Toll-like receptors (TLRs) play an important role between innate and adaptive immunity as one of the pattern recognition receptors (PRRs). Both immune cells and tumor cells express TLRs, and the same TLR molecule is expressed in different cells with different roles. TLR activation in the tumor microenvironment mostly has a dual role in tumor progression during chronic inflammation. Clinically, the therapeutic efficacy of most cancer immunotherapy strategies is restricted by the suppressive immune infiltrative environment within the tumor. Therefore, activation of TLRs in innate immune cells has the potential to eradicate tumors lacking T-cell infiltration. TLR agonists have served as important immunomodulators of cancer immunotherapy through immune responses and reprogramming the tumor suppressive microenvironment. Meanwhile, considering the complex interaction of TLRs with the tumor microenvironment, a combined approach of cancer immunotherapy and nanotechnology has been adopted to improve cancer immunotherapy not only by combining multiple drug combinations, but also by targeting the tumor microenvironment using nanoparticles. Many clinical trials are underway to improve antitumor activity through combination with other immunotherapies. In this review, we provide a comprehensive and detailed overview of the immunotherapeutic implications of TLRs activation in tumor microenvironment, highlighting its great potential to be an important tool for cancer immunotherapy. Toll-like receptors (TLRs) are mainly expressed in immune cells and are involved in the innate immune response triggered by the recognition of multiple pathogen components, as well as in the induction and regulation of adaptive immune responses ,2. TLRs T-cell activation is impaired in cancer patients due to immunosuppression, loss of tumor antigen expression, and antigen-presenting cell dysfunction. The search for therapeutic modalities that promote T-cell infiltration into human tumors and achieve optimal clinical efficacy requires additional combination regimens. In addition, most tumors may exhibit drug resistance. Methods to resensitize drug-resistant tumors to immunotherapy include direct and indirect modulation of immunogenicity. A rational combination of microenvironment-targeted therapies and immune checkpoint inhibitors or cellular therapies will be the next generation of immune-based cancer therapies .TLR agonist treatment enhances the activity of anti-cancer effector cells while blocking the activity of immunosuppressive cells. It also affects the recruitment of immune cells to tumors. Based on these unique properties, TLR agonists can be used as monotherapy or combination modality strategies for cancer treatment ,8.TLRs are transmembrane pattern recognition receptors and belong to one of several pattern recognition receptors (PRRs), and others include RIG-I-like receptors, NOD-like receptors and C-type lectin receptors. Their activation occurs early in the inflammatory cascade response . They plTLR’s function in innate immunity was firstly reported in Drosophila that Drosophila mutants in the Toll gene are highly susceptible to fungal infection. Subsequently, ten TLR family members were identified in humans, while thirteen TLR family members were found in mice. Among them, TLR1, TLR2, TLR5 and TLR6 are located on the cell membrane, TLR3, TLR7, TLR8 and TLR9 are located on the endosomal membrane, while TLR4 is distributed on both cell and endosomal membranes . They arTLRs consist of an extracellular leucine-rich repeat (LRR) recognition domain and an intracellular Toll/IL-1 receptor (TIR) domain that transmits downstream signals. Activation of TLRs proceeds by dimerization of TLRs to form TIR-TIR structures that recruit intracellular adaptor proteins that also contain TIR domains to activate NF-κB, p38 and JNK signaling transduction . TLR-actDysregulation of TLR signaling is associated with many diseases, including cancer , infectiThe close interaction between tumor cells and stromal cells forms the tumor microenvironment (TME). These stromal cells include various immune cells, cancer-associated fibroblasts and endothelial cells . In addiTumor immune microenvironment (TIME) mainly refers to the composition of immune cells and the characteristics of inflammatory response in tumors. The development of resolution technology has revealed the heterogeneity in immune composition, spatial distribution and function within tumors, which has laid the foundation for the classification of TIME. TIME, in which cytotoxic lymphocytes are distributed at the tumor margin with relatively little infiltration in the tumor core, is called infiltrative exclusion (I-E) TIME. (I-E) TIME is less immunogenic and is referred to as a “cold” tumor, an immune state in which adaptive immunity cannot be activated. In contrast, infiltrative inflammatory (I-I) TIMEs are considered immunologically “hot” tumor, characterized by a high infiltration of cytotoxic T lymphocytes expressing PD-1 and leukocytes and tumor cells expressing the immunosuppressive PD-1 ligand, PD-L1 . Cancer Cancer immunotherapies can be broadly classified into five categories: vaccine immunotherapy, cytokine immunotherapy, immune checkpoint blockade immunotherapy, adoptive cell transfer immunotherapy, and small molecule immunotherapy. Immune checkpoint inhibitors, such as antibodies targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) have been used in advanced melanoma, Merkel cell carcinoma, non-small cell lung cancer, cutaneous squamous cell carcinoma, urothelial cancer, renal cancer, refractory Hodgkin lymphoma, hepatocellular carcinoma, gastric cancer, and triple-negative breast cancer, etc. The success of therapeutic suppression with immune checkpoint inhibitors has been limited to “hot” tumors characterized by preexisting T-cell infiltration, while “cold” tumors lacking T-cell infiltration have not achieved lasting benefit. The inability of “cold” tumors to produce spontaneous immune infiltration is due to the generation of an immunosuppressive TME . T cellsTLRs are mainly expressed in human immune cells, however, they are also expressed in many tumor cell lines or tumor tissues, especially in cancers of epithelial origin, and the type of TLRs expressed on different tumor cells varies and is related to tumor progression .In the TME, TLRs are expressed on both immune cells and tumor cells and play a dual role, triggering both anti-tumor (innate and adaptive immunity) and pro-tumor  responses ,34. DAMPTLR signaling also mediates the interaction of cellular energy metabolism molecules of tumor cells and tumor-infiltrating immune cells in the TME. Among them, tumor-derived metabolites maintain an effective tumor suppressive microenvironment . Ubiquit(1)Dendritic cellsDendritic cells (DCs) act as an important anti-tumor component, though they are only a small part of the TME, because their ability to promote T-cell immunity and immunotherapeutic responses is very powerful . NormallTLR signaling induces DC differentiation and affects its interaction with T cell. TLR ligands stimulate transcription of cytokines and costimulatory factors in DC, and TLR activation induces a series of cell biological changes in DC, including increased endocytic activity induced by the membrane vesicle system, regulation of the cytoskeleton and influence on their antigen presentation through protein translation and degradation mechanisms, further enhancing the quality, quantity and detectability of peptide-MHC complex formation in mature DC . Signali+ cDC1 and CD11b+ cDC2 are the only TLR3-expressing groups at tumor sites. DCs produce the Th1-type cytokines IFN-β and IL-12 via the TLR3-TICAM-1 pathway, which contributes to the induction of anti-tumor immunity in functional cytotoxic T lymphocyte. Thus, tumor-specific cytotoxic T lymphocytes can be generated by targeting TLR3 signaling in DCs [CD103g in DCs ,51. Compg in DCs .(2)Natural killer cellsNatural killer (NK) cells are important innate cytotoxic lymphocytes with the ability to rapidly and efficiently recognize and kill tumor cells. NK cells directly regulate T-cell activity through the production of IFN-γ and also shape the DC response to cancer through the induction of DC maturation and the recruitment of DC in the TME. NK cells are also regulated by many soluble cytokines in the TME. Cytokines including IL-2, IL-12, IL-15, IL-18 and IL-21 enhance NK cell activation, survival, proliferation and maturation upon binding to their cognate receptors. Strategies currently in preclinical and clinical development include adoptive transfer therapy, recruitment of NK cells to the TME, blockade of inhibitory receptors that limit NK cell function, and therapeutic modulation of the TME to enhance antitumor NK cell function .The results of the adoptive transfer of autologous or allogeneic activated NK cells for the treatment of hematologic malignancies are encouraging, and the use of CAR-modified NK cells (CAR-NK) may be safer than CAR-T cell therapy. However, solid tumor is more difficult because NK cells are difficult to transport and penetrate to the tumor site and the TME impairs NK cell function. NK cell proliferation and antitumor activity are inhibited by the secretion of various immunosuppressive factors by tumor cells, including prostaglandin E2, indoleamine 2,3-dioxygenase (IDO), IL-10, TGF-β and vascular endothelial growth factor (VEGF) .brightCD16− and CD56dimCD16+ NK cell subsets, but TLR7/8 (R848), TLR3 (Poly I:C) and TLR9 (ODN2395) ligands promote NK cell function only in the presence of cytokines, including interleukins . Among them, R848 activates CD56brightCD16− NK cells mainly by promoting cell proliferation, cytokine production and cytotoxic activity. R848, which normally activates both TLR7 and TLR8 on DCs, macrophages and neutrophils, activates the CD56brightCD16− NK cell subpopulation only via TLR8, highlighting the potential role of TLR8-targeted infiltrating TME-NK cells as a novel tumor immunotherapy [TLRs in NK cells induce innate immune responses against bacterial and viral infections by inducing NK cytotoxicity and cytokine production . NK cell(3)Tumor-associated macrophagesTumor-associated macrophages are the most extensively infiltrated immune cells in TME . Based oThe TLR-triggered early immune response plays an important role in polarizing macrophages . AlthougTumor-associated macrophages activation inhibits TLR-mediated M1 polarization . Blood-dNanoparticles can interrupt these biological interactions within the tumor by altering the tumor-associated macrophage phenotype. Many types of nanoparticles preferentially accumulate in macrophages after systemic administration, and macrophages recognize nanoparticles as foreign bodies and take them up by endocytosis or phagocytosis. The macrophage response to nanoparticles may depend on factors such as dose, route of administration, size, nanoparticle composition, and nanoparticle surface properties . For exa(4)T cells+ leukemia or mesothelin+ solid tumors in vitro and in vivo, respectively [+ T cells; at the same time, imiquimod induces the expression of CXCR3, a homologous chemokine receptor on peripheral lymphocytes, which triggers the infiltration and accumulation of CD4+ T cells within the tumor and is essential for tumor rejection [Adoptive cell transfer studies have demonstrated that TLR functions as an important co-stimulatory and regulatory molecule within T cells themselves. By interacting with T cells, TLR agonists reduce the threshold for TCR activation, enhance T cell proliferation and cytokine production, promote memory T cell formation, and alter the suppressive function of Treg cells . Human Tectively . The TLRejection .(5)Mast cellsMast cells are unique tissue-resident immune cells in the myeloid lineage. Mast cells exhibit three modes of behavior: (1) direct antigen presentation capacity; (2) regulation of DC migration and effector T cell initiation efficiency; and (3) recruitment of effector T cell subsets to areas of inflammation or infection, and in situ activation of homing T cells to drive an effective inflammatory response ,79. Over+ T cells against brain tumor [TLR activation in immune cells and cancer cells in the TME usually prevents tumor formation and growth. However, excessive or low activation of TLR supports tumor survival and metastasis. The pro- or anti-tumor effects of TLR signaling depend on the specific TLR molecules being stimulated, the cell type with activated signaling, and the downstream signaling cascade in the activated cells ,82,83 S. TLR3 isin tumor ,87. Simiin tumor .TLR agonists trigger the innate immune system and generate antigen-specific T-cell responses against tumors. In addition to these immunostimulatory functions, TLR agonists contribute to the reprogramming of the immunosuppressive TME. Immunomodulatory approaches of TLR agonists can be divided into: (1) molecular modulation by chemical structural modifications: the splicing of key binding components of TLR agonists significantly reduces the immunostimulatory properties of TLR agonists. Molecular coupling of TLR to other therapeutic agents shows a more synergistic immune response than non-coupled equivalent models; and (2) macroscopic modulation by engineered drug delivery systems: efficient delivery of TLR agonists to secondary lymphoid organs rich in immune cells, specific targeting and delivery to immune cells or endosomes, and reduction of toxicity by focused and sustained delivery of TLR agonists . TLR inhChemical structure studies suggest that TLR agonists and antagonists share an overlapping binding region. hTLR8 agonists and antagonists have similar structures but have completely opposite biological effects. Hierarchical clustering analysis and molecular docking showed that hydrogen bonding and hydrophobic properties are key features that distinguish hTLR8 agonists from antagonists. Compared with antagonists, agonists have stronger specific hydrogen-bonding properties, while antagonists have stronger non-specific hydrophobic properties ,116.A variety of TLR agonists and TLR inhibitors have been developed for use as cancer immunotherapy tools. Some classical TLR agonists have been approved by the FDA for the treatment of cancer patients, while others have been developed for application in preclinical trials to explore the ability of this small molecule modulator to coordinate anti-tumor immunity .TLR agonists approved by the FDA for the treatment in cancer patients include Bacillus Calmette-Guérin (BCG) , monophosphoryl lipid (MPL) (TLR4 agonist) and imiquimod (TLR7 agonist) ,118,119.In addition, various common TLR agonists include: the TLR2 agonist Pam3CSK4 , the TLR+ T-cell infiltration of the tumor, DC expansion, and antibody production. And topical delivery of TLR9 agonists enhances the efficacy of immune checkpoint inhibition [In humans, TLR9 is expressed only on the endosomal membrane of B cells and pDC and induces the recruitment of immune effector cells by activating an inflammatory-like innate response and inhibits the abundant presence of MDSC around the tumor mass. Several synthetic CpG oligodeoxynucleotides (ODNs) have been developed as TLR9 agonists. Among them, the first generation CpG ODN mimics molecules with natural CpG motifs; and the second generation CpG ODN has unique modifications to its sequence and structure to form immunomodulatory oligonucleotides (IMO). TLR9 agonists have been accepted for the treatment of hematologic and solid tumors. Specifically, CpG ODN can be used as monotherapy to enhance immune response and induce tumor regression in early clinical trials, but the effect when treating patients with advanced solid tumors was not evident ,130,131.hibition ,135. Chehibition . Notablyhibition .Many TLR agonists and inhibitors undergo preclinical studies for their ability to coordinate anti-tumor immunity. The antitumor response is largely attributed to their ability to stimulate antigen presenting cells, which in turn activate tumor-specific T cell responses .The most obvious advantage of nanoparticles is their plasticity, allowing the modification of factors such as particle size, surface charge, composition, hydrophobicity and morphology to optimize the overall immunostimulatory properties. Nanoparticles can be combined with other therapies, including chemotherapy, radiotherapy, and photodynamic therapy. However, nano-delivery systems may not be applicable to all types of tumors due to the heterogeneity of different tumors and individuals ,169.+ T cell-specific nanoparticles are generated by binding anti-CD8a F(ab’)2 fragments to the surface of the particles. Targeted delivery of R848 recruits CD8+ T cells into the tumor and also sensitizes the tumor to anti-PD-1 [Here are some examples to illustrate that nano-delivery system is an important method for TLR modulators. Chemotherapy with TLR 7/8 agonists as adjuvants induced immunogenic cell death (ICD) and in situ vaccination effects, but indoleamine 2,3-dioxygenase (IDO) significantly increased the number of microenvironmental TGF-β, IL-10, MDSCs and Treg cells in tumors, counteracting activated antitumor immunity. The use of an assemblable immune modulating suspension (AIMS) containing an ICD inducer (paclitaxel) and a supra-adjuvant increased cytotoxic T lymphocytes in TME and alleviated IDO-associated immunosuppression. The supra-adjuvant refers to the synergistic combination of R848 and IDO inhibitor (epacadostat) . Mesopornti-PD-1 . As an inti-PD-1 . For difnti-PD-1 .+ Breg cells through TLR2/4 and MAPK signaling pathways, which express high levels of the immunosuppressive cytokine IL-10 and exhibit strong inhibitory activity against CD8+ T cells [+ T cells via the TLR2-MyD88-NF-κB signaling cascade. IL-6 further promotes IL-10 and IL-21 secretion by CD4+ T cells via STAT3. Autophagosomes-induced CD4+ T cells in an IL-6- and IL-10-dependent manner suppress CD4+ and CD8+ effector T cell functions and induce IL-10-producing Breg cells via IL-6, IL-10 and IL-21, thereby promoting tumor growth and metastasis [Tumor cells participate in the shaping of the TME by secreting exosomes. Tumor-derived small extracellular vesicles (SEVs) induce pro-inflammatory cytokine expression and PD-L1 upregulation in M0 macrophages through IL-6/STAT3 and TLR4 signaling pathways, contributing to an immunosuppressive microenvironment . TLR4 ac T cells . Gastric T cells . Heat shtastasis . Exosomatastasis . Epsteintastasis .Recent studies develop a localized drug delivery system with a step-by-step cell internalization capacity based on hierarchical structural fiber devices, which would be internalized by actively targeting tumor cells to induce ICDs. The TLR7 agonist imiquimod can be released from these particles in the cytoplasm to reprogram M2-like tumor-associated macrophage. Loading the drug into the targeted particles can result in doses of antibodies and small molecules that are less than one-tenth of the standard dose, thereby improving efficacy and possibly mitigate potential toxicity .Liposomes are a well-established drug delivery system that enables payload stability and enhanced cellular activity during internalization. Cytoplasmic delivery of antigens can be achieved through surface modification of liposomes by pH-responsive or fusion materials, leading to cross-presentation of exogenous antigens via the “cytosolic pathway”. The introduction of TLR agonists into liposomes enhances their immune-inducing ability .Co-encapsulation of antigenic ovalbumin with TLR9 and STING ligands in liposomes revealed that pH-sensitive liposomes co-encapsulating CpG ODN and cGAMP induced synergistic innate immune responses by increasing type I and type II interferon levels, and that ovalbumin caused specific Th1-biased immune enhancement and induced reversal of the immunosuppressive TME while enhancing effective anti-tumor immune responses . LiposomRadiation therapy is one of the most effective treatments for various solid tumors. Combining novel immune interventions with radiation therapy enhances the expression of tumor-associated antigens, induces immune-mediated tumor stromal targeting, and decreases Treg cell activity. Radiation therapy can also enhance the adaptive immune response to cancer by activating innate immune effectors through TLR-dependent mechanisms . Novel aThe combination of photothermal therapy (PTT) and TLR-mediated immunotherapy triggers antitumor immunity and modulates the immunosuppressive TME. The combination of integrated adjuvant and PTT using nanomaterials allows the production of high-quality anticancer vaccine delivery systems for elderly patients .+ T cells in the TME, while the anti-OX40 antibody triggers a T-cell immune response that is specific for the antigen of the injected tumor [Combination of TLR9 ligand (CpG) and anti-OX40 antibodies may cure many types of cancer and prevent spontaneous gene-driven cancers. Low doses of CpG injected into tumors induce OX40 expression on CD4ed tumor .Cancer vaccines are used to break immune tolerance and enhance the innate and adaptive immune response to cancer. Cancer vaccines can be cancer cells, cell lysates, proteins, peptides or genetic material encoding certain cancer antigens. Given the poor immunogenicity of the aforementioned tumor antigens, the antitumor activity of such vaccine systems remains unsatisfactory. Therefore, the vaccine system must be enhanced by the incorporation of certain vaccine adjuvants. Due to their strong immunogenicity, TLR agonists have been extensively investigated as vaccine adjuvants to enhance the biological activity of anti-cancer immunotherapies . Immunoapoly-ICLC, a multifunctional immunomodulator for cancer treatment, is a double-stranded RNA complex that consists of poly(I:C) (polyinosinic:polycytidylic acid) stabilized by poly-L-lysine. This synthetic dsRNA mimetic stimulates TLR3 and MDA5, affects IFN-I and IL-15 production, enhances T-cell infiltration into tumor parenchyma, and serves as a strong adjuvant for peptide cancer vaccines . CarbohyBoth TLR agonists and antagonists can be used for cancer treatment by enhancing innate immunity, T-cell immunity and cytotoxic antibody function . Given tWhether TLRs promote tumor development needs to be observed in the specific TME. Various types of cells in the TME interact with each other through cytokines or chemokines, which in turn lead to changes in the distribution and cell infiltration within the tumor. Both immune cells and tumor cells express TLRs, and the same TLR molecule is expressed in different cells with different roles. Despite these conflicting roles, TLR agonists have been established as anticancer agents that activate immune cells in the TME and promote the expression of cytokines that permit anti-tumor lymphocyte infiltration and inhibit oncogenic signaling pathways ,208.This review focuses on the link between TLR activation and the TME mainly through pro-inflammatory mechanisms, where TLR activation affects the level of immune cell infiltration in the TME and consequently the development of tumors. Many TLR agonists have been used in the treatment of malignant tumors, the most important applications being: (1) as vaccine adjuvants; (2) in combination with other therapeutic modalities to achieve optimal efficacy; and (3) in combination with different delivery systems to enhance the ability to target tumor cells.Nowadays, several clinical trials on TLR agonists and TLR inhibitors are underway, especially in combination with immunotherapy and conventional therapies. As a small molecule modulator, TLR can provide an effective anti-tumor response by activating cytotoxic T lymphocytes, reshaping the tumor immunosuppressive environment, polarizing neutrophils and tumor-associated macrophages, etc. Combination therapy or targeted therapy could better address the serious problems of drug resistance, excessive toxicity and strong immunogenicity. Therefore, development of TLR modulators has a great potential to be an important tool for cancer immunotherapy.


Public health authorities around the world have been disseminating messages to support mental health and psychosocial well-being during the COVID-19 pandemic. Based on the Portuguese guidelines, we have developed the Adherence Scale to the Recommendations for Mental Health during the COVID-19 Pandemic (ASR-MH-COVID19) to better understand this health behaviour.To analyse the relationship between sociodemographics, personality traits, Adherence  and psychological distress.413 individuals  completed an on-line survey, in September-December 2020, including sociodemographic questions, ASR-MH-COVID19, NEO-FFI-20 and Depression Anxiety Stress Scale (DASS-21) and Health Perception Scale.Adherence scores did not significantly differ by gender, age and years of education. Women presented higher DASS and Neuroticism scores (p<.01). Adherence were negatively correlated with Neuroticism (r=-.247) and with Depression/Anxiety/Stress , positively with Openness to Experience (r=.174), Conscientiousness (r=.194) and Perceived Health  . Mediation analysis (Hays’ Macro Process - Model 4) revealed that Adherence is a partial mediator between Openness and DASS and Conscientiousness and DASS; when considering Neuroticism, only the direct effect was significant. The effect of Perceived Health  on DASS was also mediated by Adherence.The Health Behaviour Model proposes a pathway linking personality and health that applies to these results about adherence and psychological distress during the COVID-19 pandemic. Personality and perceived health  influence both adherence to mental health behaviours and psychological distress. Understanding personality is vital for health care providers.No significant relationships.


This editorial presents 10 contributing articles on the Research Topic “Breast Milk and Passive Immunity during the COVID-19 Pandemic.” First, we describe the passive immunity from the placenta to the fetus after mRNA COVID-19 vaccination. Second, we evaluate the risk of transplacental transmission of SARS-CoV-2 to the fetus. Third, we elucidate that breast milk is not a vector of viral SARS-CoV-2 that can infect breastfed infants. Fourth, we discuss the maternal antibody response specific to SARS-CoV-2 after the two mRNA COVID-19 vaccine and the booster dose. Fifth, we report the safety of the mRNA COVID-19 vaccine and booster shot during breastfeeding. Lastly, we describe the relationship between maternal stress and antibody response in lactating mothers.Neonates are born with an immature immune system, including a lack of IgG and secretory IgA (SIgA) production by plasma cells. Thus, newborns rely on the passive transfer of antibodies via the placenta to the fetal bloodstream . Whether newborns with positive COVID-19 are due to the transplacental transmission of SARS-CoV-2 or infection after delivery is still not well-understood. The placenta provides a protective barrier to the fetus against maternal infections. However, vertical transmission of SARS-CoV-2 from the placenta to the fetus can happen when the virus-induce apoptosis and vascular damage in the placenta. SARS-CoV-2 can spread in maternal endothelium into fetal capillaries and then be aspirated through amniotic fluids. The presence of SARS-CoV-2 in blood samples from newborns was ~1%, suggesting the risk of transplacental transmission of COVID-19 to the fetus is low.Most neonates born from COVID-19-infected mothers did not test positive for COVID-19, while few cases of newborns tested positive and presented early-onset symptoms . Breast skin contaminated with SARS-CoV-2 was associated with the presence of maternal caught and other family members in the household with COVID-19 infection. These findings explain why some breast milk samples in previous studies have detected positive results for SARS-CoV-2 RNA using RT-qPCR. Breast milk is likely not a potential source of viral RNA SARS-CoV-2 when mothers wash their breast skin before breastfeeding.A few articles have reported the detection of low titers of viral SARS-CoV-2 RNA in breast milk samples. Lactating mothers and health professionals have been worried about the potential transfer of viral SARS-CoV-2 from breast milk to the infant. To confirm the safety of breastfeeding during maternal COVID-19 infection, breast milk was collected before and after washing the breast skin in lactating mothers with COVID-19 infection. Some breast skin swabs collected before washing the breast detected SARS-CoV-2 RNA in milk samples, while SARS-CoV-2 RNA was absent in all milk samples after washing the breast skin . After the second vaccine, all mothers had detectable anti-SARS-CoV-2 IgG1 and IgA in breast milk, whereas IgM was present in 87% of milk samples. Neutralizing antibodies increased after the second dose in serum and breast milk compared to the first dose  receptor and provide protection against COVID-19 infection  elevated antibody secretion in lactating mothers. The titers of IgG and IgA specific to SARS-CoV-2 were higher in breast milk after a third booster dose of mRNA COVID-19 than the peak after the first and second vaccines . A slight reduction of IgA titers in serum relative to paired breast milk samples was detected 120 days after the second vector vaccine dose, suggesting a more sustained IgA production in mucosal secretion. In contrast, IgG levels in serum and breast milk (paired samples) decreased from 45 to 120 days after the second vaccine.Like the mRNA vaccine, vector-based vaccines stimulated antibody response in lactating mothers. Paired longitudinal samples taken at 45 and 120 days after the second dose of COVID-19 vector-based vaccines showed that IgG levels waned over time in breast milk, while IgA levels remained stable in 100% of lactating women . Vaccine-related PEGylated protein concentrations did not increase in breast milk after COVID-19 vaccination. These results suggest that the mRNA-COVID-19 vaccine administered in lactating women did not lead to detectable immunogenicity in the infant's blood. In addition, low levels of intact mRNA vaccine were detected in maternal serum and breast milk samples, while infants' serum had no trace of mRNA molecule or serological evidence of infant sensitization . These findings confirm the safety of continuing breastfeeding during maternal mRNA COVID-19 vaccination.Some lactating mothers are hesitant to receive a COVID-19 vaccine due to the lack of knowledge on the immunogenicity of mRNA-based vaccines on nursing infants, as lactating mothers were excluded from initial clinical trials of mRNA vaccination. Paired blood and milk samples from lactating mothers and their infants were collected after the maternal mRNA vaccine (2 doses) to evaluate the immunogenicity of the mRNA molecule. Severe side effects were absent in infants breastfed from mothers vaccinated with mRNA COVID-19 . Another mini-review article assessed that the safety and efficacy of the developed mRNA COVID-19 vaccines were comparable between pregnant, lactating, and non-pregnant women . The administration of mRNA COVID-19 vaccination in these groups promoted the production of neutralizing antibodies against SARS-CoV-2 in mothers and passive immunity in their infants.A systemic review article demonstrated that lactating women receiving 2 doses of the mRNA COVID-19 vaccine are safe for them and their breastfed infants . However, maternal lifetime stressors were negatively correlated with breast milk IgA specific to SARS-CoV-2. Breastfed infants of mothers with high chronic stress levels might ingest lower breast milk SIgA/IgA.Antibody response is influenced by several factors related to maternal background and confounding factors, including psychological stress. Maternal stress could be elevated by stressful events, including the COVID-19 pandemic. Interestingly, the stress levels of lactating women were comparable between pre-pandemic and during the pandemic COVID-19  enhance antibody responses during vaccination.VD-M wrote the first draft of the manuscript, sections of the editorial article, revised, read, and approved the submitted version.


Modularity, small-worldness and average participation coefficient were calculated on the global scale. We used a data-driven consensus community approach to extract a partition of five subnetworks involving thalamic subregions and calculate the within- and between-subnetwork functional connectivity and topology. Multiple linear regression models were fitted to model the relationship between OCS and functional brain measures. No significant associations were found when using our preregistered definition of probable OCS. However, post-hoc analyses showed that children endorsing at least one OCS (compared with controls) had higher modularity, lower connectivity between frontoparietal, limbic and visual networks as well as altered participation of the lateral prefrontal thalamus node. Our results suggest that network characteristics of OCS in children from the general population are partly symptom-specific and severity-dependent. Thorough assessment of symptom dimensions can deepen our understanding of OCS-related brain networks.While functional brain characteristics of obsessive-compulsive disorder have been extensively studied, literature on network topology and subnetwork connectivity related to obsessive-compulsive symptoms (OCS) is sparse. Here we investigated the functional brain characteristics of OCS in children from the general population using a multiscale approach. Since we previously observed OCS-related differences in thalamus morphology, we also focused on the network participation of thalamic subregions. The study included 1701 participants (9–12 years) from the population-based Generation R study. OCS were measured using the Short Obsessive-Compulsive Disorder Screener. We studied the brain network at multiple scales: global network topology, subnetwork connectivity and network participation of thalamic nodes (pre-registration: The online version contains supplementary material available at 10.1007/s11682-022-00732-8. Functional and structural imaging studies of obsessive-compulsive disorder (OCD) have implicated dysfunction of cortico-striato-thalamo-cortical (CSTC), fronto-limbic, fronto-parietal and cerebellar circuity in its pathophysiology  scale. We used a graph theoretical approach to quantify network organization at the global and local level  rated by the primary caregiver  pipeline, that includes head motion correction, slice-timing correction and ICA-AROMA to remove motion artifacts . Nuisance regression was performed by using the ICA-AROMA components and eight physiological regressors: mean signal in the white matter and cerebrospinal fluid, their squares, temporal derivatives and derivatives squared. Aggressive denoising was applied in ICA-AROMA, which involves regressing out all variance associated with noise components, including shared variance with signal components. The functional scans were registered to the anatomical T1-weighted images and we parcellated the anatomical scan into 246 nodes using the Brainnetome atlas, which contains the caudal temporal, rostral temporal, medial pre-frontal, lateral pre-frontal, pre-motor, posterior parietal, occipital and sensory thalamus parcellations  using a standard 8-channel head coil. We obtained structural T1-weighted images using an inversion recovery fast spoiled gradient-recalled echo sequence (IR-FSPGR). Echo planar imaging was used to acquire 200 volumes of RS-fMRI data . One scanning run per participants was acquired that lasted 5 min and 52 s per scan. The resting state images were preprocessed using the standardized The global graph measures calculated for analyses were average participation coefficient (PC), modularity and small-worldness based on weighted connectivity matrices . All statistical analyses were performed in R, version 4.0.3  , leading to a final sample of 1701 participants. Sample characteristics are displayed in Table We found no significant associations between OCS and average PC, modularity and small-worldness in either continuous or case-control analyses (Table S1). Distributions of the graph measures and subnetwork functional connectivity are presented Tables S2-S4 and Figures S2-S4). In the subnetwork analyses, no significant associations were found between OCS and subnetwork connectivity in the continuous (Table S5) and case-control analyses (Table S6). The thalamic node analyses did not show any significant associations between OCS and thalamic node PC or within-module degree Z-score in the continuous (Table S7-S8) or case-control analyses (Table S9-S10). Results were similar when repeating the analyses with individuals with a SOCS > 0 (data not shown). Finally, in our NBS analysis no significant components between the probable OCD and control groups.2 = 7.60, p = 0.0058). Excessive hand washing therefore seems to drive the results. Indeed, children that scored high on the hand washing item had significantly higher modularity  and lower subnetwork connectivity between FPN/DAN and LIM  and between the FPN/ DAN and VIS subnetwork  compared with low scoring children.Children with high OCS (n = 133) compared with controls (n = 1568) had higher modularity compared with controls  , lower connectivity between the frontoparietal-dorsal attention network (FPN/DAN) and visual network (VIS)  ) and lower connectivity between the FPN-DAN and limbic network (LIM) . Finally, high OCS was associated with lower within-module degree z-score of the lateral prefrontal thalamus node . We compared the ‘probable OCD’ group, ‘high OCS’ group and remaining controls on demographic characteristics, OCS scores and CBCL subscale scores (Table S11). The high OCS group compared with the probable OCD group had a significantly larger proportion of children scoring high on the hand cleaning item of the SOCS .We added the results in the Supplementary tables (Table S12-S15).We studied the network characteristics and subnetwork functional connectivity associated with obsessive-compulsive symptoms (OCS) in 9–12 year old children from the population-based Generation R study. Specifically, we examined global network topology, between-subnetwork connectivity of five data-derived subnetworks that included thalamic nodes, and network participation of the thalamic nodes in relation with OCS. In our preregistered analyses, we found no OCS-related differences in network topology nor in subnetwork functional connectivity, both in the continuous analysis and ‘probable OCD’ analysis. However, when comparing children endorsing at least one OCS with controls to align with recent literature, we found that the ‘high OCS’ group had higher modularity, lower FPN/DAN-LIM and FPN/DAN-VIS connectivity and lower lateral prefrontal within-module degree Z-score compared with controls.The discrepancy between the preregistered and posthoc analysis is striking. We performed the ‘high OCS’ analysis to better align with Alexander-Bloch et al., where resting-state characteristics were also compared between youths endorsing at least one symptom compared with controls . Also, in the resting-state sample we found significant relationships of sex or IQ with network topology that have been previously reported  protocol to ensure the quality of the resting-state data (White et al., Future work should improve our understanding of the functional characteristics of OCD and OCS, in particular pertaining to children and thalamic connectivity. The ENIGMA-OCD working group is in preparation of a large-scale mega-analysis of resting-state functional MRI related to OCD across different age groups, which will partly solve the methodological restrictions of individual studies. A more thorough assessment of symptom dimensions could further disentangle the symptom-specific functional characteristics of OCS. To overcome the gap between structure and function, network characteristics of OCD could be assessed using structural connectivity or structural covariance analyses. A structural covariance analysis of ENIGMA-OCD already demonstrated lower small-worldness, modularity and clustering related to OCD in adults and adolescents (Yun et al., To summarize, thalamic morphology differences related to OCS in children do not translate to overall global network topological differences. Rather, children endorsing at least one symptom show differences in modularity, subnetwork connectivity and prefrontal thalamus network participation. Drawing from our results and existing literature, functional network characteristics seem to show symptom-specificity and may depend on the severity of OCS.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


In humans, the hemochorial placenta is a unique temporary organ that forms during pregnancy to support fetal development, gaseous exchange, delivery of nutrition, removal of waste products, and provides immune protection, while maintaining tolerance to the HLA-haploidentical fetus. In this review, we characterize decidual and placental immunity during maternal viral (co)-infection with HIV-1, human cytomegalovirus (HCMV), and Zika virus. We discuss placental immunology, clinical presentation, and epidemiology, before characterizing host susceptibility and cellular tropism, and how the three viruses gain access into specific placental target cells. We describe current knowledge on host-viral interactions with decidual and stromal human placental macrophages or Hofbauer cells, trophoblasts including extra villous trophoblasts, T cells, and decidual natural killer (dNK) cells. These clinically significant viral infections elicit both innate and adaptive immune responses to control replication. However, the three viruses either during mono- or co-infection (HIV-1 and HCMV) escape detection to initiate placental inflammation associated with viral transmission to the developing fetus. Aside from congenital or perinatal infection, other adverse pregnancy outcomes include preterm labor and spontaneous abortion. In addition, maternal HIV-1 and HCMV co-infection are associated with impaired fetal and infant immunity in postnatal life and poor clinical outcomes during childhood in exposed infants, even in the absence of vertical transmission of HIV-1. Given the rapidly expanding numbers of HIV-1-exposed uninfected infants and children globally, further research is urgently needed on neonatal immune programming during maternal mono-and co-infection. This review therefore includes sections on current knowledge gaps that may prompt future research directions. These gaps reflect an emerging but poorly characterized field. Their significance and potential investigation is underscored by the fact that although viral infections result in adverse consequences in both mother and developing fetus/newborn, antiviral and immunomodulatory therapies can improve clinical outcomes in the dyad. Healthy human placentation is vital for fetal viability and development in pregnancy. In order to optimize this process, hemochorial placental changes occur during gestation, which address a myriad of functions specific to survival of the fetus. Ongoing adaptations include gaseous and nutrient exchange, removal of waste products, and immune protection while maintaining tolerance to the HLA-haploidentical fetus , 2. The Many of the studies addressing viral infection of the placenta have focused on characterizing the presence and role of decidual natural killer (dNK) cells and macrophages, trophoblasts including extra villous trophoblasts EVTs), T cells in the decidua and villi, and placental macrophages or Hofbauer cells (HCs) located in underlying stromal tissue –E. It isVTs, T ceThe known underlying mechanisms of pathogenesis, protection, and transmission at the MFI to HIV-1, HCMV, and ZIKV are discussed in this article following summaries on embryology and placental immunity. In general, most of the articles cited reflect human studies unless otherwise stated. Discrepancies have arisen when comparing human with murine or other animal placentae. Identifying an ideal animal model system for congenital HCMV infection has remained a significant challenge. At the end of each section, current knowledge gaps that may prompt future research directions are also discussed.Three main placental types are recognized according to the cell layers comprising the interhemal area: (1) epitheliochorial type , (2) endotheliochorial type (carnivores) and 3) hemochorial type . The number of cell layers and placental permeability in the interhemal area modifies the exchange of nutrients between maternal and fetal blood and is one of the important factors differentiating mammalian species  hemochor. The TE − folate receptor 2 (FOL2)+. The group also identified a newly characterized population of placenta-associated maternal macrophages (PAMM1a) that may be involved in tissue repair  by single-cell RNA sequencing (scRNAseq) and profiling the transcriptomes of ~70,000 cells from first-trimester placentae with matched maternal blood and decidual cells by Vento-Tormo et al.  identifidim T cells and 5% CD4+ CD25bright FOXP3+ Treg cells. In addition, ~40% of decidual CD8+ cells have an effector-memory phenotype with reduced perforin and granzyme B release compared to peripheral CD8+ T cells ]. It is important to note that chronic villitis, specifically VUE, has been observed in placentae from term infants with normal outcomes as well as in infants born following complicated gestations . There rAgainst this background and after summarizing clinical presentations and epidemiology, the rest of this article will discuss the role of decidual macrophages and NK cells, and then focus on trophoblasts (including EVTs), HCs and T cells, and their relationship with HIV-1, HCMV, and ZIKV. We will address how each virus as mono- or co-infection (HIV-1 and HCMV) interacts with decidual and placental immune cells elucidating mechanisms of viral entry to the fetal side, characterizing their intracellular effects, and discuss potential mechanisms of protection.Mechanisms of protection have evolved at the MFI over millennia to restrict retroviral infection following exposure. Endogenous retroviruses (ERVs) are molecular remnants of ancient retroviruses that point to an ongoing relationship between lentiviruses and vertebrate hosts that began hundreds of millions of years ago. Retroviruses may have acted on vertebrates as agents of selection, driving the evolution of host genes that block viral infection. Furthermore, through a process of endogenization, retroviruses have contributed an abundance of genetic novelty to host genomes, including unique protein-coding genes and cis-acting regulatory elements. In the placenta, syncytins are glycoproteins of retroviral origin that fulfill cellular functions involving receptor-mediated membrane fusion, promoting merging of CTBs to form the multinucleate STB layer. Their potential role in HIV-1 transmission is discussed below .In general, the primary target for HIV-1 are thought to be activated memory CD4+ T cell populations, but many other cell types, including naïve CD4+ T cells, macrophages, dendritic cells, and monocytes can also be infected , 23. TheGlobally, over 60 million people have been infected by HIV-1. Maternal HIV-1 infection during pregnancy is associated with vertical or mother-to-child transmission (MTCT). MTCT of HIV-1 occurs most often intrapartum, during labor and delivery, and then through breastfeeding. In the absence of clinical interventions, the risk of intrauterine transmission is estimated at 5–10%. Current United States (US) guidelines from the Department of Health and Human Services recommend administration of ART throughout pregnancy to maintain maternal plasma viral concentrations beneath the level of detection and the avoidance of breast feeding . Other iIn order to develop strategies for prevention of MTCT of HIV-1, it is important to characterize target cells in the female reproductive tract mucosae and identify effective innate responses. Women acquire HIV-1 mainly through sexual intercourse. However, low transmission rates per sexual act suggests local immune mechanisms contribute to prevention. Neutrophils mediate mucosal protection against bacterial and fungal pathogens including by release of neutrophil extracellular traps (NETs). These DNA fragments associate with antimicrobial granular proteins. Stimulation of human genital neutrophils with HIV-1-viral-like particles (HIV-1-VLPs) induced NET release following viral exposure, through reactive oxygen species-independent mechanisms resulting in entrapment of HIV-1-VLPs. Incubation of infectious HIV-1 with pre-formed genital NETs prevented infection of susceptible cells through irreversible viral inactivation. HIV-1 inactivation by NETs from genital neutrophils may represent a previously unrecognized form of mucosal protection against viral acquisition .highFoxP3+ T regulatory cells  HCs sequester virus and may permit intracellular neutralization in virus-containing compartments (Major findings from our research include: 1) HCs exhibit reduced ability to replicate HIV-1 artments  and ARV artments . 4) In aartments  29). 5). 5)in viMost recently we have also shown that HCs early in gestation express higher levels of CCR5 and exhibit a more activated phenotype yet are less permissive to HIV-1 replication than term HCs with downregulated CCR5 levels. This observation may reflect that early/mid gestation HCs display overall increased production of anti-inflammatory cytokines, chemokines, and an IFN-dependent antiviral immune response. In contrast, term HCs exhibit attenuated IFN-induced Signal Transducer And Activator Of Transcription (STAT)1 and STAT2 activation. Treatment of early/mid gestation and term HCs, with type I IFNs or RIG-I-like receptor (RLR) agonists reduced HIV-1 replication in both populations, underscoring the importance of IFN and RLR signaling in inducing an antiviral state. Viral recognition and antiviral responses in early gestation HCs may prevent intrauterine HIV-1 infection, whereas diminished antiviral responses at term may facilitate MTCT of HIV-1 .in vitro data associating HCMV with MTCT of HIV-1  and of HIV-1 , 35, 36.nfection . We prevnfection . ConsistCytotrophoblasts (CTBs) have not been documented as target cells for HIV-1 in the scientific literature, although Tang et al. recently described a potential route of CTB infection by cell-to-cell fusion. They detected HIV-1 RNA and cDNA in placental tissue, including CTBs from pregnant HIV-1-infected women receiving ART. They suggested syncytin, the envelope glycoprotein of human ERV family W1 expressed on CTBs, triggered membrane fusion with syncytin receptors present on HIV-1-infected CD4+ T cells, leading to production of two different types of progeny HIV-1 virion  38)..38).in vitro through cellular contact and release of soluble factors with IFN-γ involved in this control  at the MFI during the first trimester. dNK cells constitute nearly 70% of the total decidual leukocyte population . Periphe control , 40. Giv control .A number of investigators have noted that even when MTCT of HIV-1 is prevented, HIV-1-exposed infants and children (HEU) exhibit impaired immunity in postnatal life associated with adverse clinical outcomes compared with HIV-unexposed/-uninfected (HUU) children. Most infection-related hospitalizations are associated with bloodstream or respiratory infections , 42, 43.HCMV has a wide range of tropism and can infect epithelial, myeloid, and endothelial cells. However, the mechanisms of fetal CMV infection are not yet fully delineated , and aniGlobally, many individuals are infected with HCMV reflecting the ubiquity of this virus, ability to spread by horizontal and vertical transmission, and to manifest with subclinical illness for nearly all individuals including pregnant women. An HCMV seroprevalence of 100% is estimated in many parts of Africa and Asia with numbers up to 86 % in women of childbearing age. The lowest seroprevalence is seen in the WHO European region at 66% , 50. HCMHCMV is one of 8 human herpesviruses that can infect humans at all ages. Once born, young children are frequently exposed to HCMV in communal settings and the Centers for Disease Control and Prevention (CDC) and WHO estimate that ~30% of children in the US are HCMV seropositive by age five , 57. ComInnate and adaptive placental immunity may promote viral entry into placental villi and cord blood. In the following sections, we will discuss viral entry in and the intracellular effects of HCMV infection in immune placental cells, which may lead to adverse birth outcomes.′)2. These results suggest CTBs are important sites for placental HCMV infection, most likely transmit virus to the fetus, and that HCMV virions could disseminate to the placenta by co-opting the receptor-mediated transport pathway for IgG  for entry into first trimester extravillous-derived (SGHPL-4) and villous-derived (HTR-8/SVneo) trophoblasts. Infection with four HCMV clinical and laboratory strains , and Merlin deletion mutants of UL128-, UL130-, and UL131A-genes, showed a cell type-dependent requirement of the viral pentameric complex for infection of trophoblasts. The viral pentameric complex was essential for infection of villous trophoblasts, but not for EVTs. Blocking of PDGFRα in EVTs, which naturally express PDGFRα, inhibited entry of pentameric complex-deficient HCMV strains, but not the entry of pentameric positive HCMV strains .We have previously demonstrated HCMV infection of term human CTBs and HCs when testing whether Modified Vaccinia Ankara (MVA)-gH-gL-pentamer complex vaccine induced neutralization titers in sera from vaccinated Rhesus Macaques to block HCMV TB40/E infection of freshly isolated HCs and CTBs. HCMV entry into both cell types is blocked by gH/gL-PC-specific neutralizing antibody , 48. HowDuring HCMV infection in pregnancy, naïve CD8+ T-cells (CCR7+CD45RA+) were reduced by 50% in maternal blood. However, there was a doubling in the proportion of CD45RA+ revertant memory cells (CCR7-CD45RA+) in seropositive donors. Moreover, seropositive women during late pregnancy demonstrated an accumulation of highly differentiated HCMV-specific T-cells . There aExtravillous trophoblasts (EVTs) appear to escape CD8+ T cell recognition through a lack of expression of major histocompatibility complex (MHC-I) receptors: HLA-A and HLA-B, which promote cytotoxic activity. CTBs express both classical (HLA-C) and non-classical (HLA-G) class I MHC-I receptors. Interestingly, HCMV did not induce a significant difference in HLA-G expression on either JEG-3 cells or primary EVTs, and only partially downregulated HLA-C. Persistence of HLA-G expression, which is associated with immune tolerance at the MFI despite HCMV infection, suggests protection of infected trophoblast cells from the effects of CD8+T cells. In this same article, however, dNK cells were able to kill HCMV-infected maternal stromal cells  Figure .NK cell diversity is based on NK receptor expression, distinct NK cell types and differentiation states, which accommodate a variety of dNK cell functions at the MFI. dNK cell antiviral activity and immunity to HCMV infection occurs in tandem with T cell activation, resulting in secretion of cytokines and interaction with EVTs. These functions may change depending on gestational age and decidual tissue type. As discussed in the previous section, first trimester dNK cells were able to clear HCMV-infected cells but failed to increase degranulation and cytokine production in response to HCMV-infected EVT cells . Vieira + NK cells after HCMV infection have been detected in children with symptomatic congenital HCMV infection   . The funIn normal early gestation, CTBs secrete matrix metalloproteinase-9 (MMP-9), which may enable cells to digest extracellular matrix to complete differentiation and invasion. MMP-9 levels gradually decrease during pregnancy. Once differentiation and invasion are complete, CTBs secrete IL-10 to modulate the immune response to protect the fetus. HCMV infection at the MFI can downregulate MMP-9 activity, so that HCMV-infected CTBs induce HCMV-IL-10 mRNA release. HCMV-IL-10 mRNA in turn, may acts as an agonist at the IL10-receptor, reducing MMP-9 activity and impairing CTB-mediated invasion remodeling, which can restrict fetal growth . HCMV caWe refer the reader to a recent review by Njue et al. who proposed mechanisms of HCMV-induced placental dysfunction that may have a role in associated still- and preterm birth, and IUGR from in vitro studies in which laboratory-adapted and low-passage strains of HCMV and various human placental models were used. Potential mechanisms identified included impairment of trophoblast progenitor stem cell differentiation and function, impairment of EVT invasiveness, dysregulation of Wnt signaling pathways in CTBs, TNF-α- mediated apoptosis of trophoblasts, HCMV-induced cytokine changes in the placenta, inhibition of indoleamine 2,3-dioxygenase activity, and downregulation of trophoblast class I MHC molecules . These iWhile HIV-1 and HCMV transmission occur through body fluids, which includes saliva for HCMV, blood, and seminal/vaginal secretions, Zika virus (ZIKV) is an arthropod-born virus (arbovirus) transmitted by Aedes aegypti and Aedes albopictus mosquitoes, which are also vectors for dengue and chikungunya viruses. Additionally, ZIKV can be transmitted sexually and vertically , 70. ZIKZIKV passes through the placental barrier and can replicate in HCs leading to viral dissemination in the fetus with signs of congenital Zika syndrome (CZS), especially following maternal infection in the 1st trimester. Clinical manifestations of CZS include fetal brain disruption sequence leading to microcephaly, subcortical calcifications, hydrocephalus, neuronal migration disorders and brainstem dysfunction. Reported CZS ocular abnormalities include macular scarring and focal retinal pigmentary mottling. Congenital Zika syndrome is also associated with contractures including arthrogryposis.Zika virus (ZIKV) belongs to the genus Flavivirus, related to many clinically significant viruses, including dengue, yellow fever, Japanese encephalitis, chikungunya, and West Nile virus. ZIKV is an enveloped virus with single-stranded positive-sense RNA enclosed by an icosahedral nucleocapsid . ZIKV enZIKV has a broad range of cell tropism and ability to infect tissue in the central nervous system, blood, retinal, and genital and reproductive tract including the placenta –77. SpecDespite the dramatic presentation of CZS, we have limited knowledge on the pathogenesis of ZIKV at the MFI, reflecting the relatively short time frame of reporting this condition from the initial outbreak in the Americas in 2015. Our group initially showed that ZIKV can infect HCs. Infected HCs may disseminate ZIKV to the fetus. ZIKV replication coincides with induction of type I IFNs, pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. These results suggest a “Trojan Horse” mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting and replicating within HCs, without direct cytotoxicity. Viral replication within HCs was mediated through FcR, TLR4 and DC-SIGN receptors. Infection appears to be augmented in HCs by IgG from prior flavivirus exposure through antibody dependent enhancement (ADE) . MultiplInnate immune activation against a given virus is associated with viral RNA recognition in the infected cytoplasm by RIG-I, with subsequent activation of IRF3 and NF-kB. IRF3 activation triggers production of type I and III IFNs. This cascade is mediated by the JAK-STAT signaling pathway to activate IFN-stimulating genes (ISGs) to further induce nearby cells to an antiviral state, specifically targeting viral replication. However, ZIKV may be able to evade the innate immune response via the non-structural protein NS5, which can act as an antagonist for the JAK-STAT pathway , 84. ZIKIn this review, we have focused on three clinically significant viral pathogens during pregnancy. Characterizing placental immunity is exceptionally challenging given that this unique organ constructed from both maternal and fetal tissue, exhibits immunological responses that are markedly different from those characterized in peripheral blood. Against this backdrop, viruses themselves exhibit uniquely specific cell and tissue tropism and evolutionary adaptation, with limited opportunity to study affected cells over gestation in vivo. At the same time, animal models infrequently recapitulate all aspects of human disease.Immunoquiescence is key to protecting the haploidentical fetus from maternal immunity during gestation. This unique placental milieu is balanced against maintenance of an adequate level of immune protection against invasive perinatal or TORCHESZ infections . Maternal viral infection during pregnancy can be self-limiting. However, if invasive HCMV infection develops during embryogenesis, teratogenic effects to the developing fetus can occur before diagnosis is established and effective antivirals administered. Furthermore, administering newer antivirals in the first and early second trimester is associated with a potential risk of antiviral-associated teratogenicity. Some antivirals, such as the protease inhibitors used for treatment of HIV-1 infection, should generally be avoided during pregnancy because of an associated risk of preterm delivery , 42, 43.in vitro experiments and point to a combination of immunoquiescent factors such as a predominance of regulatory cytokines (IL-10 and TGF-β) at the MFI that are important in not only supporting tolerance to the haploidentical fetus but also limiting inflammation and viral replication during maternal infection. Despite expression of CD4, CCR5, CXCR4, and DC-SIGN, HCs kinetically exhibit slow transcription and replication of HIV-1 compared to MDMs. We postulate that these cells have evolved to capture and contain HIV-1 and other retroviruses over millennia and can limit viral replication through intracellular neutralization and endogenous production and secretion of IL-10 and TGF-β, as shown in The modest amount of information of human host-placental cell-viral interaction that has been published are mainly from There is emerging clinical data associating HCMV with MTCT of HIV-1 , 36, 86,CTBs are important sites for placental HCMV infection and most likely transmit virus to the fetus. Cell entry may involve interaction between viral pentameric complex gH/gL/pUL128-pUL131A and cellular platelet-derived growth factor receptor-α (PDGFRα) expressed on placental trophoblasts. EVTs appear to escape CD8+ T cell recognition through a lack of expression of MHC-I receptors. However, HCs appear to be involved in light of HCMV infection of these macrophages, although their specific role in pathogenesis at the MFI during maternal primary infection or reactivation requires further characterization. First trimester dNK cells were able to clear HCMV-infected cells with activating dNK cell receptors including KIR2DS1, KIR2DS2, KIR2DS5 and KIR3DS1 exhibiting antiviral activity and cytotoxicity. However, term dNK cells exhibited reduced efficacy when responding to HCMV-infected cells.The dramatic appearance of a ZIKV pandemic in the Americas in 2015 raises as many questions as to why and how cases so precipitously decreased in a relatively short space of time. Perhaps this broad and difficult question is beyond the scope of this review. What we learned in terms of the key role of HCs is their ability to sequester ZIKV following infection with minimal cell death, and how ZIKV is able to evade the innate immune response. These observations speak to evolutionary adaptation of ZIKV with immune cells at the MFI. There are glaring omissions in this review on topics that merit further investigation. Because ZIKV can infect many different cell types, it is challenging to characterize the mechanisms by which this virus gains access to the fetus. Further research is needed to identify a putative route of vertical transmission. Vaccination may be a future consideration, however, since ZIKV is closely related to other Flavivirus families, vaccine-induced cross-reactive antibody and antibody-dependent enhancement may cause unexpected and unwanted outcomes.The importance of studying mechanisms of pathogenesis of viral infections at the MFI in detail is to enable targeted therapeutic interventions including vaccines to mitigate the adverse outcomes of these infections during pregnancy. Characterizing the complexity of interactions at the MFI across gestation with modulation by viral pathogens can also promote improved understanding of the long-term effects of maternal infection during pregnancy on neonatal and childhood immunity.As a pediatric infectious disease specialist (RC) from the 1990’s, one of the most rewarding outcomes to personally witness has been our ability to reduce MTCT of HIV-1 to just 1–2%. This contrasts with the large number of infants that were becoming HIV-1-infected prior to the landmark Pediatric AIDS Clinical Trial Group (PACTG) 076 study . It is t


Colorectal cancer (CRC) is a malignancy that seriously threatens human health, and metastasis from CRC is a major cause of death and poor prognosis for patients. Studying the potential mechanisms of small extracellular vesicles (sEVs) in tumor development may provide new options for early and effective diagnosis and treatment of CRC metastasis. In this review, we systematically describe how sEVs mediate epithelial mesenchymal transition (EMT), reconfigure the tumor microenvironment (TME), modulate the immune system, and alter vascular permeability and angiogenesis to promote CRC metastasis. We also discuss the current difficulties in studying sEVs and propose new ideas. Colorectal cancer (CRC) is a malignancy of the gastrointestinal tract with a high incidence and mortality rate. Recent statistics show that approximately 9,300,000 new cases of CRC occur worldwide each year, and that it has the second highest mortality rate 9.4%) of all cancers . Metasta% of all Small extracellular vesicles (sEVs) are the major component of extracellular vesicles (EVs), which range in size from 30 to 150 nm. sEVs originate from endosomes and are formed by the fusion of multivesicular bodies (MVBs) with the plasma membrane. MVBs contain intraluminal vesicles that are released into the extracellular lumen when these MVBs fuse with the plasma membrane ,11. The Several studies have shown that differential expression of sEV content is closely associated with CRC metastasis and plays an important role in the multi-step and multi-linked metastatic process ,21,22. TCRC cells undergo a series of complex and highly regulated processes as they metastasize to surrounding and distant organs and tissues. The TME plays a crucial role in the process of tumor metastasis. The TME refers to the surrounding microenvironment where tumor cells exist, and includes surrounding blood vessels, immune cells, fibroblasts, inflammatory cells, various signaling molecules, and the extracellular matrix (ECM) . The funIt was found that sEVs derived from highly metastatic CRC cells enriched in miR-181a-5p-activated hepatic stellate cells (HSCs) by activating the IL6/STAT3 signaling pathway, acting directly on suppressor of cytokine signaling 3 (SOCS3) and causing TME remodeling. Activated HSCs release C-C motif chemokine ligand 20 (CCL20), which upregulates miR-181a-5p expression in CRC via the ERK/Elk-1 pathway. In addition, sEVs enable a positive feedback loop between CRC cells and HSCs, ultimately leading to CRC liver metastasis (CRLM) . ExposurCRC cells originate from sEVs that highly express circPACRGL. CircPACRGL has been shown to enhance CRC cell proliferation, migration, and invasion via the miR-142-3p/miR-506-3p-TGF-β1 axis. It also modulates N1-N2 neutrophil differentiation and regulates the differentiation of tumor-associated neutrophils (TANs) in the TME, allowing immunosuppression to occur and providing an important basis for CRC metastasis . CRC celHypoxic CRC cells secrete sEVs that promote the migration and invasion of normoxic CRC cells. These hypoxic CRC cell-derived sEVs were found to contain Wnt4, which affects hypoxic extracellular vesicle-mediated migration and invasion of normoxic CRC cells, enhances pro-metastatic behaviors, and further mediates CRC metastasis . sEVs deEpithelial mesenchymal transition (EMT) plays a key role in tumorigenesis and metastasis. During EMT, epithelial tumor cells undergo marked morphological and phenotypic changes, including loss of tight junctions, cell polarity, and cytoskeletal reorganization, including a more aggressive phenotype . In addiCancer-associated fibroblasts (CAFs) are key stromal cells that play a dominant role in tumor progression. Researchers have co-cultured sEVs derived from CAFs with CRC cells. CRC cells internalized sEVs containing miR-92a-3p, leading to a significant increase in the level of miR-92a-3p in CRC cells. In addition, sEVs from CAFs with high miR-92a-3p expression activated the Wnt/βcatenin pathway in CRC cells, inhibiting mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1 expression and promoting EMT in CRC cells, thus further facilitating metastasis .Another study showed that sEVs derived from metastatic SW620 CRC cells targeted RAS p21 protein activator 1 (RASA1) via miR-335-5p. Downregulation of RASA1 in CRC cells activated signaling downstream of RAS and induced EMT, further promoting CRC cell invasion and metastasis . High leStudies have shown that co-culture of LO2 hepatocytes and phosphatase of regenerating liver-3 (PRL-3) overexpressing CRC cells induced the re-expression of E-cadherin in CRC cells. The re-expression of E-cadherin is an important marker for the development of EMT in CRC. In addition, Src plays an important role in regulating EMT through activation of epidermal growth factor receptor (EGFR) . The aboHigh expression of integrin alpha6 (ITGA6) activated TGF-1 in CRC cells to promote CRC progression and metastasis. MiR-3940-5p in mesenchymal stem cell-derived sEVs promoted the progression and metastasis of CRC by downregulating ITGA6 in CRC cells. Reduced ITGA6 levels disrupted the TGF-β1 signaling pathway, inhibiting CRC cell invasion and EMT, and ultimately reducing tumor growth and metastatic capacity . HCT116 Tumor-associated macrophages are derived from circulating peripheral blood monocytes, which are derived from the bone marrow. These monocytes are recruited into tumor tissues and undergo local differentiation in response to various cytokines, chemokines and growth factors produced by stromal and tumor cells in the TME, affecting tumor progression . TAMs haTumor-derived sEVs high in miR-934 can promote CRLM by regulating the interaction between CRC cells and TAMs. Studies have shown that tumor–TAM interactions in the metastatic microenvironment are mediated by tumor-derived sEVs and affect CRLM. Phosphatase and tensin homolog (PTEN) expression and activation of the PI3K/AKT signaling pathway induce M2 macrophage polarization. Polarized M2 macrophages can induce pre-metastatic niche formation and promote CRLM by secreting C-X-C motif chemokine ligand 13 (CXCL13) . CRC celIn addition to CRC cell-derived sEVs, CRC cells with EMT function can also promote CRC metastasis by causing M2 polarization in macrophages. It was found that EMT-CRC cell-derived extracellular vesicles highly expressing microRNA-106b-5p promoted M2 polarization of macrophages by directly inhibiting activation of the phosphatidylinositol 3-kinase (PI3K) γ/AKT/mTOR signaling pathway at the post-transcriptional level of programmed cell death 4 (PDCD4). Activated M2 macrophages promoted EMT-mediated migration, invasion and metastasis of CRC cells in a positive feedback manner . In addiTumor growth, invasion, and metastasis depend on angiogenesis for adequate oxygen and nutrient supply to the tumor cells. Moreover, disruption of vascular integrity and the consequent increase in vascular permeability allows EMT tumor cells to enter the vasculature and subsequently metastasize. sEVs secreted by cancer cells have been reported to be taken up by neighboring or distant receptor cells to promote tumor development and metastasis. They play an important role in tumor angiogenesis and vascular leakage ,73,74. TAngiopoietin-like protein 1 (ANGPTL1) protein levels were found to be significantly downregulated in sEVs from CRC tissues compared to paired normal tissues. sEVs with downregulated ANGPTL1 decreased matrix metallopeptidase 9 (MMP9) levels in Kupffer cells (KCs) by inhibiting the JAK2-STAT3 signaling pathway, preventing hepatic vascular leakage and ultimately inhibiting the development of liver metastasis in CRC . Zeng etIn addition, researchers found that sEVs secreted by EMT-CRC cells further disrupted endothelial cell junctions by transferring miR-27b-3p into vascular endothelial cells and attenuating vascular endothelial cadherin (VE-Cad) and p120-catenin (p120) expression in a post-transcriptional manner. This negatively affected cell integrity, increased vascular permeability and ultimately induced cancer metastasis . The adeBecause sEVs are formed by the fusion of multivesicular bodies (MVBs) with the plasma membrane, they serve as important carriers of intercellular information and participate in a variety of physiological activities of the body, playing a crucial role in pathological states. Most studies have focused on the role of sEVs in the development of cancer, diagnosis, drug delivery, and prognosis. During the development of malignant tumors, sEVs play a role in the regulation of the local microenvironment by carrying and transporting tumor cell content, tumor-associated fibroblasts, and tumor-associated immune cells, which are important to the regulation of tumor development in target cells. They release their content after binding to target cells, causing an intracellular cascade reaction. Cancer metastasis is a process involving the spread of cancer cells from primary lesions to distant organs and is a major cause of cancer lethality. Numerous studies have demonstrated that sEVs contribute to tumor metastasis by promoting EMT of tumor cells, inducing angiogenesis, promoting vascular leakage, establishing a pre-metastatic microenvironment, immunomodulation and forming a pre-metastatic ecological niche during cancer metastasis ,82. BeinRegarding the function of sEVs in promoting cancer metastasis through immunomodulation, previous researchers have mainly focused on cancer-derived sEVs that exert tumor–host immunosuppressive functions and promote tumor progression by binding to and altering the biological functions of surface receptors on natural killer cells, dendritic cells, T and B lymphocytes, and mast cells ,89,90. RWe have summarized mechanistic studies showing how sEVs induce macrophages to undergo M2 polarization, and thus disrupt anti-tumor immunity and enable CRC to metastasize. We have also discussed sEV-induced inhibition of M2 polarization to reduce colorectal cancer metastasis. Therefore, sEVs are a promising new target that may be used to reduce colorectal cancer metastasis. This will be the focus of our future research.In this review, we provide a detailed summary of how sEVs mediate the epithelial-mesenchymal transition, reconfigure the tumor microenvironment, modulate the immune system, and alter vascular permeability and angiogenesis to promote CRC metastasis. Importantly, these studies have identified several sEVs components such as microRNA, circRNA, lncRNA, and proteins highly associated with CRC metastasis. This work improves our understanding of the role of sEVs in the development of CRC and provides a basis for subsequent studies. Greater attention to the mechanisms of small extracellular vesicles in tumor progression and efforts in translational medicine research will provide a richer foundation for the clinical role of sEVs in the early diagnosis and treatment of colorectal cancer.In conclusion, sEVs play an important role in CRC metastasis, and an in-depth exploration of the function of sEVs in metastasis will help identify early therapeutic approaches for CRC. Through summarizing the currently available studies, we have increased the understanding of how sEVs promote metastasis and organotropism. The translation of this knowledge is very useful for clinical cancer treatment. Future studies should focus on examining whether sEVs from metastatic CRC sites facilitate further metastatic tumor formation in other distant organs. Seeking answers to these questions will not only increase our understanding of sEVs in CRC biology but also provide important evidence for much-needed novel therapies for advanced CRC. Furthermore, increasing the number of studies focused on sEVs and CRC metastasis will facilitate the development of sEVs as a biomarker for metastatic CRC.


Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The resulting biotinylated proteins can then be isolated and identified. A combination of programmable DNA targeting and proximity labeling that maps proteomic landscape at DNA elements with dCas9-APEX2 has been established in living cells. However, defining interactome at RNA elements has lagged behind. In combination with RNA-targeting CRISPR-Cas13, proximity labeling can also be used to identify proteins that interact with specific RNA elements in living cells. From this viewpoint, we briefly summarize the latest advances in CRISPR-guided proximity labeling in studying RNA–protein interactions, and we propose applying the most recent engineered proximity-labeling enzymes to study RNA-centric interactions in the future. RNA–protein interactions are pervasive in regulatory RNAs and RNA-mediated processes in cells ,2,3. A gRecently, researchers have applied the RNA-targeting CRISPR-Cas13 system to proximity labeling to achieve programmable RNA-interacting proteins biotinylation ,12,13,14Taken together, these new methods expedite the analysis of RNA interactome in living cells. Compared with previous RBPL or RaPID labeling, CRISPR-guided proximity labeling method is programmable and easy-to-use, which enables arbitrary RNA targeting and the biotinylation of RNA-associated proteins. Moreover, researchers can build inducible CRISPR-guided proximity labeling enzyme fusion cell lines, allowing the spatiotemporal control of proximity labeling activity and the mitigation of high background of biotinylation. CRISPR-guided proximity labeling methods are usually validated with well-known RNA–protein interactions first and then the unknown RNA interactome was explored with mass spectrometry to expedite the analysis of endogenous RNA–protein interactions. Similarly, Zhang et al. reported a new proximity labeling system by using PUT-IT to label proximate proteins of specific RNAs . HoweverAlthough bioID and APEX2 are useful tools for proximity labeling, they have some common drawbacks. For example, bioID takes 18~24 h of reaction time to obtain enough labeled materials for quantitative proteomics ,20. SuchE. coli birA-derived enzyme for protein–protein interaction detection [Recently, Kohki et al. reported AirID, an etection . In thisetection . UltraIDetection , was defAlthough numbers of methods have been developed to probe RNA activity, improving RNA-centric targeting affinity and sensitivity is still in early development. It is also expected that a programmable RNA-targeting system could broadly target cellular low-abundance transcripts. The combination of evolved proximity-labeling enzymes and RNA-targeting technology would further expedite the functional analysis of endogenously expressed RNAs in the future.The proximity labeling method employs promiscuous biotin ligases or peroxidases to label proximate proteins. This method is most commonly used to probe protein–protein interactions and the constituents of subcellular compartments refractory to biochemical isolation ,25. HoweProximity labeling is particularly suitable for probing insoluble or inaccessible subcellular molecules and for detecting weak or transient intermolecular interactions ,22,23,25


Coronavirus disease 2019 (COVID-19), an infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that has high incidence rates, spreads rapidly, and has caused more than 6.5 million deaths globally to date. Currently, several drugs have been used in the clinical treatment of COVID-19, including antivirals , monoclonal antibodies , protease inhibitors , and glucocorticoids . Increasing evidence suggests that circulating microRNAs (miRNAs) are important regulators of viral infection and antiviral immune responses, including the biological processes involved in regulating COVID-19 infection and subsequent complications. During viral infection, both viral genes and host cytokines regulate transcriptional and posttranscriptional steps affecting viral replication. Virus-encoded miRNAs are a component of the immune evasion repertoire and function by directly targeting immune functions. Moreover, several host circulating miRNAs can contribute to viral immune escape and play an antiviral role by not only promoting nonstructural protein (nsp) 10 expression in SARS coronavirus, but among others inhibiting NOD-like receptor pyrin domain-containing (NLRP) 3 and IL-1β transcription. Consequently, understanding the expression and mechanism of action of circulating miRNAs during SARS-CoV-2 infection will provide unexpected insights into circulating miRNA-based studies. In this review, we examined the recent progress of circulating miRNAs in the regulation of severe inflammatory response, immune dysfunction, and thrombosis caused by SARS-CoV-2 infection, discussed the mechanisms of action, and highlighted the therapeutic challenges involving miRNA and future research directions in the treatment of COVID-19. Coronavirus disease 2019 (COVID-19) is an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has spread worldwide and caused more than 6.5 million deaths globally to date MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18~25 nucleotides in length that regulate gene expression at the posttranscriptional level SARS-CoV-2 is a novel variant of the SARS-CoV virus and is also closely related to the Middle East respiratory syndrome coronavirus (MERS-CoV), which caused similar acute respiratory infections SARS-CoV-2 uses the spike glycoprotein (S) to bind to the ACE2 receptor on the surface of the host cell and internalize it through endocytosis Virus-encoded miRNAs perform their functions in two ways. The first is to interact with specific regions of the own genome or transcript, and the interaction of functional genes or gene regulatory regions may lead to changes in gene expression, thereby affecting viral replication and infection It is estimated that 2,300 mature miRNAs exist in humans, and nearly half of them have been annotated in the miRNA database miRbase. Furthermore, 2,083 miRNAs were screened in human plasma using EdgeSeq miRNA whole-transcriptome analysis, which together accounted for 91% of the miRNome in vitroThe hypersensitivity of circulating miRNAs was also observed in asymptomatic patients. Calderon-Dominguez et al. showed that serum hsa-miR-32-5p and hsa-miR-1246 could significantly discriminate between critical COVID-19 patients and asymptomatic IgG-positive individuals Inflammation is a key defense mechanism against viral infection Mitochondria have an important role in pro-inflammatory signaling that cause the activation of the immune system and generate inflammatory responses The immune system has evolved in host-virus confrontation for self-protection. Viruses have evaded host immune surveillance through evolutionary selection to achieve immune escapes As the largest IFN family in the human body, type I interferon (IFN-I) is an important mediator of antiviral immunity and homeostatic immune system regulation Several studies have shown that circulating miRNAs are a novel class of regulatory molecules that mediate host-virus interactions. Host-induced circulating miRNAs can regulate immune responses through different mechanisms, indirectly or directly acting on viral infection Increasing evidences show miRNAs regulate the expression of genes coding proteins via binding mRNAs transcribed from their downstream target genes at the posttranscriptional level in COVID-19, and are also regulated by upstream transcription factors (TFs) and non-coding RNAs (such as lncRNA and circRNA) Most patients with SARS-CoV-2 are asymptomatic or develop mild to moderate illness, such as fever, sore throat, cough, chest, and muscle pain, but 15-20% develop pneumonia and lung injury Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 infection mainly attacks the lungs Patients with COVID-19 also have a relatively high incidence of cardiac complications  -/- and WT mice. The results showed that the number of DNA- citH3-positive cells in carotid artery thrombi in miR-146a -/- mice was significantly higher than that in WT mice -/- mice, the ratio of citH3-positive and total nucleated cells found in carotid artery thrombi was significantly higher than that in WT mice, suggesting that miR-146a deficiency may be involved in thrombosis through NETs Thrombotic microangiopathy has been mentioned in autopsy reports of COVID-19 patients. Despite the anticoagulant therapy in COVID-19 patients, microthrombus still exists in the lungs and kidneys In addition, there have been numerous reports of circulating miRNAs associated with other COVID-19-related diseases, including hypertension, diabetes, obesity, and cerebrovascular diseases Extracellular vesicles (EVs) are membranous structures released by biological cells, which contain DNA, miRNA, lncRNA, and other biologically active components derived from mother cells in vitro neutralizing properties against SARS-CoV-2 in the serum of COVID-19 recovered patients with high anti-spike IgG titers Exosomes are one of the smallest EVs released from cells, and they carry specific functional RNAs (such as miRNAs) from donor to recipient cells SARS-CoV-2 gene product, spike, can modify the host exosomal cargo, which gets transported to distant uninfected tissues and organs and can initiate a catastrophic immune cascade Currently, SARS-CoV-2 continues to mutate and COVID-19 continues to spread globally Circulating miRNAs can resist degradation by endogenous RNases, are characteristically expressed in different diseases, and can be detected using liquid biopsy The RNAi-based nucleic acid molecules have attracted considerable attention as compelling therapeutics providing safe and competent delivery systems are available ACE2 is the initial cellular target of HCoV-NL63, MERS-CoV, and SARS-CoV, as well as the target of SARS-CoV-2 With the advancement of bioinformatics and high-throughput sequencing technologies, many circulating miRNAs are differentially expressed in COVID-19, and the important regulation of circulating miRNAs in virus-host interactions has also been found EVs may be an alternative therapeutic strategy in the future owing to their advantages of being cell-free, immunologically harmless, nonteratogenic, and free of foreign agents. EV-miRNA can simultaneously restore viral infection through multiple pathways in vitro transcriptional techniques to rapidly produce a highly stable circRNA RBD vaccine encoding the circRNA of SARS-CoV-2 spike protein trimer RBD. The study found that the vaccine elicited effective and sustained neutralizing antibodies that targeted the Th1 cells in mice. More importantly, the study found that the circRNA vaccine against the Delta variant has broad-spectrum protection and is effective against both the Delta and Omicron variants, while the vaccine against Omicron has narrower protection and cannot resist Delta invasion Additionally, efforts are underway to develop emerging vaccines against SARS-CoV-2 variants in various regions of the world. According to the latest research, Qu et al. used