Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection.

BACKGROUND: Human cytomegalovirus (CMV) infection is an important risk factor in the post-transplant (Tx) recovery phase for both hematopoietic stem cell Tx (HSCT) and solid organ Tx (SOT) recipients. CMV infection may be prevented or controlled by simultaneously inducing both CMV-specific neutralizing antibody (nAb) and cellular immunity. Soluble (s) UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) are immunodominant in eliciting both CMV nAb and cellular immunity. An attenuated poxvirus, modified vaccinia Ankara (MVA) was selected to develop this vaccine strategy in Tx recipients, because of its clinical safety record, large foreign gene capacity, and capability to activate strong humoral and cellular immune responses against recombinant antigens. OBJECTIVES: A subunit vaccine that targets multiple CMV antigens will be used to gain maximal coverage and protective function against CMV infection. rMVA simultaneously expressing sUL55, UL83 and UL123/e4 will be generated, and humoral and cellular immunity it elicits will be characterized, after murine immunization and in vitro to amplify clinical recall responses. STUDY DESIGN: rMVA will be constructed in two steps using UL123/e4-pLW22 followed by sUL55-UL83-pLW51 transfer plasmids. Western blots will be used to characterize expression levels of each antigen. Primary immunity will be evaluated in mouse models, while recall responses to the virally expressed CMV antigens will be assessed in human peripheral blood. RESULTS: We generated CMV-MVA via homologous recombination, and demonstrated high expression levels of sUL55, UL83 and UL123/e4 by Western blot. CMV-MVA immunization potently induced both humoral and cellular immunity to sUL55, UL83 and UL123 after murine immunization, and cellular immunity to UL83 and UL123 by in vitro amplification of T cell recall responses in human PBMC. CONCLUSIONS: rMVA promotes high level expression of three immunodominant CMV antigens, which is reflected in results of immunization studies in which high titers of UL55-specific antibodies and CD4+ T-help are detected, as well as high levels of UL83-specific and moderate levels of UL123-specific CD8+ CTL.

Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus.

Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.

Relative dominance of HLA-B*07 restricted CD8+ T-lymphocyte immune responses to human cytomegalovirus pp65 in persons sharing HLA-A*02 and HLA-B*07 alleles.

CD8(+) T-cell responses to three human cytomegalovirus (CMV) pp65 epitopes were studied in panels of healthy seropositive HLA-A*02/HLA-B*07 individuals, and HLA-A*02 donors mismatched for HLA-B*07. The majority of the latter had significant responses to a HLA-A*02-restricted epitope within the CMV pp65 antigen. By contrast, the strongest responses to CMV in the first group were to HLA-B*07-restricted epitopes. Similar immunodominance of HLA-B*07 over HLA-A*02 was found in two immunocompromised HIV-infected HLA-A*02/HLA-B*07 patients, and in the reconstituting immune system of three stem cell transplant recipients. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from two immunocompetent HLA-A*02/HLA-B*07 individuals indicated that cytotoxic T lymphocyte (CTL) precursors specific for both HLA-A*02 and HLA-B*07 restricted epitopes were present and could be expanded by stimulation with the cognate peptides. However, if stimulation was performed by antigen presenting cells infected with recombinant vaccinia expressing full-length native pp65, only HLA-B*07 epitope-specific cells were seen. In one patient the HLA-B*07 dominance was partially broken by using recombinant vaccinia expressing ubiquitinated pp65, suggesting that enhanced protein processing can reveal weaker immune responses. Our results indicate that CMV-specific cellular immune responses restricted by HLA-B*07 dominate those restricted by HLA-A*02 in both immunocompetent and immunocompromised individuals. This may have significant consequences for the design of epitope-specific vaccines.

Preclinical development of an adjuvant-free peptide vaccine with activity against CMV pp65 in HLA transgenic mice.

Epitope vaccines have shown promise for inducing cellular immune responses in animal models of infectious disease. In cases where cellular immunity was augmented, peptide vaccines composed of covalently linked minimal cytotoxic T-lymphocyte (CTL) and T-helper (T(H)) epitopes generally showed the most efficacy. To address a clinical vaccine strategy for cytomegalovirus (CMV) in the context of HCT (hematopoietic cell transplantation), we observed that linking the synthetically derived pan-DR epitope peptide (PADRE) or one of several tetanus T(H) epitopes to the immunodominant human leukocyte antigen (HLA) A*0201-restricted CTL epitope from CMV-pp65 to create a fusion peptide caused robust cytotoxic cellular immune responses in HLA A*0201/K(b) transgenic mice. Significantly, the fusion peptides are immunogenic when administered in saline solution by either subcutaneous or intranasal routes. CpG-containing single-stranded DNA (ss-oligodeoxynucleotide [ODN]) added to the fusion peptides dramatically up-regulated immune recognition by either route. Notably, target cells that either expressed full-length pp65 protein from vaccinia viruses or were sensitized with the CTL epitope encoded in the vaccine were recognized by splenic effectors from immunized animals. Visualization of murine peptide-specific CTL by flow cytometry was accomplished using an HLA A*0201 tetramer complexed with the pp65(495-503) CTL epitope. T(H)-CTL epitope fusion peptides in combination with CpG ss-ODN represent a new strategy for parenteral or mucosal delivery of vaccines in a safe and effective manner that has applicability for control or prophylaxis of infectious disease, especially in situations such as vaccination of donors or recipients of HCT, where highly inflammatory adjuvants are not desired.