Biology subjects

Khalil, A. M.

Publications and source records attributed to Khalil, A. M..

7 recordsLinked to original sources

bioRxiv2025

Identification and characterization of a SARS-CoV-2 Mpro G23 deletion ensitrelvir-resistant mutant

Ensitrelvir is an antiviral drug that specifically targets the main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been approved for the treatment of coronavirus disease 2019 (COVID-19) due to the conservation of its target protein which is essential in the viral lifecycle. However, SARS-CoV-2 could introduce mutations in the viral proteins to confer resistance to antivirals. Thus, screening for drug-resistant SARS-CoV-2 mutants and elucidating their resistant mechanisms are critical for guiding the selection of effective antiviral therapies. Here, we utilized a luminescent attenuated SARS-CoV-2 ({Delta}3a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a G23 deletion (G23del) in Mpro with high resistance (>1,000 fold) to ensitrelvir, but not to the other Mpro inhibitor (nirmatrelvir) or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del was confirmed by generating a recombinant luminescent attenuated SARS-CoV-2 containing G23del in the non-structural protein 5 (NSP5) gene ({Delta}3a7b-Nluc G23del). {Delta}3a7b-Nluc G23del exhibited significant resistance to ensitrelvir in both cultured cells an in K18 hACE2 transgenic mice. Binding affinity revealed that G23del mutation substantially altered Mpro binding affinity for ensitrelvir but not nirmatrelvir. In conclusion, our results demonstrate that G23del in Mpro can confer high resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating infections with SARS-CoV-2 containing G23del with these other approved antivirals. SIGNIFICANCEThe clinical use of SARS-CoV-2 antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for FDA-approved antivirals. Our study addresses this by employing a luminescent attenuated virus platform ({Delta}3a7b-Nluc WT) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we have identified a G23 deletion (G23del) in SARS-CoV-2 Mpro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer more than 1,000-fold increased resistance to ensitrelvir, SARS-CoV-2 containing G23del remained sensitive to other Mpro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using {Delta}3a7b-Nluc to safely identify and characterize drug resistant viruses without the biosafety concern of using virulent SARS-CoV-2 and advance the design of next-generation antiviral drugs.

microbiology↗

bioRxiv2025

Structural Basis for Childhood Antibody Recognition of The Human Metapneumovirus Fusion Protein

Human metapneumovirus (hMPV) is a significant cause of acute respiratory illness in children and adults, with the majority of children being seropositive for hMPV by five years of age. Infants, older adults, and immunocompromised individuals are more susceptible to severe hMPV infections that can lead to hospitalization and death. The hMPV fusion (F) protein is the sole target of neutralizing antibodies, and while the most common neutralizing epitopes on the hMPV F protein targeted by B cells in hMPV-infected adults have been previously determined, the antibody response in hMPV-infected children remains undefined. We isolated a panel of human monoclonal antibodies (mAbs) from children previously infected with hMPV (MPV498, MPV499, MPV510, MPV511, and MPV513), and the mAbs were assessed for binding avidity, neutralization potency, epitope specificity, and in vivo efficacy. All mAbs were neutralizing, and epitope binning revealed the presence of four different epitopes targeted by the mAbs. We determined the cryo-EM structures of four mAbs in complex with the hMPV F protein, which revealed epitopes located on the hMPV F trimer surface as well as an intratrimer epitope located completely within the hMPV F trimer interface. Furthermore, we determined the prophylactic efficacy of the mAbs in protection against hMPV challenge in mice. Overall, our data reveal new insights into the immunodominant antigenic epitopes on the hMPV F protein in children and identify new mAb therapies for hMPV F disease prevention.

immunology↗

bioRxiv2025

Structure and function of a cross-neutralizing influenza neuraminidase antibody that accommodates recent N2 NA Asn245 glycosylation

Monoclonal antibodies (mAbs) that recognize and inhibit a diverse range of influenza viruses, although relatively rare, have been isolated following infection or vaccination. Study of their ontology and mechanisms of action informs universal vaccine and therapeutic development. We have previously described a potent and broad neuraminidase (NA)-neutralizing human mAb, 1122A11, that neutralizes a wide range of H3N2 viruses. Here, further characterization of 1122A11 reveals reactivity to cross-group influenza A virus NAs, including group-1 N1 and N8, and group-2 N2 and N3 NAs. Recent H3N2 viruses have acquired Asn245 glycosylation on the active site rim. Crystal structures of an N2 NA from A/Singapore/INFIMH-16-0019/2016 (H3N2) at 2.3 [A] (apo) and 2.2 [A] (Fab bound) resolution showed that 1122A11 binding causes local changes to the periphery of NA active site to accommodate the glycan. The CDRH3 of 1122A11 inserts into the active site and mimics the substrate sialic acid. We then determined that the ability of 1122A11 to protect from lethal challenge in mice is not dependent on Fc-effector function. These results highlight the therapeutic potential of 1122A11 as a broad protective anti-viral and reinforce pursuit of immunogen development of NA antibodies toward achieving more universal influenza protection.

immunology↗

bioRxiv2025

A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 {Delta}3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. IMPORTANCESmall-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins ({Delta}3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

microbiology↗

bioRxiv2024

A potently neutralizing and protective human antibody targeting antigenic site V on RSV and hMPV fusion glycoprotein

Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are frequent drivers of morbidity and mortality in susceptible populations, most often infantile, older adults, and immunocompromised. The primary target of neutralizing antibodies is the fusion (F) glycoprotein on the surface of the RSV and hMPV virion. As a result of the structural conservation between RSV and hMPV F, three antigenic regions are known to induce cross-neutralizing responses: sites III, IV, and V. Leveraging LIBRA-seq, we identify five RSV/hMPV cross-reactive human antibodies. One antibody, 5-1, potently neutralizes all tested viruses from the major subgroups of RSV and hMPV and provides protection against RSV and hMPV in a mouse challenge model. Structural analysis reveals that 5-1 utilizes an uncommon genetic signature to bind an epitope that spans sites O, II and V, defining a new mode of antibody cross-reactivity between RSV and hMPV F. These findings highlight the molecular and structural elements influencing RSV and hMPV cross-reactivity as well as the potential of antibody 5-1 for translational development.

immunology↗

bioRxiv2024

Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1[A] resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

immunology↗

bioRxiv2020

Myeloid-derived suppressor cell subsets drive glioblastoma growth in a sex-specific manner

Myeloid-derived suppressor cells (MDSCs) that block anti-tumor immunity are elevated in glioblastoma (GBM) patients. However, the distinct contribution of monocytic (mMDSC) versus granulocytic (gMDSC) subsets has yet to be determined. We observed that mMDSCs were enriched in the male tumor microenvironment, while gMDSCs were elevated in the circulation of female GBM models. Depletion of peripheral gMDSCs extended the survival only in female mice. Using gene expression signatures coupled with network medicine analysis, we demonstrated in pre-clinical models that mMDSCs could be targeted with anti-proliferative agents in males, whereas gMDSC function in females could be inhibited by IL-1{beta} blockade. Analysis of patient data confirmed that proliferating mMDSCs were the predominant population in male tumors, and that a high gMDSC/IL-1{beta} gene signature correlated with poor prognosis of female patients. These findings demonstrate that MDSC subsets differentially drive immune suppression in a sex-specific manner and can be leveraged for therapeutic intervention in GBM. Statement of SignificanceSexual dimorphism at the level of MDSC subset prevalence, localization and gene expression profile comprises a therapeutic opportunity. Our results indicate that chemotherapy can be used to target mMDSC in males, while IL-1 pathway inhibitors can provide benefit to females through blockade of gMDSC function.

immunology↗