Long-term COVID-19: Tackling Tough Questions About the Body’s Immune Response and Brain Symptoms

When SARS-CoV-2 began to surface in early 2020, not much was known about the COVID-19, the brand-new disease caused by the virus—except that it was highly contagious and spreading rapidly. As the virus raged on and was classified as a global pandemic, there were many questions about COVID-19. Some individuals had severe cases and were hospitalized with life-threatening symptoms and later discharged with lingering health conditions, including brain fog, memory problems, and other mysterious symptoms. Many other patients died. Yet, others were asymptomatic. Why was it presenting itself in such extreme cases? And who was at risk, and why?

Laboratories quickly shifted their focus to the new coronavirus. Scientists and researchers began studying the disease, the immune response, and associated risk factors for severe disease.

The Yale School of Medicine (YSM) lab run by Akiko Iwasaki, PhD, Waldemar Von Zedtwitz Professor of Immunobiology and Molecular, Cellular and Developmental Biology; professor of molecular, cellular and developmental biology; and Investigator, Howard Hughes Medical Institute, was no different. As an immunobiologist, Iwasaki’s research is focused on the mechanisms of immune defense against viruses at the mucosal surfaces. The Iwasaki laboratory’s focus is on understanding how viruses are recognized and how that information is used to generate protective adaptive immunity.

In April 2021 , the Department of Internal Medicine hosted a live discussion on Clubhouse to discuss the findings of the Iwasaki Lab since transitioning to research for COVID-19. Manisha Juthani, MD, associate professor of medicine (infectious diseases) and of epidemiology (microbial diseases), moderated the event to discuss the clinical manifestations of the disease. In addition to Iwasaki, Juthani was joined by two student researchers in Iwasaki’s lab—Alice Lu-Culligan and Julio Silva, both MD/PhD candidates at YSM.

Over the last two decades, the Iwasaki Lab has studied a variety of different virus infections—from genital herpes, to rhinovirus, to the Zika virus.

“We've been interested in this question of host immune response against viruses, and what are protective responses versus what are immuno-pathologic responses. We were primed and had the tools to be able to tackle what might be going on in the COVID-19 patients.” said Iwasaki.

Iwasaki remembers the first meeting in January 2020 with experts spanning several departments across YSM. The meeting included Ellen Foxman, MD, PhD; Nathan Grubaugh, PhD; Albert Ko, MD; Marie-Louise Landry, MD; and Saad Omer, MBBS, MPH, PhD. This led to the launch of one of the most influential bio-repositories in COVID-19 research called IMPACT Yale (Implementing Public Health Action Against Coronavirus in Connecticut), where they met to discuss testing, surveillance, and research around the new virus.

“We started collecting bio-specimen from patients and healthcare workers and analyzing their viral replication and immune response in real time, and I've never been so excited and so tired at the same time,” admitted Iwasaki. “As immunologists, we started seeing things that we weren't used to seeing happening in patients. We started analyzing patients that came through the hospital by collecting their blood and saliva to begin analyzing their immune responses in real time.

“We collected these mono-nuclear cell fractions from the blood and ran flow cytometry assays, which tell us about what kinds of cells are in the blood and how activated they become in these patients.”

The process involved data analysis in several laboratories. RNA data was collected from patient saliva by the Grubaugh Lab. The Iwasaki Lab then performed a PCR analysis on the saliva, while researchers in the Ring Lab, led by Aaron Ring, MD, PhD, assistant professor of immunobiology, ran ELISA on the patient sera for antibody levels.

“It was a great, well-oiled machine that kept churning out data day in and day out. The outcome of this research showed that in severe COVID-19 patients, there was a defect in clearing the virus and the sustained high levels of viral load over time. We saw inflammatory cytokines and also cytokines that belong to defense systems, usually reserved for fighting other types of pathogens like fungi and parasites. This study showed that the maladaptive responses are correlating with the severity of COVID-19,” explained Iwasaki.

Iwasaki adds that they are still learning what makes individuals succumb to disease, but notes that a key factor is an individual’s gender.

A study from Iwasaki’s lab found some key differences between male and female patients who were not treated with immuno modulatory agents. While many factors were similar between the sexes, male patients had elevated interleukin 8 and IL-18, inflammatory cytokines that trigger further inflammation.

Another difference: female patients had more activated T-cells than male patients. “We found that there is an age-dependent decline in T-cell activation only in male patients, but not in female patients,” said Iwasaki. “This study emphasizes the need to develop sex disaggregated data for research, because the biological sex has such an important impact on the immune system and other organ systems that this is a real key aspect to be studying in infectious disease.”

Throughout the progression of the pandemic, it was reported that patients were experiencing a widespread of symptoms beyond the respiratory system.

Juthani treated many COVID-19 patients at Yale New Haven Hospital and added, “What has been interesting as an infectious disease physician is the spectrum of this virus and how it has been able to impact such a broad spectrum of systems within the human body, and one, in particular, is the impact on the nervous system and the brain, in particular. And for respiratory viruses, in particular, it is a little bit unusual.”

This leads to another notable discovery out of the Iwasaki Lab—the connection between the brain and the immune system. They investigated the possibility of whether SARS-CoV-2 can infect cells in the brain with three separate approaches. The first system was human brain organoids, miniature brains that grow in tissue culture, where they were able to infect the tissue culture to study neuroinvasiveness. The second model used a mouse model of COVID-19 to study tissue-specific infection in the mouse model, and the third approach used human autopsies from COVID-19 patients who died of this infection. In all three cases, they found there was a clear infection of neurons by the virus in the organoid. They saw that neurons around the infected cells actually died of lack of oxygen.

“It's kind of eerie to think that within the organ, the infected cells are soaking up the oxygen that they need to replicate the virus, while other cells around them are being deprived of oxygen and dying of hypoxia. We examined three patients' autopsy samples, and one of them had clear evidence of infection in the brain, and particularly the cortical neurons were infected, and that was accompanied also by the microinfarctions that were happening in the brain,” said Iwasaki.

When neurons die, they cannot be replaced . This explains the reports of “brain fog” and lack of memory, described by many recovering COVID-19 patients. “This could happen because of a direct infection of the neurons, or other inflammatory cytokines could act upon the endothelial cells in the brain, making a blood-brain barrier leaky, and that could also result in some of the brain-specific organ dysfunction.”

Another peculiar symptom seen in some COVID-19 patients is the development of symptoms that are similar to those caused by arthritis or syndromes that resemble autoimmune disease .

“This is not something I'm used to seeing with the run-of-the-mill respiratory viruses,” commented Juthani.

Iwasaki agreed. ”What we found was quite striking. In collaboration with Dr. Aaron Ring’s lab, we found numerous autoantibodies against autoantigens that are found in many different places in the body. The extent of this autoantibody development was comparable or even more severe than what he found in lupus patients, and so some of these were against immune proteins and immune cells, which impair the antiviral defense system altogether.”

Iwasaki’s lab found an autoantibody against type 1 interferons, the very cytokines that block virus replication. About 20% of severe COVID-19 patients had this autoantibody. This also correlated with their lack of ability to control the viral load in the nasopharynx and the saliva. Curiously, many of these people turned out to be men, said Iwasaki. A team of researchers at The Rockefeller University led by Jean-Laurent Casanova had already reported similar types of anti-interferon antibody in severe COVID-19 patients. In this case, 94% were male.

“In addition to these kinds of autoantibodies that block immune function, we also saw many that are against the tissue antigens,” said Iwasaki. Researchers are still trying to understand whether they cause dysfunction in some of the organs. Iwasaki and her team are interested in understanding the link between the antibodies and potential long-hauler disease.

When the pandemic hit, not much was known about COVID-19’s impact on pregnant women.

Lu-Culligan’s research interests prior to the pandemic were focused on the impact of viruses on the immune system, antiviral responses during pregnancy and the way that pregnancy is executed, and how the fetal immune response can impact the development of the neural system.

“An early focus of work in the field was trying to figure out, does SARS-CoV-2 actually infect the placenta? Can it cross the placenta, and can it infect babies in utero?” said Lu-Culligan. “We had the opportunity in the Iwasaki Lab to contribute to a very early study on the pandemic that focused on a woman who was diagnosed with a very severe SARS-CoV-2 infection in the second trimester of pregnancy, and very sadly, the case ultimately ended in fetal loss.”

During this case, the researchers were able to acquire the placenta and found that the SARS-CoV-2 virus is capable of invading the placenta. “The next question that comes after that is can it cross and infect the child in utero?” said Lu-Culligan. “In our early studies, we were analyzing how common this was, when does it happen, and what is the impact on the fetus.”

They worked with clinical colleagues in the Department of Obstetrics, Gynecology & Reproductive Sciences, as well as Yale New Haven Hospital’s labor and delivery floor to collect placental specimens from women who were admitted to labor and delivery and had a positive SARS-CoV-2 nasal swab.

As mothers delivered full-term third-trimester babies in the ward, the lab collected the placentas to analyze how common a placental infection was, and what the risk of transplacental infection was.

“Women who are infected during pregnancy with SARS-CoV-2 are at higher risk for poor outcomes. They typically are a high risk for severe COVID-19, for hospitalization, intubation, and unfortunately, poor outcomes to the fetus. In most of these cases, the placenta isn't directly infected, and further studies by not only our group, but other groups have shown that most of the time babies are not actually infected in utero with this virus, which is great news in general,” said Lu-Culligan.

From there, they were interested in how the immune system can actually impact pregnancy outcomes and fetal outcomes and began studying the placentas from women who had a respiratory SARS-CoV-2 infection.

“What we found is that even when the virus does not directly attack the placenta, the placenta mounts a very robust immune response and actually shows signs of physiological stress and damage, including oxidative damage and metabolic stress,” said Lu-Culligan. “We think that this actually has a lot to do with why pregnant women are so at risk for severe COVID-19.”

In addition, there are other risk factors that play a role in severe cases of COVID-19 comorbidities. Comorbidities are the presence of two or more diseases or health conditions.

“What we found was that there was an interesting correlation between the number of risk factors that an individual had and the overall viral load that they would carry, especially, in the initial stages of the disease,” said Silva, who worked on the study. “For example, if a patient has diabetes, in addition to chronic lung disease, or maybe they have some hypertension as well, they were more likely to actually have a higher viral replication seen in their saliva.

”We can imagine that these conditions, because of their chronic state, tend to exert inflammation throughout the body chronically, and so there may be some avenue in which both the hindered immune response as well as this sort of chronic state of inflammation sort of presets you to be less able to fight the infection” said Silva.

One of the many remaining and perplexing questions is the “why” behind those who continue to experience long-term effects of COVID-19. These patients—also known as “long-haulers”—have symptoms such as brain fog, shortness of breath, and more.

While there are findings of such recurring symptoms and effects, scientists are unsure of the reasons why. Compared to the acute disease, there’s little known about the underlying cause of long-term COVID-19. It’s possible that a virus infection or remnants of virus remain in some people, leading to chronic inflammation.

“The other possibility is that long-haulers develop autoantibodies or auto-reactive T-cells,” said Silva. There’s evidence that autoantibodies develop during acute COVID-19. About 40% of COVID-19 long-haulers report feeling better after vaccination. “I got very excited because it might give us a window through which we can study the long-haul disease because we know what the vaccine does,” said Silva. “The vaccine induces immunity against the spike protein, which can remove the source of these long-term, persistent infections or remnants of the virus, and that could be making the improvement in these people.” But Silva thinks another possibility could be that the inflammatory cytokines that are induced by vaccination could have an impact on these auto-reactive cells.

As more studies and research are conducted on COVID-19 patients, more data will help the medical community have a better understanding of the virus, and ultimately, a better understanding of how to treat the complicated disease.

Originally published August 11, 2021; updated May 10, 2022.