Biomedical subjects

Andreas Mörner

Publications and source records attributed to Andreas Mörner.

6 recordsLinked to original sources

PubMed2006

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot.

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.

Animals↗

PubMed2006

Elevated levels of serum perforin in chronic HIV-1 and acute SIV/SHIV infection.

The impaired functional activity of cytotoxic T lymphocytes and natural killer cells during HIV-1 infection has recently been attributed to decreased intracellular levels of perforin and granzyme B. In sera from individuals chronically infected with HIV-1 we report increased levels of extracellular perforin compared with uninfected individuals. Increased perforin was also observed during experimental SIV/SHIV infection. The combination of reduced intracellular perforin levels and an increased serum level indicates that HIV infection induces aberrant perforin secretion.

Acute Disease↗

PubMed2006

Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques.

Monitoring of viral load in macaques has usually been carried out using in-house PCR-based methods. A novel viral load (VL) kit (ExaVir Load) based on the measurement of lentivirus reverse transcriptase (RT) activity provides a potential alternative to methods that measure plasma viral RNA. RT is a fundamental and conserved activity of all retroviruses and the method should theoretically detect RT from all lentiviruses. To test this we compared VL measured by a commercially available RT kit with an in-house QC RT-PCR in macaques infected with SIV and SHIV. Both RT and RNA levels were measured over time in both sets of macaques. Results indicated that the relationship between both tests was strong for SIV and SHIV (r = 0.95 and r = 0.92, p < 0.0001, respectively). The VL trends also followed each other, indicating that both techniques measured the same process of viral replication. Furthermore, the RT load obtained using standardized control plasma samples supplied by NIBSC gave values close to the designated VL. However, when comparing RT load with QC RT-PCR a consistently three to five time higher level was obtained with the RT assay, highlighting potential differences in assay calibration. Even so, the data suggest that the RT assay is both sensitive and robust for use in the SIV/SHIV macaque model, particularly where molecular-based assays for SIV VL determinations are not easily available. The assay is also a commercially available kit and hence has the potential to reduce the variability seen between laboratories using in-house PCR.

Animals↗

PubMed2003

Productive HIV-2 infection in the brain is restricted to macrophages/microglia.

OBJECTIVES: HIV-2 can use a broader range of co-receptors than HIV-1 in vitro, and is less dependent on CD4 for infection. The aim of this study was to detect productive HIV-2 infection in the brain and investigate whether HIV-2 has an expanded tropism for brain cells in vivo, in comparison with HIV-1, which productively infects macrophages/microglia. DESIGN: Brain samples taken at autopsy from eight patients who died from AIDS, six HIV-2 and two HIV-1/HIV-2 dually seropositive, with HIV encephalitis (HIVE), collected in Abidjan, Côte d'Ivoire in 1991, were examined for the presence and localization of productive HIV-2 infection. METHODS: Using immunohistochemistry, the presence of HIV-2 p26 in formalin-fixed, wax-embedded brain tissue sections was investigated. Double-staining with glial fibrillary acidic protein (GFAP), CD45- and CD68-specific antibodies was performed to identify infected cell types. RESULTS: HIV-2 p26 was detected in brain tissue from four of the HIV-2 cases and one of the dually infected individuals. The productively infected cells were either microglia or infiltrating macrophages. CONCLUSIONS: The productively infected cells in the brains of HIV-2 infected individuals are macrophages/microglia. No evidence was found for productive infection of astrocytes, neurons or oligodendrocytes. Thus, the broader in vitro cell tropism, promiscuous coreceptor usage and relative independence of CD4 by HIV-2 compared to HIV-1 does not broaden its range of target cells in the brain.

AIDS Dementia Complex↗

PubMed2002

CCR5 or CXCR4 is required for efficient infection of peripheral blood mononuclear cells by promiscuous human immunodeficiency virus type 2 primary isolates.

Primary human immunodeficiency virus type 2 (HIV-2) isolates are characterized by their ability to use a broad range of coreceptors, including CCR5, CXCR4, and several alternative coreceptors. However, the in vivo relevance of this in vitro promiscuity in coreceptor usage remains unclear. We set out to evaluate the relative importance of CCR5 and CXCR4 for infection of activated peripheral blood mononuclear cells (PBMC). PBMC from donors homozygous for wild-type CCR5 (CCR5(+/+) or CCR5Delta32 (CCR5(-/-)) were tested for their susceptibility to infection with 10 primary HIV-2 isolates with known coreceptor usage by parallel 50% tissue culture infectious dose (TCID50) titrations. Although all isolates, except one, were able to establish productive infection in CCR5(-/-) PBMC, the infection of these cells was inefficient for all isolates that were unable to use CXCR4. For CXCR4-using isolates there were only minor differences in TCID50 between CCR5(+/+) and CCR5(-/-) PBMC. When we compared the replication kinetics in PBMC from donors of the two genotypes we observed an average delay in replication onset of 9 days in the CCR5(-/-) PBMC. This study shows that HIV-2 can use alternative coreceptors for infection of PBMC, but that this infection is much less efficient than infection mediated by CCR5 or CXCR4. Thus, CCR5 and CXCR4 appear to be the major coreceptors for HIV-2 infection of PBMC.

Cells, Cultured↗

PubMed1999

Human neutralizing human immunodeficiency virus type 2-specific Fab molecules generated by phage display.

A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.

Amino Acid Sequence↗