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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.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19.In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.HighlightsLY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic fever disease in humans. Currently, no licensed CCHF vaccines exist, and the protective epitopes remain unclear. Previously, we tested a DNA vaccine expressing the M-segment glycoprotein precursor gene of the laboratory CCHFV strain IbAr 10200 (CCHFV-M10200). CCHFV-M10200 provided >60% protection against homologous CCHFV-IbAr 10200 challenge in mice. Here, we report that increasing the dose of CCHFV-M10200 provides complete protection from homologous CCHFV challenge in mice, and significant (80%) protection from challenge with the clinically relevant heterologous strain CCHFV-Afg09-2990. We also report complete protection from CCHFV-Afg09-2990 challenge following vaccination with a CCHFV-Afg09-2990 M-segment DNA vaccine (CCHFV-MAfg09). Finally, we show that the non-structural M-segment protein, GP38, influences CCHF vaccine immunogenicity and provides significant protection from homologous CCHFV challenge. Our results demonstrate that M-segment DNA vaccines elicit protective CCHF immunity and further illustrate the immunorelevance of GP38.

The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs., A highly lethal murine infection model for SARS-CoV-2 using mice transgenic for the human ACE2 protein is described.

Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne viruses in the Americas that cause central nervous system (CNS) disease in humans and equids. In this study, we directly characterized the pathogenesis of VEEV, EEEV, and WEEV in cynomolgus macaques following subcutaneous exposure because this route more closely mimics natural infection via mosquito transmission or by an accidental needle stick. Our results highlight how EEEV is significantly more pathogenic compared to VEEV similarly to what is observed in humans. Interestingly, EEEV appears to be just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies. In contrast, subcutaneous exposure of cynomolgus macaques with WEEV caused limited disease and is contradictory to what has been reported for aerosol exposure. Several differences in viremia, hematology, or tissue tropism were noted when animals were exposed subcutaneously compared to prior aerosol exposure studies. This study provides a more complete picture of the pathogenesis of the encephalitic alphaviruses and highlights how further defining the neuropathology of these viruses could have important implications for the development of medical countermeasures for the neurovirulent alphaviruses.

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.

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. We describe a novel approach to immunodiagnostics based on virus-like particles (VLPs) attached to magnetic beads. This flexible, innovative immunoassay system, based on the MAGPIX® platform, improves sensitivity by up to 2-logs and has faster sample-to-answer time over traditional methods. As a proof of concept, a retroviral-based VLP, that presents the Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was generated and coupled to magnetic beads to create VLP-conjugated microspheres (VCMs). Using these VCMs, IgG and IgM antibodies were detectable in nonhuman primate (NHP) and human clinical serum samples at dilutions of 1 × 104 and greater. We extended the VCM methodology to two other New-World alphaviruses, eastern and western equine encephalitis viruses, as well as an Old-World alphavirus, Chikungunya virus, demonstrating the flexibility of this approach toward different VLP architectures. When multiplexed on the MAGPIX® platform, the VCMs provided differential diagnosis between Old-World and New-World alphaviruses and well as a route toward assessing the humoral response to both natural infection and vaccination. This VCM system will allow more rapid and efficient detection of endemic and emerging viral pathogens in human populations.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7–10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV., Author summary Crimean-Congo hemorrhagic Fever Virus (CCHFV) is a tick-borne virus capable of causing lethal human disease against which there are currently no approved vaccines. In this study, we compared the immunogenicity and protective efficacy of a candidate DNA vaccine expressing the glycoprotein precursor gene of CCHFV in two mouse models. In addition to the recently established IFNAR-/- mouse pathogenesis model, we also tested the vaccine in a novel murine system in which the interferon (IFN) α/β signaling response of immunocompetent mice is transiently suppressed. We found that the DNA vaccine elicited high humoral immune responses and provided significant protection against challenge with CCHFV in both mouse models. These findings further our understanding of the requirements for a CCHFV vaccine and provide a new mouse model for the development of CCHFV countermeasures.

Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.

We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.

To determine if DNA vaccines for two hantaviruses causing hemorrhagic fever with renal syndrome, Hantaan virus and Puumala virus, are immunogenic when given in combination, we delivered them to hamsters separately or as mixtures by gene gun or by electroporation. Both vaccines elicited neutralizing antibodies when given alone but when they were delivered as a mixture, antibodies to only one of the two hantaviruses could be detected. In contrast, if the DNAs were given as separate vaccinations to a single animal, responses to both were observed. These studies suggest that the two DNA vaccines will need to be given as separate administrations.

The Ebola virus (EBOV) envelope glycoprotein (GP) is the primary target of protective immunity. Mature GP consists of two disulfide-linked subunits, GP1 and membrane-bound GP2. GP is highly glycosylated with both N- and O-linked carbohydrates. We measured the influences of GP glycosylation on antigenicity, immunogenicity, and protection by testing DNA vaccines comprised of GP genes with deleted N-linked glycosylation sites or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP. Our data indicate that this is likely due to the inability of GP2 and GP1 to dimerize at the cell surface and suggest that glycosylation at this site is required for achieving the conformational integrity of GP2 and GP1. In contrast, mutation of two N-linked sites on GP1, which flank previously defined protective antibody epitopes on GP, may enhance immunogenicity, possibly by unmasking epitopes. We further showed that although deleting the mucin region apparently had no effect on antigenicity in vitro, it negatively impacted the elicitation of protective immunity in mice. In addition, we confirmed the presence of previously identified B-cell and T-cell epitopes in GP but show that when analyzed individually none of them were neither absolutely required nor sufficient for protective immunity to EBOV. Finally, we identified other potential regions of GP that may contain relevant antibody or T-cell epitopes.

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

The identification of an effective and tolerable delivery method is a necessity for the success of DNA vaccines in the clinic. This manuscript describes the development and validation of a multi-headed intradermal electroporation device which would be applicable for delivering multiple DNA vaccine plasmids simultaneously but spatially separated. Reporter gene plasmids expressing green and red fluorescent proteins were used to demonstrate the impact of spatial separation on DNA delivery to increase the number of transfected cells and avoid interference through visible expression patterns. To investigate the impact of plasmid interference on immunogenicity, a disease target was investigated where issues with multi-valent vaccines had been previously described. DNA-based Hantaan and Puumala virus vaccines were delivered separately or as a combination and the effect of multi-valence was determined by appropriate assays. While a negative impact was observed for both antigenic vaccines when delivered together, these effects were mitigated when the vaccine was delivered using the multi-head device. We also demonstrate how the multi-head device facilitates higher dose delivery to the skin resulting in improved immune responses. This new multi-head platform device is an efficient, tolerable and non-invasive method to deliver multiple plasmid DNA constructs simultaneously allowing the tailoring of delivery sites for combination vaccines. Additionally, this device would allow the delivery of multi-plasmid vaccine formulations without risk of impacted immune responses through interference. Such a low-cost, easy to use device platform for the delivery of multi-agent DNA vaccines would have direct applications by the military and healthcare sectors for mass vaccination purposes.

For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors., Author Summary Entry into a cell is an essential stage of the viral replication cycle. Enveloped viruses require fusion of viral and cellular membranes for the viral genome to enter the cell cytoplasm. This entry is mediated by a viral fusion protein. Here, we synthesized peptides based on the Rift Valley fever virus (RVFV) fusion protein stem region and tested the peptides for their ability to prevent RVFV infection of cell cultures. We found that one of these peptides was able to inhibit RVFV infectivity by preventing the fusion process and that this peptide had broad activity against other RNA viruses including Ebola, Andes, and vesicular stomatitis viruses.

We have developed a rapid, reliable, and sensitive quantitative flow cytometric assay to measure the in vitro potency and stability of DNA vaccines to be delivered either by particle-mediated epidermal delivery (PMED) or by electroporation. The method involves transfecting cells with test DNA and comparing the measured antigen expression to that generated with expression from known quantities of reference material DNA. The assay was adapted for performance under Good Laboratory Practice (GLP) guidelines and was successfully utilized to perform potency testing in support of a Phase I study for two hantavirus DNA vaccines delivered by gene gun. The results from the potency assays conducted over a 24-month period using this method proved to be highly reproducible with high signal-to-noise ratios. The assay was also adapted to assess the in vitro potency and stability of a DNA vaccine for Venezuelan equine encephalitis virus that will be delivered by electroporation. Our results indicate that this assay can be readily applied to support potency and stability testing of numerous DNA vaccines delivered by various methods, including multiagent vaccines.