Judie B. Alimonti

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses, may be a feature shared by the virulent influenza viruses.

Successful Treatment of Ebola Virus–Infected Cynomolgus Macaques with Monoclonal Antibodies

Macaques survived infection with Ebola virus when treated starting at 24 hours after infection with mix of three neutralizing monoclonal antibodies. A Race Against Time Although rare, Ebola infection figures prominently in the public’s fear of an infectious disease outbreak because of its marked, rapid, and fatal manifestation. This fear is fueled by our complete helplessness when it comes to fighting Ebola—there’s no vaccine, and any treatment options we do have only work if administered within minutes—or at most hours—after infection. Qiu et al. address this impotence head-on by demonstrating that administration of a three-antibody cocktail to macaques within 24 hours of infection yields 100% survival. The authors treated the macaques 24 or 48 hours after Ebola virus challenge with a virus-neutralizing antibody cocktail (ZMab). The three antibodies in the mix each bind to distinct regions of the Ebola envelop glycoprotein (GP) and show efficacy in small-animal models. When the cocktail was given at 24 hours after infection, 100% of the monkeys survived; if the same dose of the cocktail was administered 48 hours after infection, the survival rate was 50%. Surviving macaques developed both Ebola-specific antibodies and T cell responses, which suggests that the passive neutralizing antibody transfer may keep the virus in check long enough for endogenous immunity to take over. Timing, dose, and composition must be optimized before this therapy moves into humans, but the new findings add sand to the hourglass and provide hope for an expanded treatment window for Ebola virus infection. Ebola virus (EBOV) is considered one of the most aggressive infectious agents and is capable of causing death in humans and nonhuman primates (NHPs) within days of exposure. Recent strategies have succeeded in preventing acquisition of infection in NHPs after treatment; however, these strategies are only successful when administered before or minutes after infection. The present work shows that a combination of three neutralizing monoclonal antibodies (mAbs) directed against the Ebola envelope glycoprotein (GP) resulted in complete survival (four of four cynomolgus macaques) with no apparent side effects when three doses were administered 3 days apart beginning at 24 hours after a lethal challenge with EBOV. The same treatment initiated 48 hours after lethal challenge with EBOV resulted in two of four cynomolgus macaques fully recovering. The survivors demonstrated an EBOV-GP–specific humoral and cell-mediated immune response. These data highlight the important role of antibodies to control EBOV replication in vivo, and support the use of mAbs against a severe filovirus infection.

Granzyme B Induces BID-mediated Cytochrome c Release and Mitochondrial Permeability Transition

Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as promotes cytochrome c (cyt c) release and loss of mitochondrial membrane potential. We investigated how GrB affects mitochondria utilizing an in vitro cell-free system and determined that cyt c release and permeability transition are initiated by distinct mechanisms. The cleavage of cytosolic BID by GrB results in truncated BID, initiating mitochondrial cytc release. BID is the sole cytosolic protein responsible for this phenomenon in vitro, yet caspases were found to participate in cyt c release in some cells. On the other hand, GrB acts directly on mitochondria in the absence of cytosolic S100 proteins to open the permeability transition pore and to disrupt the proton electrochemical gradient. We suggest that GrB acts by two distinct mechanisms on mitochondria that ultimately lead to mitochondrial dysfunction and cellular demise.

A Recombinant Vesicular Stomatitis Virus Ebola Vaccine - Preliminary Report.

Background The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication‐competent, recombinant vesicular stomatitis virus (rVSV)–based vaccine candidate designed to prevent EVD. Methods We conducted two phase 1, placebo‐controlled, double‐blind, dose‐escalation trials of an rVSV‐based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV‐ZEBOV vaccine (3 million plaque‐forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. Results The most common adverse events were injection‐site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme‐linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV‐Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. Conclusions This Ebola vaccine candidate elicited anti‐Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV&Dgr;G‐ZEBOV‐GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408.)

Pathogenesis and Immune Response of Crimean-Congo Hemorrhagic Fever Virus in a STAT-1 Knockout Mouse Model

ABSTRACT Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe hemorrhagic syndrome in humans but not in its vertebrate animal hosts. The pathogenesis of the disease is largely not understood due to the lack of an animal model. Laboratory animals typically show no overt signs of disease. Here, we describe a new small-animal model to study CCHFV pathogenesis that manifests clinical disease, similar to that seen in humans, without adaptation of the virus to the host. Our studies revealed that mice deficient in the STAT-1 signaling molecule were highly susceptible to infection, succumbing within 3 to 5 days. After CCHFV challenge, mice exhibited fever, leukopenia, thrombocytopenia, and highly elevated liver enzymes. Rapid viremic dissemination and extensive replication in visceral organs, mainly in liver and spleen, were associated with prominent histopathologic changes in these organs. Dramatically elevated proinflammatory cytokine levels were detected in the blood of the animals, suggestive of a cytokine storm. Immunologic analysis revealed delayed immune cell activation and intensive lymphocyte depletion. Furthermore, this study also demonstrated that ribavirin, a suggested treatment in human cases, protects mice from lethal CCHFV challenge. In conclusion, our data demonstrate that the interferon response is crucial in controlling CCHFV replication in this model, and this is the first study that offers an in-depth in vivo analysis of CCHFV pathophysiology. This new mouse model exhibits key features of fatal human CCHF, proves useful for the testing of therapeutic strategies, and can be used to study virus attenuation.

Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses

Background Zaire ebolavirus (ZEBOV) produces a lethal viral hemorrhagic fever in humans and non-human primates. Methodology/Principal Findings We demonstrate that the VSVΔG/ZEBOVGP vaccine given 28 days pre-challenge either intranasally (IN), orally (OR), or intramuscularly (IM) protects non-human primates against a lethal systemic challenge of ZEBOV, and induces cellular and humoral immune responses. We demonstrated that ZEBOVGP-specific T-cell and humoral responses induced in the IN and OR groups, following an immunization and challenge, produced the most IFN-γ and IL-2 secreting cells, and long term memory responses. Conclusions/Significance We have shown conclusively that mucosal immunization can protect from systemic ZEBOV challenge and that mucosal delivery, particularly IN immunization, seems to be more potent than IM injection in the immune parameters we have tested. Mucosal immunization would be a huge benefit in any emergency mass vaccination campaign during a natural outbreak, or following intentional release, or for mucosal immunization of great apes in the wild.

IL-7Rα expression on CD4+ T lymphocytes decreases with HIV disease progression and inversely correlates with immune activation

Many factors can influence the rate of HIV disease progression, including those that maintain T cell homeostasis. One key homeostatic regulator is the IL‐7 receptor (IL‐7R). Previous studies have shown IL‐7R expression levels decrease in HIV infection, but effects on memory subtypes, CD4+ T cells, and cell function have not been explored. The present study examined the expression of the IL‐7Rα chain on naïve and memory T lymphocyte subsets of both HIV‐positive and HIV‐negative individuals from Nairobi, Kenya to assess the role of IL‐7Rα in HIV disease. Expression of IL‐7Rα was significantly reduced in all CD4+ and CD8+ T cell subsets in HIV‐positive individuals. This reduction was further enhanced in those with advanced HIV progression. Expression of IL‐7Rα was inversely correlated to immune activation, and apoptosis, and was positively correlated with CD4 count in both bivariate and multivariate analysis. Expression of IL‐7Rα did not correlate with HIV viral loads, indicating the elevated immune activation seen in HIV‐infected individuals may be impacting expression of IL‐7Rα, independent of viral loads. Signaling via the IL‐7R is essential for T cell homeostasis and maintenance of T cell memory. Reduction of this receptor may contribute to the homeostatic disruption seen in HIV.

Immune Parameters Correlate with Protection Against Ebola Virus Infection in Rodents and Nonhuman Primates

Levels of specific antibodies induced by vaccination correlate with survival in Ebola-infected animals. Protecting Against the Zombie Apocalypse Halloween can wreak havoc on the imagination. The advent of seasonal symptoms—spontaneous moaning, slow jerky movements, and the desire to eat the brains of your co-workers—can convince you that you’re turning into a zombie. While there are no treatments for the prevention of zombieism, when the holiday passes, symptoms of this fictional disease fade. But dire diseases—real ones—with equally horrific symptoms persist after trick-or-treat, because there are no corresponding vaccines or drug treatments. Now, Wong et al. show that for ebolavirus infections, which can cause a nightmarish fever frightening enough to be Halloween-worthy, researchers are making progress. There are currently no licensed vaccines or treatments that prevent or cure infection with Zaire ebolavirus (ZEBOV) in human patients. Experimental vaccines have been successful in animal models of hemorrhagic fever. But how can one test the efficacy of these vaccines in people without exposing them to a potentially fatal disease? Wong et al. report on an important translational step toward vaccine testing. The authors detected specific antibody responses to ZEBOV that correlate with survival in multiple animal models (mouse, guinea pig, and nonhuman primates). Much work remains to be done to determine whether these observations hold true in people. But if they do, these immune responses may be used as surrogates for testing a successful vaccine. Ebola virus causes severe hemorrhagic fever in susceptible hosts. Currently, no licensed vaccines or treatments are available; however, several experimental vaccines have been successful in protecting rodents and nonhuman primates (NHPs) from the lethal Zaire ebolavirus (ZEBOV) infection. The objective of this study was to evaluate immune responses correlating with survival in these animals after lethal challenge with ZEBOV. Knockout mice with impaired ability to generate normal T and/or B cell responses were vaccinated and challenged with ZEBOV. Vaccine-induced protection in mice was mainly mediated by B cells and CD4+ T cells. Vaccinated, outbred guinea pigs and NHPs demonstrated the highest correlation between survival and levels of total immunoglobulin G (IgG) specific to the ZEBOV glycoprotein (ZGP). These results highlight the relevance of total ZGP-specific IgG levels as a meaningful correlate of protection against ZEBOV exposure.

mAbs and Ad-Vectored IFN-α Therapy Rescue Ebola-Infected Nonhuman Primates When Administered After the Detection of Viremia and Symptoms

Monoclonal antibodies and adenovirus-vectored IFN-α extend the treatment window for Zaire ebolavirus in macaques. Every Day Counts Ebola virus (EBOV) infections cause a deadly hemorrhagic disease for which there are no currently licensed vaccines or treatments. Recent studies have demonstrated the potential of antibody therapy cocktails (ZMAb) for treating EBOV infections; however, there is a limited time window for these therapies to be effective. Because early clinical symptoms of EBOV infection resemble other common pathogens, it is critical to extend this treatment window until positive cases can be confirmed. Now, Qui et al. combine ZMAb therapy with adenovirally delivered interferon-α (Ad-IFN) to extend the EBOV treatment window in nonhuman primates. The authors dosed macaques that had received a lethal dose of EBOV with a combination of ZMAb and Ad-IFN. Combination therapy with Ad-IFN and ZMAb was 75 and 100% protective in cynomolgus and rhesus macaques, respectively, when administered together at 3 days post-infection. Fifty percent of cynomolgus macaques were protected at 4 days post-infection when Ad-IFN was administered 1 day post-infection. These results suggest that the Ad-IFN and ZMAb combination treatment could be effective after confirmation of EBOV infection as well as could substantially reduce mortality rates of cases diagnosed early after symptom onset. ZMAb is a promising treatment against Ebola virus (EBOV) disease that has been shown to protect 50% (two of four) of nonhuman primates (NHPs) when administered 2 days post-infection (dpi). To extend the treatment window and improve protection, we combined ZMAb with adenovirus-vectored interferon-α (Ad-IFN) and evaluated efficacy in EBOV-infected NHPs. Seventy-five percent (three of four) and 100% (four of four) of cynomolgus and rhesus macaques survived, respectively, when treatment was initiated after detection of viremia at 3 dpi. Fifty percent (two of four) of the cynomolgus macaques survived when Ad-IFN was given at 1 dpi, followed by ZMAb starting at 4 dpi, after positive diagnosis. The treatment was able to suppress viremia reaching ~105 TCID50 (median tissue culture infectious dose) per milliliter, leading to survival and robust specific immune responses. This study describes conditions capable of saving 100% of EBOV-infected NHPs when initiated after the presence of detectable viremia along with symptoms.

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.