Cell Host Microbe 28, 586601.e586 (2020). Krause, P., Fleming, T. R., Longini, I., Henao-Restrepo, A. M. & Peto, R. COVID-19 vaccine trials should seek worthwhile efficacy. PubMed b Trimeric pre-fusion spike with one RBD in up position. a Schematic of replication-incompetent adenoviral vector particle and its DNA. b Schematic of a lipidnanoparticle (LNP) used for delivery of mRNA vaccines. Zhang, Y. et al. Science 370, 950 (2020). Natl Acad. Head-to-head comparisons of vaccinated cohorts will be especially insightful, considering the profound differences of antigen presentation and principles of action of current COVID-19 vaccines. Sci. Lancet 397, 20432046 (2021). Such effects have been specifically shown for the ionizable lipid component in LNPs124. and JavaScript. Vogel, A. Dis. Huang, Q. More or less shedding of S1 and conversion of S2 into its post-fusion structure (S2*) may occur in the absence of stabilizing mutations. 9, e42e43 (2021). Fund. Two pertinent references do not indicate specific steps of purification after inactivation and removal of cell debris88,89, but one figure in a publication by Wang et al.90 suggests a step of chromatography, albeit without providing details of this process and the purity of the vaccine. Preprint at https://www.medrxiv.org/content/10.1101/2020.11.09.20228551v1 (2020). They use derivatives of different adenoviruses as vectors for reasons more specifically discussed in section Distinguishing features of vaccines independent of immunogen, as follows: CanSinohuman adenovirus 561, Janssen-Johnson&Johnsonhuman adenovirus 2619,62,63, Oxford-AstraZenecachimpanzee adenovirus Y2564,65,66; Gamaleya Institutehuman adenovirus 26 for the first vaccination and human adenovirus 5 for the second67,68. Quantitative recovery of adenoviral vector particles involves lysis of the cells by detergents (Fig. and K.S. McMahan, K. et al. Other components of cellular immunity, such as CD8 T cells, also contribute to immune responses after SARS-CoV-2 infection or vaccination, although their role in COVID-19 infections and protection from disease is still incompletely resolved12,13. & Fox, G. J. Efficient formation of such antibodies by B cells requires helper functions of CD4 T cells that are specifically stimulated by peptides derived from the same antigen in complex with MHCII molecules. There is evidence that the Oxford-AstraZeneca vaccine might induce higher levels of specific T cells, whereas mRNA vaccines might induce higher antibody titers115,116,117. Our case report highlights the difficulty of distinguishing SARS-CoV-2 infection in the context of PAP, as the chest CT features are similar. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Currently, a slightly but significantly increased risk of thrombotic events (including cerebral venous sinus thrombosis) was reported after vaccination with Oxford-AstraZeneca and Janssen adenovirus vector vaccines and has raised considerable concern74,75. Nat. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Head-to-head comparisons of current mRNA vaccines with respect to possible differences in the efficiency of protein translation, stability or the stimulation of innate responses are not available in the literature. Variations include (but are not limited to) the type of adenovirus used as a vector, genetic modifications of the vector, the cell lines used for vaccine production, procedures for purification, and the specific design of the gene for expressing S (Table1). The RBD oscillates between an up and down position, and interaction with the cellular receptor (ACE2) is only possible with the transiently exposed RBD in the up position20,21. Therefore, it is urgent to know the epidemic . Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory. 29 January 2023. Several of the 291 candidates listed in the COVID-19 vaccine pipeline by WHO (184 pre-clinical and 107 in clinical development) (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines, accessed on July 9, 2021), have already reached the market and are used for mass immunization. The study, published in Frontiers in Public Health, finds that a distinguishing feature of COVID-19 is the order in which symptoms first appear. Science. The energy gained by the formation of this hairpin-like structure, in which the fusion peptide is juxtaposed to the C-terminal membrane anchor, is the driving force for viral membrane fusion during entry22. Google Scholar. Earle, K. A. et al. Graham, C. et al. PubMed Central PubMedGoogle Scholar. Both the Pfizer and Moderna vaccines work by giving the body instructions for how to make a harmless protein that is a distinguishing feature of the actual virus so that when or if the vaccinated person gets exposed to the virus, their body already knows what to do to fight it off. SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19) People around the world commonly get infected with human coronaviruses 229E, NL63, OC43, and HKU1. AZD1222-induced neutralising antibody activity against SARS-CoV-2 Delta VOC. N. Engl. Each dose contains 51010 or 101010 adenoviral particles (Table1), which corresponds to 8 or 16g of adenoviral protein (for calculation see ref. Immunol. Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. People become infected when they then touch their hands to their mouth, nose or eyes. Information on cellular impurities are so far restricted to ChAdOx1 and comparative analyses of all adenovector vaccines are not yet available. An infection often resolves quickly and can appear similar to food poisoning. . Protein-based vaccines such as inactivated whole-virus vaccines or subunit vaccines are usually not sufficiently immunogenic on their own and require the addition of adjuvants. Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination. 5b). Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. Our review is limited to those vaccines in current use for which phase 3 clinical efficacy data have been reported, and for which published information on the nature and manufacturing process exists. In the Oxford-AstraZeneca vaccine, an extended form of the tPA signal sequence (containing the tPA propeptide) was engineered in front of the authentic S protein signal sequence65,77,78 (Fig. It was suggested that the acquisition of the furin-cleavage site in the SARS-CoV-2 S protein was essential for zoonotic transfer to humans. Thiagarajan, K. Covid-19: India is at centre of global vaccine manufacturing, but opacity threatens public trust. Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV152: a double-blind, randomised, phase 1 trial. Recently, data from a phase 3 clinical trial became available, showing a relatively low efficacy of 47% at preventing disease (https://www.curevac.com/en/2021/06/16/curevac-provides-update-on-phase-2b-3-trial-of-first-generation-covid-19-vaccine-candidate-cvncov/), well below the efficacies reported for the BionTech-Pfizer and Moderna vaccines46,47 and below the requirement of at least 50% efficacy proposed by WHO (https://www.who.int/publications/m/item/considerations-for-the-assessment-of-covid-19-vaccines-for-listing-by-who). Nat. Ther. Comparative analyses of antibody and T cell responses and their fine specificities will allow indirect but important conclusions to be drawn. van Doremalen, N. et al. b Schematic of the process using an additional N-terminal leader sequence (signal peptide and propeptide of tPA), as used in the vaccine of Oxford-Astra Zeneca, based on reference. 10, 28752884 (2014). In this review, we discuss the biosynthesis and relevant structural features of the viral spike as a basis for understanding differences of its presentation in current COVID-19 vaccines. Sci. Curr. However, we would like to emphasize that there is an enormous pipeline of further developments (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines), including subunit vaccines that contain only parts of the S protein, in some instances combined with components of other viral proteins. Vaccines (Basel) 9, 326 (2021). Published information about the production process is available for two inactivated whole-virus vaccines manufactured by the Chinese companies Sinopharm and Sinovac (Table1). Control. Vaccine 39, 21902200 (2021). Ther. Get full coverage of the Coronavirus pandemic including the latest news, analysis, advice and explainers from across the UK and around the world. 17, 13331339 (2009). a Schematic of the vaccine mRNA in BionTech-Pfizer and Moderna vaccines. ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. classical inactivated whole-virus and innovative subunit vaccines, which contain S in different forms and combinations with adjuvants (Sections: Protein-based vaccinesgeneral, Inactivated vaccines, Subunit vaccines). Buschmann, M. D. et al. In the CanSino vaccine, the signal peptide of S is replaced by that of human tPA (https://patents.google.com/patent/CN111218459B/en). The coronavirus disease 2019 (COVID-19) pandemic is currently spreading worldwide and contributing to widespread mortality. Coronavirus Rumor Control. Although in vitro model studies with one of the current adenovirus vector vaccines (ChAdOx1 nCoV-19; Table1) have shown that S-coding transcripts dominate the transcription patterns, rare aberrant splicing or polyadenylation site usage were observed72. 11, 576622 (2020). SPsignal peptide; SRPsignal recognition particle; tPAtissue plasminogen activator; ERendoplasmic reticulum; C-terC terminus; N-terN terminus. Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum. It has been speculated that pre-existing PEG antibodies might be involved in these allergic events124. Nat. Dolgin, E. CureVac COVID vaccine let-down spotlights mRNA design challenges. Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor. A plethora of viral mutants arose and spread since the emergence of SARS-CoV-2104,105. Microbiol. ACS Central Sci. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 11, 909 (2020). Logunov, D. Y. et al. 1b). They are called "corona" because of crown-like spikes on the surface of the virus. Call the COVID-19 Hotline at. Literature data indicate that production of the Sinovac vaccine includes several steps of virus purification, leading to a product that contains primarily the viral proteins and consists of essentially pure viral particles86,87. Hasanpourghadi, M., Novikov, M. & Ertl, H. C. J. COVID-19 vaccines based on adenovirus vectors. Gao, Q. et al. In addition, background expression of remaining adenoviral genes has been demonstrated in this as well as in other studies with human adenovirus-based vectors72,76. distinguishing feature of COVID-19 compared with other infective pneumonias and its association with disease severity Meera Mehta ,1 Hakim Ghani ,1 Felix Chua,2,3 Adrian Draper,4 Sam Calmonson,1 Meghna Prabhakar,1 Rijul Shah,1 Alessio Navarra,1 Tejal Vaghela,1 Andrew Barlow,1 Rama Vancheeswaran1 Most of the allergens are proteins, which are not contained in these chemically defined vaccines (section mRNA vaccines). Science 367, 1260 (2020). The precise mechanisms of how the RNA is taken up by different cells after vaccination and escapes from LNPs and intracellular vesicles is incompletely resolved53,55. Vogel, G. Mixing COVID-19 vaccines appears to boost immune responses. 133). J. Med. The results from a hydrogeological study at LithiumBank Resources Corp (TSX-V:LBNK)'s 100%-owned Park Place lithium brine project, located in Alberta are "surprisingly favorable," according to analysts at Noble Capital.. They are known to cause diseases including the common cold, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) in humans.In January 2020, China saw an outbreak of a new coronavirus strain now named SARS-CoV-2. Ella, R. et al. After completion of translation, the protein remains attached to the ER membrane through a C-terminal membrane anchor, trimerizes and moves to the ER-Golgi intermediate compartment (ERGIC) where virus assembly occurs by budding into the ERGIC lumen (Fig. Lancet 396, 479488 (2020). We also address the grey matter of additional variables, such as ill-defined downstream production processes and purity of vaccines as well as differences in triggering sensors of innate immunity. It is believed (but not systematically studied and formally shown) that muscle cells, fibroblasts, endothelial cells, and/or immune cells such as dendritic cells contribute to the expression of S after intramuscular vaccination40,41,42. Studies are emerging that address antibody formation to the different domains of S and analyze the ratio of neutralizing and non-neutralizing antibodies as an important parameter of vaccine performance145,146. In this case, seropositivity is negligible in Europe (zero in the UK,64) and low in Africa (9% in Gambian adults,64,131). Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity. Several lines of evidence suggest that BPL-inactivation in combination with purification processes can indeed lead to the formation of the post-fusion spike and the concomitant dissociation of S125,92. Extensive modifications by N- and O-glycosylation occur in the compartments encountered by S during its intracellular transport18. 1b) (as in mRNA and adenovirus vector vaccines as well as for production of recombinant subunit vaccines), the pathway of biosynthesis is very similar. Delrue, I., Verzele, D., Madder, A. Investig. SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD and S2. The variant gambit: COVID-19s next move. SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription. A comprehensive review of the global efforts on COVID-19 vaccine development. Heath, P. T. et al. Brun, J. et al. The findings of the MIT team suggest that interferon's potential role in fighting Covid-19 may be complex. Adv. Distinct conformational states of SARS-CoV-2 spike protein. Safety and efficacy of NVX-CoV2373 Covid-19 vaccine. Pardi, N., Hogan, M. J. Pharm. ACS Central Sci. Res. Coronaviruses have enveloped virions (virus particles) that measure approximately 120 nm (1 nm = 10 9 metre) in diameter. The structures were generated with PyMol, using protein data bank (PDB) files 7KRR and 7KRS96 for the pre-fusion forms, 6XRA for the post-fusion form25. Head-to-head comparisons of mRNA vaccines will be informative to identify and evaluate differences of innate and adaptive responses as well as reactogenicity between representatives of this class of COVID-19 vaccines. 1; Black students are more likely than White students to receive harsher consequences . JCI Insight 4, e123158 (2019). This enormous progress was achieved with fundamentally different vaccine technologies used in parallel. Corbett, K.S. Google Scholar. Lancet 397, 18191819 (2021). N. Engl. The domains were colored according to reference.20. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. Med. 5b), based on a previous study with Middle East respiratory syndrome coronavirus (MERS-CoV79 (Fig. The SARS-CoV-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens. Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial. ; Approval of final manuscript: F.X.H. Persistence of RNA and its expression after different routes of application (including intramuscular) appears to be short (at least in mice), with a maximum of 10 days60. The vaccine produced by Bharat is indeed based on a seed virus containing this mutation97 (Table1). 4B, right) and further downstream processes for the removal of cellular components and free viral DNA71. Amanat, F. et al. Immunol. mRNA, adenoviral vector as well as inactivated whole-virus vaccines are now in widespread use, and a subunit vaccine is in a final stage of authorization. The coronavirus at the root of COVID-19 is the newest known member of this family. In one study they collected 15 false and 15 true headlines about Covid-19. (Image credit: Daedalean) Daedalean is looking to get its AI certified with both the FAA (Federal Aviation Administration) and EASA (European Union Aviation Safety Agency . Results from a phase 1 clinical trial with the Curevac vaccine had indeed already shown relatively low titers of neutralizing antibodies induced by the dose used in the phase 3 clinical trial56,59. Xia, S. et al. Research on school discipline disparities has demonstrated three key trends across the country: Black students are more likely than White students to be referred for disciplinary action for subjective infractions such as disruption or defiance compared to objective infractions such as tardiness or truancy. COVID-19 dynamics after a national immunization program in Israel. npj Vaccines 5, 91 (2020). Lancet https://doi.org/10.1016/s0140-6736(21)01462-8 (2021). Virus Res. Ebright helped The Washington Post debunk a claim that the COVID-19 outbreak can somehow be tied to bioweapons activity, a conspiracy theory that's been promoted or endorsed by the likes of US Sen. Tom Cotton, Iran's supreme leader, and others. Solar deities have been integral in the development of cultures across the world. Do your part to the stop the spread of rumors by doing three easy things: Find trusted sources of information. Zost, S. J. et al. McCoy, K. et al. In addition, approaches to cope with the problem of the lability of the viral S protein cause variation across all current vaccines. The signal sequence is cleaved off by signal peptidase attached to the inner ER membrane, generating the final N-terminus of the viral spike protein (14-QCVNL). A review of the challenges assessing the clinical efficacy of vaccines against SARS-CoV-2. Cell 182, 713721.e719 (2020). Wearing a mask over your mouth and nose helps to stop your water . Emary, K. R. W. et al. Nat. Planas, D. et al. Kowalczyk, A. et al. The team ran the headlines through a rigorous fact checking. Mol. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Biosci. 1, 2) in tissues after intramuscular application. 21, 83100 (2021). Barouch, D. H. et al. Science 370, 1089 (2020). The components used for LNP formulation include phospholipids, cholesterol, special cationic (ionizable) lipids and polyethylene glycol (PEGylated) lipids that are mixed in a sophisticated and critical production step (parts of which are not documented in the published literature) to yield the final vaccine53. Kids face the same risk of catching it as adults, although the disease is usually milder in children. In Plotkins Vaccines 7th edn (eds Orenstein, W. A., Offit, P. A. Nature 586, 567571 (2020). They are known to infect the neurological, respiratory, enteric, and hepatic systems. Xia, S. et al. 8, 654866 (2021). Cell 183, 10241042.e1021 (2020). Barnes, C. O. et al. Xu, C. et al. BMJ 373, n969 (2021). Igyrt, B. Ma, J. et al. & Stiasny, K. Profiles of current COVID-19 vaccines. Google Scholar. 27, 10551061 (2021). Lancet 397, 99111 (2021). Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Section navigation Coronavirus disease (COVID-19) Feature stories Situation reports; Media resources. The development of COVID-19 vaccines was extremely fast and successful, with several manufacturers having obtained market authorization for their products within the first year from the identification of the virus (SARS-CoV-2). The RNA in this vaccine is also codon-optimized and contains modifications to improve its performance, butdifferent from the BioNTech-Pfizer and Moderna mRNA vaccines described aboveit does not contain the m1 nucleoside modifications57,58. It infects persons of any race, ethnicity, or community. Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351 Variants. Thank you for visiting nature.com. The risk of immediate serious infection is thought to be very low. CrossRef Google Scholar 4 Baker, MA, Sands, KE, Huang, SS, et al. 21, 195197 (2021). Hasan, T., Beardsley, J., Marais, B. J., Nguyen, T. A. Although corresponding data do not yet exist in the public domain for the other adenoviral vector vaccines, the problem of cellular impurities may be similar, because they all depend on the lysis of production cells for releasing the engineered vector particles (section Adenovirus-vector vaccines). 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PubMed Central JAMA 325, 12411243 (2021). The severe consequences of the COVID-19 pandemic have created a pressing need for vaccines that not only prevent serious disease but preferentially also transmission. COVID-19 Hotline. Efforts are also underway to replace existing vaccine strains and corresponding sequences for those of the most relevant circulating strains114. https://doi.org/10.1007/82_2020_217 (2020). Dyer, O. Covid-19: Chinese vaccines may need changes to improve efficacy, admits official. The two mRNA vaccines in current widespread application (BioNTech-Pfizer and Moderna) (Table1) are technologically very similar. Vaccine 34, 38823893 (2016). Vaccin Immunother. & Lor, K. Immune responses induced by mRNA vaccination in mice, monkeys and humans. Vaccine 29, 52035209 (2011). Med. Front. The impact of COVID-19 on healthcare-associated infections. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Despite the absence of S2-stabilizing mutations, structural studies of the S protein expressed in HeLa cells from the Oxford-AstraZeneca ChAdOx1 nCoV-19 vaccine provided evidence for proper folding and presentation of the trimeric pre-fusion conformation at the cellular plasma membrane78. 6, eaaz6893 (2020). Indeed, substantially reduced neutralization titers against some VOCs were observed with sera after immunization with mRNA and adenovirus vector vaccines106,107,108,109, pointing to the importance of the problem. 2c)22,23,24. Immunol. The coronavirus and its variants are very contagious. 21, 950961 (2021). Immunol. Distinguishing feature definition: If you can distinguish one thing from another or distinguish between two things, you can. B. et al. 1a)15. Lancet Infect. Nat. Teijaro, J. R. & Farber, D. L. COVID-19 vaccines: modes of immune activation and future challenges. . The degree of purity of the Sinopharm vaccine (referred to as BBIBP-CorV; Table1) is less clear. On average a coronavirus particle has 74 surface spikes. Coronavirus is mostly spread through the air, when people are in close contact with each other. 12 October 2022. The impact of coronavirus disease 2019 (COVID-19) on healthcare-associated infections in 2020: a summary of data reported to the National Healthcare Safety Network. Preprint at https://www.biorxiv.org/content/10.1101/2021.04.14.439844v1 (2021). Med. Cell 181, 281292.e286 (2020). High rates of seropositivity against adenovirus 5 (the pioneer of adenovirus vector development) have been reported in the population126,127, and a number of studies have shown that pre-existing vector immunity can impair the response to the vaccine antigen128,129,130. Acute allergic reactions to mRNA COVID-19 vaccines. In both instances, the virus is grown in Vero cells and inactivated by beta-propiolactone (BPL), which is used as an inactivating agent for other viral vaccines, including rabies vaccines85. N. Engl. Thess, A. et al. Immunol. Lancet Infect. PEGpolyethyleneglycol. Nanomaterial delivery systems for mRNA vaccines. Potently neutralizing and protective human antibodies against SARS-CoV-2. Preprint at https://www.researchsquare.com/article/rs-440461/v1 (2021). 65, 1420 (2020). Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology. & Yan, J. SARS-CoV-2 virus: vaccines in development. Nance, K. D. & Meier, J. L. Modifications in an emergency: the role of N1-methylpseudouridine in COVID-19 vaccines. As a consequence of these changes, the S1 subunits dissociate from the trimer, releasing S2 from its constraints in the pre-fusion conformation to allow an irreversible conversion into a characteristic elongated post-fusion structure (Fig. Nature 586, 583588 (2020). Fan, X., Cao, D., Kong, L. & Zhang, X. Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein. Article Donors making a difference in Pakistan's flood crisis. So far, no data are available that would allow a direct comparison of the various vaccines and their effectiveness against the different VOCs. The total amount of protein per dose was found to be 35 to 40g, most of which can be assumed to be cellular protein, because the protein of 51010 adenovirus particles per dose would account for only about 8g (for calculation see122). A total of six species have been identified to cause disease in humans. 888-535-6136. The extended N-terminal leader sequence results in two consecutive signal peptides separated by an intervening stretch of tPA propeptide (Fig. Hum. J. Med. Curr. Nature 594, 483 (2021). Details of these processes, affecting the purity and quality of the final vaccines (containing at least 51010 particles per dose), are not accessible in the published literature (see section Contaminations from cell substrates). Sadoff, J. et al. Capone, S. et al. These considerations of vector immunity also prompted the development of non-human adenovirus vectors such as ChAdOx1 derived from chimpanzee adenovirus Y2564, now used in the Oxford-AstraZeneca vaccine77. 7, 512533 (2021). The authors speculate that such secreted forms may bind to ACE2-expressing endothelial cells and could contribute to thrombotic events via antibody-mediated mechanism as observed after vaccination with adenovirus vector COVID-19 vaccines74,75 (see also section Reactions due to vaccine constituents other than the immunogen). Some people infected with the coronavirus have mild COVID-19 illness, and others have no symptoms at all.
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