Eva Gönczöl

Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis.

Although first generation recombinant adenoviruses, deleted of sequences spanning E1a and E1b, have been useful for in vivo applications of gene therapy, expression of the recombinant gene has been transient and often associated with the development of inflammation. We show that with first generation adenovirus–mediated gene transfer to the mouse lung, viral proteins are expressed leading to destructive cellular immune responses and repopulation of the lung with nontransgene containing cells. Second generation E1 deleted viruses further crippled by a temperature sensitive mutation in the E2a gene were associated with substantially longer recombinant gene expression and less inflammation. Stable expression of human CF transmembrane conductance regulator has been achieved in lungs of CF mice instilled with a second generation virus.

A Canarypox Vector–Expressing Cytomegalovirus (CMV) Phosphoprotein 65 Induces Long-Lasting Cytotoxic T Cell Responses in Human CMV-Seronegative Subjects

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8(+). Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.

Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10

Chlamydiapneumoniae is a common cause of pulmonary infection, with serum positivity in at least 50% of the general population. In this study, we report that human PBMCs exposed to C. pneumoniae are resistant to apoptosis induced by the potent photoactivated chemotherapeutic agents 8-methoxypsoralen and hypericin. In contrast, PBMCs treated with a heat-inactivated inoculum exhibit normal susceptibility to apoptosis. We also observed that human PBMCs are responsive to C. pneumoniae infection by secretion of key immune regulatory cytokines, including IL-12 and IL-10. While IL-12 may play an important role in limiting C. pneumoniae proliferation within cells, IL-10 serves an anti-inflammatory function by down-regulating proinflammatory cytokines such as IL-12 and TNF-α. Depletion of endogenous IL-10, but not of IL-12, abolished the apoptosis resistance of C. pneumoniae-infected PBMCs. Furthermore, addition of exogenous IL-10, but not IL-12, significantly increased the resistance of control inoculum-treated PBMCs to photoactivated 8-methoxypsoralen- and hypericin-induced apoptosis. Therefore, we conclude that C. pneumoniae possesses an antiapoptotic mechanism. The resistance to apoptosis observed in PBMCs exposed to C. pneumoniae is due, at least partially, to the IL-10 induced during C. pneumoniae infection.

A Canarypox Vector Expressing Cytomegalovirus (CMV) Glycoprotein B Primes for Antibody Responses to a Live Attenuated CMV Vaccine (Towne)

To develop a vaccine against cytomegalovirus (CMV), a canarypox virus (ALVAC) expressing CMV glycoprotein (gB) was evaluated alone or in combination with a live, attenuated CMV vaccine (Towne). Three doses of 106.5 TCID50 of ALVAC-CMV(gB) induced very low neutralizing or ELISA antibodies in most seronegative adults. However, to determine whether ALVAC-CMV(gB) could prime for antibody responses, 20 seronegative adults randomly received either 106.8 TCID50 of ALVAC-CMV(gB) or 106.8 TCID50 of ALVAC-RG, expressing the rabies glycoprotein, administered at 0 and 1 month, with all subjects receiving a dose of 103.5 pfu of the Towne vaccine at 90 days. For subjects primed with ALVAC-CMV(gB), neutralizing titers and ELISA antibodies to CMV(gB) developed sooner, were much higher, and persisted longer than for subjects primed with ALVAC-RG. All vaccines were well tolerated. These results demonstrate that ALVAC-CMV(gB) primes the immune system and suggest a combined-vaccine strategy to induce potentially protective levels of neutralizing antibodies.

Preclinical evaluation of an ALVAC (canarypox)-human cytomegalovirus glycoprotein B vaccine candidate

Successful vaccination against the human cytomegalovirus (HCMV) requires induction of both neutralizing antibody and cytotoxic T lymphocyte (CTL) responses. The HCMV glycoprotein B (gB, UL55) would be one of the most important immunogens to induce neutralizing antibodies. We tested the immunogenicity of an ALVAC (canarypox)-HCMV-gB (ALVAC-gB) recombinant in mice and guinea pigs in order to provide preclinical data for a phase I clinical trial of a HCMV vaccine candidate. ALVAC is an attenuated vaccine strain of canarypox virus which replicates productively in avian species but abortively in mammalian cells. The ALVAC-gB recombinant inoculated subcutaneously in mice and intramuscularly in guinea pigs induced HCMV-specific neutralizing antibodies and gB-specific CTL responses. Ultraviolet irradiation of the ALVAC-gB recombinant before immunization diminished CTL responses, indicating that intracellular expression and processing of gB-protein were necessary for CTL induction. Prior immunity to vaccinia virus did not decrease immunogenicity of the ALVAC-gB recombinant in mice. Thus, despite its host range restriction, ALVAC-gB is potentially capable of inducing both humoral and cell-mediated immune responses to HCMV in both vaccinia-immune and non-immune individuals.

Induction of human cytomegalovirus (HCMV)-glycoprotein B (gB)-specific neutralizing antibody and phosphoprotein 65 (pp65)-specific cytotoxic T lymphocyte responses by naked DNA immunization

Plasmids expressing the human cytomegalovirus (HCMV) glycoprotein B (gB) (UL55) or phosphoprotein 65 (pp65) (UL83) were constructed and evaluated for their ability to induce immune responses in mice. The full-length gB as well as a truncated form expressing amino acids 1-680 of gB, and lacking the fragment encoding amino acids 681 907 including the transmembrane domain of gB (gB680) were evaluated. Immunization of mice with plasmids coding for gB or gB680 induced ELISA and neutralizing antibodies, with the highest titres in mice immunized with the gB680 plasmid. Mice immunized with the gB plasmid predominantly produced IgG2a gB-specific antibody, while the gB680 plasmid raised mostly IgG1 anti-gB antibody. Mice immunized with the pp65 plasmid developed pp65-specific cytotoxic T lymphocytes (CTL) and ELISA antibodies. Immunization with a mixture of both gB and pp65 plasmids raised antibodies to both proteins and pp65-specific CTL, indicating a lack of interference between these two plasmids. These results suggest that DNA immunization is a useful approach for vaccination against HCMV disease.

Isolated gA/gB glycoprotein complex of human cytomegalovirus envelope induces humoral and cellular immune-responses in human volunteers

Three human cytomegalovirus (HCMV) seronegative individuals were immunized with a single dose of HCMV envelope; two individuals developed neutralizing antibodies. Two naturally HCMV seropositive and three HCMV seronegative human volunteers were immunized with a major glycoprotein complex, gA/gB, of HCMV that had been purified by immunoadsorbent column chromatography. After a single injection of the gA/gB preparation, the naturally seropositive individuals developed higher titres of neutralizing antibodies and temporarily higher HCMV-specific lymphocyte proliferation (HCMV-LP) responses in vitro. The seronegative individuals developed neutralizing antibodies after the third injection of gA/gB, which were present only transiently, but showed a rapid reappearance and increase in titre after the fourth injection. At 1 year after the first injection, the neutralizing antibody titres were still comparable with those of the naturally seropositive individuals. HCMV-LP responses to HCMV in the initially seronegative individuals developed after the second or third injection with the gA/gB preparation and remained positive during the 1-year observation period. These results show that the gA/gB protein induces both humoral and cellular immune responses in humans, and might serve as the basis of a subunit vaccine.

Early atherosclerotic plaques in the aorta following cytomegalovirus infection of mice

We show here that BALB/c mice inoculated with murine cytomegalovirus (MCMV) express viral antigens in the endothelial and smooth muscle cells of the aortic wall, and that accumulation of inflammatory cells in the aortic lumen, similar to that seen in early atherosclerotic lesions in humans, colocalizes with the site of virus antigen expression. Immunosuppression of the mice at the time of virus infection increased the expression of viral antigens and the size of early atherosclerotic lesions in the intima. The percentage of the low-density lipoprotein cholesterol (LDL-C), the major lipid contributor to atherosclerotic plaques, was significantly increased in the serum of MCMV-infected mice, whether or not the mice were fed a high cholesterol diet. Human cytomegalovirus (HCMV) significantly increased the esterified cholesterol component of the total cholesterol in a human arterial smooth muscle cell line infected in vitro with HCMV. These results suggest that CMV infection is involved in two of the major mechanisms that lead to development of atherosclerosis, i.e., immune injury and high LDL-C.

A rapid microneutralization assay for cytomegalovirus

A rapid, simple and reproducible microneutralization test for human cytomegalovirus is described. The results can be read in 1-2 days and the neutralization titer detected in human and guinea pig sera and in monoclonal antibody-containing supernatants is consistent with that derived by the plaque-reduction neutralization test.

High expression of human cytomegalovirus (HCMV)-gB protein in cells infected with a vaccinia-gB recombinant: the importance of the gB protein in HCMV immunity

The human cytomegalovirus (HCMV), Towne strain, glycoprotein B (gB) gene was cloned into a vaccinia vector (Copenhagen strain) under the control of the H6 early and late vaccinia promoters (Vac-gB recombinant). The gB protein was expressed in a high percentage of the Vac-gB-infected cells throughout the virus replication cycle. Cytosine-arabinoside (ara-C) did not influence the expression of the gB protein early after infection (5 h), but did inhibit it later in viral replication (7-29 h). The Vac-gB recombinant induced HCMV neutralizing antibodies in guinea-pigs. Cells infected with the Vac-gB recombinant absorbed 50-88% of neutralizing activity of human sera obtained from volunteers previously inoculated with the Towne or Toledo strains and from naturally seropositive individuals.