The development of broad-spectrum antiviral medical countermeasures to treat viral hemorrhagic fevers caused by natural or weaponized virus infections.

The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir (GS-5734, Veklury, Gilead Sciences) was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir's clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule. The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA's established Animal Rule (21 CFR 314.600-650 for drugs; 21 CFR 601.90-95 for biologics). The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents.

Co-administration of tecovirimat and ACAM2000™ in non-human primates: Effect of tecovirimat treatment on ACAM2000 immunogenicity and efficacy versus lethal monkeypox virus challenge.

Naturally occurring smallpox has been eradicated but research stocks of variola virus (VARV), the causative agent of smallpox, still exist in secure laboratories. Clandestine stores of the virus or resurrection of VARV via synthetic biology are possible and have led to concerns that VARV could be used as a biological weapon. The US government has prepared for such an event by stockpiling smallpox vaccines and TPOXX®, SIGA Technologies' smallpox antiviral drug. While vaccination is effective as a pre-exposure prophylaxis, protection is limited when administered following exposure. Safety concerns preclude general use of the vaccine unless there is a smallpox outbreak. TPOXX is approved by the FDA for use after confirmed diagnosis of smallpox disease. Tecovirimat, the active pharmaceutical ingredient in TPOXX, targets a highly conserved orthopoxviral protein, inhibiting long-range dissemination of virus. Although indications for use of the vaccine and TPOXX do not overlap, concomitant use is possible, especially if the TPOXX indication is expanded to include post-exposure prophylaxis. It is therefore important to understand how vaccine and TPOXX may interact. In studies presented here, monkeys were vaccinated with the ACAM2000TM live attenuated smallpox vaccine and concomitantly treated with tecovirimat or placebo. Immune responses to the vaccine and protective efficacy versus a lethal monkeypox virus (MPXV) challenge were evaluated. In two studies, primary and anamnestic humoral immune responses were similar regardless of tecovirimat treatment while the third study showed reduction in vaccine elicited humoral immunity. Following lethal MPXV challenge, all (12 of 12) vaccinated/placebo treated animals survived, and 12 of 13 vaccinated/tecovirimat treated animals survived. Clinical signs of disease were elevated in tecovirimat treated animals compared to placebo treated animals. This suggests that TPOXX may affect the immunogenicity of ACAM2000 if administered concomitantly. These studies may inform on how vaccine and TPOXX are used during a smallpox outbreak.

Lack of response to exogenous interferon-alpha in the liver of chimpanzees chronically infected with hepatitis C virus.

UNLABELLED: The mechanism of the interferon-alpha (IFNalpha)-induced antiviral response is not completely understood. We recently examined the transcriptional response to IFNalpha in uninfected chimpanzees. The transcriptional response to IFNalpha in the liver and peripheral blood mononuclear cells (PBMCs) was rapidly induced but was also rapidly down-regulated, with most interferon-alpha-stimulated genes (ISGs) returning to the baseline within 24 hours. We have extended these observations to include chimpanzees chronically infected with hepatitis C virus (HCV). Remarkably, using total genome microarray analysis, we observed almost no induction of ISG transcripts in the livers of chronically infected animals following IFNalpha dosing, whereas the response in PBMCs was similar to that in uninfected animals. In agreement with this finding, no decrease in the viral load occurred with up to 12 weeks of pegylated IFNalpha therapy. The block in the response to exogenous IFNalpha appeared to be HCV-specific because the response in a hepatitis B virus-infected animal was similar to that of uninfected animals. The lack of a response to exogenous IFNalpha may be due to an already maximally induced ISG response because chronically HCV-infected chimpanzees already have a highly up-regulated hepatic ISG response. Alternatively, negative regulation may block the response to exogenous IFNalpha, yet it does not prevent the continued response to endogenous ISG stimuli. The IFNalpha response in chronically HCV-infected chimpanzees may be mechanistically similar to the null response in the human population. CONCLUSION: In chimpanzees infected with HCV, the highly elevated hepatic ISG expression may prevent the further induction of ISGs and antiviral efficacy following an IFNalpha treatment.

Genomic response to interferon-alpha in chimpanzees: implications of rapid downregulation for hepatitis C kinetics.

The mechanism of the interferon-alpha (IFN-alpha)-induced antiviral response during hepatitis C virus (HCV) therapy is n o t completely understood. In this study,we examined the transcriptional response to IFN-alpha in uninfected chimpanzees after single doses of chimpanzee, human, or human-pegylated IFN-alpha. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours postinoculation, a time when high levels of circulating pegylated IFN-alpha were still present. The rapid downregulation of the IFN-alpha response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN-alpha therapy in HCV-infected patients. The response to all three forms of IFN-alpha was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN-alpha were not due to species specificity of IFN-alpha. The response to IFN-alpha was partially tissue-specific. A total of 1778 genes were altered in expression by twofold or more by IFN-alpha, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN-alpha and IFN-gamma responses in primary chimpanzee and human hepatocytes were compared as well. IFN-alpha and IFN-gamma induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN-alpha is largely tissue-specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response.

Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons.

The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.

Hepatitis C virus (HCV) infections represent a global health problem and are a major contributor to end-stage liver disease including cirrhosis and hepatocellular carcinoma. An improved understanding of the parameters involved in disease progression is needed to develop better therapies and diagnostic markers of disease manifestation. To better understand the dynamics of host gene expression resulting from persistent virus infection, DNA microarray analyses were conducted on livers from 10 chimpanzees persistently infected with HCV. A total of 162 genes were differentially regulated in chronically infected animals compared to uninfected controls. Many genes exhibited a remarkable consistency in changes in expression in the 10 chronically infected animals. A second method of analysis identified 971 genes altered in expression during chronic infection at a 99% confidence level. As with acute-resolving HCV infections, many interferon (IFN)-stimulated genes (ISGs) were transcriptionally elevated, suggesting an ongoing response to IFN and/or double-stranded RNA which is amplified in downstream ISG expression. Thus, persistent infection with HCV results in a complex and partially predictable pattern of gene expression, although the underlying mechanisms regulating the different pathways are not well defined. A single genotype 3-infected animal was available for analysis, and this animal exhibited reduced levels of ISG expression compared to levels of expression with genotype 1 infections and increased expression of a number of genes potentially involved in steatosis. Gene expression data in concert with other observations from HCV infections permit speculation on the regulation of specific aspects of HCV infection.

Cross-genotype immunity to hepatitis C virus.

Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.