L. Weseslindtner

Prospective Analysis of Human Cytomegalovirus DNAemia and Specific CD8+ T Cell Responses in Lung Transplant Recipients

In lung transplant recipients (LuTRs), human cytomegalovirus (HCMV) DNAemia may be associated with HCMV disease and reduced survival of the allograft. Because T cells are essential for controlling HCMV replication, we investigated in this prospective study whether the kinetics of plasma HCMV DNA loads in LuTRs are associated with HCMV‐specific CD8+ T cell responses, which were longitudinally assessed using a standardized assay. Sixty‐seven LuTRs were monitored during the first year posttransplantation, with a mean of 17 HCMV DNA PCR quantifications and 11.5 CD8+ T cell tests performed per patient. HCMV‐specific CD8+ T cell responses displayed variable kinetics in different patients, differed significantly before the onset of HCMV DNAemia in LuTRs who subsequently experienced episodes of DNAemia with high (>1000 copies/mL) and low plasma DNA levels (p = 0.0046, Fishers exact test), and were absent before HCMV disease. In HCMV‐seropositive LuTRs, high‐level DNAemia requiring preemptive therapy occurred more frequently when HCMV‐specific CD8+ T cell responses fluctuated, were detected only after HCMV DNA detection, or remained undetectable (p = 0.0392, Fishers exact test). Thus, our data indicate that HCMV‐specific CD8+ T cells influence the magnitude of HCMV DNAemia episodes, and we propose that a standardized measurement of CD8+ T cell immunity might contribute to monitoring the immune status of LuTRs posttransplantation.

CD4+ T Cell Responses in Patients with Chronic Hepatitis C Undergoing Peginterferon/Ribavirin Therapy Correlate with Faster, but Not Sustained, Viral Clearance

T cell immune responses may be important for the elimination of chronic hepatitis C virus (HCV) infection during antiviral treatment. In the present study, the kinetics of T cell responses to HCV antigens (NS3-4 and core) were prospectively assessed and were correlated with virologic outcome in 31 patients with chronic HCV infection undergoing peginterferon- alpha 2a/ribavirin therapy. NS3-4--directed T helper cell type 1 (Th1) responses were detected in 77% of patients with a significant decline in viremia at treatment week 4 but were not detected not in those with a slower viral decline. The detectability of NS3-4--directed Th1 responses was associated with faster viremia clearance, was short-lived, and did not seem to be associated with the final treatment outcome.

Human cytomegalovirus infection in lung transplant recipients triggers a CXCL-10 response.

Human cytomegalovirus (HCMV) causes significant morbidity in lung transplant recipients (LTRs). The clinical effects of HCMV replication are determined partly by a type 1 T‐helper cell (Th1) response. Because the chemokine interferon‐inducible protein of 10 kilodaltons (IP‐10, CXCL‐10) induces a Th1 response, we investigated whether HCMV triggers IP‐10 in LTRs. The IP‐10 concentration and HCMV DNA load were determined in 107 plasma and 46 bronchoalveolar lavage fluid (BALF) samples from 36 LTRs. Initial HCMV detection posttransplantation was significantly associated with increased plasma IP‐10, regardless of whether the patients showed HCMV DNAemia (p = 0.001) or HCMV replication only in the allograft (p < 0.0001). In subsequent episodes of HCMV detection, plasma IP‐10 increased regardless of whether HCMV was detected in blood (p = 0.0078) or only in BALF (p < 0.0001) and decreased after successful antiviral therapy (p = 0.0005). Furthermore, levels of HCMV DNA and IP‐10 correlated statistically (p = 0.0033). Increased IP‐10 levels in HCMV‐positive BALF samples were significantly associated with severe airflow obstruction, as indicated by a decrease in forced expiratory volume in one second (FEV1). Our data indicate that HCMV replication in LTRs evokes a plasma IP‐10 response and that, when an IP‐10 response is observed in BALF, it is associated with inflammatory airway obstruction in the allograft.

Association of human cytomegalovirus DNAaemia and specific granzyme B responses in lung transplant recipients.

In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNAaemia could be associated with HCMV disease and reduced allograft survival. In the present study we analysed whether or not HCMV‐specific granzyme B (Grz‐B) responses indicating CD8+ T cell cytotoxicity exert an impact on HCMV DNAaemia and relate to specific interferon (IFN)‐γ secretion. HCMV‐specific Grz‐B responses were quantitated by enzyme‐linked immunosorbent assay (ELISA) in 70 samples from 39 HCMV seropositive LTRs who were prospectively investigated for HCMV DNA plasma levels and IFN‐γ kinetics using a standardized CD8+ T cell assay (QuantiFERON®‐CMV assay). In all LTRs who were protected from HCMV DNAaemia by early and persistent IFN‐γ responses, Grz‐B responses were also detected. In LTRs who developed episodes of HCMV DNAaemia, the Grz‐B responses which were detected prior to viral DNA detection differed significantly in patients who experienced episodes with high (exceeding 1000 copies/ml) and low plasma DNA levels (P = 0·0290, Fishers exact test). Furthermore, the extent of Grz‐B release prior to viral DNAaemia correlated statistically with the detected levels of IFN‐γ (P < 0·0001, Spearmans rank test). Of note, simultaneous detection of Grz‐B and IFN‐γ secretion was associated significantly with protection from high HCMV DNA plasma levels during the subsequent follow‐up (P = 0·0057, Fishers exact test), and this association was stronger than for IFN‐γ detection alone. We conclude that, in addition to IFN‐γ responses, Grz‐B secretion by CD8+ T cells is essential to control HCMV replication and a simultaneous measurement of IFN‐γ and Grz‐B could contribute to the immune monitoring of LTRs.

Acute infection with a single hepatitis C virus strain in dialysis patients: analysis of adaptive immune response and viral variability.

BACKGROUND/AIMS While the adaptive immune response is crucial for spontaneous resolution of acute hepatitis C virus (HCV) infection, it also constitutes the driving force for viral escape. For acutely HCV-infected dialysis patients, little is known about the host response and its impact on viral evolution. METHODS Four haemodialysis patients accidentally infected with the same HCV strain were prospectively investigated with respect to the clinical course, CD4+ and CD8+ T-cell responses, neutralizing antibodies, viral kinetics and sequence variability. RESULTS In one patient, a robust CD4+ T-cell response was associated with transient control of infection, while in the other patients, weak responses correlated with persistently high viremia. Despite the presence of CD8+ T-cell effectors in the first patient, no sequence differences were detected in targeted regions of the viral genome in any of the patients when viral persistence was established. Genetic stability in the envelope genes, including the hypervariable regions, correlated with low-level or absent neutralizing antibodies in all of the patients. CONCLUSIONS The establishment of viral persistence in the special patient group of dialysis patients is due to a failure of the adaptive immune system, as shown by the absence of significant T-cell and antibody responses, as well as viral variability.

High CXCL-16 Levels Correlate With Symptomatic Disease in Lung Transplant Recipients With Human Cytomegalovirus Replication in the Allograft

Human cytomegalovirus (HCMV) is an important pathogen in lung transplant recipients (LTRs). In LTRs, HCMV may replicate in the transplanted lung, and this is indicated by HCMV DNA detection in the bronchoalveolar lavage fluid (BALF). Local replication may occur without causing clinical symptoms or, in some patients, it may lead to symptomatic HCMV disease. In the present study, we analyzed whether HCMV replication in the allograft induces CXCL‐16, a chemokine that may play a key role in the regulation of mucosal immunity, and investigated whether CXCL‐16 levels in BALF can be used to differentiate LTRs with asymptomatic HCMV replication from patients who simultaneously develop disease. In total, BALF samples from 57 LTRs, of whom 8 developed HCMV disease, were assessed for CXCL‐16 levels using a quantitative enzyme‐linked immunosorbent assay. We found that HCMV replication in the lung triggered a significant rise in CXCL‐16 levels in the BALF (p < 0.001, Wilcoxon signed‐rank test). Furthermore, the CXCL‐16 increase, induced by HCMV, was significantly lower in LTRs who did not develop HCMV disease (p < 0.001, Mann–Whitney U‐test). Thus, CXCL‐16 is a potential marker that may contribute to identify those LTRs in whom local HCMV replication in the lung remains asymptomatic.

Association of CMV-Specific T Cell-Mediated Immunity with CMV DNAemia and Development of CMV Disease in HIV-1–Infected Individuals

Background Among HIV-1–infected individuals, cytomegalovirus (CMV) reactivation and disease occur in the setting of advanced immunosuppression. The value of a standardized assessment of CMV-specific T-cell mediated immunity by the CMV QuantiFERON assay (CMV-QFT) has not yet been thoroughly investigated in HIV-1–infected subjects. Methods Prospective, longitudinal study in 153 HIV-1–infected subjects with a CD4+ T cell count < 350/μL who simultaneously underwent CMV-QFT, CMV serology testing and CMV-DNA quantification. Factors associated with CMV-QFT were evaluated. Clinical screening for CMV manifestations was then performed every 3 months. Results Among the 141 CMV IgG-seropositive individuals the CMV-QFT assay yielded reactive results in 84% (118/141), negative results in 15% (21/141) and indeterminate (negative mitogen IFN-gamma response) results in 1% (2/141) of subjects. The mean actual CD4+ T cell count was significantly higher in CMV-QFT reactive subjects, when compared to CMV-QFT non-reactive individuals (183 ± 102 vs. 126 ± 104 cells/μL, P = 0.015). A significantly lower proportion of CMV-QFT reactive vs. non-reactive patients displayed CMV DNAemia > 100 copies/mL (23% (27/118) vs. 48% (11/23), P = 0.02). Furthermore, a statistically significant inverse association between mitogen IFN-gamma response and CMV-DNAemia > 1000 copies/mL was observed (P < 0.001). During the observational period, 5 CMV end-organ manifestations were observed. In three of the CMV cases the CMV-QFT yielded indeterminate results. Conclusions While CMV-QFT reactivity indicates CMV-specific immunity, indeterminate results due to negative mitogen IFN-gamma response might reflect HIV-1-induced immunodeficiency. Thus, dependency upon CD4+ T cell count should be considered when interpreting CMV-QFT results.

Analysis of plasma surfactant protein D levels in lung transplant recipients

In lung transplant recipients (LTRs), severe clinical complications, such as microbial infections of the lung or transplant rejection, may occur. Surfactant protein D (SP‐D) is a C‐type lectin that is mainly produced in alveolar type II cells. Plasma SP‐D levels are usually low, but may increase when the lung–blood barrier is impaired. In this study, we analyzed whether plasma SP‐D concentrations reflect rejection or infection of the lung allograft. An enzyme‐linked immunosorbent assay was used to measure SP‐D levels in plasma samples from 58 LTRs during intervals without pathologic respiratory findings and during episodes of acute cellular rejection (ACR), microbial colonization, and microbial pneumonia. Median plasma SP‐D levels were significantly increased during episodes of microbial pneumonia, but not in the absence of pathologic respiratory findings, during microbial colonization, or during ACR up to grade A2–A3 (P < 0.05). During pneumonia, an increased plasma SP‐D level was detected in 60% of LTRs and this was further associated with a significantly higher risk for the patients to develop stage III bronchiolitis obliterans syndrome (BOS III) or to die within the subsequent 6 months after pneumonia (P = 0.0093). All patients with a plasma SP‐D level of >300 ng/mL during pneumonia developed BOS III and/or died within 6 months of follow‐up (P = 0.001). The determination of SP‐D levels in plasma during pneumonia in LTRs may be of prognostic value and warrants further evaluation.

Intrapulmonary Human Cytomegalovirus Replication in Lung Transplant Recipients Is Associated With a Rise of CCL-18 and CCL-20 Chemokine Levels.

Background In lung transplant recipients (LTRs), human cytomegalovirus (HCMV) DNA detection in the bronchoalveolar lavage fluid (BALF) indicates HCMV replication in the pulmonary compartment. Such local HCMV replication episodes may remain asymptomatic or may lead to symptomatic HCMV disease. Here, we investigated LTRs with intrapulmonary HCMV replication for the chemokines CCL-18 and CCL-20. In particular, we analyzed whether these chemokines rise in the allograft and/or the blood and are associated with HCMV disease. Methods CCL-18 and CCL-20 levels were quantitated by ELISA in BALF and serum samples from 60 LTRs. During the posttransplant follow-up, these LTRs displayed HCMV DNA detection in the BALF by PCR, whereas other infectious agents were undetectable. Furthermore, we investigated samples from 10 controls who did not display any HCMV replication episode during the follow-up. Results HCMV replication in the allograft was associated with a significant increase of CCL-18 and CCL-20 BALF levels (P < 0.001, Wilcoxon signed-rank test) and a significant rise of CCL-20 (P < 0.0001, Wilcoxon signed-rank test) but not of CCL-18 in the blood. In controls, no such chemokine increase was observed. Furthermore, CCL-18 BALF levels were significantly higher in 8 LTRs who additionally developed HCMV disease, as compared with the other 52 patients in whom HCMV replication remained asymptomatic (P < 0.001, Mann–Whitney U test). Conclusions HCMV replication in the allograft causes an intrapulmonary increase of CCL-18 and CCL-20 and a systemic rise of CCL-20 serum levels. Strong intrapulmonary CCL-18 responses are associated with symptomatic HCMV disease, proposing that CCL-18 BALF levels could serve as a marker.

The chemokine CXCL-10 is a marker for the infection stage in individuals with Parvovirus B19 DNAemia.

Background Accurate diagnosis of Parvovirus B19 (B19V) infection requires the differentiation between acute and past infection, which is especially important when B19V DNAemia is detected in pregnancy. Here, we explored the ability of the chemokine CXCL-10 in combination with molecular and serological assays to discriminate between acute and past B19V infection. Methods B19V DNA positive serum samples from 222 immunocompetent individuals were analyzed for (a) viral DNA loads, (b) anti-B19V IgM and IgG, (c) anti-VP1 IgG avidity, (d) anti-VP-2 epitope type specificity (ETS) as well as for (e) CXCL-10 serum levels. Results Using anti-B19V IgM and IgG, avidity and ETS assays, individuals with B19V DNAemia were staged for acute or past infection. Acute B19V infection caused a significant increase of the CXCL-10 serum concentration from uninfected baseline. Higher CXCL-10 serum levels were furthermore detected in acute B19V infection as compared to past infection. As a marker, CXCL-10 could discriminate between acute and past B19V infection, with an excellent discriminatory capacity when CXCL-10 and B19V DNA levels were used as combined parameters. Conclusion Acute B19V infection is associated with increased CXCL-10 production and measurement of CXCL-10 serum levels thus allows for the staging of B19V infection in individuals with B19V DNAemia.