COVID‐19 is disastrous to global health and the economy. SARS‐CoV‐2 infection exhibits similar clinical symptoms and immunopathological sequelae to SARS‐CoV infection. Therefore, much of the developmental progress on SARS‐CoV vaccines can be utilized for the development of SARS‐CoV‐2 vaccines. Careful antigen selection during development is always of utmost importance for the production of effective vaccines that do not compromise recipient safety. This holds especially true for SARS‐CoV vaccines, as several immunopathological disorders are associated with the activity of structural and nonstructural proteins encoded in the virus's genetic material. Whole viral protein and RNA‐encoding full‐length proteins contain both protective and “dangerous” sequences, unless pathological fragments are deleted. In light of recent advances, peptide vaccines may present a very safe and effective alternative. Peptide vaccines can avoid immunopathological pro‐inflammatory sequences, focus immune responses on neutralizing immunogenic epitopes, avoid off‐target antigen loss, combine antigens with different protective roles or mechanisms, even from different viral proteins, and avoid mutant escape by employing highly conserved cryptic epitopes. In this review, an attempt is made to exploit the similarities between SARS‐CoV and SARS‐CoV‐2 in vaccine antigen screening, with particular attention to the pathological and immunogenic properties of SARS proteins. Careful antigen selection is essential for development of an effective and safe SARS‐CoV‐2 vaccine. Considering recent advances, peptide vaccines can be ideal substitutes for protein and RNA/DNA‐based vaccines. Peptide vaccines can avoid immunopathological sequences, focus immune responses on neutralizing B‐cell and on T‐cell epitopes, combine antigens from different viral proteins, and avoid mutant escape by employing conserved epitopes. The overall mortality rate of the infection has been estimated to be around 2.1%. Currently (May 2021), the heaviest infection burden is in the United States, India, Brazil, France, Turkey, Russia, United Kingdom , Italy, Spain, Germany, Argentina, Columbia, Poland, Iran, Mexico, and Ukraine: each of these localities has more than 2 million infected cases. Positively, infections are mostly under control in some countries, including China, Canada, Australia, and Taiwan. However, it needs to be noticed that world infection burden is changing rapidly. The economic losses from the “lockdown” of cities and whole countries, the severely impacted tourism and transport industries, and the rapid shutdown of services provided by a diversity of small businesses across the globe have been estimated to be in the hundreds‐of‐billions of dollars.SARS‐CoV‐2 (abbreviated here as SARS‐2) infection is the causative factor underlying coronavirus disease of 2019—more generally known as COVID‐19. The novel SARS‐2 outbreak, which initially presented in Wuhan in the Hubei province of China, rapidly transitioned into a worldwide pandemic, as declared by the WHO on March 11, 2020. In contrast, containment efforts for SARS‐2 failed, and global spread ensued. However, the extensive research conducted on MERS‐CoV and SARS‐1 infections provided solid background for the development of treatments for SARS‐2 and facilitated rapid advancement of several vaccine candidates and antiviral drugs to clinical trials. That being said, at present, there are still no approved therapeutic vaccines against any of the coronavirus infections: SARS‐1, MERS‐CoV, or SARS‐2. Recently, the DNA vaccine, nCoV‐19 ChAdOx‐1 (AstraZeneca/Oxford), and mRNA vaccines, mRNA‐1273 (Moderna), and BNT‐621 b1/b2 (Pfizer/Biontech), were approved for prophylactic human use. Several other vaccines, such as Ad26.COV2.S (Johnson & Johnson), Sputnik V , and CoronaVac (Sinovac) have also been approved, but only for use in certain countries. Furthermore, the only approved antiviral medication  has very modest efficacy . Likewise, it failed to demonstrate significant benefits in treatment of moderate or severe illness in small clinical trials.Three different zoonotic coronavirus epidemics have occurred in the past two decades. In addition to COVID‐19 disease, an outbreak of severe acute respiratory syndrome disease (SARS), which was caused by SARS‐1 virus, occurred in 2002 in the Guangdong province of China, and Middle East respiratory syndrome diseased, which was caused by MERS‐CoV virus, occurred in 2012 in Saudi Arabia (KSA). These viruses resulted in mortality rates of around 10% and 35%, respectively. The two previous outbreaks were well contained and, in each case, fatalities were limited to a few hundred.Notably, peptide‐based vaccines against these coronaviruses have been overlooked. However, they may hold great potential in providing safe and protective immune responses against SARS‐2 infections. Therefore, this review summarizes the immunogenicity and protective capacity of SARS antigens, as well as the pathological dangerous sequences mapped within the highly similar SARS proteome. In addition, adjuvant choice, animal models of infection, SARS vaccination approaches with relative efficacy, and potential adverse responses are discussed.1.1Betacoronavirus genera, Coronaviridae family. SARS‐2 (Accession no. MN908947.3) has a similar overall genome identity to several other coronaviruses, such as bat coronavirus RaTG13  and SARS‐CoV (SARS‐1) . RaTG13 has 99% genome coverage, while SARS‐1 has 88% coverage of the SARS‐2 genome. MERS‐CoV (Accession no. NC_019843.3), the causative agent of MERS coronavirus infections, appears to the most divergent of the group, with only 30% genome coverage of SARS‐2, as calculated using the Blast‐n server.SARS‐2 virus is a positive‐sense (+) ssRNA‐enveloped virus of the Figure  S‐protein plays a virulent role mediating viral attachment and fusion into host cells. E‐protein plays a key role in viral life cycle contributing to assembly and budding, and functions as ion‐channeling viroporin. The open reading frames (ORFs: portions of RNA sequence encoding amino acids without stop codons) encode several NSPs that play various roles in viral replication and the disruption of host immune responses. NSP‐1 inhibits host mRNA translation, while viral RNA helicases and polymerases, encoded in ORF1‐NSPs 7–16, transcribe viral RNA. Viral proteases, for example, PLpro and 3CLpro, are encoded in ORF1‐NSPs 2–6, cleave the polyprotein precursor directly translated from viral RNA. Finally, in addition to NSPs, nine accessory factors are also encoded in viral RNA within several ORFs, that is, ORF 1, ORF 3, ORFs 6–9, and ORF 10, which interfere with host interferon production , membrane (M), envelope (E) and nuclear (N) proteins, and 16 nonstructural proteins (NSPs)  is homologous to SARS‐1 S‐protein (SARS‐1‐S) and RaTG13 S‐protein, with identity similarities of 75% and 98%, respectively, as calculated by the ClustalW server.S‐protein is a homotrimeric transmembrane class‐I fusion glycoprotein that coats the surface of the SARS‐2 viral membrane. S‐protein is responsible for binding to the host cell surface receptor, fusing, and then entering into the host cell to initiate replication  contains a receptor binding domain (RBD) located close to the S1/S2 cleavage site, while the S2 subunit contains fusion peptide , which is expressed by lung type‐II pneumocytes and alveolar macrophages. It is also worth noting that ACE2 is expressed in several other tissues, including muscles, adipose tissue, and the brain, kidney, and intestinal tract.Similar to other e Figure . A1.3 It is an important part of the blood pressure regulation renin angiotensin system (RAS), as it cleaves angiotensin II (Ag‐II) and converts it into angiotensin (1–7), which reduces blood pressure. While SARS‐1 and SARS‐2 S‐proteins bind to ACE2 receptors to gain entry into a host cell, MERS‐CoV instead employs dipeptidyl peptidase‐4 (DPP‐4). ACE2 expression reflects viral host and tissue tropism. ACE2 is highly expressed in several human tissues, like lung, kidney. and heart tissue, but has far lower expression in other tissues, for example, in the upper respiratory tract. This explains the very mild effects of SARS infections on the upper respiratory tract compared to the lower respiratory tract. The high ACE2 expression in type‐II pneumocytes in the lower respiratory tract and in heart ventricular tissues explains the severe illness sequelae, including elevated viral RNA load in the ventricles of deceased patients. In the lungs, where the infection generally takes the strongest hold, SARS‐1 not only infects pneumocytes II, but also macrophages, as they too express ACE2. This causes damage to lung tissues as well as alveolar macrophages.ACE2 is a member of the dipeptidyl peptidase enzyme‐receptor family.1.3.1 The RBD contains several amino acids that are able to form hydrogen bonds, and cationic amino acids that form electrostatic interactions or salt bridges with the anionic amino acids of the ACE2 ridge. The binding mode of RBDs to ACE2 is similar for both SARS viruses, and neither require glycosylation of the S‐protein. CBRs for SARS‐2‐RBD are K417, G446, Y449, Y453, L455, F456, F486, A475, N487, Y489, Q493, G496, Q498, T500, N501, G502, and Y505. These residues are located in the RBM region, position 438 to 506, in the S1‐subunit of the S‐ectodomain; they carry similar amino acids to SARS‐1. However, this does not mean that noncontact residues are unimportant; they impart the structural conformation necessary for the contact residues to bind effectively.The binding of both SARS viruses, through RBDs, occurs at the anionic ridge of ACE2 at the entrance to the catalytic site (position 636–717), but not within the catalytic site itself.1.3.2 The presence of this receptor affects tissue tropism in the presence of ACE2. The relative infectivity is 1.6‐fold higher with a combined presence of ACE2 and NRP‐1, compared to ACE2 alone in human embryonic kidney cells. The NRP‐1 receptor binds to the C‐terminus of RBD after S1/S2 cleavage by host enzymes. Therefore, S‐protein priming is critical for NRP‐1 to function as an entry receptor. Since priming occurs after conformational alteration to the S‐protein, which takes place due to ACE2 binding, NRP‐1 can enhance infectivity only in the presence of ACE2. NRP‐1 is expressed mainly in smooth muscles, lung cells, cardiac myocytes, and dendritic cells (DCs). It has been suggested that the presence of this alternative virus entry receptor, especially in the upper lung airways, could explain the more severe symptoms associated with SARS‐2 disease compared to SARS‐1 infections. However, if this were the case, then the increased tissue tropism and virulence would be dependent on the co‐expression of ACE2 and NRP‐1, as well as the priming enzymes, in the tissues. Since ACE2 is only highly co‐expressed with NRP‐1 in a few tissues, the overall effect would not be highly significant, that is, in the absence of ACE2. Still, it could be beneficial to include immunogenic epitopes near or at the cleavage site of S1/S2 in peptide vaccine to prevent NRP‐1 from binding to the C‐terminus of RBD after priming.Recently, SARS‐2 S‐protein was found to be able to bind to neuropilin‐1 (NRP‐1) receptors.2 Unfortunately, no similar studies have been published on the immunopathology of sequences within SARS‐2 proteins so far. Coronavirus infections, especially SARS‐1 and SARS‐2, are associated with the suppression of type‐I interferons (IFNs‐I) in patients showing severe illness (compared to those with mild illness) and the induction of pro‐inflammatory cytokine and chemokine production. IFNs‐I (IFN‐α and IFN‐β) can stimulate the immune system to generate appropriate virus‐specific adaptive immune responses that counter and locally contain infection within afflicted tissues. Inflammatory cytokines, on the other hand, can deteriorate patient clinical status rapidly and even more severely than viral replication itself. This is supported by the deterioration of SARS‐1‐infected patients’ clinical conditions, characterized with severe coagulopathy, coinciding with the increase in inflammatory cytokine level and infiltration of monocytes and neutrophils, rather than the level of viral load, that is, replication‐associated damage. This highlights the essential importance of careful antigen and adjuvant selection in order to control the cytokine release profile and develop a safe adaptive immune response.The SARS‐1 proteome comprises several structural and NSPs that play immunopathological roles. SARS‐2 infection is characterized by leaky vasculature and infiltration of inflammatory neutrophils. In COVID‐19 severe illness, the spread of immunopathology radiates in the lower respiratory tract, resulting in fibrosis, diffuse alveolar damage, and pneumonia from the accumulation of cell debris, fluids and fibrils, thus compromising the gas exchange processes. The cytokine profile of SARS‐1‐infected patients is characterized by elevated expression of IL‐1ß, IL‐6, IL‐8, MCP‐1, and IP‐10 (CXCL‐10); this is similar to the elevated pro‐inflammatory cytokine expression of SARS‐2‐infected patients. Elevated IL‐6 cytokine level, which is prominent in both infections, induces vascular permeability and the production of IL‐8, MCP‐1, and more IL‐6. In young patients, moderate levels of pro‐inflammatory cytokines and IFN‐γ have been reported; this could be a contributing factor to the comparatively low fatality rate of infected children.Since most of the influential immunopathological sequences in SARS‐1 viral proteins are similar  to those of SARS‐2, it is expected that they play the same role in infection. This is also supported by similar clinical symptoms and cytokine release profiles exhibited by both types of SARS infections  signals a macrophage inflammatory response. These latter immune cells contribute to immunopathology, for example, neutrophil infiltration, as reported in severe illness symptoms and in deceased patients. Inflammatory cell recruitment results in acute damage to the lungs. Therefore, immunopathology‐causing sequences and viral components should be avoided for the production of safe vaccines.COVID‐19, like SARS disease, shifts the immune system toward an innate Th2 hyperreactivity response, while inhibiting Th1 responses by blocking IFN‐I signaling and production pathways. As a result, cytokine storm chemotaxis recruits inflammatory cell responses, which induce tissue‐damaging inflammatory immune responses, for example, IL‐8 recruits neutrophils, and CCL3  viral challenge following early depletion of alveolar macrophages.Alveolar macrophages and respiratory dendritic cells are vital antigen presenting cells (APCs), which are normally responsible for pathogen sampling and phagocytosis, as well as the development of adaptive immune responses through the production of necessary Th1 or Th2 interleukins, or both. The infection of alveolar macrophages, albeit not well investigated to date, may play a key role in pro‐inflammatory cytokine storm release.2.1α and IFN‐β, is important in developing strong immune responses against viral infections. The pathways of IFN‐I production and signaling are blocked by SARS viral proteins. The production of IFNs‐I occurs through two main processes that start with either 1) the toll‐like receptor (TLR) pathway, or 2) the retinoic acid‐inducible gene 1 (RIG‐1) and melanoma differentiation‐associated protein 5 (MDA‐5) pathway  adaptor molecules are signal transduction adaptors of most TLRs. They initiate the transformational growth factor‐β‐activated kinase 1 (TAK‐1) cascade that activates nuclear transcription factors IRF‐3 and IRF‐7 by phosphorylation and translocation into the nucleus. This leads to the transcription of IFN‐I genes. In contrast, RIG‐1 and MDA‐5 employ adaptor protein mitochondrial antiviral signaling proteins (MAVs) that activate the complex formation of TNF receptor‐associated factor‐3 (TRAF‐3), along with several other factors and activators. These then phosphorylate IRF‐3 and IRF‐7, which results in their transport to the nucleus for IFN‐I transcription  transcription genes occurs through interferon stimulating gene factor‐3 (ISGF‐3). This signaling pathway is initiated by the activation of Janus kinases  and signal transducers and activators of transcription (STAT‐1 and ‐2). Together, this is known as the JAK/STAT pathway  to their counterparts in SARS‐2, except for ORF‐3b, which is absent in SARS‐2. It is likely that they play similar immunopathological roles in SARS‐2 infections.Several viral proteins, or their domains/peptidic fragments, inhibit the aforementioned IFN‐1 production and signaling pathways. Therefore, the incorporation of such fragments/epitopes as vaccine antigens should be avoided to prevent training the immune system to develop a pro‐inflammatory response upon viral infection. SARS‐1 NSP‐1,] Figure ; most of2.2 It was found that administration of SARS‐1 S‐protein downregulates ACE2 expression, induces inflammatory responses, and exacerbates acute lung injuries in vivo in mice. Low ACE2 expression levels lead to an increase in Ag‐II, which increases blood pressure, thrombus formation, reactive oxygen species, and upregulation of matrix metalloproteinase‐2 and ‐9, as well as inflammatory cytokines, for example, IL‐6. Increased levels of Ag‐II in the blood are associated with acute pulmonary tissue damage and inflammatory cytokine  release. These effect were reversible upon addition of recombinant rACE2, which resulted in lower IL‐6 and Ag‐II levels and an increase in angiotensin 1–7 levels.ACE2 plays a major role in tissue repair, homeostasis, and fluid balance, while increased levels of Ag‐II are associated with acute tissue damage. All COVID‐19 mortalities have shown pneumonia and acute respiratory distress syndrome, characterized by severe inflammation and diffuse alveolar damage; a third of fatalities have additionally displayed pulmonary artery emboli. The damage afflicted in the lower respiratory tract and heart tissue of patients, especially in the ventricular myocytes, can result in hypoxic respiratory failure and ventricular remodeling leading to deteriorating condition and increased risk of death. The transcription levels of viral RNA in heart tissue of SARS‐deceased patients were associated with a decrease in tissue ACE2‐expression, that is, below normal levels, an increase in macrophage infiltration, and a higher extent of tissue damage. Therefore, inhibiting SARS‐S binding to ACE2 should not only relieve the symptoms but also arrest the replication cycle of the virus by interrupting the fusion process, thus protecting deeper tissue and systemic invasion.Clinical evidence supports the role of ACE2 in tissue infection tropism and severe illness sequelae in both SARS‐1 and SARS‐2 infections. Loss of RAS system balance, fluid balance, and high blood pressure have been reported in severe COVID‐19 illness.2.3 Furthermore, inflammation at the immunization site and within the liver was reported after injection of Vaccinia Ankara virus bearing S‐protein in ferrets, which was not present in blank virus negative controls. The inflammatory component of the S‐protein sequence explains the high immunopathological scores in preclinical trials associated with S‐protein‐based subunit vaccines, and even S‐protein‐encoded RNA as vaccine antigen against SARS‐1 in mice, ferrets, and nonhuman primates. Since a variety of studies have shown that S‐protein induces immunopathological reactions when used as antigen, studying the immunopathology of this protein and its fragments is critical for the development of a safe and effective peptide vaccine.S‐protein is the most surface‐exposed SARS‐2 protein. It is also the most immunogenic, as 90% of antibodies (Abs) in patients infected with SARS‐2 are directed against it. However, when full‐length S‐protein or DNA‐encoding S‐protein were employed as vaccine antigen against SARS‐1 in low dose, pro‐inflammatory responses were observed in mice and ferrets.α, and IL‐8 in lung epithelial cells, in peripheral blood monocytes, and in murine macrophages, in vitro through AP‐1 and NF‐κB pathways  have proven to be protective, immunogenic, and inclusive of neutralizing epitopes. Immunization against SARS‐1 using the RBD as vaccine antigen provided potent Abs in mice that neutralized SARS‐2‐S protein in vitro. In a later study, RBD‐based vaccine did not cause immunopathology in animal models. Thus, an alternative explanation of the immunopathological role of the S‐protein 324–488 sequence is needed.SARS‐1‐S stimulated the production of IL‐6, TNF‐s Figure . The side effects were absent in the placebo group, indicating immunopathology of the encoded RBD antigen. Therefore, while the induction of Th1‐type immune responses is important toward safe vaccine development, it is not sufficient on its own to produce safe vaccine, as demonstrated by these RNA‐based examples.More recently, Pfizer released another mRNA‐based vaccine product (BNT162b1) that has passed clinical trials and approved for human use.4.2 Thus, it is rather difficult to identify continuous peptide epitopes that are both protective/neutralizing and that maintain native structure conformation. The RBD is an obvious target for epitope screening for SARS‐2 vaccine development. Epitopes should be a) protein‐surface exposed, b) contain a sufficiently long sequence to maintain conformational features, for example, full helices, to preserve native conformation, and c) contain some but not all of the CBRs necessary to bind RBD to ACE2. This ensures recognition by B‐cell receptors in native conformation. Epitopes should also be tested for their affinity toward ACE2 binding, which should be minimal. Other groups of promising epitopes are those at or near cleavage sites, thus preventing priming by TMPRSS2 or furin action at the S1/S2 or S2ʹ sites.B‐cell epitopes are mostly (90%) conformational, which implies that 90% of epitopes are discontinuous. For example, a vaccine antigen triggered Ab response against influenza virus and generated a response that protected more mice from viral infection challenge, compared to the native fusion protein. Improved immunogenicity was also observed when a similar approach was employed for gp120 of the HIV virus. Therefore, focusing the immune response against fewer, but highly effective or neutralizing epitopes as a general strategy can be an effective approach that affords superior protection. Peptide‐based epitope combined with strong adjuvant that recruits APCs effectively, therefore, is a more direct and often potent approach compared to a large protein‐based antigen that contains hundreds of obsolete epitopes—most of which provide no or undesired immunity.Focusing immune response against known neutralizing or opsonizing epitopes has been found to be extremely effective in generating protective responses against several pathogens, especially viruses. Interestingly, immunodominant epitopes might not always be the most effective antigens for peptide‐based vaccine development. Cryptic epitopes are sequences that are initially less exposed and located at low profile sites that become exposed when structural conformation alterations take place during protein movements (breathing), binding, or functioning. These epitopes often remain highly conserved across pathogen strains and mutations. However, it is highly unlikely that completely buried epitopes could be made more immunogenic by insertion of multiple epitope copies in protein antigen. In contrast, the peptide‐based vaccine approach guarantees exposure of epitopes, and therefore their recognition by B‐cell receptors. Cryptic epitopes, once administered with a strong adjuvant, may neutralize a virus or trigger ADCC mechanisms.Exposed epitopes are often more immunodominant compared to less accessible, buried sequences. Insertion of multiple copies of a cryptic epitope at different positions within a fusion protein sequence did not help to improve the immunogenicity of HIV protein‐based vaccine due to a lack of exposure of these epitopes.m. We explored the binding of peptide fragments of RBD to ACE2 (unpublished data) with the i‐tasser SPRING template server of Michigan University using protein–protein complex interaction reported by Guerler et al. When we compared RBD/ACE2 binding to the binding of potentially neutralizing epitopes from the RBM region (pep1: 445VGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV483), (pep2: 469STEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY508), and one highly conserved cryptic epitope outside of the RBM region but within the RBD sequence (pep3: 366SVLYNSASFSTFKCYGVSPTKLNDLCFTNV395), the binding scores were 2‐ to 4‐fold lower than full‐length SARS‐2‐RBD; resulting in scores of 3.8, 6.8, 4.7, and 13.2 for pep1, pep2, pep3, and RBD, respectively . Thus, these epitopes are too small to bind effectively to ACE2, as they do not possess enough CBRs or are conjugated to bulky carriers (often used for peptide epitope delivery), which creates steric hindrance. Therefore, the administered antigen is available for B‐cell receptors without dose reduction associated with binding to ACE2, and without triggering the adverse effects associated with such binding. The binding affinity of SARS‐2‐RBD to ACE2 is around 15 n New epitopes specific for SARS‐2‐RBM (has 59% sequence similarity to SARS‐1‐RBM) within SARS‐2‐S‐RBD were mapped and found to overlap within the above mentioned Pep1‐3 sequences. However, highly neutralizing anti‐SARS‐1‐S nAbs, such as m396, CR3022, 80R, and S230 were found to be ineffective against SARS‐2, and CR3014 was found to bind to SARS‐2‐S with rather low competitive affinity to ACE2. Yet, in a recent study, two old anti‐SARS‐1‐S neutralizing Abs were found to bind to SARS‐2‐RBM CBRs noncompetitively with highly neutralizing capacity; they lowered viral load titers by 103‐fold in mice. Sera Abs from both SARS and COVID‐19 recovered mice and humans were found to bind to the RBD of S‐protein and block SARS‐2 cell entry.Several recent observations support the theory that SARS‐2 behaves similarly to SARS‐1 and that humoral response remains the key to recovery. Similar to SARS‐1 infections, 90% of Abs produced against SARS‐2 in COVID‐19 patients are directed against S‐protein. which carry CBRs to ACE2 receptors. SARS‐2‐S binds to ACE2 in an identical manner to SARS‐1‐S, and has similar CBRs. The relative importance of each of the binding residues in SARS‐1‐RBD were determined by single residue mutations  as in the Pep1 sequence, or  as in the Pep2 sequence  after recovery from SARS‐2 infection. Several cases of reinfection have been reported. Naturally acquired immunity against SARS‐2 typically decays rapidly after recovery and vaccinations may need to be repeated annually. Vaccines should aim to train the immune system more efficiently than natural infection through use of suitable adjuvants. Thus, immunization, and not natural infection, may be critical for generating and maintaining immunity.Anti‐RBD Abs constitute about 90% of all the neutralizing Abs against SARS infection; thus, they are considered to be highly protective and neutralizing.+ T‐cell epitopes are also recommended for synergistic and superior protective immune responses. Recently mapped in SARS‐1‐S and SARS‐2‐S, CD4+, and CD8+ T‐cell epitopes are nonconformational 5“DYNYKLPDDFTGCVIAWNS,” S2T(h)6“RVVVLSFELLHAP,” and ST(h)25“NYNYKYRYLRGKLRPF.” The most potent CD8+ T‐cell epitopes were ST8 “NYNYLYRY,” ST9 “VNFNFNGL,” and ST10 “CYGVSPTKL.” Other immunodominant T‐cell epitopes were recently mapped in SARS‐2, and they were similar to those identified previously in SARS‐1. Further, HLA‐I A*02:01 restricted epitopes showed superior protection against SARS‐1 in lethal infection challenge using transgenic mice. HLA‐I A*02:01 is one of the most common human MHC‐I allele. Among these CTL‐epitopes, the most potent were S2T8269YLQPRTFLL277, T3S363VADYSVLYNS372, T2S958ALNTLVKQL966, S2T10976VLNDILSRL984, S2T181000RLQSLQTYV1008, and T1S1192NLNESLIDL1200 from SARS‐2‐S, Orf12T13183FLLNKEMYL3191, and Orf12T21637TTDPSFLGRY1646 from SARS‐2‐ORF‐1, and N2T3322MEVTPSGTWL331 and NT1246TVTKKSAAEA255 from SARS‐2‐N. N‐protein, ORF‐1, and S‐protein‐based T‐cell epitopes were found to induce IFN‐γ production from T‐cells of COVID‐19 recovered patients in vitro via Elispot stimulation. In these experiments, stimulation of CD4+ T‐cells by the helper T‐cell epitopes, for example, N2T(h)181DDQIGYYRRATRRIR95 produced IFN‐γ. CD8+ T‐cells also produced IFN‐γ via stimulation with T‐cell epitopes, for example, N2T3322MEVTPSGTWL331. Interestingly, CD8+ T‐cell epitopes were able to protect 80–100% of either humanized HLA transgenic mice or BALB/c mice against lethal infection against SARS‐1. Since these epitopes are immunodominant in both SARS infections, it is highly likely that they play a similar protective role. Therefore, combining these T‐cell epitopes to support the principal protective humoral response may provide synergistic results and significantly improve host protection.Helper T‐cell epitopes should also be taken into account during vaccine development to generate immunological memory and reproducibility of immune responses. In addition, CD8 Similar reductions in efficacy were also found in the context of the South African variants, for example, B1.351 with a N501Y mutation. Further, the mRNA‐1273 vaccine (Moderna) was found to have a 5‐ to 10‐fold reduced efficacy against the B.1.351 variant in nonhuman primates and in sera isolated from immunized humans. However, it still provides sufficient protection against the B.1.1.7 lineage (based on pseudovirion nAb titers of >300). The P1 variant, which is common in Japan and Brazil, possesses similar mutations (T20J and N501Y) to those reported to be highly resistant to vaccination with formulations utilizing the original SARS‐2‐S sequence.The neutralization efficacies of the currently approved vaccines dropped significantly when they were tested against recently emerged SARS‐2 variants. To date, mutations have been observed in viral structural and NSPs, including ORF‐1, S‐, E‐, and N‐proteins. Importantly, mutations in the RBD sequence have also been reported. SARS‐2‐S mutations have occurred in the P1 lineage , the B.1.1.7 lineage , and the B.1.351 lineage . All approved vaccines have efficacies of ≥85% against the original SARS‐2 virus strain. However, the efficacy of the ChAdOx‐1 nCoV‐19 (AstraZeneca/Oxford) vaccine dropped to 70% and 81% against the B.1.1.7 (T20I and N501Y) and non‐(B.1.1.7) lineages (“UK variants”), respectively.N‐terminus domain (NTD) of the S‐protein has been shown to include potent neutralizing epitopes and mutations in this region have also impaired vaccine efficacy. Immune sera against the original SARS‐2‐S were effective against the D614G mutation (nAb titers of 1800) in nonhuman primates suggesting that this mutation is less detrimental to S‐protein‐based vaccine efficacy. However, it is important to note that these outcomes are mainly resultant from in vitro neutralization‐based assays. Contrary to humoral (antibody‐based) responses, convalescent patient T‐cells continue to recognize mutant strains, as most T‐cell epitopes have remained conserved between variants. Combining cellular and humoral responses could protect vaccine efficacy against future emerging variants, even if the main neutralizing epitopes are altered. Therefore, vaccine efficacy against mutants cannot be determined by simple serum‐based naturalization assays; instead, infection challenge studies in mice and nonhuman primates are required, and, more importantly, longitudinal studies on post‐vaccinated human subjects.The main reason for reduced vaccine efficacy has been mutation at or near the SARS‐2‐S binding motif. However, the 4.3 This not only implies that macrophages are vulnerable to infection in the lungs, but also that S‐protein and its RBD when used as vaccine antigen could preferentially bind to macrophage surface‐expressed ACE2. All subunit‐based vaccine formulations contain an adjuvant and B‐cell epitope/s (possibly in the form of whole protein) intended to target pathogen recognition receptors on APCs, for example, dendritic cells, and B‐cells, respectively. The binding of S‐protein or its antigenic fragments to macrophage‐ACE2 can be an alternative to antigen binding to B‐cell receptors for the production of Abs. This leads to off‐target loss of antigen. Simultaneously, the effects from ACE2‐downregulation due to RBD‐ACE2 binding causes a switch toward inflammatory responses and delay in the development of adaptive immune responses due to the combined effects of antigen underdosing and ACE2‐downregulation. DNA‐ and RNA‐based vaccines may initially circumvent this major drawback of off‐target dose wasting and preserve the S‐protein in prefusion conformation.Interestingly, one type of APCs: macrophages, express ACE2. This effect was also observed when low‐dose S‐protein was utilized as vaccine antigen. However, relatively low nAb titers were observed and immunopathology was not completely prevented. While the low efficacy of DNA and RNA vaccines results from limited transfection efficacy, the use of nontoxic and safe transfection agents, for example, lipoplexes, reduces the chance of side‐effects. In contrast, more effective nonreplicating viral vectors induce fever and have safety concerns. These critical drawbacks can be completely avoided by employing peptide‐based subunit vaccines combined with suitable adjuvants to stimulate a specific effective immune response.However, after the translation and secretion of S‐protein, nonspecific ACE2 binding could occur during antigen presentation. The gradual release of RNA‐translated S‐protein demonstrated much milder immunopathology compared to protein‐based vaccine antigens and developed the required noninflammatory adaptive immune response necessary for protection.4.4 For example, as an adjuvant, alum normally generates IgG1 as the dominant subclass with very minor IgG2a,c levels, while Th1‐supporting adjuvants, poly I:C and CPG ODN, generate IgG2a,c as the dominant subclass. Recently, SARS‐2‐S protein was tested for its efficacy in generating nAbs against SARS‐2 in combination with adjuvants, such as AS03, AS37, Alum, and Alum‐CPG1018. AS03 followed by Alum‐CPG1018 outperformed the other adjuvants tested in terms of generating stable, high levels of nAb titers > 103 (n = 5–6 mice per group).Antibody subclass has an important role in protection against SARS infections, as each subclass varies in its involvement in inducing pro‐inflammatory responses, recruitment of inflammatory cells, and in opsonization by antibody‐dependent cytotoxic effects. Danger signals recognized by APCs drive interleukin production. The interleukin profile then drives immunoglobulin class‐switching, for example, IL‐10 drives immunoglobulin class‐switching to IgG4. Hence, proper adjuvant choice is one of the most crucial steps in vaccine development. For example, carbohydrate antigens almost exclusively result in IgG2 responses. The natural ratio of IgG subtypes IgG1/IgG2 is 2:1, unless a strong, specific, class‐switching adjuvant is employed. In early viral infection stages, the levels of IgG3 are initially elevated and class‐switching follows to the less inflammatory IgG1 subclass. APCs bear FCγR and FcRn receptors, which amplify signaling and interleukin production, including pro‐inflammatory cytokines  in mice facing infection challenge, although only total neutralizing IgG titers were evaluated in the study. Similar IgG2 mediated protective responses with minimal immunopathology were induced by influenza vaccine in mice.Soluble and transmembrane protein antigens recognized by B‐cells trigger the production of IgG1 and IgG3, while IgG4 and IgE production results from repeated exposure to allergens, which provokes inflammatory allergenic or anti‐inflammatory responses, respectively. In summary, IgG2 has prolonged circulation half‐life, contributes to mucosal immunity, and has reduced inflammatory effects, due to low C1q and FCγR binding, but retains the protective functions of opsonization and neutralization of Abs.IgG1 and IgG2 also bind to the FcRn receptor, which is widely expressed on epithelia, including respiratory epithelia and myeloid cells, thus complementing the role of IgA in mucosal immunity. Another inflammatory mechanism—binding of IgG to C1q protein—is also triggered by complement activation. IgG2 is the weakest C1q binder, compared to IgG3 and IgG1. CCR1 triggers pro‐inflammatory responses in early infection; however, it also induces protective innate responses to some extent in early infection. In contrast to innate responses, early recruitment of professional DCs and the avoidance of pro‐inflammatory cell recruitment alleviated symptoms and generated a more protective adaptive immune response. Therefore, vaccine against SARS‐2 should preferably induce balanced Th1/Th2 or even biased Th1 and Th17 responses at the expense of Th2‐type responses. Therefore following Th1‐biased adjuvants have been suggested as promising immune stimulant for SARS vaccine formulations: a) IC30, cationic polypeptide with TLR9 agonist (Th1); b) GLA SE, a squalene‐based water‐in‐oil emulsion with TLR4 lipid agonist (Th1); c) CAF01, a cationic liposome with mincle receptor agonist (Th1/Th17); d) poly I:C, a double‐stranded RNA as a TLR3 agonist, (Th1/Th17); and d) MF59, a squalene‐based oil‐in‐water emulsion . Adjuvancy power, expressed as total IgG humoral responses, varies among these adjuvants. When hemagglutinin was applied as model antigen the level of Ab titers decreased in following order MF59 > GLA SE > CAF01 > alum, after two subcutaneous immunizations in mice. The most common adjuvant used in vaccine formulations worldwide, alum, was not only the weakest among them but it also stimulates Th2 rather than desired Th1 immune responses.Careful adjuvant selection is important in helping to develop adaptive immune responses; as engineering of the produced interleukin profile can directly drive maturation and recruitment of immune cells. APCs express several pathogen recognition receptors, including TLRs, for which MyD88 is an adaptor signaling protein and key for interleukin production. MyD88 signaling in APCs was found to be essential for protection against SARS‐1 infection in mice. In a recent study, MyD88 −/− C57BL/6 mice were more susceptible to infection by SARS‐1 MA15 strain (>90% of mice died), whereas all of the wild‐type black mice survived. In addition, CCR1 is the MCP‐1 cytokine receptor expressed in APCs, T‐cells and neutrophils. CCR1‐deficient mice exhibited exacerbated immunopathology, with only 60% survival.4.5Several in vitro tests were developed to investigate potential protection against SARS infections via humoral responses. Pseudovirion cell entry inhibition, antigen‐Ab binding affinity, for example, by surface plasmon resonance, and competitive ELISA tests have all been used to assess specific inhibitory activities of produced Abs against viral fusion proteins. Binding affinity tests assess when generated Abs have a strong enough binding affinity to S‐protein or RBD to block S‐protein binding to ACE2. In competitive ELISA tests, plates are either coated with ACE2 or RBD followed by immune sera, then the other protein  is added to explore how binding was affected or inhibited by the sera.R2 = 0.83); this supports the suitability of employing simple in vitro tests, such as competitive ELISA, in preclinical vaccine candidate screening in early development stages. However, these tests, including pseudovirion entry, often fail to assess the true protective efficacy of Abs with nonneutralizing protective mechanisms, for example, opsonizing Abs. For example, Abs generated against several conserved epitopes in the influenza fusion protein (hemagglutinin) were protective in vivo, but did not exhibit cell entry inhibition in vitro. Similarly, this assay failed to assess the neutralization of a protective Ab (7D10) against a conserved neutralizing epitope at the NTD of MERS‐CoV‐S. The test failed, as 7D10 binds to the NTD of MERS‐CoV‐S only when the latter is bound to DPP‐4 receptor and the epitope is accessible. In such cases, competitive ELISA tests are incompatible; however, pseudovirion cell entry test may assess efficacy depending on design. Several studies demonstrated the protective power of nonneutralizing Abs against lethal SARS and influenza infections, even though they failed to demonstrate protection via in vitro pseudovirion cell entry inhibition tests. Therefore, depending on the mechanisms of protection offered by antigen‐specific Abs, the efficacy of in vitro assessment testing for evaluating protective role will vary.Pseudovirion cell entry inhibition provides the closest simulation of cell infection. ACE2‐transfected HEK cells are often employed with a mixture of animal sera at different dilutions and pseudo‐virus particles (lentivirus) bearing S‐protein added to ACE2 cells. This allows for exploration of binding inhibition between S‐protein and ACE2 in an environment that mimics in vivo conditions. Recently, a strong correlation was demonstrated between nAb titers and the pseudovirion entry test  to enable quantification of viral RNA titers, and to exhibit histopathological symptoms, pneumonia, and lung lesions. Viral RNA titers peak 2–6 days post‐infection, then viral clearance occurs by day 14 post‐infection. Ferrets are also immune to reinfection. To date, only two SARS‐2 infection challenge studies have utilized ferrets for the purpose of examining vaccine efficacy. In contrast, Syrian hamsters have been recently employed as a suitable infection challenge animal model for SARS‐2; this model is susceptible to infection using relatively low SARS‐2 dose (103.5 PFU). Importantly, viral RNA titers are detectable, days 2–7 post‐infection, in nasal turbinates, duodenum, feces, brain, and kidneys. Infected hamsters exhibit similar transmissibility and histopathology to infected patients, including pneumonia, lung lesions, and inflammatory cells infiltration. The infection lasts for 14 days post‐infection, when infection is cleared, humoral immune responses protects the animals against reinfections. Hamsters have been recently used to evaluate vaccine efficacy. Thus, hamster represents a promising highly susceptible infection challenge model for SARS‐2.A variety of distinct animal models have been used to study SARS infections. Models were selected based on their ability to mimic pathological symptoms and the lethality of human infection. A few studies employed ferrets as animal models for SARS‐1 infection challenges; Upon SARS‐1 infection, mice produce a wave of pro‐inflammatory cytokines  and chemokines  by day 2 post‐infection. In response to chemotaxis, NK‐cells, immature macrophages, and DCs are recruited to the lungs. On day 7 post‐infection, a second wave of cytokines and chemokines are produced, in addition to IFN‐γ, IL‐2, IL‐5, and CXCL‐9. Similar acute lung injury with diffuse alveolar damage occurs due to immunopathology. However, SARS‐1 infection in BALB/c mice is not highly lethal, which reduces the applicability of these mice for demonstrating the protection efficacy of vaccine candidates.BALB/c mice are the most common models for SARS‐infection challenge, due to compatibility with pro‐inflammatory pathological responses in early stages of infection. Transgenic hACE2 mice were able to mimic the infectivity of SARS‐cell entry observed in primary hosts. Infection of hACE2‐transgenic mice with TCID50 SARS‐2 doses of 3 × 104 and 7 × 105 resulted in 50% and 100% lethality, respectively, by fifth day post‐infection (n = 4–6). Thus feasible lethal infection challenge model was created. Alternatively, lungs of regular mice can also be transfected/transduced, with hACE2 via adenovirus serotype‐5 (Ad5) encoding hACE2, by intranasal administration, thus rendering them susceptible to SARS‐2 infection. Infection of the hACE2‐Ad5‐transduced BALB/c or C57BL/6 mice with 2.5 × 108 PFU virus dose resulted in steady viral RNA lung titers of 1010 g−1, and PFU g−1of 105, at fourth to eighth day post‐infection. Additionally, this transduced mouse model exhibited sensitivity to neutralization by nAbs, detectable lung histopathological damage by hematoxylin and eosin tissue staining, and significant increase in cytokine storm, that is, IL‐6, CXCL‐10, and IL‐28a/b, with infection progression and a decrease in body weight. The second approach involved developing a special SARS‐virus strain that was more suited for mouse lethal infection. MA15 SARS‐1 strain infected both black C57BL/6 and BALB/c mice with SARS‐1. Recently, a similar approach was adopted for SARS‐2 infection in old and young BALB/c mice, on third day post‐infection with the N501Y mutant SARS‐2 strain, the infected mice had steady SARS‐2 viral RNA lung titers of 1011 g−1, detectable cytokine storm, a decrease in body weight with infection progression, and detectable lung histopathological damage via hematoxylin and eosin tissue staining. Therefore, similar pathology in animal models is important to allow for not only vaccine efficacy, but also safety. Ultimately, both of these approaches achieved the required degree of susceptibility to immunopathology and lethality in control mice, thus rendering them sensitive and suitable animal models for comparing the protective efficacy of vaccines while monitoring immunopathological adverse events, especially after infection challenge.Two approaches were adopted to develop more sensitive mouse models to mimic lethality and pathological symptoms. The first approach employed transgenic mice expressing human ACE2 (hACE2). They possess highly homologous ACE2 to humans, which is expressed in similar tissues. The rhesus monkey model was recently employed to provide proof‐of‐concept on the safety and efficacy of S‐protein‐encoded DNA‐loaded chimpanzee adenovirus (Chadox‐1) vector. These animal models are considered adequate for establishing preclinical efficacy and safety before proceeding to phase I human clinical trials.Rhesus macaque monkeys are a common final preclinical model with close physiological, histological, immunological, and immunopathological responses to human infection by SARS‐1 and SARS‐2.6Even after the current COVID‐19 pandemic disaster is curbed, spillover from viral reservoir animal hosts to humans could occur again. In order to be as prepared as possible, it is extremely important to progress extensive research in vaccine development, molecular pathology, and surrogate in vitro efficacy tests to fast‐track effective medical interventions.It is highly likely—and possibly essential—that a peptide‐based subunit vaccine is produced against SARS‐2, as this type of vaccine has several advantages over protein and RNA‐based vaccines. Peptide‐based vaccine can a) incorporate epitopes from different proteins in one immunogenic construct, thus exceeding the efficacy of a single epitope, b) support the initiation of different protective immune mechanisms simultaneously, and c) avoid unnecessary immunopathological side effects. Moreover, peptide vaccines are easy to chemically synthesize, process and purify; are scalable for mass production to meet current global demand; are stable as dry, sterile powder; can be stored/transported without needing to be kept cold; and are devoid of biological contaminants. Furthermore, combining peptide vaccine with proper Th1 and Th17 interleukin‐inducing adjuvants is achievable to avoid hyperreactive Th‐2 responses that exacerbate immunopathology.Peptide‐based vaccines can enclose opsonic or neutralizing epitopes, or both. Neutralizing B‐cell epitopes that contain several CBRs prevent mutant escape; as mutations of these moieties will also reduce virus infectivity. Convalescent patient sera, even with low nAb concentration, can neutralize the virus. Three neutralizing epitopes (pep1‐3) are suggested here as potential neutralizing peptide vaccine antigens. Moreover, the incorporation of cytotoxic T‐cell epitopes and B‐cell epitopes in the vaccine may provide synergistic protective effects.In conclusion, peptide‐based vaccines against coronaviruses have been somewhat overlooked, but they hold great potential for delivering safe and protective immune responses against SARS‐2 infections.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Dear Editor,It has been reported that up to half but as low as 3% of COVID‐19 patients had gastrointestinal symptoms p < .05, Figure p < .05, Table Escherichia coli, Salmonella enterica, Staphylococcus auricularis, Klebsiella pneumoniae and Enterococcus faecium, and decreased presence of 279 species, including Bacteroides vulgatus, Faecalibacterium prausnitzii and Eubacterium eligens , and 15 non‐COVID‐19 pneumonia controls (NC‐PCs) Table . Decreass Figure . OverallEscherichia coli, and Bacterial invasion of epithelial cells as well as depletion of 86 MetaCyc pathways, most of which related to the metabolism of amino acids, lipids, and carbohydrates  concentrations, and red blood cells (RBC); Bacteroides and Veillonella displayed positive association with haemoglobins, RBC, and CD3, respectively, as did Enterococcus with plasma concentration of carbon dioxide. The expression levels of bacteria virulence factors  were negatively correlated with CD8  and an increase of COVID‐19‐depleted genera , respectively  showed prominent influences  and Klebsiella pneumoniae (75%)), whereas 22/279 of the species that had COVID‐19‐associated transcriptional reduction exhibited at least two‐fold increase in more than 65% of the COVID‐19 patients  , 7‐day posttreatment (T1), and 14‐day post‐treatment (T2) for each patient. Of the 12 recruited COVID‐19 patients, stool samples both before and after the therapy could only be collected from eight individuals Figure . At the %) Table . An impo%) Table . For exaFaecalibacterium prausnitzii and Roseburia hominis, and decreased transcriptional activities of Escherichia coli and Klebsiella pneumoniae). These findings provided new insights into the gut and airway microbiome characteristics of COVID‐19 that may have significant clinical implications.In conclusion, we identified substantial COVID‐19‐associated gut and upper airway microbiota shifts, which were characterized by great inter‐personal and inter‐timepoint variations. In addition to resolving the respiratory symptoms in COVID‐19 patients, a probiotics‐assisted therapy correlated with partial recovery of the microbiota perturbations (e.g., increased transcriptional activities of Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Phodopus sungorus, Cricetidae) to overcome harsh environmental conditions comprises of behavioral, morphological, and physiological adjustments, including spontaneous daily torpor, a metabolic downstate. These acclimatizations are triggered by short photoperiod and orchestrated by the hypothalamus. Key mechanisms of long‐term photoperiodic acclimatizations have partly been described, but specific mechanisms that acutely control torpor remain incomplete. Here, we performed comparative transcriptome analysis on hypothalamus of normometabolic hamsters in their summer‐ and winter‐like state to enable us to identify changes in gene expression during photoperiodic acclimations. Comparing nontorpid and torpid hamsters may also be able to pin down mechanisms relevant for torpor control. A de novo assembled transcriptome of the hypothalamus was generated from hamsters acclimated to long photoperiod or to short photoperiod. The hamsters were sampled either during long photoperiod normothermia, short photoperiod normothermia, or short photoperiod‐induced spontaneous torpor with a body temperature of 24.6 ± 1.0 °C, or. The mRNA‐seq analysis revealed that 32 and 759 genes were differentially expressed during photoperiod or torpor, respectively. Biological processes were not enriched during photoperiodic acclimatization but were during torpor, where transcriptional and metabolic processes were reinforced. Most extremely regulated genes (those genes with |log2(FC)| > 2.0 and padj < 0.05 of a pairwise group comparison) underpinned the role of known key players in photoperiodic comparison, but these genes exhibit adaptive and protective adjustments during torpor. Targeted analyses of genes from potentially involved hypothalamic systems identified gene regulation of previously described torpor‐relevant systems and a potential involvement of glucose transport.The energy‐saving strategy of Djungarian hamsters ( Phodopus sungorus) use morphological and physiological changes to overcome extreme environmental conditions. These acclimatizations include spontaneous daily torpor. To uncover the mechanisms behind torpor, samples were collected from nontorpid and torpid hamsters during summer‐ or winter‐like photoperiod. The pipeline comprised a de novo transcriptome assembly, a mapping against Mus musculus, and several analyses to identify the most relevant data. Analysis of the hypothalamus transcriptome confirms known and suggests new mechanisms that might orchestrate seasonal acclimation and metabolic downstates in photoperiodic rodents.Djungarian hamsters ( HThypothermialog2(FC)logarithm of fold change to the base 2LPsummer‐like long photoperiodNTnormothermiapadjP‐valueadjusted SPwinter‐like short photoperiodTbcore body temperatureZTZeitgeber‐timePhodopus sungorus, Cricetidae) are photoperiodic mammals  varied between 1.4 and 1.8 (1.7 ± 0.1). The mRNA was isolated and the library prepared . RNA‐Seq was performed in 2020 on an Illumina NextSeq 500 .Hypothalamic tissue was homogenized in 100 µL buffer RLT with beta‐ME using prefilled 2‐mL beadbug tubes containing Zirconium beads  on a microtube homogenizer . Purification of total RNA was performed using the RNeasy Midi Kit . The concentration of total RNA ranged between 15 and 86 ng·µLde novo transcriptome assembly containing 868,657 transcripts using Trinity 2.8.5  > 2.0. These strict parameters were chosen because they were able to pick up the known key players of physiologically relevant gene expression changes in the photoperiodic comparison. Applying the same thresholds in the torpor comparison, we identified genes that appear to rather reflect adaptive mechanisms of the brain.Dio3 was among the upregulated genes. Dio3 is a T3 catabolic enzyme, converting the bioactive hormone into inactive or less active derivates. Upregulation in SP is consistent with previous data, demonstrating that Dio3‐dependent reduction of hypothalamic T3 availability is a crucial driver of SP‐acclimation in Djungarian hamsters [Comparison between photoperiods revealed seven most extremely regulated genes. In SP, hamsters , 50, 51.Tshb, encoding for the thyrotropin beta subunit, was downregulated in normothermic SP‐acclimated hamsters. Downregulation of Tshb in Pars tuberalis has previously been identified as crucial interface of melatonin signaling and hypothalamic gene expression in many species [ species , 53, 54. species , 57, 58.Cga had, with a log2(FC) of −4.6, the most extreme (down)regulation during SP‐acclimation , such as kisspeptin and Qrfp. Kisspeptin plays a role in the photoperiodic control of reproduction, while Qrfp is relevant for hypometabolism in mice [Npvf was previously found to be downregulated in hypothalamus during early SP acclimation (SP08) [ in mice , 64, 65.n (SP08) .Cspg4b and upregulated in SP‐acclimated hamsters, is related to diverse topics in several fields. As structural macromolecules of connective tissues’ extracellular matrix, Cspg4b might be related to brain plasticity. Cdc26, downregulated in SP‐acclimated hamsters, has been studied in Chinese hamster ovary cells only, but might contribute as part of the anaphase‐promoting complex to brain structure and metabolism [The other most extremely regulated genes were not directly linked to photoperiodic acclimation. The uncharacterized protein LOC408066, also known as chondroitin sulfate proteoglycan 4B at locus Rbm3 was upregulated. Studies on natural and enforced hypothermia in the hibernating 13‐line ground squirrels and in cooled mice suggest that Rbm3 and other cold‐inducible RNA‐binding proteins within the brain might enable RNA stability and protein synthesis at low body temperatures, which is important for synaptic regeneration and therefore structural plasticity [Using the same thresholds, we picked up five regulated genes during torpor that appear to reflect adaptive mechanisms. During torpor, the cold‐shock protein asticity , 68, 69.Egln1 was downregulated during torpor. Although its biological relevance is still unclear, it has been linked to ischemia and hypoxia in mouse and human due to, for example, its suppression of hypoxia‐inducible factor 1α (HIF‐1α) [(HIF‐1α) , 71, 72.(HIF‐1α) , 74.Grhl2 appears to play a role in hearing loss. The expression of Grhl2 and HIF‐1α was linked [Gm16500, also known as predicted gene 16500 or short coiled‐coil protein‐like.The genes upregulated during torpor have not been in focus of torpor research so far. Adipolin has been studied regarding diabetes, obesity, and cardiovascular diseases such as ischemia. It increases insulin sensibility and favors glucose tolerance and uptake, which might play a role for energy gain and storage in the torpid hypothalamus . Grhl2 as linked . At thisOverall, the most regulated genes during torpor also reflect major structural changes, rather than distinct signaling mechanisms. This was already concluded from our previous study, which analyzed gene expression during torpor entrance, although time point of sampling, bioinformatic approach, reference organism, and consequently the actual regulated genes were different .In a more targeted approach to identify mechanisms involved in torpor control, we specifically screened a number of indicator genes from potentially relevant hypothalamic systems Table  as revieDio3 to be upregulated with an extremely high log2(FC) of 5.1 (see paragraph ‘Most extremely regulated genes’). By contrast, Per3 was upregulated with high significance in SP‐normothermia. Per3 is a photoinducible clock gene oscillating with circadian rhythm [Pomc was downregulated in SP‐acclimated hamsters’ hypothalamus, which is consistent with earlier studies on the hamsters’ energy homeostasis. Pomc might be involved in controlling the photoperiodic body mass acclimation of Djungarian hamsters [Four of 68 indicator genes were differentially regulated by photoperiod Table . Among tn rhythm , 78. Sinhamsters , 80.Gpr50, downregulated in SP‐acclimated hamsters. This photoperiodic downregulation of Gpr50 has previously been demonstrated [Gpr50 augments fasting‐induced torpor and suppresses leptin responsiveness as well as thyrotropin‐release hormone [Gpr50 might be a torpor‐permissive factor, although the gene is not regulated during individual torpor bouts. Furthermore, Gpr50 seems to play a major role in overall energy metabolism, as knockout mice had a higher food intake, a higher locomotor activity, and higher basal metabolic rate, yet a lower body mass and fat mass [Gpr50 is likely to be located on the X‐chromosome. Although irrelevant for this NT: SP vs LP comparison with two males and two females in each group, the location of a gene on sex chromosomes might play a role for the interpretation of the results of the other comparison. In SP: HT vs NT, three males and one male sampled in torpor were used to compare their gene expression with those of two males and two females sampled on a torpor‐free day.The gene with the most extreme significance of this study was nstrated , 81. Bes hormone . Thus, afat mass . Gpr50 iSstr1 was upregulated. It has previously been demonstrated that the GH axis is regulated by photoperiod and involved in torpor control [Seven of 68 indicator genes were differentially expressed during torpor Table . The som control . Downreg control .Glut3, the most important glucose and galactose transporter in the brain, was upregulated during torpor. This might be related to an enhanced hexose transport to the brain. Plasma glucose levels are reduced during torpor, while hypoglycemia seems to be rather a consequence than a requisite for torpor [Glut3 expression can be mediated by HIF‐1α [r torpor , 87. Eitr torpor . Besidesr torpor . Furthery HIF‐1α , 91, whi, Pml is annotated to the mouse’ circadian clock. A loss of Pml causes a downregulation of Per2, Per1, Cry1, Bmal1, and Npas2 expression in the mouse’ suprachiasmatic nucleus [Pml, like in the Tgf‐beta signaling pathway [Although uninvestigated in the Djungarian hamster so far nucleus . Its upr pathway .Jun and c‐Jun1 , Bhlhe40 and Ppp1cc were downregulated in torpor as well, both with proven functions in the mouse’ circadian clock with unclear consequences. Bhlhe40 regulates the phase of several clock genes, perhaps in a novel feedback loop different from the positive regulator CLOCK/BMAL and the negative regulator PER/CRY [Ppp1cc is an enzyme with countless substrates, which plays a role in as much biological processes including energy metabolism of cells [Besides a downregulation of the immediate early genes  PER/CRY , 95. Pppof cells  and reguof cells .Mus musculus but were absent in this study’s data on hypothalamic mRNA expressed by the Djungarian hamster , statistics (pairwise group comparison), principal component analysis, heatmap, setup of the r‐software and installed packages.Fig. S1. Core body temperature patterns of SP‐acclimated hamsters.Table S1. Background information on hamsters.Table S2. Quality control of RNA‐Seq data and accession numbers of data depositories.Click here for additional data file.


The hygiene hypothesis suggests that decrease in early life infections due to increased societal‐level hygiene standards subjects one to allergic and autoimmune diseases. In this report, we have studied the effect of sterilized forest soil and plant‐based material on mouse immune system and gut microbiome.Inbred C57Bl/6 mice maintained in normal sterile environment were subjected to autoclaved forest soil‐derived powder in their bedding for 1 h a day for 3 weeks. Immune response was measured by immune cell flow cytometry, serum cytokine enzyme‐linked immunoassay (ELISA) and quantitative polymerase chain reaction (qPCR) analysis. Furthermore, the mouse gut microbiome was analyzed by sequencing.When compared to control mice, mice treated with soil‐derived powder had decreased level of pro‐inflammatory cytokines namely interleukin (IL)−17F and IL‐21 in the serum. Furthermore, splenocytes from mice treated with soil‐derived powder expressed less IL‐1b, IL‐5, IL‐6, IL‐13, and tumor necrosis factor (TNF) upon cell activation. Gut microbiome appeared to be stabilized by the treatment.These results provide insights on the effect of biodiversity on murine immune system in sterile environment. Subjecting mice to soil‐based plant and microbe structures appears to elicit immune response that could be beneficial, for example, in type 2 inflammation‐related diseases, that is, allergic diseases. Autoclaved forest soil extract was added to the bedding of standard laboratory mice. The immune response of mice was measured indicating decreased pro‐inflammatory cytokine production in soil‐subjected mice. We study here, how the immune system of sterile laboratory animals is affected by exposure to autoclaved antigens from boreal forest soil, containing environmental plant, yeast, bacteria, and virus particles. We found that soil‐treated mice showed decreased pro‐inflammatory cytokine expression, both in soil‐subjected mice and in in vitro activated splenocytes from soil‐subjected mice. In particular, the decreased expression of allergic inflammation‐related cytokines in response to splenocyte activation from soil‐treated mice is of interest; this would imply that nonpathogenic microbial encounters would generate an antiallergic environment. In total, these results offer one explanation, why biodiversity is important in regulating tolerance of the immune system.2Epidemiological studies have associated the epidemic of immune‐mediated disorders, such as allergies and autoimmune diseases, with the adoption of a modern, urban lifestyle.The biodiversity hypothesis has further expanded the hygiene hypothesis, underlining the importance of environmental biodiversity and its impact on the gut and skin microbiota in the development of a healthy immune system.Therapeutic interventions that aim to provide immunomodulatory, “protective” microbiota have been under intense investigation lately. Most studies have investigated the effect of probiotics, containing only one or few strains of bacteria in the prevention or treatment of conditions varying from allergy to obesity and cancer.Inbred mouse strains living in a clean environment are traditionally used for immunological studies. The sterile environment of animal facilities greatly helps in the evaluation of the function of the immune system and its genetic regulation, but the exclusion of external microbes that are present in the natural mouse environment may also modulate the immune response. Here, we have studied the effect of autoclaved soil and plant‐based material (aka soil powder) on the immune system of C57Bl/6 mice. We prepared an organic powder with high microbiological diversity from microbially diverse organic soil and plant materials. We then subjected mice externally to the autoclaved powder and measured subsequently the immune response in these mice. Interestingly, we found a reduced expression of pro‐inflammatory cytokines, IL‐17F, and IL‐21, in the serum of mice treated with the soil powder. CD4 + T cells from soil‐treated mice showed decreased expression of CD69 activation marker upon stimulation, as well as decreased expression of IL‐1β, IL‐5, IL‐6, IL‐13, and tumor necrosis factor (TNF). Overall, autoclaved soil powder might provide an additional tool to provide safe immunoregulation by down‐tuning harmful immune responses.33.1n = 8) to the powder while leaving the control group (n = 8) with normal bedding. We added the soil powder to the bedding of mice for one hour, 5 days in a row, for 3 weeks , and 422.0 pg/ml and 53.1 pg/ml for IL‐21 (p = .02), respectively. Meanwhile no differences were found in other lymphocyte‐related cytokines such as IL‐5 and IL‐13  was performed using flow cytometry Figure . No sign‐CD62L+ naïve (NA), CD44+CD62L− effector memory (EM) and CD44+CD62L+ central memory (CM) cells in CD4 and CD8 T cells  was upregulated in the soil‐treated animals  did not differ between the intervention and control groups  in secondary lymphoid organs, and organs directly exposed to the soil powder via ingestion or inhalation . In the mLN s Figure . In sples Figure .Tbet and Gata3 were downregulated in soil powder‐exposed animals compared to the control group , while Il10, Il13, and Tgfβ remained unchanged   and the difference was linked to an early drop of diversity in control mice, which later leveled off   compared to control mice but the difference leveled off later due to a decrease of abundance in the exposed mice . Similarly, the abundance phylum Deferribacteres was higher in the exposed mice after a week of exposure (adjusted p = .01) but later the difference leveled out  Figure . However) Figure . The gut) Figure . The abudy Table . The abuut Table . All in 4The activation of immune system is regulated by environmental factors. Down‐tuning the immune response could be beneficial in conditions where overactivated immune system can cause tissue damage and trigger and/or accelerate autoimmune or other immune‐mediated diseases. In developed societies, urbanization has been suggested to be linked to increase in allergies and autoimmune diseases.Though the relationship between urban lifestyle and allergies is likely a complex phenomenon, any low‐cost, safe, and efficient immunomodulatory tools would be of value in clinical trials aiming to identify new treatment and prevention modalities. As there is clear evidence that, for example, touching biodiverse soil increases skin microbiota,Tbet in mLN from soil‐exposed animals meanwhile its expression was reduced in colon  could be attributed trafficking of T cells that have been activated by the ingested extract in the gut into the draining lymph node (mLN).Subjecting mice to the autoclaved material in sterile environment has important difference to our previous study of nonautoclaved soil powder on humans.n Figure . Tbet isImportantly, we found that PMA/Ionomycin‐activated splenocytes from mice that had been treated with soil powder showed a significantly decreased expression of TNF, IL‐1β, IL‐5, IL‐6, and IL‐13. Soluble TNF selectively binds TNFR1, which induces pro‐inflammatory signals and acts as a potent neutrophil chemoattractant.Actinobacteria in the gut after the whole exposure period. In addition, the treatment balanced the fluctuations in the microbial abundances and diversity during the treatment period as such fluctuations were more pronounced in the control mice (phyla Proteobacteria and Deferribacteres). Since the exposure material was microbially inactivated the changes in the gut microbial compositions were expected to be subtle. In principle, the bacterial particles in the material could serve as a source of energy for other bacteria which then could be enriched in the gut. However, no such enrichment was observed. Thus, in this experimental setting, the bacterial PRR ligands acting as immunostimulants seem to be the major drivers of the observed immunological changes, not the living bacterial component of the gut microbiota.An interesting observation is that mice treated with the microbial powder had a lower abundance of phylum Lactobacillus plantarum.Tgf‐β expression in mLN of soil‐treated animals  and organs were subsequently collected for analysis.A total of 16 female C57Bl/6J (Jax line 00664) mice from The Jackson Laboratory , aged 7–8 weeks at the beginning of the exposure, were used in this study. Ethical approval for all animal work was provided by the Regional State Administrative Agency . Animals were housed in individual ventilated cages in the Tampere University animal facility. They were separated in groups of four animals per cage and exposed for 1 h to 50 ml inactivated powder, sprinkled on top of clean bedding in a new, clean cage. The dose was chosen based on visual verification that the mice became exposed to the powder. The powder was observed on furs, noses, and ears of the mice as well as a visible powder on cage bedding. The exposure was repeated on five consecutive days per week, for 3 weeks. Control animals were kept in a new cage with clean bedding for 1 h. The animals from different groups were fed with the same food and drinking water ad libitum but kept in different rooms and handled by different researchers to avoid cross‐contamination. At Day 21, mice were euthanized in CO5.3−/−  supplemented with 1% heat‐inactivated FBS  and 2 mM ethylenediaminetetraacetic acid (EDTA). After centrifugation, red blood cells (RBCs) were lysed with 1 min ACK (Lonza) treatment and then washed with PBS containing 1% FBS and 2 mM EDTA. Cells were resuspended in PBS and stained with viability dye FVS510 (Becton Dickinson Biosciences) for 20 min, RT, followed by washing with FBS buffer. Fc receptors were blocked with Rat Anti‐Mouse CD16/CD32 antibodies (Mouse BD Fc Block™) for 5 min at 4°C before staining. Antibodies for flow cytometry were purchase from BD . Staining was performed at 4°C for 20 min and cells were washed twice with fetal bovine serum (FBS) buffer before analyzing them. Foxp3 intracellular staining was performed after surface staining using Mouse Foxp3 Buffer set according to manufacturer instructions. CD4 was stained together with Foxp3 postpermeabilization. All samples were run with BD FACSAria Fusion and analyzed with FlowJo software (BD).mLNs and spleen were collected from animals at Day 21. Single‐cell suspensions were prepared by mechanic dissociation of the organs by first mincing them and then straining them through a 40 µm cell strainer (Fisher Scientific) into PBS5.4RNA extraction was performed for spleen, lymph nodes, lung, small intestine, and colon samples. For spleen, small intestine, and colon TissueRuptor II was used according to manufacturer instructions (Qiagen). Briefly, 5–20 mg of tissue was collected in 2 ml Eppendorf tube containing 1 ml of TRIzol (Thermo Fisher Scientific). Using TissueRuptor II, the tissue was homogenized by five cycles of 40–50 Hz. Then, RNA extraction was conducted following RNeasy Mini Kit (Qiagen). Lung and lymph node samples were homogenized using ceramic beads with PowerLyzer® 24 homogenizer  and the RNA was extracted using RNeasy Plus Universal Mini Kit . Using iScript™ cDNA Synthesis Kit (Biorad) cDNA synthesis from RNA samples was performed. Gene expression was assessed using RT‐qPCR and used primers are listed in Table 5.5Serums were collected on Day 21 after euthanasia of mice. Cytokines were measured using MILLIPLEX MAP Mouse TH17 Magnetic Bead Panel  following manufacturer instructions. Fluorescence was analyzed with Bio‐Plex® 200 system (Bio‐Rad Laboratories) and data were collected with Bio‐Plex Manager software . If cytokine concentrations were below detection limit for some serum samples, lowest detected concentration/2 (LOD/2) value of the specific cytokine was used for these samples in the statistical analysis.5.6l‐glutamine (Lonza), and 1% penicillin/streptomycin (Lonza). The next day splenocytes were stimulated with PMA 50 ng/ml (Thermo Fisher Scientific) and Ionomycin 1 ug/ml (Thermo Fisher Scientific) or left untreated as control. Cells were harvested at 4 h poststimulation and cells analyzed by flow cytometry as described above. Supernatants were collected at 24 h poststimulation and stored at −80°C for cytokine measurement using the Meso Scale Discovery U‐plex kit Biomarker Group 1 , which include the following analytes: IFN‐ɣ, IL‐12p70, IL‐13, IL17A, IL‐17E/IL‐25, IL‐1β, IL‐5, IL‐6, TNF, IL‐17F, and IL‐33. Samples were also analyzed using MILLIPLEX MAP Mouse TH17 Magnetic Bead Panel  as mentioned previously.Spleen from animals was collected after euthanasia and single‐cell suspension was obtained as described in the previous section. After single‐cell suspension was obtained cells were rested overnight in RPMI 1640 with 10% FBS (Thermo Fisher Scientific), 1% 5.7Total DNA from the stool samples was extracted using DNeasy PowerSoil Kit (QIAGEN GmbH). The V4 region of the bacterial 16S rRNA gene was amplified as described earlier.The 16S rRNA gene read pairs were demultiplexed and reads were merged using USEARCH, version 10 with default parameters.5.8α = 0.05). Nonparametric data were analyzed using a Mann−Whitney U test , parametric data were analyzed using a two tailed t test, as appropriate (α = 0.05). All immunological data sets were analyzed using GraphPad Prism 8.0 (GraphPad Inc.). The statistical analyses for the gut microbiota were performed in Rphyloseq.Normal distribution was assessed for each individual data set using Anderson–Darling tests or Shapiro–Wilk test when data sets did not fulfil the requirements , Department of Medicine, Karolinska Institutet, Huddinge, Sweden;Mika Saarenpää: University of Helsinki, Helsinki, Finland; Yan Sun: University of Helsinki, Helsinki, Finland; Olli H Laitinen: Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; Marja I Roslund: University of Helsinki, Helsinki, Finland; Juho Rajaniemi: Faculty of Built Environment, Tampere University, Tampere, Finland; Heikki Hyöty: Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; Aki Sinkkonen: Horticulture Technologies, Natural Resources Institute Finland, Turku, Finland.H. H., O. H. L., and A. S. are stakeholders and members of the board of Uute Scientific LtD., which develops biodiversity‐based interventions for the prevention of immune‐mediated disease. A. S., H. H. O. H. L., N. N., and S. O. have been named as inventors in a patent application “immunomodulatory compositions” submitted by the University of Helsinki (patent application number 20165932 at Finnish Patent and Registration Office). A. P., M. I. R., and A. S. have been named as inventors in a patent application “Immunomodulatory gardening and landscaping material” submitted by the University of Helsinki (patent application number 175196 at Finnish Patent and Registration Office). None of the inventors have received royalties from the patent applications.Martín Ignacio González‐Rodríguez, Noora Nurminen, Laura Kummola, Olli H. Laitinen, Sami Oikarinen, Anirudra Parajuli, Tanja Salomaa, Iida Mäkelä, Marja I. Roslund planned and performed the experiments, Aki Sinkkonen, Heikki Hyöty and Ilkka S. Junttila conceived the idea, planned the experiments, analyzed the data, wrote the MS, and oversaw the project.Supporting information.Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting cortical and spinal motor neurons. There is a lack of optimal biomarkers to diagnose and prognosticate the ALS patients. C‐reactive protein (CRP), an inflammatory marker, has shown promising results in ALS patients.PubMed, Embase, and Google Scholar databases were searched from 2000 to June 1, 2021 for suitable studies showing the relationship between CRP and ALS. The concentration of CRP levels was assessed between ALS patients and controls. Further, end outcomes like ALS functional rating scale (ALSFRS‐R), survival status, and mortality risks were assessed in relation to CRP levels.Eleven studies including five case–control, five cohorts, and one randomized control study were assessed. There were 2785 ALS patients and 3446 healthy controls. A significant increment in CRP levels among ALS patients in comparison with healthy controls were seen in most of the studies. ALSFRS‐R and disease progression were found to be significantly correlated with CRP levels. Overall accuracy of CRP in CSF was 62% described in a single study.Although CRP has shown promise as a prognostic biomarker, extensive cohort studies are required to assess its prognostic value and accuracy in diagnosing ALS taking into account the confounding factors. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects cortical and spinal motor neurons. The underlying neuroinflammatory process in the pathogenesis of ALS has researchers interested in C‐reactive protein (CRP) as a possible disease biomarker. We used a comprehensive review to investigate the link between CRP levels and disease progression in ALS patients versus healthy controls. The concentration of CRP levels was assessed between ALS patients and controls. Further, end outcomes like ALS functional rating scale, survival status, and mortality risks were assessed in relation to CRP levels. CRP is 22.1amyotrophic lateral sclerosis, motor neuron disease, Lou Gehrig's disease, ALS, MND, C‐reactive protein, and CRP. In addition, references of selected studies were hand searched. The related abstracts and conference proceedings in journals and preprint servers were also searched where available. All the studies were selected and reviewed by two independent investigators (S. K. and R. O.). Any disagreements were resolved by the third author.We identified studies from PubMed, Embase, and Google Scholar . The search was limited to only English language. The following keywords or MeSH terms were used: The flowchart for the selection process is shown in Figure 2.2The inclusion criteria for selection of studies should have fulfilled the following criteria: (1) patients diagnosed with definite or probable ALS according to revised El Escorial criteria; (2) data showing relationship between serum/CSF CRP levels and disease status in ALS patients; (3) data with end point assessment like disease progression over time, ALSFRS‐R, survival outcome, or mortality risk; and (4) use of standardized method to quantify CRP levels.The exclusion criteria were as follows: (1) animal or in vitro studies; (2) insufficient data; (3) duplicate article; (4) case reports, reviews, or meta‐analysis; (5) non‐English studies.2.3Data were extracted by two independent investigators (S. K. and R. O.) in an Excel spreadsheet (Microsoft Corp) using a standardized data extraction form, and results were compiled to complete the following fields: author, year of publication, study site, study design, number of patients (ALS and controls), age of patients, source of sample, CRP subtypes, CRP assays used, and end point assessment/main outcomes. A third reviewer (V. P. K.) was consulted to resolve inconsistencies when consensus could not be reached. In case of confusions and incomplete data, corresponding authors of the article were emailed.2.4https://training.cochrane.org/handbook/current) Studies with scores greater than 5  were included in our study. Two independent authors discussed and assessed the quality of studies.Quality assessment for the included observational studies was performed using the Newcastle–Ottawa quality assessment scale, which consists of three domains: (a) selection of the participants; (b) comparability between the groups; and (c) ascertainment of the outcome in cohort studies. While for randomized controlled trial (RCTs) studies, the risk of bias was evaluated by the tool described in the Cochrane Handbook for Systematic Reviews of Interventions.  score, survival status, and mortality risks.33.1Initially, the search retrieved 124 articles, from which 74 articles remained after removal of duplicates. After screening the titles and abstract, we shortlisted 30 articles. Further, 19 articles were excluded according to predetermined inclusion and exclusion criteria. Therefore, 11 studies including 2785 ALS patients and 3446 HCs were included in our review. Figure Among the selected literature, five were cohort studies ; however, no difference was seen among slow progressing patients and HCs (p = .075). Overall patients (fast and slow progressing) had elevated CRP compared with HCs (p < .008). In second cohort, all ALS patients (fast and slow progressing) CRP was elevated compared with HCs (p < .001). Huang et al. (2020) also showed no significant increment of CRP in ALS patients when compared with controls (p = .76); however, increasing trend was noted among the C9orf72 positive ALS patients. The pooled frequency analysis showed 53% of ALS patients had higher level of CRP when compared with the HCs.There were five case–control studies that compared the CRP levels among ALS patients and HCs. Keizman et al.  showed sA study by Cui et al.  showed t3.6Ryberg et al.  was the 3.7There were seven studies that assessed CRP as a prognostic biomarker as compared with the disease progression (ALSFRS‐R and AALS score) and survival/mortality rate. Only five studies showed positive association between CRP levels and disease progression/survival rate.Chełstowska & Kuźma‐Kozakiewicz  studied p < .001). They also stated that high levels of wide‐range CRP were observed as disease progressed. Similarly, logistic regression analysis showed OR  for each unit increase in wide‐range CRP. The CRP level was also found to be a valuable preclinical predictor for the subsequent development of an overt respiratory tract infection in this study.Keizman et al.  showed ar = −0.14818; p = .004) at initial evaluation and after 1 year follow‐up. The study included 50 patients of NEMO cohort and they also proposed that the correlation was significant when age, sex, body mass index (BMI), and smoking status were adjusted. Similarly, serum CRP levels were correlated with patient survival in NEMO group  and independent cohort groups .Lunetta et al.  conductep < .001, r = 0.420) and patient's disease progression rate  in the second cohort of patients.Beers et al.  studied In a study by Sun et al. (2020), they found patients with a higher than median level of log CRP  had high risk of mortality. Elevated levels of CRP were found in very fast (death with 1 year of diagnosis) and medium progression (death within 1–3 years of diagnosis) groups compared with the slow progression (death after 3 years of diagnosis) group.There were two negative studies that showed no association between CRP levels and survival rate.p = .4196). A population‐based registry study in Germany by Nagel et al. , fused in sarcoma and C9orf72 induced RNA processing defect, whereas both genetic and environmental factors have been linked to sporadic ALS. Gordon,  Protein Several inflammatory biomarkers apart from CRP, like serum Cystatin C and transtyrectin  in ALS patients requiring noninvasive ventilation and tracheostomy‐invasive ventilation compared with intake clinic patients while was also correlated with ALSFRS‐Rt (p = .0018). As CRP levels were significantly (p = .0313) reduced after riluzole therapy, it may be considered as a potential biomarker for treatment responsiveness. (Sanjak, A RCT by Miller et al.  assessin Sanjak,  Currentl5Our review suggested that CRP is a reliable prognostic biomarker of ALS that correlated with the disease progression and therapeutic response. CRP was elevated in majority of the ALS cases compared with the HCs. Further studies are needed for assessment of the accuracy of serum CRP levels in the diagnosis of ALS taking in account of the confounding factors.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.None.https://publons.com/publon/10.1002/brb3.2532The peer review history for this article is available at Appendix 1: Search strategy used in the current systematic review.Appendix 2: Quality assessment of the included observational articles.Click here for additional data file.


KGF, VEGF, and GM-CSF expression in HaCaT cells and NHDF. Physiological COL1A1 and COL3A1 gene expression by NHDF was observed under DACC treatment with no observable effect on S100A7 and RNASE7 levels in HaCaT cells. Overall, the DACC coating was found to be safe and may positively influence the wound healing outcome.Wound dressings that exert an antimicrobial effect in order to prevent and treat wound infections can be harmful to the wound healing process. Dressings with hydrophobic coatings, however, have been suggested to both reduce the microbial load and promote the healing process. Therefore, the potential effects of a dialkylcarbamoyl chloride (DACC)-coated dressing on fibroblasts and keratinocytes in wound healing were studied using mechanical scratch wounding of confluent cell layers as an in vitro model. Additionally, gene expression analysis by qRT-PCR was used to elucidate the longitudinal effects of the DACC-coated dressing on cell responses, specifically inflammation, growth factor induction and collagen synthesis. DACC promoted cell viability, did not stick to the cell layers, and supported normal wound healing progression in vitro. In contrast, cells became attached to the uncoated reference material, which inhibited scratch closure. Moreover, DACC slightly induced  The increasing antimicrobial resistance seen worldwide  demands This study is the first to explore the potential effects of the DACC-coated dressing on fibroblasts as well as its influence on the human keratinocyte cell line HaCaT employing the mechanical scratch wounding of confluent monolayers as a model to study wound healing in vitro. Scratch wound models allow the direct measurement of cell migration and cell layer regeneration comparable to healing processes that occur at the wound margins . These m® compress) or kept uncoated and used as reference material (RM). Both, the DACC-coated and uncoated reference material  were sterilized by gamma sterilization. Several material aspects are identical for the Sorbact® compress and the reference fabric, i.e., both fabrics consist of a sparse mesh allowing fluid absorption into secondary layers and conform easily to the wound bed. However, after DACC-coating, the fabric features a high hydrophobicity and low absorbing properties, similar to the polypropylene stress control. In contrast, the reference fabric is hydrophilic and absorbs water to some extent.Green-colored, cellulose acetate fabric was either treated in an impregnation step to become DACC-coated  at 37 °C and in a humidified atmosphere containing 5% CO2 .Cell adherence to material surface of samples removed from the NHDF monolayer after 1, 24, and 48 h from the previous experiment (see “Determination of the effect on cell viability”) was also investigated. Cell presence was ascertained using a luminometric adenosine triphosphate (ATP) assay  control. Cell scratches were incubated for 1, 8, 24, and 48 h. Material samples and stress controls were then removed and cells were stained with hematoxylin and eosin for evaluation. Microscopic assessment was done using the VHX 950F digital microscope (KEYENCE DEUTSCHLAND GmbH) and images were obtained. Scratch width was determined using the VHX 950F software (KEYENCE DEUTSCHLAND GmbH).Scratch wound closure under the DACC-coated dressing and the uncoated RM samples was determined . Brieflyg for 2 min. Subsequently, automatic RNA purification was performed using the RNeasy® Mini Kit and the QIAcube (Qiagen). RNA concentration was determined at 260 nm using the SPECTROstar® Omega with an UV/Vis plate (BMG Labtech GmbH). For reverse transcription the High Capacity cDNA Reverse Transcription Kit by Applied Biosystems (ThermoScientific™) was used according to the manufacturers’ instructions. The PCR protocol was run using the Mastercycler® gradient thermal cycler (Eppendorf), which included primer annealing for 10 min at 25 °C, reverse transcription for 120 min at 37 °C, and termination for 5 min at 85 °C. The cDNA samples were stored at −80 °C until further use. After reverse transcription, cDNA was diluted to obtain a final test concentration of 0.5 ng/ml. Gene expression was analyzed by RTqPCR using the QuantiNova™ SYBR Green PCR Kit (Qiagen) following the manufacturers’ instructions. Briefly, master mix prepared on ice contained forward and reverse primer (each test concentration 0.5 µM) and cDNA or Yellow Template Dilution Buffer as a no template control. Using the qTOWER3G  the real-time amplification protocol was set for polymerase heat activation at 95 °C for 3 min, and 40 cycles with three steps: denaturation at 95 °C for 5 s, annealing at 57 °C for 10 s, and elongation at 72 °C for 10 s. Signals were detected at λex/λem 470 nm/520 nm. Finally, a melting curve from 65 °C to 95 °C served as amplicon control. All primer sequences or ordering IDs are shown in Table Gene expression analysis of specific inflammatory cytokines, growth factors, antimicrobial peptides, and collagen was performed at 4, 8, 24, and 48 h . In briep < 0.05. Asterisks indicate significant deviations at the respective time point from the untreated control  unless otherwise stated.Experiments were performed in triplicate. Measurements were carried out in quadruplicate (scratch assay) or duplicate  and all values expressed as the mean ± SD (standard deviation). Statistical significance was determined using one-way analysis of variance . A differences was considered statistically significant at p < 0.001) and almost 25 times the concentration after 48 h (p < 0.001).Viability of HaCaT cells Fig.  and NHDFp < 0.05). During treatment with the DACC-coated dressing, the HaCaT keratinocytes showed normal cell proliferation and migration, resulting in 70% scratch closure over 48 h , and residual scratches at 48 h remained larger than those of the untreated control and those covered with DACC . In contrast, uncoated RM was associated with decreased healing progression already at 24 h (p < 0.01) and resulted in only ~73% scratch wound healing at 48 h (p < 0.001) with scratches remaining open . In NHDF, the increase of IL1A transcripts was more moderate and limited to the first 8 h . In contrast, transcript levels were significantly augmented in NHDF by approximately four-fold under DACC and over tenfold under the uncoated RM (p < 0.01). Similar observations were made for CXCL8 and CXCL1 gene expression under DACC and uncoated RM in NHDF, with levels particularly increased over 24 h (p < 0.05). In contrast, CXCL8 levels were not affected in HaCaT cells and while CXCL1 gene expression was found to be slightly elevated at 4 h, it was significantly decreased under DACC and uncoated RM at 48 h (p < 0.05) compared to the untreated control . In NHDF there was an early and significant (p < 0.05) increase of KGF, FGF2, and VEGF gene expression . TGFB1, PDGFA, and EGF gene expression was hardly affected by incubation with the DACC-coated dressing . In contrast, the expression of FGF2 (p < 0.001), VEGF (p < 0.05), PDGF-A, and PDGF-C (p < 0.05) in HaCaT was decreased under treatment with uncoated RM compared to the untreated control at 48 h , FGF2 (p < 0.001), VEGF (p < 0.001), PDGF-A (p < 0.001), PDGF-C (p < 0.001), and EGF (p < 0.05) after treatment of NHDF with uncoated RM at 24 to 48 h . The gene expression of COL1A1 and COL3A1 by NHDF was not negatively affected by DACC compared to the untreated control . In contrast, these two collagen genes were distinctly less expressed in uncoated RM treated samples at both 24 h (p < 0.01) and 48 h (p < 0.001).In addition to observation of scratch wound closure, the wound healing process was monitored by gene expression analysis. Here, effects of DACC-coated and uncoated RM samples on cell responses as regards inflammation, induction of growth factor and collagen transcripts, as well as gene expression connected to antimicrobial defense were evaluated in vitro. Expression of s p < 0.0. In NHDFs p < 0.0. In NHDFp < 0.001) and open scratches persisted, probably due to cell attachment to the hydrophilic fiber surface. The DACC-coating did not affect the healing processes negatively and supported normal wound closure in vitro. The wound healing process was also monitored by gene expression studies using RT-PCR. Here, the effects of DACC-coated samples and uncoated RM on cell responses associated with inflammation, growth factor induction and collagen synthesis, as well as antimicrobial defense were evaluated in vitro. Controls showed increased expression of the pro-inflammatory cytokine gene IL1A over time in HaCaT keratinocytes, while its expression was found to decrease over time in fibroblasts. Similarly, CXCL1 and CXCL8 expression declined over the observation period in both cell types. IL6 expression showed no significant changes in HaCaT keratinocytes yet was diminished in fibroblasts. Studies have shown an immediate elevation of inflammatory mediator gene expression of, such as IL6, IL1A, or CXCL8, after wounding in vivo [CXCL8 in keratinocytes up to 4 h after treatment and a subsequent decline in gene expression [IL1A, IL6, CXCL8, and CXCL1 by HaCaT cells and fibroblasts was found to be significantly increased by the DACC-coated dressing as well as the uncoated RM compared to the untreated control and also exceeded that of the stress control. Therefore, it is unlikely that positive effects on wound healing observed here stem from a decreased inflammatory response. The interchange between keratinocytes and fibroblasts is regulated in a paracrine manner via growth factors and plays a particularly vital role for completion of wound closure [TGFB1 and FGF2 levels in keratinocytes in response to the untreated or stress control. A slight increase in fold expression (2- to 4 times) was observed for KGF, PDGFA, PDGFC and VEGF. Mechanical stress further induced the early increase in GM-CSF transcript levels. Growth factor gene expression in fibroblasts differed to some extent from that of keratinocytes with no distinct changes observed for TGFB1, VEGF, and PDGFC over time while decreased levels of KGF, FGF2, GM-CSF, and PDGFA gene expression were noted. Interestingly, after 48 h a return to starting levels of gene expression at 4 h was found for KGF and PDGFA. An induction of EGF expression was also observed over time. Timely and spatially differential dispersion of growth factors plays a major role in the proliferative process during wound healing and is regulated in both, a cytokine-dependent and paracrine fashion [TGFB1 expression in fibroblasts by the stress control at a late time point, which was accompanied by a slight increase of COL1A1 and a significant rise in COL3A1 expression corresponding to the reformation of the fibroblast layer in the scratch wound assay. Increased collagen expression has been implicated in the formation of keloid scars [KGF, VEGF, and FGF2 expression in HaCaT keratinocytes and fibroblasts while TGFB1, PDGFA, PDGFC, and EGF were less affected. Overall, growth factor gene expression profiles in both cell types, as well as collagen gene expression in fibroblasts, showed no negative effects of the DACC-coated dressing on the wound healing process emphasizing its high cell compatibility. Moreover, small positive effects on growth factor gene expression were observed that could explain the clinically observed supportive effects of DACC-coated dressings in wound treatment. In contrast, COL1A1 and COL3A1 expression was distinctly decreased in cells treated with the uncoated RM; this either explains the inhibited scratch closure or is due to the observed scratch progression inhibition. Moreover, significantly diminished levels of FGF2, VEGF, PDGF-A, and PDGF-C were observed in both cell types after 48 h of treatment with uncoated RM. The gene expression of KGF, TGFB1, and EGF was further reduced in NHDF treated with uncoated RM, which could account for the inhibited scratch healing progression. Finally, and importantly, gene expression of the antimicrobial peptides S100A7 and RNASE7 by HaCaT keratinocytes was monitored under the influence of the DACC-coated dressing and uncoated RM. Both genes are upregulated upon wounding, and the antimicrobial peptides are responsible for the protection against evading pathogens and have also been implicated directly in the wound healing process [In this study, the effect of a DACC-coated wound dressing on wound healing was assessed using an in vitro scratch wound assay with human keratinocytes (HaCaT cells) and NHDF. Fibroblasts together with keratinocytes ensure maintencance of the barrier function of normal skin. When the skin defense is broken, fibroblasts, and keratinocytes enter the wound area to reform skin tissue. The mechanical scratch wounding of confluent monolayers, often referred to as the scratch wound assay, serves as a model to study cell migration at the wound margins, as well as by de novo synthesis and deposition of newly formed matrix components . Such mo in vivo –29 as we in vivo , which i in vivo , 15. Fib in vivo . In contpression . Downregpression , 34. Henpression , 26 migh closure , 36. The fashion . For ins fashion . Other c fashion , which s fashion . Another fashion . However fashion . Here, wid scars , but no  process , 45. In Physical binding of microorganisms by wound dressings presents a novel concept on preventing wound infection, which may be crucial in the light of increasing global antimicrobial resistance . Coating