Identification of a broad sarbecovirus neutralizing antibody targeting a conserved epitope on the receptor-binding domain.

Omicron, as the emerging variant with enhanced vaccine tolerance, has sharply disrupted most therapeutic antibodies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subgenus Sarbecovirus, members of which share high sequence similarity. Herein, we report one sarbecovirus antibody, 5817, which has broad-spectrum neutralization capacity against SARS-CoV-2 variants of concern (VOCs) and SARS-CoV, as well as related bat and pangolin viruses. 5817 can hardly compete with six classes of receptor-binding-domain-targeted antibodies grouped by structural classifications. No obvious impairment in the potency is detected against SARS-CoV-2 Omicron and subvariants. The cryoelectron microscopy (cryo-EM) structure of neutralizing antibody 5817 in complex with Omicron spike reveals a highly conserved epitope, only existing at the receptor-binding domain (RBD) open state. Prophylactic and therapeutic administration of 5817 potently protects mice from SARS-CoV-2 Beta, Delta, Omicron, and SARS-CoV infection. This study reveals a highly conserved cryptic epitope targeted by a broad sarbecovirus neutralizing antibody, which would be beneficial to meet the potential threat of pre-emergent SARS-CoV-2 VOCs.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Corrigendum: XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants.

[This corrects the article DOI: 10.3389/fimmu.2021.761250.].

Comparing the incubation period, serial interval, and infectiousness profile between SARS-CoV-2 Omicron and Delta variants.

In January 2022, the SARS-CoV-2 Omicron variants initiated major outbreaks and dominated the transmissions in Hong Kong, displacing an earlier outbreak seeded by the Delta variants. To provide insight into the transmission potential of the emerging variants, we aimed to compare the epidemiological characteristics of the Omicron and Delta variants. We analyzed the line-list clinical and contact tracing data of the SARS-CoV-2 confirmed cases in Hong Kong. Transmission pairs were constructed based on the individual contact history. We fitted bias-controlled models to the data to estimate the serial interval, incubation period and infectiousness profile of the two variants. Viral load data were extracted and fitted to the random effect models to investigate the potential risk modifiers for the clinical viral shedding course. Totally 14 401 confirmed cases were reported between January 1 and February 15, 2022. The estimated mean serial interval (4.4 days vs. 5.8 days) and incubation period (3.4 days vs. 3.8 days) were shorter for the Omicron than the Delta variants. A larger proportion of presymptomatic transmission was observed for the Omicron (62%) compared to the Delta variants (48%). The Omicron cases had higher mean viral load over an infection course than the Delta cases, with the elder cases appearing more infectious than the younger cases for both variants. The epidemiological features of Omicron variants were likely an obstacle to contact tracing measures, imposed as a major intervention in settings like Hong Kong. Continuously monitoring the epidemiological feature for any emerging SARS-CoV-2 variants in the future is needed to assist officials in planning measures for COVID-19 control.

Genetic diversity and molecular epidemiology of Middle East Respiratory Syndrome Coronavirus in dromedaries in Ethiopia, 2017-2020.

Middle East respiratory syndrome coronavirus (MERS-CoV) is enzootic in dromedary camels and causes zoonotic infection and disease in humans. Although over 80% of the global population of infected dromedary camels are found in Africa, zoonotic disease had only been reported in the Arabia Peninsula and travel-associated disease has been reported elsewhere. In this study, genetic diversity and molecular epidemiology of MERS-CoV in dromedary camels in Ethiopia were investigated during 2017-2020. Of 1766 nasal swab samples collected, 61 (3.5%) were detected positive for MERS-CoV RNA. Of 484 turbinate swab samples collected, 10 (2.1%) were detected positive for MERS-CoV RNA. Twenty-five whole genome sequences were obtained from these MERS-CoV positive samples. Phylogenetically, these Ethiopian camel-originated MERS-CoV belonged to clade C2, clustering with other East African camel strains. Virus sequences from camel herds clustered geographically while in an abattoir, two distinct phylogenetic clusters of MERS-CoVs were observed in two sequential sampling collections, which indicates the greater genetic diversity of MERS-CoV in abattoirs. In contrast to clade A and B viruses from the Arabian Peninsula, clade C camel-originated MERS-CoV from Ethiopia had various nucleotide insertions and deletions in non-structural gene nsp3, accessory genes ORF3 and ORF5 and structural gene N. This study demonstrates the genetic instability of MERS-CoV in dromedaries in East Africa, which indicates that the virus is still actively adapting to its camel host. The impact of the observed nucleotide insertions and deletions on virus evolution, viral fitness, and zoonotic potential deserves further study.

Neutralizing Antibody Response to Sarbecovirus Is Delayed in Sequential Heterologous Immunization.

Antigenic imprinting, which describes the bias of the antibody response due to previous immune history, can influence vaccine effectiveness. While this phenomenon has been reported for viruses such as influenza, there is little understanding of how prior immune history affects the antibody response to SARS-CoV-2. This study provides evidence for antigenic imprinting through immunization with two Sarbecoviruses, the subgenus that includes SARS-CoV-2. Mice were immunized subsequently with two antigenically distinct Sarbecovirus strains, namely SARS-CoV-1 and SARS-CoV-2. We found that sequential heterologous immunization induced cross-reactive binding antibodies for both viruses and delayed the emergence of neutralizing antibody responses against the booster strain. Our results provide fundamental knowledge about the immune response to Sarbecovirus and important insights into the development of pan-sarbecovirus vaccines and guiding therapeutic interventions.

XAV-19, a Swine Glyco-Humanized Polyclonal Antibody Against SARS-CoV-2 Spike Receptor-Binding Domain, Targets Multiple Epitopes and Broadly Neutralizes Variants.

Amino acid substitutions and deletions in the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBMs), in direct contact sites with the angiotensin converting enzyme-2 (ACE-2). Therefore, in Spike/ACE-2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study on Vero E6 cells conducted over 1 month, no mutation was associated with the addition of increasing doses of XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE-2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (COVID-19) including the different variants of concern identified so far.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2 in humans and mice.

The increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the results of SARS-CoV-2 RBD, the serological response of SARS-CoV-2 NTD is less cross-reactive with SARS-CoV, a pandemic strain that was identified in 2003. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers a supplementary strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID-19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrates, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3-galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate-N-acetylneuraminic acid hydroxylase and α1,3-galactosyl-transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor binding domain to produce glyco-humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3-galactose epitopes. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized spike/angiotensin converting enzyme-2 interaction at a concentration <1 µg/mL, and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. We also found that pig GH-pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage-dependent exacerbated inflammatory responses and a possible antibody-dependent enhancement. These data and the accumulating safety advantages of using GH-pAbs in humans warrant clinical assessment of XAV-19 against COVID-19.

Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections.

The World Health Organization has recently declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, as pandemic. There is currently a lack of knowledge in the antibody response elicited from SARS-CoV-2 infection. One major immunological question is concerning the antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by using plasma from patients infected by SARS-CoV-2 or SARS-CoV, and plasma obtained from infected or immunized mice. Our results show that while cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses is rare, indicating the presence of non-neutralizing antibody response to conserved epitopes in the spike. Whether these non-neutralizing antibody responses will lead to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding the antigenicity differences between SARS-CoV-2 and SARS-CoV, but also has important implications in vaccine.

Cross-reactive Antibody Response between SARS-CoV-2 and SARS-CoV Infections.

The World Health Organization has declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, a pandemic. There is currently a lack of knowledge about the antibody response elicited from SARS-CoV-2 infection. One major immunological question concerns antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by analyzing plasma from patients infected by SARS-CoV-2 or SARS-CoV and from infected or immunized mice. Our results show that, although cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses may be rare, indicating the presence of a non-neutralizing antibody response to conserved epitopes in the spike. Whether such low or non-neutralizing antibody response leads to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding antigenicity differences between SARS-CoV-2 and SARS-CoV but also has implications for immunogen design and vaccine development.

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore determined the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein at 3.1-angstrom resolution. CR3022 targets a highly conserved epitope, distal from the receptor binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBDs on the trimeric S protein are in the "up" conformation and slightly rotated. These results provide molecular insights into antibody recognition of SARS-CoV-2.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1µg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

Cross-reactive Antibody Response between SARS-CoV-2 and SARS-CoV Infections.

The World Health Organization has declared the ongoing outbreak of COVID-19, which is caused by a novel coronavirus SARS-CoV-2, a pandemic. There is currently a lack of knowledge about the antibody response elicited from SARS-CoV-2 infection. One major immunological question concerns antigenic differences between SARS-CoV-2 and SARS-CoV. We address this question by analyzing plasma from patients infected by SARS-CoV-2 or SARS-CoV and from infected or immunized mice. Our results show that, although cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the live viruses may be rare, indicating the presence of a non-neutralizing antibody response to conserved epitopes in the spike. Whether such low or non-neutralizing antibody response leads to antibody-dependent disease enhancement needs to be addressed in the future. Overall, this study not only addresses a fundamental question regarding antigenicity differences between SARS-CoV-2 and SARS-CoV but also has implications for immunogen design and vaccine development.

Influenza A Virus Infections in Dromedary Camels, Nigeria and Ethiopia, 2015-2017.

We examined nasal swabs and serum samples acquired from dromedary camels in Nigeria and Ethiopia during 2015-2017 for evidence of influenza virus infection. We detected antibodies against influenza A(H1N1) and A(H3N2) viruses and isolated an influenza A(H1N1)pdm09-like virus from a camel in Nigeria. Influenza surveillance in dromedary camels is needed.

Diversity of Dromedary Camel Coronavirus HKU23 in African Camels Revealed Multiple Recombination Events among Closely Related Betacoronaviruses of the Subgenus Embecovirus.

Genetic recombination has frequently been observed in coronaviruses. Here, we sequenced multiple complete genomes of dromedary camel coronavirus HKU23 (DcCoV-HKU23) from Nigeria, Morocco, and Ethiopia and identified several genomic positions indicative of cross-species virus recombination events among other betacoronaviruses of the subgenus Embecovirus (clade A beta-CoVs). Recombinant fragments of a rabbit coronavirus (RbCoV-HKU14) were identified at the hemagglutinin esterase gene position. Homolog fragments of a rodent CoV were also observed at 8.9-kDa open reading frame 4a at the 3' end of the spike gene. The patterns of recombination differed geographically across the African region, highlighting a mosaic structure of DcCoV-HKU23 genomes circulating in dromedaries. Our results highlighted active recombination of coronaviruses circulating in dromedaries and are also relevant to the emergence and evolution of other betacoronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV).IMPORTANCE Genetic recombination is often demonstrated in coronaviruses and can result in host range expansion or alteration in tissue tropism. Here, we showed interspecies events of recombination of an endemic dromedary camel coronavirus, HKU23, with other clade A betacoronaviruses. Our results supported the possibility that the zoonotic pathogen MERS-CoV, which also cocirculates in the same camel species, may have undergone similar recombination events facilitating its emergence or may do so in its future evolution.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface.