Quantification of antibody responses against multiple antigens of the two infectious forms of Vaccinia virus provides a benchmark for smallpox vaccination


Smallpox was eradicated without an adequate understanding of how vaccination induced protection. In response to possible bioterrorism with smallpox, the UK government vaccinated 300 health care workers with vaccinia virus (VACV) strain Lister. Antibody responses were analyzed using ELISA for multiple surface antigens of the extracellular enveloped virus (EEV) and the intracellular mature virus (IMV), plaque reduction neutralization and a fluorescence-based flow cytometric neutralization assay. Antibody depletion experiments showed that the EEV surface protein B5 is the only target responsible for EEV neutralization in vaccinated humans, whereas multiple IMV surface proteins, including A27 and H3, are targets for IMV-neutralizing antibodies. These data suggest that it would be unwise to exclude the B5 protein from a future smallpox vaccine. Repeated vaccination provided significantly higher B5-specific and thus EEV-neutralizing antibody responses. These data provide a benchmark against which new, safer smallpox vaccines and residual immunity can be compared.

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Figure 1: Antibody responses in humans (n = 92) after smallpox vaccination.
Figure 2: Mean residual neutralizing activity in human sera (n = 8) after antibody depletion.


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We thank all the UK health care workers who participated in the study; the people at the regional Occupational Health Departments who supervised and monitored the study; the people at the Health Protection Agency, Colindale, UK, in particular C. Auckland and R. Gopal, for the sample collection; E. Montomoli (University of Siena) for the serum samples of unvaccinated teenagers; D.H. Davies and D. Huynh (Protein Microarray Laboratory, University of California Irvine) for the gift of purified recombinant H3 protein (NIH/National Institute of Allergy and Infectious Diseases (NIAID) grant support #R41AI058365 to P. Felgner, Principal Investigator); H.-P. Su and D. Garboczi (NIH/NIAID) for the gift of purified recombinant L1 protein; and E. Kulinskaya (Statistical Advisory Service, Imperial College, London) for advice on the statistical analysis. VIG was provided by T. Monath (Acambis Inc.). M.M.P. and C.M.M. received financial support from the Department of Health and M.L. from the Wellcome Trust. G.L.S. is a Wellcome Trust Principal Research Fellow.

Author information




M.M.P., M.L. and G.L.S. designed the study. M.L. established the expression and purification of EEV glycoproteins. C.M.M. performed the expression and purification of IMV proteins. M.M.P. implemented the antigen-specific ELISAs. C.M.M. set up the IMV ERN assay. M.M.P. and C.M.M. carried out the serological assays and performed statistical analysis. The manuscript was written by M.M.P. and G.L.S. with input from all the other investigators. M.M.P. and C.M.M. contributed equally to this work.

Corresponding author

Correspondence to Geoffrey L Smith.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Residual antibody titers observed at day 0 in comparison to time since most recent vaccination. (PDF 46 kb)

Supplementary Fig. 2

Comparative seropositivity rates in groups of participants with different vaccination history. (PDF 29 kb)

Supplementary Fig. 3

Comparison of antibody titers obtained at day 0 and at day 7. (PDF 60 kb)

Supplementary Fig. 4

Effect of vaccination history on seroconversion and boosting following vaccination. (PDF 25 kb)

Supplementary Fig. 5

Effect of vaccination history on antibody stability following vaccination. (PDF 22 kb)

Supplementary Table 1

Correlation analysis between data sets from different assays. (PDF 14 kb)

Supplementary Table 2

Amino acid (and nucleotide) identities between antigens of VACV WR and other orthopoxviruses. (PDF 14 kb)

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Pütz, M., Midgley, C., Law, M. et al. Quantification of antibody responses against multiple antigens of the two infectious forms of Vaccinia virus provides a benchmark for smallpox vaccination. Nat Med 12, 1310–1315 (2006). https://doi.org/10.1038/nm1457

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