Quantitative assay to analyze neutralization and inhibition of authentic Middle East respiratory syndrome coronavirus.

To date, there is no licensed vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV). Therefore, MERS-CoV is one of the diseases targeted by the Coalition for Epidemic Preparedness Innovations (CEPI) vaccine development programs and has been classified as a priority disease by the World Health Organization (WHO). An important measure of vaccine immunogenicity and antibody functionality is the detection of virus-neutralizing antibodies. We have developed and optimized a microneutralization assay (MNA) using authentic MERS-CoV and standardized automatic counting of virus foci. Compared to our standard virus neutralization assay, the MNA showed improved sensitivity when analyzing 30 human sera with good correlation of results (Spearman's correlation coefficient r = 0.8917, p value < 0.0001). It is important to use standardized materials, such as the WHO international standard (IS) for anti-MERS-CoV immunoglobulin G, to compare the results from clinical trials worldwide. Therefore, in addition to the neutralizing titers (NT50 = 1384, NT80 = 384), we determined the IC50 and IC80 of WHO IS in our MNA to be 0.67 IU/ml and 2.6 IU/ml, respectively. Overall, the established MNA is well suited to reliably quantify vaccine-induced neutralizing antibodies with high sensitivity.

Passive Transfer of Animal-Derived Polyclonal Hyperimmune Antibodies Provides Protection of Mice from Lethal Lassa Virus Infection.

BACKGROUND: Lassa virus (LASV) can cause severe acute systemic infection in humans. No approved antiviral drugs or vaccines are currently available. Antibody-based therapeutics are considered a promising treatment strategy in the management of LASV disease. METHODS: We used chimeric Ifnar-/- C57BL/6 (Ifnar-/- Bl6) mice, a lethal LASV mouse model, to evaluate the protective efficacy of polyclonal antibodies purified from sera of rabbits hyperimmunized with virus-like particles displaying native-like LASV glycoprotein GP spikes. RESULTS: Polyclonal anti-LASV GP antibodies provided 100% protection against lethal LASV infection in a pre- and post-exposure treatment setting and prevented LASV disease. Treatment also significantly lowered viremia level and virus load in organs. When treatment was initiated at the onset of symptoms, the hyperimmune antibodies provided partial protection and increased the survival rate by 80%. CONCLUSIONS: Our findings support the consideration of animal-derived hyperimmune antibodies targeting GP as an effective treatment option for highly pathogenic LASV.

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies.

Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.

Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses.

Lassa fever continues to be a major public health burden in endemic countries in West Africa, yet effective therapies or vaccines are lacking. The isolation of potent and protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccines candidates have generally been unsuccessful in doing so and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron-microscopy based epitope mapping pipeline that enables high-resolution structural characterization of polyclonal antibodies to GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization which involve epitopes of the GPC-C, GPC-A, and GP1-A competition clusters. Furthermore, by identifying previously undescribed immunogenic off-target epitopes, we expose challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.

Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection.

The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.

Induction of robust cellular and humoral immunity against SARS-CoV-2 after a third dose of BNT162b2 vaccine in previously unresponsive older adults.

Here we compared SARS-CoV-2-specific antibody and T-cell responses between older adults (>80 years old, n = 51) and a younger control group (20-53 years old, n = 46) after receiving two doses of BNT162b2. We found that responses in older adults were generally lower, and we identified 10% low-/non-responders. After receiving a third vaccination with BNT162b2, 4 out of 5 low-/non-responders showed antibody and T-cell responses similar to those of responders after two vaccinations.

Polymer microarrays rapidly identify competitive adsorbents of virus-like particles.

The emergence of SARS-CoV-2 highlights the global need for platform technologies to enable the rapid development of diagnostics, vaccines, treatments, and personal protective equipment (PPE). However, many current technologies require the detailed mechanistic knowledge of specific material-virion interactions before they can be employed, for example, to aid in the purification of vaccine components or in the design of a more effective PPE. Here, we show that an adaption of a polymer microarray method for screening bacterial-surface interactions allows for the screening of polymers for desirable material-virion interactions. Nonpathogenic virus-like particles including fluorophores are exposed to the arrays in an aqueous buffer as a simple model of virions carried to the surface in saliva/sputum. Competitive binding of Lassa and Rubella virus-like particles is measured to probe the relative binding properties of a selection of copolymers. This provides the first step in the development of a method for the discovery of novel materials with promise for viral binding, with the next being development of this method to assess absolute viral adsorption and assessment of the attenuation of the activity of live virus, which we propose would be part of a material scale up step carried out in high containment facilities, alongside the use of more complex media to represent biological fluids.