Biomedical subjects

Andreas Meyerhans

Publications and source records attributed to Andreas Meyerhans.

At least 19 recordsLinked to original sources

PubMed2007

In a mixed subtype epidemic, the HIV-1 Gag-specific T-cell response is biased towards the infecting subtype.

OBJECTIVES: Southwest Tanzania is affected by an HIV-1 epidemic consisting of subtypes A, C, and D, and their recombinant forms. This study was designed to assess whether the Gag- and Nef-specific T-cell response is biased towards recognizing the infecting subtype. METHODS: The infecting subtypes were characterized with a Multi-hybridization assay that discriminates between subtypes A, C and D. The interferon-gamma ELISPOT assay was used to detect the Gag- and Nef-specific T-cell responses in freshly isolated peripheral blood mononuclear cells in 56 seropositive patients. To study the HIV-specific T-cell responses, isolate-based Gag and Nef peptide sets representative of the locally occurring subtypes were used. The results were analysed at the total protein and single peptide level. RESULTS: In the study population, 35% were infected with a pure C subtype, 24% and 23% with ACD or AC recombinant forms, respectively. The total magnitude (P < 0.01) and breadth (P < 0.01) of the Gag-specific T-cell response detected with the subtype C-Gag peptide set was significantly greater than that detected with either the subtype A-Gag or D-Gag peptide sets. No significant difference was observed in the Nef-specific response. In 85% of responses targeting the most immunodominant Gag epitopes with subtype-specific sequence differences, the best recognized epitope variant corresponded to the infecting subtype. CONCLUSIONS: The Gag-specific T-cell response had a preference for recognizing peptides related to the infecting subtype.

Amino Acid Sequence↗

PubMed2006

CD8 T-cell recognition of multiple epitopes within specific Gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients.

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P = 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = -0.36; P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

CD8-Positive T-Lymphocytes↗

PubMed2006

Improved efficiency in detecting cellular immunity towards M. tuberculosis in patients receiving immunosuppressive drug therapy.

BACKGROUND: Reactivation of a latent Mycobacterium tuberculosis infection in immunocompromised individuals is associated with significant morbidity and mortality. The limited sensitivity of the established tuberculin skin-test in identifying latently infected patients on immunosuppressive drug therapy represents a major obstacle to better tuberculosis control after transplantation. METHODS: In this study, a quantitative flow-cytometric whole-blood assay and the skin-test were comparatively evaluated towards both diagnostic power and practicability in 117 long-term renal transplant recipients (age 53.1+/-14.8 years; 7.0+/-5.0 years after transplantation) in a low-prevalence region. RESULTS: Among the aforementioned renal transplant recipients, a high proportion (52.14%) had purified protein-derivative (PPD)-specific T-cell-immunity in vitro. Despite immunosuppression, prevalence as well as median frequencies of PPD-specific T-cells (0.22%; >0.05-4.71%) were as high as previously reported for immunocompetent individuals and haemodialysis patients. In contrast to in vitro testing, skin testing was less practicable in an ambulatory setting. Moreover, skin-test reactivity was significantly reduced as only 50.0% of patients with PPD-reactivity in vitro were skin-test positive. T-cell reactivity towards early secretory antigenic target-6 (ESAT-6), a protein specific for M. tuberculosis but absent from the bacillus Calmette-Guerin BCG-vaccine strain, was found in 52.9% of all individuals with PPD-reactivity in vitro. CONCLUSIONS: In conclusion, the whole-blood assay reveals a high prevalence of latent tuberculosis infection in renal transplant recipients. It may represent a valuable alternative to skin testing as the test result is not adversely affected by immunosuppression. Moreover, reactivity towards ESAT-6 allows the distinction of a latent infection from BCG-induced reactivity. The assay is well-suited for use in screening programmes and may facilitate the management of tuberculosis infection in immunocompromised individuals.

Adult↗

PubMed2006

The Autographa californica nuclear polyhedrosis virus AcNPV induces functional maturation of human monocyte-derived dendritic cells.

The initiation of an adaptive immune response is critically dependent on the activation of dendritic cells (DCs). Therefore, vaccination strategies targeting DCs have to ensure a proper presentation of the immunogen as well as an activation of DCs to accomplish their full maturation. Viral vectors can achieve gene delivery and a subsequent presentation of the expressed immunogen, however, the immunization efficiency may be hampered by an inhibition of DC activation. Here we report that the insect born Autographa californica nuclear polyhedrosis virus (AcNPV), which is already used for genetic immunization, is able to activate human monocyte-derived DCs. This activation induces the production of tumor necrosis factor alpha (TNF-alpha), an up-regulation of the surface molecules CD83, CD80, CD86, HLA-DR and HLA-I and increases the T cell stimulatory capacity of DCs. Thus, AcNPV represents a promising vector for vaccine trials.

Antibodies, Blocking↗

PubMed2006

Cross-presentation of HLA class I epitopes from influenza matrix protein produced in Saccharomyces cerevisiae.

Here we report that genetically engineered yeast of the strain Saccharomyces cerevisiae expressing full-length influenza matrix protein (IMP) attached to the yeast cell wall are a very versatile host for antigen delivery. Feeding of dendritic cells with either intact yeast expressing IMP protein or soluble IMP protein cleaved off the cell wall resulted in protein uptake, processing and cross-presentation of IMP-derived peptides. This process was analysed using previously established T-cell lines recognizing the immuno-dominant 58-66 peptide when presented by HLA-A2*0201 complexes. In addition, IMP(58-66)/HLA-A2*0201-specific antibodies were selected from a naive phage library which confirmed that peptide presentation was an active process of endocellular uptake and not just a result of external peptide loading. Moreover, MHC peptide antibodies could block the recognition of peptide-presenting dendritic cells by IMP(58-66)-specific T-cells in a dose dependent manner. There was no difference in T-cell recognition when either intact yeast or yeast cell extracts were used for DC feeding. Together, these data demonstrate that yeast derived proteins either in their soluble form or as part of a whole yeast vaccine are taken up, processed and presented by dendritic cells in HLA class I context.

Antibody Specificity↗

PubMed2006

Saccharomyces cerevisiae: a useful model host to study fundamental biology of viral replication.

Understanding the fundamental steps of virus life cycles including virus-host interactions is essential for the design of effective antiviral strategies. Such understanding has been deferred by the complexity of higher eukaryotic host organisms. To circumvent experimental difficulties associated with this, systems were developed to replicate viruses in the yeast Saccharomyces cerevisiae. The systems include viruses with RNA and DNA genomes that infect plants, animals and humans. By using the powerful methodologies available for yeast genetic analysis, fundamental processes occurring during virus replication have been brought to light. Here, we review the different viruses able to direct replication and gene expression in yeast and discuss their main contributions in the understanding of virus biology.

Animals↗

PubMed2006

Antigen-specific T cell responses: determination of their frequencies, homing properties, and effector functions in human whole blood.

Several prevalent and life-threatening agents enter the organism via the mucosa. In this case, a mucosal cellular immune response is essential for protection and is therefore considered the main objective of vaccination. The frequency of antigen-specific CD4+ and CD8+ T cells can be determined directly in human whole blood by a combination of surface marker and intracellular cytokine staining. Immune cells primed in the mucosal compartment also migrate through the blood and can be identified by expression of the gut-specific homing receptor alpha4beta7. Simultaneously, these lymphocytes can be functionally characterized regarding their differentiation status by analysis of CD45RO and CD27 expression and effector functions by measuring intracellular perforin or granzyme B content. Thus, the technique described in the paper is a powerful tool for clinical monitoring of the total cellular immune response to complex antigens during infection or vaccination.

Antigens↗

PubMed2005

Replication of M-tropic HIV-1 in activated human intestinal lamina propria lymphocytes is the main reason for increased virus load in the intestinal mucosa.

The gastrointestinal tract is the site of early abundant HIV replication and associated marked CD4 T-cell depletion. The aim of this study was to characterize the basis for the increased HIV replication in this compartment. Isolated mononuclear cells of the peripheral blood (PBMCs), the intestinal lamina propria (LPMCs), and purified gut lamina propria CD4 T-cell subpopulations (LP T cells) were isolated, phenotypically characterized, and infected in vitro with 2 different HIV-1 strains. T-cell subpopulations were analyzed by fluorescence-activated cell sorter. HIV-1 core protein p24 was determined in supernatants after in vitro infection. Furthermore the effect of T-cell stimulation on the replication of M- and T-tropic HIV strains was studied. In vitro replication of HIV-1 was significantly increased in CD69 compared with CD69 CD4 LP T cells, while there was no difference between CD103 and CD103 CD4 LP T cells. Experimental stimulation of LPMCs, which mimics activation by intestinal pathogens frequently present in the bowel of HIV-infected patients, further dramatically enhances HIV replication (24.5-fold) compared with nonstimulated LPMCs. M-tropic HIV-1 showed a preferential replication in LPMCs, while T-tropic HIV-1 strain showed a preferential replication in PBMCs. Thus, the elevated activation state of target cells in the intestine and not the expression of the homing marker CD103 is directly linked to massive HIV production.

Antigens, CD↗

PubMed2005

A genetic-algorithm approach to simulating human immunodeficiency virus evolution reveals the strong impact of multiply infected cells and recombination.

It has been previously shown that the majority of human immunodeficiency virus type 1 (HIV-1)-infected splenocytes can harbour multiple, divergent proviruses with a copy number ranging from one to eight. This implies that, besides point mutations, recombination should be considered as an important mechanism in the evolution of HIV within an infected host. To explore in detail the possible contributions of multi-infection and recombination to HIV evolution, the effects of major microscopic parameters of HIV replication (i.e. the point-mutation rate, the crossover number, the recombination rate and the provirus copy number) on macroscopic characteristics (such as the Hamming distance and the abundance of n-point mutants) have been simulated in silico. Simulations predict that multiple provirus copies per infected cell and recombination act in synergy to speed up the development of sequence diversity. Point mutations can be fixed for some time without fitness selection. The time needed for the selection of multiple mutations with increased fitness is highly variable, supporting the view that stochastic processes may contribute substantially to the kinetics of HIV variation in vivo.

Algorithms↗

PubMed2005

Differences in CMV-specific T-cell levels and long-term susceptibility to CMV infection after kidney, heart and lung transplantation.

Patients after kidney, heart and lung transplantation differ in their immunosuppressive drug regimens and in susceptibility to infectious complications with cytomegalovirus (CMV). In this study, CMV-specific T-cell responses were characterized in long-term transplant recipients and associated with the frequency of infectious complications. CMV-reactive CD4 T cells from 50 healthy controls, 68 renal, 14 heart and 24 lung transplant recipients were flow cytometrically quantified by the induction of cytokines after specific stimulation. Moreover, the immunosuppressive effect of calcineurin inhibitors on specific T-cell reactivity was quantified in vitro and compared with responses in vivo. Median CMV-specific T-cell frequencies in long-term renal (1.48%; range 0.06-17.26%) and heart transplant recipients (0.90%; 0.13-12.49%) did not differ from controls (1.82%; 0.26-21.00%). In contrast, CMV-specific T-cell levels were significantly lower in lung transplant recipients (0.50%; <0.05-4.98%) and showed a significant correlation with the frequency of infectious episodes (r =-0.57, p = 0.005). The differences within the groups were associated with increasing dosages of immunosuppressive drugs, as exemplified for calcineurin inhibitors that dose dependently reduced specific T-cell reactivity in vitro. In conclusion, monitoring CMV-specific CD4 T cells may serve as a measure for long-term disease susceptibility and may contribute to an improved management of CMV complications after lung transplantation.

Adult↗

PubMed2004

Gut-homing (alpha(4)beta(7)(+)) Th1 memory responses after inactivated poliovirus immunization in poliovirus orally pre-immunized donors.

Mucosal infections are prevented by a specialized local immune system. Immune cells of this compartment can also be found in the blood and are characterized by the expression of mucosa-specific homing molecules. Here, the cellular immune responses after inactivated poliovirus immunization (IPV) in poliovirus orally pre-immunized donors were investigated. Subcutaneous IPV induced a transient increase in the proliferative response against poliovirus antigen and in the number of poliovirus-specific CD4(+) T cells in the blood of the vaccinees. These cells were characterized to be of the effector memory type (CD45RA(-)/CD45RO(+)/CCR7(-)/CD27(+)) and expressed the homing molecule alpha(4)beta(7), indicating their origin from the gut. Together these data show the recurrence of gut-derived poliovirus-specific cells upon IPV and evaluate the whole-blood assay as a powerful tool for monitoring the success of a vaccination.

Administration, Oral↗

PubMed2003

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans.

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.

Adult↗

PubMed2003

Specific activation of CMV-primed human T lymphocytes by cytomegalovirus pp65 expressed in fission yeast.

Threatening virus infections constantly illustrate the growing need for novel vaccines that specifically induce efficient T cell-mediated immune responses. In this study, we used a human whole blood assay to determine the activation of antigen-specific human T lymphocytes by a viral antigen of human cytomegalovirus (HCMV). The major HCMV tegument protein pp65, recombinantly expressed in fission yeast (Schizosaccharomyces pombe), specifically activated antigen-specific CD4- and CD8-positive memory T cells in blood of HCMV seropositive donors. Moreover, the immune response against recombinant pp65, in particular that of CD8 class I major histocompatibility complex-restricted cytotoxic T cells, was similar to the response against the intact HCMV. Since fission yeast cells per se did not activate a significant number of human T lymphocytes ex vivo, the system described here might represent a novel approach in vaccine development as well as in the identification of vaccine candidates directly from human whole blood.

Antibodies, Viral↗

PubMed2003

The non-clonal and transitory nature of HIV in vivo.

From a posteriori analyses of genetic variation, recombination can only be identified when the parental genomes are distinct. For viruses like HIV-1, this requires the producer cell to be infected by more than one virus. Using fluorescence in situ hybridisation, the provirus copy numbers in splenocytes from two HIV-1 patients were determined. More than 75% of infected splenocytes harboured two or more proviruses, range 1-8, with a mean of approximately 3-4 per cell. Sequencing of amplified DNA from single laser micro-dissected cells showed an extraordinary degree of diversity while numerous recombinants were evident. Given the dynamics of HIV-1 turnover in vivo and a recombination rate of approximately 3 cross-overs per cycle, some genomes from a fifteen year old infection may have undergone as many cross-overs as bases in the genome. Thus, recombination profoundly influences HIV evolution and gives it a non-clonal and transitory nature in vivo.

HIV Infections↗

PubMed2003

Network analysis of human and simian immunodeficiency virus sequence sets reveals massive recombination resulting in shorter pathways.

The intrinsic recombination rate of human immunodeficiency virus (HIV) exceeds the point mutation rate by a factor of 10. As the majority of infected cells in vivo harbour multiple proviruses, the stage is set for rampant recombination. Therefore, it may be presumed that phylogenic relationships and mutation frequencies will probably be affected by recombination. However, the proportion of homoplasies arising from recombination and mutation is not known. By studying the evolution of the hypervariable regions of the simian immunodeficiency virus envelope gene among four macaques, it is shown that homoplasies arise more from recombination than from point mutation. When recombination is accounted for, the minimum number of substitutions in a sequence set may be reduced by as much as 45 %. In fact, the true number of point mutations in a set of HIV sequences tends to the number of discrete substitutions. Hence, lineages are younger than anticipated previously, although not in proportion to the ratio of the intrinsic recombination/point mutation rate. Recombination also inflates codon polymorphisms.

Animals↗

PubMed2002

The fraction of perforin-expressing HIV-specific CD8 T cells is a marker for disease progression in HIV infection.

OBJECTIVE: Perforin is an important component of the death machinery of cytotoxic T cells (CTL). To evaluate functional differences between HIV- and cytomegalovirus (CMV)-specific CTL of coinfected patients, the frequencies of the respective perforin-expressing T cells were analysed in a rapid whole blood assay. METHODS: Whole blood of HIV- and CMV-infected individuals was specifically stimulated by HIV-1 Pr55(gag) or complete CMV antigen, and activation-induced intracellular cytokine and perforin expression in CD8 T cells was analysed by flow cytometry. RESULTS: Perforin-expressing HIV-1- and CMV-specific CD8 T cells can be quantified simultaneously. Within a patient, the frequency of such HIV-specific CD8 T cells in peripheral blood was lower than the frequency of the respective CMV-specific cells. The number of the perforin-expressing HIV-specific CD8 T cells inversely correlated with the peripheral blood CD4 T cell count. CONCLUSIONS: The differential fractions of perforin-expressing virus-specific CD8 T cells in HIV and CMV double infection might be caused by differences in priming and trafficking to or from replication sites. However, without knowing the underlying mechanism, the fraction of perforin-expressing HIV-specific CD8 T cells provides another surrogate marker for disease progression.

Adult↗

PubMed2002

Recombination: Multiply infected spleen cells in HIV patients.

The genome of the human immunodeficiency virus is highly prone to recombination, although it is not obvious whether recombinants arise infrequently or whether they are constantly being spawned but escape identification because of the massive and rapid turnover of virus particles. Here we use fluorescence in situ hybridization to estimate the number of proviruses harboured by individual splenocytes from two HIV patients, and determine the extent of recombination by sequencing amplified DNA from these cells. We find an average of three or four proviruses per cell and evidence for huge numbers of recombinants and extensive genetic variation. Although this creates problems for phylogenetic analyses, which ignore recombination effects, the intracellular variation may help to broaden immune recognition.

Amino Acid Sequence↗

PubMed2002

Dominance of virus-specific CD8 T cells in human primary cytomegalovirus infection.

Cellular immune responses are of high importance in initiating and maintaining immunity against virus infections. Whereas the cellular immune response during persistent cytomegalovirus (CMV) infection is well assessable, the individual contribution of CD4 and CD8 T cell responses during primary infection has not been described. A novel whole-blood assay, which relies on the flow-cytometric detection of antigen-induced cytokine expression, was used to characterize CMV-specific CD4 and CD8 T cell responses during primary infection of CMV seronegative recipients of a renal allograft from a CMV seropositive donor. These T cell responses were compared with long-term CMV-positive patients with known history of transplantation-related seroconversion. Results were further correlated to CMV load and serum IgG and IgM. The long-term seroconverted patients consistently showed a dominant CMV-specific CD4 T cell response (median frequencies: CD4, 1.12% [range, 0.35 to 8.10%] versus CD8 0.13% [range, <0.05 to 0.55%]). In contrast, during primary infection, the cellular immune response is strongly dominated by CMV-specific CD8 T cells (median peak frequencies: CD4, 1.24% [range, 0.21 to 1.60%] versus CD8, 2.47% [range, 1.34 to 6.67%]). Upon receipt of ganciclovir, viral load as well as CMV-specific CD8 responses decreased. The frequency of the respective CD4 T cells fluctuated during decrease of CMV load and became dominant over CMV-specific CD8 T cell responses. These results are consistent with the view of an effective direct antiviral activity of CD8 T cells, which is most critical during periods of high viremia. Later on during persistent infection, CD4 T cells dominate the immune response to support the state of antiviral immunity.

Adult↗