Community composition of black flies during and after the 2020 vesicular stomatitis virus outbreak in Southern New Mexico, USA.

BACKGROUND: Vesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies (Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel. METHODS: We randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification. RESULTS: DNA barcoding of adult black flies detected four species in 2020: Simulium meridionale (N = 158), S. mediovittatum (N = 83), S. robynae (N = 26) and S. griseum/notatum (N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum, 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections. CONCLUSIONS: Our results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted.

Intranasal mRNA-LNP vaccination protects hamsters from SARS-CoV-2 infection.

Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)-lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding [immunoglobulin G (IgG) and IgA] and neutralizing antibodies. Intranasally vaccinated hamsters also had decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. Together, this study demonstrates successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.

A Lassa virus mRNA vaccine confers protection but does not require neutralizing antibody in a guinea pig model of infection.

Lassa virus is a member of the Arenaviridae family, which causes human infections ranging from asymptomatic to severe hemorrhagic disease with a high case fatality rate. We have designed and generated lipid nanoparticle encapsulated, modified mRNA vaccines that encode for the wild-type Lassa virus strain Josiah glycoprotein complex or the prefusion stabilized conformation of the Lassa virus glycoprotein complex. Hartley guinea pigs were vaccinated with two 10 µg doses, 28 days apart, of either construct. Vaccination induced strong binding antibody responses, specific to the prefusion conformation of glycoprotein complex, which were significantly higher in the prefusion stabilized glycoprotein complex construct group and displayed strong Fc-mediated effects. However, Lassa virus-neutralizing antibody activity was detected in some but not all animals. Following the challenge with a lethal dose of the Lassa virus, all vaccinated animals were protected from death and severe disease. Although the definitive mechanism of protection is still unknown, and assessment of the cell-mediated immune response was not investigated in this study, these data demonstrate the promise of mRNA as a vaccine platform against the Lassa virus and that protection against Lassa virus can be achieved in the absence of virus-neutralizing antibodies.

Overview of Experimental Vaccines and Antiviral Therapeutics for Henipavirus Infection.

Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic paramyxoviruses, which have emerged in recent decades and cause sporadic outbreaks of respiratory and encephalitic disease in Australia and Southeast Asia, respectively. Over two billion people currently live in regions potentially at risk due to the wide range of the Pteropus fruit bat reservoir, yet there are no approved vaccines or therapeutics to protect against or treat henipavirus disease. In recent years, significant progress has been made toward developing various experimental vaccine platforms and therapeutics. Here, we describe these advances for both human and livestock vaccine candidates and discuss the numerous preclinical studies and the few that have progressed to human phase 1 clinical trial and the one approved veterinary vaccine.

Nonhuman Primate Models for Nipah and Hendra Virus Countermeasure Evaluation.

Hendra and Nipah viruses are henipaviruses that have caused lethal human disease in Australia and Malaysia, Bangladesh, India, and the Philippines, respectively. These viruses are considered Category C pathogens by the US Centers for Disease Control. Nipah virus was recently placed on the World Health Organization Research and Development Blueprint Roadmaps for vaccine and therapeutic development. Given the infrequent and unpredictable nature of henipavirus outbreaks licensure of vaccines and therapeutics will likely require an animal model to demonstrate protective efficacy against henipavirus disease. Studies have shown that nonhuman primates are the most accurate model of human henipavirus disease and would be an important component of any application for licensure of a vaccine or antiviral drug under the US FDA Animal Rule. Nonhuman primate model selection and dosing are discussed regarding vaccine and therapeutic studies against henipaviruses.

Ferret Models for Henipavirus Infection.

Henipaviruses are emerging zoonotic viruses that can cause outbreaks of severe respiratory and neurological disease in humans and animals such as horses. The mechanism by which these viruses can cause disease remain largely unknown and to date there are no therapeutics or vaccines approved for use in humans. Nipah virus is listed on the World Health Organization R & D Blueprint list of epidemic threats. In order to advance the availability of effective therapeutics and vaccines and medicines that can be used to save lives and avert large scale crises, animal models are required which recapitulate the disease progression in humans. Ferrets are highly susceptible to infection with henipaviruses and develop both severe respiratory and neurological disease. Therefore, the ferret model is highly suitable for studies into both the pathogenesis of henipaviruses, as well as pre-clinical evaluation of intervention strategies.

The Mucin-Like Domain of the Ebola Glycoprotein Does Not Impact Virulence or Pathogenicity in Ferrets.

BACKGROUND: Ebola virus (EBOV) is considered among the most dangerous viruses with case fatality rates approaching 90% depending on the outbreak. While several viral proteins (VPs) including VP24, VP35, and the soluble glycoprotein are understood to contribute to virulence, less is known of the contribution of the highly variable mucin-like domain (MLD) of EBOV. Early studies have defined a potential role in immune evasion of the MLD by providing a glycan shield to critical glycoprotein residues tied to viral entry. Nonetheless, little is known as to what direct role the MLD plays in acute EBOV disease (EVD). METHODS: We generated an infectious EBOV clone that lacks the MLD and assessed its virulence in ferrets compared with wild-type (WT) virus. RESULTS: No differences in growth kinetics were observed in vitro, nor were there any differences in time to death, viremia, or clinical picture in ferrets infected with recombinant EBOV (rEBOV)-WT or rEBOV-Δmucin. CONCLUSIONS: The EBOV MLD does not play a critical role in acute pathogenesis of EVD in ferrets.

Design and evaluation of neutralizing and fusion inhibitory peptides to Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.

A single intranasal dose of human parainfluenza virus type 3-vectored vaccine induces effective antibody and memory T cell response in the lungs and protects hamsters against SARS-CoV-2.

Respiratory tract vaccination has an advantage of needle-free delivery and induction of mucosal immune response in the portal of SARS-CoV-2 entry. We utilized human parainfluenza virus type 3 vector to generate constructs expressing the full spike (S) protein of SARS-CoV-2, its S1 subunit, or the receptor-binding domain, and tested them in hamsters as single-dose intranasal vaccines. The construct bearing full-length S induced high titers of neutralizing antibodies specific to S protein domains critical to the protein functions. Robust memory T cell responses in the lungs were also induced, which represent an additional barrier to infection and should be less sensitive than the antibody responses to mutations present in SARS-CoV-2 variants. Following SARS-CoV-2 challenge, animals were protected from the disease and detectable viral replication. Vaccination prevented induction of gene pathways associated with inflammation. These results indicate advantages of respiratory vaccination against COVID-19 and inform the design of mucosal SARS-CoV-2 vaccines.

Hemorrhagic Fever Viruses: Pathogenesis and Countermeasures.

Before December 2019 and the COVID-19 pandemic, the general public was to some extent aware that zoonotic viruses can spill over into the human population and cause a disease outbreak [...].

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

Effective treatments against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Monoclonal antibodies have shown promising results in patients. Here, we evaluate the in vivo prophylactic and therapeutic effect of COVA1-18, a neutralizing antibody highly potent against the B.1.1.7 isolate. In both prophylactic and therapeutic settings, SARS-CoV-2 remains undetectable in the lungs of treated hACE2 mice. Therapeutic treatment also causes a reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg-1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 shows very strong antiviral activity in the upper respiratory compartments. Using a mathematical model, we estimate that COVA1-18 reduces viral infectivity by more than 95% in these compartments, preventing lymphopenia and extensive lung lesions. Our findings demonstrate that COVA1-18 has a strong antiviral activity in three preclinical models and could be a valuable candidate for further clinical evaluation.

Comparison of Zaire and Bundibugyo Ebolavirus Polymerase Complexes and Susceptibility to Antivirals through a Newly Developed Bundibugyo Minigenome System.

Members of the genus Ebolavirus cause lethal disease in humans, with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV, and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small-molecule inhibitors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3'-untranslated region) and replication (5'-untranslated region). Various levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus, resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3'-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, posttransfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV than EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex is a viable target for therapeutics. IMPORTANCE Ebolavirus disease is a viral infection and is fatal in 25 to 90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species, but there is still limited information regarding the viral mechanism for these differences. Previous studies suggested that BDBV replicates slower than EBOV, but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system that can be used in a biosafety level 2 laboratory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

Single dose rVSVΔG-JUNVGP vaccine protects guinea pigs against lethal Junin virus challenge.

Junin virus (JUNV) is a pathogen of biodefense importance due to its potential for aerosol transmission and mortality rates reaching 30%. Currently, there are no JUNV vaccines licensed by the United States Food and Drug Administration (FDA) for at-risk individuals. A vaccine based on recombinant vesicular stomatitis virus (rVSV) has been effectively used to prevent Ebola virus disease in humans. Here, we evaluated the protective efficacy of a rVSV expressing the JUNV glycoprotein (rVSVΔG-JUNVGP) in a guinea pig model of lethal JUNV disease. Two groups of guinea pigs, one prime and one prime-boost, were vaccinated with rVSVΔG-JUNVGP; six control animals remained unvaccinated. Survival for prime and prime-boost vaccinated animals was 100% while the challenge virus was uniformly lethal in all control animals. Animals in both vaccine groups developed robust, high avidity IgG antibody titers post-vaccination as well as detectable neutralizing antibodies while control animals failed to develop detectable antibody responses. This study demonstrates for the first time that rVSV expressing the JUNV GP fully protects guinea pigs from lethal JUNV challenge with a single injection vaccine.

Attenuated activation of pulmonary immune cells in mRNA-1273-vaccinated hamsters after SARS-CoV-2 infection.

The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

Ebola vaccine-induced protection in nonhuman primates correlates with antibody specificity and Fc-mediated effects.

Although substantial progress has been made with Ebola virus (EBOV) vaccine measures, the immune correlates of vaccine-mediated protection remain uncertain. Here, five mucosal vaccine vectors based on human and avian paramyxoviruses provided nonhuman primates with varying degrees of protection, despite expressing the same EBOV glycoprotein (GP) immunogen. Each vaccine produced antibody responses that differed in Fc-mediated functions and isotype composition, as well as in magnitude and coverage toward GP and its conformational and linear epitopes. Differences in the degree of protection and comprehensive characterization of the response afforded the opportunity to identify which features and functions were elevated in survivors and could therefore serve as vaccine correlates of protection. Pairwise network correlation analysis of 139 immune- and vaccine-related parameters was performed to demonstrate relationships with survival. Total GP-specific antibodies, as measured by biolayer interferometry, but not neutralizing IgG or IgA titers, correlated with survival. Fc-mediated functions and the amount of receptor binding domain antibodies were associated with improved survival outcomes, alluding to the protective mechanisms of these vaccines. Therefore, functional qualities of the antibody response, particularly Fc-mediated effects and GP specificity, rather than simply magnitude of the response, appear central to vaccine-induced protection against EBOV. The heterogeneity of the response profile between the vaccines indicates that each vaccine likely exhibits its own protective signature and the requirements for an efficacious EBOV vaccine are complex.

Broad and potently neutralizing monoclonal antibodies isolated from human survivors of New World hantavirus infection.

New World hantaviruses (NWHs) are endemic in North and South America and cause hantavirus cardiopulmonary syndrome (HCPS), with a case fatality rate of up to 40%. Knowledge of the natural humoral immune response to NWH infection is limited. Here, we describe human monoclonal antibodies (mAbs) isolated from individuals previously infected with Sin Nombre virus (SNV) or Andes virus (ANDV). Most SNV-reactive antibodies show broad recognition and cross-neutralization of both New and Old World hantaviruses, while many ANDV-reactive antibodies show activity for ANDV only. mAbs ANDV-44 and SNV-53 compete for binding to a distinct site on the ANDV surface glycoprotein and show potently neutralizing activity to New and Old World hantaviruses. Four mAbs show therapeutic efficacy at clinically relevant doses in hamsters. These studies reveal a convergent and potently neutralizing human antibody response to NWHs and suggest therapeutic potential for human mAbs against HCPS.

Broadly neutralizing antibody cocktails targeting Nipah virus and Hendra virus fusion glycoproteins.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses (HNVs) causing respiratory illness and severe encephalitis in humans, with fatality rates of 50-100%. There are no licensed therapeutics or vaccines to protect humans. HeV and NiV use a receptor-binding glycoprotein (G) and a fusion glycoprotein (F) to enter host cells. HNV F and G are the main targets of the humoral immune response, and the presence of neutralizing antibodies is a correlate of protection against NiV and HeV in experimentally infected animals. We describe here two cross-reactive F-specific antibodies, 1F5 and 12B2, that neutralize NiV and HeV through inhibition of membrane fusion. Cryo-electron microscopy structures reveal that 1F5 and 12B2 recognize distinct prefusion-specific, conserved quaternary epitopes and lock F in its prefusion conformation. We provide proof-of-concept for using antibody cocktails for neutralizing NiV and HeV and define a roadmap for developing effective countermeasures against these highly pathogenic viruses.

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge.

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.