Zugl.: Giessen, Univ., Diss., 2009

Background: In times of limited organs for transplantation, anti-HBc positive organs can be accepted for lung transplantation to increase the number of donors. Transplant recipients should be vaccinated against hepatitis B to prevent HBV infection. However, response after HBV vaccination has only been poorly evaluated in patients with end-stage pulmonary disease. Material/Methods: Anti-HBs titers of 40 anti-HBc negative patients with end-stage pulmonary disease evaluated for lung transplantation were analyzed with the Architect® system (Abbott, Germany). Responders, partial responders, or non-responders after HBV vaccination were defined by anti-HBs titers >100 IU/L, 10–100 IU/L, and <10 IU/L, respectively. Results: There were 34/40 individuals (85%) vaccinated against hepatitis B, and 6 were not vaccinated. Response, partial response, and non-response after vaccination were observed in 10/34 (29.4%), 11/34 (32.4%), and 13/34 (38.2%) of patients, respectively. Response to vaccination did not correlate with sex, pulmonary disease, comorbidities, immunosuppressive therapy, or smoking status. Conclusions: Although 85% of patients evaluated for lung transplantation were vaccinated against hepatitis B, 38.2% did not show an anti-HBs titer >10 IU/L. Thus, anti-HBs titers should be regularly monitored. Nonresponders should be considered for booster vaccinations, alternative vaccination schedules, or prophylactic treatment with a nucleos(t)ide analogue in case of transplantation of an anti-HBc–positive organ.

Background: In times of limited organs for transplantation, anti-HBc positive organs can be accepted for lung transplantation to increase the number of donors. Transplant recipients should be vaccinated against hepatitis B to prevent HBV infection. However, response after HBV vaccination has only been poorly evaluated in patients with end-stage pulmonary disease. Material/Methods: Anti-HBs titers of 40 anti-HBc negative patients with end-stage pulmonary disease evaluated for lung transplantation were analyzed with the Architect® system (Abbott, Germany). Responders, partial responders, or non-responders after HBV vaccination were defined by anti-HBs titers >100 IU/L, 10–100 IU/L, and <10 IU/L, respectively. Results: There were 34/40 individuals (85%) vaccinated against hepatitis B, and 6 were not vaccinated. Response, partial response, and non-response after vaccination were observed in 10/34 (29.4%), 11/34 (32.4%), and 13/34 (38.2%) of patients, respectively. Response to vaccination did not correlate with sex, pulmonary disease, comorbidities, immunosuppressive therapy, or smoking status. Conclusions: Although 85% of patients evaluated for lung transplantation were vaccinated against hepatitis B, 38.2% did not show an anti-HBs titer >10 IU/L. Thus, anti-HBs titers should be regularly monitored. Nonresponders should be considered for booster vaccinations, alternative vaccination schedules, or prophylactic treatment with a nucleos(t)ide analogue in case of transplantation of an anti-HBc–positive organ.

HINTERGRUND: Die Reaktivierung des Humanen Cytomegalievirus (HCMV) ist immer noch eine häufige Ursache für Morbidität und Mortalität unter immunsupprimierten Patienten. Eine effiziente T-Zell-Antwort vermag die unkontrollierte Ausbreitung des Virus zu verhindern. Vieles über diese T-Zell-Antwort ist aber noch unklar. Im Rahmen dieser Studie wurden daher bei HCMV-seropositiven herz- (n = 17) und lungentransplantierten (n = 3) Patienten Epitope in zwei wichtigen T-Zell-Zielen, den HCMV-Proteinen IE-1 (UL123) und pp65 (UL83), identifiziert, die Frequenzen der für diese Epitope spezifischen T-Zellen gemessen und die Klonalität ausgewählter starker CD8+ T-Zell-Antworten untersucht. METHODEN: Dazu wurden Pentadecapeptide, die die gesamte Aminosäure-Sequenz von IE-1 bzw. pp65 umfassten und sich um jeweils 11 Aminosäurereste überlappten, in Pools von 25 bis 30 Peptiden so zusammengefasst, dass jedes Peptid in einer einzigartigen Kombination von drei Pools enthalten war. PBMC der Patienten wurden dann mit den Peptid-Pools stimuliert und die resultierenden T-Zell-Reaktionen durch Färbung von intrazellulär zurückgehaltenem Interferon-gamma durchflusszytometrisch sichtbar gemacht. Immunogene Peptide konnten anhand der jeweiligen drei Pools, die zu IFN-gamma-Produktion führten, eindeutig identifiziert werden. Einige dieser T-Zell-Populationen wurden durch einen IFN-gamma-Sekretions-Assay, magnetische Zellseparation und durchflusszytometrische Feinsortierung aus PBMC isoliert und ihre Klonalität mit Hilfe einer Polymerase-Kettenreaktion zum Nachweis klonal expandierter gamma-T-Zell-Rezeptor-Rearrangements (TCR-PCR) und anschliessender Fragmentanalyse fluoreszenzmarkierter PCR-Amplifikate untersucht. ERGEBNISSE: Bei den Patienten bestanden grosse Unterschiede hinsichtlich des jeweils immundominanten Proteins, der Dominanz von CD4+ bzw. CD8+ T-Zell-Subpopulation, der antigenen Determinanten, der gemessenen Peptid-spezifischen T-Zell-Frequenzen sowie der Anzahl der identifizierten Epitope. Zehn zuvor noch nicht beschriebene Epitope wurden eben-falls identifiziert und die präsentierenden HLA-Allele der meisten in der Patientengruppe identifizierten Epitope bestimmt. Die mittels TCR-PCR untersuchten CD8+ T-Zell-Reaktionen waren auf einen oder wenige Klone fokussiert. Die Korrelation der experimentellen Daten mit den klinischen Verläufen der Patienten hinsichtlich HCMV-Reaktivierung und -Erkrankung erbrachte jedoch keine Hinweise auf einen konkreten Zusammenhang. SCHLUSSFOLGERUNGEN: Zusammenfassend ermöglichen die hier vorgestellten Methoden die Untersuchung des Langzeitverlaufes der CD4+ und CD8+ T-Zell-Antwort gegen immundominante Proteine auf Epitop-Ebene nach initialer Identifizierung der antigenen Determinanten, die direkte Bestimmung der Frequenzen der Epitop-spezifischen T-Zellen sowie die Untersuchung der Klonalität dieser Reaktionen aus ca. 2 x 20 ml Blut. Die Langzeit-Untersuchung von Patienten mit hohem Risiko für HCMV-Reaktivierung und -Erkrankung kann so zu einem besseren Verständnis der komplexen HCMV-spezifischen T-Zell-Anwort und damit möglicherweise auch zur Verbesserung von Diagnose, Prophylaxe und Therapie dieser Patienten beitragen. / BACKGROUND: Human cytomegalovirus (HCMV) reactivation is still a leading cause of morbidity and mortality among immunosuppressed patients. Uncontrolled viral spread is prevented by an efficient T-cell response. However, little is known about the nature of this T-cell response. In this study we identified epitopes in two immunodominant HCMV-proteins, IE-1 (UL123) and pp65 (UL83), measured the frequencies of T-cells specific for these, and studied the clonotypic composition of selected T-cell responses in a group of HCMV-seropositive heart (n = 17) and lung (n = 3) transplant patients. METHODS: For both proteins overlapping pentadecapeptides covering the entire respective amino acid sequences were arranged in pools of 25 peptides each in such a way that every peptide was contained in exactly 3 pools. PBMC were stimulated with the resulting 15 pools for IE-1 or 16 pools or pp65, respectively, as well as with pools containing all peptides of the corresponding protein. Individual peptides leading to a positive T-cell response were identified by flow cytometric detection of intracellular interferon-gamma, each single peptide corresponding to a unique combination of 3 peptide pools. Selected T-cell populations specific for the previously identified single peptides were purified by performing an IFN-gamma secretion assay prior to magnetic cell separation and subsequent fluorescence-activated cell sorting. The clonality of these highly purified peptide-specific T-cell populations was then investigated by a T-cell receptor-gamma rearrangement-PCR and subsequent fragment analysis of fluorescence-labelled PCR amplificates. RESULTS: We observed broad heterogeneity among the patients in terms of the immunodominant protein, number of epitopes, predominance of CD4 or CD8 T-cell responses, and epitope-specific T-cell frequencies. 10 previously unknown epitopes were identified, and the HLA-restriction of most of the identified epitopes could be determined. The investigated T-cell responses showed a high degree of clonal focussing. These data were correlated to the patients episodes of HCMV reactivation, but a correlation between differences in the T-cell responses and a different clinical outcome in terms of HCMV-reactivation could not be established. CONCLUSIONS: In summary, this novel approach allows the rapid identification of epitopes contained in a given protein, direct determination of T-cell frequencies, and investigation of the T-cell clonality in the CD4 and CD8 T-cell subsets from as little as 2 times 20 ml of blood. Long-term follow-up of patients at risk for HCMV reactivation and disease may thus allow a more detailed insight into the complexity of the T-cell response to HCMV and may thus lead to improved diagnosis, prophylaxis and therapy.