Seroprevalence of Arboviruses in a Malaria Hyperendemic Area in Southern Mali.

Diagnostics for febrile illnesses other than malaria are not readily available in rural sub-Saharan Africa. This study assessed exposure to three mosquito-borne arboviruses-dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV)-in southern Mali. Seroprevalence for DENV, CHIKV, and ZIKV was analyzed by detection of IgG antibodies and determined to be 77.2%, 31.2%, and 25.8%, respectively. Among study participants, 11.3% were IgG-positive for all three arboviruses. DENV had the highest seroprevalence rate at all sites; the highest seroprevalence of CHIKV and ZIKV was observed in Bamba. The seroprevalence for all three arboviruses increased with age, and the highest seroprevalence was observed among adults older than 50 years. The prevalence of Plasmodium spp. in the cohort was analyzed by microscopy and determined to be 44.5% (N = 600) with Plasmodium falciparum representing 95.1% of all infections. This study demonstrates the co-circulation of arboviruses in a region hyperendemic for malaria and highlights the needs for arbovirus diagnostics in rural sub-Saharan Africa.

Detection of mpox virus in wastewater provides forewarning of clinical cases in Canadian cities.

Wastewater-based surveillance (WBS) has shown to be an effective tool in monitoring the spread of SARS-CoV-2 and has helped guide public health actions. Consequently, WBS has expanded to now include the monitoring of mpox virus (MPXV) to contribute to its mitigation efforts. In this study, we demonstrate a unique sample processing and a molecular diagnostic strategy for MPXV detection that can inform on the epidemiological situation of mpox outbreaks through WBS. We conducted WBS for MPXV in 22 Canadian wastewater treatment plants (WWTPs) for 14 weeks. Three MPXV qPCR assays were assessed in this study for the detection of MPXV which include the G2R assays (G2R_WA and G2R_G) developed by the Centers for Disease Control and Prevention (CDC) in 2010, and an in-house-developed assay that we have termed G2R_NML. The G2R_NML assay was designed using reference genomes from the 2022 MPXV outbreak and provides a larger qPCR amplicon size to facilitate Sanger sequencing. Results show that all three assays have similar limits of detection and are able to detect the presence of MPXV in wastewater. The G2R_NML assay produced a significantly greater number of Sanger sequence-confirmed MPXV results compared to the CDC G2R assays. Detection of MPXV was possible where provincial surveillance indicated overall low caseloads, and in some sites forewarning of up to several weeks was observed. Overall, this study proposes that WBS of MPXV provides additional information to help fill knowledge gaps in clinical case-surveillance and is potentially an essential component to the management of mpox.

Protective Efficacy of Lyophilized Vesicular Stomatitis Virus-Based Vaccines in Animal Model.

We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.

Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.

Bivalent VSV Vectors Mediate Rapid and Potent Protection from Andes Virus Challenge in Hamsters.

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human-human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination.

The rVSV-EBOV vaccine provides limited cross-protection against Sudan virus in guinea pigs.

Recombinant vesicular stomatitis viruses (rVSVs) engineered to express heterologous viral glycoproteins have proven to be remarkably effective vaccines. Indeed, rVSV-EBOV, which expresses the Ebola virus (EBOV) glycoprotein, recently received clinical approval in the United States and Europe for its ability to prevent EBOV disease. Analogous rVSV vaccines expressing glycoproteins of different human-pathogenic filoviruses have also demonstrated efficacy in pre-clinical evaluations, yet these vaccines have not progressed far beyond research laboratories. In the wake of the most recent outbreak of Sudan virus (SUDV) in Uganda, the need for proven countermeasures was made even more acute. Here we demonstrate that an rVSV-based vaccine expressing the SUDV glycoprotein (rVSV-SUDV) generates a potent humoral immune response that protects guinea pigs from SUDV disease and death. Although the cross-protection generated by rVSV vaccines for different filoviruses is thought to be limited, we wondered whether rVSV-EBOV might also provide protection against SUDV, which is closely related to EBOV. Surprisingly, nearly 60% of guinea pigs that were vaccinated with rVSV-EBOV and challenged with SUDV survived, suggesting that rVSV-EBOV offers limited protection against SUDV, at least in the guinea pig model. These results were confirmed by a back-challenge experiment in which animals that had been vaccinated with rVSV-EBOV and survived EBOV challenge were inoculated with SUDV and survived. Whether these data are applicable to efficacy in humans is unknown, and they should therefore be interpreted cautiously. Nevertheless, this study confirms the potency of the rVSV-SUDV vaccine and highlights the potential for rVSV-EBOV to elicit a cross-protective immune response.

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.

Experimental Infection of North American Deer Mice with Clade I and II Monkeypox Virus Isolates.

The global spread of monkeypox virus has raised concerns over the establishment of novel enzootic reservoirs in expanded geographic regions. We demonstrate that although deer mice are permissive to experimental infection with clade I and II monkeypox viruses, the infection is short-lived and has limited capability for active transmission.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

An Outbred Guinea Pig Disease Model for Lassa Fever Using a Host-Adapted Clade III Nigerian Lassa Virus.

Nigeria experiences annual outbreaks of Lassa fever (LF) with high case numbers. At least three clades of Lassa virus (LASV) have been documented in Nigeria, though recent outbreaks are most often associated with clade II or clade III viruses. Using a recently isolated clade III LASV from a case of LF in Nigeria in 2018, we developed and characterized a guinea pig adapted virus capable of causing lethal disease in commercially available Hartley guinea pigs. Uniform lethality was observed after four passages of the virus and was associated with only two dominant genomic changes. The adapted virus was highly virulent with a median lethal dose of 10 median tissue culture infectious doses. Disease was characterized by several hallmarks of LF in similar models including high fever, thrombocytopenia, coagulation disorders, and increased inflammatory immune mediators. High viral loads were noted in all solid organ specimens analyzed. Histological abnormalities were most striking in the lungs and livers of terminal animals and included interstitial inflammation, edema, and steatosis. Overall, this model represents a convenient small animal model for a clade III Nigeria LASV with which evaluation of specific prophylactic vaccines and medical countermeasures can be conducted.

In vitro and in vivo efficacy of tecovirimat against a recently emerged 2022 monkeypox virus isolate.

The recent emergence of the monkeypox virus (MPXV) in non-endemic countries has been designated a Public Health Emergency of International Concern by the World Health Organization. There are currently no approved treatments for MPXV infection in the United States or Canada. The antiviral drug tecovirimat (commonly called TPOXX), previously approved for smallpox treatment, is currently being deployed for treatment of MPXV infections where available based on previously accrued data. We tested the efficacy of TPOXX both in vitro and in vivo against a clade 2 Canadian 2022 isolate of MPXV isolated during the current outbreak. TPOXX prevented MPXV replication in vitro with an effective concentration in the nanomolar range. To evaluate TPOXX efficacy in vivo, we first characterized the CAST/EiJ mouse model with the same 2022 Canadian isolate. Unlike previous descriptions of this model, the Canadian isolate was not lethal in CAST/EiJ mice, although it replicated efficiently in the respiratory tract after intranasal infection. Subsequent experiments demonstrated that daily oral TPOXX treatment markedly reduced viral titers in the tissues 1 and 2 weeks after infection. Our data indicate that TPOXX is highly effective against currently circulating MPXV strains and could be an important contributor to curbing the ongoing outbreak.

An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

AAV-monoclonal antibody expression protects mice from Ebola virus without impeding the endogenous antibody response to heterologous challenge.

Filoviruses cause severe hemorrhagic fever with case fatality rates as high as 90%. Filovirus-specific monoclonal antibodies (mAbs) confer protection in nonhuman primates as late as 5 days after challenge, and FDA-approved mAbs REGN-EB3 and mAb114 have demonstrated efficacy against Ebola virus (EBOV) infection in humans. Vectorized antibody expression mediated by adeno-associated virus (AAV) can generate protective and sustained concentrations of therapeutic mAbs in animal models for a variety of infectious diseases, including EBOV. Here we demonstrate that AAV6.2FF-mediated expression of murine IgG2a EBOV mAbs, 2G4 and 5D2, protects from mouse-adapted (MA)-EBOV infection with none of the surviving mice developing anti-VP40 antibodies above background. Protective serum concentrations of AAV6.2FF-2G4/AAV6.2FF-5D2 did not alter endogenous antibody responses to heterologous virus infection. AAV-mediated expression of EBOV mAbs 100 and 114, and pan-ebolavirus mAbs, FVM04, ADI-15878, and CA45, as human IgG1 antibodies conferred protection against MA-EBOV at low serum concentrations, with minimum protective serum levels as low as 2 µg/mL. Vectorized expression of murine IgG2a or human IgG1 mAbs led to sustained expression in the serum of mice for >400 days or for the lifetime of the animal, respectively. AAV6.2FF-mediated mAb expression offers an alternative to recombinant antibody administration in scenarios where long-term protection is preferable to passive immunization.

Temporal analysis of Lassa virus infection and transmission in experimentally infected Mastomys natalensis.

Little is known about the temporal patterns of infection and transmission of Lassa virus (LASV) within its natural reservoir (Mastomys natalensis). Here, we characterize infection dynamics and transmissibility of a LASV isolate (Soromba-R) in adult lab-reared M. natalensis originating from Mali. The lab-reared M. natalenesis proved to be highly susceptible to LASV isolates from geographically distinct regions of West Africa via multiple routes of exposure, with 50% infectious doses of < 1 TCID50. Postinoculation, LASV Soromba-R established a systemic infection with no signs of clinical disease. Viral RNA was detected in all nine tissues examined with peak concentrations detected between days 7 and 14 postinfection within most organs. There was an overall trend toward clearance of virus within 40 days of infection in most organs. The exception is lung specimens, which retained positivity throughout the course of the 85-day study. Direct (contact) and indirect (fomite) transmission experiments demonstrated 40% of experimentally infected M. natalensis were capable of transmitting LASV to naïve animals, with peak transmissibility occurring between 28 and 42 days post-inoculation. No differences in patterns of infection or transmission were noted between male and female experimentally infected rodents. Adult lab-reared M. natalensis are highly susceptible to genetically distinct LASV strains developing a temporary asymptomatic infection associated with virus shedding resulting in contact and fomite transmission within a cohort.

Pandemic 1918 Influenza Virus Does Not Cause Lethal Infection in Rhesus or Cynomolgus Macaques.

The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy. IMPORTANCE The world remains at risk of an influenza pandemic, and the development of new therapeutic and preventative modalities is critically important for minimizing human death and suffering during the next influenza pandemic. Animal models are central to the development of new therapies and vaccine approaches. In particular, nonhuman primates like rhesus and cynomolgus macaques are highly relevant preclinical models given their physiological and immunological similarities to humans. Unfortunately, there remains a scarcity of macaque models of pandemic influenza with which to test novel antiviral modalities. Here, we demonstrate that even at the highest doses tested, 1918 influenza was not lethal in these two macaque species, suggesting that they are not ideal for the development and testing of novel pandemic influenza-specific vaccines and therapies. Therefore, other physiologically relevant nonhuman primate models of pandemic influenza are needed.

Experimental Infection of Peromyscus Species Rodents with Sin Nombre Virus.

We demonstrate that 6 distinct Peromyscus rodent species are permissive to experimental infection with Sin Nombre orthohantavirus (SNV). Viral RNA and SNV antibodies were detected in members of all 6 species. P. leucopus mice demonstrated markedly higher viral and antibody titers than P. maniculatus mice, the established primary hosts for SNV.

Japanese encephalitis virus persists in the human reproductive epithelium and porcine reproductive tissues.

Japanese encephalitis virus (JEV) is the emerging and geographically expanding flavivirus and the major causative agent of encephalitis in humans in Asia. There are risks of JEV introduction into the Americas given a large population of amplifying hosts-pigs and wild boars, and insect vectors-Culex mosquitoes. There are emerging concerns about vector-free ways of flavivirus transmission, for example sexual and transplacental Zika virus transmissions, which may change flavivirus epidemiology and expand the geographical range to territories with no insect vectors. It is unknown whether JEV has tropism in the female lower reproductive tract and the potential for sexual transmission in humans. While clinical outcomes of transplacental JEV infection are described in humans and pigs, cellular targets and tissue tropism in the upper reproductive tract are also unknown. Here, we studied JEV infection phenotypes and host transcriptional responses in human reproductive epithelial cells. We found that JEV caused persistent infection and cytopathology in the vaginal epithelium, endometrial epithelium, and trophoblast. Human vaginal epithelial cells infected with JEV had altered transcriptional responses associated with inflammation and disruption of epithelial barrier function. Also, using pigs-the native amplifying host for JEV, we confirmed JEV tropism in the female lower and upper reproductive tracts. We discovered that JEV persists in the vaginal mucosa for at least 28 days and pigs shed the virus in vaginal secretions. We also found JEV persistence in the endometrium and placenta with transplacental and fetal infections. Altogether, we discovered that JEV targets the vaginal epithelium and has the potential for sexual transmission in humans. We also contributed to a better understanding of JEV pathogenesis during transplacental infection. Further studies are needed to better understand the interactions of JEV with reproductive tissues, how persistent infection affects female reproductive functions, and the risks for non-vector transmission.

Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice.

Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.

The fusion peptide is the only universally conserved epitope in both IAV and IBVMono-specific universal antibody induces strong ADCC against human and avian IAVMono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBVThe antibody has bi-functional effector functions against several influenza viruses.

Single Immunization with Recombinant ACAM2000 Vaccinia Viruses Expressing the Spike and the Nucleocapsid Proteins Protects Hamsters against SARS-CoV-2-Caused Clinical Disease.

Increasing cases of SARS-CoV-2 breakthrough infections from immunization with current spike protein-based COVID-19 vaccines highlight the need to develop alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins based on a novel vaccinia virus (VACV) ACAM2000 platform (rACAM2000). In this platform, the vaccinia virus host range and immunoregulatory gene E3L was deleted to make the virus attenuated and to enhance innate immune responses, and another host range gene, K3L, was replaced with a poxvirus ortholog gene, taterapox virus 037 (TATV037), to make virus replication competent in both hamster and human cells. Following a single intramuscular immunization, the rACAM2000 coexpressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and shorter recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titer, and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate, and further studies will investigate if the rACAM2000 vaccine candidate can induce a long-lasting immunity against infection by SARS-CoV-2 variants of concern. IMPORTANCE Continuous emergence of SARS-CoV-2 variants which cause breakthrough infection from the immunity induced by current spike protein-based COVID-19 vaccines highlights the need for new generations of vaccines that will induce long-lasting immunity against a wide range of the variants. To this end, we investigated the protective efficacy of the recombinant COVID-19 vaccine candidates based on a novel VACV ACAM2000 platform, in which an immunoregulatory gene, E3L, was deleted and both the SARS-CoV-2 spike (S) and nucleocapsid (N) antigens were expressed. Thus, it is expected that the vaccine candidate we constructed should be more immunogenic and safer. In the initial study described in this work, we demonstrated that the vaccine candidate expressing both the S and N proteins is superior to the constructs expressing an individual protein (S or N) in protecting hamsters against SARS-CoV-2 challenge after a single-dose immunization, and further investigation against different SARS-CoV-2 variants will warrant future clinical evaluations.