Biomedical subjects

Reinhard Kurth

Publications and source records attributed to Reinhard Kurth.

25 records · Page 2Linked to original sources

PubMed2003

Neutralizing antibodies against conserved domains of p15E of porcine endogenous retroviruses: basis for a vaccine for xenotransplantation?

Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, a goat antiserum against the recombinant ectodomain of the transmembrane envelope protein p15E was found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, E1 (GPQQLEK) and E2 (FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, antibodies isolated from AIDS patients and specific for sequences of HIV-1 partially homologous with E2 (Mab4E10, LWNWFN) or located in close proximity to E2 (Mab2F5, ELDKWA) are known to neutralize several strains of HIV-1. It is the first report showing epitope mapping of gammaretrovirus-specific neutralizing antibodies and demonstrating similarity to corresponding epitopes in HIV. These domains of the transmembrane proteins of different retroviruses are an effective target for neutralizing antibodies and may be a useful antigen to create an antiretroviral vaccine.

Amino Acid Sequence↗

PubMed2003

Porcine endogenous retroviruses: no infection in patients treated with a bioreactor based on porcine liver cells.

BACKGROUND: Acute liver failure (ALF) remains a disease with high mortality. Bioartificial liver support systems, which combine living cells of the liver in an extracorporeal circuit, have been successfully used in first clinical trials. The shortage of human organs to be used for bioreactors and the lack of safe and effective human liver cell lines have resulted in pigs becoming an important hepatic cell source. However, using these cells may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs). PERVs are present in the genome of all pigs and are able to infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. OBJECTIVES: To detect antibodies directed against specific epitopes from PERVs in seven individuals who were treated with porcine liver cell bioreactor therapy prior to liver transplantation. METHODS: Sera from seven patients treated with a hybrid liver support system based on porcine liver cells for ALF who survived the treatment and were discharged from hospital were investigated for antibodies against PERV. For this in addition to methods already reported (Xenotransplantation (2001) 125), new immunological detection methods were developed. RESULTS: PERV-specific antibodies were found in none of the patients using Western blot assays based on purified virus or recombinant viral core and envelope proteins or ELISA based on synthetic diagnostic peptides. CONCLUSION: The assays used are specific and sensitive, and correlated in their diagnostic value. The data indicate that no PERV infection had occurred in none of the patients treated with the CellModule bioreactor containing porcine cells.

Adult↗

PubMed2003

Retroviral safety: analyses of phylogeny, prevalence and polymorphisms of porcine endogenous retroviruses.

Porcine endogenous retroviruses (PERV) are discussed as putative infectious agents in xenotransplantation (XTx). PERV classes A, B, and C harboring different envelope proteins and two types of long terminal repeat (LTR) structures exist. One type of LTR contains a distinct repeat structure in U3, while the other is repeat-less and confers poor transcriptional activity. As the different LTR structures were found to be distributed unequally among the proviruses, we were interested in determining which LTR is the ancestor. Since replication-competent PERV can still be found today suggesting an evolutionary recent origin, we investigated the distribution and prevalence of six well-characterized and chromosomally assigned PERV in individuals of five different pig (Suidae, Sus scrofa) subspecies. Our studies revealed a heterogenous distribution of replication-competent PERV among individuals as well as among subspecies. The age of PERV was calculated to be 7.6 x 10(6) years, whereby the repeat-less LTR type evolved approximately 3.4 x 10(6) years ago being the phylogenetically younger structure. The age correlates with the time of separation between pigs and their closest relatives, american-borne peccaries (Tayassuidae, Pecari tajacu), 7.4 x 10(6) years ago.

Animals↗

PubMed2002

Simian immunodeficiency viruses with defective nef genes show increased susceptibility to the noncytotoxic antiviral activity of CD8+ lymphocytes.

The noncytotoxic soluble factor produced by CD8+ T cells inhibits replication of HIV and SIV in vitro and is thought to play a crucial role in combatting infection in vivo. We determined the effect of human CD8+ lymphocytes on the in vitro replication potential of both wild-type and nef-defective mutants of the simian immunodeficiency virus SIVmac251. Although replication of wild-type SIVmac251 in unstimulated human PBMC supplemented with IL-2 was unaffected by the presence of CD8+ T cells, the nef mutants were susceptible to the inhibitory effects. The effect of exogenous IL-2 depended upon the culture conditions: (i) in nonstimulated human PBMC depleted of CD8+ T cells, addition of IL-2 had a positive effect on the growth of the nef-defective viruses; (ii) in total human PBMC, IL-2 appeared to reinforce the CD8+ T-cell-dependent inhibition of the same mutant viruses. This strongly suggests that IL-2 stimulates the noncytotoxic anti-HIV/SIV response of CD8+ cells present in PBMC cultures. PHA stimulation of unfractionated human PBMC overrode the suppression of viral replication by CD8+ T cells. Depletion of activated T cells expressing the IL-2 receptor alpha-chain (CD25+ T cells), present in small amounts in these primary T cell cultures, dramatically reduced viral replication, indicating that the depleted cell population harbors the target cells permissive for viral replication. Furthermore, using neutralizing antibodies we could show that inhibition by the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES and the inhibitory effect of CD8+ lymphocytes on nef mutant SIVmac viruses are harbored on different levels.

Animals↗

PubMed2002

Induction of anti-simian immunodeficiency virus cellular and humoral immune responses in rhesus macaques by peptide immunogens: correlation of CTL activity and reduction of cell-associated but not plasma virus load following challenge.

Lipopeptides which carry the N-terminal moiety tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl (P(3)CSS) have been shown to have effective adjuvant and transmembrane carrier properties. To test the ability of these constructs to immunize against simian immunodeficiency virus [(SIV)(mac)] infection, rhesus macaques, prescreened for expression of the Mamu-A*01 MHC class I molecule, were immunized at regular intervals with lipopeptides corresponding to known SIV(mac) CTL epitopes alone or in combination with multiple antigenic peptides corresponding to neutralizing epitopes. Both humoral and CTL responses were elicited and the monkeys, along with non-immunized control animals, were challenged intravenously with 20 MID(50) of the homologous, uncloned SIV(mac251-32H) grown in rhesus monkey PBMC. Although none of the monkeys were protected from infection, most demonstrated an anamnestic CTL response with epitope-specific CTL precursor frequencies reaching as high as 1 in 20 total PBMC as measured by limiting dilution CTL assay or 25% of all CD8(+) T-cells using tetrameric MHC-I/peptide complexes. A significant inverse correlation between the levels of CTLp and the number of infected cells in circulation was observed. However, no such correlation with the plasma viral load (RNA copies/ml) was evident.

Amino Acid Sequence↗

PubMed2002

Immunological changes in simian immunodeficiency virus (SIV(agm))-infected African green monkeys (AGM): expanded cytotoxic T lymphocyte, natural killer and B cell subsets in the natural host of SIV(agm).

The African green monkey (AGM) model system for simian immunodeficiency virus (SIV(agm)) has been used to examine why prolonged infection with the relevant virus does not result in the development of immunodeficiency in its natural host. Blood lymphocyte subset values were determined in uninfected (n=88) and naturally SIV(agm)-infected AGMs (n=74). A number of blood cell subsets, such as CD8alpha(+)CD3(+)CD28(neg), CD8alpha(+)CD3(neg) and CD20(+) cells, were expanded significantly in clinically asymptomatic animals carrying a relatively high plasma load of viral RNA (10(4)-10(7) RNA copies/ml plasma). The expanded CD8alpha(+)CD3(+)CD28(neg) subpopulation (1094 +/- 986 cells/microl blood in infected animals versus 402 +/- 364 cells/microl blood, P=0.03) comprised cells that resembled terminally differentiated effector CD8 T cells (CD27(neg) and CD11a(+)). In SIV(agm)-infected animals, the expanded CD8alpha(+)CD3(neg) cell subset shared identity with the CD16(+) population (natural killer cells). These results demonstrate for the first time that apathogenic SIV(agm) infection causes significant changes in the immune system of its natural host. Although previous studies had indicated that noncytotoxic mechanisms might play an important role in the suppression of virus replication in the natural host of SIV(agm), this study sheds new light on the possible role of cytotoxic T lymphocytes, the innate immune system and double-positive T helper cells (CD4(+)CD8alpha(+)CD3(+)) in suppressing virus replication in this animal model of AIDS.

Animals↗

PubMed2002

Vaccine development using the simian immunodeficiency virus model for AIDS.

OBJECTIVE: A number of trials in primates using a wide range of putative vaccines based on simian immunodeficiency virus (SIV) have been performed and are summarised here. METHODS: Rhesus macaques and African green monkey (AGMs) were immunised with the test vaccines and challenged with live virus to test the efficacy of the induced or transferred immune responses to protect from infection or disease development. RESULTS: In initial studies, successful protection from challenge by whole inactivated virus vaccines was subsequently shown to be mediated by immune responses to human cell rather than viral proteins. Passive transfer of neutralising antibodies failed to protect against challenge. The induction of SIV-specific cytotoxic T lymphocytes (CTLs) using lipopeptides also failed to protect from infection, and whereas the frequency of post-infection CTLs (as measured by limiting dilution CTL assay and MHC/tetramer staining) correlated inversely with the cell-associated virus load, no correlation with the plasma virus load was observed. No immunological correlation of protection could be identified in macaques immunised with live attenuated SIV, with sterilising immunity being induced as early as 10 weeks after infection with the attenuated virus. Similarly, whole inactivated virus and passive IgG transfer failed to protect the natural host AGM species from challenge with apathogenic SIVagm, although live attenuated SIVagm afforded some protection despite the lack of overt vaccine virus replication. CONCLUSIONS: 'Traditional' types of vaccine are either ineffective or inappropriate for use in humans. Current efforts are therefore focusing on the rapidly evolving field of genetic vaccines based on vector DNA and recombinant, attenuated viral and bacterial vectors.

Animals↗