David Liu Smallpox: Gone but not forgotten Antiviral Treatment Is More Effective Than Smallpox Vaccination Upon Lethal Monkeypox Virus Infection Stittelaar, et al. Nature. 2006 Feb 9;439(7077):745-8

History: Vaccination and Eradication Variola Major The first vaccine in history was developed for smallpox by Edward Jenner in 1796. By the 1960s, smallpox was limited to developing countries—completely eradicated from the West. Vaccinia vaccine. 1967 WHO global vaccination campaign Last natural case in 1977 In 1978, all known stocks were transferred to either the CDC in Atlanta or the VECTOR Institute in the USSR.

Possible: bioterrorism Smallpox is: Easily transmissible High mortality rate Disfiguring Vaccination has stopped virgin population Pox Americana Hypothetical illicit sources. Known weaponization of smallpox in the former Soviet Union Flat-type smallpox: Mortality rate > 96% Hemorrhagic-type smallpox: Mortality rate > 98%

CDC response plan Overall plan: 1) Identify and isolate smallpox cases to prevent further disease spread, 2) Identify, vaccinate, and monitor contacts of cases and household contacts of contacts, to prevent secondary cases. Stance on post-exposure: “if [the vaccine is] administered within 3 days after exposure to smallpox virus, it may prevent disease, or decrease the severity of disease and risk of death.” Newest version 3.0: The CDC Smallpox Response Plan and Guidelines were last updated on September 23, 2002.

Antiviral treatment: nucleotide analogues (De Clerq, 2004) Acyclic nucleotide phosphonates

Research Question What would be the efficacy of post-exposure vaccination versus antiviral treatment in the event of poxvirus re-emergence, e.g. a bioterrorist attack? Proof of concept study

Overview of methods Infect monkeys with a lethal respiratory dose of monkeypox virus Why not smallpox? Determine treatment efficacy in drug-treated, vaccinated, and control groups Five parameters (independent variables): survival, O2 profile, lesions, viral load, immune response (antibodies/T-cells) Measurements made on days 1, 3, 5, 7, 10, 13, 18, 21, 26 Throat swabs, plasma samples, O2 readings, observation

Setting up. Infection of monkeys with 107 pfu of MPXV Segregation into experimental groups: I : Control (n=17) (n=6) II: Elstree-RIVM (vaccinia-virus vaccine) (n=6) III: Cidofovir (5 doses): Days 1, 3, 7, 10, 13 (n=6) IV: Cidofovir (6 doses): Days 1, 3, 5, 7, 10, 13 (n=6) V: HPMPO-DAPy (5 doses) (n=4) VI: HPMPO-DAPy (6 doses) (n=6) Already proven that pre-exposure vaccination is effective against smallpox infection (Stittelaar 2005) Drugs given in dosages of 5 mg kg-1 Monkeys in group III — VI were also given probenecid

Methods for Parameter 1: Survival Kaplan-Meier Plot Commonly used in epidemiological studies Accounts for “lost” subjects Depicts the probability of survival at time t Approximate true survival curve Plasma creatinine levels

Results for Parameter 1: Survival Figure 1.

Methods for Parameter 2/3: Blood O2 levels and lesions Complications of smallpox most commonly arise in the respiratory system, usually around day 8 Dyspnea/anorexia Blood O2 levels measured using pulse oximeter Lesions are an obvious sign of the severity of the virus

Results for Parameter 2/3: Blood O2 levels and lesions Figure 2.

Methods for Parameter 4: Viral Load Real-time PCR with monkey plasma/throat swabs for MXPV DNA (dsDNA virus) Simultaneous amplification and quantification Fluorescent nucleotides HA gene primer The golden standard of viral studies

Results for Parameter 4: Viral Load Figure 3.

Methods for Parameter 5: Immune Response ELISA with RK13 cells as a reference MPXV- specific IgG Antibodies Interferon-γ ELISPOT assay MPXV-specific T-Cells 80% of peripheral blood is T-cells

Results for Parameter 5: Immune Response Supplementary Figure 1.

Summary of Results Post-exposure vaccination not effective Cidofovir and HPMPO-DAPy can protect macaques against MXPV when administered early 6 doses better than 5 HPMPO-DAPy performed better than Cidofovir Drug treatment allows for development of antibody resistance

Possible Limitations Better smallpox vaccines Possible toxicity (monkey 23) Small sample sizes Proof of concept Monkey model

Implications Need to rethink efficacy of post-exposure vaccination strategies Integration of antiviral drugs into CDC emergency response plan

Future Research In vitro experimentation with antiviral drugs in Variola Major Combining vaccine and drug therapy Changing dosage schedules

Debate Proposed destruction in 1986. Destruction postponed to 1993, and has been a resurfacing issue ever since. Last visitation was in 2011 Will be revisited again in 2014

References http://www.emergency.cdc.gov/agent/smallpox/response-plan/files/exec-sections-i-vi.pdf http://www.vassarstats.net/survival.html http://www.ncbi.nlm.nih.gov/pubmed/16341204 http://en.wikipedia.org/wiki/Smallpox http://en.wikipedia.org/wiki/Vaccinia http://www.cdc.gov/ncidod/monkeypox/factsheet2.htm http://www.nature.com/nature/journal/v439/n7077/extref/nature04295-s2.pdf http://en.wikipedia.org/wiki/Dyspnea http://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130682/pdf/0386.pdf