Structures and distributions of SARS-CoV-2 spike proteins on intact virions.

SARS-CoV-2 mRNA vaccine induces robust specific and cross-reactive IgG and unequal neutralizing antibodies in naive and previously infected people

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Long-term, infection-acquired immunity against the SARS-CoV-2 Delta variant in a hamster model.

The rapidly spreading Omicron variant is highly resistant to vaccines, convalescent sera, and neutralizing antibodies (nAbs), highlighting the urgent need for potent therapeutic nAbs. Here, a panel of human nAbs from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients show diverse neutralization against Omicron, of which XMA01 and XMA04 maintain nanomolar affinities and excellent neutralization (half maximal inhibitory concentration [IC50]: ∼20 ng/mL). nAb XMA09 shows weak but unattenuated neutralization against all variants of concern (VOCs) as well as SARS-CoV. Structural analysis reveals that the above three antibodies could synergistically bind to the receptor-binding domains (RBDs) of both wild-type and Omicron spikes and defines the critical determinants for nAb-mediated broad neutralizations. Three nAbs confer synergistic neutralization against Omicron, resulting from the inter-antibody interaction between XMA04 and XMA01(or XMA09). Furthermore, the XMA01/XMA04 cocktail provides synergistic protection against Beta and Omicron variant infections in hamsters. In summary, our results provide insights for the rational design of antibody cocktail therapeutics or universal vaccines against Omicron.

Severe acute respiratory syndrome (SARS) first emerged in Guangdong province, China in November 2002. During the following 3 months, it spread rapidly across the world, resulting in approximately 800 deaths. In 2004, subsequent sporadic cases emerged in Singapore and China. A novel coronavirus, SARS-CoV, was identified as the etiological agent of SARS.1,2 This virus belongs to a family of large, positive, single-stranded RNA viruses. Nevertheless, genomic characterization shows that the SARS-CoV is only moderately related to other known coronaviruses.3 In contrast with previously described coronaviruses, SARS-CoV infection typically causes severe symptoms related to the lower respiratory tract. The SARS-CoV genome includes 14 putative open reading frames encoding 28 potential proteins, and the functions of many of these proteins are not known.4 A number of complete and partial autopsies of SARS patients have been reported since the first outbreak in 2003. The predominant pathological finding in these cases was diffuse alveolar damage (DAD). This severe pulmonary injury of SARS patients is caused both by direct viral effects and immunopathogenetic factors.5 Many important aspects of the pathogenesis of SARS have not yet been fully clarified. In this article, we summarize the most important mechanisms involved in the complex pathogenesis of SARS, including clinical characters, host and receptors, immune system response and genetic factors. CLINICAL PATHOLOGY CHARACTERS OF SARS Patients with SARS-CoV infection have a wide spectrum of disease, varying from a self limiting illness to a fatal outcome.6,7 This disease consists of two phases, including prodromal influenza-like symptoms characterized by myalgia, malaise, chills and fever, and the onset of respiratory and gastrointestinal symptoms.8 Fever was the most common and the earliest symptom.9 The clinical picture is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome (ARDS). Upper-respiratory-tract symptoms are not prominent9 but gastrointestinal symptoms were common.10,11 A majority of the patients admitted to the hospital showed pulmonary X-ray abnormalities varying from bilateral interstitial infiltrates to focal consolidation.6,12 Autopsies of SARS cases indicated that the lungs were edematous and increased in weight.13,14 In some SARS cases organizing features, like dense septal and alveolar fibrosis, were demonstrated.15,16 The longer the disease persists, the more extensive becomes the fibrous organization of the lung tissue.17 Fibrin and collagen were found deposited in the alveolar space.18 Morphological changes identified were bronchial epithelial denudation, loss of cilia and squamous metaplasia. DAD is the most consistent finding in the lungs of SARS patients in the terminal stage.9 In SARS postmortem samples viral RNA has been localized by in situ hybridization to cells of the conducting airways and alveoli.19 The infection and release of virus was close to the pulmonary capillary bed, which might allow systemic spread of virus to distant organs, especially in the context of inflammation and alveolar capillary leak.20 In many cases, cellular infiltration has been observed, including macrophages, neutrophils and CD8+ T cells. Macrophages are a prominent component of the cellular exudates in the alveoli and lung interstitium.9,21 In addition, immunohistochemistry, in situ hybridization and electron microscopy examination of tissue upon autopsy or tissue biopsy showed that SARS-CoV replicates in pneumocytes and macrophages.22 The replication of SARS in macrophages suggests a passive role for macrophages as scavengers, rather than being the primary target.23 A disproportionate scarcity of inflammatory cells has been noted.5,13 Mononuclear infiltrates increased in the interstitium. Large multinucleated cells have been frequently observed in the lungs of SARS patients.5,14 The presence of hemophagocytosis supports the contention that cytokine dysregulation may account for the severity of the clinical disease. The lack of a prominent inflammatory response is also distinctive. Such changes reflect the combined effects of primary infection, host immune responses and therapeutic interventions. A substantial number of SARS patients have diarrhea.11,24 In the intestine, little pathology is observed at the light microscopy level, either in biopsies taken during early phases11 or in autopsy specimens.13,19 Severe depletion of mucosal lymphoid tissue in the small intestines and appendix has been described.25 Both extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have also been found.14,26,27 A sharp decrease in the number of periarterial sheaths in the spleen have been demonstrated. CD4+ lymphocytes, CD8+ lymphocytes, CD20+ lymphocytes, dendritic cells, macrophages, and natural killer cells in the spleen showed a decrease of 78, 83, 90, 80, 39 and 48%, respectively. The average size of macrophages was found to be increased by more than 100%. T lymphocytes and macrophages in the spleen have been detected to be infected by SARS-CoV.27,28 Lymph nodes usually show atrophy and reduction of lymphocytes with loss of germinal centers.28 Focal necrotic inflammation of hilar lymph nodes has been found in some cases.29 Evidence of hemophagocytosis in lymph nodes was observed in a limited number of cases.30 High viral loads have been detected in lymph nodes, whereas viral isolation was negative.26 T lymphocytes and macrophages in lymph nodes have also confirmed SARS-CoV infection.28 Several observations suggest that SARS-CoV is also capable of causing an infection of the central nervous system. Cerebrospinal fluid, brain tissue specimens and neurons were detected to have SARS-CoV infection.28,31 Kidneys of autopsied SARS patients have shown focal necrosis and vasculitis of small veins in the renal interstitial tissue.14 High viral loads have been detected in the renal tissue specimens and the distal convoluted tubules, which suggest that urine may be an additional source of sewage contamination.32 In addition, monocytic infiltration, acute tubular necrosis and other nonspecific changes, such as glomerular fibrosis and nephrosclerosis, have been all observed.33 A high proliferative index has been demonstrated in hepatocytes in the liver in some cases. But viral particles are not detected by electron microscopy (EM).34 In both the liver and the kidney signals for SARS-CoV were detected by both immunohistochemical (IHC) and in situ hybridization (ISH),35 yet EM failed to reveal recognizable viral particles. This raises the question that whether the virus exists in a non-packaged form.25 Destruction of epithelial cells with significant changes in the follicular architecture was present in the thyroid glands. Both myofiber necrosis and atrophy were observed in the limited number of skeletal muscle tissue specimens of SARS autopsies examined.36 Edema of the walls of small veins and arteries has also been reported. The few available studies on adrenal glands described the presence of necrosis and vasculitis of the medulla with monocytic and lymphocytic infiltration. Edema of both myocardial stroma, as well as vascular walls, and atrophy of cardiac muscle fibers all have been demonstrated.14,22 SARS-CoV genomic sequences and antigens have also been detected in sweat glands and pancreatic islet cells.35,37 The presence of virus in the sweat glands suggests that SARS may be spread via contact with the skin.25 In some cases, evidence of reactive hemophagocytosis or bone marrow hypoplasia was present.29 Raised creatine kinase, thrombocytopenia, an increase in lactate dehydrogenase and a decrease in absolute lymphocyte counts are the most common laboratory findings. The majority of SARS patients showed a transient increase in serum alanine aminotransferase levels during the course of their disease.38 In some autopsy cases fatty degeneration were observed. These findings suggest that SARS is a systemic disease with widespread extrapulmonary dissemination, resulting in viral shedding in respiratory secretions, stools, urine and possibly even in sweat. The organ damage in patients with SARS could be due to both local viral replication and the immunopathologic consequences of the host response, hence it is important to delineate what human cells the SARS-CoV can infect and replicate in as well as the subsequent host immune response.35,39 HOST AND RECEPTORS SARS-CoV was isolated from Himalayan palm civets found in a live-animal market in Guangdong, China in 2003. The full-length genome sequences had 99.8% homology to the SARS-CoV genomic found in humans. So primarily, palm civets were suspected as the origin of the SARS outbreak in 2003. Subsequently, many other animals have also been found to be a host or to be infected by the virus.40 Bats and swine were also reported as natural carriers of SARS virus.41,42 Recently, horseshoe bats were designated as the natural reservoir for SARS-CoV-like virus and civets were identified as the amplification host. This highlights the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.43 SARS-CoV spreads via droplet and contact transmission and via the fecal-oral route.44 Through these routes, SARS-CoV can be transmitted from animal to human or from human to human. The primary target of SARS-CoV is epithelial cells in the respiratory and intestinal tract.18 Epithelia are primary barrier to infection by microorganisms entering their host via body cavities. Epithelial cells are organized in a polarized fashion that involves the separation of the plasma membrane into an apical and a basolateral domain. The polarity of these cells affects both the early and late stages of infection, i.e. viruses may enter into and exit from a cell either via the apical membrane facing the external environment or via the basolateral membrane directed to the internal milieu of the organism. An important determinant of the virus infection is the presence of suitable receptors on the cell surface that allow attachment to and penetration through the plasma membrane.20 Angiotensin-converting enzyme 2 (ACE2), a protector of lung damage, has been identified as the primary functional receptor for SARS-CoV.45 ACE2 is a membrane-associated aminopeptidase.46 A region of the extracellular portion of ACE2 that includes the first α-helix and lysine 353 and proximal residues of the N terminus of β-sheet 5 interacts with high affinity to the receptor binding domain of the SARS-CoV S glycoprotein.47 The N terminal half of the S protein (S1) contains the receptor binding domain whereas the C-terminal half (S2) is the membrane-anchored membrane-fusion subunit, which contains two heptad repeat regions (HR1 and HR2).48 After binding to ACE2 on the target cells, the transmembrane S protein changes conformation by association between the HR1 and HR2 regions to form a six helix oligomeric complex, leading to fusion between the viral and target-cell membranes. Apart from direct membrane fusion at the target cell surface, SARS-CoV might gain cell entry via pH-dependent endocytosis, which is also mediated by the S protein.49 In addition to being a cellular receptor, ACE2 may contribute to the pathogenesis of DAD in SARS through its role in the tissue rennin-angiotensin system (RAS).8 ACE2 is a negative regulator of the RAS and has a negative effect on the formation of angiotensin II. Angiotensin II appears to be one of the elements of the RAS that contributes to exacerbation of acute lung injury.50 With respect to SARS-related lung injury, binding of SARS-CoV Spike proteins to ACE2 has been found to reduce ACE2 expression, thus inducing acute lung edema.51 Based on animal experiments, ACE2 may protect against respiratory failure and down-regulation of ACE2 may cause acute lung injury. The insert/deletion genotype of the ACE gene was associated with DAD after SARS-CoV infection in a small cohort of 44 patients.52 ACE2 protein is reportedly present in type 1 and type 2 pneumocytes, enterocytes in all parts of the small intestine, the brush border of the proximal tubular cells of the kidney, as well as the endothelial cells of small and large arteries and veins and arterial smooth muscle cells.46 This localization of ACE2 explains the tissue tropism of SARS-CoV for the lung, small intestine and kidney. Theoretically, all tissues and cell types expressing ACE2 may be potential targets of SARS-CoV infection. However, notable discrepancies were found including virus replication in colonic epithelium, which has no ACE2, and no virus infection in endothelial cells, which have ACE2. Despite the fact that SARS-CoV can infect the lung and intestine the tissue responses in these two organs are different. Furthermore, studies in a new human cell culture model have indicated that the presence of ACE2 alone is not sufficient for maintaining viral infection.39,53 Other findings indicate that ACE2 expression positively correlated with the differentiation state of the epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected.18 It is apparent that the effect of SARS-CoV infection is different in different cell types and it is possible that the virus may utilize different receptors, or involve various co-receptors, in these different cells. C-type lectins, including CD209 and CD209L, were identified as alternative SARS-COV receptors.53 CD209, also known as dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN), is mainly expressed in certain types of dendritic cells (DCs) and alveolar macrophages.54 However, in the lung tissue of SARS autopsies, CD209 has been localized to pneumocytes.55In vitro, CD209 was also inducible in lung epithelial and monocytic cells after SARS-CoV infection,55 which confirmed that SARS infection is capable of inducing CD209 expression. The glycosylated S protein has been shown to bind to the CD209 expressed on the DCs; these cells then mediate SARS-CoV infection in trans of cells that express human ACE2. CD209L, also known as L-SIGN or DC-SIGNR, is generally found in lymph nodes and liver sinusoidal cells. By IHC it has been demonstrated that CD209L is also expressed on type II pneumocytes and endothelial cells. CD209L can also bind to S protein and mediate virus entry.56,57 Although SARS-CoV does not replicate in DCs, these cells may act as a reservoir and distribute the virus to other cell types.58 This is an attractive concept and similar biological behaviours have been proposed for human immunodeficiency virus I (HIV I).59In vitro experiments have demonstrated that cells expressing CD209 or CD209L without ACE-2 are not, or are only partially, susceptible to SARS-CoV infection. This would imply that these molecules are much less efficient receptors than ACE2 as specific receptors and may therefore merely enhance infection of permissive cells.49,56,57 SARS-CoV infection of ACE2-expressing cells also seems to be dependent on the proteolytic enzyme cathepsin L. Cathepsin L is poorly expressed in endothelial cells which may explain the low infection rate of these cells despite the high expression of ACE2. SARS-CoV infection seems to be pH-dependent because the activation of cathepsin L is pH sensitive. Differential expression of cathepsin L in various cell types may explain the differences in viral distribution in relation to the ACE2 expression pattern.60,61 CYTOKINES AND CHEMOKINES Both cytokines and chemokines are soluble proteins with a key function in the innate immune system. Dysregulation of these proteins may result in immunemediated injury.5 High levels of cytokines and chemokines, triggered by the host immune response to SARS coronavirus (SARS-CoV), are believed to contribute to the progressive pulmonary infiltration of macrophages,9 polymorphonuclear leukocytes, T cells,62 eventual DAD and fibrosis.6 This assumption is supported by the clinical deterioration of many patients in the second week of the disease's course, despite decreasing viral loads.9,63 Increased serum levels of several cytokines were found in major SARS patients.51,57 Most cytokines showed only transient and short-lived activation in patients after SARS-CoV infection.64 Even in patients who developed DAD, most cytokine concentrations were not significantly increased.65 In contrast, circulating concentrations of several chemokines, including chemokine C-X-C motif ligand 9 or monokine induced by γ-interferon (CXCL9), chemokine C-X-C motif ligand 10 or interferoninducible protein-10 (CXCL10) and C-C motif ligand 2 or monocyte chemoattractant protein-1 (CCL2), were markedly increased in SARS patients.62,64,66 Recent studies have focused on the role of chemokines rather than cytokines in SARS infection.5 The chemokines are a family of small-molecule proteins that important in intercellular and Based on their protein are into with a common C-X-C of residues the which interacts with and with a C-C with In the lung tissues from SARS patients who chemokines and were markedly and a chemokine C-X-C motif ligand were also markedly A number of chemokines, including and were increased 1 after to the Most for the receptor for and was in the lungs of of gene expression changes in cells from in vitro with show an early activation of the innate in the first including expression of receptor 9 chemokines and their receptors with and and macrophages in the lungs express In in vitro cells chemoattractant protein 1 and after with SARS-CoV and expressed and vascular cell adhesion SARS-CoV induced cells to express and which and T cells in a The showed low expression of a and of cytokines necrosis and but significant of inflammatory chemokine It activation of T cell to express and and also to be significantly increased in lung tissue and lymphoid tissue of autopsied SARS which was confirmed by IHC with An increased has been found to be an of SARS-CoV, through a with lung epithelial cells and monocytic cells, an environment to immune cell and that to lung The lack of cytokine response against a of chemokine could a of immune by A model to explain cellular infiltration may result in SARS was pulmonary epithelial cells infected by SARS-CoV express adhesion molecules and high levels of and which macrophages and and macrophages by with SARS-CoV and a of chemokines that more and neutrophils as well as T viral effects are also to contribute to the pulmonary injury resulting from SARS-CoV infection. In during the first 10 of the disease, virus replication is viral effects to an important The presence of multinucleated cells in SARS lungs may be the result of viral The virus is also capable of causing effects in both renal epithelial cells and epithelial cells in and formation are in infected studies have reported evidence of in cells of the thyroid cells, epithelial cells, pneumocytes, lymphocytes and vitro experiments indicate that expression of certain proteins may in several cell expression of a protein by SARS-CoV, can via a dependent in cell from different organs, including lung, liver and kidney. of may be one of the mechanisms for the pathogenesis of SARS-CoV SARS protein also appears to be important in in some cell It is into the and may also act as one of the Through an host cells SARS have increased expression of Furthermore, SARS-CoV proteins also have the to in SARS-CoV proteins may be involved in through of proteins Increased expression of has been detected in infected alveolar and bronchial epithelial cells. is an of cell of this cytokine may also account for of such cells. OF SARS and viral in the first 10 of SARS immune by The innate immune system the first of the immune against viruses and involves several cellular and soluble factors. T lymphocytes and are the key immune cells that are infected by vitro infected cells have shown viral replication for to In from SARS SARS-CoV has also been found to infect and replication was Both T lymphocytes and were found in the circulating lymph nodes, lungs and in SARS These findings may a partial for the and the widespread of spleen and lymphoid tissue in the majority SARS immune cells may cause widespread to various and T cells are involved in both the innate and immune the of such cells may result in a immune with a decrease in both CD4+ and CD8+ T cells is common during the acute of SARS and may be associated with an Several other viral such as virus and respiratory virus are also associated with severe However, in these direct infection and subsequent of lymphocytes are generally as to account for the severe lymphocyte In for only a small of the are infected during acute with the high infection of in SARS immune syndrome with respect to the fact that both are viral that result in However, SARS various immune cells and rapidly whereas human immunodeficiency virus mainly CD4+ lymphocytes and is In addition, SARS-CoV seems to the of macrophages, which may SARS patients to pulmonary SARS-CoV also causes and functional of dendritic cells in vitro, resulting in a of cytokines and an T cells may in the to pulmonary injury. In viral in are and by infected cells. These cells cause cells to that to viral to other SARS-CoV is not capable of inducing significant or gene expression in infected macrophages, or in infected dendritic Furthermore, in contrast to patients with was a lack of expression of for the and in cells of patients with studies have found that from SARS patients could be an of of serum and against SARS-CoV and by against the that most patients after onset of can be detected as early as after the onset of The for and most of the is to In a of SARS the levels at and were for on the of 14 patients showed that the was in for a from developed a and and of the not The indicated that specific and could be in the could the SARS-CoV and protect cells from SARS-CoV The low response of SARS-CoV has been identified in which proposed a question that whether the serve as a host for were SARS cases that in Guangdong in In contrast to in these cases with clinical and caused a lower and transient immune The of the cases to a at at and then rapidly a of This that SARS-CoV may have to during the These can the particles the S protein from different SARS-CoV that these are and that the S protein is the S the other proteins, such as or is the only significant SARS-CoV and with as the major The on the S was also developed may also be involved in the pathogenesis of Both and immune responses to animal have been identified to be capable of the disease or causing new are with and coronavirus a of by with the SARS-CoV of were to the specific of the while of the could of the or These may have some of the that were to the and characterized the binding of SARS-CoV by an detected specific for the of an human serum In cohort of SARS patients immune against antigens from lung epithelial cell and endothelial cell was found in some approximately 1 after high levels of these in the were shown to be to lung epithelial cells and endothelial cells in may be to the of against specific SARS-CoV against the domain 2 of the protein have been found to with pulmonary epithelial that possibly explains is the of caused by organ has been a the of since some antigens in the may that enhance viral infection rather than also to a role in the pathogenesis of a of the was associated with severity of SARS This association has not been for certain Nevertheless, in the a association was demonstrated between and and an increased to SARS infection. is a serum protein that can bind to the of various for immune of a specific is capable of binding to the glycosylated SARS-CoV S protein and SARS-CoV in In a of SARS patients and have been shown to be associated with increased of In contrast, CD209L to have a significantly lower of SARS infection. The in genetic between is for by In the context of to after at the of were associated with high concentrations of in the plasma and a In addition to other genetic such as enzyme are also associated with severity and of Although ACE2 as its receptor and ACE2 is known to be an important protector of lung damage in no association between of the two ACE and and the severity of after SARS infection was is in the of different and the of in clinical is studies are to fully the genetic for both to infection and the after infection with the The pathogenesis of SARS appears to be and The most and possible appears to of a direct injury to the target cells by the virus and an injury mediated by subsequent immune system By droplet SARS-CoV the respiratory and the epithelial cells of the and infection and replication in target cells causes direct damage to the respiratory tract. inflammatory changes the of the barrier and increase the of the capillary of in the formation of membranes. The infection and associated inflammation acute injury of type II alveolar cells, decreasing the of alveolar resulting in alveolar the SARS-CoV and circulating immune cells. The infected immune cells mainly macrophages and T cells. immune cells the virus to other organs, including the spleen and the lymph The of immune cells with extensive damage to the white pulp in A immune the infection and replication of the virus in the lungs and viral damage to the respiratory resulting in respiratory The proposed mechanisms of SARS have significant for the and on this emerged disease. Although much has been of SARS since its many with respect to the pathogenesis of SARS is a that SARS for the of at 2 known animal in civets and horseshoe bats that are found in the as well as and in China. are

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

SARS-CoV-2 infection and ACE2 inhibition.

Humoral Response to SARS-CoV-2 in Hemodialysis Patients

D614G mutation eventuates in all VOI and VOC in SARS-CoV-2: Is it part of the positive selection pioneered by Darwin?

By May 15, 2020, 65 days after the World Health Organization (WHO) declared the novel coronavirus disease 2019 (COVID-19) pandemic, 4.2 million individuals were confirmed as being infected with the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and 294,000 people had died from the disease1. These numbers are almost certainly an underestimation of the true morbidity and mortality of the disease to this point. From the beginning of this public health crisis, attention has focused on the development of vaccines against SARS-CoV-2. Many believe that immunization is the key advance in the war against COVID-19 and that control of SARS-CoV-2 allowing approximations of pre-pandemic social conditions will not be possible without a viable vaccine2. Until a viable vaccine is developed, extensive diagnostic testing, quarantining, and social distancing are the only control methods that we have3. This article was written for musculoskeletal physicians and scientists without immunology backgrounds. It seeks to provide a concise but comprehensive understanding of vaccine development with a special emphasis on efforts to establish immunization directed against SARS-CoV-2. This will help to inform readers of the challenges to be hurdled and allow them to track milestones reached as the global community labors toward an effective vaccine. In-depth reviews of complex immunology topics, viral epidemiology, the myriad historic aspects of vaccine development, and the economics of developing and utilizing different types of vaccines are beyond the scope of this work. Background SARS-CoV-2 To understand the process of vaccine development, one must start with a basic understanding of the pathogen against which it will be directed. SARS-CoV-2 is a beta coronavirus4. The virus has been found to be similar to another coronavirus whose usual hosts are bats. It is believed that wild animals being sold at a market in Wuhan, Hubei Province, the People's Republic of China, led to the transmission of SARS-CoV-2 to humans5. Members of the Coronaviridae family have a positive-sense, single-stranded RNA ([+]ssRNA) genome6. The (+)ssRNA can serve as messenger RNA (mRNA) in the host cell, allowing utilization of host ribosomal machinery to translate viral proteins. This coronavirus genome is highly conserved6. The first gene (open reading frame [ORF]1a and ORF1b) is involved in replication and transcription. Subsequent genes are related to structural proteins (Fig. 1). The spike protein (S) has an exposed location on the virion and is necessary for entry into the cell. Blocking of the spike protein epitopes that interact with angiotensin-converting enzyme 2 (ACE2), the host cellular receptor for SARS-CoV-2, should lead to neutralization (Fig. 2). ACE2 degrades angiotensin II (AngII), downregulating the renin-angiotensin-aldosterone system. ACE2 is distinct from the angiotensin type-I receptor, which leads to activating signal transduction by AngII7.Fig. 1: Illustration of the SARS-CoV-2 genome showing expressed structural component proteins and their location within the virion. The open reading frame (ORF) codes for nonstructural viral proteins participating in viral genome replication, transcription, and protein processing. The spike protein (S) binds to the ACE2 receptor, allowing virus entry into host cells. The envelope protein (E) has been found to bind to host gene regulatory proteins and putatively influences host cell gene expression. The membrane protein (M) seems to cooperate with S during binding and entry into the cell. The nucleocapsid protein (N) coils the viral mRNA inside the viral particle, organizing and protecting it.Fig. 2: SARS-CoV-2 virion entry into the host cell. The virus enters the host cell via the S protein interacting with ACE2. It is important to generate antibodies to the S protein rather than ACE2. This allows ACE2 to remain open to receive the host ligand and to avoid the possibility that the blocking antibody interferes with its normal physiologic function.COVID-19 is usually characterized by fever, cough, dyspnea, fatigue, and sore throat. Radiographic findings have demonstrated pneumonia with infiltrates on chest imaging8. Typically, radiographic changes appear on presentation. In Wuhan, 76% of diagnosed patients demonstrated changes on chest radiographs9. It is unknown whether radiographic changes arise prior to the onset of symptoms. Currently, there are no known curative treatments for COVID-19. Treatment is centered around supportive care while the afflicted individual clears the infection. In some cases, supplemental oxygen needs to be administered to help those with the associated pneumonia to maintain their oxygen saturation. Severe cases may benefit from antiviral medications, dexamethasone10, and ventilatory support with or without extracorporeal membrane oxygenation11,12. In March 2020, the WHO estimated the global mortality rate for those confirmed to have a SARS-CoV-2 infection to be 4.3%, although this varies by region13. Vaccines Vaccines meet the definition of a drug because they are substances other than food used in the prevention, diagnosis, alleviation, treatment, or cure of a disease14. They are administered to healthy individuals to prevent diseases caused by infectious agents to which they might be exposed in the future or, in some cases, to which they have been recently exposed. Immunization is the process of presenting antigens to a live host to induce an immune response. Vaccines can be utilized therapeutically and prophylactically. Most vaccines are developed as prophylaxis against infection. A primary goal of prophylaxis is to generate neutralizing antibodies that prevent viral entry into cells. However, in some cases, vaccines can be administered for short-term protection. This short-term protection is termed passive immunization. A smaller proportion of vaccines are developed for the therapeutic treatment of infections that have already become initiated. An example of this is the vaccine used after exposure to rabies15. Epidemiology of Infectious Disease and Vaccination During infectious disease outbreaks, vaccines have the ability to break chains of transmission, drastically slowing the propagation of infection and eventually eradicating the disease. The vast majority of individuals in modernized societies are immunized against a host of pathogens as children. This global public health effort has resulted in the worldwide elimination of smallpox16 and substantial progress toward the elimination of polio17. These accomplishments required concerted international efforts that were sustained over many decades. The epidemiologic dynamics of vaccination, especially in a population with a disease presence (either endemic or epidemic), can be described mathematically. The basic reproduction number (R0) is the intrinsic measure of transmissibility of a pathogen. It describes the mean number of secondary infections resulting from a single infection within a fully susceptible population (Fig. 3). For SARS-CoV-2, R0 has been estimated to be between 2 and 318.Fig. 3: Illustration of the basic reproduction number (R0) values for representative infectious diseases, demonstrating increasing infectivity with increasing values of R0. HIV = human immunodeficiency virus.Once substantial immunity, through either vaccination or natural exposure to the disease, starts to be established in the population, R0 becomes a less accurate measure of transmissibility, and a second number, the effective reproduction number (Re), becomes more representative of transmissibility. Re represents the actual number of transmissions per infection. For an explanation of how these numbers are calculated and their effect on viral epidemiology (Fig. 4), please see Appendix I.Fig. 4: The relationship between the basic reproduction number (R0) and the effective reproduction number (Re) of a pathogen. Re changes based on the percentage of the population that is immune, demonstrated by the different lines on the chart. For a disease with an R0 of 3.5, immunization of 70% of the population will be necessary to bring Re below 1.0. This can be compared with an immunization level of only 50% needed to bring Re below 1.0 for a disease with an R0 of 2.0. If Re, a measure of disease transmissibility, is <1, a disease outbreak will end without further measures being taken.Herd Immunity Herd immunity is a condition achieved in a population when the indirect protection of susceptible individuals against a specific pathogen is conferred by immune individuals. In effect, those with immunity within a population shield those without immunity from transmission, thereby limiting the spread of the disease19 (Fig. 5). A further explanation of how R0 and Re affect herd immunity (Figs. 6 and 7) is found in Appendix II.Fig. 5: Illustration demonstrating herd immunity. The spread of a hypothetical illness within a population is shown under 3 different conditions. The top row is a time early after the introduction of the pathogen within a population. The second row represents 1 replication cycle and the third row represents 2 replication cycles following the time represented in the top row. In the left column, the population is fully susceptible (no naturally acquired immunity or immunity through vaccination) to the pathogen. The middle column represents an intermediate state between full susceptibility and achievement of the herd immunity threshold (Pcrit). The column on the right represents how spread is hindered in a population that has already achieved Pcrit.Fig. 6: The relationship between the basic reproduction number of a virus, R0, and the proportion of the population that needs to be immunized to enter into a state in which herd immunity takes effect, Pcrit. R0 is unitless and Pcrit is measured as the percentage of the population. The slope of the curve is greatest between R0 values of 1 and 4, indicating that a large increase in the proportion of immune individuals within the population is needed to achieve Pcrit for an infectious disease with an R0 that is just slightly larger than another. The estimated R0 for SARS-CoV-2 is between 2 and 3, which is demarcated in the area between the dotted lines on the graph. This R0 range indicates that the percentage of the population that needs to become immune to reach Pcrit, either through infection or vaccination, is between 50% and 66.7%.Fig. 7: Vaccination affects the achievement of herd immunity. This graph shows vaccination affecting herd immunity to diphtheria. Diphtheria-tetanus-pertussis (DTP3) immunization coverage is expressed as a percentage of the population from 1982 to 2016. The red line on this graph represents the number of diphtheria cases reported worldwide by year. The blue line represents the immunization coverage as a percentage of the population by year. The dashed orange line represents the herd immunity threshold (Pcrit) for diphtheria assuming that it has an R0 of 6. The graph demonstrates how achieving immunization levels just slightly above Pcrit can drive the number of cases down to just a few thousand per year.SARS-CoV-2 Vaccines Under Development It is through recombinant nucleic acid technology that most efforts against COVID-19 are progressing. Theoretically, vaccines using recombinant nucleic acid technology can be developed and, therefore, deployed more quickly than those using more traditional means. However, a few are based on immunologic techniques used widely in the mid-twentieth century for the development of vaccines used in standard childhood immunization schedules. Table I summarizes the types of vaccines as well as their advantages and disadvantages. By June 2, 2020, >130 candidate vaccines targeting SARS-CoV-2 were under development worldwide according to the WHO20. Ten of these candidate vaccines have entered into human trials. Table II summarizes the SARS-CoV-2 vaccines currently in development. TABLE I - Advantages and Disadvantages of Different Vaccine Types* Type of Vaccine Example Vaccine(s) Advantages Disadvantages Potential Solutions to Development Problems Attenuated Sabin polio (host-range mutant) Smallpox (Jennerian method) Reliable Confer both humoral and cell-mediated immunity Limited need for booster doses Relatively inexpensive technology May yield less protective mutant viruses if passages are performed in non-human cell lines63 Can cause disease in immunocompromised individuals Enhanced storage and maintenance requirements (e.g., refrigeration, culture media) Inactivated Typhoid Salk polio Rabies Influenza Relatively fast Easy to scale up Relatively inexpensive development Alternate routes of administration (e.g., oral) Inactivation can sometimes damage key epitopes Can still theoretically cause disease (if not fully inactivated) Labor-intensive process to ensure no viable virion remains after inactivation Greater amounts of inoculum required to achieve immunity Immune response is less durable, requiring booster doses64 Titration of inactivation methods to prevent overtreatment, maintaining key epitopes Subunit Hepatitis B surface antigen Safe Easy to use Requires large amounts of isolated antigen Immunopotentiation2,65 Recombinant technology has made large-scale production possible Adjuvants can be added to vaccine preparations66 mRNA29 HIV† Zika† Influenza† Rabies† Can replicate using host machinery Highly potent Relatively fast Easy to scale up • Relative low cost of manufacturing Safe Confer both humoral and cell-mediated immunity dsRNA byproducts of production are PAMPs‡ and lead to immune recognition and degradation29 Inefficient delivery to target cells Instability of mRNA Modified nucleosides avoid host immune recognition Purification to eliminate dsRNA Multiple delivery methods devised to improve efficiency Inclusion of upstream and downstream untranslated sequences improves stability of mRNA DNA plasmid Prostate cancer† Melanoma† Can replicate using host machinery Highly potent Relatively fast Easy to scale up • Safe Confer both humoral and cell-mediated immunity Inefficient delivery to target cells Coupled treatments (e.g., electroporation) to improve DNA plasmid entry into human cells34 and enhance immune response32–33- Vector-based recombinant (e.g., adenovirus vector) Malaria† HIV† Ebola (in development) Confer both humoral and cell-mediated immunity Enter host cells via ubiquitously expressed cell surface receptors Can incorporate additional transgenes for biological adjuvants37,67 Highly potent Relatively fast Easy to scale up in low-resource settings68 Safe Easier, alternate routes of administration favoring IgA production (e.g., oral, intranasal)69,70 Vector immunity due to host recognition of adenovirus71 If replication-competent viral vectors are utilized, could cause adenovirus infection Administration via mucosa may require dose escalation Repeated immunizations can diminish neutralizing antibodies to adenovirus vectors, thereby decreasing vector immunity71 Utilize different adenovirus strains to evade vector immunity70,72,73 Utilize chimpanzee adenovirus vectors to evade vector immunity due to highly conserved genome74 Use of replication-competent viral vectors can boost immunogenicity *HIV = human immunodeficiency virus, dsRNA = double-stranded RNA, PAMP = pathogen-associated molecular pattern, and IgA = immunoglobulin A.†In trials.‡These are present and invariant in the pathogen but not in the potential host organism; PAMPs allow early recognition and immune activation by the host organism75. TABLE II - SARS-CoV-2 Candidate Vaccines Currently in Clinical Development* Vaccine Type Clinical Phase of Development Trial No. (Location) Chimpanzee adenovirus vector-based (nonreplicating) Phase 2b/3 2020-001228-32 (EU) mRNA (lipid nanoparticle encapsulated) Phase 2: scheduled to conclude September 2021 NCT04405076 (USA) Adenovirus-5 vector-based (nonreplicating) Phase 2: scheduled to conclude January 2021 ChiCTR2000031781 (China) Subunit (recombinant spike protein) Phase 1 and 2: scheduled to conclude July 2021 NCT04368988 (USA) mRNA (lipid nanoparticle encapsulated) Phase 1 and 2: scheduled to conclude June 2021 NCT04368728 (USA), 2020-001038-36 (EU) Inactivated with aluminum adjuvant Phase 1 and 2: scheduled to conclude July/August 2020 NCT04383574 (USA), NCT04352608 (USA) Inactivated Phase 1 and 2: scheduled to conclude November 2021 ChiCTR2000031809 (China) Inactivated Phase 1 and 2: scheduled to conclude November 2021 ChiCTR2000032459 (China) DNA plasmid (with electroporation) Phase 1: scheduled to conclude July 2021 NCT04336410 (USA) Inactivated Phase 1 China *As of June 2, 2020. Attenuated Vaccines Vaccines that use a weakened but viable form of the pathogen to establish an immune response are termed attenuated vaccines. The establishment of attenuated strains is an empiric rather than a designed process, the duration of which is influenced by the scale of the process and the time necessary to complete the passage of the virus in the cell culture. In rare cases, the attenuated variant can revert to a virulent form, resulting in the establishment of the disease in the immunized cohort. There are 4 different types of attenuated vaccines. At least 3 different groups are seeking to develop attenuated vaccines against COVID-1920. The most common form of an attenuated vaccine is the host range mutant. This type is usually achieved by the passage of the virus in the cell culture to generate nonvirulent mutants. Over multiple passages, the host range mutant acquires mutations, rendering it harmless while retaining the necessary antigens to generate immunity. The second type is naturally attenuated. This method requires empiric identification and recovery of variant or minimally variant strains during periods of outbreak21. The third type of attenuated vaccine is the temperature-sensitive mutant. This method isolates mutants that at of strains that can be at is the found at the respiratory but become nonvirulent at the or The type of attenuated termed is not being in the COVID-19 vaccine development Inactivated Vaccine vaccine requires large amounts of viral to be in and by either or (Fig. The of inactivation is to the virus nonvirulent without the epitopes to which an immune response is and can virus A needs to be achieved in the of vaccines. will lead to and of key to a neutralizing antibody response that is and protective with a live However, if complete inactivation not of the disease can from the vaccination through some vaccine types that are being developed against SARS-CoV-2. Inactivated viral are in and through or means. The of viral allows the of an immune response without of infection. Subunit viral proteins are and These proteins are into patients to be The proteins are by the host cells and the antigens are that an antibody response is mRNA mRNA the viral S protein is in the and is using technology as These mRNA are and are into viral proteins using host and the antigens are that an antibody response is DNA plasmid DNA for the viral S protein is into a DNA This plasmid enters host and machinery viral proteins. These viral proteins generate an antibody response. vector-based the gene for the viral S protein is into an adenovirus genome in These adenovirus vectors with entry into the host cell and the host machinery to viral proteins that generate an antibody Vaccines Subunit vaccines generate immunity by administration of a virus antigen (Fig. These antigens are by the immune to generate an antibody response. Typically, antigens are that will generate an immune response. For SARS-CoV-2, one of the candidate antigens for vaccine development is the spike A number of the of and are on developing spike protein mRNA Vaccines In this mRNA for the SARS-CoV-2 spike protein is into the host cells to it (Fig. proteins made by host cells will be as by the immune which will generate both an antibody and the response to mRNA vaccines have had that effective but have of these mRNA vaccine delivery and the of the and methods are standard at this 1 developing an mRNA vaccine against SARS-CoV-2 has that it could be as an measure to as early as DNA Vaccine the of the of SARS-CoV-2, the development of a DNA vaccine the (+)ssRNA genome of SARS-CoV-2, DNA sequences can be These sequences can be into or double-stranded DNA that for antigen DNA (Fig. can replicate using host is into host it can viral antigens that are as the host immune to generate an antibody and the response. This method of vaccine development advantages similar to those for mRNA vaccines nucleic are that the potential with protein which can recombinant multiple have demonstrated that can enter host cells and can in a immune This technology has been shown to be and in to there have been no DNA vaccines for use in Recombinant Vaccines Recombinant viral vectors have been as vaccine over the few for their ability to antigens and their immunogenicity (Fig. are DNA of recombinant adenovirus vector-based vaccines is that they can be made and highly effective through recombinant The of recombinant adenovirus vector-based vaccines is by rendering the virion replication This is achieved by the antigen in the of the adenovirus genome for viral replication, thereby its ability to most vaccines are administered either or adenovirus vector-based vaccines provide the possibility of This is a more administration than For these many different recombinant adenovirus vector-based vaccines targeting SARS-CoV-2 are currently under development. Immunization immunization may be achieved by administration of to a or recently infected protection against pathogens within the immunized The protective of the are eventually down or this the protective effect is because not or the immune to provide immunization. There are with and patients develop the at a of infection. Currently, there are a number of for the treatment of patients with Immunity to COVID-19 There and should a to develop a vaccine against SARS-CoV-2. health measures will need to the development, and administration of a vaccine against SARS-CoV-2 to a large proportion of the population. with these morbidity and mortality will The potential rate increase from to between and of the population while the the herd immunity threshold (Pcrit) may be to A number of potential immunity, immunity, and vaccine could the of a COVID-19 vaccine TABLE - and the of COVID-19 Vaccine Immunization on COVID-19 Vaccine Immunization immunity are of Immunity in individuals may require vaccination individuals may not be fully immune Vaccine is only effective May require vaccination or vaccination of more individuals than Pcrit Vaccine or use of vaccines to achieve Pcrit for development for vaccine development and is 4 the development can only if necessary the first Vaccine takes to develop similar efforts have of vaccine development, we still not have a vaccine Vaccine takes to develop molecular techniques more effective of recombinant Development Vaccines or manufacturing complete of vaccine development to from and Over worldwide on vaccine Development Vaccine to by the is especially A by the for that of not and were if they when a vaccine becomes A of found that were at and an additional were in a COVID-19 is an that could Pcrit or lead to the of COVID-19 The COVID-19 vaccine will not be the This was with polio and both of which through in the and types that were used when or were in the early The vaccines which groups to they are administered and toward and their delivery methods will with limiting vaccine is immunity. illness are of the This that SARS-CoV-2 may exposed individuals. may be due to either antibody and cell or The need for vaccination could the establishment of Pcrit. possibility is that only some individuals have full immunity. immunity is still it the numbers of infected individuals and those requiring care on the level of an care The of developed vaccines will most be by of blocking Vaccines that have entered into have already been for blocking antibodies in their The administration of different vaccines has been in achieving immunity. attenuated and polio vaccines administered have a for viruses with substantial as immunizations help to neutralizing antibody for conserved There is a complex of and on immune In antibody to vaccines are in while both humoral immune to novel that are related to multiple affect the time for immunity to a is 2 to 3 following the Problems that are of in developing a vaccine to RNA viruses are the of and is the of the viral disease rather than its This has led to and in immunized has been with a number of those against is the of infection. of infection when viruses are by cells via their or This has been shown to in human cells with It is not that vaccine administration will have a musculoskeletal However, the time to testing, and will have for the and of care It is standard methods or will be during the production and of viable as many of the currently being used have resulted in doses at that multiple vaccines and their will be to more and effective and administration to the Immunization with candidate vaccines should not lead to should be to allow the establishment of an immune response and of severe morbidity and mortality associated with The for the development of effective vaccines should be with the that the to achieve will be There will be many and we must to this The protective measures that we have social protective and the use of testing, will be a of COVID-19 has morbidity and Pcrit will be in to a of social Pcrit will be through immunization. an early and response toward developing a COVID-19 remain in Recombinant techniques may this A between the community and should the need for vaccine development with Appendix by the is with the of this article as a at

Facing the challenge of viral mutations in the age of pandemic: Developing highly potent, broad-spectrum, and safe COVID-19 vaccines and therapeutics.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome (MERS-CoV), which cause acute respiratory distress syndrome and case fatalities. COVID-19 disease severity is worse in older obese patients with comorbidities such as diabetes, hypertension, cardiovascular disease, and chronic lung disease. Cell binding and entry of betacoronaviruses is via their surface spike glycoprotein; SARS-CoV binds to the metalloprotease angiotensin-converting enzyme 2 (ACE2), MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4), and recent modeling of the structure of SARS-CoV-2 spike glycoprotein predicts that it can interact with human DPP4 in addition to ACE2. DPP4 is a ubiquitous membrane-bound aminopeptidase that circulates in plasma; it is multifunctional with roles in nutrition, metabolism, and immune and endocrine systems. DPP4 activity differentially regulates glucose homeostasis and inflammation via its enzymatic activity and nonenzymatic immunomodulatory effects. The importance of DPP4 for the medical community has been highlighted by the approval of DPP4 inhibitors, or gliptins, for the treatment of type 2 diabetes mellitus. This review discusses the dysregulation of DPP4 in COVID-19 comorbid conditions; DPP4 activity is higher in older individuals and increased plasma DPP4 is a predictor of the onset of metabolic syndrome. DPP4 upregulation may be a determinant of COVID-19 disease severity, which creates interest regarding the use of gliptins in management of COVID-19. Also, knowledge of the chemistry and biology of DPP4 could be utilized to develop novel therapies to block viral entry of some betacoronaviruses, potentially including SARS-CoV-2.

Tackling COVID19 by Exploiting Pre-existing Cross-Reacting Spike-Specific Immunity

Coronavirus disease 2019 (COVID-19) has had a major effect on kidney and other solid organ transplant recipients.1 In addition to public health measures, improved access to testing, and therapeutic developments, vaccination has emerged as a key tool for controlling the ongoing pandemic. In December 2020, multiple regulatory agencies worldwide authorized the use of two mRNA vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and several other vaccine platforms are in advanced-stage clinical trials.2,3 Individuals who have received a transplanted kidney or other solid organ have been identified as high-risk populations and prioritized for vaccination in public health guidelines, but unfortunately have been excluded from major SARS-CoV-2 vaccine clinical trials.1,2 Thus, studies are urgently needed to characterize the safety, immunogenicity, and ultimately, efficacy of SARS-CoV-2 vaccines for such patients. Below, we provide an overview of SARS-CoV-2 vaccines and highlight key concepts that should be considered in evaluating their safety in solid organ transplant recipients. Despite the theoretical concerns described below, we emphasize that available evidence from studies demonstrates safety and efficacy in the general population. Because of the known substantial risks of COVID-19–associated morbidity and mortality in recipients of kidney and other solid organs, and the long track record of safety of other vaccinations in such recipients, we anticipate the benefits of selected SARS-CoV-2 vaccines will far outweigh risks of vaccination. Accordingly, current guidance from multiple professional organizations recommend vaccination for all eligible organ transplant recipients.2,4,5 Each vaccine platform has distinct safety considerations for kidney transplant recipients. Live (replication-competent) vaccines are generally contraindicated in immunocompromised individuals because of a risk of vaccine-acquired disease.6 The SARS-CoV-2 candidate vaccines that are furthest along in development do not contain replication-competent SARS-CoV-2 virus, and therefore do not carry risk of SARS-CoV-2 infection (Table 1). Table 1. - Major SARS-CoV-2 platforms in developmenta Vaccine Platform Vaccine Name (Manufacturer) Vehicle Phase of Development Adjuvant Safety and Efficacy in the General Population Specific Considerations for Kidney Transplant Recipients mRNA BNTb162b2 (Pfizer/BioNTech) mRNA encapsulated in lipid nanoparticles Authorized for emergency use in the United States and other countries Unadjuvanted, but lipid nanoparticles possess natural adjuvant activity7 95% efficacy in phase 3 trials.1 Anaphylaxis has been reported. Avoid in patients with a known allergy to a vaccine component (e.g., polyethylene glycol). Close monitoring after administration for patients with a history of anaphylaxis to any food or drug.3 Does not contain live virus. No evidence of vaccine-induced off-target immune responses in large phase 3 clinical trials.2,3 mRNA-1273 (Moderna) Replication-defective viral vectors AZD122 (Oxford/AstraZeneca) Human-chimpanzee adenovirus (ChAdOx1) Phase 3 Unadjuvanted 70%–90% efficacy depending on dose in phase 3 trials.8 Transverse myelitis reported.8 Removal of genes necessary for replication reduces risk of vaccine-associated AdV disease.9 Theoretical risk of emergence of new AdV type with replicative potential through homologous recombination, although this has never been demonstrated to occur with AdV-vectored vaccines.9 JNJ78436735/Ad26.COV2.S (Janssen) Human adenovirus (Ad26) Phase 3 Unadjuvanted Unknown Convidecia (Ad5-nCov) Human adenovirus (Ad5) Approved for limited use in China Unadjuvanted Unknown Sputnik V (Gamaleya) Human adenovirus (Ad5 and Ad26 in consecutive doses) Early use in Russia, Belarus, and Argentina Unadjuvanted Unknown Protein subunit NVX-CoV2373 (Novavax) Recombinant spike glycoprotein Phase 3 Matrix-M1 system plus an additional, unnamed adjuvant Unknown Does not contain live virus. Matrix-M1 contains the same QS21 saponin as the AS01B adjuvant system contained in the recombinant varicella zoster vaccine.7 SARS-CoV-2 recombinant protein formulation (GSK/Sanofi) Recombinant spike protein Phase 2 AS03 adjuvant Unknown High incidence of anti-HLA antibodies in KTR vaccinated with AS03-adjuvanted influenza vaccines, but no association between AS03 exposure and rejection.3,10 EpiVacCorona (Vector Institute) Peptide epitope Early use in Russia Unknown Limited data available Whole-inactivated (killed) BBIBP-CoV (Sinopharm) CoronaVac (SinoVac) Whole-inactivated SARS-CoV-2 viral particles Limited use in China and other countries Unknown Unknown Does not contain live virus. Limited data available in peer-reviewed literature. GSK, GlaskoSmithKline; KTR, kidney transplant recipients.aDoes not include all candidate vaccines or platforms under investigation; limited to platforms in advanced stages of clinical development or authorized for use as of December 31, 2020. However, viral vector–based vaccines that incorporate viruses other than SARS-CoV-2 are in advanced-phase studies, including adenovirus (AdV) vector–based vaccines that have been licensed in Europe. These vaccines consist of intact virions that are engineered to include the gene encoding the SARS-CoV-2 spike protein, a technique that leverages the viral vector's ability to efficiently infect cells and enhances spike gene delivery. Vaccines that use viral vectors contain either replication-deficient or replication-competent viruses (Table 1). The majority of viral-vectored vaccines in the most advanced phases of development have been rendered replication-deficient through deletion of genes essential for replication.8 By limiting vector replication, the potential for vaccine-associated AdV disease is greatly diminished. There are, however, theoretical mechanisms by which replication-deficient viral vector–based vaccines could become replication competent and cause disease, especially in immunocompromised individuals. For example, in cells concurrently infected with two different AdVes, homologous recombination of genetic elements could occur and result in the emergence of new, pathogenic, replication-competent AdV types.9 This has been observed in patients with advanced HIV disease during natural AdV infections, and is theoretically possible with AdV vector–based vaccines in patients who are immunocompromised with a concurrent wild-type AdV infection.9 Although infrequent, severe AdV infections, including allograft nephritis, can occur in kidney transplant recipients during natural infection.9 Notably, vaccine-associated AdV disease has not been reported, albeit there is little experience in immunocompromised populations. It should be emphasized that, despite the theoretical concerns with replication-deficient viral vector–based vaccines, immunosuppression is not considered a contraindication to their use.11 Replication-competent viral-vectored vaccines carry a greater risk of vaccine-derived vector infection in patients who are immunocompromised and should only be administered under carefully controlled circumstances (specifically, clinical trials). Other vaccine candidates that are in advanced stages of development, including mRNA, protein subunit, or whole virus–inactivated SARS-CoV-2 vaccines, do not contain intact virus and thus do not carry a risk of vaccine-associated infection.3 Induction of generalized systemic inflammation by either the vaccine antigen or an associated adjuvant, or by more specific cellular and humoral crossreactivity between vaccine epitopes and allograft antigens, theoretically could promote undesired allograft-directed immune responses. AdV vectors elicit potent innate immune responses through complement activation and induce a diverse cytokine repertoire.10 Although this phenomenon is most prominent at the site of AdV-vector inoculation, systemic inflammation could promote vaccine-associated allograft rejection. Concern for autoimmunity related to the SARS-CoV-2 vaccine on the basis of a modified chimpanzee AdV vector (ChAdOx1) arose after two vaccine recipients developed transverse myelitis, although the possibility of an unrecognized preexisting demyelinating condition has raised questions about the significance of one of these events.8In vitro reactivity between spike protein antibodies and human collagen has been demonstrated, but molecular mimicry has not been identified as a primary mechanism of kidney injury in COVID-19.12 Available data suggest acute allograft rejection is uncommon during COVID-19, despite frequent reduction in immunosuppression as a therapeutic strategy.1 In the absence of an observed association between natural SARS-CoV-2 infection and acute allograft rejection in kidney transplant recipients, it is unlikely that vaccine antigens would precipitate clinically significant immune responses to renal allografts. In general, adjuvants used to enhance vaccine immunogenicity also elicit nonspecific inflammatory responses, and thus have the potential to induce acute allograft rejection. Concern about adjuvant safety in organ transplant recipients arose from observations of an unusually high incidence of anti-HLA antibodies in kidney transplant recipients who received the 2009 influenza A(H1Na1)pdm09 vaccine, which contained the squalene-based AS03 adjuvant system.6,7 However, only a fraction of these anti-HLA antibodies were donor specific, and a subsequent investigation of >10,000 solid organ transplant recipients found no definitive association between the AS03 adjuvant system and acute allograft rejection.6 The AS01B adjuvant used in the recombinant varicella zoster virus vaccine contains a combination of monophosphoryl lipids and QS21, a saponin.13 This adjuvant induces a potent innate immune response and associated concerns for precipitating acute allograft rejection in kidney transplant recipients. Several recombinant spike protein SARS-CoV-2 vaccines contain adjuvants, such as AS03 and the novel Matrix M1 adjuvant, which contains the same QS21 saponin found in the recombinant varicella zoster vaccine.3 Viral-vectored and mRNA vaccines do not generally contain adjuvants, although lipid nanoparticle delivery devices used in the mRNA vaccines have natural adjuvant activity.13 Postmarketing surveillance will be essential to assess any potential association between SARS-CoV-2 vaccine components and acute allograft rejection. In the interim, theoretical concerns associated with any vaccine must be weighed against the benefits of preventing or mitigating the severity of a life-threatening infection in a vulnerable population. We emphasize that a broad range of vaccines have a substantial track record of safety in kidney and other solid organ transplant recipients. Furthermore, no definitive association between any vaccine or adjuvant and allograft rejection has been identified to date.2,6 Immunosuppression in kidney transplant recipients is anticipated to reduce the immunogenicity of SARS-CoV-2 vaccines, and immunogenicity may vary by vaccine platform. Available data across a broad range of vaccines in solid organ transplant recipients suggest they have relative humoral response rates that are approximately 50%–70% of those seen in nontransplant populations.6,7 Patients with ESRD may have more a robust response to vaccines before rather than after kidney transplant,6 and when possible, SARS-CoV-2 vaccines should be given before transplantation.2 In the post-transplant setting, age >65 years, more recent transplantation, use of mycophenolate and mammalian target of rapamycin inhibitors, and lower graft function are associated with decreased serologic responses to influenza vaccines.6,7 Despite the effects of lymphocyte-depleting immunosuppression in the early period after transplant or treatment for rejection, the benefits of even modest SARS-CoV-2 protection might outweigh the risk of delaying immunization during a pandemic.2 In general, vaccines are not recommended immediately post-transplant due to a presumed decrease in immunogenicity after recent high-level immunosuppression. Expert opinion advises that delaying SARS-CoV-2 vaccination of vaccine-naive transplant recipients until 3 months after transplant or receipt of T cell or B cell ablative therapies may be appropriate; for patients who received a first dose before transplant, administration of the second dose should be delayed until at least 4 weeks post-transplant.2 Higher doses, booster doses, adjuvants, and intradermal delivery have all been used with variable success to improve immunogenicity of commonly administered vaccines in solid organ transplant recipients.6,7 If immunogenicity of standard regimens in kidney transplant recipients is suboptimal, these alternative approaches should be considered. The diversity of vaccine platforms and phased vaccine allocation present unique challenges for assessing the safety, immunogenicity, and efficacy of SARS-CoV-2 vaccination in kidney transplant recipients. Surveillance for adverse events related to each specific formulation through prospective multicenter registries of vaccinated solid organ transplant recipients is one potential approach to assessing safety, especially given that large population-specific studies of kidney transplant recipients may not be feasible. Prospective clinical trials should directly compare different SARS-CoV-2 vaccine platforms, assess the magnitude and durability of humoral and cellular responses, and utilize the same functional laboratory immunogenicity assays as the vaccine trials to facilitate direct comparisons between transplant recipients and general populations. It is hoped these investigations will identify relevant differences among the various vaccine platforms to guide future studies of alternative vaccination strategies, if warranted. Because immunosuppression may increase SARS-CoV-2 viral load and prolong the duration of SARS-CoV-2 viral shedding and transmissibility, studies to monitor both symptomatic and asymptomatic infection in vaccinated kidney transplant recipients—with quantitation of viral loads, viral culture, or both—would complement studies of safety and immunogenicity. Large prospective studies of vaccine efficacy in kidney transplant recipients that include a placebo arm are likely not feasible and may not be ethically appropriate, depending on the final results of ongoing phase 3 studies. Case-control trials of kidney transplant recipients with COVID-19 that examine the effect of prior vaccination on disease severity, viral load, and duration of viral persistence, although less definitive, may offer a more practical approach. SARS-CoV-2 vaccines have significant potential to reduce COVID-19–associated morbidity and mortality among recipients of solid organ transplants, including kidney transplants. Because transplant recipients' responses to vaccines may be suboptimal, continued emphasis on nonvaccine preventive measures—use of face covers, hand hygiene, and physical distancing—will be needed, even after vaccination.2 Although the vaccines' ability to disrupt viral transmission in either immunocompetent or immunocompromised individuals is not yet established, vaccination of caregivers and close contacts of kidney transplant recipients, recommended for influenza vaccination, would be an important strategy to reduce the risk of infection.6 Assessment of vaccine efficacy against emerging SARS-CoV-2 variants is necessary to establish optimal vaccine strategies for both immunocompetent and immunocompromised populations. Future evaluations of SARS-CoV-2 vaccine platforms in kidney transplant recipients are imperative to confirm safety and immunogenicity, but the expectation is that SARS-CoV-2 vaccines will add to the armamentarium of vaccines that have safely protected transplant recipients from serious infectious diseases for decades. Disclosures A. Limaye reports having consultancy agreements with AlloVir, Amplyx, GSK, Merck, NovaNordisk, Novartis, and Sana Biotech and being a scientific advisor or member with NobelPharma and Novartis. M.R. Heldman reports receiving speaking honoraria from CignaLife Source, outside the scope of the submitted work. Funding This work was supported by the National Institute of Allergy and Infectious Diseases (T32AI118690 to M.R. Heldman). The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Mechanic Forces Promote Brain Endothelial Activation by SARS-CoV-2 Spike Protein.