Rüdiger Pipkorn

Single-tube nested quantitative PCR: a rational and sensitive technique for detection of retroviral DNA. Application to RERV-H/HRV-5 and confirmation of its rabbit origin

It was reported earlier that a few patients suffering from non-Hodgkins lymphoma had low amounts of DNA from the so-called fifth human exogenous retrovirus, HRV-5. A sensitive and rational method for large-scale screening for HRV-5 DNA was therefore developed. It is a single-tube nested quantitative PCR (stnQPCR), which uses two functionally isolated primer pairs and one probe target distinct from related endogenous retroviral sequences, yet encompassing known HRV-5 variation, allowing optimal use of sequence conservation. DNA from lymphoma, myeloma, and follicular dendritic cell lines was tested for HRV-5 positivity, as was DNA from whole blood of blood donors, non-Hodgkins lymphoma and systemic lupus erythematosus patients, as well as DNA from lymph node biopsies of rheumatoid arthritis patients with lymphoma. One blood donor, one systemic lupus erythematosus patient, two previously known positive non-Hodgkins lymphoma patients, and one rheumatoid arthritis lymphoma patient, came out positive. They had 24, 2, 148, 480 and 30 proviral copies per microg of DNA from PBMC or lymphoma tissue, respectively. During the completion of this work it was reported that HRV-5 is a rabbit endogenous retrovirus (RERV-H), and that HRV-5 positivity was due to presence of rabbit DNA. DNA from six RERV-H/HRV-5 positive samples was therefore retested. Three also contained rabbit mitochondrial DNA. A search for HRV-5 antibodies using synthetic peptides was negative in sera from three RERV-H/HRV-5 positive individuals, as well as in 144 other sera, according with a noninfectious origin of the RERV-H/HRV-5 DNA in human samples. A search for possible sources of rabbit DNA contamination was negative. Methods for prevention of PCR contamination were strictly adhered to. Three samples from RERV-H/HRV-5 positive individuals positive at the Uppsala laboratory were retested at one or two other laboratories, and all three were positive. Two other samples, which were positive in the Riga laboratory, were tested also in London and also found positive. One non-Hodgkins lymphoma patient was RERV-H/HRV-5 positive in four consecutive samples, showing that positivity was a property of that patient. It is concluded that the stnQPCR developed to detect and quantify minute amounts of RERV-H/HRV-5 DNA is a principle which can be applied widely and HRV-5 is a RERV-H. Its presence in a few human blood samples could not be explained.

Antibodies to a putative HIV gp41 immunosuppressive peptide, pHIVIS (583-599), do not correlate significantly with outcome in HIV infection.

We have studied the prevalence of antibodies to peptides derived from the transmembrane protein of HIV, gp41. Previous work has suggested that the presence of antibodies to the gp41 peptide known as pHIVIS (env 583-599) is associated with protection from immunosuppression in HIV infection. We studied 171 sequential sera from 55 HIV-1-infected people in various clinical stages of disease. There was no significant association between antibodies to pHIVIS and clinical status in this study. Although pHIVIS has sequence similarity to the putative immunosuppressive region of the C-type oncornaviruses (p15E), antibodies to this peptide do not appear to be associated with protection from immunosuppression in natural HIV infection.

Increased immunoglobulin G, but not M, binding to endogenous retroviral antigens in HIV-1 infected persons.

The modes of interaction between products of human endogenous retroviral (HERV) sequences and the immune system are largely unknown. In HIV infected persons, an exogenous retrovirus adds further complexity to the situation. Therefore, 14 synthetic peptides with sequences derived from conserved regions of various endogenous retroviruses (ERVs) and from related exogenous retroviruses were used to search for IgG and IgM antibodies that bind to such antigens in 15 HIV‐1 seropositive and 17 seronegative immunosuppressed patients. IgG binding to three peptides, namely, the C‐terminal half of murine leukemia virus (MLV) capsid protein, the conserved portion of HERV‐H transmembrane protein, and the Pol region of human mouse mammary tumor virus (MMTV)‐like (HML3) sequence, was observed in both groups. Binding was, however, more frequent and more firm in HIV‐1 positive samples (P<0.0001, Wilcoxon rank sum test). IgM binding to the same peptides showed no significant differentiation between the two groups of patients. Binding to both immunoglobulin isotypes was sometimes variable over time in both groups. No correlation of either IgG or IgM peptide binding with progression to AIDS in HIV‐1 infected individuals was observed. Inhibition studies using analogous endogenous and exogenous retroviral peptides, including HIV‐1, demonstrated specificity of the IgG antibodies for a narrow range of MLV‐ and MMTV‐like retroviral antigens, and excluded cross‐reactivity of antibodies to HIV‐1 as a cause of these observations. Thus, unlike IgG, IgM binding to retroviral antigens was ubiquitous. It is suggested that anti‐HERV IgM belong to a class of natural antibodies and might serve as primers in the mediation of humoral immune responses to more or less related exogenous retroviruses. Increased IgG binding in HIV‐1 infected individuals could result from such priming, or reflect higher HERV antigen expression. J. Med. Virol. 62:435–444, 2000.

Phylogeny-directed search for murine leukemia virus-like retroviruses in vertebrate genomes and in patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome and prostate cancer

Gammaretrovirus-like sequences occur in most vertebrate genomes. Murine Leukemia Virus (MLV) like retroviruses (MLLVs) are a subset, which may be pathogenic and spread cross-species. Retroviruses highly similar to MLLVs (xenotropic murine retrovirus related virus (XMRV) and Human Mouse retrovirus-like RetroViruses (HMRVs)) reported from patients suffering from prostate cancer (PC) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) raise the possibility that also humans have been infected. Structurally intact, potentially infectious MLLVs occur in the genomes of some mammals, especially mouse. Mouse MLLVs contain three major groups. One, MERV G3, contained MLVs and XMRV/HMRV. Its presence in mouse DNA, and the abundance of xenotropic MLVs in biologicals, is a source of false positivity. Theoretically, XMRV/HMRV could be one of several MLLV transspecies infections. MLLV pathobiology and diversity indicate optimal strategies for investigating XMRV/HMRV in humans and raise ethical concerns. The alternatives that XMRV/HMRV may give a hard-to-detect “stealth” infection, or that XMRV/HMRV never reached humans, have to be considered.

A survey of synthetic HIV-1 peptides with natural and chimeric sequences for differential reactivity with Zimbabwean Tanzanian and Swedish HIV-1-positive sera.

ObjectiveTo determine whether the known sequence differences between African and non-African HIV-1 strains are reflected in the serological response. Design and methodsWe investigated the antibody reactivity of 34 Swedish, 30 Tanzanian and 42 Zimbabwean HIV-1-positive sera to 67 synthetic peptides with sequences from North American and African HIV-1 isolates, mostly derived from regions of gag and env known to be antigenic. Not all sera were tested against all peptides. ResultsDifferences in frequency of reactivity were noted with peptides covering the entire third variable domain (V3), which is a primary neutralization determinant, and the carboxyl terminus of gp120, in two regions of gp41, and the carboxyl terminus of p24. In env Tanzanian sera reacted preferentially with a V3 peptide from the strain JY1 (Zaire). Gradual substitutions in the central motif in V3 of ELI from GLGQ to GPGR, typical of many non-African strains, led to a gradual increase in reactivity of many Swedish sera, but did not affect Tanzanian and Zimbabwean sera, suggesting that the major epitopes recognized by these African sera are outside GPGR. V3 peptides from the MN and Z3 strains reacted with most sera, but missed 30% of those of Tanzanian origin. In the carboxyl terminus of gp120 both sets of African sera reacted preferentially with peptides from strains JY1 and MAL. Swedish sera reacted strongest with analogues from strains Z321 and HXB2. In gp41, Swedish sera showed a weak preference for reactivity with HXB2-derived peptides in the immunodominant region (amino acids 590–620), and further towards the carboxyl terminus (amino acids 620–665). ConclusionThe differences in serological reactivity were as great between Zimbabwe and Tanzania as between the two African sets and the Swedish. The geographical differences in the pattern of reactivity with HIV peptides probably depend on both host and viral variation and may be developed into a seroepidemiological tool, useful for optimization of future HIV vaccines.

A cluster of continuous antigenic structures in the transmembrane protein of HIV-1: Individual patterns of reactivity in human sera

We investigated the antigenicity of a highly conserved region in the transmembrane protein of the human immunodeficiency virus type 1 (HIV-1). In order to identify antigenically important residues, amino-acid sequences of synthetic peptides representing this region were varied systematically: single residues were omitted from the sequence of HIV-env 583-599; threonines were substituted for pairs of residues in HIV-env 581-599; the sequences of heptadeca-peptides were shifted by single residues. The peptides were tested in an enzyme immuno-assay against fourteen HIV-1 antibody-positive human sera, which were previously found to react with HIV-env 583-599, and against rabbit antisera to the peptides HIV-env 583-599 and 586-606. Substitutions as well as deletions in the sequence 589-596 (AVERYLKD) aborgated the antigenicity of the peptides with most of the human sera. Changes outside this sequence affected the reactivities differentially. Six overlapping dodeca-peptides, shifted in the sequence by single residues, lacked antigenicity in a competition assay, suggesting antigenic dependence on an ordered peptide conformation, which the longer peptides may preferentially assume. 19- and 21-mers with overlapping sequences competed to different extents with each other for binding to the antibodies of 3 human sera, illustrating that more than one antigenic structure in this narrow region can be recognized by a single polyclonal serum.

Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays

Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays.

Three-dimensional structure and antigenicity of transmembrane-protein peptides of the human immunodeficiency virus type 1: Effects of a neutralization-escape substitution

A point mutation (Ala‐589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV‐1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV‐1 specific antibodies [(1990) J. Virol. 64, 3240‐3248]. Here 17‐residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV‐1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol‐water solutions approximately 60% α‐helix coula be induced. The three‐dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies.

Structural Prerequisites for Intersubtype B and D Antigenicity of the Third Variable Envelope Region (V3) of Human Immunodeficiency Virus Type 1

To elucidate the structural requirements for intersubtype antigenicity of human immunodeficiency virus type 1 (HIV-1) third variable envelope region (V3), synthetic peptides were used in enzyme immunoassays (EIAs) with serum samples from persons with proven or probable subtype B and D infections. Mathematical analyses of results from EIAs with singly substituted V3 peptides revealed important residues determining overall N-terminal V3 peptide antigenicity. This information was used to design V3 immunogens, rabbit antiserum to which were tested in EIA and for in vitro neutralization of molecular clones of HIV-1(MN) and HIV-1(MAL). Intersubtype-reactive epitopes were distributed toward the N-terminal half of the V3 loop. Lysine at position 310, arginine at position 311, and isoleucine at position 314, all derived from the MN primary sequence, were major determinants of intersubtype V3 antigenicity. Combinations of residues that enhanced antigenicity often contained lysine at position 310. Threonine at position 308 was common in the least advantageous combinations. V3 immunogens modified to achieve optimal antigenicity induced antiserum with augmented cross-neutralization of virus from MAL and MN molecular clones, suggesting one approach to subunit vaccine development.