Development of ST-246® for Treatment of Poxvirus Infections

ST-246 (Tecovirimat) is a small synthetic antiviral compound being developed to treat pathogenic orthopoxvirus infections of humans. The compound was discovered as part of a high throughput screen designed to identify inhibitors of vaccinia virus-induced cytopathic effects. The antiviral activity is specific for orthopoxviruses and the compound does not inhibit the replication of other RNA- and DNA-containing viruses or inhibit cell proliferation at concentrations of compound that are antiviral. ST-246 targets vaccinia virus p37, a viral protein required for envelopment and secretion of extracellular forms of virus. The compound is orally bioavailable and protects multiple animal species from lethal orthopoxvirus challenge. Preclinical safety pharmacology studies in mice and non-human primates indicate that ST-246 is readily absorbed by the oral route and well tolerated with the no observable adverse effect level (NOAEL) in mice measured at 2000 mg/kg and the no observable effect level (NOEL) in non-human primates measured at 300 mg/kg. Drug substance and drug product processes have been developed and commercial scale batches have been produced using Good Manufacturing Processes (GMP). Human phase I clinical trials have shown that ST-246 is safe and well tolerated in healthy human volunteers. Based on the results of the clinical evaluation, once a day dosing should provide plasma drug exposure in the range predicted to be antiviral based on data from efficacy studies in animal models of orthopoxvirus disease. These data support the use of ST-246 as a therapeutic to treat pathogenic orthopoxvirus infections of humans.     

Risk of lymphoma and leukaemia after bacille Calmette-Guérin and smallpox vaccination: A Danish case-cohort study

Vaccines may have non-specific effects as suggested mainly in mortality studies from low-income countries. The objective was to examine the effects of BCG and smallpox vaccinations on subsequent risk of lymphoma and leukaemia in a Danish population experiencing rapid out-phasing of these vaccines. In a background cohort (N = 47,622) from the Copenhagen School Health Records Register, cases of leukaemia (N = 20) and lymphoma (N = 51) were identified through the Danish Cancer Registry. The vaccination status of the cases was compared with the vaccination status of a 5% random sample (N = 2073) of the background cohort and analysed in a case-cohort design. BCG vaccination reduced the risk of lymphomas (HR = 0.49 (95% CI: 0.26–0.93)), whereas smallpox vaccination did not (HR = 1.32 (0.56–3.08)). With the small number of leukaemia cases, the analysis of leukaemia had limited power (BCG vaccination HR = 0.81 (0.31–2.16); smallpox vaccination HR=1.32 (0.49–3.53)). The present study with very reliable vaccine history information indicates a beneficial effect of BCG vaccination on the risk of lymphomas.     

Discovery of naturally processed and HLA-presented class I peptides from vaccinia virus infection using mass spectrometry for vaccine development

An important approach for developing a safer smallpox vaccine is to identify naturally processed immunogenic vaccinia-derived peptides rather than live whole vaccinia virus. We used two-dimensional liquid chromatography coupled to mass spectrometry to identify 116 vaccinia peptides, encoded by 61 open reading frames, from a B-cell line (homozygous for HLA class I A*0201, B*1501, and C*03) after infection with vaccinia virus (Dryvax). Importantly, 68 of these peptides are conserved in variola, providing insight into the peptides that induce protection against smallpox. Twenty-one of these 68 conserved peptides were 11 amino acids long or longer, outside of the range of most predictive algorithms. Thus, direct identification of naturally processed and presented HLA peptides gives important information not provided by current computational methods for identifying potential vaccinia epitopes.     

Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 10⁶ pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.     

Bioterrorism and electron microscopic differentiation of poxviruses from herpesviruses: dos and don'ts

With increased threat of terrorism, much attention is being directed toward readiness for biodefense. Smallpox virus, a deadly and much feared organism, is among possible bioterrorism agents. Herpesviruses, such as the one that causes chickenpox and shingles, produce skin lesions that may resemble those seen early in smallpox infection. Electron microscopy (EM) is a rapid and reliable method for differentiating poxviruses from herpesviruses. However, before becoming involved in the monitoring of potential smallpox cases, a laboratory must consider several issues, including expertise in virus identification, capacity for handling biohazards, and health and immune status of laboratory staff.     

Immunity from Smallpox Vaccine Persists for Decades: A Longitudinal Study

Purpose

The threat of smallpox resulting from bioterrorist action has prompted a reassessment of the level of immunity in current populations.

Methods

We have examined the magnitude and duration of antiviral antibody immunity conferred by smallpox vaccination in 246 participants of the Baltimore Longitudinal Study of Aging. Of this population, 209 subjects were vaccinated one or more times 13 to 88 years before this evaluation, and stored serum samples were available at various intervals after vaccination. An additional 8 subjects who had documented childhood smallpox infection and 29 subjects with no history of infection or vaccination were included. We quantified the total vaccinia IgG and neutralizing antibody titers in each of these subgroups of participants over time.

Results

Vaccinated participants maintained antivaccinia IgG and neutralizing antibody titers above 3 natural logs essentially indefinitely. The absolute titer of antivaccinia antibody was only slightly higher after multiple vaccinations. In 97% of the participants, no decrease in vaccinia-specific antibody titers was noted with age over a follow-up period of up to 88 years. Moreover, Baltimore Longitudinal Study of Aging participants who survived active smallpox infections in their youth retained antivaccinia antibody titers that were similar to the levels detected in vaccinated subjects.

Conclusion

These data suggest that multiple or recent vaccinations are not essential to maintain vaccinia-specific antibody responses in human subjects. Scarce vaccine supplies should be applied first to individuals who have not previously been vaccinated.     

Evaluation of orally delivered ST-246 as postexposure prophylactic and antiviral therapeutic in an aerosolized rabbitpox rabbit model

Orthopoxviruses, such as variola and monkeypox viruses, can cause severe disease in humans when delivered by the aerosol route, and thus represent significant threats to both military and civilian populations. Currently, there are no antiviral therapies approved by the U.S. Food and Drug Administration (FDA) to treat smallpox or monkeypox infection. In this study, we showed that administration of the antiviral compound ST-246 to rabbits by oral gavage, once daily for 14 days beginning 1h postexposure (p.e.), resulted in 100% survival in a lethal aerosolized rabbitpox model used as a surrogate for smallpox. Furthermore, efficacy of delayed treatment with ST-246 was evaluated by beginning treatment on days 1, 2, 3, and 4 p.e. Although a limited number of rabbits showed less severe signs of the rabbitpox disease from the day 1 and day 2 p.e. treatment groups, their illness resolved very quickly, and the survival rates for these group of rabbits were 88% and 100%, respectively. But when the treatment was started on days 3 or 4 p.e., survival was 67% and 33%, respectively. This work suggests that ST-246 is a very potent antiviral compound against aerosolized rabbitpox in rabbits and should be investigated for further development for all orthopoxvirus diseases.     

Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate

Recombinant and non-recombinant modified vaccinia virus Ankara (MVA) strains are currently in clinical trials as human immunodeficiency virus-1 (HIV) and attenuated smallpox vaccines, respectively. Here we tested the ability of a recombinant MVA delivered by alternative needle-free routes (intramuscular, intradermal, or into the palatine tonsil) to protect against immunodeficiency and orthopoxvirus diseases in a non-human primate model. Rhesus macaques were immunized twice 1 month apart with MVA expressing 5 genes from a pathogenic simian human immunodeficiency virus (SHIV)/89.6P and challenged intrarectally 9 months later with the pathogenic SHIV/89.6P and intravenously 2.7 years later with monkeypox virus. Irrespective of the route of vaccine delivery, binding and neutralizing antibodies and CD8 responses to SHIV and orthopoxvirus proteins were induced and the monkeys were successively protected against the diseases caused by the challenge viruses in unimmunized controls as determined by viral loads and clinical signs. These non-human primate studies support the clinical testing of recombinant MVA as an HIV vaccine and further demonstrate that MVA can provide long-term poxvirus immunity, essential for use as an alternative smallpox vaccine.     

Phase II randomized,double-blinded comparison of a single high dose (5 × 10 TCID50) of modified vaccinia Ankara compared to a standard dose (1 × 10 TCID50) in healthy vaccinia-naïve individuals

Introduction

Reintroduction of Variola major as an agent of bioterrorism remains a concern. Time to seroconversion and plaque reduction neutralizing antibody titers (PRNT) of 1 or 2 standard doses (SD) were compared to a single high dose (HD) of modified vaccinia Ankara (MVA).

Methods

Ninety subjects were randomized 1:1 to receive 1 HD or 2 SD of MVA subcutaneously on Days 0 and 28 in a placebo-controlled trial. Serum was collected for PRNT and ELISA. Subjects were followed for safety for the entire study.

Results

The HD was well-tolerated. Using Bavarian Nordic's ELISA, subjects in both groups achieved seroconversion by Study Day 15 (HD) and Day 28 (SD). Before second vaccination, the hazard rate of seroconverting for the HD group was 1.7 times the SD group with a median time for seroconversion of 14 days for both groups. The peak titer of one HD vaccine was superior to one dose of SD vaccine but inferior to the peak titer after the second dose of the SD vaccination regimen.Using Saint Louis University's PRNT, peak titers were 95.8 and 65.2 for the HD and SD groups, respectively, prior to second vaccination. Non-inferiority of the SD group was not established. The proportions of positives were 93.3% (42/45) and 82.2% (37/45) for the HD and SD groups, respectively. The peak titer after two standard doses was superior to that of the HD.

Conclusions

HD MVA was safe and well-tolerated. While the hazard rate for seroconverting was significantly higher in the HD group before second dose, the effect was small as the median time to seroconversion was identical. When comparing PRNT, non-inferiority of one SD was not established and the peak titers were low for both groups. The HD peak response was inferior to the standard two-dose regimen response based on ELISA and PRNT.     

A short history of biological warfare

Biological weapons have been used in war from the start of recorded history. This article reviews the history of the subject, including the outbreak of the Black Death and the use of smallpox against American Indians. The new science of microbiology was misused from soon after its start and, despite the 1925 Geneva Protocol, the Japanese experimented extensively on prisoners in China. The Allies carried out extensive research during the Second World War, notably the United Kingdom into anthrax on Gruinard Island and the United States into a variety of agents. Despite the 1972 Biological Weapons Convention (BWC), a major programme continued in the former Soviet Union (leading to an accidental outbreak of anthrax). Most recently Iraq was revealed as having an extensive programme, with weaponization of large amounts of various agents, and several terrorists groups have attempted to use biological agents as weapons. Modern developments in biotechnology could lead to even more serious developments, and effective preventive measures, including strengthening of the BWC, are imperative.