Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Case of Human Orthohantavirus Infection, Michigan, USA, 2021.

Orthohantaviruses cause hantavirus cardiopulmonary syndrome; most cases occur in the southwest region of the United States. We discuss a clinical case of orthohantavirus infection in a 65-year-old woman in Michigan and the phylogeographic link of partial viral fragments from the patient and rodents captured near the presumed site of infection.

ICTV Virus Taxonomy Profile: Filoviridae 2024.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.

Wastewater threshold as an indicator of COVID-19 cases in correctional facilities for public health response: A modeling study.

Although prison facilities are not fully isolated from the communities in which they are located, most of the population is confined and requires high levels of health vigilance and protection. This study aimed to examine the dynamic relationship between facility-level wastewater viral concentrations and the probability of at least one positive COVID-19 case within the facility. The study period was from January 11, 2021 to May 8, 2023. Wastewater samples were collected and analyzed for SARS-CoV-2 (N1) and pepper mild mottle virus (PMMoV) three times weekly across 14 prison facilities in Kentucky (USA). Positive clinical case reports were also provided. A hierarchical Bayesian facility-level temporal model with a latent lagged process was developed. We modeled facility-specific SARS-CoV-2 (N1) normalized by the PMMoV wastewater concentration ratio threshold associated with at least one COVID-19 clinical case at an 80 % probability. The threshold differed among facilities. Across the 14 facilities, our model demonstrates a mean capture rate of 94.95 % via the N1/PMMoV ratio threshold with pts≥0.5. However, as the pts threshold was set higher, such as at ≥0.9, the mean capture rate of the model was reduced to 60 %. This robust performance underscores the effectiveness of the model for accurately detecting the presence of positive COVID-19 cases among incarcerated people. The findings of this study provide a facility-specific threshold model for public health response based on frequent wastewater surveillance.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

Fine-tuning spatial-temporal dynamics and surface receptor expression support plasma cell-intrinsic longevity.

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4 + helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines.

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Prefusion-stabilized SARS-CoV-2 S2-only antigen provides protection against SARS-CoV-2 challenge.

Ever-evolving SARS-CoV-2 variants of concern (VOCs) have diminished the effectiveness of therapeutic antibodies and vaccines. Developing a coronavirus vaccine that offers a greater breadth of protection against current and future VOCs would eliminate the need to reformulate COVID-19 vaccines. Here, we rationally engineer the sequence-conserved S2 subunit of the SARS-CoV-2 spike protein and characterize the resulting S2-only antigens. Structural studies demonstrate that the introduction of interprotomer disulfide bonds can lock S2 in prefusion trimers, although the apex samples a continuum of conformations between open and closed states. Immunization with prefusion-stabilized S2 constructs elicits broadly neutralizing responses against several sarbecoviruses and protects female BALB/c mice from mouse-adapted SARS-CoV-2 lethal challenge and partially protects female BALB/c mice from mouse-adapted SARS-CoV lethal challenge. These engineering and immunogenicity results should inform the development of next-generation pan-coronavirus therapeutics and vaccines.

Engineering, structure, and immunogenicity of a Crimean-Congo hemorrhagic fever virus pre-fusion heterotrimeric glycoprotein complex.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause severe disease in humans with case fatality rates of 10-40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we used structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-EM structure of this complex provides the molecular basis for GP38's association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development.

A VHH single-domain platform enabling discovery and development of monospecific antibodies and modular neutralizing bispecifics against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.

Distinct pathway for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses.

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles' heels that could be targeted with countermeasures.

Antibodies targeting Crimean-Congo hemorrhagic fever virus GP38 limit vascular leak and viral spread.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen transmitted by tick bites, with no vaccines or specific therapeutics approved to date. Severe disease manifestations include hemorrhage, endothelial dysfunction, and multiorgan failure. Infected cells secrete the viral glycoprotein GP38, whose extracellular function is presently unknown. GP38 is considered an important target for vaccine and therapeutic design as GP38-specific antibodies can protect against severe disease in animal models, albeit through a currently unknown mechanism of action. Here, we show that GP38 induces endothelial barrier dysfunction in vitro, and that CCHFV infection, and GP38 alone, can trigger vascular leak in a mouse model. Protective antibodies that recognize specific antigenic sites on GP38, but not a protective neutralizing antibody binding the structural protein Gc, potently inhibit endothelial hyperpermeability in vitro and vascular leak in vivo during CCHFV infection. This work uncovers a function of the secreted viral protein GP38 as a viral toxin in CCHFV pathogenesis and elucidates the mode of action of non-neutralizing GP38-specific antibodies.

Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38.

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, exclusive to Nairoviridae, is a target of protective antibodies and is a key antigen in preclinical vaccine candidates. Here, we isolate 188 GP38-specific antibodies from human survivors of infection. Competition experiments show that these antibodies bind across 5 distinct antigenic sites, encompassing 11 overlapping regions. Additionally, we show structures of GP38 bound with 9 of these antibodies targeting different antigenic sites. Although these GP38-specific antibodies are non-neutralizing, several display protective efficacy equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and may inform the development of broadly effective CCHFV antibody therapeutics.

Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38.

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, unique to Nairoviridae, is a target of protective antibodies, but extensive mapping of the human antibody response to GP38 has not been previously performed. Here, we isolated 188 GP38-specific antibodies from human survivors of infection. Competition experiments showed that these antibodies bind across five distinct antigenic sites, encompassing eleven overlapping regions. Additionally, we reveal structures of GP38 bound with nine of these antibodies targeting different antigenic sites. Although GP38-specific antibodies were non-neutralizing, several antibodies were found to have protection equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and inform the development of broadly effective CCHFV antibody therapeutics.