforts to long Covid.

“It seems to have been like, well, if we don’t do any­thing, maybe no one will notice,” said Güthe. “It’s so impor­tant to do an eval­u­a­tion of what was promised. What’s been accom­plished, and what hasn’t?”

A huge chunk of fund­ing to study a chron­ic ill­ness like long Covid is rare, so any clin­i­cal tri­als that the NIH choos­es to run are cru­cial choic­es — and some doc­tors and advo­ca­cy groups have voiced seri­ous con­cerns about the selec­tion of one clin­i­cal tri­al in par­tic­u­lar.

That tri­al would test exer­cise as a poten­tial long Covid treat­ment, despite years of research sug­gest­ing that exer­cise could harm patients and set back fur­ther study.

Many peo­ple with long Covid have sim­i­lar symp­toms to peo­ple with myal­gic encephalomyelitis or chron­ic fatigue syn­drome (ME/CFS), a debil­i­tat­ing con­di­tion that often fol­lows viral infec­tion. The defin­ing fea­ture of ME/CFS is intense fatigue and wors­en­ing of oth­er health issues after phys­i­cal or men­tal activ­i­ty. This symp­tom, known as post-exer­tion­al malaise, often occurs with a lag, which can make it tough for doc­tors to diag­nose — and even for patients to rec­og­nize them­selves.

“What often hap­pens is, peo­ple will go for a walk, they may not feel it for a day or two, and then sud­den­ly, they feel ill on the third day,” said Adam Lowe, an ME/CFS patient and one of the founders of MEAc­tion UK, the U.K. branch of the Myal­gic Encephalomyelitis Action Net­work. Patients might sud­den­ly become bed-bound and have trou­ble focus­ing, he said.

This wors­en­ing of symp­toms hap­pens because a patient isn’t pro­duc­ing and using ener­gy in the same way as a healthy per­son, said Todd Dav­en­port, a pro­fes­sor at Uni­ver­si­ty of the Pacif­ic who hasstud­ied exer­cise and this con­di­tion. It’s an inter­nal change sim­i­lar to the whole-body exhaus­tion that a marathon run­ner might expe­ri­ence at the fin­ish line of their race.

A num­ber of past stud­ies and sur­veys of patients have demon­strat­ed how dan­ger­ous exer­cise can be for peo­ple with ME/CFS. Many patients told to exer­cise by their doc­tors lat­er dropped out of stud­ies or treat­ment reg­i­mens, cit­ing wors­en­ing symp­toms. One infa­mous tri­al that point­ed to exer­cise as a poten­tial treat­ment was lat­er dis­cred­it­ed as deeply flawed.

Study­ing exer­cise as a treat­ment could “frame long Covid as some­thing that can be over­come with grit and hard work,” said Jaime Seltzer, the direc­tor of sci­en­tif­ic and med­ical out­reach at MEAc­tion, argu­ing that such fram­ing is “unsound and eth­i­cal­ly trou­bling.”

Not all patients with long Covid expe­ri­ence post-exer­tion­al malaise, and those who don’t could find exer­cise help­ful, Dav­en­port said. In those cas­es, slow and care­ful exer­cise through a reha­bil­i­ta­tion or phys­i­cal ther­a­py pro­gram might help repair ener­gy sys­tems that have fall­en out of shape.

But it may be dif­fi­cult to dis­tin­guish between these dif­fer­ent groups of patients, unless a clin­i­cal tri­al is set up with the utmost cau­tion. “Ide­al­ly, what you would want is a very coher­ent, very spe­cif­ic set of inclu­sion and exclu­sion cri­te­ria,” Dav­en­port said. Oth­er­wise, the study would risk pro­duc­ing results that over­sim­pli­fy long Covid, he added, lead­ing doc­tors to wide­ly pre­scribe a treat­ment that doesn’t work for some or many.

Sci­en­tists and patient advo­cates respon­si­ble for advis­ing RECOVER have warned that an exer­cise tri­al could harm patients, but received mixed respons­es. Patients involved in the study sent emails and social media posts demand­ing that RECOVER stop the planned tri­al, while MEAc­tion sent a pub­lic let­ter to NIH lead­ers.

[Click to read the entire doc­u­ment.]

Sci­en­tists and clin­i­cians on an NIH advi­so­ry com­mit­tee focused on reha­bil­i­ta­tion sim­i­lar­ly sug­gest­ed that post-exer­tion­al malaise could be a dan­ger­ous result of the tri­al, accord­ing to inter­nal emails shared with STAT and Muck­Rock. In response, NIH pro­gram offi­cer Antonel­lo Pun­turi­eri pushed back on the con­cerns. Pun­turi­eri cit­ed clin­i­cal guide­lines from the World Health Orga­ni­za­tion and a U.K. agency, even though both rec­om­mend against exer­cise for peo­ple with ME/CFS.

In response to these con­cerns, RECOVER set up inter­nal meet­ings includ­ing researchers in charge of the exer­cise study, patient rep­re­sen­ta­tives, and the initiative’s top advi­so­ry com­mit­tee. “Work is now under­way to fur­ther revise that pro­to­col” based on these meet­ings, the Duke Clin­i­cal Research Insti­tute said.

[Click to read the entire doc­u­ment.]

The study’s planned revi­sions will address con­cerns about patient safe­ty, such as mon­i­tor­ing for post-exer­tion­al malaise after exer­cise. But it’s unclear how the researchers will do this screen­ing, or whether ME/CFS doc­tors will be involved.

Even with revi­sion, experts and patient advo­cates remain con­cerned that the exer­cise study takes resources away from oth­er research and could lead to harm­ful rec­om­men­da­tions from doc­tors. If RECOVER finds exer­cise is help­ful for some patients, asked JD Davids, an advo­cate with Long COVID Jus­tice and author of a peti­tion ask­ing the NIH to stop this tri­al, “What are the chances that doc­tors would cor­rect­ly under­stand how lim­it­ed this rec­om­men­da­tion is? I think it’s very low.”

It’s not like there aren’t plen­ty of poten­tial treat­ment options worth study­ing.

Topol and oth­er researchers com­piled a full table of oth­er treat­ment can­di­dates for a review paper pub­lished in Nature in Jan­u­ary. Experts on one of RECOVER’s advi­so­ry com­mit­tees com­piled a sim­i­lar list, for a paper pub­lished in March.

Giv­en “the num­ber of oth­er can­di­date treat­ments out there, I can’t imag­ine why you would choose grad­ed exer­cise ther­a­py,” said Julia Moore Vogel, a researcher at the Scripps Trans­la­tion­al Insti­tute liv­ing with long Covid, and co-author of the Nature review paper. Vogel is lead­ing a study of wear­able devices for long Covid, which will start with about 500 par­tic­i­pants despite plan­ning for up to 100,000.

One study has even report­ed results already, via a preprint shared by The Lancet in ear­ly March. The tri­al found that met­formin, a com­mon treat­ment for dia­betes that also has antivi­ral prop­er­ties, low­ered Covid patients’ risk of devel­op­ing long-term symp­toms by about 42%.

This research group actu­al­ly didn’t set out to study long Covid, said David Boul­ware, one of the sci­en­tists and an infec­tious dis­ease physi­cian at the Uni­ver­si­ty of Min­neso­ta Med­ical School. The ini­tial goal was to eval­u­ate poten­tial treat­ments for acute Covid-19, but the team added long Covid track­ing part­way through the tri­al.

And it’s unlike­ly to get fur­ther study with­out some kind of gov­ern­ment assis­tance. The ini­tial study relied on phil­an­thropic fund­ing, and addi­tion­al grants would be need­ed to keep study­ing this gener­ic drug.

“It’s a great drug, it’s cheap, it’s avail­able world­wide,” Boul­ware said, “but there’s no prof­it mar­gin for any­one to study it.”

There may be sim­i­lar con­cerns for research into low-dose nal­trex­one, an off-label use of the addic­tion drug that has become com­mon for long Covid and oth­er chron­ic dis­eases. In low dos­es, nal­trex­one can help reduce inflam­ma­tion in the immune and neu­ro­log­i­cal sys­tems, poten­tial­ly alle­vi­at­ing long Covid symp­toms.

But because the drug has been wide­ly avail­able for decades, phar­ma­ceu­ti­cal com­pa­nies aren’t moti­vat­ed to fund large tri­als. A few small clin­i­cal tri­als are under­way, accord­ing to report­ing by Rolling Stone.

The lack of help from NIH has left biotech exec­u­tives frus­trat­ed.

“You have to under­stand what you’re try­ing to tack­le, so we sup­port that, of course. But as patients will tell you, we want inter­ven­tion, not obser­va­tion,” said Axcel­la CEO Bill Hin­shaw. His Mass­a­chu­setts-based com­pa­ny has gone all in on test­ing a drug can­di­date to treat long Covid symp­toms, with­out any help from NIH.

Tonix Phar­ma­ceu­ti­cals, which is devel­op­ing a fibromyal­gia med­ica­tion that the com­pa­ny is hop­ing could be an effec­tive treat­ment for long Covid symp­toms, didn’t receive any fund­ing from NIH either, despite putting in an appli­ca­tion.

“I hope there are more ther­a­peu­tics tri­als. And I think that the ther­a­peu­tics tri­als can go hand in hand with the nat­ur­al his­to­ry kind of stud­ies like RECOVER,” Tonix CEO Seth Led­er­man said.

Patients and experts fear that if RECOVER is the extent of fed­er­al effort to study long Covid, the con­di­tion could fall into the long­stand­ing pat­tern of apa­thy and lack of urgency that has made break­throughs in chron­ic ill­ness treat­ment chal­leng­ing.

“It’s clear that there are a lot of peo­ple at the NIH who are ded­i­cat­ed and deter­mined, try­ing to fig­ure this out,” said Char­lie McCone, a patient rep­re­sen­ta­tive at RECOVER. As a result, “patients are con­fused” why only a hand­ful of clin­i­cal tri­als have been planned and none of those have launched yet, he said.

...

————-

“The NIH has poured $1 bil­lion into long Covid research — with lit­tle to show for it” By Rachel Cohrs and Bet­sy Ladyzhets; Stat News; 04/20/2023

“There’s no sense of urgency to do more or to speed things up, either. The agency isn’t ask­ing Con­gress for any more fund­ing for long Covid research, and STAT and Muck­Rock obtained doc­u­ments show­ing the NIH refus­es to use its own mon­ey to change course.”

Over $1 bil­lion invest­ed into...what exact­ly? Where did the mon­ey go? That’s part of the mys­tery here. But only part. There’s also gen­er­al mys­tery of who is even run­ning the fed­er­al­ly-fund­ed RECOVER pro­gram, which is espe­cial­ly prob­lem­at­ic when you want to get an answer as to why the pro­gram has­n’t done any of the things it was man­dat­ed to do but instead appears to just be putting up new obsta­cles and delays. Some­how this pro­gram was set up in a man­ner where no one appears to be account­able:

...
The Nation­al Insti­tutes of Health hasn’t signed up a sin­gle patient to test any poten­tial treat­ments — despite a clear man­date from Con­gress to study them. And the few tri­als it is plan­ning have already drawn a firestorm of crit­i­cism, espe­cial­ly one inter­ven­tion that experts and advo­cates say may actu­al­ly make some patients’ long Covid symp­toms worse.

Instead, the NIH spent the major­i­ty of its mon­ey on broad­er, obser­va­tion­al research that won’t direct­ly bring relief to patients. But it still hasn’t pub­lished any find­ings from the patients who joined that study, almost two years after it start­ed.

...

“So far, I don’t think we’ve got­ten any­thing for a bil­lion dol­lars,” said Ezekiel Emanuel, a physi­cian, vice provost for glob­al ini­tia­tives, and co-direc­tor of the Health­care Trans­for­ma­tion Insti­tute at the Uni­ver­si­ty of Penn­syl­va­nia. “That is just unac­cept­able, and it’s a seri­ous dys­func­tion.”

Eric Topol, the founder and direc­tor of the Scripps Research Trans­la­tion­al Insti­tute, said he expect­ed the NIH would have launched many large-scale tri­als by now, and that test­ing treat­ments should have been an urgent pri­or­i­ty when Con­gress first gave the agency mon­ey in late 2020.

“I don’t know that they’ve con­tributed any­thing except more con­fu­sion,” Topol said.

Patients and researchers have already raised alarms about the glacial pace of the NIH’s ear­ly long Covid efforts. But a new inves­ti­ga­tion from STAT and the non­prof­it news orga­ni­za­tion Muck­Rock, based on inter­views with near­ly two dozen gov­ern­ment offi­cials, experts, patients, and advo­cates, and inter­nal NIH cor­re­spon­dence, let­ters, and pub­lic doc­u­ments, under­scores that the NIH hasn’t picked up the pace — instead, the delays have com­pound­ed.

It’s dif­fi­cult to pin­point exact­ly why progress is so stalled, experts and patients involved in the project empha­sized, because the NIH has obscured both who is in charge of the long Covid efforts and how it spent the mon­ey. The broad­er Biden admin­is­tra­tion has also missed oppor­tu­ni­ties for over­sight and account­abil­i­ty of the effort — despite the president’s lofty promis­es to focus on the dis­ease.

The NIH’s blun­ders have mas­sive ram­i­fi­ca­tions for the more than 16 mil­lion Amer­i­cans suf­fer­ing from long Covid, in addi­tion to those with oth­er, sim­i­lar chron­ic dis­eases. As the biggest gov­ern­ment-fund­ed study on this top­ic, the NIH ini­tia­tive, dubbed RECOVER, sets prece­dents for future research and clin­i­cal guide­lines. It will dic­tate how doc­tors across the coun­try treat their patients — and, in turn, impact people’s abil­i­ty to access work accom­mo­da­tions, dis­abil­i­ty ben­e­fits, and more.
...

And, again, note that the $1.2 bil­lion ear­marked by Con­gress for the RECOVER pro­gram was explic­it­ly ear­marked to fund both stud­ies that exam­ine the dis­ease pathol­o­gy of long COVID but also treat­ments. And yet, at this point there has­n’t been a sin­gle treat­ment test­ed. And the only treat­ment approved so far will focus on Pfiz­er’s Paxlovid. This is a good time to recall how the rel­a­tive­ly expen­sive new drug Paxlovid appeared to work in a sim­i­lar man­ner to the much cheap­er exist­ing drug lopinavir, and yet, as we saw, no tri­als of lopinavir’s effi­ca­cy against SARS-CoV­‑2 was ever con­duct­ed at the time of Paxlovid’s roll out. And even that Paxlovid tri­al is behind sched­ule, but keep in mind that Paxlovid is already approved so it’s not like this delay is harm­ing Pfiz­er’s bot­tom line. It’s a per­va­sive pat­tern:

...
In 2020, Con­gress made an invest­ment of $1.2 bil­lion to learn more about the mys­te­ri­ous ongo­ing symp­toms that were afflict­ing some peo­ple infect­ed with Covid-19. That sort of mon­ey to fund research into a chron­ic con­di­tion like long Covid was vir­tu­al­ly unheard of.

The mon­ey was explic­it­ly ear­marked to fund both research to under­stand the dis­ease and clin­i­cal tri­als to test treat­ments that could bring patients relief. But more than two years in, the agency hasn’t start­ed test­ing a sin­gle treat­ment. Nor is it plan­ning to test many in the future. Instead, it’s focused on obser­va­tion­al research — and that, too, has pro­duced few insights.

The NIH is plan­ning five clin­i­cal tri­als, each of which will test treat­ments that may help with a major cat­e­go­ry of long Covid symp­toms. Some of these treat­ments will be drugs, while oth­ers will be behav­ioral ther­a­pies, such as cog­ni­tive retrain­ing. Each tri­al will include 300 to 900 patients, select­ed based on their symp­toms, accord­ing to details shared dur­ing a webi­nar in mid-April.

The only tri­al to be for­mal­ly announced so far will focus on Paxlovid, test­ing whether the drug alle­vi­ates symp­toms by mit­i­gat­ing any ongo­ing viral infec­tion in patients’ bod­ies. The study was sup­posed to start recruit­ing in Jan­u­ary.

But as of April, RECOVER hasn’t signed up a sin­gle patient for any of those clin­i­cal tri­als. And the time­line has slipped over and over again.
...

And as the arti­cle notes, this is all in stark con­trast with the US gov­ern­men­t’s “wild­ly suc­cess­ful part­ner­ship with the phar­ma­ceu­ti­cal indus­try to get Covid-19 vac­cines to mar­ket”. Indeed, it’s been quite the con­trast when it comes to cheap­er exist­ing drug alter­na­tives like met­formin or nal­trex­one, where the NIH just could­n’t be both­ered to help. Except it’s not real­ly in con­trast if you oper­at­ing on the assump­tion that max­i­miz­ing the phar­ma­ceu­ti­cal indus­try’s prof­it mar­gins is the top pri­or­i­ty. It’s all quite con­sis­tent with reg­u­la­to­ry cap­ture, whether we’re talk­ing about vac­cines or drug treat­ments:

...
The crawl­ing pace of the government’s long Covid efforts stand in stark con­trast with the government’s wild­ly suc­cess­ful part­ner­ship with the phar­ma­ceu­ti­cal indus­try to get Covid-19 vac­cines to mar­ket in less than 12 months. There are no ongo­ing efforts to sup­port inde­pen­dent pri­vate-sec­tor com­pa­nies or researchers try­ing to study treat­ments for long Covid through the NIH, even though some have proved promis­ing. Just this month, the White House left long Covid out of a $5 bil­lion effort to research next-gen­er­a­tion Covid-19 treat­ments and vac­cines.

...

One study has even report­ed results already, via a preprint shared by The Lancet in ear­ly March. The tri­al found that met­formin, a com­mon treat­ment for dia­betes that also has antivi­ral prop­er­ties, low­ered Covid patients’ risk of devel­op­ing long-term symp­toms by about 42%.

This research group actu­al­ly didn’t set out to study long Covid, said David Boul­ware, one of the sci­en­tists and an infec­tious dis­ease physi­cian at the Uni­ver­si­ty of Min­neso­ta Med­ical School. The ini­tial goal was to eval­u­ate poten­tial treat­ments for acute Covid-19, but the team added long Covid track­ing part­way through the tri­al.

And it’s unlike­ly to get fur­ther study with­out some kind of gov­ern­ment assis­tance. The ini­tial study relied on phil­an­thropic fund­ing, and addi­tion­al grants would be need­ed to keep study­ing this gener­ic drug.

“It’s a great drug, it’s cheap, it’s avail­able world­wide,” Boul­ware said, “but there’s no prof­it mar­gin for any­one to study it.”

There may be sim­i­lar con­cerns for research into low-dose nal­trex­one, an off-label use of the addic­tion drug that has become com­mon for long Covid and oth­er chron­ic dis­eases. In low dos­es, nal­trex­one can help reduce inflam­ma­tion in the immune and neu­ro­log­i­cal sys­tems, poten­tial­ly alle­vi­at­ing long Covid symp­toms.

But because the drug has been wide­ly avail­able for decades, phar­ma­ceu­ti­cal com­pa­nies aren’t moti­vat­ed to fund large tri­als. A few small clin­i­cal tri­als are under­way, accord­ing to report­ing by Rolling Stone.

The lack of help from NIH has left biotech exec­u­tives frus­trat­ed.

“You have to under­stand what you’re try­ing to tack­le, so we sup­port that, of course. But as patients will tell you, we want inter­ven­tion, not obser­va­tion,” said Axcel­la CEO Bill Hin­shaw. His Mass­a­chu­setts-based com­pa­ny has gone all in on test­ing a drug can­di­date to treat long Covid symp­toms, with­out any help from NIH.
...

And that brings us to this remark­able claim by Tonix Phar­ma­ceu­ti­cals: Tonix ulti­mate­ly received NO FEDERAL fund­ing for its POTENTIALLY BLOCKBUSTER DRUG. Recall the intrigu­ing sto­ry of Oya­Gen, the Rochester, NY-based com­pa­ny that claimed to have found a well-estab­lished safe old drug was effec­tive­ly ster­il­iz­ing SARS-CoV­‑2. These find­ings were derived in part­ner­ship with US gov­ern­ment researchers as Fort Det­rick. The founder of Oya­Gen even went to the media call­ing for some sort of atten­tion to be drawn to these find­ings and per­mis­sion for emer­gency clin­i­cal tri­als. Noth­ing came of that. Instead, as we lat­er saw, Oya­Gen basi­cal­ly sold the rights to Oya‑1 to Tonix Phar­ma­ceu­ti­cals. And now we’re learn­ing that the US gov­ern­ment has con­tin­ued to refuse to sup­port the devel­op­ment of this drug...an old drug that the phar­ma­ceu­ti­cal indus­try sim­ply won’t be able to charge exor­bi­tant prices to use:

...
Tonix Phar­ma­ceu­ti­cals, which is devel­op­ing a fibromyal­gia med­ica­tion that the com­pa­ny is hop­ing could be an effec­tive treat­ment for long Covid symp­toms, didn’t receive any fund­ing from NIH either, despite putting in an appli­ca­tion.

“I hope there are more ther­a­peu­tics tri­als. And I think that the ther­a­peu­tics tri­als can go hand in hand with the nat­ur­al his­to­ry kind of stud­ies like RECOVER,” Tonix CEO Seth Led­er­man said.

Patients and experts fear that if RECOVER is the extent of fed­er­al effort to study long Covid, the con­di­tion could fall into the long­stand­ing pat­tern of apa­thy and lack of urgency that has made break­throughs in chron­ic ill­ness treat­ment chal­leng­ing.

“It’s clear that there are a lot of peo­ple at the NIH who are ded­i­cat­ed and deter­mined, try­ing to fig­ure this out,” said Char­lie McCone, a patient rep­re­sen­ta­tive at RECOVER. As a result, “patients are con­fused” why only a hand­ful of clin­i­cal tri­als have been planned and none of those have launched yet, he said.
...

But it’s not sim­ply that RECOVER is pri­or­i­tiz­ing the study the long COVID dis­ease over the study of treat­ments. Even the stud­ies of the dis­ease are run­ning behind sched­ule. It’s like we have a $1.2 bil­lion smoke­screen:

...
Rather than pri­or­i­tiz­ing treat­ments from the start, the NIH used much of its long Covid fund­ing on a large-scale study to track long Covid symp­toms and learn how the dis­ease works. This choice has frus­trat­ed patients because thou­sands of oth­er stud­ies have already answered many major ques­tions about the con­di­tion.

“We didn’t need to recre­ate” exist­ing stud­ies that already answered these ques­tions, said Cole, the long Covid patient. Researchers have been com­pil­ing lists of com­mon symp­toms since sum­mer 2020, she said. For Cole, fatigue and brain fog are the most debil­i­tat­ing aspects of the con­di­tion.

And even the symp­tom study is mov­ing slow­ly, in part because the ini­tia­tive has failed to bring in healthy peo­ple who could be com­pared against long Covid patients. RECOVER quick­ly filled its slots for peo­ple who had Covid more than 30 days pri­or to their recruit­ment, but is still look­ing for peo­ple who were infect­ed recent­ly, study lead Leo­ra Hor­witz said in a state­ment. Most study sites closed enroll­ment for long Covid patients at the end of August 2022.

The major­i­ty of the sci­en­tif­ic find­ings to emerge from RECOVER so far have been based on small groups of patients or on elec­tron­ic health records, rather than on the thou­sands of peo­ple who signed up to par­tic­i­pate.
...

Final­ly, we get to this very inter­est­ing tie in to anoth­er long-stand­ing med­ical scan­dal: the refusal to rec­og­nize chron­ic-fatigue syn­drome (CFS). Keep in mind the sor­did his­to­ry tying the emer­gence of CFS to Lyme dis­ease, its direct ties US biowar­fare research, and the decades of appar­ent coverup of that research in rela­tion to Lyme dis­ease and CFS. And here we find the CFS com­mu­ni­ty rais­ing major alarm bells about the lack of any CFS-relat­ed con­sid­er­a­tions in the long COVID pop­u­la­tion despite the enor­mous symp­to­matic over­lap. Are we look­ing at anoth­er biowar­fare-relat­ed CFS coverup in action?

...
Many peo­ple with long Covid have sim­i­lar symp­toms to peo­ple with myal­gic encephalomyelitis or chron­ic fatigue syn­drome (ME/CFS), a debil­i­tat­ing con­di­tion that often fol­lows viral infec­tion. The defin­ing fea­ture of ME/CFS is intense fatigue and wors­en­ing of oth­er health issues after phys­i­cal or men­tal activ­i­ty. This symp­tom, known as post-exer­tion­al malaise, often occurs with a lag, which can make it tough for doc­tors to diag­nose — and even for patients to rec­og­nize them­selves.

...

This wors­en­ing of symp­toms hap­pens because a patient isn’t pro­duc­ing and using ener­gy in the same way as a healthy per­son, said Todd Dav­en­port, a pro­fes­sor at Uni­ver­si­ty of the Pacif­ic who hasstud­ied exer­cise and this con­di­tion. It’s an inter­nal change sim­i­lar to the whole-body exhaus­tion that a marathon run­ner might expe­ri­ence at the fin­ish line of their race.

A num­ber of past stud­ies and sur­veys of patients have demon­strat­ed how dan­ger­ous exer­cise can be for peo­ple with ME/CFS. Many patients told to exer­cise by their doc­tors lat­er dropped out of stud­ies or treat­ment reg­i­mens, cit­ing wors­en­ing symp­toms. One infa­mous tri­al that point­ed to exer­cise as a poten­tial treat­ment was lat­er dis­cred­it­ed as deeply flawed.

Study­ing exer­cise as a treat­ment could “frame long Covid as some­thing that can be over­come with grit and hard work,” said Jaime Seltzer, the direc­tor of sci­en­tif­ic and med­ical out­reach at MEAc­tion, argu­ing that such fram­ing is “unsound and eth­i­cal­ly trou­bling.”

...

Sci­en­tists and clin­i­cians on an NIH advi­so­ry com­mit­tee focused on reha­bil­i­ta­tion sim­i­lar­ly sug­gest­ed that post-exer­tion­al malaise could be a dan­ger­ous result of the tri­al, accord­ing to inter­nal emails shared with STAT and Muck­Rock. In response, NIH pro­gram offi­cer Antonel­lo Pun­turi­eri pushed back on the con­cerns. Pun­turi­eri cit­ed clin­i­cal guide­lines from the World Health Orga­ni­za­tion and a U.K. agency, even though both rec­om­mend against exer­cise for peo­ple with ME/CFS.

In response to these con­cerns, RECOVER set up inter­nal meet­ings includ­ing researchers in charge of the exer­cise study, patient rep­re­sen­ta­tives, and the initiative’s top advi­so­ry com­mit­tee. “Work is now under­way to fur­ther revise that pro­to­col” based on these meet­ings, the Duke Clin­i­cal Research Insti­tute said.

[Click to read the entire doc­u­ment.]

The study’s planned revi­sions will address con­cerns about patient safe­ty, such as mon­i­tor­ing for post-exer­tion­al malaise after exer­cise. But it’s unclear how the researchers will do this screen­ing, or whether ME/CFS doc­tors will be involved.

...

Topol and oth­er researchers com­piled a full table of oth­er treat­ment can­di­dates for a review paper pub­lished in Nature in Jan­u­ary. Experts on one of RECOVER’s advi­so­ry com­mit­tees com­piled a sim­i­lar list, for a paper pub­lished in March.

Giv­en “the num­ber of oth­er can­di­date treat­ments out there, I can’t imag­ine why you would choose grad­ed exer­cise ther­a­py,” said Julia Moore Vogel, a researcher at the Scripps Trans­la­tion­al Insti­tute liv­ing with long Covid, and co-author of the Nature review paper. Vogel is lead­ing a study of wear­able devices for long Covid, which will start with about 500 par­tic­i­pants despite plan­ning for up to 100,000.
...

It looks like the CFS com­mu­ni­ty is going to be grow­ing quite a bit in com­ing decades. Let’s hope this now-COVID-fueled patient pop­u­la­tion is able to have more suc­cess than the Lyme Dis­ease CFS suf­fer­ers in wran­gling the truth of this dis­ease and its ori­gins. Of course, there’s a poten­tial dark flip-side to this: the need to cov­er up the biowar­fare ori­gins of Lyme Dis­ease are even more urgent now that we have this whole new COVID CFS mega-scan­dal and relat­ed biowar­fare his­to­ry. Might we already be see­ing that cov­er up in action? It sure would explain the near com­plete lack of progress. It’s also a grim reminder that when we’re talk­ing about inex­plic­a­ble delays and obsta­cles put in front of cru­cial research need­ed to answer vital ques­tions, we should­n’t nec­es­sar­i­ly assume the answers to those cru­cial ques­tions aren’t already qui­et­ly known. And already qui­et­ly iden­ti­fied as too scan­dalous to pub­licly answer.

It’s hard to think of a scari­er update about the nature of the SARS-CoV­‑2 virus than the recent study reveal­ing the exten­sive DNA dam­age caused by the virus. But as we seem­ing­ly always dis­cov­er, things can always get worse and scari­er. And that brings us to the fol­low­ing pair of stud­ies that fur­ther elu­ci­date both the scope of the dam­age inflict­ed upon cel­lu­lar DNA and the unusu­al nature of this dam­age.

First, it turns out the DNA dam­age caused by SARS-CoV­‑2 was iden­ti­fied back in Sep­tem­ber in a study that com­pared the hearts of patients who died from COVID vs those who died from influen­za. As we should now expect, they found exten­sive signs of DNA dam­age in the hearts of the SARS-CoV­‑2 patients. As the researchers put it, the DNA dam­age pre­sent­ed was sim­i­lar to the way chron­ic dis­eases such as dia­betes or even can­cer pre­sent­ed. Inter­est­ing­ly, this DNA dam­age was much low­er in the influen­za patients.

Anoth­er intrigu­ing find­ing was that the inflam­ma­to­ry sig­nals in the hearts of the COVID patients appeared to be sup­pressed. Keep in mind that we’ve seen an asso­ci­a­tion between myocardi­tis — inflam­ma­tion of the heart — in both COVID patients and also peo­ple who have just received the vac­cine. Also keep in mind that the peo­ple used in this study obvi­ous­ly had severe cas­es of COVID since they died and may not be rep­re­sen­ta­tive of the impact of the virus on rel­a­tive­ly healthy peo­ple. It’s part of what will make this all a very inter­est­ing avenue of fur­ther inves­ti­ga­tion.

Next, there was a study pub­lished back in March that looks at a dif­fer­ent form of dam­age inflict­ed upon the cel­l’s nucle­us: a dra­mat­ic repro­gram­ming of the cel­l’s 3d-struc­tu “epigenome”. Put sim­ply, while all cells pos­sess all of your genes, only some genes are made “avail­able” in each cell-type and SARS-CoV­‑2 appears to change which genes are “avail­able” or not in a high­ly chaot­ic man­ner. It’s, again, the kind of phe­nom­e­na we expect to see in can­cer. Oh, and it turns out this epi­ge­net­ic repro­gram­ming isn’t seen in the oth­er com­mon-cold coro­n­avirus­es they test­ed. We just keep find­ing SARS-CoV­‑2 nov­el ‘fea­tures’.

Ok, first, here’s as look at that Sep­tem­ber 2022 paper find­ing exten­sive DNA dam­age in the hearts of SARS-CoV­‑2 patients, but not in influen­za patients, sug­gest­ing that there’s some­thing unusu­al about how SARS-CoV­‑2 attacks the cel­l’s DNA. But not entire­ly unusu­al. It’s the same kind of DNA dam­age sig­nal seen in dia­betes and can­cer:

Bris­bane Times

Unlike flu, COVID-19 attacks DNA in the heart: new research

By Stu­art Layt
Sep­tem­ber 29, 2022 — 8.07pm

Direct research on the hearts of COVID-19 patients who have died from the dis­ease has revealed they sus­tained DNA dam­age in a way com­plete­ly unlike how influen­za affects the body.

...

The work was an inter­na­tion­al col­lab­o­ra­tion led by researchers from Aus­tralia, with Dr Arutha Kulas­inghe from the Uni­ver­si­ty of Queensland’s Dia­man­ti­na Insti­tute and Ning Liu from Victoria’s Wal­ter and Eliza Hall Insti­tute of Med­ical Research co-lead authors.

Kulas­inghe said the research is only a pre­lim­i­nary step because the sam­ple size was so small – the hearts of just sev­en COVID-19 patients from Brazil were stud­ied for the paper, along with hearts from peo­ple who had died from influen­za and a con­trol group.

But he said their find­ings gave them insight for the first time as to why the heart was being affect­ed by COVID-19 but not but severe influen­za.

“We found a lot of DNA dam­age that was unique to the COVID-19 patients which wasn’t present in the flu patients,” he said.

“So in this study, COVID-19 and flu look very dif­fer­ent in the way they affect the heart.”

The fact that COVID-19 affects people’s hearts has been observed both anec­do­tal­ly and in a num­ber of stud­ies since the start of the pan­dem­ic, but the under­ly­ing cause is still yet to be deter­mined.

One rea­son is that it is extreme­ly dif­fi­cult to get tis­sue sam­ples from a liv­ing heart, so researchers have to wait for peo­ple to die from the dis­ease and for their heart to be able to be stud­ied.

Kulas­inghe said he and his col­leagues found that rather than extreme inflam­ma­tion which they had expect­ed to find, inflam­ma­tion sig­nals had been sup­pressed in the hearts of the COVID-19 patients, while mark­ers for DNA dam­age and repair were much high­er than in peo­ple who had died from the flu.

“So the indi­ca­tions here are that there’s DNA dam­age here, it’s not inflam­ma­tion, there’s some­thing else going on that we need to fig­ure out,” Kulas­inghe said.

He said the way the DNA dam­age pre­sent­ed was sim­i­lar to the way chron­ic dis­eases such as dia­betes or even can­cer pre­sent­ed, with the tis­sues of the heart putting out DNA dam­age sig­nals.

...

A fed­er­al sen­ate inquiry into the issue is cur­rent­ly tak­ing sub­mis­sions, with the inquiry’s Chair, south-west­ern Syd­ney Labor MP Dr Mike Free­lander, say­ing say­ing ear­li­er this month that he was “very con­cerned” about high­er-than-nor­mal deaths in the pan­dem­ic and that more research was need­ed to ascer­tain why.

Kulas­inghe said they hoped their find­ings could be built upon to devel­op meth­ods to estab­lish who was at risk of seri­ous com­pli­ca­tions from COVID-19, so they could be treat­ed ear­ly.

“Ide­al­ly in the future if you have car­dio­vas­cu­lar dis­ease, if you’re obese or have oth­er com­pli­ca­tions, and you’ve got a sig­na­ture in your blood that indi­cates you are at risk of severe dis­ease, then we can risk-strat­i­fy patients when they are diag­nosed,” he said.

“Our chal­lenge now is to draw a clin­i­cal find­ing from this, which we can’t at this stage, but it’s a real­ly fun­da­men­tal bio­log­i­cal dif­fer­ence we’re observ­ing [between COVID-19 and flu] which we need to val­i­date with larg­er stud­ies.”

The research has been pub­lished in the jour­nal Immunol­o­gy.

————

“Unlike flu, COVID-19 attacks DNA in the heart: new research” By Stu­art Layt; Bris­bane Times; 09/29/2022

“The fact that COVID-19 affects people’s hearts has been observed both anec­do­tal­ly and in a num­ber of stud­ies since the start of the pan­dem­ic, but the under­ly­ing cause is still yet to be deter­mined.”

A con­nec­tion between COVID and heart trou­bles isn’t new. We’ve repeat­ed­ly heard such anec­dotes. What is new is the dis­cov­ery that this heart dam­age is not seen in influen­za patients and is direct­ly relat­ed to DNA dam­age. It’s fur­ther evi­dence that DNA dam­age is part of what is dri­ving a num­ber of COVID’s unusu­al fea­tures. At least unusu­al for a viral infec­tion. It’s the same kind of DNA dam­age seen in chron­ic dis­eases asso­ci­at­ed with chron­ic inflam­ma­tion like dia­betes and can­cer:

...
Kulas­inghe said the research is only a pre­lim­i­nary step because the sam­ple size was so small – the hearts of just sev­en COVID-19 patients from Brazil were stud­ied for the paper, along with hearts from peo­ple who had died from influen­za and a con­trol group.

But he said their find­ings gave them insight for the first time as to why the heart was being affect­ed by COVID-19 but not but severe influen­za.

“We found a lot of DNA dam­age that was unique to the COVID-19 patients which wasn’t present in the flu patients,” he said.

“So in this study, COVID-19 and flu look very dif­fer­ent in the way they affect the heart.”

...

One rea­son is that it is extreme­ly dif­fi­cult to get tis­sue sam­ples from a liv­ing heart, so researchers have to wait for peo­ple to die from the dis­ease and for their heart to be able to be stud­ied.

Kulas­inghe said he and his col­leagues found that rather than extreme inflam­ma­tion which they had expect­ed to find, inflam­ma­tion sig­nals had been sup­pressed in the hearts of the COVID-19 patients, while mark­ers for DNA dam­age and repair were much high­er than in peo­ple who had died from the flu.

“So the indi­ca­tions here are that there’s DNA dam­age here, it’s not inflam­ma­tion, there’s some­thig else going on that we need to fig­ure out,” Kulas­inghe said.

He said the way the DNA dam­age pre­sent­ed was sim­i­lar to the way chron­ic dis­eases such as dia­betes or even can­cer pre­sent­ed, with the tis­sues of the heart putting out DNA dam­age sig­nals.

...

“Our chal­lenge now is to draw a clin­i­cal find­ing from this, which we can’t at this stage, but it’s a real­ly fun­da­men­tal bio­log­i­cal dif­fer­ence we’re observ­ing [between COVID-19 and flu] which we need to val­i­date with larg­er stud­ies.”
...

How many oth­er tis­sues is SARS-CoV­‑2 induc­ing this kind of DNA dam­age? Don’t for­get that one of the dis­turb­ing fea­tures of SARS-CoV­‑2 was how wide­spread the infec­tion could get across a wide vari­ety of tis­sues. DNA dam­age in the heart might kill you in the short run, but that does­n’t mean there isn’t all sorts of long-term DNA dam­age in oth­er tis­sues tak­ing place. How about in the tes­ti­cles and ovaries? Any DNA dam­age tak­ing place there? Let’s hope some­one is inves­ti­gat­ing that.

But as the fol­low­ing arti­cle about research pub­lished back in March reminds us, there’s more than one way for SARS-CoV­‑2 to dam­age your cel­l’s DNA. Because it turns out SARS-CoV­‑2 has anoth­er DNA-dam­ag­ing trait: dra­mat­ic epige­nom­ic repro­gram­ming. Yes, SARS-CoV­‑2 appears to sig­nif­i­cant­ly dis­rupt the 3D chro­matin struc­ture inside the cell nucle­us that deter­mines which genes are “avail­able” inside a cell. Keep in mind that ALL cells pos­sess ALL genes, but WHICH gene is ‘avail­able’ is deter­mined by the cell type. In oth­er words, if you dra­mat­i­cal­ly change which genes are ‘turned on’ in a cell you’re effec­tive­ly scram­bling its mode of oper­a­tion and turn it into anoth­er kind of dys­func­tion­al cell. Some­thing much clos­er to a can­cer cell, which has epige­nom­ic repro­gram­ming as one of its hall­mark fea­tures. So as we learn more and more about the dam­age SARS-CoV­‑2 is going to the genet­ic con­tents of the cells it attacks, keep in mind that it’s not just induc­ing DNA muta­tions. It’s also scram­bling the high­er-lev­el reg­u­la­to­ry genet­ic struc­ture:

News-Medical.net

Epigenome repro­gram­ming after SARS-CoV­‑2 infec­tion

By Neha Math­ur Mar 27 2023
Reviewed by Lily Ram­sey, LLM

In a recent arti­cle in pub­lished in the jour­nal Nature Micro­bi­ol­o­gy, researchers in Texas, Unit­ed States (US) per­formed a three-dimen­sion­al (3D) eval­u­a­tion of severe acute res­pi­ra­to­ry syn­drome coro­n­avirus 2 (SARS-CoV­‑2) infect­ed human cells to show a direct cell-autonomous effect elicit­ed by SARS-CoV­‑2 on the host chro­matin.

The study aimed at improv­ing the under­stand­ing of coro­n­avirus dis­ease 2019 (COVID-19)-related per­tur­ba­tions in the genome and epigenome of a host cell.

Back­ground

The 3D fold­ing of chro­matin in mam­mals, includ­ing humans, influ­ences deoxyri­bonu­cle­ic acid (DNA) repli­ca­tion, recom­bi­na­tion, DNA dam­age repair, and tran­scrip­tion. It is a key deter­mi­nant of how human cells act and func­tion. Virus­es, includ­ing SARS-CoV­‑2, antag­o­nize host defense by rewiring their chro­matin archi­tec­ture, which typ­i­cal­ly has sev­er­al lay­ers, e.g., A/B com­part­ments, chro­matin loops, and topo­log­i­cal asso­ci­at­ing domains (TADs).

The A and B com­part­ments super­im­pose tran­scrip­tion­al­ly active euchro­matin and rel­a­tive­ly inac­tive het­e­rochro­matin, respec­tive­ly. How­ev­er, stud­ies have bare­ly inves­ti­gat­ed these effects.

In addi­tion, epi­ge­net­ic alter­ations impact gene expres­sion and result­ing phe­no­types in the long term. Thus, a sneak peek into the inter­ac­tions between the virus, host chro­matin, and epigenome could help find nov­el meth­ods to fight SARS-CoV­‑2 in the acute phase. In addi­tion, it could unrav­el the mol­e­c­u­lar basis of post-acute SARS-CoV­‑2 seque­lae or long COVID and sub­se­quent­ly mit­i­gate it.

About the study

At 24 hours post-infec­tion (24 hpi), human A549 cells express­ing angiotensin-con­vert­ing enzyme 2 (ACE2), infect­ed with SARS-CoV­‑2 at a mul­ti­plic­i­ty of infec­tion (MOI) of 0.1, had high lev­els of infec­tion. This was shown by ribonu­cle­ic acid-sequenc­ing (RNA-seq). Immuno­flu­o­res­cence of the SARS-CoV­‑2 spike (S) gly­co­pro­tein also sub­stan­ti­at­ed an ele­vat­ed infec­tion ratio.

...

Results

The Hi‑C analy­sis showed exten­sive alter­ations in the hosts’ 3D genome after SARS-CoV­‑2 infec­tion. The researchers also plot­ted a Pear­son cor­re­la­tion map of their Hi‑C analy­sis that reaf­firmed these changes along­side indi­cat­ing mod­i­fied chro­matin com­part­men­tal­iza­tion.

A focused view of the ~0.7 Mb region showed a weak­en­ing of the rec­tan­gle-shaped chro­matin domains and dereg­u­la­tion of chro­matin loops. While SARS-CoV­‑2 prompt­ed a glob­al decline in near-diag­o­nal short-range chro­matin con­tacts (28 megabas­es) were often dereg­u­lat­ed.

Fur­ther, a P(s) curve showed that SARS-CoV­‑2 elicit­ed mod­est and enhanced inter­ac­tions in mid­dle-to-long-dis­tance con­tacts (~560 kb to 8.9 Mb) and far-posi­tioned regions, respec­tive­ly.

Fold changes in inter-chro­mo­so­mal inter­ac­tions or trans-vs-cis con­tact ratios also depict­ed the effect of SARS-CoV­‑2 infec­tion on inter-chro­mo­so­mal con­tacts. The enhance­ment of inter- and intra-chro­mo­so­mal inter­ac­tions indi­cat­ed changes in chro­matin com­part­men­tal­iza­tion. Con­se­quent­ly, prin­ci­pal com­po­nent analy­sis (PCA) of a 100-kb bin on Hi‑C back­ground showed notice­able defects of chro­matin com­part­men­tal­iza­tion in virus-infect­ed cells.

The total PCA E1 scores quan­ti­fy­ing E1 changes in ~30% of genom­ic regions showed a wide­spread dimin­ish­ing of the A com­part­ment, A‑to‑B switch­ing, or strength­en­ing of the B com­part­ment post-SARS-CoV­‑2 infec­tion.

Among all, A to weak­er A changes were the most com­mon and occurred in ~18% of the genome, which indi­cat­ed that SARS-CoV­‑2 exten­sive­ly weak­ened the host euchro­matin.

Fur­ther analy­sis showed that the ‘B‑ing’ and ‘A‑ing’ genom­ic regions were his­tor­i­cal­ly enriched in active chro­matin mark­ers (e.g., H3K27ac) and repres­sive his­tone mark­ers, espe­cial­ly H3K27me3. Unex­pect­ed­ly, SARS-CoV­‑2 infec­tion selec­tive­ly mod­i­fied the H3K4me3 mark­er of phy­tochrome inter­act­ing fac­tors (PIF) gene pro­mot­ers, sug­gest­ing unap­pre­ci­at­ed mech­a­nisms at these pro­mot­ers that con­fer devi­at­ing inflam­ma­tion in COVID-19.

A flawed chro­matin com­part­men­tal­iza­tion like­ly caused the his­tor­i­cal­ly well-par­ti­tioned A or B com­part­ments to lose their iden­ti­ty. A sad­dle plot illus­trat­ing inter-com­part­ment chro­matin inter­ac­tions across the genome showed these glob­al changes.

The authors also not­ed weak­ened com­part­men­tal­iza­tion between chro­mo­somes. For instance, in chro­mo­somes 17 & 18, while A–B inter­ac­tions were ampli­fied, A–A/B–B homo­typ­ic inter­ac­tions appeared to have become com­pro­mised.

More­over, SARS-CoV­‑2 infec­tion mech­a­nis­ti­cal­ly deplet­ed the cohesin com­plex in a per­va­sive but selec­tive man­ner from intra-TAD regions. These changes pro­vid­ed a mol­e­c­u­lar expla­na­tion for the weak­en­ing of intra-TAD inter­ac­tions.

It sup­port­ed the notion that defec­tive cohesin loop extru­sion inside TADs releas­es this chro­matin to engage in long-dis­tance asso­ci­a­tions. Intrigu­ing­ly, chro­matin in SARS-CoV-2-infect­ed cells exhib­it­ed a high­er fre­quen­cy of long-dis­tance intra-chro­mo­so­mal and inter-chro­mo­so­mal inter­ac­tions.

Con­clu­sions

SARS-CoV­‑2 infec­tion marked­ly restruc­tured 3D host chro­matin, fea­tur­ing wide­spread com­part­ment A weak­en­ing and A–B mix­ing and glob­al reduc­tion in intra-TAD chro­matin con­tacts.

How­ev­er, it is still unknown exact­ly how SARS-COV‑2 infec­tion restruc­tures host chro­matin. Like­ly, open read­ing frame 8 (ORF8) dis­rupts the host epigenome, sug­gest­ing that some viral fac­tors are involved in host chro­matin rewiring.

It also altered the host epigenome, includ­ing a glob­al reduc­tion in active chro­matin mark H3K27ac and a spe­cif­ic increase in H3K4me3 at pro-inflam­ma­to­ry gene pro­mot­ers. Intrigu­ing­ly, all these host chro­matin alter­ations were unique to SARS-CoV­‑2 infec­tion, and oth­er com­mon-cold coro­n­avirus­es or immune stim­uli did not elic­it these changes.

Jour­nal ref­er­ence:

Wang, R. et al. (2023) “SARS-CoV­‑2 restruc­tures host chro­matin archi­tec­ture”, Nature Micro­bi­ol­o­gy. doi: 10.1038/s41564-023–01344‑8. https://www.nature.com/articles/s41564-023–01344‑8

———–

“Epigenome repro­gram­ming after SARS-CoV­‑2 infec­tion” By Neha Math­ur; News-Medical.net; 03/27/2023

“The 3D fold­ing of chro­matin in mam­mals, includ­ing humans, influ­ences deoxyri­bonu­cle­ic acid (DNA) repli­ca­tion, recom­bi­na­tion, DNA dam­age repair, and tran­scrip­tion. It is a key deter­mi­nant of how human cells act and func­tion. Virus­es, includ­ing SARS-CoV­‑2, antag­o­nize host defense by rewiring their chro­matin archi­tec­ture, which typ­i­cal­ly has sev­er­al lay­ers, e.g., A/B com­part­ments, chro­matin loops, and topo­log­i­cal asso­ci­at­ing domains (TADs).”

It’s not just dam­age to the DNA sequences. SARS-CoV­‑2 also induces mas­sive alter­ations to the cel­l’s epigenome by essen­tial­ly repro­gram­ming the chro­matin that deter­mines what DNA in a cell is “avail­able” to be con­vert­ed into pro­teins or oth­er­wise inter­act with the cel­l’s reg­u­la­to­ry path­ways. In oth­er words, SARS-CoV­‑2 intro­duces a kind of reg­u­la­to­ry chaos at the cel­lu­lar lev­el:

...
Results

The Hi‑C analy­sis showed exten­sive alter­ations in the hosts’ 3D genome after SARS-CoV­‑2 infec­tion. The researchers also plot­ted a Pear­son cor­re­la­tion map of their Hi‑C analy­sis that reaf­firmed these changes along­side indi­cat­ing mod­i­fied chro­matin com­part­men­tal­iza­tion.

...

Fur­ther analy­sis showed that the ‘B‑ing’ and ‘A‑ing’ genom­ic regions were his­tor­i­cal­ly enriched in active chro­matin mark­ers (e.g., H3K27ac) and repres­sive his­tone mark­ers, espe­cial­ly H3K27me3. Unex­pect­ed­ly, SARS-CoV­‑2 infec­tion selec­tive­ly mod­i­fied the H3K4me3 mark­er of phy­tochrome inter­act­ing fac­tors (PIF) gene pro­mot­ers, sug­gest­ing unap­pre­ci­at­ed mech­a­nisms at these pro­mot­ers that con­fer devi­at­ing inflam­ma­tion in COVID-19.

...

The authors also not­ed weak­ened com­part­men­tal­iza­tion between chro­mo­somes. For instance, in chro­mo­somes 17 & 18, while A–B inter­ac­tions were ampli­fied, A–A/B–B homo­typ­ic inter­ac­tions appeared to have become com­pro­mised.

More­over, SARS-CoV­‑2 infec­tion mech­a­nis­ti­cal­ly deplet­ed the cohesin com­plex in a per­va­sive but selec­tive man­ner from intra-TAD regions. These changes pro­vid­ed a mol­e­c­u­lar expla­na­tion for the weak­en­ing of intra-TAD inter­ac­tions.

It sup­port­ed the notion that defec­tive cohesin loop extru­sion inside TADs releas­es this chro­matin to engage in long-dis­tance asso­ci­a­tions. Intrigu­ing­ly, chro­matin in SARS-CoV-2-infect­ed cells exhib­it­ed a high­er fre­quen­cy of long-dis­tance intra-chro­mo­so­mal and inter-chro­mo­so­mal inter­ac­tions.
...

Intrigu­ing­ly, this restruc­tur­ing of the cel­l’s epigenome isn’t seen as a con­se­quence for the com­mon-cold coro­n­avirus­es they also test­ed. So like the DNA dam­age, this epigenome restruc­tur­ing appears to be anoth­er unusu­al ‘fea­ture’ of this virus:

...
Con­clu­sions

SARS-CoV­‑2 infec­tion marked­ly restruc­tured 3D host chro­matin, fea­tur­ing wide­spread com­part­ment A weak­en­ing and A–B mix­ing and glob­al reduc­tion in intra-TAD chro­matin con­tacts.

How­ev­er, it is still unknown exact­ly how SARS-COV‑2 infec­tion restruc­tures host chro­matin. Like­ly, open read­ing frame 8 (ORF8) dis­rupts the host epigenome, sug­gest­ing that some viral fac­tors are involved in host chro­matin rewiring.

It also altered the host epigenome, includ­ing a glob­al reduc­tion in active chro­matin mark H3K27ac and a spe­cif­ic increase in H3K4me3 at pro-inflam­ma­to­ry gene pro­mot­ers. Intrigu­ing­ly, all these host chro­matin alter­ations were unique to SARS-CoV­‑2 infec­tion, and oth­er com­mon-cold coro­n­avirus­es or immune stim­uli did not elic­it these changes.
...

This is a good time to recall how one of the big hopes from the out­set of this pan­dem­ic was that we might even­tu­al­ly see this virus evolve into a rel­a­tive­ly harm­less com­mon-cold coro­n­avirus as has hap­pened in the past. Also keep in mind that when we’re assess­ing the results of these stud­ies we should dif­fer­en­ti­ate between severe­ly ill patients and those with just a mild infec­tion. in oth­er words, it’s not clear at this point if the kind of pro­found DNA dam­age we’re see­ing in these stud­ies is spe­cif­ic to severe cas­es of COVID. And it’s cer­tain­ly pos­si­ble that the DNA dam­age real­ly is lim­it­ed to severe cas­es. Let’s hope that’s the case. But if not, it’s going to real­ly hard to get a sense of the long-term dam­age cre­at­ed by this virus. Again, are testis and ovaries impact­ed too? Because we real­ly don’t want to see DNA dam­age in those tis­sues. More stud­ies are clear­ly nec­es­sary. And, depend­ing on the find­ings, per­haps a few more ‘can­cer moon­shots’.

@Pterrafractyl–

Bril­liant and ter­ri­fy­ing!

Keep up the mag­nif­i­cent work!

Best,

Dave

We just got anoth­er myocardi­tis-relat­ed COVID update. The head­line find­ings are great. But as we should expect, upon clos­er exam­i­na­tion not only are the find­ings not great but appear to be fur­ther con­fir­ma­tion of anoth­er COVID-relat­ed night­mare sce­nario. A night­mare sce­nario not dri­ven by the SARS-CoV­‑2 virus itself but rather the lipid nanopar­ti­cle (LNP) deliv­ery vehi­cles used in the Pfiz­er and Mod­er­na mRNA vac­cines:

Researchers at Yale just pub­lished a study based on 23 young men who expe­ri­ence myocardi­tis — inflam­ma­tion of the heart — fol­low­ing their COVID vac­ci­na­tions. Accord­ing to their find­ings, no signs of an autoim­mune response attack­ing the heart was seen. That’s the good news. Based on those find­ings, it is now being report­ed as a con­fir­ma­tion that the mRNA vac­cines aren’t the cause of the myocardi­tis

And indeed it is good to not find signs of an autoim­mune response attack­ing the heart. But as we’re going to see, it’s not like the study did­n’t see signs of a vac­cine-induced inflam­ma­to­ry response in the hearts of these patients. They just did­n’t see a spe­cif­ic autoim­mune response gen­er­at­ed by the vac­cine. They still saw an gen­er­al innate immune response and in fact that’s what they attrib­uted the myocardi­tis to: an over­ac­tive innate immune response some­how trig­gered by the vac­cine.

Beyond that, as we’re going to see when we look close­ly at the Dis­cus­sion sec­tion of this new study, we find that the authors explic­it­ly spec­u­late that some sort of “com­pound role of the adju­vant deliv­ery plat­form in syn­er­gy with vac­cine-vec­tored anti­gens is more like­ly the dri­ver of an exag­ger­at­ed immune cytokine response dri­ving car­diac pathol­o­gy after vac­ci­na­tion in sus­cep­ti­ble indi­vid­u­als.” A “com­pound role of the adju­vant deliv­ery plat­form in syn­er­gy with vac­cine-vec­tored anti­gens” is anoth­er way of say­ing that the com­bined effects of the immune sys­tem response to both the vac­cine-spe­cif­ic tar­get (the COVID spike pro­tein, in this case) and the immune response to the LNP deliv­ery vehi­cle. So this new study that is being tout­ed as a val­i­da­tion of the safe­ty of mRNA vac­cines mere­ly found that the side effects aren’t exclu­sive­ly trig­gered by an autoim­mune response but are instead also heav­i­ly dri­ven by some sort of “adju­vant” innate immune response trig­gered by the LNPs.

This is a good time to recall that 2016 STAT News arti­cle describ­ing the shift in Mod­er­na’s mRNA ther­a­peu­tics research towards vac­cine devel­op­ment. As we saw, a num­ber of indus­try observers com­ment­ed on how the shift like­ly reflect­ed long-stand­ing indus­try frus­tra­tions with the side-effects of mRNA deliv­ery mak­ing vac­cines — where the side effects of an immune response are desired and hypo­thet­i­cal­ly as lit­tle as a sin­gle shot — mak­ing vac­cines an obvi­ous path to final­ly bring­ing an mRNA prod­uct to mar­ket. This is also a good time to recall how the US gov­ern­ment cur­rent­ly ONLY allows the mRNA vac­cines for any boost­er shots after your first boost­er.

Final­ly, as we’re also going to see, the sig­nal pro­file of innate inflam­ma­to­ry sig­nals that this Yale team found in the these patients — ele­vat­ed lev­els of cir­cu­lat­ing inter­leukins (IL-1ß, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix met­al­lo­pro­teas­es (MMP1, MMP8, MMP9, and TIMP1) — sure looks an awful lot like the sig­nal pro­file of inflam­ma­to­ry respons­es induced by LNPs alone found in mice pub­lished in a study back in Decem­ber of 2021. That’s part of the con­text of this lat­est study: it’s fol­low­ing a grow­ing num­ber of stud­ies show­ing the pow­er­ful inflam­ma­to­ry response to LNPs alone. When the Yale authors refer to the “adju­vant deliv­ery plat­form”, that pow­er­ful immune response to LNPs is what they are talk­ing about.

So if you want to pro­tect your­self from the DNA-dam­ag­ing effects of COVID and you hap­pen to be in the US, you can roll the immuno­log­i­cal dice with the mRNA vac­cines or you can go with­out pro­tec­tion. Those are the only two options cur­rent­ly avail­able. Best of luck with that:

STAT News

What explains rare heart con­di­tion among young men after Covid vac­cines? A new study offers clues

By Elaine Chen May 5, 2023

A new study sheds light on what may be caus­ing very rare cas­es of heart inflam­ma­tion in young men after receiv­ing an mRNA Covid vac­cine.

Ear­ly hypothe­ses as to what was dri­ving the heart inflam­ma­tion, called myocardi­tis, includ­ed an aller­gic response to the vac­cine, vac­cine-induced anti­bod­ies, or an autoim­mune response in which the immune sys­tem attacks the body’s own healthy tis­sue by mis­take. But the study, pub­lished Fri­day in Sci­ence Immunol­o­gy, doesn’t find evi­dence for any of these mech­a­nisms.

Instead, when look­ing at cas­es in 23 patients, the researchers found signs of a revved-up immune sys­tem dri­ven by inflam­ma­to­ry pro­teins.

“We were a lit­tle relieved that what we found was the inflam­ma­tion-induced myocardi­tis,” said Akiko Iwasa­ki, one of the authors and a pro­fes­sor of immuno­bi­ol­o­gy at Yale Uni­ver­si­ty. In par­tic­u­lar, if the cas­es were dri­ven by an autoim­mune response, they “would be a lit­tle bit more dif­fi­cult to treat and deal with.”

...

The researchers behind the study explored each of the lead­ing pos­si­bil­i­ties as to the root cause of myocardi­tis after vac­ci­na­tion. When they looked at blood sam­ples, the researchers didn’t see greater anti­body or neu­tral­iz­ing anti­body lev­els in patients who had devel­oped myocardi­tis com­pared with a con­trol group, sug­gest­ing the heart con­di­tion wasn’t dri­ven by vac­cine-induced anti­bod­ies.

The researchers then looked for signs of an autoim­mune response. They didn’t find any autoan­ti­bod­ies, and the pat­tern of immune cells that pro­lif­er­at­ed also didn’t look like the pat­tern typ­i­cal­ly seen in an autoim­mune response.

They then pro­ceed­ed to do more detailed analy­sis of the patients’ blood sam­ples. They found ele­vat­ed lev­els of inflam­ma­to­ry pro­teins, such as ones called inter­leukin-15, and also found a pro­lif­er­a­tion of tis­sue-dam­ag­ing immune cells that looked to be acti­vat­ed by inflam­ma­to­ry pro­teins. Tak­en togeth­er, the find­ings sup­port the idea that the myocardi­tis cas­es were dri­ven by an over­ac­tive immune sys­tem.

...

But it’s still unclear why the rare cas­es of myocardi­tis are con­cen­trat­ed in young men, said Daniela Cihako­va, a pro­fes­sor of pathol­o­gy at Johns Hop­kins Uni­ver­si­ty who is unaf­fil­i­at­ed with the study. “What we need to under­stand in a broad way is — how is the response of that age cat­e­go­ry and that sex cat­e­go­ry dif­fer­ent from oth­er cat­e­gories in vac­ci­na­tion?”

————

“What explains rare heart con­di­tion among young men after Covid vac­cines? A new study offers clues” by Elaine Chen; STAT News; 05/05/2023

““We were a lit­tle relieved that what we found was the inflam­ma­tion-induced myocardi­tis,” said Akiko Iwasa­ki, one of the authors and a pro­fes­sor of immuno­bi­ol­o­gy at Yale Uni­ver­si­ty. In par­tic­u­lar, if the cas­es were dri­ven by an autoim­mune response, they “would be a lit­tle bit more dif­fi­cult to treat and deal with.””

It’s a relief! That’s the head­line find­ing in this new­ly pub­lished Yale study based on 23 patients. They did­n’t find signs of an autoim­mune response gen­er­at­ed by the mRNA vac­cines. Nope. Instead, they found signs of a revved up immune sys­tem dri­ven by inflam­ma­to­ry pro­teins. So they did find an immune response, just not an autoim­mune response. That’s the basis for the col­lec­tive “phew!”

...
Ear­ly hypothe­ses as to what was dri­ving the heart inflam­ma­tion, called myocardi­tis, includ­ed an aller­gic response to the vac­cine, vac­cine-induced anti­bod­ies, or an autoim­mune response in which the immune sys­tem attacks the body’s own healthy tis­sue by mis­take. But the study, pub­lished Fri­day in Sci­ence Immunol­o­gy, doesn’t find evi­dence for any of these mech­a­nisms.

Instead, when look­ing at cas­es in 23 patients, the researchers found signs of a revved-up immune sys­tem dri­ven by inflam­ma­to­ry pro­teins.

...

>The researchers behind the study explored each of the lead­ing pos­si­bil­i­ties as to the root cause of myocardi­tis after vac­ci­na­tion. When they looked at blood sam­ples, the researchers didn’t see greater anti­body or neu­tral­iz­ing anti­body lev­els in patients who had devel­oped myocardi­tis com­pared with a con­trol group, sug­gest­ing the heart con­di­tion wasn’t dri­ven by vac­cine-induced anti­bod­ies.

The researchers then looked for signs of an autoim­mune response. They didn’t find any autoan­ti­bod­ies, and the pat­tern of immune cells that pro­lif­er­at­ed also didn’t look like the pat­tern typ­i­cal­ly seen in an autoim­mune response.

They then pro­ceed­ed to do more detailed analy­sis of the patients’ blood sam­ples. They found ele­vat­ed lev­els of inflam­ma­to­ry pro­teins, such as ones called inter­leukin-15, and also found a pro­lif­er­a­tion of tis­sue-dam­ag­ing immune cells that looked to be acti­vat­ed by inflam­ma­to­ry pro­teins. Tak­en togeth­er, the find­ings sup­port the idea that the myocardi­tis cas­es were dri­ven by an over­ac­tive immune sys­tem.
...

But what was it that cre­at­ed the over­ac­tive innate immune response in the first place? Was it just some­thing about these patients? Or was the vac­cine also play­ing a role? That’s all dis­missed in the report­ing we’re see­ing on this study. And yet, when we look more close­ly at the study, we find they do actu­al­ly address the immune respons­es trig­gered by LNPs as pos­si­bly play­ing a role in myocardi­tis. As they put it in the study’s Dis­cus­sion sec­tion, “a com­pound role of the adju­vant deliv­ery plat­form in syn­er­gy with vac­cine-vec­tored anti­gens is more like­ly the dri­ver of an exag­ger­at­ed immune cytokine response dri­ving car­diac pathol­o­gy after vac­ci­na­tion in sus­cep­ti­ble indi­vid­u­als”:

Sci­ence Immunol­o­gy

Cytokinopa­thy with aber­rant cyto­tox­ic lym­pho­cytes and profi­brot­ic myeloid response in SARS-CoV­‑2 mRNA vaccine–associated myocardi­tis

Anis Bar­ma­da, Jon Klein, Anjali Ramaswamy, Nina N. Brod­sky, Jil­lian R. Jay­cox, Has­san Sheikha, Kate M. Jones, Vic­to­ria Habet, Melis­sa Camp­bell, Tomokazu S. Sum­i­da, Amy Kon­torovich, Dusan Bogunovic, Car­los R. Oliveira, Jere­my Steele, E. Kevin Hall, Mario Pena-Her­nan­dez, Val­ter Mon­teiro, Car­oli­na Lucas, Aaron M. Ring, Saad B. Omer, Akiko Iwasa­ki, Inci Yildirim, and Car­rie L. Lucas

05/05/2023 Vol 8, Issue 83

Abstract

Rare immune-medi­at­ed car­diac tis­sue inflam­ma­tion can occur after vac­ci­na­tion, includ­ing after SARS-CoV­‑2 mRNA vac­cines. How­ev­er, the under­ly­ing immune cel­lu­lar and mol­e­c­u­lar mech­a­nisms dri­ving this pathol­o­gy remain poor­ly under­stood. Here, we inves­ti­gat­ed a cohort of patients who devel­oped myocardi­tis and/or peri­cardi­tis with ele­vat­ed tro­ponin, B‑type natri­uret­ic pep­tide, and C‑reactive pro­tein lev­els as well as car­diac imag­ing abnor­mal­i­ties short­ly after SARS-CoV­‑2 mRNA vac­ci­na­tion. Con­trary to ear­ly hypothe­ses, patients did not demon­strate fea­tures of hyper­sen­si­tiv­i­ty myocardi­tis, nor did they have exag­ger­at­ed SARS-CoV‑2–specific or neu­tral­iz­ing anti­body respons­es con­sis­tent with a hyper­im­mune humoral mech­a­nism. We addi­tion­al­ly found no evi­dence of car­diac-tar­get­ed autoan­ti­bod­ies. Instead, unbi­ased sys­tem­at­ic immune serum pro­fil­ing revealed ele­va­tions in cir­cu­lat­ing inter­leukins (IL-1ß, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix met­al­lo­pro­teas­es (MMP1, MMP8, MMP9, and TIMP1). Sub­se­quent deep immune pro­fil­ing using sin­gle-cell RNA and reper­toire sequenc­ing of periph­er­al blood mononu­clear cells dur­ing acute dis­ease revealed expan­sion of acti­vat­ed CXCR3+ cyto­tox­ic T cells and NK cells, both phe­no­typ­i­cal­ly resem­bling cytokine-dri­ven killer cells. In addi­tion, patients dis­played sig­na­tures of inflam­ma­to­ry and profi­brot­ic CCR2+ CD163+ mono­cytes, cou­pled with ele­vat­ed serum-sol­u­ble CD163, that may be linked to the late gadolin­i­um enhance­ment on car­diac MRI, which can per­sist for months after vac­ci­na­tion. Togeth­er, our results demon­strate up-reg­u­la­tion in inflam­ma­to­ry cytokines and cor­re­spond­ing lym­pho­cytes with tis­sue-dam­ag­ing capa­bil­i­ties, sug­gest­ing a cytokine-depen­dent pathol­o­gy, which may fur­ther be accom­pa­nied by myeloid cell–associated car­diac fibro­sis. These find­ings like­ly rule out some pre­vi­ous­ly pro­posed mech­a­nisms of mRNA vaccine–-associated myoperi­cardi­tis and point to new ones with rel­e­vance to vac­cine devel­op­ment and clin­i­cal care.

...

Dis­cus­sion

...

Although the LNP com­po­nent of the vac­cine alone was found to be high­ly inflam­ma­to­ry, such respons­es cen­tered on IL‑6 and IL-1ß (41, 123). IL-1ß was ele­vat­ed in our cohort of patients and togeth­er with upstream NLRP3 inflam­ma­some acti­va­tion and asso­ci­at­ed cytokines may play a role in the patho­gen­e­sis of myocardi­tis (32, 39). How­ev­er, IL-1ß induc­tion by lipid-for­mu­lat­ed RNA vac­cines, which can then stim­u­late var­i­ous proin­flam­ma­to­ry cytokines, was also shown to be depen­dent on both the RNA and lipid for­mu­la­tion in human immune cells (124). Thus, a com­pound role of the adju­vant deliv­ery plat­form in syn­er­gy with vac­cine-vec­tored anti­gens is more like­ly the dri­ver of an exag­ger­at­ed immune cytokine response dri­ving car­diac pathol­o­gy after vac­ci­na­tion in sus­cep­ti­ble indi­vid­u­als. What caus­es cer­tain indi­vid­u­als, notably ado­les­cent and young adult males, to be more sus­cep­ti­ble to these car­diac-relat­ed adverse events is not clear but like­ly not unique to vac­cine-induced patho­gen­e­sis. A bias toward younger males is sim­i­lar­ly seen in com­mu­ni­ty-acquired myocarditis/pericarditis, where many large-scale epi­demi­o­log­i­cal and clin­i­cal stud­ies have demon­strat­ed that patients are much more fre­quent­ly males (65 to 84% of patients) and sig­nif­i­cant­ly younger than female patients (125–136), poten­tial­ly sug­gest­ing height­ened immune and/or inflam­ma­to­ry respons­es in these demo­graph­ic groups.

...

———–

“Cytokinopa­thy with aber­rant cyto­tox­ic lym­pho­cytes and profi­brot­ic myeloid response in SARS-CoV­‑2 mRNA vaccine–associated myocardi­tis” by Anis Bar­ma­da, et al; Sci­ence Immunol­o­gy; 05/05/2023 Vol 8, Issue 83

“Instead, unbi­ased sys­tem­at­ic immune serum pro­fil­ing revealed ele­va­tions in cir­cu­lat­ing inter­leukins (IL-1ß, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix met­al­lo­pro­teas­es (MMP1, MMP8, MMP9, and TIMP1).”

Take note of that list of cytokines, chemokines, and inter­luekins they found at ele­vat­ed lev­els in these myocardi­tis patients, along with their rea­son­able spec­u­la­tion that “a com­pound role of the adju­vant deliv­ery plat­form” is play­ing a role here. Because that “adju­vant deliv­ery plat­form” is anoth­er way of describ­ing the LNPs and the innate immune response they elic­it. And as we’re going to see in the fol­low­ing arti­cle pub­lished in Decem­ber of 2021 explor­ing that innate response on mouse mod­els, the LNP’s alone are capa­ble of illic­it a very sim­i­lar look­ing immune response:

iScience

The mRNA-LNP plat­for­m’s lipid nanopar­ti­cle com­po­nent used in pre­clin­i­cal vac­cine stud­ies is high­ly inflam­ma­to­ry

Sonia Nde­u­pen, Zhen Qin, Sanya Jacob­sen, Aure­lie Bouteau, Hen­ri Estanbouli, Botond Z. Igyártó

Vol­ume 24, Issue 12, 17 Decem­ber 2021, 103479

High­lights

• Lipid nanopar­ti­cles (LNPs) used for pre­clin­i­cal stud­ies are high­ly inflam­ma­to­ry
• The LNPs acti­vate mul­ti­ple inflam­ma­to­ry path­ways and induce IL-1ß and IL‑6
• The LNPs’ inflam­ma­to­ry prop­er­ties stem from their ion­iz­able lipid com­po­nent
• The LNPs could be respon­si­ble for adju­van­tic­i­ty and some of the side effects

Sum­ma­ry

Vac­cines based on mRNA-con­tain­ing lipid nanopar­ti­cles (LNPs) are a promis­ing new plat­form used by two lead­ing vac­cines against COVID-19. Clin­i­cal tri­als and ongo­ing vac­ci­na­tions present with vary­ing degrees of pro­tec­tion lev­els and side effects. How­ev­er, the dri­vers of the report­ed side effects remain poor­ly defined. Here we present evi­dence that Acuitas’ LNPs used in pre­clin­i­cal nucle­o­side-mod­i­fied mRNA vac­cine stud­ies are high­ly inflam­ma­to­ry in mice. Intra­der­mal and intra­mus­cu­lar injec­tion of these LNPs led to rapid and robust inflam­ma­to­ry respons­es, char­ac­ter­ized by mas­sive neu­trophil infil­tra­tion, acti­va­tion of diverse inflam­ma­to­ry path­ways, and pro­duc­tion of var­i­ous inflam­ma­to­ry cytokines and chemokines. The same dose of LNP deliv­ered intranasal­ly led to sim­i­lar inflam­ma­to­ry respons­es in the lung and result­ed in a high mor­tal­i­ty rate, with mech­a­nism unre­solved. Thus, the mRNA-LNP plat­forms’ poten­cy in sup­port­ing the induc­tion of adap­tive immune respons­es and the observed side effects may stem from the LNPs’ high­ly inflam­ma­to­ry nature.

Intro­duc­tion

The nucle­o­side-mod­i­fied mRNA-LNP vac­cine plat­form used by Pfizer/BioNTech and Mod­er­na in their SARS-CoV­‑2 vac­cines has been wide­ly test­ed in pre­clin­i­cal stud­ies, and its effec­tive­ness in sup­port­ing Tfh cells and pro­tec­tive humoral immune respons­es match­es or sur­pass­es oth­er vac­cines (Alameh et al., 2020). These vac­cines’ mRNA com­po­nent is nucle­o­side mod­i­fied to decrease poten­tial innate immune recog­ni­tion (Karikó et al., 2005; Karikó et al., 2008). The LNP was cho­sen as a car­ri­er vehi­cle to pro­tect the mRNA from degra­da­tion and aid intra­cel­lu­lar deliv­ery and endo­so­mal escape. The LNPs con­sist of a mix­ture of phos­pho­lipids, cho­les­terol, PEGy­lat­ed lipids, and cation­ic or ion­iz­able lipids. The phos­pho­lipids and cho­les­terol have struc­tur­al and sta­bi­liz­ing roles, where­as the PEGy­lat­ed lipids sup­port pro­longed cir­cu­la­tion. The cationic/ionizable lipids are includ­ed to allow the com­plex­ing of the neg­a­tive­ly charged mRNA mol­e­cules and enable the exit of the mRNA from the endo­some to the cytosol for trans­la­tion (Samari­dou et al., 2020). The ion­iz­able lipids were devel­oped to decrease the high­ly inflam­ma­to­ry and cyto­tox­ic effects of some of the per­ma­nent­ly charged cation­ic lipids (Kulka­rni et al., 2018). A pre­clin­i­cal study showed that nucle­o­side-mod­i­fied mRNA com­plexed with Acuitas Ther­a­peu­tics’ LNPs con­tain­ing pro­pri­etary ion­iz­able lipid has adju­vant activ­i­ty (Par­di et al., 2018a). How­ev­er, the poten­tial inflam­ma­to­ry nature of these LNPs was not assessed (Alameh et al., 2020; Par­di et al., 2018a).

The human clin­i­cal tri­als of the Pfizer/BioNTech and Mod­er­na vac­cines have report­ed side effects such as pain, swelling, fever, and sleepi­ness (Jack­son et al., 2020; Sahin et al., 2020; Walsh et al., 2020). Under the pre­sump­tion that this vac­cine plat­form is non­in­flam­ma­to­ry, some of the clin­i­cians and pub­lic health com­mu­ni­ca­tors inter­pret­ed these report­ed acute side effects as the vac­cine being potent and gen­er­at­ing an adap­tive immune response. These side effects, how­ev­er, are more in line with acute inflam­ma­to­ry respons­es induced by the vac­cine. Still, no stud­ies have been under­tak­en to char­ac­ter­ize the imme­di­ate innate inflam­ma­to­ry reac­tions induced by this vac­cine plat­form that could poten­tial­ly cause the local and sys­temic side effects. There­fore, in this study, we took a sys­tem­at­ic approach, focus­ing our atten­tion on the injec­tion site and ana­lyz­ing the inflam­ma­to­ry reac­tions caused by the LNPs used for pre­clin­i­cal vac­cine stud­ies (Awasthi et al., 2019; Laczkó et al., 2020; Led­er­er et al., 2020; Par­di et al., 2017, Par­di et al., 2018a, Par­di et al., 2018b). Using com­ple­men­tary tech­niques, we show that in mice intra­der­mal, intra­mus­cu­lar, or intranasal deliv­ery of LNPs used in pre­clin­i­cal stud­ies trig­gers inflam­ma­tion char­ac­ter­ized by leuko­cyt­ic infil­tra­tion, acti­va­tion of dif­fer­ent inflam­ma­to­ry path­ways, and secre­tion of a diverse pool of inflam­ma­to­ry cytokines and chemokines. Thus, the inflam­ma­to­ry milieu induced by the LNPs could be par­tial­ly respon­si­ble for report­ed side effects of mRNA-LNP-based SARS-CoV­‑2 vac­cines in humans and are pos­si­bly con­trib­u­to­ry to their report­ed high poten­cy for elic­it­ing anti­body respons­es.

Results

Intra­der­mal and intra­mus­cu­lar inoc­u­la­tion with LNPs induces robust inflam­ma­tion

mRNAs com­bined with LNPs were used in many pre­clin­i­cal stud­ies and are key com­po­nents of the recent Pfizer/BioNTech and Mod­er­na SARS-CoV­‑2 vac­cine (Alameh et al., 2020; Jack­son et al., 2020; Sahin et al., 2020; Walsh et al., 2020). The mech­a­nism of action of this mRNA-LNP plat­form is not well defined. The mRNA com­po­nent is mod­i­fied to decrease the engage­ment of innate immune sen­sors (Karikó et al., 2005; Karikó et al., 2008), but the mRNA com­plexed with LNPs was shown to have adju­vant activ­i­ty (Par­di et al., 2018a). The mRNA-LNP plat­form pro­motes robust humoral immune respons­es, and humans receiv­ing the vac­cine often pre­sent­ed with typ­i­cal acute side effects of inflam­ma­tion, such as pain, swelling, and fever (Jack­son et al., 2020). Based on these obser­va­tions, we hypoth­e­sized that mRNA-LNP adju­vant activ­i­ty and the report­ed side effects in humans could stem from the LNPs’ inflam­ma­to­ry prop­er­ties. mRNAs com­plexed with LNPs were used in pre­clin­i­cal stud­ies at dos­es rang­ing from 3 to 30 µg/mouse (Laczkó et al., 2020; Par­di et al., 2018a). We inject­ed 10 µg (4 spots; 2.5 µg/spot) of these emp­ty LNPs for­mu­lat­ed in phos­phate buffered saline (PBS) or con­trol PBS intra­der­mal­ly into adult wild-type (WT) C57BL/6 (B6) mice. We sac­ri­ficed the mice at dif­fer­ent time points post-injec­tion, and ~1 cm² skin sam­ples from the injec­tion sites were col­lect­ed. The LNP-inject­ed skin sam­ples macro­scop­i­cal­ly showed signs of intense inflam­ma­tion, such as red­ness and swelling (Fig­ure 1A). Sin­gle-cell sus­pen­sions were pre­pared from these sam­ples and ana­lyzed for infil­trates using flow cytom­e­try (Fig­ures 1B and S1A). Flow cytom­e­try revealed mas­sive and rapid leuko­cyt­ic infil­trates dom­i­nat­ed by neu­trophils that slow­ly resolved by day 14 (Fig­ure 1B). Removal of the ion­iz­able lipid com­po­nent from the LNPs abol­ished vis­i­ble skin inflam­ma­tion Fig­ure 1C) and the leuko­cyt­ic infil­tra­tion (Fig­ure 1D). Thus, LNPs used in pre­clin­i­cal stud­ies pro­mote swift inflam­ma­to­ry respons­es at the injec­tion site, which depends on the ion­iz­able lipid com­po­nent.

[see Fig­ure 1]

*****

The pre­clin­i­cal ani­mal vac­cine stud­ies with this mRNA-LNP plat­form were car­ried out through intra­mus­cu­lar and intra­der­mal inoc­u­la­tions. Both deliv­ery routes led to robust humoral immune respons­es (Laczkó et al., 2020; Par­di et al., 2018a). The cur­rent mRNA-LNP-based human SARS-CoV­‑2 vac­cines are deliv­ered intra­mus­cu­lar­ly, which might be moti­vat­ed by clin­i­cal prac­ti­cal­i­ty (Ols et al., 2020). To test whether intra­mus­cu­lar deliv­ery also leads to inflam­ma­tion, we inject­ed ani­mals intra­mus­cu­lar­ly with 10 µg of LNPs in 30 µL of PBS or PBS alone. Twen­ty-four hours lat­er, the inject­ed mus­cles were col­lect­ed for analy­sis. Sim­i­lar to the skin, we observed robust neu­trophil infil­tra­tion to the mus­cle sam­ples that received LNPs (Fig­ures 1E and S1B). The mus­cles inject­ed with LNPs also showed a sig­nif­i­cant increase in weight (Fig­ure 1F). Thus, these find­ings col­lec­tive­ly indi­cate mas­sive local inflam­ma­tion that could account for some of the report­ed local side effects.

To acquire a more in-depth under­stand­ing of the glob­al changes trig­gered by the injec­tion of the LNPs, we repeat­ed the intra­der­mal exper­i­ments pre­sent­ed above with LNPs com­plexed with con­trol, non­cod­ing poly-cyto­sine mRNA. Skin sam­ples har­vest­ed one day after injec­tion were split into two and ana­lyzed using Luminex and bulk RNA-seq (Fig­ure 2A). The Luminex data cor­rob­o­rat­ed the flow cytom­e­try find­ings and demon­strat­ed the pres­ence of a vari­ety of inflam­ma­to­ry cytokines and chemokines (Fig­ures 2B, 2C and S2), in com­par­i­son to con­trol sam­ples. Chemokines that attract neu­trophils and mono­cytes and pro­mote their func­tions, such as CCL2, CCL3, CCL4, CCL7, CCL12, CXCL1, and CXCL2, dom­i­nat­ed the pan­el (Fig­ure 2B). We fur­ther found large amounts of inter­leukin-1ß (IL-1ß), gran­u­lo­cyte-macrophage colony-stim­u­lat­ing fac­tor (GM-CSF), and IL‑6, the sig­na­ture cytokines of inflam­ma­to­ry respons­es (Fig­ure 2C). Our RNA-seq analy­sis revealed that thou­sands of genes were upreg­u­lat­ed (Fig­ure 2D) upon LNP injec­tion. With p < 0.05 and FDR<0.05, 9,508 genes and 8,883 genes, respec­tive­ly, were dif­fer­en­tial­ly expressed. More impor­tant­ly, con­firm­ing our flow cytom­e­try and Luminex data, the genes asso­ci­at­ed with monocyte/granulocyte devel­op­ment, recruit­ment, and func­tion (Cxcl1, Cxcl2, Cxcl5, Cxcl10, Ccl2, Ccl3, Ccl4, Ccl7, Ccl12, Csf2, and Csf3) and inflam­ma­tion (Il1b and Il6) showed the high­est fold increas­es over the con­trol sam­ples (Fig­ure 2E). We also observed sig­nif­i­cant upreg­u­la­tion of gene tran­scripts asso­ci­at­ed with acti­va­tion of inflam­ma­somes, such as Il1b and Nlrp3, and down­reg­u­la­tion of Nlrp10, which is known to inhib­it inflam­ma­somes (Fig­ure 2E). Gene set enrich­ment analy­ses (GSEA) showed the acti­va­tion of many dif­fer­ent inflam­ma­to­ry path­ways, includ­ing, but not lim­it­ed to, viral infec­tions, RIG‑I, NOD-like, and Toll-like recep­tor sig­nal­ing (Fig­ure 2F). Pro-apop­tot­ic and necrop­tot­ic gene sets were also sig­nif­i­cant­ly upreg­u­lat­ed, as well as inter­fer­on sig­nal­ing Fig­ure 2F).

[see Fig­ure 2]

****

In sum­ma­ry, using dif­fer­ent tech­niques, we show that LNPs, alone or com­plexed with con­trol non­cod­ing poly-cyto­sine mRNA, are high­ly inflam­ma­to­ry in mice, like­ly through the engage­ment and acti­va­tion of var­i­ous dis­tinct and con­ver­gent inflam­ma­to­ry path­ways.

Intranasal inoc­u­la­tion with LNPs leads to robust inflam­ma­to­ry respons­es in the lung

Res­pi­ra­to­ry infec­tions, includ­ing with SARS-CoV­‑2, induce a dom­i­nant mucosa-pro­tec­tive anti­body response and local T‑cell-medi­at­ed immu­ni­ty (Grau-Expósi­to et al., 2021; Rus­sell et al., 2020; Ster­lin et al., 2021). There­fore, to achieve an immune response that mim­ic nat­ur­al infec­tion and might con­fer more pro­tec­tion against SARS-CoV­‑2 at the lev­els of the res­pi­ra­to­ry tract (Lund and Ran­dall, 2021; Rus­sell et al., 2020; Zens et al., 2019), researchers are active­ly work­ing on devel­op­ing SARS-CoV­‑2 vac­cines that could be admin­is­tered intranasal­ly (Buschmann et al., 2021; Chav­da et al., 2021; van Dore­malen et al., 2021). There­fore, we first test­ed whether the LNPs can reach the lungs upon intranasal deliv­ery. For this pur­pose, we inoc­u­lat­ed mice intranasal­ly with PBS or 10 µg of DiI-labeled LNPs. Six hours lat­er his­tol­ogy showed a homo­ge­neous dis­tri­b­u­tion of the LNPs in the lung tis­sue (Fig­ure 3A). Next, we test­ed whether intranasal deliv­ery leads to inflam­ma­tion in the lungs. For this, we intranasal­ly inoc­u­lat­ed adult WT B6 mice with PBS or 10 µg of LNPs in PBS/mouse. Lung sam­ples from PBS- and 10 µg LNP-treat­ed mice were pre­pared for macro­scop­ic analy­ses 9 and 24 h post-inoc­u­la­tion and for flow cytom­e­try 9 h post-inoc­u­la­tion. In a mat­ter of hours, the lungs turned red in col­or in the LNP-inoc­u­lat­ed group (Fig­ure 3B). As was observed for the skin and mus­cle, flow cyto­met­ric analy­ses revealed sig­nif­i­cant leuko­cyt­ic infil­tra­tion dom­i­nat­ed by neu­trophils and eosinophils and a decrease in macrophages and cer­tain den­drit­ic cell (DC) sub­sets Fig­ure 3C and S3). Thus, intranasal deliv­ery of LNPs leads to mas­sive inflam­ma­tion in the lungs.

[see Fig­ure 3]

******

With the exper­i­ments pre­sent­ed ear­li­er, we observed a high mor­tal­i­ty rate among the mice inoc­u­lat­ed with LNPs. These find­ings prompt­ed us to per­form a dose-response exper­i­ment to deter­mine the amounts of LNPs that might be safe to use for mucos­al vac­ci­na­tion. We intranasal­ly inoc­u­lat­ed adult WT B6 mice with LNPs rang­ing from 2.5 µg to 10 µg/mouse and mon­i­tored their health and weight for up to 8 days. We found that ~80% of mice treat­ed with 10 µg of LNP died in less than 24 h (Fig­ure 3D). The 5 µg dose killed ~20% of the mice by that time, where­as all the 2.5 µg-treat­ed mice sur­vived and showed no weight drop (Fig­ure 3D) and no sig­nif­i­cant clin­i­cal signs of dis­tress (Fig­ure 3E). For the 5 and 10 µg dos­es, the sur­viv­ing mice showed notable clin­i­cal scores of dis­tress, such as shaking/shivering, and they lost weight sig­nif­i­cant­ly dur­ing the first 2 days of treat­ment (Fig­ure 3E and 3F). After the first ~3 days, these mice did not con­tin­ue to show sig­nif­i­cant clin­i­cal scores any­more, and their weight slow­ly start­ed to nor­mal­ize (Fig­ure 3E). These data sug­gest that care­ful opti­miza­tion of the LNP dose might allow the use of the mRNA-LNP plat­form for intranasal vac­ci­na­tion.

Dis­cus­sion

Here we show that the LNPs used for many pre­clin­i­cal stud­ies (Freyn et al., 2020; Laczkó et al., 2020; Led­er­er et al., 2020; Par­di et al., 2017, Par­di et al., 2018a, Par­di et al., 2018b) are high­ly inflam­ma­to­ry. This could explain their potent adju­vant activ­i­ty and their supe­ri­or­i­ty, com­pared with oth­er adju­vants, in sup­port­ing the induc­tion of adap­tive immune respons­es.

Pre­vi­ous pre­clin­i­cal mouse data sug­gest­ed that mRNA com­plexed with LNPs have adju­vant activ­i­ty (Par­di et al., 2018a). The mRNA is nucle­o­side mod­i­fied and puri­fied to decrease acti­va­tion of cer­tain innate inflam­ma­to­ry path­ways (Karikó et al., 2005; Karikó et al., 2008, 2011). Our injec­tion-site-focused analy­ses revealed the inflam­ma­to­ry nature of these LNPs, which like­ly pro­vides a basis for their adju­vant prop­er­ties. The cation­ic lipid com­po­nent of the lipid car­ri­ers is often inflam­ma­to­ry and cyto­tox­ic (Fil­ion and Phillips, 1997, 1998; Lv et al., 2006; Samari­dou et al., 2020; Sedic et al., 2018; Tao et al., 2011). The ion­iz­able lipids were devel­oped to over­come the inflam­ma­to­ry nature of the per­ma­nent­ly charged cation­ic lipids (Kulka­rni et al., 2018). How­ev­er, we found that the pro­pri­etary ion­iz­able lipid com­po­nent of these LNPs is inflam­ma­to­ry (Fig­ures 1C and 1D). The inflam­ma­to­ry respons­es trig­gered by the LNPs alone or com­plexed with mRNAs were inde­pen­dent of the deliv­ery route and were dom­i­nat­ed by neu­trophil infil­tra­tion. Despite the induc­tion of phe­no­typ­i­cal­ly sim­i­lar innate inflam­ma­to­ry respons­es, unlike intra­der­mal and intra­mus­cu­lar inoc­u­la­tions, intranasal deliv­ery of 2.5 µg of mRNA-LNP cod­ing for PR8 HA did not lead to detectable lev­els of adap­tive immune respons­es (unpub­lished obser­va­tion). Thus, slight dif­fer­ences are like­ly to exist between the route of admin­is­tra­tion, which war­rants fur­ther inves­ti­ga­tion to opti­mize this plat­form for intranasal use. Under­stand­ing the cel­lu­lar and mol­e­c­u­lar dif­fer­ences between intradermal/intramuscular and intranasal deliv­ery sites that under­line this dis­sim­i­lar­i­ty in adap­tive immune respons­es might also help to deci­pher why cer­tain peo­ple do not react to the intra­mus­cu­lar­ly deliv­ered mRNA-LNP vac­cines.

Humans present var­i­ous side effects, most often pain, swelling, fever, and chills, after intra­mus­cu­lar vac­ci­na­tion with the Pfizer/BioNTech or Mod­er­na vac­cines (Jack­son et al., 2020; Sahin et al., 2020; Walsh et al., 2020). These are typ­i­cal symp­toms asso­ci­at­ed with inflam­ma­tion trig­gered by cytokines such as IL-1ß and IL‑6 (Dinarel­lo, 2018; Tana­ka et al., 2014). Along with caus­ing local inflam­ma­to­ry respons­es, these cytokines also act as major endoge­nous pyro­gens (Con­ti, 2004) and instruct the hypo­thal­a­mus to increase the body’s tem­per­a­ture (fever) to help over­come pos­si­ble infec­tions. In con­cor­dance with this, the intra­der­mal inoc­u­la­tion of LNPs in mice led to the secre­tion of large amounts of major and minor pyro­gens, IL-1ß/IL‑6 and macrophage inflam­ma­to­ry protein‑a (CCL3) and macrophage inflam­ma­to­ry protein‑ß (CCL4), respec­tive­ly (Fig­ures 2B and 2C). Fur­ther­more, the observed acti­va­tion of oth­er inflam­ma­to­ry path­ways and cell death could fur­ther accen­tu­ate the expe­ri­enced side effects. How­ev­er, fur­ther stud­ies will be need­ed to deter­mine the exact nature of the inflam­ma­to­ry respons­es trig­gered by mRNA-LNP vac­cines in humans and how much over­lap there might be with the inflam­ma­to­ry sig­na­tures doc­u­ment­ed here for mice.

It remains to be deter­mined how these LNPs or their ion­iz­able lipid com­po­nent acti­vate dis­tinct inflam­ma­to­ry path­ways. In the­o­ry, LNPs could acti­vate mul­ti­ple path­ways or alter­na­tive­ly engage only one that would ini­ti­ate an inflam­ma­to­ry cas­cade. Some cationic/ionizable lipids bind and acti­vate TLRs (Lonez et al., 2012, 2014; Samari­dou et al., 2020; Tana­ka et al., 2008). Our GSEA analy­ses are con­sis­tent with TLR engage­ment by these pro­pri­etary LNPs, among oth­ers (Fig­ure 2F). We also observed upreg­u­la­tion of inflam­ma­some com­po­nents such Nlrp3 and enrich­ment of genes involved in necrop­to­sis. Inflam­ma­to­ry cell death, such as necrop­to­sis and pyrop­to­sis, could cause the release of DAMPs and the fur­ther enhance­ment of inflam­ma­tion. The syn­thet­ic ion­iz­able lipid in the Pfiz­er SARS-CoV­‑2 vac­cine has been spec­u­lat­ed to have approx­i­mate­ly 20–30 days of half-life in humans (Comir­naty, 2021). Whether the long-term pres­ence of ion­iz­able lipid could lead to low lev­els of chron­ic inflam­ma­tion and immune exhaus­tion (Wher­ry and Kurachi, 2015) remain to be deter­mined.

Intranasal inoc­u­la­tion with high­er dos­es of LNPs led to a sig­nif­i­cant mor­tal­i­ty rate. Hypo­thet­i­cal­ly, the mas­sive innate inflam­ma­to­ry respons­es induced in the lung (Fig­ure 3) and chem­i­cal dis­rup­tion of the pul­monary sur­fac­tant by cation­ic lipids (Bon­cuk et al., 1997), i.e., reduced gas exchange, cyto­tox­i­c­i­ty to alve­o­lar cells, or oth­er mech­a­nisms, could all have con­tributed to ani­mal death (Dok­ka et al., 2000). Thus, fur­ther exper­i­men­ta­tion is need­ed to inves­ti­gate the mech­a­nism of the tox­i­c­i­ty. Mate­ri­als of dif­fer­ent nature includ­ing nanopar­ti­cles inoc­u­lat­ed intranasal­ly read­i­ly enter the cen­tral ner­vous sys­tem (CNS), and it is the pre­ferred method to over­come the blood-brain bar­ri­er and deliv­er active sub­stances to the brain (Cos­ta et al., 2021; Elder et al., 2006; Gao et al., 2006; Han­son et al., 2013; Lock­man et al., 2004). LNPs as lipid par­ti­cles can quick­ly dif­fuse (Fig­ure 3A) (Par­di et al., 2015) and, through intranasal deliv­ery, could poten­tial­ly gain access to the CNS at high enough lev­els to induce sig­nif­i­cant inflam­ma­tion. Whether these LNPs reach the CNS and induce lev­els of inflam­ma­tion that could have con­tributed to the ani­mal death observed at high­er dos­es remains to be deter­mined. Inter­est­ing­ly, it has been report­ed that the mRNA from Mod­er­na’s mRNA-LNP vac­cine inject­ed intra­mus­cu­lar­ly could be detect­ed in very low lev­els in the brain, poten­tial­ly indi­cat­ing that the mRNA-LNP plat­form might cross the blood-brain bar­ri­er and reach the CNS (Mod­er­na, 2021). How­ev­er, in this case, the scant amounts detect­ed in the CNS would like­ly not induce sig­nif­i­cant inflam­ma­tion but might trig­ger hypo­thal­a­mus-dri­ven side effects such as fever, nau­sea, and sleepi­ness. Fur­ther stud­ies will be need­ed to deter­mine whether these min­i­mal amounts of vac­cine com­po­nents detect­ed in the CNS are phys­i­o­log­i­cal­ly rel­e­vant and define whether they could exac­er­bate pre­ex­ist­ing inflam­ma­to­ry respons­es report­ed in a small case report with MS autoim­mune patients (Khay­at-Khoei et al., 2021). It is impor­tant to note that a medi­um-sized obser­va­tion­al study did not find an increased risk of relapse activ­i­ty after mRNA-LNP vac­ci­na­tion in MS patients (Ach­i­ron et al., 2021), where­as oth­ers have not­ed a slight but increased risk of hem­or­rhag­ic stroke. How­ev­er, the risk of these com­pli­ca­tions was greater fol­low­ing a pos­i­tive SARS-CoV­‑2 test (Patone et al., 2021).

Peo­ple often present with more severe and sys­temic side effects after the boost­er shot. This rais­es the pos­si­bil­i­ty that the adap­tive immune response might some­how ampli­fy side effects induced by the vac­cine. One cul­prit iden­ti­fied so far is PEG, which is immuno­genic. Anti­bod­ies formed against PEG have been report­ed to sup­port a so-called ana­phy­lac­toid, com­ple­ment acti­va­tion-relat­ed pseudoal­ler­gy (CARPA) reac­tion (Koz­ma et al., 2020; Szebeni, 2005, Szebeni, 2014). Of note, because PEG is a com­pound fre­quent­ly used in cos­met­ics and tooth­paste, many indi­vid­u­als could have anti-PEG anti­bod­ies. We have dis­cussed oth­er pos­si­ble mech­a­nisms in our recent opin­ion arti­cle (Igyártó et al., 2021). Briefly, although mRNA main­ly trans­fects cells near the injec­tion site, it could hypo­thet­i­cal­ly reach any cell in the body (Maugeri et al., 2019; Par­di et al., 2015). The result­ing trans­lat­ed pro­tein could be pre­sent­ed on MHC‑I in the form of pep­tides or dis­played as a whole pro­tein in the cell mem­brane. In both cas­es, cells with the vac­cine peptide/protein on their sur­faces could be tar­get­ed and killed by cells of the adap­tive and innate immune sys­tem, CD8+ T and nat­ur­al killer (NK) cells (via anti­body-depen­dent cel­lu­lar tox­i­c­i­ty [ADCC]), respec­tive­ly. In line with this, the so-called “Covid-arm,” a delayed-type hyper­sen­si­tiv­i­ty reac­tion that devel­ops in some patients sev­er­al days after vac­ci­na­tion (Blu­men­thal et al., 2021), or the myocarditis/pericarditis recent­ly report­ed with some of the vac­ci­nat­ed peo­ple (Abu Mouch et al., 2021; Diaz et al., 2021; Mar­shall et al., 2021; Mont­gomery et al., 2021; Shay et al., 2021), or the CNS inflam­ma­tion observed in a small num­ber of vac­ci­nat­ed MS patients (Khay­at-Khoei et al., 2021) could be indeed an indi­ca­tion of immune respons­es tar­get­ing the cells express­ing the vac­cine-derived peptides/proteins.

In sum­ma­ry, the first vac­ci­na­tion’s side effects, except for CARPA, are like­ly asso­ci­at­ed with robust innate inflam­ma­tion induced by the LNPs. In con­trast, after the sec­ond vac­ci­na­tion, side effects could be fur­ther exac­er­bat­ed by immune respons­es tar­get­ing cells express­ing the vac­cine pro­tein or its pep­tide deriv­a­tives. Whether innate mem­o­ry respons­es (Netea et al., 2011) to LNPs also con­tribute to ampli­fy­ing the side effects remains to be deter­mined (Fig­ure 4). Over­all, the robust inflam­ma­to­ry milieu induced by LNPs, com­bined with pre­sen­ta­tion of the vac­cine-derived peptides/protein out­side of anti­gen-pre­sent­ing cells, might cause tis­sue dam­age and exac­er­bate side effects. Because self-anti­gen pre­sen­ta­tion in an inflam­ma­to­ry envi­ron­ment has been linked to autoim­mune dis­ease devel­op­ment (Janeway et al., 2001), this mer­its fur­ther inves­ti­ga­tion.

[see Fig­ure 4]

The side effects observed with the SARS-CoV­‑2 vac­cine’s first dose are like­ly asso­ci­at­ed with the LNPs’ inflam­ma­to­ry prop­er­ties. LNPs acti­vate dif­fer­ent inflam­ma­to­ry path­ways that will lead to the pro­duc­tion of inflam­ma­to­ry cytokines, such as IL-1ß and IL‑6, that can ini­ti­ate and sus­tain local and sys­temic inflam­ma­tions and side effects. The dashed line indi­cates the pos­si­bil­i­ty that the LNPs might also dif­fuse from the periph­ery and reach any organs in the body, includ­ing CNS (hypo­thal­a­mus) where they could direct­ly induce side effects. PEG is wide­ly used as a food and med­i­cine addi­tive, and many of us devel­op anti­bod­ies to PEG. There­fore, the LNPs’ PEGy­lat­ed lipids can induce CARPA in humans with pre­ex­ist­ing PEG-spe­cif­ic anti­bod­ies. Humans often expe­ri­ence more severe side effects with the sec­ond dose. Here we posit that might be due to mul­ti­ple rea­sons. First­ly, innate immune mem­o­ry against the LNPs might form after the first vac­ci­na­tion and that could lead to even more robust inflam­ma­to­ry respons­es upon the sec­ond vac­ci­na­tion. Sec­ond­ly, after the first vac­ci­na­tion, adap­tive immune respons­es are formed tar­get­ing the viral pro­tein cod­ed by the mRNA. As such, cells (shown as red shape) express­ing the viral pro­tein-derived pep­tides or pro­tein itself can become the tar­get of CD8+ T- or NK-cell-medi­at­ed killing (ADCC), respec­tive­ly. Because the LNPs could dif­fuse through­out the body and trans­fect any cell in their path with the mRNA, and the mRNA could also be fur­ther dis­trib­uted through extra­cel­lu­lar vesi­cles (Maugeri et al., 2019), the tar­get pop­u­la­tion could poten­tial­ly be vast and diverse.

...

———–

“The mRNA-LNP plat­for­m’s lipid nanopar­ti­cle com­po­nent used in pre­clin­i­cal vac­cine stud­ies is high­ly inflam­ma­to­ry” by Sonia Nde­u­pen, Zhen Qin, Sanya Jacob­sen, Aure­lie Bouteau, Hen­ri Estanbouli, Botond Z. Igyártó; iScience; Vol­ume 24, Issue 12, 17 Decem­ber 2021

“The human clin­i­cal tri­als of the Pfizer/BioNTech and Mod­er­na vac­cines have report­ed side effects such as pain, swelling, fever, and sleepi­ness (Jack­son et al., 2020; Sahin et al., 2020; Walsh et al., 2020). Under the pre­sump­tion that this vac­cine plat­form is non­in­flam­ma­to­ry, some of the clin­i­cians and pub­lic health com­mu­ni­ca­tors inter­pret­ed these report­ed acute side effects as the vac­cine being potent and gen­er­at­ing an adap­tive immune response. These side effects, how­ev­er, are more in line with acute inflam­ma­to­ry respons­es induced by the vac­cine. Still, no stud­ies have been under­tak­en to char­ac­ter­ize the imme­di­ate innate inflam­ma­to­ry reac­tions induced by this vac­cine plat­form that could poten­tial­ly cause the local and sys­temic side effects. There­fore, in this study, we took a sys­tem­at­ic approach, focus­ing our atten­tion on the injec­tion site and ana­lyz­ing the inflam­ma­to­ry reac­tions caused by the LNPs used for pre­clin­i­cal vac­cine stud­ies (Awasthi et al., 2019; Laczkó et al., 2020; Led­er­er et al., 2020; Par­di et al., 2017, Par­di et al., 2018a, Par­di et al., 2018b). Using com­ple­men­tary tech­niques, we show that in mice intra­der­mal, intra­mus­cu­lar, or intranasal deliv­ery of LNPs used in pre­clin­i­cal stud­ies trig­gers inflam­ma­tion char­ac­ter­ized by leuko­cyt­ic infil­tra­tion, acti­va­tion of dif­fer­ent inflam­ma­to­ry path­ways, and secre­tion of a diverse pool of inflam­ma­to­ry cytokines and chemokines. Thus, the inflam­ma­to­ry milieu induced by the LNPs could be par­tial­ly respon­si­ble for report­ed side effects of mRNA-LNP-based SARS-CoV­‑2 vac­cines in humans and are pos­si­bly con­trib­u­to­ry to their report­ed high poten­cy for elic­it­ing anti­body respons­es.”

As the authors point out, the assump­tion that the report­ed side effects from the mRNA vac­cines were all a con­se­quence of the adap­tive respons­es induced by the mRNA in the vac­cine is just that, an assump­tion. A wide­ly held assump­tion but a rather ques­tion­able one giv­en that the side effect symp­toms — pain, swelling, fever, and sleepi­ness, etc — are more in line with the non-adap­tive imme­di­ate innate immune response to the LNP vac­cine plat­form itself. In oth­er words, is it the chunk of the SARS-CoV­‑2 spike pro­tein embed­ded in the mRNA stuffed into the LNP bub­bles that is dri­ving these side effects or is it the LNP bub­bles them­selves dri­ving this? That’s the ques­tion these authors set out to address in this study pub­lished in Decem­ber of 2021. Mice had LNP expose their skin, inject­ed into their mus­cles, and exposed intranasal­ly in order to explore the inflam­ma­to­ry impact of the LNP par­ti­cles alone. They also exam­ined LNPs with mRNA spiked in (mRNA that does­n’t code for any pro­tein), to sim­u­late the gen­er­ate expo­sure of mRNA and LNPs non-spe­cif­ic to the SARS-CoV­‑2 spike pro­tein’s immuno­log­i­cal effects. And as we can see in their pub­lished results, the observed inflam­ma­tion induced by the LNPs in these mice was pro­found, whether the LNPs were exposed to the skin, inject­ed into mus­cle, or deliv­ered through the nose where inflam­ma­tion of the lungs was wide­spread short­ly after expo­sure.

And here we get to the very inter­est­ing results that direct­ly tie in to the recent Yale study’s find­ings: When they spiked in non-pro­tein mRNA into the LNPs to more close­ly sim­u­late the mRNA vac­cines, they found a vari­ety of inflam­a­to­ry cytokines and chemokines such as CCL2, CCL3, CCL4, CCL7, CCL12, CXCL1, and CXCL2 and large amounts of inter­leukin-1ß (IL-1ß), gran­u­lo­cyte-macrophage colony-stim­u­lat­ing fac­tor (GM-CSF), and IL‑6, the sig­na­ture cytokines of inflam­ma­to­ry respons­es. Recall what the Yale authors found: “Con­trary to ear­ly hypothe­ses, patients did not demon­strate fea­tures of hyper­sen­si­tiv­i­ty myocardi­tis, nor did they have exag­ger­at­ed SARS-CoV‑2–specific or neu­tral­iz­ing anti­body respons­es con­sis­tent with a hyper­im­mune humoral mech­a­nism. We addi­tion­al­ly found no evi­dence of car­diac-tar­get­ed autoan­ti­bod­ies. Instead, unbi­ased sys­tem­at­ic immune serum pro­fil­ing revealed ele­va­tions in cir­cu­lat­ing inter­leukins (IL-1ß, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix met­al­lo­pro­teas­es (MMP1, MMP8, MMP9, and TIMP1).” Now, those two lists of cytokines and chemokines don’t over­lap exact­ly, but they are both point­ing towards the same gen­er­ate innate immune response. All of this research demon­strat­ing how LNPs induce the same kind of immune response that the Yale team was observ­ing was just left out of their analy­sis:

...
To acquire a more in-depth under­stand­ing of the glob­al changes trig­gered by the injec­tion of the LNPs, we repeat­ed the intra­der­mal exper­i­ments pre­sent­ed above with LNPs com­plexed with con­trol, non­cod­ing poly-cyto­sine mRNA. Skin sam­ples har­vest­ed one day after injec­tion were split into two and ana­lyzed using Luminex and bulk RNA-seq (Fig­ure 2A). The Luminex data cor­rob­o­rat­ed the flow cytom­e­try find­ings and demon­strat­ed the pres­ence of a vari­ety of inflam­ma­to­ry cytokines and chemokines (Fig­ures 2B, 2C and S2), in com­par­i­son to con­trol sam­ples. Chemokines that attract neu­trophils and mono­cytes and pro­mote their func­tions, such as CCL2, CCL3, CCL4, CCL7, CCL12, CXCL1, and CXCL2, dom­i­nat­ed the pan­el (Fig­ure 2B). We fur­ther found large amounts of inter­leukin-1ß (IL-1ß), gran­u­lo­cyte-macrophage colony-stim­u­lat­ing fac­tor (GM-CSF), and IL‑6, the sig­na­ture cytokines of inflam­ma­to­ry respons­es (Fig­ure 2C). Our RNA-seq analy­sis revealed that thou­sands of genes were upreg­u­lat­ed (Fig­ure 2D) upon LNP injec­tion. ...

...

It’s also inter­est­ing that, as the authors note, peo­ple often expe­ri­ence their most severe side effects fol­low­ing a boost­er shot, and not the first shot, which might indeed point towards an inter­play with the adap­tive immune response and the fact that, while inject­ed mRNA vac­cine are pri­mar­i­ly trans­fect the cells near the injec­tion site, they can hypo­thet­i­cal­ly reach any cell in the body. And this could, in turn, explain the observed inci­dents of myocardi­tis fol­low­ing vac­ci­na­tions. In oth­er words, if the LNPs inject­ed into your arm are mak­ing their way to your heart, an adap­tive response could hypo­thet­i­cal­ly tar­get the trans­fect­ed heart cells:

...
... Briefly, although mRNA main­ly trans­fects cells near the injec­tion site, it could hypo­thet­i­cal­ly reach any cell in the body (Maugeri et al., 2019; Par­di et al., 2015). The result­ing trans­lat­ed pro­tein could be pre­sent­ed on MHC‑I in the form of pep­tides or dis­played as a whole pro­tein in the cell mem­brane. In both cas­es, cells with the vac­cine peptide/protein on their sur­faces could be tar­get­ed and killed by cells of the adap­tive and innate immune sys­tem, CD8+ T and nat­ur­al killer (NK) cells (via anti­body-depen­dent cel­lu­lar tox­i­c­i­ty [ADCC]), respec­tive­ly. In line with this, the so-called “Covid-arm,” a delayed-type hyper­sen­si­tiv­i­ty reac­tion that devel­ops in some patients sev­er­al days after vac­ci­na­tion (Blu­men­thal et al., 2021), or the myocarditis/pericarditis recent­ly report­ed with some of the vac­ci­nat­ed peo­ple (Abu Mouch et al., 2021; Diaz et al., 2021; Mar­shall et al., 2021; Mont­gomery et al., 2021; Shay et al., 2021), or the CNS inflam­ma­tion observed in a small num­ber of vac­ci­nat­ed MS patients (Khay­at-Khoei et al., 2021) could be indeed an indi­ca­tion of immune respons­es tar­get­ing the cells express­ing the vac­cine-derived peptides/proteins.
...

Do you take the shot? Or roll the dice with the virus? Which immune sys­tem gam­ble is right for you? That’s the trag­ic health dilem­ma fac­ing the pop­u­lace. Again, no non-mRNA vac­cines are approved in the US after you’ve had your first boost­er. If you want fur­ther boosts you HAVE to get one of these mRNA shots, despite the exis­tence of both the J&J and Novavax that some­how just can’t ever get full approval. Why is that? And how is it that we can have stud­ies pub­lished in peer reviewed jour­nals rais­ing major ques­tions about the safe­ty of LNPs and it’s like it nev­er hap­pened? More ques­tions to add to the ever-grow­ing pile of unan­swered ques­tions. Or, in some cas­es, ques­tions with answers no one wants to hear.