Smallpox was eradicated without an adequate understanding of how vaccination induced protection. In response to possible bioterrorism with smallpox, the UK government vaccinated approximately 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.

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 Å resolution. The protein has a globular β sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI–chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (μM–nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine–chemokine receptor interactions.

Vaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.

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Decades after smallpox was eradicated and vaccination discontinued, the level of residual immunity in today's population is largely unknown. This study describes an epidemiological assessment in Italians of antibodies against the intracellular mature virus (IMV) and extracellular envelope virus (EEV) forms of Vaccinia virus. Serum samples (n=642) were taken in 1993 and 2003 from people between 11 and 102 years old. Most citizens >27 years old were positive for antibodies to IMV and EEV. These antibodies were long-lasting and similar titres were present in citizens between 30 and 100 years old. Serum samples from 1993 and 2003 displayed very similar EEV-and IMV-specific antibody titres. By using these data and demographic considerations, it was predicted that, in 2003, 46 % of the Italian population were positive for both IMV and EEV, 42 % were negative for both and 12 % were positive for one antigen.Variola virus, the causative agent of smallpox, was eradicated in 1977 after widespread vaccination with Vaccinia virus (VACV) (Fenner et al., 1988). In Italy and many other European countries, smallpox vaccination used predominantly VACV strain Lister/Elstree. Vaccination was compulsory in Italy until 1976(Tagarelli et al., 2004 although, after 1974, public-health services were less stringent in their vaccination policy. Mass smallpox vaccination was abandoned in most European countries in the early 1970s and production of the vaccine was discontinued in the early 1980s.Understanding of the immune responses induced after vaccination with VACV is incomplete and concern about the deliberate release of Variola virus (Smith & McFadden, 2002) has prompted investigation of how much immunity remains in populations today. It was generally accepted that vaccination would protect most vaccinees for 5-10 years (Fenner et al., 1988), although the World Health Organization (WHO) recommended revaccination every 3 years for those in areas where smallpox was endemic. Recent studies have shown that humoral (Crotty et al., 2003;Frey et al., 2003;Gallwitz et al., 2003;Hammarlund et al., 2003;Hatakeyama et al., 2005;Viner & Isaacs, 2005) and cellular (Hammarlund et al., 2003;Amara et al., 2004;Combadiere et al., 2004;Kennedy et al., 2004) forms of immunity are long-lived after VACV immunization, but it is unknown whether this remaining immunity will protect against smallpox. Although analysis of historical data suggests that protection after vaccination might last for several decades (Hanna & Baxby, 2002;Eichner, 2003a), the last smallpox fatality, in Birmingham, UK, in 1978, occurred despite the patient having been vaccinated twice previously, once as a child and once 12 years before contracting the disease (Shooter, 1980). VACV produces two morphologically and antigenically distinct infectious forms of virus, called intracellular mature virus (IMV) and extracellular envelope virus (EEV) . Most infectious virus particles remain in the cell as IMV until cell lysis; this form is very stable and so is likely to be responsible for host-to...