Reference preparations in the standardisation of HIV-1 PCR--an international collaborative study.

Following the recent developments in diagnostic polymerase chain reaction (PCR) technology, we have assessed a set of HIV-1 DNA reference standards using the first commercial diagnostic test kit for the detection of HIV-1 (Amplicor, Roche) in an international collaborative study. Nineteen laboratories in 11 countries analysed a set of ten (re-coded) HIV-1 DNA reference standards, whose performance had been validated in a previous collaborative study (Bootman and Kitchin, 1992). Results from the current study show that, using the diagnostic kit, 84% of laboratories (16/19) obtained correct diagnoses for all ten test samples. One additional laboratory failed only to detect the sample containing ten copies of target template. Test results from the remaining two laboratories were declared void in accordance with the Amplicor quality control guidelines.

The assessment of nucleotide sequence diversity by the polymerase chain reaction is highly reproducible.

The polymerase chain reaction (PCR) is often used to assess the diversity of viral nucleotide sequences present in various biological samples (e.g., blood and tissues). However, it is not clear how reproducible this approach may be. DNA was extracted from the peripheral blood lymphocytes of a macaque that had been infected, experimentally, with SIVmac251-32H 6 months previously. The nef gene was then amplified by the PCR on three separate occasions from this same template preparation. A panel of clones was prepared from the product of each PCR, the entire nef gene was sequenced and the sequences obtained compared with each other. Phenogram analysis revealed that within each panel the same degree of sequence diversity was observed between clones. Furthermore, when the sequences obtained from all three panels were compared, the overall sequence diversity observed was no greater than that observed for each panel individually. These data indicate that the analysis of sequence diversity in SIV 'quasi-species' populations by the PCR is reliable and, more important, reproducible.

An international collaborative study to assess a set of reference reagents for HIV-1 PCR.

An international collaborative study was performed to evaluate a set of PCR reference reagents for HIV diagnosis. Twenty-six laboratories from 9 countries analysed a proficiency panel of 10 coded DNA samples using the PCR reference reagents and protocols. For comparison, these coded samples were then assessed using a laboratory's own 'in-house' reagents and methodologies. The objectives of the study were: (i) to assess inter-laboratory variation of PCR sensitivity, (ii) to evaluate the DNA 'carryover' problem and frequency of false negative results and (iii) to examine the utility of the complete set of reagents and templates to act as reference preparations for HIV PCR. Using the reference reagents, 46% of laboratories reported no false positive results in any of their assays of the negative controls. The remaining laboratories all reported a false positive result(s) in at least one assay. The overall false positive result rate for the study was 9.3%. In contrast, an overall false negative result rate of 7.4% was observed, with some laboratories recording negative results even for samples containing 10,000 molecules of target DNA. The level of absolute sensitivity may be assessed accurately only from the 12 laboratories that obtained no false positive results. All 12 laboratories detected the sample containing 10 molecules of template DNA and 9 out of the 12 laboratories detected the sample containing 1 molecule. This is in close agreement with the theoretical detection rate based on a statistical probability model for the detection of a single molecule. These characterised reference reagents were at least as sensitive as any of the 'in-house' reagents and methodologies applied, including nested PCR. The complete set of characterised reference reagents is now available for quality control assessment of HIV-1 PCR from the MRC ADP.

Intrarectal challenge of macaques vaccinated with formalin-inactivated simian immunodeficiency virus.

Macaques can be protected from intravenous infection with simian immunodeficiency virus (SIV) by vaccination with chemically inactivated virus. However, protection against infection via a mucosal surface has not been demonstrated. We vaccinated four rhesus macaques with formalin-inactivated SIV given intramuscularly. These monkeys, which had remained virus free for 10 months after intravenous challenge with SIV, were given a further dose of vaccine and together with four unvaccinated controls were challenged intrarectally with SIV. Subsequently, virus was isolated from all control animals on five successive occasions, but the vaccinated animals remained free of virus. Proviral DNA could not be detected in peripheral blood mononuclear cells from the vaccinated animals. Preliminary data indicate that vaccinated animals make a local antibody response.

Histopathological changes in simian immunodeficiency virus infection.

The histological lesions were studied in seven rhesus and three cynomolgus monkeys infected with simian immunodeficiency virus for periods ranging from nine weeks to 18 months. Lymphoreticular changes included hyperplasia, follicular involution and depletion, and one animal had amyloidosis of the spleen. Hyperplastic changes also took place in mucosa-associated lymphoid tissue and infiltrations occurred in the vaginal mucosa of one animal, which could be significant in sexual transmission of the infection. The range of opportunistic infections was small compared with that in human AIDS patients, although two monkeys had Pneumocystis carinii pneumonia. Enterocolitis was a common finding and brown adipose tissue was transformed into a large vacuolated type. Lesions of the central nervous system were found in five of nine monkeys, and consisted of foci of glial activity and perivascular and meningeal lymphocytic infiltration. A lymphoma involving the lumbar spinal cord developed in one animal.

The use of the polymerase chain reaction for the detection of Simian immunodeficiency virus in experimentally infected macaques.

A rapid, non-radioactive assay for the detection of proviral Simian immunodeficiency virus (SIV) in tissue-culture cells is described. The assay is based on the co-amplification of the SIV env and gag genes by the polymerase chain reaction (PCR). When the gag PCR product is blotted onto a nylon membrane and hybridised to a radioactive oligonucleotide probe, the assay can also be used to detect the SIV gag gene in DNA isolated directly from experimentally infected cynomolgus macaque lymphocytes. This provides a valuable assay for the presence of proviral SIV during animal trials of AIDS vaccines and chemotherapeutics.

HIV p24-specific helper T cell clones from immunized primates recognize highly conserved regions of HIV-1.

We have investigated Th cell recognition of the HIV core protein p24 by using CD4+ T cell clones derived from cynomolgus macaques immunized with hybrid HIV p24: Ty virus-like particles (VLP). T cell lines from two immunized animals responded to p24: Ty-VLP, control Ty-VLP, purified p24, and whole inactivated HIV, indicating the presence of T cells specific for p24 as well as the Ty carrier protein. The HIV determinants recognized by the T cell lines were identified by using a series of overlapping peptides synthesized according to the sequence of p24. Both T cell lines recognized peptide 11 (amino acids 235-249) and peptide 14 (amino acids 265-279). In addition, one T cell line also responded to peptide 9 (amino acids 215-229). Definitive identification of two T cell epitopes on p24 was confirmed at the clonal level: from a total of four T cell clones generated from one of the T cell lines, two respond specifically to peptide 11 and two to peptide 14. The T cell clones were CD4+ and MHC class II-restricted and secreted IL-2 in response to stimulation with purified p24, inactivated HIV or a single synthetic peptide. The specificity of the Th clones for variant peptides demonstrated cross-reactivity with two simian immunodeficiency virus isolates, but only limited responses to HIV-2 sequences. However, the Th cell epitopes identified on p24 are highly conserved between 12 HIV-1 isolates and were recognized by both of the immunized primates. These sequences may therefore be useful for priming a broadly reactive immune response to HIV-1.

The morphology of simian immunodeficiency virus as shown by negative staining electron microscopy.

Negative staining electron microscopy was used to study sucrose gradient-purified preparations of the simian immunodeficiency virus (SIVmac251). Both isolated and aggregated virus particles were observed together with some free-lying virus cores. The cores were 110 nm long and 25 to 50 nm wide and were mainly conical or wedge-like in shape. Surface projections were seen on the envelope membrane of many of the virus particles; the knobs were approximately 6 nm in length, 10 nm wide and from an end-on view they had a Y or triangular-shaped morphology.

A highly bent fragment of Crithidia fasciculata kinetoplast DNA.

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.

Visualization of the bent helix in kinetoplast DNA by electron microscopy.

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.

Intermediates in the replication of kinetoplast DNA minicircles.

Kinetoplast DNA of Crithidia fasciculata and other trypanosomatids is in the form of a network of thousands of minicircles and a few dozen maxicircles. Minicircles replicate as free molecules after release from the network, and their progeny subsequently reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). The minicircles just released from the network are covalently closed and apparently completely relaxed. After Cairns-type (theta) replication, the two minicircle progeny have different structures. One has a nascent H (heavy) strand which initially is in the form of 20-110 nucleotide fragments that are separated by gaps (Kitchin, P. A., Klein, V. A., Fein, B. I., and Englund, P. T. (1984) J. Biol. Chem. 259, 15532-15539). The other initially has a full-size (2.5 kilobase) nascent L (light) strand. During the time between formation of these progeny molecules and network reattachment, the nascent L strand is nicked (or gapped) and nascent H strand is partially repaired. Therefore, both progeny, at the time of reattachment, have several nicks (or gaps) in their nascent strand. Minicircle progeny with a nascent L strand reattach to the network quickly, whereas those with a nascent H strand reattach more slowly. Once reattached to the network, the nicks or gaps in the minicircles are repaired until finally covalent closure occurs.

Gapped Minicircles. A novel replication intermediate of kinetoplast DNA.

Kinetoplast DNA, the mitochondrial DNA in trypanosomatids, is a network of thousands of interlocked circles. Most of these circles are minicircles and a few are maxicircles. Minicircles replicate, after decatenation from the network, by a Cairns-type mechanism. The minicircle progeny then reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). We have now discovered a novel intermediate in Crithidia fasciculata minicircle replication. It is a highly gapped 2.5-kilobase free minicircle with nascent fragments of only 20 to 110 nucleotides. These fragments are nonligasable, and some remain nonligasable even after gap filling with DNA polymerase. Solution hybridization studies show that the nascent fragments are predominantly, if not exclusively, heavy strand.

Presence of a bent helix in fragments of kinetoplast DNA minicircles from several trypanosomatid species.

Some restriction fragments of kinetoplast minicircles from several trypanosomatid species (Leishmania tarentolae, Trypanosoma brucei, T. equiperdum, Herpetomonas muscarum, Crithidia fasciculata, but not T. cruzi) migrate anomalously on polyacrylamide gels. This behavior is probably due to a natural curvature of the helix. Bent helices appear to be a common property of kinetoplast minicircles, and may be important for minicircle function. In the case of T. equiperdum, we present evidence that each minicircle has a single bent region which resides in or near the 'conserved sequence.'