Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production

Viral protein glycosylation represents a successful strategy employed by the parasite to take advantage of host–cell machinery for modification of its own proteins. The resulting glycans have unneglectable roles in viral infection and immune response. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which presents on the surface of matured virion and mediates viral entry into the host, also undergoes extensive glycosylation to shield it from the human defense system. It is believed that the ongoing COVID-19 pandemic with more than 90,000,000 infections and 1,900,000 deaths is partly due to its successful glycosylation strategy. On the other hand, while glycan patches on S protein have been reported to shield the host immune response by masking “nonself” viral peptides with “self-glycans,” the epitopes are also important in eliciting neutralizing antibodies. In this review, we will summarize the roles of S protein glycans in mediating virus–receptor interactions, and in antibody production, as well as indications for vaccine development.

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Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins

10.1101/2020.06.09.141580 ◽

2020 ◽

Author(s):

Yuejun Shi ◽

Jiale Shi ◽

Limeng Sun ◽

Yubei Tan ◽

Gang Wang ◽

...

Keyword(s):

Public Health ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Neutralizing Antibody ◽

Immune Recognition ◽

Global Public Health ◽

S Protein ◽

Specific Antibodies ◽

Major Target

AbstractCoronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. However, no commercial vaccines against the coronaviruses that can infect humans are available. The spike (S) homotrimers bind to their receptors through the receptor-binding domain (RBD), which is believed to be a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs were observed to adopt two different conformational states (lying or standing). Then, structural and immunological analyses were used to explore differences in the immune response with RBDs among these coronaviruses. Our results showed that more RBD-specific antibodies were induced by the S trimer with the RBD in the “standing” state (SARS-CoV and SARS-CoV-2) than the S trimer with the RBD in the “lying” state (HCoV-229E), and the affinity between the RBD-specific antibodies and S trimer was also higher in the SARS-CoV and SARS-CoV-2. In addition, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was much lower and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus. Our findings will provide important implications for future development of coronavirus vaccines.ImportanceOutbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in lying and standing state, respectively. Here, we tested the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that Beta-CoVs RBDs are in a standing state, and their S proteins can induce more neutralizing antibodies targeting RBD. However, HCoV-229E RBD is in a lying state, and its S protein induces a low level of neutralizing antibody targeting RBD. Our results indicate that Alpha-coronavirus is more conducive to escape host immune recognition, and also provide novel ideas for the development of vaccines targeting S protein.

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Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

10.1101/2021.01.15.426463 ◽

2021 ◽

Author(s):

Yasunori Watanabe ◽

Luiza Mendonça ◽

Elizabeth R. Allen ◽

Andrew Howe ◽

Mercede Lee ◽

...

Keyword(s):

Immune Response ◽

Neutralizing Antibodies ◽

Viral Antigen ◽

Vaccine Development ◽

Spike Glycoprotein ◽

S Protein ◽

Platform Technology ◽

Adenovirus Vectors ◽

S Proteins

AbstractVaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

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Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins

Journal of Virology ◽

10.1128/jvi.02284-20 ◽

2021 ◽

Author(s):

Yuejun Shi ◽

Jiale Shi ◽

Limeng Sun ◽

Yubei Tan ◽

Gang Wang ◽

...

Keyword(s):

Public Health ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Global Public Health ◽

Subunit Vaccines ◽

S Protein ◽

Specific Antibodies ◽

Antibody Titers ◽

Major Target

Abstract Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. The spike (S) homotrimers bind to their receptors via the receptor-binding domain (RBD), which is a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs exist two different conformational states (lying or standing) in the prefusion S-trimer structure. Then, the differences in the immune responses to RBDs from these coronaviruses were analyzed structurally and immunologically. Our results showed that more RBD-specific antibodies (antibody titers: 1.28×105; 2.75×105) were induced by the S-trimer with the RBD in the “standing” state (SARS-CoV and SARS-CoV-2) than the S-trimer with the RBD in the “lying” state (HCoV-229E, antibody titers: <500), and more S-trimer-specific antibodies were induced by the RBD in the SARS-CoV and SARS-CoV-2 (antibody titers: 6.72×105; 5×105) than HCoV-229E (antibody titers:1.125×103). Besides, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was lower than S-trimer, and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus. Our findings will provide important implications for future development of coronavirus vaccines. Importance Outbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in “lying” and “standing” states in the prefusion S-trimer structure. Here, we evaluated the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that the S-trimer and RBD are both candidates for subunit vaccines in Beta-coronavirus (SARS-CoV and SARS-CoV-2) with a RBD “standing” state. However, for Alpha-coronavirus (HCoV-229E) with a RBD “lying” state, the S-trimer may be more suitable for subunit vaccines than the RBD. Our results will provide novel ideas for the development of vaccines targeting S protein in the future.

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SARS-CoV-2 Biology Insights, Part IV.Antibody-Dependent Enhancement (ADE) and the tricky “Herd Immunity”: narrative review (Preprint)

10.2196/preprints.21599 ◽

2020 ◽

Author(s):

Laura Lafon-Hughes

Keyword(s):

Viral Infection ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Whooping Cough ◽

Common Knowledge ◽

Herd Immunity ◽

Life Quality ◽

Host Cells ◽

System P

BACKGROUND It is common knowledge that vaccination has improved our life quality and expectancy since it succeeded in achieving almost eradication of several diseases including chickenpox (varicella), diphtheria, hepatitis A and B, measles, meningococcal, mumps, pneumococcal, polio, rotavirus, rubella, tetanus and whooping cough (pertussis) Vaccination success is based on vaccine induction of neutralizing antibodies that help fight the infection (e.g. by a virus), preventing the disease. Conversely, Antibody-dependent enhancement (ADE) of a viral infection occurs when anti-viral antibodies facilitate viral entry into host cells and enhance viral infection in these cells. ADE has been previously studied in Dengue and HIV viruses and explains why a second infection with Dengue can be lethal. As already reviewed in Part I and Part II, SARS-Cov-2 shares with HIV not only 4 sequences in the Spike protein but also the capacity to attack the immune system. OBJECTIVE As HIV presents ADE, we wondered whether this was also the case regarding SARS-CoV-2. METHODS A literature review was done through Google. RESULTS SARS-CoV-2 presents ADE. As SARS, which does not have the 4 HIV-like inserts, has the same property, ADE would not be driven by the HIV-like spike sequences. CONCLUSIONS ADE can explain the failure of herd immunity-based strategies and will also probably hamper anti-SARS-CoV-2 vaccine development. As reviewed in Part I, there fortunately are promising therapeutic strategies for COVID-19, which should be further developed. In the meantime, complementary countermeasures to protect mainly the youth from this infection are presented to be discussed in Part V Viewpoint.

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Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

Journal of Virology ◽

10.1128/jvi.79.6.3289-3296.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3289-3296 ◽

Cited By ~ 59

Author(s):

Choong-Tat Keng ◽

Aihua Zhang ◽

Shuo Shen ◽

Kuo-Ming Lip ◽

Burtram C. Fielding ◽

...

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Antiviral Agents ◽

Host Cells ◽

Heptad Repeat ◽

Antigenic Determinants ◽

Amino Acid Residues ◽

S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

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Lipopolysaccharide: a tool and target in enterobacterial vaccine development

Biological Chemistry ◽

10.1515/bc.2008.056 ◽

2008 ◽

Vol 389 (5) ◽

Cited By ~ 30

Author(s):

Gábor Nagy ◽

Tibor Pál

Keyword(s):

Immune Response ◽

Protective Immunity ◽

Vaccine Development ◽

The Other ◽

Live Vaccine Strain ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Vaccination Strategies ◽

Optimal Balance ◽

Alternative Approaches

AbstractLipopolysaccharide (LPS) is an essential component of Gram-negative bacteria. While mutants exhibiting truncated LPS molecules are usually over-attenuated, alternative approaches that affect the extent or timing of LPS expression, as well as its modification may establish the optimal balance for a live vaccine strain of sufficient attenuation and retained immunogenicity. On the other hand, a specific immune response to LPS molecules in itself is capable of conferring protective immunity to certain enterobacterial pathogens. Therefore, purified LPS derivatives could be used as parenteral vaccines. This review summarizes various LPS-based vaccination strategies, as well as approaches that utilize LPS mutants as whole-cell vaccines.

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Conformational States of a Soluble, Uncleaved HIV-1 Envelope Trimer

Journal of Virology ◽

10.1128/jvi.00175-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 9

Author(s):

Yuhang Liu ◽

Junhua Pan ◽

Yongfei Cai ◽

Nikolaus Grigorieff ◽

Stephen C. Harrison ◽

...

Keyword(s):

Viral Entry ◽

Clinical Studies ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Recombinant Form ◽

Soluble Form ◽

Compact Structure ◽

Antigenic Properties ◽

Hiv 1 ◽

Gp140 Trimer

ABSTRACT The HIV-1 envelope spike [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers—the uncleaved ectodomains of (gp160)3—from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20 Å) electron cryomicroscopy (cryoEM) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with a truncated cytoplasmic tail (EnvΔCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env. IMPORTANCE Development of any HIV vaccine with a protein component (for either priming or boosting) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications, including an engineered disulfide. This construct, which is difficult to produce in large quantities, has yet to induce better antibody responses than those to other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and have an inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.

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The Emergence and Spread of Novel SARS-CoV-2 Variants

Frontiers in Public Health ◽

10.3389/fpubh.2021.696664 ◽

2021 ◽

Vol 9 ◽

Author(s):

Huaimin Yi ◽

Jin Wang ◽

Jiong Wang ◽

Yuying Lu ◽

Yali Zhang ◽

...

Keyword(s):

Immune Response ◽

Severe Acute Respiratory Syndrome ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Neutralizing Antibodies ◽

Whole Genome ◽

Distribution Characteristics ◽

S Protein ◽

The World ◽

Viral Genes

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in late 2019, laboratories around the world have widely used whole genome sequencing (WGS) to continuously monitor the changes in the viral genes and discovered multiple subtypes or branches evolved from SARS-CoV-2. Recently, several novel SARS-CoV-2 variants have been found to be more transmissible. They may affect the immune response caused by vaccines and natural infections and reduce the sensitivity to neutralizing antibodies. We analyze the distribution characteristics of prevalent SARS-CoV-2 variants and the frequency of mutant sites based on the data available from GISAID and PANGO by R 4.0.2 and ArcGIS 10.2. Our analysis suggests that B.1.1.7, B.1.351, and P.1 are more easily spreading than other variants, and the key mutations of S protein, including N501Y, E484K, and K417N/T, have high mutant frequencies, which may have become the main genotypes for the spread of SARS-CoV-2.

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SARS-CoV-2 Infection, COVID-19, and long covid: Saga of erratic immune response, waning immunity, and immune system failure

Journal of Pulmonology and Respiratory Research ◽

10.29328/journal.jprr.1001030 ◽

2021 ◽

Vol 5 (1) ◽

pp. 077-086

Author(s):

Nikhra Vinod

Keyword(s):

Immune Response ◽

Immune System ◽

Disease Transmission ◽

Neutralizing Antibodies ◽

Evolutionary Dynamics ◽

Immune Escape ◽

Clinical Manifestations ◽

System Failure ◽

S Protein ◽

Adaptive Mutations

Introduction - evolution of SARS-CoV-2 variants: With the unrestrained pandemic for over last one-and-half year, SARS-CoV-2 seems to have adapted to its habitat, the human host, through mutations that facilitate its replication and transmission. The G variant incorporating D614G mutation, potently more transmissible than the ancestral virus arose during January 2020 and spread widely. Since then, various SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) with higher infectivity or virulence or both, have evolved on the background of G variant, and spread widely. SARS-CoV-2 infection and the immunodynamics: As the virus becomes more transmissible, its lethality may drop. Apart from the humoral immunity, T-cell recognition from a previous SARS-CoV-2 infection or vaccination may modify the disease transmission correlates and its clinical manifestations. On the other hand, the immunity generated may reduce probability of re-infection as well as limit evolution of adaptive mutations, and emergence of highly infectious and immune-escape variants. There are complex issues related to the SARS-CoV-2 evolutionary dynamics and host’s immunodynamics. Trending etiopathoimmunological correlates: The evolution potential of SARS-CoV-2 is limited because of proofreading function of nsp14. The S protein mutations affect transmissibility, virulence, and vaccine efficacy. The D614G mutation in G variant with higher infectivity has turned the Chinese epidemic into a pandemic. Other SARS-CoV-2 variants, such as Alpha, Beta, Gamma, and Delta seem to have evolved as result of adaptation to selective pressures during periods of prolonged infections and subsequent transmission. Further, there is issue of convergent association of mutations. Basics of immunity and immune system failure: The nature of the immune response after natural SARS-CoV-2 infection is variable and diverse. There are pre-existing neutralizing antibodies and sensitized T cells elicited during previous infection with seasonal CoVs influencing the disease susceptibility and course. The virus has evolved adaptive mechanisms to reduce its exposure to IFN-I and there are issues related to erratic and overactive immune response. The altered neutralizing epitopes in the S protein in SARS-CoV-2 variants modify the immune landscapes and clinical manifestations. Conclusion: current scenarios and prospects: Presently, the SARS-CoV-2 infection is widespread with multiple evolving infectious variants. There is probability of its transition from epidemic to endemic phase in due course manifesting as a mild disease especially in the younger population. Conversely, the pandemic may continue with enhanced disease severity due to evolving variants, expanded infection pool, and changing immunity landscape. There is need to plan for the transition and continued circulation of the virus during the endemic phase or continuing pandemic for indefinite period.

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Recombinant Antigens Based on Non-Glycosylated Regions from RBD SARS-CoV-2 as Potential Vaccine Candidates against COVID-19

Vaccines ◽

10.3390/vaccines9080928 ◽

2021 ◽

Vol 9 (8) ◽

pp. 928

Author(s):

Leandro Núñez-Muñoz ◽

Gabriel Marcelino-Pérez ◽

Berenice Calderón-Pérez ◽

Miriam Pérez-Saldívar ◽

Karla Acosta-Virgen ◽

...

Keyword(s):

Immune Response ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Polyclonal Antibodies ◽

Igg Antibodies ◽

Mice Model ◽

Strong Immune Response ◽

Recombinant Antigens ◽

Glycosylation Sites ◽

Potential Vaccine

The Receptor-Binding Domain (RBD) of the Spike (S) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has glycosylation sites which can limit the production of reliable antigens expressed in prokaryotic platforms, due to glycan-mediated evasion of the host immune response. However, protein regions without glycosylated residues capable of inducing neutralizing antibodies could be useful for antigen production in systems that do not carry the glycosylation machinery. To test this hypothesis, the potential antigens NG06 and NG19, located within the non-glycosylated S-RBD region, were selected and expressed in Escherichia coli, purified by FPLC and employed to determine their immunogenic potential through detection of antibodies in serum from immunized rabbits, mice, and COVID-19 patients. IgG antibodies from sera of COVID-19-recovered patients detected the recombinant antigens NG06 and NG19 (A450 nm = 0.80 ± 0.33; 1.13 ± 0.33; and 0.11 ± 0.08 for and negatives controls, respectively). Also, the purified antigens were able to raise polyclonal antibodies in animal models evoking a strong immune response with neutralizing activity in mice model. This research highlights the usefulness of antigens based on the non-N-glycosylated region of RBD from SARS-CoV-2 for candidate vaccine development.

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