SARS-CoV-2 neutralizing antibody epitopes are overlapping and highly mutated which raises the chances of escape variants and requires development of broadly reactive vaccines.

The rapid adaptation of SARS-CoV-2 within the host species and the increased viral transmission triggered the evolution of different SARS-CoV-2 variants. Though numerous monoclonal antibodies (mAbs) have been identified as prophylactic therapy for SARS-CoV-2, the ongoing surge in the number of SARS-CoV-2 infections shows the importance of understanding the mutations in the spike and developing novel vaccine strategies to target all variants. Here, we report the map of experimentally validated 74 SARS-CoV-2 neutralizing mAb binding epitopes of all variants. The majority (87.84%) of the potent neutralizing epitopes are localized to the receptor-binding domain (RBD) and overlap with each other, whereas limited (12.16%) epitopes are found in the N-terminal domain (NTD). Notably, 69 out of 74 mAb targets have at least one mutation at the epitope sites. The potent epitopes found in the RBD show higher mutations (4-10aa) compared to lower or modest neutralizing antibodies, suggesting that these epitopes might co-evolve with the immune pressure. The current study shows the importance of determining the critical mutations at the antibody recognition epitopes, leading to the development of broadly reactive immunogens targeting multiple SARS-CoV-2 variants. Further, vaccines inducing both humoral and cell-mediated immune responses might prevent the escape of SARS-CoV-2 variants from neutralizing antibodies.

SARS-CoV-2 Cellular Entry Is Independent of the ACE2 Cytoplasmic Domain Signaling.

Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH4Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development.

BACKGROUND: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. METHODS: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. FINDINGS: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. INTERPRETATION: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.

Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein.

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro, and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs.IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro; (iii) seven of the nonessential proteins affect viral growth in vitro, and two affect virulence in vivo; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins.

Identification of HCV Resistant Variants against Direct Acting Antivirals in Plasma and Liver of Treatment Naïve Patients.

Current standard-of-care treatment of chronically infected hepatitis C virus (HCV) patients involves direct-acting antivirals (DAA). However, concerns exist regarding the emergence of drug -resistant variants and subsequent treatment failure. In this study, we investigate potential natural drug-resistance mutations in the NS5B gene of HCV genotype 1b from treatment-naïve patients. Population-based sequencing and 454 deep sequencing of NS5B gene were performed on plasma and liver samples obtained from 18 treatment- naïve patients. The quasispecies distribution in plasma and liver samples showed a remarkable overlap in each patient. Although unique sequences in plasma or liver were observed, in the majority of cases the most dominant sequences were shown to be identical in both compartments. Neither in plasma nor in the liver codon changes were detected at position 282 that cause resistance to nucleos(t)ide analogues. However, in 10 patients the V321I change conferring resistance to nucleos(t)ide NS5B polymerase inhibitors and in 16 patients the C316N/Y/H non-nucleoside inhibitors were found mainly in liver samples. In conclusion, 454-deep sequencing of liver and plasma compartments in treatment naïve patients provides insight into viral quasispecies and the pre-existence of some drug-resistant variants in the liver, which are not necessarily present in plasma.

Genetic diversity of hepatitis C virus in Ethiopia.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.

Middle East respiratory syndrome coronavirus vaccines: current status and novel approaches.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. The re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. Ongoing efforts to develop MERS-CoV vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. Extending on our current knowledge of MERS, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish MERS-CoV transmission to humans. In addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next CoV outbreak.

Naturally occurring recombination in ferret coronaviruses revealed by complete genome characterization.

Ferret coronaviruses (FRCoVs) exist as an enteric and a systemic pathotype, of which the latter is highly lethal to ferrets. To our knowledge, this study provides the first full genome sequence of a FRCoV, tentatively called FRCoV-NL-2010, which was detected in 2010 in ferrets in The Netherlands. Phylogenetic analysis showed that FRCoV-NL-2010 is most closely related to mink CoV, forming a separate clade of mustelid alphacoronavirus that split off early from other alphacoronaviruses. Based on sequence homology of the complete genome, we propose that these mustelid coronaviruses may be assigned to a new species. Comparison of FRCoV-NL-2010 with the partially sequenced ferret systemic coronavirus MSU-1 and ferret enteric coronavirus MSU-2 revealed that recombination in the spike, 3c and envelope genes occurred between different FRCoVs.

Deletion Variants of Middle East Respiratory Syndrome Coronavirus from Humans, Jordan, 2015.

We characterized Middle East respiratory syndrome coronaviruses from a hospital outbreak in Jordan in 2015. The viruses from Jordan were highly similar to isolates from Riyadh, Saudi Arabia, except for deletions in open reading frames 4a and 3. Transmissibility and pathogenicity of this strain remains to be determined.

Cross host transmission in the emergence of MERS coronavirus.

Coronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact that closely related viruses have been isolated from this but not any other animal species. Given the widespread geographical distribution of dromedaries found seropositive for MERS-CoV, continued transmission may likely occur in the future. Therefore, a further understanding of the cross host transmission of MERS-CoV is needed to limit the risks this virus poses to man.

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels.

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor--dipeptidyl peptidase 4 (DPP4)--is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.

Molecular epidemiology and genetic diversity of hepatitis B virus in Ethiopia.

Although hepatitis B virus (HBV) infection is hyperendemic in Ethiopia and constitutes a major public health problem, little is known about its genetic diversity, genotypes, and circulation. The aim of this study was to determine the molecular epidemiology and genetic diversity of HBV in Ethiopia, using 391 serum samples collected from HBsAg-positive blood donors living in five different geographic regions. The HBV S/pol gene was amplified, sequenced, and HBV genotypes, subgenotypes, serotypes, and major hydrophilic region (MHR) variants were determined. Phylogenetic analysis of 371 samples (95%) revealed the distribution of genotypes A (78%) and D (22%) in Ethiopia. Further phylogenetic analysis identified one subgenotype (A1) within genotype A, and 4 subgenotypes within genotype D (D1; 1.3%, D2; 55%, D4; 2.5%, and D6; 8.8%). Importantly, 24 isolates (30%) of genotype D formed a novel phylogenetic cluster, distinct from any known D subgenotypes, and two A/D recombinants. Analysis of predicted amino-acid sequences within the HBsAg revealed four serotypes: adw2 (79%), ayw1 (3.1%), ayw2 (7.8%), and ayw3 (11.6%). Subsequent examination of sequences showed that 51 HBV isolates (14%) had mutations in the MHR and 8 isolates (2.2%) in the reverse transcriptase known to confer antiviral resistance. This study provides the first description of HBV genetic diversity in Ethiopia with a predominance of subgenotypes A1 and D2, and also identified HBV isolates that could represent a novel subgenotype. Furthermore, a significant prevalence of HBsAg variants in Ethiopian population is revealed.

An orthopoxvirus-based vaccine reduces virus excretion after MERS-CoV infection in dromedary camels.

Middle East respiratory syndrome coronavirus (MERS-CoV) infections have led to an ongoing outbreak in humans, which was fueled by multiple zoonotic MERS-CoV introductions from dromedary camels. In addition to the implementation of hygiene measures to limit further camel-to-human and human-to-human transmissions, vaccine-mediated reduction of MERS-CoV spread from the animal reservoir may be envisaged. Here we show that a modified vaccinia virus Ankara (MVA) vaccine expressing the MERS-CoV spike protein confers mucosal immunity in dromedary camels. Compared with results for control animals, we observed a significant reduction of excreted infectious virus and viral RNA transcripts in vaccinated animals upon MERS-CoV challenge. Protection correlated with the presence of serum neutralizing antibodies to MERS-CoV. Induction of MVA-specific antibodies that cross-neutralize camelpox virus would also provide protection against camelpox.