Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).

Escape from adaptive T cell immunity through transmutation of viral antigenic structure is a cardinal feature in the pathogenesis of SIV/HIV infection and a major obstacle to antiretroviral vaccine development. However, the molecular determinants of this phenomenon at the T cell receptor (TCR)-antigen interface are unknown. Here, we show that mutational escape is intimately linked to the structural configuration of constituent TCR clonotypes within virus-specific CD8(+) T cell populations. Analysis of 3416 SIV-specific TCR sequences revealed that polyclonal T cell populations characterized by highly conserved TCRB CDR3 motifs were rendered ineffectual by single residue mutations in the cognate viral epitope. Conversely, diverse clonotypic repertoires without discernible motifs were not associated with viral escape. Thus, fundamental differences in the mode of antigen engagement direct the pattern of adaptive viral evolution. These findings have profound implications for the development of vaccines that elicit T cell immunity to combat pathogens with unstable genomes.

IntroductionThe induction and maintenance of immune responses to antigens is tightly regulated. Activation of T cells requires the interaction between the T-cell receptor and the antigen presented on the surface of an antigen-presenting cell (APC; first signal) and engagement of CD28 (second signal) by the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). 1,2 Costimulation is particularly important for the initial T-cell response, promoting proliferation and survival. Following antigen stimulation, both CD28 and its negative regulatory, cytotoxic T lymphocyte antigen-4 (CTLA-4), are up-regulated on the cell surface and compete for their ligands, B7-1 and B7-2. CTLA-4 binds to both B7-1 and B7-2 with higher (10-to 20-fold) affinity than CD28, 3,4 and, in contrast to CD28, CTLA-4 suppresses T-cell activation. However, competition with CD28 for the costimulatory molecules is not likely to be the main mechanism responsible for CTLA-4 immunoregulatory activity. [5][6][7] Several lines of evidence suggest that expression of CTLA-4 by CD25 ϩ CD4 ϩ regulatory T (T reg ) cells plays a role in controlling peripheral T-cell tolerance and differentiation. 8,9 T reg cells are CD4 ϩ T lymphocytes that express high levels of the interleukin-2 (IL-2) receptor ␣-chain (CD25), and constitutively express CTLA-4. T reg cells inhibit the proliferation of T cells through contactdependent or cytokine-mediated (IL-10, transforming growth factor-␤ [TGF-␤]) inhibition of T-cell responses. 10 T reg cells can induce activation of the enzyme IDO in APCs via CTLA-4-mediated ligation of CD80/CD86. 11,12 IDO confers immunosuppressive activity to APCs. 11,12 Two mechanisms have been suggested as mediators of the T-cellsuppressive action of IDO 13 : degradation and consequent reduction of tryptophan, an essential amino acid required for T-cell proliferation; and generation of inhibitory tryptophan metabolites. Blocking CTLA-4 signals with monoclonal antibodies may provide an important tool to influence the host immune response in clinical settings. For example, synergy between anti-CD25 and anti-CTLA-4 monoclonal antibodies has been shown to be effective in antitumor therapy. 14,15 T reg cells may suppress a potentially successful adaptive host immune response to a pathogen. [16][17][18][19][20][21] In the case of HIV infection, CTLA-4 expression is higher in patients with advanced clinical symptoms compared with asymptomatic individuals, 22,23 and the frequency of CTLA-4-expressing CD25 ϩ CD4 ϩ T reg cells is increased in lymphoid tissues in untreated individuals infected with HIV-1. 24 Primary infection of macaques with simian immunodeficiency virus (SIV) is associated with an increase in T reg cells, IDO, TGF-␤, and IL-10. 25 Similarly, chronic SIV infection is associated with an accumulation of T reg cells in lymphoid tissues, including the gut, and an increased level of immunosuppressive cytokines (A. B., M. V., A. H., D. F., J. N., Valentina Cecchinato, G. F., G. M. S., and C. C., our unpublished results, October 2006).Here, we examined...

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20–155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.