Rolf Kaiser

Decreased frequency of HCV core-specific peripheral blood mononuclear cells with type 1 cytokine secretion in chronic hepatitis C

BACKGROUND/AIMnSince the outcome of hepatitis C infection appears to be correlated with the immune response to the HCV core protein, the aim of this study was to investigate the T cell response to hepatitis C virus core and core-derived antigens.nnnMETHODSnAs this response may be regulated importantly by differential secretion of cytokines, we determined the number of peripheral blood mononuclear cells (PBMC) that secreted IL-2, IL-4, IL-10, and IFN-gamma in response to a recombinant HCV core protein and a panel of 19 core-derived peptides, using the ELI-Spot-technique. Two groups of patients were studied: group A: 11 patients with previously self-limited HCV infection; group B: 12 patients with chronic hepatitis C.nnnRESULTSnIn group B significantly less IFN-gamma spot forming cells (SFC) could be detected, both after stimulation with the core protein (0.083+/-0.083 SFC vs. 1.3+/-0.4 SFC/10(5) PBMC; p = 0.005 and with the core-derived peptides (1.3+/-0.5 vs. 4.4+/-1.1 SFC SFC/10(5) PBMC; p = 0.007). By analyzing the cytokine response to each single peptide, we found IFN-gamma responses to peptides aa 39-63 and aa 148-172 in group A but not in group B (p<0.03). In group B also, fewer IL-2 secreting cells were found after peptide stimulation (p = 0.04). Whereas subjects of group B showed IL-10-specific responses to HCV peptides more frequently than patients with self-limiting hepatitis C (p = 0.03), the number of IL-4-producing cells was not different between the two groups.nnnCONCLUSIONSnThe data suggest that patients with persistent viremia and chronic liver disease (group B) have less PBMC showing type 1 cytokine (IL-2, IFN-gamma) responses to HCV core protein than patients with self-limited HCV infection (group A).

Role of HIV-1 phenotype in viral pathogenesis and its relation to viral load and CD4+ T-cell count

The predictive value of HIV‐1 phenotype in peripheral blood mononuclear cell (PBMC) coculture and the relation among viral phenotype, viral load, and CD4+ T‐cell count were examined in two studies. In study A, 132 HIV‐1–infected individuals were examined retrospectively for the relation between the result of their initial HIV cultivation in PBMC coculture and survival rate 6 years later. In study B, 176 patients were examined since 1994 for markers of HIV disease progression. HIV‐1 phenotype was determined by PBMC cocultivation, viral load by NASBA HIV RNA QT System, and CD4+ T‐cell count by flow cytometry. In study A, the percentage of survival for patients with initial negative virus culture was significantly higher (95%) than in patients with nonsyncytia‐inducing (NSI) isolates (78%) and syncytia‐inducing (SI) isolates (21%) (P < 0.05 and P < 0.0001, respectively). When SI phenotype was subdivided into moderately cytopathogenic and highly cytopathogenic, significant differences in the rate of survival between these subgroups could be observed (45% vs. 14%; P < 0.05). In study B, progression from negative virus culture to the isolation of NSI variants was associated with increasing viral load (P < 0.0001) but did not affect CD4+ T‐cell count significantly (P > 0.07), whereas the switch from NSI to SI virus was accompanied by significant decline of CD4+ T‐cells (P < 0.0001) but no change in viral load (P > 0.21). Thus, isolation and phenotyping of HIV represents an additional striking predictive marker for progression of HIV infection. J. Med. Virol. 56:259–263, 1998.

Maternal and fetal hepatitis C virus exposure by intrauterine transfusion

We report a case of accidental exposure to hepatitis C virus by an intrauterine transfusion that resulted in infection of the mother but not the child.

394 MUTATIONS IN THE HBS-ANTIGEN INFLUENCE THE RESULTS OF HBSAG QUANTIFICATION ASSAYS

Methods: We have been conducting sentinel surveillance for acute viral hepatitis by 28 national hospitals located nationwide for over 30 years. Total 547 sera from AHB patients collected between 1991 and 2009 were studied. HBV genomes were analyzed to determine genotypes (gts) and subgenotypes (sub-gts) and phylogenetic analysis based on preS1/S2/S region was performed. The investigation period was divided into three phases: 1st (1991– 1996); 2nd (1997–2002) and 3rd (2003–2008). Results: Gts were A in 136 (25%) patients, B in 48 (9%), C in 359 (66%), and others in 4 (0.8%). The proportion of gtA was low (6%) in the 1st phase and has been remarkably increasing thereafter (1st (9/150) vs. 2nd (19/123 (15.4%)), P < 0.05; 2nd vs. 3rd (88/226 (38.9%)), P < 0.0001). Of the 114 gtA isolates, 101 (88.6%) were classified into sub-gtAe, and 13 (11.4%) were into Aa, while of 43 gtB, 33 (76.7%) were into Ba and 10 (23.3%) were into Bj. Sequences of 65 (64%) isolates of Ae, 3 (23%) of Aa, 5 (15%) of Ba, and none of Bj were completely homologous with each other within each sub-gt. The proportion of isolates those have sequences homologous with some of the others’ was significantly higher in Ae than Aa or Bj or Ba (Ae vs. Aa, P < 0.001; Ae vs. Ba, P < 0.0001; Ae vs. Bj, P < 0.0001). No patients with gtB or gtC became chronic, whereas 4 with gtA (3.3%) did (A vs. C, P < 0.05). Conclusions: Unlike gtB or gtC, gtA infection has a potential to lead chronic disease. The prevalence of gtA has been spreading explosively. It is mostly via invasion of extremely similar isolates of sub-gtAe.

HIV colonizing peripheral blood monocytes follows lymphocytic isolates in shifting from NSI to SI genotype

SummaryNon-syncytium inducing (NSI) and syncytium inducing (SI) variants of human immunodeficiency virus (HIV) isolated from peripheral blood mononuclear cells (PBMC) could be definitely typed by sequence analysis of the env-gene V3 region. It was thus possible to compare the genotypes of viral variants isolated from PBMC and accompanying monocyte cultures and those derived directly from the patients blood cells prior to cultivation. Within the investigated group of patients it was shown that HIV variants colonizing monocytes displayed a similar shift from NSI to SI as observed previously for PBMC, i.e. lymphocyte derived isolates. Lymphocytic SI variants could be isolated from the blood of patients, while simultaneously the predominant provirus in both blood and monocytic isolate was NSI. Consequently, we observed a delayed switch in the predominant provirus genotype found in blood which was associated with a synchronous change in the genotype of the corresponding monocytic isolate. The results show that monocytes/macrophages can be colonized by heterogeneous HIV variants in vivo and can therefore also function as carriers for the spread of highly virulent SI variants into the tissues.

Etravirine in Protease Inhibitor-Free Antiretroviral Combination Therapies

Etravirine (ETR) is a next generation non‐nucleoside reverse transcriptase inhibitor (NNRTI). The studies for ETR EMA approval were almost exclusively performed together with the protease inhibitor (PI) darunavir. However the fact that ETR can be active against NNRTI‐pretreated HIV variants and that it is well tolerated suggests its application in PI‐free antiretroviral combination therapies. Although approved only for PI‐containing therapies, a number of ETR treatments without PIs are performed currently. To evaluate the performance of ETR in PI‐free regimens, we analyzed the EURESIST database. We observed a total of 70 therapy switches to a PI‐free, ETR containing antiretroviral combination with detectable baseline viral load. 50/70 switches were in male patients and 20/70 in females. The median of previous treatments was 10. The following combinations were detected in the EURESIST database: ETR+MVC+RAL (20.0%); ETR+FTC+TDF (18.6%); 3TC+ETR+RAL (7.1%); 3TC+ABC+ETR (5.7%); other combinations (31.4%). A switch was defined as successful when either ≤50 copies/mL or a decline of the viral load of 2 log10, both at week 24 (range 18–30) were achieved. The overall success rate (SR) was 77% (54/70), and for the different combinations: ETR+MVC+RAL=78.6% (11/14); ETR+FTC+TDF=92.3% (12/13); 3TC+ETR+RAL =80.0% (4/5), 3TC+ABC+ETR=100% (SR 4/4); and for other combinations=67.6% (23/34). These SR values are comparable to those for other therapy combinations in such pretreated patients.

Determination of HIV-1 Coreceptor Usage in German Patients - Comparison of Genotypic Methods with the TROFILE Phenotypic Assay

Address: 1Medizinisches Labor Dr. Berg, Berlin, Germany, 2University of Cologne, Cologne, Germany, 3PZB Aachen, Aachen, Germany, 4Medizinisches Labor Dr. Thiele, Kaiserslautern, Germany, 5NRZ fur Retroviren, Erlangen, Germany, 6Labor Lademannbogen, Hamburg, Germany, 7MUC-Research, Munich, Germany, 8Labor Dr. Fenner, Hamburg, Germany, 9University of Frankfurt, Frankfurt, Germany and 10MPI for Bioinformatics, Saarbrucken, Germany * Corresponding author

Elucidation of an HIV -1 Transmission from Mother to Child in West Africa by Sequence Analysis*

A pregnant woman living in Germany went to Ghana for several months, where she received 4 blood transfusions. Her newborn child also received one blood transfusion in West Africa. After return to Germany, HIV-1 infection was detected in both of them. Serotyping with V3 peptides revealed that the sera reacted only poorly with the subtype B-specific antigens. To investigate whether the child had been infected by vertical or parenteral transmission, we amplified different proviral HIV-1 gene segments from samples obtained 1-3 years after infection. Sequence analysis of the hypervariable regions V1 and V2 of the proviral env gene was misleading, since the viral population of the mother was highly heterogeneous, whereas only one predominant viral variant was found in the child. In contrast, sequences of the gag p17 gene and the regulatory genes nef and vif were homogeneous and revealed a very high homology, suggesting that the child had been infected by the mother. This was confirmed by phylogenetic tree analysis showing that sequences of mother and child clustered together and that both were infected by HIV-1 subtype A which is common in West Africa. The results suggest that sequence analysis of the hypervariable regions V1 and V2 alone can lead to unclear results, especially if not single genomes are analysed but a mixture of quasi-species. It is recommended that investigations into HIV transmission should be based on sequence analysis of several HIV genes.

HIV2EU: supporting standardized HIV-2 drug resistance interpretation in Europe

Various items are complicating the treatment of HIV‐2 infected patients. Compared to HIV‐1 there is much less treatment experience, no evidence from randomized control trials, a reduced number of effective drugs and no broadly available test for viral load monitoring. In case of treatment failure there is only limited guidance and presently no easy accessible tool for nucleic acid sequence interpretation available. To solve this problem, we initiated an expert workshop to address some of these problems. A panel of experts from four different European countries voted on a rule set for interpretation of mutations in the HIV‐2 protease, reverse transcriptase and integrase. Rules were proposed by each member and were then modified during discussion by considering data gained from HIV‐1 and accumulated experience of the follow up of HIV‐2‐infected patients. Based on the HIV‐GRADE internet‐tool an online tool was developed to make the rule set easily accessible and usable. Rules were laid down for the interpretation of HIV‐2 drug resistance to NRTIs, PIs and INIs (integrase inhibitors). Due to natural resistance of HIV‐2, usage of NNRTIs and T‐20 was not recommended as part of an antiretroviral regimen for HIV‐2. These rules were then translated in a machine interpretable format (algorithm specification interface, ASI) and the HIV‐GRADE tool was extended for usage of HIV‐2 sequences. Further consensus sequences were generated from the reference sequence data set provided by Los Alamos National Laboratories. In contrast to HIV‐1, mutations were compared to a group specific consensus sequence (Group A or Group B) and not to a consensus sequence from the most predominant HIV‐2 Group A. This change was necessary due to significant differences between the various HIV‐2 strains. We developed a rule set and an automated tool for HIV‐2 drug resistance analyses. This tool and the rules will be freely available on the internet. Access to the pre‐publication versions can be granted by each of the group members. To keep the algorithm rules up‐to‐date it will be actualized on a yearly basis.

Efficacy of first-line antiretroviral therapy: differences between hospital-based outpatient units and private practices in Germany

Purpose of the study : The efficacy of highly active antiretroviral therapy of HIV-infection (HAART) is influenced by factors like potency of applied drugs, adherence of the patient, or resistance-associated mutations. Up to now, there is little evidence about the impact of the therapeutic setting. Methods: Since 2001, the prospective multicenter study RESINA examines the epidemiology of primary HIV drug resistance in Nordrhein-Westfalen, the biggest federal state of Germany. Longitudinal data of all participants are followed in a cohort. Parameters of patients treated in hospital-based outpatient units were compared to those of participants treated in private practices. All 36 centers were specialized in the care for HIV and AIDS patients. Summary of results: Altogether, 1591 patients were enrolled from 2001 to 2009 with a follow-up until the end of 2010. 1099 cases were treated in hospital-based units, 492 were treated in private practices. Significant differences were found considering baseline characteristics. Patients with further progressed disease and more non-European individuals were cared for in hospital units. Only median age and the rate of primary drug resistance were not significantly different. After 48 weeks, 81.9% of patients in hospital units and 85.9% in private practices had a viral load below the limit of detection (p=0.12). A similar result was seen after 96 weeks (p=0.54). A comparable increase of CD4 cell-count was determined in both groups. Conclusions : The RESINA study covers a large cohort treated in different specialized facilities. We found significant differences in baseline characteristics with more unfavourable parameters in cases at hospital-based units compared to private practices. Nevertheless, treatment outcome was similar up to 2 years after therapy initiation. In conclusion, adminstration of HAART is highly efficacious regardless of treatment setting in Germany.