Reconstitution of cytomegalovirus-specific T-cell response in allogeneic hematopoietic stem cell recipients: the contribution of six frequently recognized, virus-encoded ORFs.

BACKGROUND: The reactivation of human cytomegalovirus (HCMV) in immunosuppressed patients is associated with significant morbidity. Testing HCMV-specific T-cell responses can help determine which patients are at high risk of HCMV disease. We optimized selection of HCMV antigens for detection of T-cell response of patients after allogeneic hematopoietic stem cell transplantation (HSCT) with the aim of identifying patients with insufficient control of HCMV reactivation. METHODS: T-cell immune response to HCMV was monitored in 30 patients during the first year after HSCT. The HSCT recipients were classified according to their anti-HCMV T-cell response and the presence of HCMV DNA in the blood. RESULTS: We observed an inverse relationship between the magnitude of HCMV-specific T-cell responses against CMV lysate, phosphoprotein (pp) 65, immediate early-1 (IE-1), UL36, and UL55, but not to US3 and US29 detected by interferon-gamma (IFNγ)- ELISPOT and the level of HCMV DNA in the blood of patients during the 30 days following sampling. The study has revealed that patients who received a graft from a seronegative donor have a lower T-cell response against HCMV and increased probability of HCMV reactivation in comparison to the patients who had received their graft from a seropositive donor. CONCLUSION: The individual peptide pools and native HCMV antigens were useful for monitoring the time course of the anti-HCMV response by IFNγ-ELISPOT, which proved to have a prognostic value. Besides widely employed peptide pools of pp65 and IE-1, the use of antigens UL36 and UL55, but not US3 or US29, increased sensitivity of the test.

Evolution of human cytomegalovirus-seronegative donor/-seropositive recipient high-risk combination frequency in allogeneic hematopoietic stem cell transplantations at Institute of Hematology and Blood Transfusion during 1995-2014.

BACKGROUND: Human cytomegalovirus (HCMV) establishes lifelong latent infection that can result in severe life-threatening disease in immunosuppressed patients after hematopoietic stem cell transplantation (HSCT). An HCMV-seropositive transplant recipient who receives a graft from a seronegative donor (R+/D-) is at high risk of recurrent HCMV reactivation. METHODS: To assess the incidence of R+/D- combination, we retrospectively evaluated HCMV-seronegative donors for 746 allogeneic HSCT treatments carried out at our center during 1995-2014. In our cohort, 20% HCMV-seronegative HSCT recipients, 21% HCMV-seronegative related graft donors, and 52% HCMV-seronegative unrelated graft donors were included. RESULTS: Analyses of the HCMV serostatus of hematopoietic stem cell donors during 2 consecutive calendar periods (1995-2005 and 2006-2014) showed a significant increase in the proportion of seronegative donors (odds ratio [OR] = 1.947). In addition, the number of HSCT treatments using an unrelated donor increased (OR = 2.376). Finally, the use of grafts from countries with a very low HCMV prevalence increased. CONCLUSION: This increase in HCMV seronegativity in unrelated donors and the increased proportion of unrelated donors were responsible for the increased occurrence of the high-risk combination R+/D- (OR = 1.680). If the reduction in the rate of HCMV-seropositive graft donors continues, an increased frequency of HCMV reactivation events in our transplant recipients can be expected, because of the increasing occurrence of the high-risk R+/D- combination.

Effect of IFN-gamma receptor gene deletion on vaccinia virus virulence.

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.

Early gene expression of vaccinia virus strains replicating (Praha) and non-replicating (modified vaccinia virus strain Ankara, MVA) in mammalian cells.

Modified vaccinia virus Ankara strain (MVA) is a safe highly attenuated non-pathogenic virus suitable as a vector for developing various vaccines. Study of expression of a reporter beta-galactosidase gene under the control of an early vaccinia virus (VV) promoter in MVA and non-modified vaccinia virus Praha strain showed that early transcription in MVA is elevated in comparison with non modified VV. This property was demonstrated in various cell cultures including CV1 cells, human lung diploid cells, chicken primary fibroblasts but not in bone marrow-derived mouse dendritic cells. There the relationship between the elevated early transcription and the permisivity of cells for MVA was not observed.

Effect of 3-beta-hydroxysteroid dehydrogenase gene deletion on virulence and immunogenicity of different vaccinia viruses and their recombinants.

3-beta-Hydroxysteroid dehydrogenase (3-beta-HSD) activity coded for by the A44L gene of vaccinia virus (VV) was demonstrated in CV-1 cultures infected by all five VV strains tested, viz. WR, Praha virus, DRYVAX Wyeth-derived virus (DD), LIVP and MVA. Deletion of the A44L gene in two Praha virus-derived clones (the moderately virulent P13 and the highly attenuated P20), the WR and DD viruses resulted in absence of 3-beta-HSD activity from infected cultures. The virulence for mice of P13 was not affected, and that of WR was only slightly decreased, by the A44L gene deletion. Recombinant VVs expressing either varicella-zoster virus glycoprotein E (VZV-gE) or hepatitis B virus preS2-S protein (HBV-preS2-S) and their respective A44L deleted mutants were used in immunogenicity tests in mice. In terms of antibody response to VV and the recombinant proteins, the deletion resulted in a lowering the immunogenicity in the moderately virulent clone P13 virus and its progenies. In the highly attenuated P20 and DD viruses and their progenies no effects were apparent.

Hepatitis B virus core-preS2 particles expressed by recombinant vaccinia virus.

Vaccinia virus (VV) recombinants expressing hepatitis B virus (HBV) surface (HBsAg) or core (HBcAg) antigens (Kunke et al., Virology 195, 132 - 139 (1993)] have been shown to raise specific antibodies in mice, nevertheless the levels of antibodies reactive with the preS2 and S antigens were low. In an attempt to enhance the immunogenicity of HBsAg-preS2, a fused C-preS2 gene was constructed. The fusion protein was expressed in E. coli and displayed both HBcAg and preS2 antigen as demonstrated by enzyme-linked immunosorbent assay (ELISA). The same gene was then expressed using recombinant VV and chimerical particles whose size and density were similar to those of native HBV core particles produced in CV-1 cells infected with recombinant VV. Unlike HBcAg, preS2 antigen could not be detected on these particles by ELISA but was revealed by immunoblot analysis only. The immunogenicity of the recombinant VV was evaluated in mice. Antibodies to HBcAg and VV antigen but not to preS2 antigen were found in sera of animals inoculated with 10(7) PFU of the recombinant VV. Presumably, HBcAg-preS2 particles produced in E. coli and in eukaryotic cells have a different conformation, and the presence of preS2 antigen on the surface of chimerical particle might be necessary for a pronounced antibody response.