COVID-19 vaccine development is getting closer to success in India

In new delhi, Scientists have identified regions of the SARS-CoV-2 virus that causes COVID-19 to target with a vaccine, by harnessing tools used for the development of cancer immunotherapies. The researchers at Children's Hospital of Philadelphia (CHOP) in the US employed the same approach used to elicit an immune response against cancer cells to stimulate an immune response against the novel coronavirus.

Using this strategy, the researchers believe a resulting vaccine would provide protection across the human population and drive a long-term immune response.

"In many ways, cancer behaves like a virus, so our team decided to use the tools we developed to identify unique aspects of childhood cancers that can be targeted with immunotherapies and apply those same tools to identify the right protein sequences to target in SARS-CoV-2," said John MMaris, a pediatric oncologist at CHOP, and a professor at the University of Pennsylvania.

"We think our approach provides a roadmap for a vaccine that would be both safe and effective and could be produced at scale," said Maris, senior author of the research published in the journal Cell ReportsMedicine.

The COVID-19 pandemic has led to an urgent need for the development of a safe and effective vaccine against SARS-CoV-2, the virus that causes the COVID-19 disease, the researchers said.

An optimally designed vaccine maximises a long-lasting immune response, while minimising adverse reactions, autoimmunity, or disease exacerbation, they said. To increase the likelihood that a vaccine is both safe and effective, the research team prioritised parameters in identifying regions of the virus to target.

The researchers looked for regions that would stimulate a memory T-cell response that, when paired with the right B cells, would drive memory B cell formation and provide lasting immunity and do so across the majority of human genomes.

They targeted regions of SARS-CoV-2 that are present across multiple related coronaviruses, as well as new mutations that increase infectivity, while also ensuring that those regions were as dissimilar as possible from sequences naturally occurring in humans to maximize safety.

The researchers propose a list of 65 peptide sequences that, when targeted, offer the greatest probability of providing population scale immunity.

The team will now test various combinations of a dozen or so of these sequences in mouse models to assess their safety and effectiveness.

Note: The above news is confirmed by newspaper, Economic Times, India.

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Coronavirus : Triggers For Membrane Fusion.

Triggers for Membrane Fusion by Coronavirus Spike Proteins:

The triggers for coronavirus spikes to undergo conformational transitions demonstrate a more complex pattern than many other class I membrane fusion proteins, probably a reflection of their unique structural features discussed above. For example, although influenza virus HA is primed by proteolysis during virus packaging, many coronavirus spikes are not. Instead, coronavirus spikes are often subjected to proteolysis later in the cell entry process, sometimes after receptor binding. Thus, proteolysis of coronavirus spikes can lead directly to membrane fusion and thereby serves as an essential trigger for membrane fusion. The host proteases that cleave coronavirus spikes mainly come from four different stages of the virus infection cycle: (a) proprotein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d) lysosomal proteases (e.g., cathepsin L and cathepsin B) after virus endocytosis in virus-targeting cells. In addition to proteolysis, traditional triggers such as receptor binding and low pH may also play a role in membrane fusion. Here, I discuss detailed triggers for membrane fusion by the spikes from three representative coronaviruses.

As the prototypic coronavirus, MHV has been extensively examined for its cell entry mechanism. The findings are complicated and in some cases contradictory. First, MHV spike is cleaved by proprotein convertases during virus packaging in the virus-producing cells . This proteolysis is critical for MHV entry into virus-targeting cells. Second, the binding of CEACAM1 triggers the conformational transition of MHV spike and hence membrane fusion. This is supported by the observations that incubation of MHV spike with recombinant soluble CEACAM1 led to enhanced hydrophobicity of MHV S2 and the appearance of a protease-resistant S2 fragment. These observations indicated the exposure of fusion peptides and formation of the six-helix bundle, respectively, in postfusion S2. Third, there have been contradictory reports about whether MHV enters target cells at the cell membrane or through endocytosis and whether MHV spike undergoes conformational transitions at low, neutral, or even elevated pH. This may depend on the MHV strains examined or experimental approaches used in the studies. The roles of pH and endocytosis in MHV entry still need to be further clarified. Interestingly, the neurotropic strain MHV-JHM can mediate virus entry into host cells that do not express CEACAM1. This receptor-independent entry by MHV-JHM is unique among viruses. Biochemical characterization of MHV-JHM spike suggested that it is more labile than the spikes from other MHV strains, meaning that it undergoes conformational transitions spontaneously in the absence of the receptor. It is believed that MHV-JHM is able to infect neural cells where CEACAM1 expression level is very low, at least in part because its spike can mediate receptor-independent entry. Taken together, the membrane fusion mechanism of MHV spike depends on both proteolysis and receptor binding, and it may or may not depend on the low pH of endosomes; in addition, receptor-independent membrane fusion by MHV-JHM spike contributes to the neutral tropism of MHV-JHM.

Research on the cell entry mechanism of SARS-CoV has led to novel findings. First, SARS-CoV spike is not cleaved by proprotein convertases during virus packaging and hence remains intact on mature virions. Instead, SARS-CoV enters host cells through endocytosis, and its spike is processed by lysosomal proteases (e.g., cathepsin L and cathepsin B). This is supported by the observation that inhibitors against either endosomal acidification or lysosomal cysteine proteases block SARS-CoV entry. However, low pH itself is not a trigger for SARS-CoV entry. This is supported by the observation that when expressed on the cell surface and cleaved by exogenous proteases, SARS-CoV spike can mediate cell-cell fusion with ACE2-expressing cells at neutral pH. Thus, the role of low pH in SARS-CoV entry is to activate lysosomal proteases, which further activate SARS-CoV spike for membrane fusion. This is different from influenza virus HA, which is activated through binding protons in the low-pH environment of endosomes. Second, in addition to lysosomal proteases, both extracellular proteases (e.g., elastases in the respiratory tract) and cell surface proteases (e.g., TMPRSS2 on the surface of lung cells) also activate SARS-CoV spike for membrane fusion. Due to their cell and tissue specificities, these proteases likely contribute to the respiratory tract and lung tropism of SARS-CoV. Third, in addition to the cleavage site at the S1/S2 boundary, a second site, S2′, has been identified at the N terminus of the internal fusion peptide within S2 . Whereas the cleavage at the S1/S2 boundary removes the structural constraint of S1 on S2, the cleavage at the S2′ site releases the internal fusion peptide for insertion into target membranes. Fourth, it is not clear whether the binding of receptor ACE2 is a trigger for SARS-CoV spike to fuse membranes. Two electron microscopy studies observed no or moderate conformational changes of SARS-CoV spike associated with ACE2 binding . However, other studies suggested that ACE2 binding triggers a conformational change in SARS-CoV spike, which exposes previously cryptic protease sites for cleavage. The role of ACE2 binding in triggering membrane fusion waits to be further investigated. Nevertheless, SARS-CoV entry does not depend on low pH, but it requires at least two protease cleavages in the spike by lysosomal proteases, extracellular proteases, or cell surface proteases.

The overall cell entry mechanism of MERS-CoV is similar to that of SARS-CoV. Like SARS-CoV spike, MERS-CoV spike must be cleaved at both the S1/S2 boundary and the S2′ site for membrane fusion to occur . MERS-CoV also enters host cells through endocytosis and is activated by lysosomal cysteine proteases for membrane fusion . Moreover, extracellular proteases and cell surface proteases help activate MERS-CoV entry. Unlike SARS-CoV, MERS-CoV spike is cleaved by host proprotein convertases during virus packaging. Interestingly, despite recognizing the same receptor DPP4, MERS-CoV and HKU4 spikes differ in their activities to mediate virus entry: HKU4 spike mediates virus entry into bat cells but not human cells, whereas MERS-CoV spike mediates virus entry into both bat and human cells. Two residue differences have been identified between MERS-CoV and HKU4 spikes that account for this functional difference ; they allow MERS-CoV spike, but not HKU4 spike, to be activated by human proprotein convertases and lysosomal cysteine proteases. Thus, the corresponding two mutations played a critical role in the transmission of MERS-CoV from its likely natural reservoir, bats, to humans, either directly or through intermediate host camels. On the other hand, HKU4 spike can be activated by bat lysosomal proteases but not human lysosomal proteases, suggesting that human and bat lysosomal proteases process viral spikes differently. These studies on MERS-CoV entry reveal that different activities of spike-processing proteases from different hosts can pose a barrier for cross-species transmissions of viruses.

In sum, proteolysis has been established as an essential trigger for coronavirus spikes to fuse membranes, as cleavages at the S1/S2 boundary and S2′ site can remove the structural constraint of S1 on S2 and release the internal fusion peptide, respectively. Among the host proteases, lysosomal proteases provide the most reliable source for spike processing because they are ubiquitous and abundant in many cell types. The availability of some other proteases (e.g., proprotein convertases, extracellular proteases, and cell surface proteases) depends on the types of cells and tissues, regulating tissue tropisms of coronaviruses. Moreover, protease activities from different host species may vary, regulating host ranges of coronaviruses. Some other triggers for coronavirus entry (e.g., receptor binding and low pH) may depend on specific coronaviruses or different strains of the same coronavirus. The overall goal of these triggers is to overcome the energy barrier for the conformational transition of coronavirus spikes.

Acknowledgement:

This work is supported by Nation Institutes of Health.

Structure, Function And Evolution of Corinavirus.

jump to introduction

Introduction:

The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells.

Coronaviruses belong to the family Coronaviridae in the order Nidovirales. They can be classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Among them, alpha-and betacoronaviruses infect mammals, gammacoronaviruses infect avian species, and deltacoronaviruses infect both mammalian and avian species.

Representative alphacoronaviruses include human coronavirus NL63 (HCoV-NL63), porcine transmissible gastroenteritis coronavirus (TGEV), PEDV, and porcine respiratory coronavirus (PRCV). Representative betacoronaviruses include SARS-CoV, MERS-CoV, bat coronavirus HKU4, mouse hepatitis coronavirus (MHV), bovine coronavirus (BCoV), and human coronavirus OC43. Representative gamma-and deltacoronaviruses include avian infectious bronchitis coronavirus (IBV) and porcine deltacoronavirus (PdCV), respectively. Coronaviruses are large, enveloped, positive-stranded RNA viruses. They have the largest genome among all RNA viruses, typically ranging from 27 to 32 kb. The genome is packed inside a helical capsid formed by the nucleocapsid protein (N) and further surrounded by an envelope. Associated with the viral envelope are at least three structural proteins: The membrane protein (M) and the envelope protein (E) are involved in virus assembly, whereas the spike protein (S) mediates virus entry into host cells. Some coronaviruses also encode an envelope-associated hemagglutinin-esterase protein (HE). Among these structural proteins, the spike forms large protrusions from the virus surface, giving coronaviruses the appearance of having crowns (hence their name; corona in Latin means crown). In addition to mediating virus entry, the spike is a critical determinant of viral host range and tissue tropism and a major inducer of host immune responses.

The coronavirus spike contains three segments: a large ectodomain, a single-pass transmembrane anchor, and a short intracellular tail. The ectodomain consists of a receptor-binding subunit S1 and a membrane-fusion subunit S2. Electron microscopy studies revealed that the spike is a clove-shaped trimer with three S1 heads and a trimeric S2 stalk. During virus entry, S1 binds to a receptor on the host cell surface for viral attachment, and S2 fuses the host and viral membranes, allowing viral genomes to enter host cells. Receptor binding and membrane fusion are the initial and critical steps in the coronavirus infection cycle; they also serve as primary targets for human inventions. In this article, I review the structure and function of coronavirus spikes and discuss their evolution.

Receptors Recognized by Corinavirus Proteins:

Coronaviruses demonstrate a complex pattern for receptor recognition. For example, the alphacoronavirus HCoV-NL63 and the betacoronavirus SARS-CoV both recognize a zinc peptidase angiotensin-converting enzyme 2 (ACE2). Moreover, HCoV-NL63 and other alphacoronaviruses recognize different receptors: other alphacoronaviruses such as TGEV, PEDV, and PRCV recognize another zinc peptidase, aminopeptidase N (APN). Similarly, SARS-CoV and other betacoronaviruses recognize different receptors: MERS-CoV and HKU4 recognize a serine peptidase, dipeptidyl peptidase 4 (DPP4) ; MHV recognizes a cell adhesion molecule, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) ; BCoV and OC43 recognize sugar. The alphacoronavirus and PEDV and the gammacoronavirus IBV also use sugar as receptors or coreceptors. Other than their role in viral attachment, these coronavirus receptors have their own physiological functions. The diversity of receptor usage is an outstanding feature of coronaviruses. To further compound the complexity of the issue, the S1 subunits from different genera share little sequence similarity, whereas those from the same genus have significant sequence similarity. Therefore, the following questions have been raised regarding receptor recognition by coronaviruses:

  (a) How do coronaviruses from different genera recognize the same receptor protein? 

  (b) How do coronaviruses from the same genus recognize different receptor proteins?

  (c) What is the molecular basis for coronavirus spikes to recognize sugar receptors and             function as viral lectins?

Two major domains in coronavirus S1, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), have been identified. One or both of these S1 domains potentially bind receptors and function as the receptor-binding domain (RBD). S1-NTDs are responsible for binding sugar , with the only known exception being betacoronavirus MHV S1-NTD that recognizes a protein receptor CEACAM1. S1-CTDs are responsible for recognizing protein receptors ACE2, APN, and DPP4. Crystal structures have been determined for a number of S1 domains complexed with their respective receptor . These structures, along with functional studies, have addressed many of the puzzles surrounding receptor recognition by coronaviruses.

Receptors Recognized by Corinavirus S1 CTDS:

The structure of betacoronavirus SARS-CoV S1-CTD complexed with human ACE2 provided the first atomic view of coronavirus S1. SARS-CoV S1-CTD contains two subdomains: a core structure and a receptor-binding motif (RBM). The core structure is a five-stranded antiparallel β-sheet. The RBM presents a gently concave outer surface to bind ACE2. The base of this concave surface is a short, two-stranded antiparallel β-sheet, and two ridges are formed by loops. The ectodomain of ACE2 contains a membrane-distal peptidase domain and a membrane-proximal collectrin domain  Several virus-binding motifs (VBMs) have been identified on the outer surface of the peptidase domain, away from the buried peptidase catalytic site. SARS-CoV binding does not interfere with the enzymatic activity of ACE2, nor does the enzymatic activity of ACE2 play any role in SARS-CoV entry.

Research on SARS-CoV-ACE2 interactions has provided novel insight into cross-species transmissions of SARS-CoV. During the SARS epidemic, highly similar SARS-CoV strains were isolated from both human patients and palm civets from nearby animal markets. Their S1-CTDs differ by only two residues in the RBM region: Asn479 and Thr487 in human viral strains become Lys479 and Ser487 in civet viral strains, respectively. However, human SARS-CoV S1-CTD binds to human ACE2 much more tightly than civet SARS-CoV S1-CTD does. Two virus-binding hot spots have been identified on human ACE2, centering on ACE2 residues Lys31 and Lys353, respectively. Both hot spots consist of a salt bridge buried in a hydrophobic environment and contribute critically to virus-receptor binding. Residues 479 and 487 in SARS-CoV S1-CTD interact closely with these hot spots and are under selective pressure to mutate. Two naturally selected viral mutations, K479N and S487T, strengthened the hot spot structures and enhanced the binding affinity of S1-CTD for human ACE2. Consequently, these two mutations played important roles in the civet-to-human and human-to-human transmissions of SARS-CoV during the SARS epidemic.  Compared to human ACE2, rat ACE2 contains two different residues that disfavor SARS-CoV binding: His353 disturbs the hot spot structure centering on Lys353, whereas Asn82 introduces an N-linked glycan, presenting steric interference with SARS-CoV binding. Mouse ACE2 also contains His353 but does not have the N-linked glycan at the 82 position. Thus, rat ACE2 is not a receptor for SARS-CoV, whereas mouse ACE2 is a poor receptor. Consequently, SARS-CoV does not infect rat cells, and it infects mouse cells inefficiently. SARS-like coronaviruses (SLCoVs) have been identified in bats, and some can infect human cells. Structural details on how these bat SLCoV S1-CTDs interact with ACE2 from different mammalian species still wait to be determined. Overall, these studies on SARS-CoV-ACE2 interactions reveal that (a) one or two mutations in viral RBDs can cause serious epidemic outcomes and (b) one or a few residue variations in receptor homologs from different animal species can form critical barriers for cross-species transmissions of viruses.

Structure of Betacoronavirus:

The structure of betacoronavirus MERS-CoV S1-CTD complexed with human DPP4, when compared with SARS-CoV, presented an interesting example of how two structurally similar viral RBDs recognize different protein receptors. Like SARS-CoV S1-CTD, MERS-CoV S1-CTD also contains two subdomains, a core structure and an RBM. The core structures of MERS-CoV and SARS-CoV S1-CTDs are similar to each other, whereas their RBMs are markedly different. In contrast to the loop-dominated and gently concave surface of SARS-CoV RBM, MERS-CoV RBM consists of a four-stranded antiparallel β-sheet, presenting a relatively flat surface to bind DPP4. On the other hand, DPP4 forms a homodimer and each monomer contains a hydrolase domain and a β-propeller domain. The VBMs are located on the outer surface of the β-propeller domain, away from the peptidase catalytic site. The variations of VBM residues on DPP4 homologs from different mammalian species pose a barrier for cross-species transmissions of MERS-CoV. For example, mouse and rat DPP4 molecules are both poor receptors for MERS-CoV because they each contain a number of VBM residues that disfavor MERS-CoV binding. Camel DPP4 is an effective receptor for MERS-CoV due to its conserved VBM residues. Indeed, MERS-CoV has been isolated from camels, suggesting a camel-to-human transmission of MERS-CoV. Several MERS-related coronaviruses have been isolated from bats . Among them, HKU4 recognizes DPP4 using a structural mechanism similar to that used by MERS-CoV, indicating a bat origin of MERS-CoV. Overall, these studies reveal that viral RBDs with a conserved core structure can recognize different receptors through structural variations in their RBM, and they also reinforce the concept that receptor recognition is a critical determinant of viral host ranges.

Structure of Alphacoronavirus:

The structure of alphacoronavirus HCoV-NL63 S1-CTD complexed with human ACE2, when compared with SARS-CoV, showed how two structurally divergent viral RBDs recognize the same protein receptor. HCoV-NL63 S1-CTD contains a core structure and three RBM loops. The core structure of HCoV-NL63 S1-CTD is a β-sandwich consisting of two three-stranded antiparallel β-sheets. It differs from the core structure of SARS-CoV S1-CTD, which consists of a single-layer, five-stranded β-sheet. The RBMs of HCoV-NL63 S1-CTD are three short, discontinuous loops. They differ from the RBM of SARS-CoV S1-CTD, which is a long, continuous subdomain. Nevertheless, HCoV-NL63 and SARS-CoV S1-CTDs share the same structural topology (connectivity of secondary structural elements). Despite their different structures, HCoV-NL63 and SARS-CoV S1-CTDs bind to the same VBMs on human ACE2 . Between the two SARS-CoV-binding hot spots on human ACE2, the hot spot centering on Lys353 also plays a critical role in HCoV-NL63 binding.  Consequently, as with SARS-CoV, entry of HCoV-NL63 into mouse cells is inefficient due to the presence of His353 on mouse ACE2. These studies demonstrate that viral RBDs with different structures can bind to a common virus-binding hot spot on the same protein receptor.

Nevertheless, HCoV-NL63 and SARS-CoV S1-CTDs share the same structural topology (connectivity of secondary structural elements). Despite their different structures, HCoV-NL63 and SARS-CoV S1-CTDs bind to the same VBMs on human ACE2 . Between the two SARS-CoV-binding hot spots on human ACE2, the hot spot centering on Lys353 also plays a critical role in HCoV-NL63 binding. Consequently, as with SARS-CoV, entry of HCoV-NL63 into mouse cells is inefficient due to the presence of His353 on mouse ACE2 . These studies demonstrate that viral RBDs with different structures can bind to a common virus-binding hot spot on the same protein receptor.

The structure of alphacoronavirus PRCV S1-CTD complexed with procine APN, when compared with HCoV-NL63, presented another example of how two similar coronavirus RBDs bind to different protein receptor. Like HCoV-NL63 S1-CTD, PRCV S1-CTD contains a β-sandwich core structure and three RBM loops. The core structures of PRCV and HCoV-NL63 S1-CTDs are similar to each other, but their RBMs are divergent, leading to different receptor specificities. The ectodomain of APN has a seahorse-shaped structure and forms a head-to-head dimer. The VBMs are located on the outer surface of APN, away from the buried APN catalytic site. Several other alphacoronaviruses, such as TGEV, PEDV, human coronavirus 229E, feline coronavirus, and canine coronavirus, recognize APN from their natural host as their receptor.  These APN-recognizing alphacoronaviruses, except for PEDV, have been shown to also recognize feline APN, suggesting transmission of feline coronavirus from cats to other mammals. These studies showed again that similar viral RBDs with a conserved core structure can recognize different protein receptors through structurally divergent RBMs.

The above studies provide insight into the evolution of coronavirus S1-CTDs. Although the core structures of alpha-and betacoronavirus S1-CTDs are a β-sandwich and a single-layer β-sheet, respectively, they share the same structural topology, suggesting a common evolutionary origin. The S1-CTDs from different genera likely have undergone extensive divergent evolution to attain different core structures. The three RBM loops of alphacoronavirus S1-CTDs might have further diverged into ACE2-binding RBMs in HCoV-NL63 and APN-binding RBMs in PRCV. The RBM subdomain of betacoronavirus S1-CTDs might also have diverged into ACE2-binding RBM in SARS-CoV and DPP4-binding RBM in MERS-CoV. Despite their different structures, alphacoronavirus HCoV-NL63 and betacoronavirus SARS-CoV S1-CTDs bind to a common region on ACE2, possibly driven by the common virus-binding hot spot on ACE2. The tertiary structures of the S1-CTDs from gamma-and deltacoronaviruses are unavailable but are likely related to the folds of alpha-and betacoronavirus S1-CTDs. The complex evolutionary relationships among the S1-CTDs from different genera reflect the heavy evolutionary pressure on this domain, which is discussed in more detail in next post.

Acknowledgement:

This work is supported by Nation Institutes of Health.