A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge.

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

Innate immune responses elicited by Sin Nombre virus or type I IFN agonists protect hamsters from lethal Andes virus infections.

Sin Nombre virus (SNV) and Andes virus (ANDV) cause hantavirus pulmonary syndrome (HPS) in humans. Both SNV and ANDV infect Syrian hamsters, but only ANDV causes lethal disease. A co-infection study was performed to determine which virus, SNV or ANDV, would dominate the survival outcome in hamsters. Infection of hamsters with SNV 1 day before ANDV challenge did not result in disease characteristic of the latter virus, and all animals survived challenge. Control animals infected solely with ANDV all succumbed by day 14. In contrast, when viruses were injected at the same site concurrently, all hamsters succumbed to HPS disease. Hantaviruses are segmented viruses; therefore we investigated which segment might be responsible for the protective phenotype of SNV by using two SNV/ANDV reassortant viruses, both with reciprocal M-segments from the other virus (denoted ASA and SAS). Both reassortants asymptomatically infect hamsters, similar to SNV. However, unlike SNV, 1 day prior preinfection with the reassortant virus did not prevent ANDV lethality. The ASA reassortant virus, but not SAS, protected hamsters from lethal ANDV infection when administered 3 days prior to ANDV challenge. Similar to SNV preinfection, the potent innate immune stimulator poly I:C administered to hamsters 1 day before ANDV challenge prevented lethal ANDV disease. Combined, these results suggest that the difference in pathogenicity of SNV and ANDV in hamsters involves differences in early host-pathogen interactions and resultant anti-viral immune responses of both the innate and adaptive immune system.

Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters.

UNLABELLED: Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS. IMPORTANCE: Hantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection, but those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like in our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses.

Human pathogenic hantaviruses and arenaviruses are maintained in nature by persistent infection of rodent carrier populations. Several members of these virus groups can cause significant disease in humans that is generically termed viral hemorrhagic fever (HF) and is characterized as a febrile illness with an increased propensity to cause acute inflammation. Human interaction with rodent carrier populations leads to infection. Arenaviruses are also viewed as potential biological weapons threat agents. There is an increased interest in studying these viruses in animal models to gain a deeper understating not only of viral pathogenesis, but also for the evaluation of medical countermeasures (MCM) to mitigate disease threats. In this review, we examine current knowledge regarding animal models employed in the study of these viruses. We include analysis of infection models in natural reservoirs and also discuss the impact of strain heterogeneity on the susceptibility of animals to infection. This information should provide a comprehensive reference for those interested in the study of arenaviruses and hantaviruses not only for MCM development but also in the study of viral pathogenesis and the biology of these viruses in their natural reservoirs.

Antiviral Biologic Produced in DNA Vaccine/Goose Platform Protects Hamsters Against Hantavirus Pulmonary Syndrome When Administered Post-exposure.

Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA80 titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD50). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.

DNA vaccine-derived human IgG produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome.

Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen.

A lethal disease model for hantavirus pulmonary syndrome in immunosuppressed Syrian hamsters infected with Sin Nombre virus.

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.

T cells are not required for pathogenesis in the Syrian hamster model of hantavirus pulmonary syndrome.

Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology.

Antigen controls IL-7R alpha expression levels on CD8 T cells during full activation or tolerance induction.

The high-affinity chain of the IL-7 receptor, IL-7Ralpha (CD127), is expressed by effector CD8 T cells that have the capacity to become memory cells. IL-7Ralpha expression is uniformly high on naive CD8 T cells, and the majority of these cells down-regulate expression upon antigenic challenge. At the peak of expansion, the fraction of effectors expressing high IL-7Ralpha varies depending on the response examined. The signals that a CD8 T cell receives during a response to Ag that lead to altered expression of IL-7Ralpha have not been fully defined. In vitro experiments demonstrated that Ag alone is sufficient to down-regulate IL-7Ralpha on all cells and most of the cells rapidly re-express the receptor upon removal from Ag. Expression was not altered by the B7.1 costimulatory ligand or when IL-12 was present to provide the signal needed for development of effector functions, indicating that TCR engagement is sufficient to regulate IL-7Ralpha expression. Consistent with this, in vivo priming with peptide Ag resulted in IL-7Ralpha expression that inversely correlated with Ag levels, and expression levels were not changed when IL-12 or adjuvant were administered with Ag. A large fraction of the cells present at the peak of expansion had re-expressed IL-7Ralpha, but most of these cells failed to survive; those that did survive expressed high IL-7Ralpha levels. Thus, Ag-dependent signals regulate IL-7Ralpha levels on responding CD8 T cells, and this occurs whether the responding cells become fully activated or are rendered tolerant by administration of peptide Ag alone.

Molecular basis for checkpoints in the CD8 T cell response: tolerance versus activation.

CD8 T cells specific for self-antigens are present in the peripheral lymphoid system and can contribute to autoimmunity or transplant rejection. Whether recognition of Ag leads to full activation, or to induction of tolerance, depends upon availability of cytokine at critical stages of the response. Signals provided by IL-12 and/or IFN-alpha/beta are required for activation of naïve CD8 T cells, and IL-2 is needed to sustain and further expand the effector cells if Ag persists. These critical signaling requirements provide new insights into the factors that regulate the CD8 T cell contributions to development of autoimmunity or rejection of transplants.

Signals required for programming effector and memory development by CD8+ T cells.

Stimulation of naïve CD8+ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin-12 (IL-12) or interferon-alpha (IFN-alpha). CD4+ T cells condition dendritic cells (DCs) to effectively present antigen to CD8+ T cells, and this conditioning involves, at least in part, CD40-dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation-induced non-responsiveness (AINR), a form of split anergy characterized by an inability to produce IL-2 to support continued expansion. If antigen remains present, IL-2 provided by CD4+ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL-2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8+ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.

Cutting edge: Bcl-3 up-regulation by signal 3 cytokine (IL-12) prolongs survival of antigen-activated CD8 T cells.

Clonal expansion of T cells requires cell division and survival during the proliferative phase of the response. Naive murine CD8 T cells responding to Ag and costimulation undergo an abortive response characterized by impaired clonal expansion, failure to develop effector functions, and long-term tolerance. A third signal provided by IL-12 is required for full expansion, activation, and establishment of memory. The enhanced survival, and thus clonal expansion, supported by IL-12 is not due to increased Bcl-2 or Bcl-x(L) expression; both are maximally activated by signals 1 and 2. In contrast, Bcl-3, recently shown to enhance survival when ectopically expressed in T cells, is increased only when IL-12 is present. Furthermore, examination of Bcl-3-deficient CD8 T cells demonstrates that the increased survival caused by IL-12 depends upon Bcl-3. The time courses of expression suggest that Bcl-2 and Bcl-x(L) promote survival early in the response, whereas Bcl-3 acts later in the response.