Gesteigerte Immunogenität von Plasmamembranen vaccinia virusinfizierter BHK-Zellen

Zusammenfassung Vacciniavirusinduzierte Zelloberflächenveränderungen können durch den zytoziden Effekt virusspezifischer Antikörper auf infizierte Zellen sowie durch die Agglutination virusinfizierter Zellen durch Concanavalin A demonstriert werden. Diese Veränderungen treten zeitlich korreliert mit einer gesteigerten Immunogenität der Wirtszelle 2–3 Stunden nach Infektionsbeginn bereits vor der Produktion reifer infektiöser Viruspartikeln auf. Eine gesteigerte Antikörperproduktion gegen Wirtszellantigene wird auch nach Immunisierung mit isolierten Plasmamembranen vacciniavirusinfizierter Zellen erreicht, während Zellkernfraktionen diesen Effekt nicht auslösen. Bei den zytotoxischen Antikörpern, die zu den 7 S-Immunglobulinen gehören, handelt es sich — wie durch Absorption mit BHK-Zellmembranen gezeigt werden kann — um spezifisch gegen BHK-Zellen gerichtete Antikörper.

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Increased immunogenicity of plasma membranes of BHK-cells infected by vaccinia virus

Summary Cell surface alterations induced by vaccinia virus can be demonstrated either by the cytocidal effect of specific antibodies against vaccinia virus on the infected cells or agglutination of the infected cells by Concanavalin A. This change occurs 2–3 hours after infection, that is before any mature particles of vaccinia virus were produced. It coincides with an increased immunogenicity of the host cell. The production of cytotoxic antibodies against host cell antigens was also increased after active immunization with plasma membranes isolated from vaccinia virus infected BHK-cells, whereas the fraction of cell nuclei did not show up this effect. The cytotoxic antibodies which proved to be 7 S immunoglobulines, are specifically directed against BHK-cells as could be shown by absorption tests with membranes of BHK-cells.

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Gefördert mit Hilfe von Forschungsmitteln des Landes Niedersachsen.     

Viral mechanisms of immune evasion

During the millions of years they have coexisted with their hosts, viruses have learned how to manipulate host immune control mechanisms. Viral gene functions provide an overview of many relevant principles in cell biology and immunology. Our knowledge of viral gene functions must be integrated into virus-host interaction networks to understand viral pathogenesis, and could lead to new anti-viral strategies and the ability to exploit viral functions as tools in medicine.     

The Sequence Alteration Associated with a Mutational Hotspot in p53 Protects Cells From Lysis by Cytotoxic T Lymphocytes Specific for a Flanking Peptide Epitope

A high proportion of tumors arise due to mutation of the p53 tumor suppressor protein. A p53 hotspot mutation at amino acid position 273 from R to H, flanking a peptide epitope that spans residues 264–272, renders cells resistant to killing by human histocompatibility leukocyte antigen (HLA)-A*0201–restricted cytotoxic T lymphocytes (CTLs) specific for this epitope. Acquisition of the R to H mutation at residue 273 of the human p53 protein promotes tumor growth in vivo by selective escape from recognition by p53.264–272 peptide-specific CTLs. Synthetic 27-mer p53 polypeptides covering the antigenic nonamer region 264–272 of p53 were used as proteasome substrates to investigate whether the R to H mutation at the P1′ position of the COOH terminus of the epitope affects proteasome-mediated processing of the protein. Analysis of the generated products by tandem mass spectrometry and the kinetics of polypeptide processing in conjunction with CTL assays demonstrate that the R to H mutation alters proteasomal processing of the p53 protein by inhibiting proteolytic cleavage between residues 272 and 273. This prevents the release of the natural CTL epitope that spans flanking residues 264–272 as well as a putative precursor peptide. These results demonstrate that mutation of p53 not only leads to malignant transformation but may also, in some instances, affect immune surveillance and should be considered in the design of cancer vaccines.     

Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli

The complete genome of the varicella-zoster virus (VZV) Oka strain has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli (E. coli) strain DH10B, the VZV BAC was stably propagated over multiple generations of its host. Human embryonic lung (HEL) cells transfected with VZV BAC DNA recovered from DH10B showed cytopathic effect (CPE), and virus spread to neighbouring cells was observed. BAC vector sequences are flanked by loxP sites and, coinfection of the reconstituted virus, with a recombinant adenovirus expressing Cre recombinase removed the bacterial sequences. The resulting recombinant rV02 grew as well as the parental virus in HEL cells. The recombinant VZV will promote VZV research and increase use of the viral genome as an investigative tool.     

Secreted virus-encoded proteins reflect murine cytomegalovirus productivity in organs.

Mouse infection with murine cytomegalovirus (MCMV) is an established model for studying human cytomegalovirus infection. In this study, the relationship was analyzed between MCMV activity in organs of infected mice and the presence of infectious virus (viremia), viral genomes (DNAemia), or secreted virus-encoded proteins in the blood. For the latter, 2 recombinant viruses were constructed that encode for the hepatitis B virus surface antigen and the secreted alkaline phosphatase, respectively, as secreted marker proteins. The secreted markers correlated better with the infection in organs than DNAemia and viremia. The marker protein assays can serve as practical and sensitive tools for longitudinal monitoring of MCMV infection in individual mice.     

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.     

Prevalence and resistance patterns of extended‐spectrum and AmpC β‐lactamase in Escherichia coli,Klebsiella pneumoniae,Proteus mirabilis,and Salmonella serovar Stanley in a Korean tertiary hospital

Park SD, Uh Y, Lee G, Lim K, Kim JB, Jeong SH. Prevalence and resistance patterns of extended‐spectrum and AmpC β‐lactamase in Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella serovar Stanley in a Korean tertiary hospital. APMIS 2010; 118: 801–8. A total of 100 clinical isolates of Escherichia coli (n = 35), Klebsiella pneumoniae (n = 63), Proteus mirabilis (n = 1), and Salmonella serovar Stanley (n = 1), showing resistance to cefoxitin, or returning positive in extended‐spectrum β‐lactamase (ESBL) by Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory method, were studied. The isolates were examined by the boronic acid (BA) disk test, polymerase chain reaction, and pulsed‐field gel electrophoresis (PFGE) to investigate genetic similarities. The concurrence rates for ESBLs by the CLSI and the BA disk test were 97% for E. coli and 96.7% for K. pneumoniae. A total of 41 isolates showing cefoxitin resistance yielded all positive by the BA disk test. All the 33 K. pneumoniae isolates, which showed positive by the BA disk test, were carrying AmpC genes. The TEM and CTX‐M types were predominant in E. coli and the SHV and the CIT and/or DHA types were predominant in K. pneumoniae. PFGE analysis showed almost 75% of genetic similarities among K. pneumoniae isolates producing ESBLs and/or AmpC β‐lactamases (AmpCs) as each K. pneumoniae carried variable genes and showed variable antibiotic patterns. Clearly, the BA disk test was a useful method for the detection of ESBLs and AmpCs. In particular, cefoxitin resistance and BA‐positive trait of K. pneumoniae do reflect the presence of AmpC genes in the organism.