Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo–election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

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Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate

10.1101/2020.08.06.234674 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sandhya Bangaru ◽

Gabriel Ozorowski ◽

Hannah L. Turner ◽

Aleksandar Antanasijevic ◽

Deli Huang ◽

...

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Structural Integrity ◽

Vaccine Candidate ◽

Full Length ◽

Spike Protein ◽

Primary Target ◽

Site Specific ◽

Vaccine Candidates ◽

Novel Interactions

AbstractVaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.

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Protective Immunity against SARS Subunit Vaccine Candidates Based on Spike Protein: Lessons for Coronavirus Vaccine Development

Journal of Immunology Research ◽

10.1155/2020/7201752 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Atin Khalaj-Hedayati

Keyword(s):

Neutralizing Antibodies ◽

Protective Immunity ◽

Subunit Vaccine ◽

Vaccine Development ◽

Spike Protein ◽

Effective Vaccine ◽

Universal Vaccine ◽

Health Security ◽

Vaccine Candidates ◽

Novel Coronavirus

The recent outbreak of the novel coronavirus disease, COVID-19, has highlighted the threat that highly pathogenic coronaviruses have on global health security and the imminent need to design an effective vaccine for prevention purposes. Although several attempts have been made to develop vaccines against human coronavirus infections since the emergence of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003, there is no available licensed vaccine yet. A better understanding of previous coronavirus vaccine studies may help to design a vaccine for the newly emerged virus, SARS-CoV-2, that may also cover other pathogenic coronaviruses as a potentially universal vaccine. In general, coronavirus spike protein is the major antigen for the vaccine design as it can induce neutralizing antibodies and protective immunity. By considering the high genetic similarity between SARS-CoV and SARS-CoV-2, here, protective immunity against SARS-CoV spike subunit vaccine candidates in animal models has been reviewed to gain advances that can facilitate coronavirus vaccine development in the near future.

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Deciphering the Role of Humoral and Cellular Immune Responses in Different COVID-19 Vaccines—A Comparison of Vaccine Candidate Genes in Roborovski Dwarf Hamsters

Viruses ◽

10.3390/v13112290 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2290

Author(s):

Jakob Trimpert ◽

Susanne Herwig ◽

Julia Stein ◽

Daria Vladimirova ◽

Julia M. Adler ◽

...

Keyword(s):

Immune Responses ◽

Nucleocapsid Protein ◽

Neutralizing Antibodies ◽

Specific Antigen ◽

T Cell Response ◽

Full Length ◽

Spike Protein ◽

Protective Effects ◽

Cellular Immune Responses ◽

Cellular Immune

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

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A SARS-CoV-2 vaccine candidate would likely match all currently circulating variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008281117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23652-23662 ◽

Cited By ~ 25

Author(s):

Bethany Dearlove ◽

Eric Lewitus ◽

Hongjun Bai ◽

Yifan Li ◽

Daniel B. Reeves ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Spike Protein ◽

Reference Sequence ◽

Viral Population ◽

Effective Vaccine ◽

Nucleotide Substitutions ◽

Bioinformatics Analyses ◽

Vaccine Candidates

The magnitude of the COVID-19 pandemic underscores the urgency for a safe and effective vaccine. Many vaccine candidates focus on the Spike protein, as it is targeted by neutralizing antibodies and plays a key role in viral entry. Here we investigate the diversity seen in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and compare it to the sequence on which most vaccine candidates are based. Using 18,514 sequences, we perform phylogenetic, population genetics, and structural bioinformatics analyses. We find limited diversity across SARS-CoV-2 genomes: Only 11 sites show polymorphisms in >5% of sequences; yet two mutations, including the D614G mutation in Spike, have already become consensus. Because SARS-CoV-2 is being transmitted more rapidly than it evolves, the viral population is becoming more homogeneous, with a median of seven nucleotide substitutions between genomes. There is evidence of purifying selection but little evidence of diversifying selection, with substitution rates comparable across structural versus nonstructural genes. Finally, the Wuhan-Hu-1 reference sequence for the Spike protein, which is the basis for different vaccine candidates, matches optimized vaccine inserts, being identical to an ancestral sequence and one mutation away from the consensus. While the rapid spread of the D614G mutation warrants further study, our results indicate that drift and bottleneck events can explain the minimal diversity found among SARS-CoV-2 sequences. These findings suggest that a single vaccine candidate should be efficacious against currently circulating lineages.

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Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant

Journal of Virology ◽

10.1128/jvi.02180-16 ◽

2017 ◽

Vol 91 (8) ◽

Cited By ~ 32

Author(s):

Kirsten Schneider-Ohrum ◽

Corinne Cayatte ◽

Angie Snell Bennett ◽

Gaurav Manohar Rajani ◽

Patrick McTamney ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Replication ◽

Rat Model ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Candidate ◽

Dose Range ◽

Vaccine Candidates ◽

Cotton Rat ◽

Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract. IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of nonlive RSV vaccine candidates. The findings presented here underscore the need to evaluate a nonlive RSV vaccine candidate during preclinical development over a wide dose range in the cotton rat RSV enhanced-disease model, as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself and to safe development of nonlive RSV vaccines for seronegative infants and children.

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RelCoVax®, a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

10.1101/2022.01.07.475330 ◽

2022 ◽

Author(s):

Abhishek Phatarphekar ◽

GEC Vidyadhar Reddy ◽

Abhiram Gokhale ◽

Gopala Karanam ◽

Pushpa Kuchroo ◽

...

Keyword(s):

Immune Responses ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Protein Vaccine ◽

Vaccine Candidates ◽

N Protein ◽

Subunit Protein ◽

Boost Dose

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax® through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax® formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-γ release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax® demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax® through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

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Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates

Science Advances ◽

10.1126/sciadv.abg7156 ◽

2021 ◽

Vol 7 (22) ◽

pp. eabg7156

Author(s):

So-Hee Hong ◽

Hanseul Oh ◽

Yong Wook Park ◽

Hye Won Kwak ◽

Eun Young Oh ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Nonhuman Primates ◽

Vaccine Candidate ◽

Statistical Significance ◽

Neutralizing Antibody ◽

Spike Protein ◽

Vaccine Candidates ◽

Cell Responses ◽

Antibody Levels ◽

Further Development

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

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Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19

Journal of Virology ◽

10.1128/jvi.00194-21 ◽

2021 ◽

Author(s):

Jiahao Ma ◽

Danmei Su ◽

Yinyan Sun ◽

Xueqin Huang ◽

Ying Liang ◽

...

Keyword(s):

Clinical Trial ◽

Receptor Binding ◽

Subunit Vaccine ◽

Vaccine Candidate ◽

Phase 1 ◽

Full Length ◽

Effective Vaccine ◽

S Protein ◽

Closed Conformation ◽

A Subunit

Within a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 100 million people worldwide with a death toll over 2 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Å and 2.6 Å respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. The tightly closed conformation is stabilized by fatty acid and polysorbate 80 binding at the receptor binding domains (RBDs) and the N terminal domains (NTDs) respectively. Additionally, we identified an important pH switch in the WT S-Trimer that shows dramatic conformational change and accounts for its increased stability at lower pH. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine. IMPORTANCE Effective vaccine against SARS-CoV-2 is critical to end the COVID-19 pandemic. Here, using Trimer-Tag technology, we are able to produce stable and large quantities of WT S-Trimer, a subunit vaccine candidate for COVID-19 with high safety and efficacy from animal and Phase 1 clinical trial studies. Cryo-EM structures of the S-Trimer subunit vaccine candidate show that it predominately adopts tightly closed pre-fusion state, and resembles that of the native and full-length spike in detergent, confirming its structural integrity. WT S-Trimer is currently being evaluated in global Phase 2/3 clinical trial. Combining with published structures of the S protein, we also propose a model to dissect the conformation change of the spike protein before receptor binding.

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Characterization of the Immune Response of MERS-CoV Vaccine Candidates Derived from Two Different Vectors in Mice

Viruses ◽

10.3390/v12010125 ◽

2020 ◽

Vol 12 (1) ◽

pp. 125 ◽

Cited By ~ 7

Author(s):

Entao Li ◽

Feihu Yan ◽

Pei Huang ◽

Hang Chi ◽

Shengnan Xu ◽

...

Keyword(s):

Immune Responses ◽

Antibody Response ◽

Rabies Virus ◽

Vaccine Development ◽

Vaccine Candidate ◽

Viral Vector ◽

Vaccine Vector ◽

Vaccine Candidates ◽

Cellular Immune Responses ◽

Cellular Immune

Middle East respiratory syndrome (MERS) is an acute, high-mortality-rate, severe infectious disease caused by an emerging MERS coronavirus (MERS-CoV) that causes severe respiratory diseases. The continuous spread and great pandemic potential of MERS-CoV make it necessarily important to develop effective vaccines. We previously demonstrated that the application of Gram-positive enhancer matrix (GEM) particles as a bacterial vector displaying the MERS-CoV receptor-binding domain (RBD) is a very promising MERS vaccine candidate that is capable of producing potential neutralization antibodies. We have also used the rabies virus (RV) as a viral vector to design a recombinant vaccine by expressing the MERS-CoV S1 (spike) protein on the surface of the RV. In this study, we compared the immunological efficacy of the vaccine candidates in BALB/c mice in terms of the levels of humoral and cellular immune responses. The results show that the rabies virus vector-based vaccine can induce remarkably earlier antibody response and higher levels of cellular immunity than the GEM particles vector. However, the GEM particles vector-based vaccine candidate can induce remarkably higher antibody response, even at a very low dose of 1 µg. These results indicate that vaccines constructed using different vaccine vector platforms for the same pathogen have different rates and trends in humoral and cellular immune responses in the same animal model. This discovery not only provides more alternative vaccine development platforms for MERS-CoV vaccine development, but also provides a theoretical basis for our future selection of vaccine vector platforms for other specific pathogens.

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