Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.

SARS-CoV-2 Aerosol and Intranasal Exposure Models in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the worldwide COVID-19 pandemic. Animal models are extremely helpful for testing vaccines and therapeutics and for dissecting the viral and host factors that contribute to disease severity and transmissibility. Here, we report the assessment and comparison of intranasal and small particle (~3 µm) aerosol SARS-CoV-2 exposure in ferrets. The primary endpoints for analysis were clinical signs of disease, recovery of the virus in the upper respiratory tract, and the severity of damage within the respiratory tract. This work demonstrated that ferrets were productively infected with SARS-CoV-2 following either intranasal or small particle aerosol exposure. SARS-CoV-2 infection of ferrets resulted in an asymptomatic disease course following either intranasal or small particle aerosol exposure, with no clinical signs, significant weight loss, or fever. In both aerosol and intranasal ferret models, SARS-CoV-2 replication, viral genomes, and viral antigens were detected within the upper respiratory tract, with little to no viral material detected in the lungs. The ferrets exhibited a specific IgG immune response to the SARS-CoV-2 full spike protein. Mild pathological findings included inflammation, necrosis, and edema within nasal turbinates, which correlated to positive immunohistochemical staining for the SARS-CoV-2 virus. Environmental sampling was performed following intranasal exposure of ferrets, and SARS-CoV-2 genomic material was detected on the feeders and nesting areas from days 2-10 post-exposure. We conclude that both intranasal and small particle aerosol ferret models displayed measurable parameters that could be utilized for future studies, including transmission studies and testing SARS-CoV-2 vaccines and therapeutics.

Seroprevalence as an Indicator of Undercounting of COVID-19 Cases in a Large Well-Described Cohort.

Introduction: Reported confirmed cases represent a small portion of overall true cases for many infectious diseases. The undercounting of true cases can be considerable when a significant portion of infected individuals are asymptomatic or minimally symptomatic, as is the case with COVID-19. Seroprevalence studies are an efficient way to assess the extent to which true cases are undercounted during a large-scale outbreak and can inform efforts to improve case identification and reporting. Methods: A longitudinal seroprevalence study of active duty U.S. military members was conducted from May 2020 through June 2021. A random selection of service member serum samples submitted to the Department of Defense Serum Repository was analyzed for the presence of antibodies reactive to SARS-CoV-2. The monthly seroprevalence rates were compared with those of cumulative confirmed cases reported during the study period. Results: Seroprevalence was 2.3% in May 2020 and increased to 74.0% by June 2021. The estimated true case count based on seroprevalence was 9.3 times greater than monthly reported cases at the beginning of the study period and fell to 1.7 by the end of the study. Conclusions: In our sample, confirmed case counts significantly underestimated true cases of COVID-19. The increased availability of testing over the study period and enhanced efforts to detect asymptomatic and minimally symptomatic cases likely contributed to the fall in the seroprevalence to reported case ratio.

Development of a specific MPXV antigen detection immunodiagnostic assay.

Human monkeypox (mpox) has recently become a global public health emergency; however, assays that detect mpox infection are not widely available, largely due to cross-reactivity within the Orthopoxvirus genus. Immunoassay development was largely confined to researchers who focus on biothreats and endemic areas (Central and West Africa) until the 2022 outbreak. As was noted in the COVID-19 pandemic, antigen detection assays, integrated with molecular assays, are necessary to help curb the spread of disease. Antigen-detecting immunoassays offer the advantage of providing results ranging from within min to h and in lateral flow formats; they can be deployed for point-of-care, home, or field use. This study reports the development of an mpox-specific antigen detection immunoassay developed on a multiplexed, magnetic-bead-based platform utilizing reagents from all research sectors (commercial, academic, and governmental). Two semi-quantitative assays were developed in parallel and standardized with infectious mpox virus (MPXV) cell culture fluid and MPXV-positive non-human primate (NHP) sera samples. These assays could detect viral antigens in serum, were highly specific toward MPXV as compared to other infectious orthopoxviruses (vaccinia virus, cowpox virus, and camelpox virus), and exhibited a correlation with quantitative PCR results from an NHP study. Access to a toolbox of assays for mpox detection will be key for identifying cases and ensuring proper treatment, as MPXV is currently a global traveler.

Induced protection from a CCHFV-M DNA vaccine requires CD8+ T cells.

Crimean-Congo hemorrhagic fever (CCHF) is a World Health Organization prioritized disease because its broad distribution and severity of disease make it a global health threat. Despite advancements in preclinical vaccine development for CCHF virus (CCHFV), including multiple platforms targeting multiple antigens, a clear definition of the adaptive immune correlates of protection is lacking. Levels of neutralizing antibodies in vaccinated animal models do not necessarily correlate with protection, suggesting that cellular immunity, such as CD8+ T cells, might have an important role in protection in this model. Using a well-established IFN-I antibody blockade mouse model (IS) and a DNA-based vaccine encoding the CCHFV M-segment glycoprotein precursor, we investigated the role of humoral and T cell immunity in vaccine-mediated protection in mice genetically devoid of these immune compartments. We found that in the absence of the B-cell compartment (µMT knockout mice), protection provided by the vaccine was not reduced. In contrast, in the absence of CD8+ T cells (CD8+ knockout mice) the vaccine-mediated protection was significantly diminished. Importantly, humoral responses to the vaccine in CD8+ T-cell knockout mice were equivalent to wild-type mice. These findings indicated that CD8+ T-cell responses are necessary and sufficient to promote protection in mice vaccinated with the M-segment DNA vaccine. Identifying a crucial role of the cellular immunity to protect against CCHFV should help guide the development of CCHFV-targeting vaccines.

A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model.

The COVID-19 pandemic has had a staggering impact on social, economic, and public health systems worldwide. Vaccine development and mobilization against SARS-CoV-2 (the etiologic agent of COVID-19) has been rapid. However, novel strategies are still necessary to slow the pandemic, and this includes new approaches to vaccine development and/or delivery that will improve vaccination compliance and demonstrate efficacy against emerging variants. Here, we report on the immunogenicity and efficacy of a SARS-CoV-2 vaccine comprising stabilized, pre-fusion spike protein trimers displayed on a ferritin nanoparticle (SpFN) adjuvanted with either conventional aluminum hydroxide or the Army Liposomal Formulation QS-21 (ALFQ) in a cynomolgus macaque COVID-19 model. Vaccination resulted in robust cell-mediated and humoral responses and a significant reduction in lung lesions following SARS-CoV-2 infection. The strength of the immune response suggests that dose sparing through reduced or single dosing in primates may be possible with this vaccine. Overall, the data support further evaluation of SpFN as a SARS-CoV-2 protein-based vaccine candidate with attention to fractional dosing and schedule optimization.

Exposure Route Influences Disease Severity in the COVID-19 Cynomolgus Macaque Model.

The emergence of SARS-CoV-2 and the subsequent pandemic has highlighted the need for animal models that faithfully replicate the salient features of COVID-19 disease in humans. These models are necessary for the rapid selection, testing, and evaluation of potential medical countermeasures. Here, we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure-combined intratracheal/intranasal and small particle aerosol-in two nonhuman primate species, rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models produced suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.

Antibody Response to SARS-CoV-2 Infection and Vaccination in COVID-19-naïve and Experienced Individuals.

Understanding the magnitude of responses to vaccination during the ongoing SARS-CoV-2 pandemic is essential for ultimate mitigation of the disease. Here, we describe a cohort of 102 subjects (70 COVID-19-naïve, 32 COVID-19-experienced) who received two doses of one of the mRNA vaccines (BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)). We document that a single exposure to antigen via infection or vaccination induces a variable antibody response which is affected by age, gender, race, and co-morbidities. In response to a second antigen dose, both COVID-19-naïve and experienced subjects exhibited elevated levels of anti-spike and SARS-CoV-2 neutralizing activity; however, COVID-19-experienced individuals achieved higher antibody levels and neutralization activity as a group. The COVID-19-experienced subjects exhibited no significant increase in antibody or neutralization titer in response to the second vaccine dose (i.e., third antigen exposure). Finally, we found that COVID-19-naïve individuals who received the Moderna vaccine exhibited a more robust boost response to the second vaccine dose (p = 0.004) as compared to the response to Pfizer-BioNTech. Ongoing studies with this cohort will continue to contribute to our understanding of the range and durability of responses to SARS-CoV-2 mRNA vaccines.

Human convalescent plasma protects K18-hACE2 mice against severe respiratory disease.

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.

Transcriptomic Analysis Reveals Host miRNAs Correlated with Immune Gene Dysregulation during Fatal Disease Progression in the Ebola Virus Cynomolgus Macaque Disease Model.

Ebola virus is a continuing threat to human populations, causing a virulent hemorrhagic fever disease characterized by dysregulation of both the innate and adaptive host immune responses. Severe cases are distinguished by an early, elevated pro-inflammatory response followed by a pronounced lymphopenia with B and T cells unable to mount an effective anti-viral response. The precise mechanisms underlying the dysregulation of the host immune system are poorly understood. In recent years, focus on host-derived miRNAs showed these molecules to play an important role in the host gene regulation arsenal. Here, we describe an investigation of RNA biomarkers in the fatal Ebola virus disease (EVD) cynomolgus macaque model. We monitored both host mRNA and miRNA responses in whole blood longitudinally over the disease course in these non-human primates (NHPs). Analysis of the interactions between these classes of RNAs revealed several miRNA markers significantly correlated with downregulation of genes; specifically, the analysis revealed those involved in dysregulated immune pathways associated with EVD. In particular, we noted strong interactions between the miRNAs hsa-miR-122-5p and hsa-miR-125b-5p with immunological genes regulating both B and T-cell activation. This promising set of biomarkers will be useful in future studies of severe EVD pathogenesis in both NHPs and humans and may serve as potential prognostic targets.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV): A Silent but Widespread Threat.

PURPOSE OF REVIEW: This review is aimed at highlighting recent research and articles on the complicated relationship between virus, vector, and host and how biosurveillance at each level informs disease spread and risk. RECENT FINDINGS: While human cases of CCHFV and tick identification in non-endemic areas in 2019-2020 were reported to sites such as ProMed, there is a gap in recent published literature on these and broader CCHFV surveillance efforts from the late 2010s. SUMMARY: A review of the complex aspects of CCHFV maintenance in the environment coupled with high fatality rate and lack of vaccines and therapeutics warrants the need for a One-Health approach toward detection and increased biosurveillance programs for CCHFV.

Development of a coronavirus disease 2019 nonhuman primate model using airborne exposure.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

Persistent Crimean-Congo hemorrhagic fever virus infection in the testes and within granulomas of non-human primates with latent tuberculosis.

Crimean-Congo hemorrhagic fever (CCHF) is the most medically important tick-borne viral disease of humans and tuberculosis is the leading cause of death worldwide by a bacterial pathogen. These two diseases overlap geographically, however, concurrent infection of CCHF virus (CCHFV) with mycobacterial infection has not been assessed nor has the ability of virus to persist and cause long-term sequela in a primate model. In this study, we compared the disease progression of two diverse strains of CCHFV in the recently described cynomolgus macaque model. All animals demonstrated signs of clinical illness, viremia, significant changes in clinical chemistry and hematology values, and serum cytokine profiles consistent with CCHF in humans. The European and Asian CCHFV strains caused very similar disease profiles in monkeys, which demonstrates that medical countermeasures can be evaluated in this animal model against multiple CCHFV strains. We identified evidence of CCHFV persistence in the testes of three male monkeys that survived infection. Furthermore, the histopathology unexpectedly revealed that six additional animals had evidence of a latent mycobacterial infection with granulomatous lesions. Interestingly, CCHFV persisted within the granulomas of two animals. This study is the first to demonstrate the persistence of CCHFV in the testes and within the granulomas of non-human primates with concurrent latent tuberculosis. Our results have important public health implications in overlapping endemic regions for these emerging pathogens.

Development of a bead-based immunoassay using virus-like particles for detection of alphaviral humoral response.

There is a pressing need for sustainable and sensitive immunodiagnostics for use in public health efforts to understand and combat the threat of endemic and emerging infectious diseases. In this proof-of-concept work, we describe an immunodiagnostic approach based on the utilization of virus-like particles (VLPs) in a magnetic bead-based platform for multiplexed detection of antiviral humoral response. A retroviral-based VLP, that presents Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was synthesized and coupled to magnetic beads to create VLP-conjugated microspheres (VCMs). Using these VCMs, IgM and IgG antibodies were detectable in nonhuman primate (NHP) and human clinical serum samples at dilutions of 1 × 10 Basile et al. [4] and greater. We also extended the VCM methodology to an Old World alphavirus, chikungunya virus, demonstrating the flexibility of this approach toward different VLP architectures. When multiplexed on the MAGPIX® platform, this method provided differential detection between Old World and New World alphaviral IgM. This flexible, immunodiagnostic method, based on the MAGPIX® platform, demonstrates compatibility of particulate antigens with bead-based assays, improves sensitivity by up to 2-logs, and has faster sample-to-answer time over traditional methods.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/µl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection.

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found an increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.

Simultaneous capture and sequential detection of two malarial biomarkers on magnetic microparticles.

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous.

Direct transfer of HRPII-magnetic bead complexes to malaria rapid diagnostic tests significantly improves test sensitivity.

BACKGROUND: The characteristic ease of use, rapid time to result, and low cost of malaria rapid diagnostic tests (RDTs) promote their widespread use at the point-of-care for malaria detection and surveillance. However, in many settings, the success of malaria elimination campaigns depends on point-of-care diagnostics with greater sensitivity than currently available RDTs. To address this need, a sample preparation method was developed to deliver more biomarkers onto a malaria RDT by concentrating the biomarker from blood sample volumes that are too large to be directly applied to a lateral flow strip. METHODS: In this design, Ni-NTA-functionalized magnetic beads captured the Plasmodium falciparum biomarker HRPII from a P. falciparum D6 culture spiked blood sample. This transfer of magnetic beads to the RDT was facilitated by an inexpensive 3D-printed apparatus that aligned the sample tube with the sample deposition pad and a magnet beneath the RDT. Biomarkers were released from the bead surface onto the lateral flow strip using imidazole-spiked running buffer. Kinetics of HRPII binding to the Ni-NTA beads as a function of blood sample volume were explored prior to determining the effect of the proposed method on the limit of detection of Paracheck RDTs. RESULTS: More than 80 % of HRPII biomarkers were extracted from blood sample volumes ranging from 25 to 250 µL. The time required to reach 80 % binding ranged from 5 to 60 min, depending on sample volume. Using 250 µL of blood and a 30-min biomarker binding time, the limit of detection of the Paracheck Pf RDT brand was improved by 21-fold, resulting in a limit of detection below 1 parasite/µL. CONCLUSIONS: This approach has the sensitivity and simplicity required to assist in malaria elimination campaigns in settings with limited access to clinical and laboratory resources.