NEWS

The study was carried out in Tel Aviv University and led by Ph.D. student Noam Ben-Shalom from the lab of Dr. Natalia Freund at the Faculty of Medicine and Ph.D. student Elad Sandbank from the Neuro-immunology Lab of Prof. Shamgar Ben-Eliyahu at The School of Psychological Sciences and the Sagol School of Neuroscience. The paper was published on July 6th in the leading scientific journal Brain, Behavior, and Immunity.

Dr. Freund explains: "In this study we examined, for the first time, the correlation between stress and the body's ability to develop an immune response following vaccination. The prevailing assumption is that the effectiveness of a vaccine is determined mainly by its own quality. However, over the years, professional literature has reported influences of other factors as well, such as the age, genetics, and microbiome of the outcomes of vaccination. Our study was the first to investigate the possible effects of acute stress. We found that this mental state has a dramatic impact – not only on the vaccine's effectiveness, but also on how it works." 

Classical 'Fight or Flight' Response

Acute stress is a mental state caused by immediate threat (either real or imagined), involving the secretion of adrenaline and stimulation. In this study, Dr. Freund and her colleagues vaccinated mice with two different vaccines: the model protein Ovalbumin and a fragment of the SARS-CoV-2 spike protein also used in the COVID-19 vaccine. Nine days later, just as the adaptive immunity became active and the production of antibodies began, the mice were subjected to a widely used behavioral paradigm simulating acute stress. Two and a half weeks after exposure to stress, namely 30 days after vaccination, the level of antibodies in the blood of vaccinated animals that had experienced stress was 70% higher compared to the control group. This phenomenon was observed in animals vaccinated with either type of vaccine.

At the same time, the researchers discovered that the immune system of the animals that had experienced stress was not cross reactive to variants of the protein used in the vaccine. In other words, following stress the immune system was focused entirely on the original vaccine, showing no response to proteins that were only slightly different – such as variants of concern (VOC) of SARS-CoV-2.

Dr. Natalia Freund

"In general, the purpose of vaccination is not only protection against a specific pathogen, but also creating a long-lasting immunological memory for protection against future mutations of that pathogen." - Dr. Natalia Freund

"Initially, we were surprised to find out that the response to the vaccine was much more effective in animals that had experienced stress," says Dr. Freund, "we would have assumed just the opposite – that stressful situations would have a negative impact on the immune system. Nevertheless, with both types of vaccines, we observed a stronger immune response after stress, both in the blood and in B cells (the lymphocytes that produce antibodies) derived from the spleen and lymph nodes of the immunized mice. The enhancement of the antibodies' activity following stress was mediated by the cellular receptor that identifies adrenaline - the beta2 adrenergic receptor. When we blocked this receptor, either pharmacologically or by means of genetic engineering, the effects of stress were completely eliminated. On the other hand, to our great surprise, the breadth of the immune response generated by the vaccine was reduced by about 50% following stress. In general, the purpose of vaccination is not only protection against a specific pathogen, but also creating a long-lasting immunological memory for protection against future mutations of that pathogen. In this sense, the vaccines appeared to lose much of their effectiveness after exposure to stress."

According to the researchers, this is in fact a classical 'fight or flight' response, however this time demonstrated at the molecular level. During stress, the immune system produces large quantities of antibodies and stronger antibodies, to address the immediate infection, and this large energetic investment in the here and now comes at the expense of future immunological memory.

Does It Apply to Humans?

Dr. Freund adds: "In the second part of the study we wanted to test whether humans also display the post-stress immune impairment observed in vaccinated mice. For this purpose, we cultured B cells obtained from blood of people who had contracted COVID-19 in the first wave. We then induced stress in these cultures using an adrenaline-like substance that stimulates the beta2 adrenergic receptor, that was identified by us in the first part of the study as a mediator of the response to stress in cells that produce antibodies in mice. B cells express a very high level of these receptors, but until now the receptors' role in producing antibodies was not known. In fact, it was unclear why these cells need the ability to respond to adrenaline."

"We discovered that just like in mice, human cells also exhibit a zero-sum game between the intensity and breadth of the immune response. When the adrenaline receptor is activated during stress, the entire immune system is stimulated, generating antibodies that are 100-fold stronger than antibodies produced in cells that had not undergone stress. But here too, the response was narrower: the diversity of antibodies was reduced by 20-100%, depending on the individual from whom the cells were taken.

"Stress 9 to 12 days after vaccination, at the time when B cells are generating high affinity antibodies, enhances short-term immunity and damages long-term memory." – Dr. Natalia Freund

RNA sequencing of the cells in which the beta 2 adrenergic receptor was activated, compared to regular cells, indicated that the receptor's activation caused antibody-producing cells to work at maximum capacity (by activating the PI3 kinase protein and phosphorylation of AKT) - at the expense of antibody breadth and diversity."

"From the evolutionary perspective," concludes Dr. Freund, "stress can be caused by different factors. We tend to think of mental stress, but physical illness also causes a form of stress. When the body contracts a virus or bacteria it experiences stress, and signals to the immune system that the top priority is getting rid of the pathogen, while investing energy in long-term immunological memory is a second priority. Therefore, stress 9 to 12 days after vaccination, at the time when B cells are generating high affinity antibodies, enhances short-term immunity and damages long-term memory."

The study was led by Dr. Meir Finkel of the Department of Archaeology and Ancient Near East Cultures, Tel Aviv University and Prof. Gonen Sharon of the MA Program in Galilee Studies, Tel-Hai College, in collaboration with Prof. Erez Ben-Yosef, Tel Aviv University, Dr. Oded Bar and Dr. Yoav Ben Dor, the Geological Survey of Israel, and Ofir Tirosh, the Hebrew University. The paper was published in Geoarchaeology.

Dr. Finkel: "The Hula Valley, located along the Dead Sea Transform Rift, is well known for its many prehistoric sites, the oldest of which date back to 750,000 years before present (YBP). The valley offered early humans rich sources of water, vegetation, and game, right on the northward migration route from Africa - the Great African Rift Valley. These early inhabitants left behind them many artifacts, including thousands of hand axes – flint stones chiseled to fit the human hand. One of the earliest and most universal tools produced by humans, the hand axe may have served as a multipurpose 'penknife' for many different tasks, from cutting game meat to digging for water and extracting roots. It was used in many different parts of the Old World, in Africa, Asia, and Europe, for about 1.5 million years."

"One of the earliest and most universal tools produced by humans, the hand axe may have served as a multipurpose 'penknife' for many different tasks, from cutting game meat to digging for water and extracting roots." -  Dr. Meir Finkel

In the present study the researchers looked for the source of the raw material used to produce thousands of hand axes found at two prehistoric sites in the Hula Valley: Gesher Benot Ya'aqov, dated to 750,000 YBP and Ma'ayan Barukh, dated to 500,000 YBP, both Acheulian.  Prof. Sharon: "Approximately 3,500 hand axes were found scattered on the ground at Ma'ayan Barukh, and several thousands more were discovered at Gesher Benot Ya'aqov. The average hand axe, a little over 10cm long and weighing about 200g, was produced by reducing stones that are five times larger – at least 1kg of raw material. In other words, to make the 3,500 hand axes found at Ma'ayan Barukh alone, early humans needed 3.5 tons of flint. But where did they obtain such a huge amount of flint? Many researchers have tried to answer this question, but our study was the first to use innovative 21st century technologies: advanced chemical analysis and an AI algorithm developed specifically for this purpose."

The Gesher Benot Ya'aqov area

20 Kilometer Hikes Across Diverse Terrain

The researchers took samples from 20 hand axes – 10 from Gesher Benot Ya'aqov and 10 from Ma'ayan Barukh, ground them into powder and dissolved the powder in acid in a clean lab. For each sample they measured the concentration of approximately 40 chemical elements, using an ICP-MS (inductively coupled plasma mass spectrometer), a state-of-the-art device that accurately measures the concentration of dozens of elements, down to a resolution of one particle per billion.

In addition, in order to locate possible flint sources available to the Hula Valley's prehistoric inhabitants, the researchers conducted a field survey covering flint exposures in the Safed Mountains, Ramim Ridge, Golan Heights, and Dishon Plateau, as well as cobbles from streams draining into the Hula Valley: the Jordan, Ayun, Dishon, Rosh Pina, and Mahanayeem. This methodical survey was combined with a comprehensive literature review led by Dr. Bar of the Geological Survey of Israel. Flint samples collected from all potential sources were then analyzed using ICP-MS technology to enable comparison with the hand axes. A novel computational approach specially adapted by Dr. Ben Dor of the Geological Survey of Israel was used for this comparison.  

Dr. Ben Dor: "The complex process, from collecting and preparing the samples to the chemical analysis, produced a very large amount of data for each sample. To enable optimal matching between data from the archaeological artifacts and data from the flint exposures, we developed a dedicated algorithm based on several computational steps, alongside machine learning models. Thus, we were able to classify the archaeological artifacts according to the database derived from the geological samples."

"To procure suitable raw materials for producing their vital hand axes, [humans living in the Hula Valley hundreds of thousands of years ago] planned and carried out 20km hikes that included an ascent from 70 to 800 meters above sea level." Prof. Erez Ben-Yosef

Dr. Finkel: "Through the computational process we discovered that all 20 archaeological artifacts were made of flint from a single source: the Dishon Plateau's flint exposures dating back to the Eocene geological epoch, about 20km west of the Gesher Benot Ya'aqov and Ma'ayan Barukh sites. At the Dishon Plateau we also found a prehistoric flint extraction and reduction complex, indicating that the place served as a flint source for hundreds of thousands of years. In addition, we demonstrated that cobbles from streams draining into the Hula Valley were too small to be used as raw material for hand axes, ruling out this possibility."

Prof. Ben-Yosef: "Our findings clearly indicate that humans living in the Hula Valley hundreds of thousands of years ago, probably hominids of the homo erectus species, possessed high cognitive and social capabilities. To procure suitable raw materials for producing their vital hand axes, they planned and carried out 20km hikes that included an ascent from 70 to 800 meters above sea level. Moreover, they passed on this important knowledge from one generation to the next, over many millennia. All these suggest a high level of sophistication and ability, which modern researchers do not usually attribute to prehistoric humans from such an early period."

From left: Dr. Meir Finkel and Prof. Erez Ben Yosef

The groundbreaking study was led by PhD student Yasmin Granot-Matok and Prof. Dan Peer, a pioneer in the development of RNA therapeutics and Head of the Nanomedicine Laboratory at The Shmunis School of Biomedicine and Cancer Research, also serving as TAU's VP R&D. The study's results were published in Theranostics.

Prof. Peer explains: "Many bacteria secrete toxins. The most famous of these is probably the botulinum toxin injected in Botox treatments. Another classic treatment technique is chemotherapy, involving the delivery of small molecules through the bloodstream to effectively kill cancer cells. However, chemotherapy has a major downside: it is not selective, and also kills healthy cells. Our idea was to deliver safe mRNA molecules encoded for a bacterial toxin directly to the cancer cells – inducing these cells to actually produce the toxic protein that would later kill them. It's like placing a Trojan horse inside the cancer cell."

Prof. Dan Peer

Impressive Results

First, the research team encoded the genetic info of the toxic protein produced by bacteria of the pseudomonas family into mRNA molecules (resembling the procedure in which genetic info of COVID-19's 'spike' protein was encoded into mRNA molecules to create the vaccine). The mRNA molecules were then packaged in lipid nanoparticles developed in Prof. Peer's laboratory and coated with antibodies - to make sure that the instructions for producing the toxin would reach their target, the cancer cells. The particles were injected into the tumors of animal models with melanoma skin cancer. After a single injection, 44-60% of the cancer cells vanished.  

"With a simple injection to the tumor bed, we can cause cancer cells to 'commit suicide', without damaging healthy cells. Moreover, cancer cells cannot develop resistance to our technology as often happens with chemotherapy – because we can always use a different natural toxin." - Prof. Dan Peer

"In our study, the cancer cell produced the toxic protein that eventually killed it," says Prof. Peer. "We used pseudomonas bacteria and the melanoma cancer, but this was only a matter of convenience. Many anaerobic bacteria, especially those that live in the ground, secrete toxins, and most of these toxins can probably be used with our method. This is our 'recipe', and we know how to deliver it directly to the target cells with our nanoparticles. When the cancer cell reads the 'recipe' at the other end it starts to produce the toxin as if it were the bacteria itself and this self-produced toxin eventually kills it. Thus, with a simple injection to the tumor bed, we can cause cancer cells to 'commit suicide', without damaging healthy cells. Moreover, cancer cells cannot develop resistance to our technology as often happens with chemotherapy – because we can always use a different natural toxin."

Other contributors to the study included: Dr. Assaf Ezra, Dr. Srinivas Ramishetti, Dr. Preeti Sharma Dr. Gonna Somu Naidu and Prof. Itai Benhar, Head of the Antibody Engineering Lab at the Shmunis School of Biomedicine and Cancer Research at TAU. The study was funded by the Shmunis Family Foundation for Biomedicine and Cancer Research.