State-of-the-Art Therapeutic Medical Countermeasures for Viral Threat Agents

Abstract

In recent years, there has been an increase in the perceived threat of biological agents being used against civilian populations. This has prompted an urgent need for the development and procurement of medical countermeasures (MCMs) against highly pathogenic viruses that can prevent morbidity and mortality from infections caused by these agents. To date, antiviral drug development has been largely focused on clinically prevalent chronic infections due to their commercial viability. This has left a huge gap in the drug development path for acute infections of biodefense importance. In this review, we discuss the antiviral research and development initiatives focusing specifically on poxviruses, filoviruses, and equine encephalitis viruses (EEV). We discuss the benefits and technical challenges in the current development strategies and the hurdles in the licensure path for MCMs against these highly pathogenic viruses under the FDA Animal Rule, and we provide recommendations for the path forward.

In this review, the authors discuss the antiviral research and development initiatives focusing on poxviruses, filoviruses, and equine encephalitis viruses. They discuss the benefits and technical challenges in the current development strategies and the hurdles in the licensure path for medical countermeasures against these highly pathogenic viruses under the FDA Animal Rule.

For the past 10 years, there has been an increase in the perceived threat of biological terrorism, and a large-scale release of biological agent(s) on a civilian population appears more plausible. The mass vaccination of civilian populations is not practical, as the risks of vaccinating the general population outweigh the benefits when framed in the context of probability of exposure. This has prompted significant interest and investment in the development of medical countermeasures (MCMs), both as prophylactics that can prevent morbidity and mortality from these diseases after individuals have been exposed and as postexposure therapeutics against threat agents after people have become symptomatic. Historically, this work was aimed at protecting military personnel in battlefield environments. While this focus resulted in the development of a number of fielded countermeasures, much of the work did not focus on providing medical emergency response options to vulnerable civilian populations.

The enormous expense of drug development—estimated at $1.2 billion (U.S.) to bring a drug from discovery to approval—necessitates that only promising strategies and products are supported.¹ In general terms, industry has been reluctant to support the research and development of antivirals for acute infections, instead focusing on developing treatments for clinically prevalent chronic infections like HIV and HCV, which have a greater degree of profitability.

Historically, antiviral drug development has been more challenging than antibacterial drug development. This is due in part to the dependence on host cellular machinery for viral propagation, which complicates the identification and exploitation of targets that will generate compounds with pathogen selective toxicities. The development pathway is even more complex for antiviral products against threat agent viruses, where clinical efficacy studies may not be feasible or ethical. In these instances, the Animal Rule by the U.S. Food and Drug Administration (FDA) allows animal efficacy data to be used to support drug approval along with human safety evaluation data and pharmacokinetic information.² Several technical challenges exist with this regulatory pathway, including the development of animal models that recapitulate the human disease. This is particularly challenging when the virus is human specific (eg, variola) or when the data on the clinical presentation of the disease in humans are limited (eg, filoviruses). Conducting animal studies according to regulatory standards under high biological safety containment represents an additional set of challenges. Therefore, it is likely that drug development costs and timelines for viral threat agents will exceed industry averages. Thus, a deliberate and methodical selection of candidate therapies must be conducted to appropriately mitigate developmental risks in this area.

Antiviral strategies have been broadly divided into 2 categories: products that specifically target the virus and products that are more active toward the virus with an acceptable toxicity to the host. Chemotherapeutics act by disrupting the pathogen lifecycle at various stages, including entry into host cells, genome replication, capsid assembly, and egress from the host cell. The earliest antivirals were developed through laborious, trial-and-error in vitro screens. The first widely successful antiviral drug discovered in this manner, acyclovir, works against herpes simplex virus by inhibiting the viral DNA polymerase.

Subsequent genomic information and pharmaceutical innovations have provided more precise tools for developing novel, effective antiviral chemistries. Although advances in the development of recombinant viruses and resulting high-throughput screening systems have enabled early discovery efforts that obviate the requirement of a biocontainment facility for highly pathogenic viruses, these advances have not accelerated the identification of more lead candidates. The concept of using chemotherapeutics to inhibit viral life cycle is not novel, yet the historical investment in this type of MCM against viral threat agents has lagged behind other measures, such as vaccines. Immunotherapeutics on the other hand modulate the host immune mechanism in an effort to combat the infection.

The evaluation of these products depends on the availability of predictive animal models that mimic the host response mechanism. The application of traditional and state-of-the-art drug development methodologies for these agents has been initiated only in the past few years. Regardless, significant progress has been made in advancing therapeutics for several agents.

In this article, we discuss candidate antivirals for viral threat agents in 2 contexts: those used for treatment—that is, after symptom onset—and those used for postexposure prophylaxis (PEP)—that is, following exposure but prior to symptom onset. This review summarizes the recent advances in the development of MCMs specifically for PEP and treatment against poxviruses, filoviruses, and the equine encephalitis viruses (EEV).

While there have been many experimental advances in the research and development of medical countermeasures for these agents with respect to viral targets, screening strategies, and animal model development, our review specifically focuses on literature published within the past 5 years.^3–6 Our intent to focus on the recent literature is to provide a current status update of the developmental pipeline. We discuss the benefits and technical challenges associated with these emerging countermeasures. Finally, we provide recommendations to enhance the productivity of the developmental pipeline.

Poxviruses

In the past decade, significant progress has been made in the research and development of therapies against poxviruses. These viruses show narrow to broad species specificity for host reservoirs. Since the most important of the poxviruses, variola, infects only humans, efficacy and safety testing of candidate compounds has been restricted to surrogate animal models. Despite these limitations, several new compounds have been reported in recent years that show antiviral activity against poxviruses. Of these, 2 candidate antiviral compounds are in clinical development for the treatment of orthopoxvirus infection, ST-246 and CMX-001. This section discusses these 2 compounds and others that are early in development (Table 1).

Table 1.

Summary of Products in Development Against Poxviruses

Compound	Mechanism of Action	Efficacy Data Shown	References
CMX001	Viral replication inhibitor	PEP and treatment of RPX in rabbits	13,14,15,16,17,18
ST-246	Viral egress inhibitor	PEP and treatment of MPX and Variola in nonhuman primates	7,8,9,10,11,12
Nigericin	Inhibits viral early gene transcription and viral DNA replication	In vitro VACV	19
CSA -13	Not known	VACV treatment in murine model (topical)	20
IFNγ195-132	Immune-modulators	VACV prophylaxis murine model	23
lipo-KIR and lipo-Tkip	Immune modulators	Prophylaxis and PEP VACV murine model	24
RNAi	RNA interference	In vitro MPX	22
anti-H3 Mab hV26 and anti B5 Mab h10	Passive immunization	Prophylaxis and PEP VACV murine model	25,26
rVIG cocktail	Passive immunization	Prophylaxis and PEP VACV murine model	27

Chemotherapeutics

ST-246 (Tecovirimat) is an inhibitor of viral egress from host cells that has demonstrated significant protective efficacy in ectromelia virus and rabbitpox challenge models.^7–9 Further, ST-246 has demonstrated 100% protection in a lethal monkeypox challenge model in nonhuman primates (NHPs) when administered up to 3 days postinfection (a time point at which lesions become evident), suggesting the drug may hold promise as a therapeutic antiviral treatment for smallpox.¹⁰ ST-246 has undergone clinical trials to establish human safety, tolerability, and pharmacokinetics.¹¹ In this phase I study, ST-246 was administered as a single daily oral dose of 250, 400, or 800 mg for 21 days to nonfasting healthy human volunteers. ST-246 was safe and very well tolerated, with no serious adverse events.¹²

CMX-001, a prodrug derivative of cidofovir, is being developed as an oral formulation. Cidofovir, a cytidine analog targeting viral DNA replication, has demonstrated PEP efficacy against a lethal monkeypox challenge in cynomolgus macaques.¹³ In addition, cidofovir is FDA approved for the treatment of cytomegalovirus (CMV) retinitis in human immunodeficiency virus (HIV) patients. However, the nephrotoxicity of cidofovir precludes its widespread use in a public health emergency. Potentially better tolerated, the prodrug CMX-001 possesses broad spectrum antiviral activity against a number of dsDNA viruses such as BK, adenovirus, and herpesviruses and is also being developed for its utility as a smallpox antiviral.^14,15 CMX-001 demonstrated 100% protection in a lethal rabbitpox virus (RPX) challenge when administered 1 day postinfection.¹⁶ In a parallel study in the same model when CMX-001 was administered at lesion onset, a dose dependent improvement in survival was observed between the treatment groups.¹⁷ A phase I clinical study has been conducted for CMX-001, and other clinical trials are in preparation.¹⁸

ST-246 and CMX-001 act synergistically when evaluated in combination against vaccinia or cowpox virus (CV) in vitro.⁸ Effects of the combination therapy were also evaluated in mice infected with cowpox virus. Treatment with the combination of ST-246 and CMX-001 at individually subtherapeutic doses of 1 and 3 mg/kg/day, respectively, was effective in reducing mortality even when the drugs were administered 6 days after lethal cowpox virus challenge. No evidence of toxicity was observed with the combination either in vitro or in vivo, a benefit that can likely be attributed to a lower effective dose of each compound being enabled. The drugs have different antiviral mechanisms; therefore, the combination may provide an additional benefit in decreasing development of resistance.

The carboxylic ionophore nigericin has shown promise in early studies. Nigericin inhibited early viral gene transcription and DNA replication in several human cell lines infected with vaccinia virus.¹⁹ Nigericin showed an IC₅₀ of 7.9 nM against vaccina virus in HeLa cell lines; however, it is also cytotoxic, and the therapeutic index is much lower than cidofovir or ST-246. Identification of an analog with an improved therapeutic index must take place before nigericin could become a useful pox virus therapeutic.

Antimicrobial peptides (AMPs) are generally considered part of the host defense mechanism and are conserved components of the innate immune response. Synthetic structural mimics of endogenous AMPs, ceragenins (CSAs), were evaluated for antiviral activity against vaccinia virus.²⁰ Topical application of one such peptide, CSA-13, to vaccinia lesions in a murine model reduced the development of satellite lesions. In addition, CSA-13 also stimulated the expression of endogenous AMPs against vaccinia virus in a SCID mouse model. The antiviral mechanism of CSAs remains to be determined. Peptide mimetics like CSAs have the advantage of being resistant to human protease degradation, which provides an opportunity to improve stability and toxicity issues associated with AMPs. In order to design new antivirals for use against biodefense pathogens, one would need to selectively enhance the specificity and potency of AMPs while reducing their toxicity.²¹

RNA interference (RNAi) has been evaluated as a potential therapy for orthopox viruses.²² Using monkeypox (MPX) as a model system, 48 siRNA constructs were screened in vitro against genes essential for viral replication or entry. These constructs were able to inhibit viral expression by 65% to 95% without apparent signs of cytotoxicity. One construct was capable of a prolonged inhibition of viral replication (7 days) at a pharmacologically relevant concentration of 10 nM. In addition to further substantiating the critical role of these genes in the viral lifecycle, this study provides in vitro proof-of-principle for the use of RNAi as a therapeutic platform for these viruses.

Immunotherapeutics

Poxviruses are able to evade IFN-γ with the help of the B8R protein, a homolog of the extracellular domain of the IFN-γ receptor. B8R binds to IFN-γ and prevents its interaction with the receptor. A peptide mimetic of IFN-γ, called IFN-γ₉₅-₁₃₂, was able to divert the decoy viral protein B8R and inhibit vaccinia virus replication in vitro, presumably by preventing depletion of IFN-γ important for innate immune response.²³ Intraperitoneal administration of the peptide mimetic completely protected mice challenged intranasally with a lethal dose of vaccinia when administered before and up to 2 days postinfection. When treatment was initiated 6 days postinfection, the peptide mimetic conferred 40% protection. The peptide mimetic was also effective in protecting mice against an intranasal lethal challenge of the virus when administered orally on days −2, −1, and 0 prior to infection. Furthermore, the peptide mimetic possessed adjuvant properties by boosting the adaptive response in vaccinated mice. Next, it will be important to study the effect of the peptide mimetic in nonhuman primates.

Structural mimetics of cellular modulators have also been studied for antipoxviral activity. A structural mimic of suppressor of cytokine signaling 1 protein (SOCS-1) was evaluated for its ability to protect against lethal vaccinia virus infection in mice.²⁴ Intraperitoneal administration of SOCS-1 mimetics lipo-KIR and lipo-Tkip provided complete protection when administered before, and at the time of, a lethal intranasal challenge of vaccinia virus and 80% protection when administered 1 day postinfection. Lipo-Tkip was protective against lethal intranasal vaccinia challenge even when administered orally.

Vaccinia immune globulin (VIG) was licensed to treat complications arising from vaccinations against smallpox with vaccinia virus. VIG has limitations, however, because it is a variable human product with poor efficacy in treating progressive vaccinia. This has stimulated the development of immune-therapeutics against poxvirus. The main hurdle in this development path is that poxvirions exist in 2 forms, the intracellular mature virions (IMV) and extracellular enveloped virions (EEV). Both virions are immunologically distinct and cannot be neutralized by a single antibody against any one of these forms. This demands the development of an effective monoclonal antibody (Mab) cocktail targeting both virion forms. A combination therapy with 2 human Mabs targeting the antigen in IMV (anti-H3 Mab hV26) and EEV (anti B5 Mab h101) has been proposed to address this issue.²⁵ Both antibodies exhibited significant efficacy against progressive vaccinia in mice compared to VIG. Combination therapy with both Mabs in mice infected with VACV_NYCBOH showed improvement in survival compared to single Mab therapy. The combination therapy also showed improved protection in a murine model of eczema vaccinatum when administered 24 hours preinfection and 12 hours postinfection.²⁶ Recently, a recombinant VIG (rVIG) cocktail comprised of 26 human Mabs targeting antigens in IMV and EEV was evaluated for its ability to protect against vaccinia virus infections using recombinant viruses WRvFire and IHD-J-Luc that express luciferase. The course of infection was monitored using whole body imaging. This model provides the opportunity to monitor virus dissemination in live animals. The rVIG cocktail was able to fully protect mice at 100 μg from WRvFire– and IHD-J-Luc–induced lethality when administered 2 days preexposure.²⁷ However, the cocktail did not prevent virus replication at the site of inoculation or dissemination to internal organs. It will be critical to study the effect of combination therapy postinfection to evaluate the usefulness of such a therapy postexposure if there were a smallpox public health emergency.

Filoviruses

Development of therapies for filoviral infections has suffered largely due to biocontainment issues in handling these highly pathogenic viruses. Recent advances in the development of replicon constructs with a readout have enabled high-throughput screening (HTS) campaigns and lead finding efforts. Another strategy to combat these infections has been to improve survival by modulating the host immune response. All products discussed in this section are early in development and are summarized in Table 2.

Table 2.

Summary of Products in Development Against Filoviruses

Compound	Mechanism of Action	Efficacy Data Shown	References
Chlorin E6	Not known	In vitro MARV	28
NSC 188491	Not known	In vitro EBOV	29
LJ001	Viral entry inhibitor	PEP in EBOV murine model	30
PAV-667	Viral capsid assembly inhibitor	Prophylaxis EBOV murine model	31
FGI-106	Viral replication inhibitor	PEP in EBOV murine model	32
rNAPc2	Immune-modulator	PEP in EBOV, MARV nonhuman primate model	33
17-AAG, AV-81, AV-1, AV-2, AV-3	HSP-90 inhibitors	In vitro	35
PMOs	Antisense therapy	Prophylaxis EBOV, MARV nonhuman primate model	36
SNALP formulated siRNAs	RNA interference	Prophylaxis EBOV guinea pig and nonhuman primate models	37, 38
rhMBL	Immune-modulator	Prophylaxis EBOV murine model	39
rhAPC	Immune-modulator	Prophylaxis EBOV nonhuman primate model	40
KZ52 and JP3K11	Passive immunization	Prophylaxis EBOV nonhuman primate model	45, 46

Chemotherapeutics

An alkylated porphyrin chlorphyllide selected from a cell-based high-throughput screening for hepatitis B virus (HBV) inhibition showed broad spectrum activity against filoviruses and other enveloped viruses including flaviviruses and arenaviruses.²⁸ One of the chemophores of chlorphyllide, chlorin E6, inhibited Marburg virus (MARV) in Vero cells at low micromolar concentrations. As yet undefined pharmacological properties and a mechanism of action for this prospective antiviral will determine whether a useful drug can be developed.

High content imaging technology has enabled cell-based high-throughput screening for inhibitors of viruses. Panchal et al reported an image-based method to screen 580 compounds against Marburg virus and Ebola virus (EBOV) using a viral replicon construct fused with the green fluorescent protein (GFP) reporter gene.²⁹ One compound, NSC 188491, a nucleoside analog, showed 70% inhibition of Ebola virus at 1.5 μM. Clearly, more potent analogs of this compound will need to be developed and yield acceptable pharmacology for a useful drug to result.

Virus pseudotypes have been used to conduct high-throughput screening for compounds capable of inhibiting viral entry.³⁰ A compound designated LJ001 was identified in a screen of approximately 80,000 compounds for inhibitors of Nipah virus entry into host cells. When tested against other viruses, LJ001 possessed the ability to inhibit the entry of a number of negative-sense enveloped RNA viruses. The compound was capable of inhibiting Ebola virus entry in vitro and protected 80% of mice when administered at the time of infection. However, once-daily injections of LJ001 initiated immediately postinfection and repeated every 24 hours for 7 days did not protect mice from a lethal challenge with Ebola virus. The authors of this study reason that this shortcoming was likely due to insufficient steady state plasma concentrations of LJ001. Given that the compound is a viral entry inhibitor, blocking establishment of infection, its utility in the treatment of symptomatic Ebola virus infection is likely low.

Host proteins that are involved in Ebola virus capsid assembly have been targeted using a cell-free based screening system.³¹ A small molecule library screened using this system identified hits with EC₅₀ in the submicromolar range and high selectivity. Mice treated with one of the compounds, PAV-667, were completely protected from a lethal challenge of Ebola virus when PAV-667 was administered once daily for days 1 to 3 preinfection in a 14-day study. The potential utility of this compound as a therapy will be determined in part by whether this compound has any efficacy when administered postinfection.

A novel antiviral compound, FGI-106, was identified in a cell-based high-throughput screening for inhibitors of Ebola virus replication and was subsequently assessed for its ability to inhibit viral infection in mouse models. FGI-106 was also tested in cell-based assays against other virus families and was found to possess inhibitory activity against a wide range of viruses, including dengue, Rift Valley fever, hepatitis C virus, and HIV.³² Given the broad spectrum antiviral activity that was observed, the authors speculate that FGI-106 targets a cellular pathway. Against a mouse-adapted strain of Ebola virus, the compound provided significant protection when given preexposure as well as 24 hours postexposure. A single dose of FGI-106 administered 24 hours postexposure provided 90% protection from lethal Ebola virus challenge in mice. Further, administration of FGI-106 reduced viral load in the kidney, liver, and spleen. Additional studies will be needed to determine if FGI-106 is effective if administered more than 24 hours postexposure, as would likely be required in a public health emergency.

Therapies targeting host inflammatory and coagulation response pathways associated with the pathology of filovirus infection are also being examined. Recombinant nematode anticoagulant protein (rNAPc2) was evaluated for PEP efficacy in a nonhuman primate filovirus challenge model.³³ RNAPc2 significantly increased survival time and rate by 33% when administered 10 minutes or 24 hours postinfection. Reductions in markers for the coagulation cascade and proinflammatory response were also observed, suggesting that the administration of rNAPc2 could provide additional supportive care to patients beyond therapies targeting viral replication. Unfortunately, rNAPc2 has not been tested at periods beyond 24 hours postinfection.

Drugs targeting cellular factors have been examined for their utility in attenuating Ebola virus replication. Heat shock protein 90 (Hsp90), a molecular chaperone, has been identified as an important factor in the replication of several negative stranded enveloped viruses.³⁴ Smith et al evaluated 7 Hsp90 inhibitors for their ability to reduce Ebola virus replication.³⁵ In the initial screen, geldanamycin, 17-AAG (a geldanamycin derivative), radicicol (a natural product), and 4 inhibitors from the benzamide class (AV-81 and its 3 derivates, AV-1, AV-2, and AV3) were evaluated for their ability to inhibit Ebola virus replication. These compounds significantly inhibited viral replication and demonstrated varying EC₅₀ values in the sub- to low-micromolar range with no cytotoxicity. Further studies in animal models will be required to evaluate the utility of these compounds.

Antisense therapies have been evaluated for their ability to suppress filovirus replication. Administration of phosphorodiamidate morpholino oligomers (PMOs) that target viral genomic sequences 30 to 60 minutes following virus challenge protected 60% of nonhuman primates against a lethal Ebola virus challenge and 100% of nonhuman primates against a Marburg virus challenge.³⁶ While this is a novel breakthrough for the ameloriation of infection with these extraordinarily pathogenic viruses, it will be necessary to improve the therapeutic window of opportunity with these candidate MCMs to truly offer utility during a military operation or public health emergency. Additionally, whether these products possess additive benefit or synergistic efficacy when combined with other candidate therapies is currently unknown. If the PMOs' efficacy is limited to 1 hour postexposure and there is no synergy with other products, PMOs will not be helpful in an emergency.

Other small interfering RNAs (siRNAs) have been evaluated for their ability to protect against Ebola virus infection. siRNAs targeting the EBOV-Zaire RNA polymerase L coding sequence formulated in stable nucleic acid-lipid particles (SNALPs) provided 100% protection of guinea pigs when administered 1 hour after a lethal Ebola virus challenge.³⁷ Further, SNALP-formulated siRNAs were evaluated for their ability to protect nonhuman primates from Ebola virus challenge.³⁸ When given intravenously 30 minutes following exposure, 2 of 3 rhesus macaques given 4 postexposure treatments of the pooled anti-EBOV-Zaire siRNAs were protected from lethal ZEBOV infection, whereas all macaques given 7 postexposure treatments exhibited 100% protection. It will be necessary to conduct dose delay studies to ascertain the therapeutic window for siRNAs and the utility of this countermeasure.

Immunotherapeutics

Therapeutic application of a recombinant human innate immune molecule, mannose binding lectin (rhMBL), was evaluated for its ability to target glycosylated viruses.³⁹ RhMBL binds to Ebola virus and Marburg virus envelope GP proteins and blocks the interaction with DC–SIGN. Mice administered with rhMBL resulting in rhMBL serum concentrations more than 7-fold above average human levels survived lethal Ebola virus infection and became immune to virus rechallenge. The ability of rhMBL to target Ebola virus in a nonhuman primate model must be demonstrated to advance this as a potential MCM.

A similar immune-modulatory molecule, recombinant human activated protein C (rhAPC), is currently licensed to treat patients with sepsis at a high risk of death. When rhAPC was administered to rhesus macaques 30 to 60 minutes postinfection with a lethal dose of EBOV-Zaire, it increased the mean survival time by 4.3 days and 2 out of 11 animals survived.⁴⁰ Whether rhAPC would be effective when administered beyond 60 minutes postinfection is unknown. Thus, it is difficult to ascertain the utility of this therapeutic in a mass public health emergency when response times to administer MCMs will go beyond 60 minutes. These immune-modulatory molecules will need to prove capable of mitigating the pathology of disease at later time points during infection; there is no therapeutic utility if they can only delay but not prevent death.

Ebola virus is a strong candidate for development of passive immunization therapies. Human survivors of Ebola virus infection elicit a strong, long-lasting humoral response, whereas nonsurvivors are characterized by a suppressed humoral response.^41,42 Passive immunization-mediated viral clearance is dependent upon antibody specificity. Neutralizing monoclonal antibodies have been shown to be protective in rodents.^43,44 KZ52, a neutralizing antibody from a human survivor, was able to protect guinea pigs but was unable to protect nonhuman primates.⁴⁵ Shedlock et al studied KZ52 and a survivor monkey antibody, JP3K11, and showed 2 distinct mechanisms to neutralize Ebola virus in vitro, in which both antibodies differed in their ability to recognize proteolytically processed surface glycoprotein (GP), thus affecting their neutralizing activity.⁴⁶ Species-specific proteolytic processing of GP has been found to be responsible for the inability of the human antibody KZ52 to protect nonhuman primates: nonhuman primates process GP into a form not generated in infected humans and thus not recognized by the human antibody. Any passive immunization therapy that is developed will have to account for these species-specific differences in epitope generation.

Equine Encephalitis Viruses (EEV)

A review of the existing literature conducted by the U.S. Department of Defense's Defense Threat Reduction Agency identified significant gaps in the data, assays, and animal models required for successful development of drugs to combat equine encephalitis viruses (EEVs), including Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV).⁴⁷ Specifically, the development of high-throughput screening assays, the resolution of the crystal structure of the nonstructural proteins, and the development of animal models to support therapeutic evaluation were cited as missing prerequisites to a successful target-based drug development program. The products discussed in this section are early in development and are summarized in Table 3.

Table 3.

Summary of Products in Development Against Equine Encephalitis Viruses (EEV)

Compound	Mechanism of Action	Efficacy Data Shown	References
Carbodine	Not known	PEP and treatment in VEEV murine model	48
siRNAs	RNA interference	In vitro	49
PMOs	Antisense therapy	Prophylaxis and PEP in VEEV murine model	50
Anti E3-Mab	Passive immunization	Prophylaxis in VEEV murine model	51
Anti E2-Mab	Passive immunization	Prophylaxis or PEP in VEEV murine model	52
hu1A3B-7	Passive immunization	Prophylaxis or PEP in VEEV murine model	53
hMAb F5 nIgG	Passive immunization	Prophylaxis or PEP in VEEV murine model	54, 55
CLDCs	Immune-modulator	Prophylaxis or PEP in VEEV murine model	56

Chemotherapeutics

Carbocyclic cytosine (carbodine) has been shown to inhibit an attenuated vaccine strain of VEEV (TC-83) in Vero cells. Inhibitory activity is conformation dependent, where the (-) enantiomer had EC₅₀ concentrations of 0.2 μg/ml in a mouse model infected with TC-83.⁴⁸ Further, pretreatment with 200 mg/kg of the (-) enantiomer significantly improved survival when administered up to 4 days postinfection. Carbodine is limited by its toxicity; hence it is necessary to develop and characterize its analogs to improve the therapeutic index of this compound.

Antiviral agents based on RNAi have also been explored against VEEV. A combination of 4 siRNAs was tested against 6 strains of VEEV in vitro.⁴⁹ The siRNAs were able to inhibit viral replication in vitro. However, the study also reports the emergence of resistance to RNAi. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) evaluated against VEEV have been reported to inhibit viral replication.⁵⁰ Vero cells infected with different strains of VEEV when treated with 5′+P7-PPMO resulted in modest reduction in viral titer at 2.5 μM and significant reduction at 5, 7.5, and 10 μM. Mice receiving 5′+P7-PPMO pre- and postinfection resulted in 100% (8/8) protection, whereas 63% (5/8) protection was observed in mice receiving only postinfection treatment.

Immunotherapeutics

Neutralizing antibodies have also been reported against VEEV. Passive immunization using monoclonal antibodies against VEEV E3 glycoprotein protected mice.⁵¹ The antibodies recognized protective epitopes within E3 that decreased viremia and provided a survival window during which the host adaptive immune response could be activated. Further, a purified humanized neutralizing antibody against the VEEV E2 protein was able to provide complete protection in mice when administered 24 hours before or after challenge with VEEV.⁵² A similar humanized Mab, hu1A3B-7, has been reported recently with broad serogroup specificity.⁵³ Hu1A3B-7 showed neutralizing activity in vitro and protected mice infected via aerosol and subcutaneous routes with the VEEV TrD strain.

Another advancement in this area has been the development of human Mabs (hMab) against VEEV by mapping the epitope of E1 and E2 proteins using phage display technology. The hMAb F5 nIgG derived from VEEV-specific Fab from human donors showed neutralizing activity against VEEV in vitro.⁵⁴ Neutralization escape variants of F5nIgG helped map the binding sites of F5nIgG. This is the first report of a human epitope map for VEEV E1 and E2 proteins. Further, hMAb F5 nIgG was able to confer protection when administered 24 hours before subcutaneous or aerosol exposure. However, in mice treated 24 hours post–aerosol exposure, hMAb F5 nIgG prevented clinical signs of disease but could not prevent infection.⁵⁵ Further studies will determine the ability of F5nIgG to confer protection in nonhuman primates.

The use of cationic lipsome-DNA complexes (CLDCs) containing noncoding plasmid DNA to treat WEEV infection has been evaluated.⁵⁶ Mice were administered CLDCs prior to infection or at the time of infection or were treated following challenge with a subcutaneous or aerosol dose of WEEV. Mice treated with CDLCs had increased cytokine levels consistent with innate immune activation, which would lead to a robust Th1 response. Pretreatment with CLDCs afforded the most protection from a uniformly lethal WEEV challenge. These data suggest that a nonspecific activation of the innate immune response can provide protection from encephalitic alphavirus exposure and/or infection.

Conclusion

Over the past 10 years, there has been significant investment in the research and development of antiviral therapeutics for viral threat agents by small companies and public research institutions, yet few compounds have moved beyond early-stage research and development. While the reasons for this are multifactorial, the rapidly escalating costs of the candidate product as it progresses in the developmental pipeline is likely a significant contributor to the dearth of leads progressing to advanced testing. As such, it will be critical to test and evaluate these candidate products in a manner that informs their use during a public health emergency as early in development as possible. It is imperative that developers test their products at time points postexposure or postchallenge that are consistent with the ability to provide an effective public health response, even if it risks revealing limited efficacy, in order to provide meaningful, actionable data on product utility.

Given the highly pathogenic nature of many of these agents, it is likely that the corresponding candidate therapeutic will have to be extraordinarily potent in its ability to inhibit viral pathogenesis or replication to provide a substantial clinical benefit. Alternatively, a combination of antiviral medications, inhibiting multiple aspects of the viral life cycle, or damage mediated by the host response will likely be required to provide substantial therapeutic benefit once disease symptoms have emerged.

Some of the research described in this article involved the development of candidate therapeutics that target host cellular processes as a means to disrupt viral pathogenesis or replication. While this approach appears potentially promising, several major limitations should temper enthusiasm for biodefense applications.

First and foremost is the concept that, given the small number of FDA approvals under the Animal Rule despite significant investment, we believe the biodefense community should practice risk aversion to technologies that have not yielded approved drug or strong drug candidates (phase III) against conventional, clinically prevalent diseases. This is due to the largely unprecedented regulatory environment the field must perform in. Given the fact that there are currently no host-directed antivirals for any viral infection, the developmental risk of pursuing an unprecedented technology in an unprecedented regulatory environment appears extremely high. Overall, the risk of reliance on emerging technologies could be reduced by validating them in other therapeutic areas prior to investment by the biodefense community if the goal is to develop FDA-approved products.

Second, given that the approval of these products would require efficacy testing in animal models, it would be imperative that the target of the host-directed therapeutic be conserved across human and experimental model animal species in order to be able to demonstrate efficacy. Depending on the target, this may represent a technical challenge.

Third, achieving appropriate levels of selective toxicity may be a technical challenge. Given that the host targets are involved in normal cellular processes, their inhibition or activation may have deleterious consequences. A determination of potential toxicity should be identified quickly in any host-directed development program.

The challenges of development and regulatory approval of therapeutics for viral threat agents are significant. One of the most notable challenges is the development of animal models that support approval under the FDA Animal Rule. These models must effectively recapitulate the human disease, a fact that is typically impossible to completely mimic with any one model. This is particularly true in developing animal models for smallpox, where the route of challenge administration, time course of disease, absence of a prodromal phase, variable endpoints, and nature of viral tropism (human specific) all complicate model development.⁵⁷ The regulatory acceptance of any model of viral threat agent infection for therapeutic evaluation has not been demonstrated. In the absence of a single or limited number of animal models that are highly compelling in their ability to recapitulate human disease, it is likely that data from multiple animal models, each with their own strengths and limitations, will be required to provide the supporting evidence for a meaningful determination of a product's therapeutic efficacy.

References

DiMasi

, Hansen

, Grabowski

. The price of innovation: new estimates of drug development costs. J Health Econ, 2003; 22,2:151–185.

U.S. Food and Drug Administration. Guidance for Industry: Animal Models—Essential Elements to Address Efficacy Under the Animal Rule. January 2009. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm078923.pdf. 2011 September 7.

Jahrling

, Fritz

, Hensley

. Countermeasures to the bioterrorist threat of smallpox. Curr Mol Med, 2005; 5,8:817–826.

Bray

, Pilch

. Filoviruses: recent advances and future challenges. Expert Rev Anti Infect Ther, 2006; 4,6:917–921.

Olinger

, Biggins

, Melanson

, Wahl-Jensen

, Geisbert

, Hensley

. Drug targets in infections with Ebola and Marburg viruses. Infect Disord Drug Targets, 2009; 9,2:191–200.

Steele

, Twenhafel

. Review paper: pathology of animal models of alphavirus encephalitis. Vet Pathol, 2010; 47,5:790–805.

Yang

, Pevear

, Davies

et al. An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge. J Virol, 2005; 79,20:13139–13149.

Quenelle

, Buller

, Parker

et al. Efficacy of delayed treatment with ST-246 given orally against systemic orthopoxvirus infections in mice. Antimicrob Agents Chemother, 2007; 51,2:689–695.

Nalca

, Hatkin

, Garza

et al. Evaluation of orally delivered ST-246 as postexposure prophylactic and antiviral therapeutic in an aerosolized rabbitpox rabbit model. Antivir Res, 2008; 79,2:121–127.

10.

Huggins

, Goff

, Hensley

et al. Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246. Antimicrob Agents Chemother, 2009; 53,6:2620–2625.

11.

SIGA Technologies Inc.

Safety, tolerability, pharmacokinetics (PK) of the anti-orthopox drug, ST-246 (246-Safety)

ClinicalTrials.gov 2000NLM Identifier NCT00907803http://clinicaltrials.gov/ct2/show/NCT00907803?term=NCT00907803&rank=1. 2011 September 7.

12.

Jordan

, Chinsangaram

, Bolken

et al. Safety and pharmacokinetics of the anti-orthopoxvirus compound ST-246 following repeat oral dosing in healthy adult subjects. Antimicrob Agents Chemother, 2010; 54,6:2560–2566.

13.

Stittelaar

, Neyts

, Naesens

et al. Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox viral infection. Nature, 2006; 439,7077:745–748.

14.

Dropulic

, Cohen

. Update on new antivirals under development for the treatment of double-stranded DNA virus infections. Clin Pharmacol Ther, 2010; 88,5:610–619.

15.

Toth

, Spencer

, Dhar

et al. Hexadecyloxypropyl-cidofovir, CMX001, prevents adenovirus-induced mortality in a permissive, immunosuppressed animal model. Proc Natl Acad Sci U S A, 2008; 105,20:7293–7297.

16.

Rice

, Adams

, Lampert

et al. Efficacy of CMX001 as a prophylactic and presymptomatic antiviral agent in New Zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans. Viruses, 2011; 3,2:63–82.

17.

Rice

, Adams

, Wallace

et al. Efficacy of CMX001 as a post exposure antiviral in New Zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans. Viruses, 2011; 3,1:47–62.

18.

Chimerix Inc. Comparative bioavailability and effect of food on CMX001 in healthy volunteers. ClinicalTrials.gov 2000NLM Identifier NCT00780182http://clinicaltrials.gov/ct2/show/NCT00780182?term=chimerix&rank=5. 2011 September 7.

19.

Myskiw

, Piper

, Huzarewich

, Booth

, Cao

, He

. Nigericin is a potent inhibitor of the early stage of vaccinia virus replication. Antivir Res, 2010; 88,3:304–310.

20.

Howell

, Streib

, Kim

et al. Ceragenins: a class of antiviral compounds to treat orthopox infections. J Invest Dermatol, 2009; 129,11:2668–2675.

21.

Dawson

, Liu

. Properties and applications of antimicrobial peptides in biodefense against biological warfare threat agents. Crit Rev Microbiol, 2008; 34,2:89–107.

22.

Alkhalil

, Strand

, Mucker

, Huggins

, Jahrling

, Ibrahim

. Inhibition of monkeypox virus replication by RNA interference. Virol J, 2009; 6:188.

23.

Ahmed

, Martin

, Johnson

. IFN mimetic as a therapeutic for lethal vaccinia virus infection: possible effects on innate and adaptive immune responses. J Immunol, 2007; 178,7:4576–4583.

24.

Ahmed

, Dabelic

, Waiboci

, Jager

, Heron

, Johnson

. SOCS-1 mimetics protect mice against lethal poxvirus infection: identification of a novel endogenous antiviral system. J Virol, 2009; 83,3:1402–1415.

25.

McCausland

, Benhnia

, Crickard

et al. Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model. Antivir Ther, 2010; 15,4:661–675.

26.

Tomimori

, Kawakami

, McCausland

et al. Protective murine and human monoclonal antibodies against eczema vaccinatum. Antivir Ther, 2011; 16:67–75.

27.

Zaitseva

, Kapnick

, Meseda

et al. Passive immunotherapies protect WRvFire and IHD-J-Luc vaccinia virus-infected mice from lethality by reducing viral loads in the upper respiratory tract and internal organs. J Virol, 2011; 85,17:9147–9158.

28.

Guo

, Pan

, Mao

et al. Alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses. Antimicrob Agents Chemother, 2011; 55,2:478–486.

29.

Panchal

, Kota

, Spurgers

et al. Development of high-content imaging assays for lethal viral pathogens. J Biomol Screen, 2010; 15,7:755–765.

30.

Wolf

, Freiberg

, Zhang

et al. A broad spectrum antiviral targeting entry of enveloped viruses. Proc Natl Acad Sci U S A, 2010; 107,7:3157–3162.

31.

Francis

, Kalina

, Warren

et al. The discovery and efficacy of a small molecule inhibitor of Ebola capsid assembly in an animal model, 2010Abstract 12323rd ICAR: San Francisco, California.

32.

Aman

, Kinch

, Warfield

et al. Development of a broad-spectrum antiviral with activity against Ebola virus. Antiviral Res, 2009; 83,3:245–251.

33.

Geisbert

, Hensley

, Jahrling

et al. Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet, 2003; 362,9400:1953–1958.

34.

Connor

, McKenzie

, Parks

, Lyles

. Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses. Virology, 2007; 362,1:109–119.

35.

Smith

, McCarthy

, Chrovian

et al. Inhibition of heat-shock protein 90 reduces Ebola virus replication. Antiviral Res, 2010; 87,2:187–194.

36.

Warren

, Warfield

, Wells

et al. Advanced antisense therapies for postexposure protection against lethal filovirus infections. Nat Med, 2010; 16,9:991–994.

37.

Geisbert

, Hensley

, Kagan

et al. Postexposure protection of guinea pigs against a lethal Ebola virus challenge is conferred by RNA interference. J Infect Dis, 2006; 193,12:1650–1657.

38.

Geisbert

, Lee

, Robbins

et al. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet, 2010; 375,9729:1896–1905.

39.

Michelow

, Lear

, Scully

et al. High-dose mannose binding lectin therapy for Ebola virus infection. J Infec Dis, 2011; 203,2:175–179.

40.

Hensley

, Stevens

, Yan

et al. Recombinant human activated protein C for the postexposure treatment of Ebola hemorrhagic fever. J Infect Dis, 2007; 196,Suppl 2:S390–399.

41.

Wauquier

, Becquart

, Gasquet

, Leroy

. Immunoglobulin G in Ebola outbreak survivors, Gabon. Emerg Infect Dis, 2009; 15,7:1136–1137.

42.

Wauquier

, Becquart

, Padilla

, Baize

, Leroy

. Human fatal Zaire Ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis. PLoS Negl Trop Dis, 2010; 4,10:e837.

43.

Parren

, Geisbert

, Maruyama

, Jahrling

, Burton

. Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody. J Virol, 2002; 76,12:6408–6412.

44.

Takada

, Ebihara

, Jones

, Feldmann

, Kawaoka

. Protective efficacy of neutralizing antibodies against Ebola virus infection. Vaccine, 2007; 25,6:993–999.

45.

Oswald

, Geisbert

, Davis

et al. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathog, 2007; 3,1:62–66.

46.

Shedlock

, Bailey

, Popernack

, Cunningham

, Burton

, Sullivan

. Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms. Virology, 2010; 401,2:28–35.

47.

Reichert

, Clase

, Bacetty

, Larsen

. Alphavirus antiviral drug development: scientific gap analysis and prospective research areas. Biosecur Bioterror, 2009; 7,4:413–427.

48.

Julander

, Bowen

, Rao

et al. Treatment of Venezuelan equine encephalitis virus infection with (-)-carbodine. Antiviral Res, 2008; 80,3:309–315.

49.

O'Brien

. Inhibition of multiple strains of Venezuelan equine encephalitis virus by a pool of four short interfering RNAs. Antivir Res, 2007; 75,1:20–29.

50.

Paessler

, Rijnbrand

, Stein

et al. Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers. Virology, 2008; 376,2:357–370.

51.

Parker

, Buckley

, Melanson

, Glass

, Norwood

, Hart

. Antibody to the E3 glycoprotein protects mice against lethal Venezuelan equine encephalitis virus infection. J Virol, 2010; 84,24:12683–12690.

52.

, Phelps

, Jager

et al. A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus. Vaccine, 2010; 28,34:5558–5564.

53.

Goodchild

, O'Brien

, Steven

et al. A humanized murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus. Antiviral Res, 2011; 90,1:1–8.

54.

Hunt

, Frederickson

, Maruyama

, Roehrig

, Blair

. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity. PLoS Negl Trop Dis, 2010; 4,7:e739.

55.

Hunt

, Bowen

, Frederickson

, Roehrig

, Blair

. Treatment of mice with human monoclonal antibody 24h after lethal aerosol challenge with virulent Venezuelan equine encephalitis virus prevents disease but not infection. Virology, 2011; 414,2:146–152.

56.

Logue

, Phillips

, Mossel

et al. Treatment with cationic liposome-DNA complexes (CLDCs) protects mice from lethal Western equine encephalitis virus (WEEV) challenge. Antiviral Res, 2010; 87,2:195–203.

57.

Jordan

, Hruby

. Smallpox antiviral drug development: satisfying the animal efficacy rule. Expert Rev Anti Infect Ther, 2006; 2:277–289.