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.

Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.

Production and Purification of Filovirus Glycoproteins.

Ebola (EBOV) and Marburg (MARV) viruses cause hemorrhagic fever disease in humans and non-human primates (NHPs) with case-fatality rates as high as 90%. The 2013-2016 Ebola virus disease (EVD) outbreak led to over 28,000 cases and 11,000 deaths and took an enormous toll on the economy of West African nations, in the absence of any vaccine or therapeutic options. Like EVD, there have been at least 6 outbreaks of MVD with ~88% case-fatality and the most recent cases emerging in Equatorial Guinea in February 2023. These outbreaks have spurred an unprecedented global effort to develop vaccines and therapeutics for EVD and MVD and led to an approved vaccine (ERVEBO™) and two monoclonal antibody (mAb) therapeutics for EBOV. In contrast to EVD, therapeutic options against Marburg and another Ebola-relative Sudan virus (SUDV) are lacking. The filovirus glycoprotein (GP), which mediates host cell entry and fusion, is the primary target of neutralizing antibodies. In addition to its pre- and post-fusion trimeric states, the protein is highly glycosylated making production of pure and homogeneous trimers on a large scale, a requirement for subunit vaccine development, a challenge. In efforts to address this roadblock, we have developed a unique combination of structure-based design, selection of expression system, and purification methods to produce uniform and stable EBOV and MARV GP trimers at scales appropriate for vaccine production.

A Flexible, Quantitative Plasmonic-Fluor Lateral Flow Assay for the Rapid Detection of Orthoebolavirus zairense and Orthoebolavirus sudanense.

Filoviruses comprise a family of single-stranded, negative-sense RNA viruses with a significant impact on human health. Given the risk for disease outbreaks, as highlighted by the recent outbreaks across Africa, there is an unmet need for flexible diagnostic technologies that can be deployed in resource-limited settings. Herein, we highlight the use of plasmonic-fluor lateral flow assays (PF-LFA) for the rapid, quantitative detection of an Ebolavirus-secreted glycoprotein, a marker for infection. Plasmonic fluors are a class of ultrabright reporter molecules that combine engineered nanorods with conventional fluorophores, resulting in improved analytical sensitivity. We have developed a PF-LFA for Orthoebolavirus zairense (EBOV) and Orthoebolavirus sudanense (SUDV) that provides estimated limits of detection as low as 0.446 and 0.641 ng/mL, respectively. Furthermore, our assay highlights a high degree of specificity between the two viral species while also maintaining a turnaround time as short as 30 min. To highlight the utility of our PF-LFA, we demonstrate the detection of EBOV infection in non-human primates. Our PF-LFA represents an enormous step forward in the development of a robust, field-deployable assay for filoviruses.

Targeting RET Solvent-Front Mutants with Alkynyl Nicotinamide-Based Inhibitors.

Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

Detection of biomarkers for filoviral infection with a silicon photonic resonator platform.

This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022).

Evidence of Neutralizing and Non-Neutralizing Anti-Glucosaminidase Antibodies in Patients With S. Aureus Osteomyelitis and Their Association With Clinical Outcome Following Surgery in a Clinical Pilot.

Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC50) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 µg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/- neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted.

Rapid detection of an Ebola biomarker with optical microring resonators.

Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.

Wall Teichoic Acids Facilitate the Release of Toxins from the Surface of Staphylococcus aureus.

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures; however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilizing a genome-wide association approach, we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA are required for the release of several cytolytic toxins from the bacterial cells. We show that this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to the process of toxin release by a major human pathogen while also demonstrating that clinical isolates can utilize WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. IMPORTANCE The production and release of cytolytic toxins is a critical aspect for the pathogenicity of many bacterial pathogens. In this study, we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aureus cells into the extracellular environment. Our findings suggest that this effect is mediated by a gradient of electrostatic charge which the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

Antigenic landscapes on Staphylococcus aureus pore-forming toxins reveal insights into specificity and cross-neutralization.

Staphylococcus aureus carries an exceptional repertoire of virulence factors that aid in immune evasion. Previous single-target approaches for S. aureus-specific vaccines and monoclonal antibodies (mAbs) have failed in clinical trials due to the multitude of virulence factors released during infection. Emergence of antibiotic-resistant strains demands a multi-target approach involving neutralization of different, non-overlapping pathogenic factors. Of the several pore-forming toxins that contribute to S. aureus pathogenesis, efforts have largely focused on mAbs that neutralize α-hemolysin (Hla) and target the receptor-binding site. Here, we isolated two anti-Hla and three anti-Panton-Valentine Leukocidin (LukSF-PV) mAbs, and used a combination of hydrogen deuterium exchange mass spectrometry (HDX-MS) and alanine scanning mutagenesis to delineate and validate the toxins' epitope landscape. Our studies identified two novel, neutralizing epitopes targeted by 2B6 and CAN6 on Hla that provided protection from hemolytic activity in vitro and showed synergy in rodent pneumonia model against lethal challenge. Of the anti-LukF mAbs, SA02 and SA131 showed specific neutralization activity to LukSF-PV while SA185 showed cross-neutralization activity to LukSF-PV, γ-hemolysin HlgAB, and leukotoxin ED. We further compared these antigen-specific mAbs to two broadly neutralizing mAbs, H5 (targets Hla, LukSF-PV, HlgAB, HlgCB, and LukED) and SA185 (targeting LukSF-PV, HlgAB, and LukED), and identified molecular level markers for broad-spectrum reactivity among the pore-forming toxins by HDX-MS. To further underscore the need to target the cross-reactive epitopes on leukocidins for the development of broad-spectrum therapies, we annotated Hla sequences isolated from patients in multiple countries for genomic variations within the perspective of our defined epitopes.

Hyperimmune Targeting Staphylococcal Toxins Effectively Protect Against USA 300 MRSA Infection in Mouse Bacteremia and Pneumonia Models.

Staphylococcus aureus has been acquiring multiple drug resistance and has evolved into superbugs such as Methicillin/Vancomycin-resistant S. aureus (MRSA/VRSA) and, consequently, is a major cause of nosocomial and community infections associated with high morbidity and mortality for which no FDA-approved vaccines or biotherapeutics are available. Previous efforts targeting the surface-associated antigens have failed in clinical testing. Here, we generated hyperimmune products from sera in rabbits against six major S. aureus toxins targeted by an experimental vaccine (IBT-V02) and demonstrated significant efficacy for an anti-virulence passive immunization strategy. Extensive in vitro binding and neutralizing titers were analyzed against six extracellular toxins from individual animal sera. All IBT-V02 immunized animals elicited the maximum immune response upon the first boost dose against all pore-forming vaccine components, while for superantigen (SAgs) components of the vaccine, second and third doses of a boost were needed to reach a plateau in binding and toxin neutralizing titers. Importantly, both anti-staphylococcus hyperimmune products consisting of full-length IgG (IBT-V02-IgG) purified from the pooled sera and de-speciated F(ab')2 (IBT-V02-F(ab')2) retained the binding and neutralizing titers against IBT-V02 target toxins. F(ab')2 also exhibited cross-neutralization titers against three leukotoxins (HlgAB, HlgCB, and LukED) and four SAgs (SEC1, SED, SEK, and SEQ) which were not part of IBT-V02. F(ab')2 also neutralized toxins in bacterial culture supernatant from major clinical strains of S. aureus. In vivo efficacy data generated in bacteremia and pneumonia models using USA300 S. aureus strain demonstrated dose-dependent protection by F(ab')2. These efficacy data confirmed the staphylococcal toxins as viable targets and support the further development effort of hyperimmune products as a potential adjunctive therapy for emergency uses against life-threatening S. aureus infections.

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort.

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 "critical" (in the intensive care unit under mechanical ventilation) and 25 "non-critical" (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Staphylococcus aureus adaptive evolution: Recent insights on how immune evasion, immunometabolic subversion and host genetics impact vaccine development.

Despite meritorious attempts, a S. aureus vaccine that prevents infection or mitigates severity has not yet achieved efficacy endpoints in prospective, randomized clinical trials. This experience underscores the complexity of host-S. aureus interactions, which appear to be greater than many other bacterial pathogens against which successful vaccines have been developed. It is increasingly evident that S. aureus employs strategic countermeasures to evade or exploit human immune responses. From entering host cells to persist in stealthy intracellular reservoirs, to sensing the environmental milieu and leveraging bacterial or host metabolic products to reprogram host immune responses, S. aureus poses considerable challenges for the development of effective vaccines. The fact that this pathogen causes distinct types of infections and can undergo transient genetic, transcriptional or metabolic adaptations in vivo that do not occur in vitro compounds challenges in vaccine development. Notably, the metabolic versatility of both bacterial and host immune cells as they compete for available substrates within specific tissues inevitably impacts the variable repertoire of gene products that may or may not be vaccine antigens. In this respect, S. aureus has chameleon phenotypes that have alluded vaccine strategies thus far. Nonetheless, a number of recent studies have also revealed important new insights into pathogenesis vulnerabilities of S. aureus. A more detailed understanding of host protective immune defenses versus S. aureus adaptive immune evasion mechanisms may offer breakthroughs in the development of effective vaccines, but at present this goal remains a very high bar. Coupled with the recent advances in human genetics and epigenetics, newer vaccine technologies may enable such a goal. If so, future vaccines that protect against or mitigate the severity of S. aureus infections are likely to emerge at the intersection of precision and personalized medicine. For now, the development of S. aureus vaccines or alternative therapies that reduce mortality and morbidity must continue to be pursued.

Cross-Strain Neutralizing and Protective Monoclonal Antibodies against EEEV or WEEV.

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Therapy for Argentine hemorrhagic fever in nonhuman primates with a humanized monoclonal antibody.

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.

The sphingosine kinase 1 activator, K6PC-5, attenuates Ebola virus infection.

Ebola virus (EBOV) is responsible for outbreaks with case fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. Here, by employing pseudotyped EBOV-GP viral particles, we uncover a critical role for sphingolipids in inhibiting viral entry. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in diverse cell lines. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells and the de novo production of viral proteins. These data present K6PC-5 as an efficient tool to inhibit EBOV infection in endothelial cells and suggest further studies to evaluate its systemic effects.

IBT-V02: A Multicomponent Toxoid Vaccine Protects Against Primary and Secondary Skin Infections Caused by Staphylococcus aureus.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Safety and Immunogenicity of a 4-Component Toxoid-Based Staphylococcus aureus Vaccine in Rhesus Macaques.

Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.