The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and ...subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.
Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from ...30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.
Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.
A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined ...by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase-antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days.
A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, ...early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.
The mechanisms used by SARS-CoV-2 to induce major adverse cardiac events (MACE) are unknown. Thus, we aimed to determine if SARS-CoV-2 can induce necrotic cell death to promote MACE in patients with ...severe COVID-19.
This observational prospective cohort study includes experiments with hamsters and human samples from patients with severe COVID-19. Cytokines and serum biomarkers were analysed in human serum. Cardiac transcriptome analyses were performed in hamsters' hearts.
From a cohort of 70 patients, MACE was documented in 26% (18/70). Those who developed MACE had higher Log copies/mL of SARS-CoV-2, troponin-I, and pro-BNP in serum. Also, the elevation of IP-10 and a major decrease in levels of IL-17ɑ, IL-6, and IL-1rɑ were observed. No differences were found in the ability of serum antibodies to neutralise viral spike proteins in pseudoviruses from variants of concern. In hamster models, we found a stark increase in viral titters in the hearts 4 days post-infection. The cardiac transcriptome evaluation resulted in the differential expression of ~ 9% of the total transcripts. Analysis of transcriptional changes in the effectors of necroptosis (mixed lineage kinase domain-like, MLKL) and pyroptosis (gasdermin D) showed necroptosis, but not pyroptosis, to be elevated. An active form of MLKL (phosphorylated MLKL, pMLKL) was elevated in hamster hearts and, most importantly, in the serum of MACE patients.
SARS-CoV-2 identification in the systemic circulation is associated with MACE and necroptosis activity. The increased pMLKL and Troponin-I indicated the occurrence of necroptosis in the heart and suggested necroptosis effectors could serve as biomarkers and/or therapeutic targets. Trial registration Not applicable.
A panel of influenza A viruses expressing chimeric hemagglutinins (cHA) with intragroup or intergroup head/stalk combinations was generated. Viruses were characterized for growth kinetics and ...preservation of stalk epitopes. With a few notable exceptions, cHA viruses behaved similarly to wild-type viruses and maintained stalk epitopes, which indicated their potential as vaccine candidates to induce stalk-specific antibodies.
Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus ...(VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples, and run-to-run variability.
For complete details on the use and execution of this protocol, please refer to Sievers et al. (2022).1
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•Procedure for the titration of SARS-CoV-2 pseudoviruses•Steps to perform a multiplexed SARS-CoV-2 pseudovirus neutralization assay•Simultaneous analysis of two distinct fluorophore-linked pseudoviruses
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus (VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples, and run-to-run variability.
Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can ...protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043.
The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.
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