key: cord-0965931-ajjc9wif authors: Hachim, Asmaa; Kavian, Niloufar; Valkenburg, Sophie A title: Antibody landscapes of SARS-CoV-2 can reveal novel vaccine and diagnostic targets date: 2021-08-20 journal: Curr Opin Virol DOI: 10.1016/j.coviro.2021.08.006 sha: b43f4f629bcfab8f21a0ee6facb7c61b9dc50007 doc_id: 965931 cord_uid: ajjc9wif SARS-CoV-2 virions are composed of structural proteins, but during virus infection, an additional 30 proteins could be expressed according to putative open reading frames (ORFs) of the viral genome. Some of these additional proteins modulate cellular processes through direct interactions, their truncations can affect disease pathogenesis and serve as antigenic targets for more specific serology. In addition to structural proteins, the ORF1a/b polyprotein and accessory proteins can stimulate antibody responses during infection. Antibodies that target non-structural proteins can impact viral infection, through Fc mediated effector functions, through interactions during virus entry, fusion, replication and egress within infected cells. Characterization of the serological responses to additional proteins, provides a snapshot of the ‘antibody landscape’, which includes the antibody magnitude, antigenic specificity and biological relevance of SARS-CoV-2 proteins. 3 16 non-structural proteins (NSP1- 16) . The NSPs are mainly involved in the replication machinery of the virus (NSP3, NSP4, NSP5, NSP6, NSP7, NSP8, NSP9, NSP10, NSP12, NSP13, NSP14, NSP15, NSP16) but take also part in the evasion of the host immune response (NSP1, NSP15 notably) (2) . At the 3' end of the SARS-CoV-2 genome are additional ORFs S, 3a, 3c, E, M, 6, 7a, 7b, 8, N and 9b which are translated and functional; ORFs 2b and 3d-2 which are translated but not functional and ORFs 3b, 3d, 9c and 10 which appear not to be translated to biologically meaningful levels (Table 1, Figure 1 ) (3)*. As the pandemic has progressed and alternate reading frame products have been defined, nomenclature of the ORF3 has been updated (3), for clarity the ORF3b we previously referred to (4) is actually now referred to as ORF3d and contains the isoform ORF3d-2, that is expressed (1) . Therefore, study of the antibody landscape can be linked to studies of protein function, interactomes and evolutionary rates to determine functional expression. Recently, a large scale interactome study of the proteins of SARS-CoV-2 infected human lung cell line revealed the systems level host proteins that directly interact with viral proteins (5)** and diverse roles of the accessory proteins and NSPs were found for immunomodulation, cellular trafficking and metabolism among many more cellular processes, whilst the S and N proteins had more limited functions within infected cells. The most prominent immunomodulation effect studied to date has been type-I interferon antagonism which is mediated by NSP1, NSP6, NSP13, NSP14 and NSP15 of ORF1a/b polyprotein (6, 7)*, as well as ORF3d (8), ORF6 (9), ORF7a (10), ORF8 (9) and N (11) ( Table 1) . Moreover, ORF7a transmembrane protein (12) has been shown to inhibit Tetherin activation circumventing blockade of viral budding (13) and ORF3a has been shown to induce apoptosis in vitro through activation of the caspase-8 pathway (14) and to block the mechanism of autophagy (15) . The accessory proteins therefore mediate immune evasion through a multitude of pathways and appear to be essential in COVID-19 pathogenesis. J o u r n a l P r e -p r o o f This is further supported by the positive selection of ORF1a/b, ORF3a and ORF8 genes which drives the evolution of SARS-CoV-2 (16) . Accessory proteins are also subject to mutations, to a lesser extent than S and N ( (17) , and could play an important role in viral fitness and pathogenesis as its deletion led to reduced COVID-19 severity (18) . However, its involvement, location and function within the virus life cycle remain to be determined. Another accessory protein which has been prone to deletions is ORF3b (and ORF3c, 3d, 3d-2), prominently due to a Q57H mutation in ORF3a (in VoC Beta, B1.351 lineage, Table 1 ) which leads to its truncation and loss of antagonism function of ORF3b (19) . Important roles are emerging for the ORF1a/b polyprotein and accessory proteins in SARS-CoV-2, highlighting their essential role in viral pathogenesis and replication, hence the need for further investigation into their location and function within the virus and its life cycle. Most vaccines in use against COVID-19 target the S protein to elicit neutralizing antibodies to block infection (20) , as the S protein, contains the receptor binding domain (RBD) which is critical for viral entry (21) . Whilst the N protein is the most abundant protein within infected cells and the virion, and is a target of routine serological tests like S. The sensitivity and specificity of S and N based serological testing is not as effective in children Protein domain specific antigens may improve the sensitivity of N testing (24) , but the same issue of waning and whole virion inactivated vaccines remains. Therefore, confirmatory serological testing with additional serological makers of SARS-CoV-2 infection will be useful when vaccine use is widespread, but waning immunity from vaccines may require further diagnosis of subsequent infection. Upon the initiation of an adaptive immune response against a pathogen, antibody targets are selected depending on their physical properties, their expression and the host's immune repertoire diversity. For SARS-CoV-2, antibodies to the Spike protein that are neutralizing were largely studied early in the pandemic by several teams (25) , but the spectrum of the antibody landscape was first described by our team in April 2020 (4, 26) . Since then, others have also confirmed that COVID-19 induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome to define cross-reactive B cells and accurate serology (27)*, identify unique epitopes by virscan (28) , and the proteome response by microarray (29)*. Antibody landscapes can also refer to the viral strain diversity recognized by the humoral response, for example influenza specific antibodies to hemagglutinin antigens over time with infection and vaccination (30) (31) (32) (33) . To characterize the SARS-CoV-2 antibody landscape we employed an unbiased and quantitative approach by luciferase immunoprecipitation system (LIPS) (34)* to assess Prior immunity did not impact the magnitude of ORF8 and ORF3b due to minimal cross-reactivity of these responses with HCoVs (4). ORF8 is not encoded by other endemic common cold corona viruses, and only found in some sarbecoronaviruses (bat coronaviruses and SARS-CoV), where it has the smallest sequence homology of all genes (1) . Whilst only the NL63 common cold -coronavirus, has an ORF3 protein which shares minimal homology with the SARS-CoV-2 derived ORF3a and ORF3b/c/d, making crossreactivity unlikely. We found the combined use of ORF3b and ORF8 was a highly sensitive and specific serological marker of infection, as early as 5 days post infection and long term up to 6 months after infection. This is consistent with the concept of 'dating' time estimates of infection based on the relative magnitude of antibodies to different antigens, as waning for different specificities can result in their unequal decline (35) and has been adopted by other fields such as malaria (36) , Ebola (37) , and influenza infection (38) . However, so far, combined antigen testing and the 'dating' of recent infection using S, RBD and N has wide ranges in estimates (23) which may undermine its utility without incorporation of additional more specific antigens like ORF8 and ORF3b. Therefore, commercial protein production should progress from the limited available accessory antigens and structural S and N, to improve the research efforts and tools widely available on the role of alternate targets of SARS- HCoV cross-reactivity (46) or new de novo responses were made (52) . Similarly, memory B cells to N and ORF8 showed a pronounced maturation over time, however monoclonal antibodies to these internal targets do not confer protection in vivo in mice (46) . Non-neutralizing antibodies may also inadvertently mediate antibody dependent enhancement (ADE), as shown in severe patients in which divalent antibodies binding to J o u r n a l P r e -p r o o f Spike-NTD allowed for a conformational change of the spike protein resulting in enhanced infectivity (55) . This could possibly also occur for other Spike-specific antibodies outside of the RBD or that bind with a lower affinity, such as the variants of concern P.1 and B.1.351 which have the N501Y mutation leading to incomplete neutralization (56) . However, although ADE did occur leading to enhanced virus infection (55, 57) , replication is not apparent in primary macrophages (58) , however modulation of the myeloid compartment is associated with disease outcomes (59) and immune activation (60) . Antibodies that target non-neutralising antigens may also mediate their effect by directly interfering with virus replication by targeting the Replicase machinery formed by the NSPs (2) (such as NSP12 which is responsible for replication and transcription of viral genome (61)), viral entry at fusion (such as S2' specific antibodies), virus budding (such as ORF7a) (2) . Therefore, antibody specificity and effector functions leading to immune cell recruitment can drive clinical outcomes. However, the antibody effector functions of nonstructural and accessory proteins remain uncharacterized currently and warrants further studies, especially as variants of concern, such as B1.1.7, B1.351, P.1 and B1.617 have alterations within these key proteins ( Table 1 ). The utility of antibodies to non-neutralising and internal proteins is gaining traction with a growing body of work for other virus infections as diagnosis, treatment and vaccines. Antibody landscapes can be used to detect spillover infection (62), whereby viruses that have limited protein tools, such as bat orthoreoviruses, have their entire genomes cloned, expressed and screened for antibody binding in population serosurveillance. Unbiased and quantitative approaches, like LIPS (34), microarray (29) , virscan (28) or cell-based ELISA (25, 63) for serosurveillance will be useful tools in the post-COVID-19 future to highlight and identify key immunogenic targets of novel viruses. 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Nat Comms 2021) This study was partly supported by the Theme based Research Grants Scheme (T11-712/19-N), Health and Medical Research Fund (HMRF COVID-190115 and COVID-190126). Figure 1 was partly made with Biorender. Papers of particular interest, published within the period of review, have been highlighted as:* of special interest ** of outstanding interest