Similar patterns of simian immunodeficiency virus env sequences are found in the blood and lymphoid tissues of chronically infected macaques.

Two cynomolgus macaques were infected with a genetically complex challenge stock of simian immunodeficiency virus (SIVmac251-32H). One animal developed SIV-induced disease and was sacrificed at 16 months postinfection. The second remained healthy until it too was sacrificed at 20 months postinfection. The polymerase chain reaction (PCR) was used to amplify env gp120-coding sequences from provirus present in samples of blood, spleen, and inguinal lymph node taken from both animals on the day of sacrifice. The proviral burden present in each of the tissue samples was also determined using a quantitative PCR assay. The proviral burdens in the blood, spleen, and inguinal lymph node of the healthy animal (I225) were similar. This was not the case for animal I227, in which the burden in the inguinal lymph node was much higher than for blood or spleen. Phenogram analysis of the hypervariable V1 region of env revealed that the diversity of nucleotide sequences recovered from each tissue of both macaques were similar and overlapping. Some selected amino acid differences were observed that were specific for a tissue or one of the macaques. However, the results do not suggest that the overall evolution of env in provirus populations recovered from lymphoid tissues is distinct from that recovered from the blood.

Molecular characterization of an HIV type 1 isolate from Burundi.

PIP: HIV infection is highly prevalent in Burundi. There are, however, few published reports on the envelope sequence of the prevailing HIV-1 strains in the country. The authors selected an isolate of HIV-1 from Burundi and characterize the envelope glycoprotein gp120 in detail. A sample of venous blood was taken from a 36-year-old HIV-1-positive female volunteer from Bujumbura classified as WHO stage IV, exhibiting clinical AIDS and pulmonary tuberculosis. Nine clones containing complete gp120 genes were derived from the isolate and were designated 91BU009D/1-9. The sequences have been submitted to the Los Alamos Database under accession numbers L35452-L35459 (two clones had a stop codon in their C1 regions and were not studied further). The viral sequences from the coculture closely reflect that circulating in the patient's peripheral blood mononuclear cells. That finding is in striking contrast to the rapid adaptation and evolution of HIV-1 passaged onto human T cell lines. The ability to isolate and culture virus in vitro without the rapid appearance and selection of mutants is important, because it enables relevant observations to be made with regard to the antigenic recognition of the virus by the host. The authors' study found that although 91BU009D clustered with the D subtype, it contained unique sequences noticeably divergent from other characterized proviruses of the D subtype. Further work to investigate and clarify the relationship between genotype and serotype of HIV-1 isolates is of the utmost importance if molecular epidemiology is to be of value in designing an effective AIDS vaccine.^ieng

The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo.

Polymerase chain reaction based assays, which amplify a region of the gag gene, have been developed for the direct detection of simian immunodeficiency virus (SIV) DNA sequences in the blood of experimentally infected cynomolgus macaques. In macaques infected with a characterised virus pool (11/88 pool SIVmac 32H), an assay employing a single round of amplification was found to be highly sensitive and specific. However, in animals infected with the SIV molecular clones J5 and C8 (Rud et al., J. Gen. Virol. 75, 529-543), it was necessary to use two rounds of amplification and nested primer pairs in order to achieve sensitivity > 90%. In order to differentiate macaques infected with either of the two genetically distinct SIV clones, J5 or C8, a third PCR based assay has been developed, which amplifies a 492 bp region of the nef gene. Sequence differences between the nef genes of the two molecular clones enabled the PCR product amplified from each virus to be distinguished by restriction analysis. These sensitive and specific assays complement virological detection of SIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

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.

Complex splicing of simian immunodeficiency virus (mac 251-32H) rev gene transcript from infected macaques.

Sequences of the simian immunodeficiency virus (SIV) rev mRNA transcripts were characterized from peripheral blood lymphocytes (PBLs) of two macaques experimentally infected with SIVmac251 (32H reisolate). This analysis has demonstrated that the complex splicing patterns observed for the rev mRNA transcripts originally identified in vitro is not an artifact of tissue culture, but is also found in vivo.

Purification of crystallizable recombinant SIVmac251-32H proteinase.

We have cloned a simian immunodeficiency virus (SIV) proteinase gene directly from proviral DNA of the infectious viral stock SIVmac251-32H (11/88 pool). The deduced amino acid sequence from this proteinase gene is similar to that for the published SIVmac239 molecular clone. SIVmac251-32H proteinase (SIV PR) and its flanking pol sequences were expressed in Escherichia coli as a fusion protein with most of the T7 bacteriophage gene 10 protein. The expressed protein formed cytoplasmic inclusion bodies which were solubilized in 8 M urea, and the recombinant SIV PR was refolded, yielding active, self-processed enzyme. The SIV PR was purified to homogeneity using a single pepstatin A affinity chromatography step, and had a specific peptidolytic activity of 20 mumol/min/mg. Enzymatic characteristics similar to those previously documented for other immunodeficiency virus proteinases (EC 3.4.23) were observed. These include an acidic pH optimum (pH 5.3), sensitivity to sodium chloride concentration, and complete inhibition by pepstatin A. In addition to these properties we have observed quantitative crystallization from low protein concentrations. We describe the first crystal habit for the proteinase from the HIV-2/SIV class of immunodeficiency virus, which is distinctly different from that for HIV-1 proteinase crystals.

Passive immunization of cynomolgus macaques with immune sera or a pool of neutralizing monoclonal antibodies failed to protect against challenge with SIVmac251.

In the first of two passive transfer experiments, three groups of four macaques were injected intraperitoneally with a normal serum pool, an immune serum pool (pool 1) collected 132-172 weeks postinfection with the 11/88 pool of SIVmac251, or with a pool of four neutralizing monoclonal antibodies (KK9, 17, 54, and 56) raised against gp120 of the 11/88 pool. Sera were given at a dose of 13 ml/kg whereas the MAb pool was given at 30 ml/kg. In a second experiment, a further four macaques were injected with an immune serum pool (pool 2) collected 12 weeks postinfection with simian-grown SIVmac251 at a dose of 19 ml/kg. Animals in both experiments were challenged with SIVmac251 grown in simian peripheral blood lymphocytes. Despite high levels of circulating antibodies in the serum of animals that received either the immune serum pools or the MAbs, all macaques became infected following challenge. The results described are in contrast to a previous report in which passive transfer of sera from animals infected with SIVsm successfully protected against challenge with the homologous virus grown in human PBMCs. Challenge with SIVmac251 grown in simian PBMCs may be the reason for these conflicting results. Nevertheless, the results suggest that in this model the presence of circulating neutralizing antibodies alone does not necessarily confer protection against challenge with SIVmac251 grown in simian cells.

Sequence variation in the env gene of simian immunodeficiency virus recovered from immunized macaques is predominantly in the V1 region.

Three cynomolgus macaques were immunized with recombinant envelope protein preparations derived from simian immunodeficiency virus (SIV). Although humoral and cellular responses were elicited by the immunization regime, all macaques became infected upon challenge with 10 MID50 of the 11/88 virus challenge stock of SIVmac251-32H. The polymerase chain reaction was used to amplify proviral SIV gp120 sequences present in the blood of both immunized and control macaques at 2 months post-infection. A comparison of the predominant sequences found in the region from V2 to V5 of gp120 failed to differentiate provirus recovered from either immunized or control animals. A detailed investigation of sequences obtained from the hypervariable V1 region identified a mixture of sequences in both immunized and control macaques. Some sequences were identical to those previously detected in the virus challenge stock, whereas others had not been detected previously. Phenogram analysis of the new V1 sequences found in immunized animals revealed that they were quite distinct from those from the virus challenge stock and that they included alterations to potential N-linked glycosylation sites. In contrast, new sequence variants recovered from the control animals were closely related to sequences from the virus challenge stock. The difference in diversity of new V1 sequences recovered from immunized and control macaques was highly significant (P < 0.001). Thus, the presence of pre-existing immune responses to SIV envelope protein is associated with greater genetic change in the V1 region of gp120. These data are discussed in relation to the epitopes of SIV gp120 that may confer protection from in vivo challenge.

Immunisation of macaques with SIV env recombinants: specificity of T cell and antibody responses and evaluation of protective efficacy.

Macaques were immunised with lentil lectin purified recombinant SIVmac (BK28) derived gp160 (rgp160) with or without live vaccinia (vac)-env (BK28) priming, followed by a final boost with solid matrix antibody antigen (SMAA)-gp160 (J5) complexes and challenged with the SIVmac molecularly cloned virus J5M. Rgp160 and vac-env plus gp160 induced strong Ab responses against the homologous virus. Live vac-env did not enhance or prolong the antibody response, however, T cell responses were stronger. Analysis of the specificity of the immune response demonstrated that sequence variation within SIVmac viruses can affect antibody and T cell recognition. A single booster immunisation with the heterologous SIVmac J5 env recombinant protein was not sufficient to protect against the molecularly cloned virus J5M. These findings further illustrate the difficulty of generating protective immunity with immunogens based on single sequence recombinants.

Simian immunodeficiency virus (mac 251-32H) transmembrane protein sequence remains conserved throughout the course of infection in macaques.

Two cynomolgus macaques were infected with a genetically complex challenge stock of simian immunodeficiency virus (SIVmac251-32H). The polymerase chain reaction (PCR) was used to amplify the env gp41, rev, and nef overlapping coding sequences from provirus present in the blood of both animals at 1, 6, and 15 months post infection (p.i.). The predominant, env sequences found in both animals at the three time points were very similar to that found in the original 11/88 challenge stock. The functionally important hydrophobic fusion and membrane-spanning domains within gp41 remained conserved throughout the course of infection. Nucleotide variation within the region corresponding to the REV response element (RRE) was limited to four positions, none of which were predicted to cause any significant disruption to the secondary structure of the RRE. Very little genetic variation was observed in and around the cluster of potential glycosylation sites of the external portion of gp41. However, the existence of a previously assigned variable region elsewhere in the cytoplasmic domain of gp41 was confirmed. The three gene loci (env, rev, and nef) examined varied independently. All changes in the predominant protein sequences were brought about by single nucleotide substitutions only. After 15 months of infection with SIV, 1 animal was sick from SIV-induced disease whereas the other remained healthy. In-frame stop codons within the transmembrane protein occurred with a much greater frequency in the healthy animal.

Studies on the specificity of the vaccine effect elicited by inactivated simian immunodeficiency virus.

Inactivated, partially purified simian immunodeficiency virus (SIVmac) protected macaques from intravenous challenge with homologous and heterologous strains of SIV that had been grown on human cells but no protection against challenge with monkey peripheral blood mononuclear cell-grown SIVmac was afforded. Human immunodeficiency virus type 1 prepared in an analogous way to the SIVmac vaccine on the C8166 human T cell line protected macaques against challenge with human cell-grown SIVmac. These results suggest that protection may be mediated by xenoimmunization with the vaccine cell substrate proteins. All vaccinated macaques had anti-cell antibodies. Major reactivity to MHC class I antigens was found as well as to a 70-kD protein detectable only under nonreducing conditions.

The genetic evolution of the envelope gene of simian immunodeficiency virus in cynomolgus macaques infected with a complex virus pool.

Two cynomolgus macaques were infected with a complex, but characterized, challenge stock of simian immunodeficiency virus (SIVmac251 32H). The polymerase chain reaction was applied in a temporal sequence analysis to determine the sequences of the gp120 region of the SIV env gene, which were present in the blood of both macaques at 1, 6, and 15 months postinfection (p.i.). At 1 month p.i. selected sequences, which had been present in the original virus challenge stock, were reisolated. At later times, new sequences emerged, which had not been detected in the original virus challenge stock. Changes in sequence were restricted to specific regions of gp120, notably those equivalent to V1, V2, V4, and V5 of HIV-1, but not V3. The diversity and the rate of appearance of new sequences in the V1 region suggest that genetic evolution occurs by mechanisms in addition to nucleotide substitutions. These results are discussed in relation to the role of the envelope protein in the generation of protective immunity against infection with immunodeficiency viruses.

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.

Protection in simian immunodeficiency virus-vaccinated monkeys correlates with anti-HLA class I antibody response.

Our earlier reports demonstrated that Cynomolgus macaques vaccinated with either inactivated partially purified simian immunodeficiency virus (SIV), fixed SIV-infected C8166 (a human T lymphoblastoid cell line) cells, or fixed uninfected C8166 cells can be protected against a challenge infection with the 32H isolate of SIVmac 251 (grown in C8166) (Stott, E. J., W. L. Chan, K. H. G. Mills, M. Page, F. Taffs, M. Cranage, P. Greenway, and P. Kitchin. 1990. Lancet. 336:1538; Stott, E. J., P. A. Kitchin, M. Page, B. Flanagan, L. F. Taffs, W. L. Chan, K. H. G. Mills, P. Silvera, and A. Rodgers. 1991. Nature [Lond.]. 353:393). Protection is correlated with the levels of antibody response to cellular antigens in the human cells from which the virus immunogen was grown. However, the mechanism of protection is unclear. We report here the analysis of sera from these protected monkeys and demonstrate that there is positive correlation of protection with antibody response to the HLA class I molecule.

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.

Protection against SIV infection in macaques by immunization with inactivated virus from the BK28 molecular clone, but not with BK28-derived recombinant env and gag proteins.

Vaccination of cynomolgus macaques with beta-propiolactone inactivated SIVmacBK28 in Freund's adjuvant induced low but detectable levels of anti-SIV envelope (env) antibodies and T-cell responses and protected against challenge with the 32H isolate of SIVmac251 grown in C8166 cells. In contrast, purified recombinant SIV env and gag proteins derived from BK28 formulated in Syntex adjuvant generated consistent and long-lived cellular and humoral immune responses to SIV env, but failed to protect against infection with the 32H virus. Thus, protection against a heterogeneous challenge stock is possible by immunization with a molecularly-cloned virus, but not with recombinant proteins from the same molecular origin. High levels of anti-cell antibodies induced by the whole virus vaccine, but not by recombinant proteins, may have contributed to the protection observed.

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.

Population sequence analysis of a simian immunodeficiency virus (32H reisolate of SIVmac251): a virus stock used for international vaccine studies.

The virus structural genes gag and env (both gp120 and gp41 regions) of the 32H isolate of SIVmac251 were amplified using the polymerase chain reaction (PCR). The proviral template used in the PCR was DNA isolated from cells used to prepare several experimental SIV vaccines, which have been tested in simians, and a standard challenge stock of virus, which has been used in international collaborative studies. The PCR products were cloned and the nucleotide sequences of several clones were determined for each gene. From a comparison of the sequences obtained the predominant amino acid sequences of gag and env were predicted and the degree of sequence heterogeneity was determined. Conserved and more variable regions of each protein were identified. The gp120 region of env was more heterogeneous than gag or the transmembrane protein of env (gp41). Within gp120, sequence variability was concentrated to specific regions equivalent to the V1, V2, and, to a lesser extent, the C1 regions identified for human immunodeficiency virus type 1 (HIV-1). In contrast the region equivalent to the hypervariable "V3-loop" of HIV-1 was highly conserved. The implications of the data is discussed in relation to the ability of this virus stock to prepare effective vaccines against SIV.

Preliminary report: protection of cynomolgus macaques against simian immunodeficiency virus by fixed infected-cell vaccine.

Cynomolgus macaques were vaccinated with inactivated simian immunodeficiency virus-infected cells and 'Quil-A' as adjuvant at 0, 4, 8, and 36 weeks or at 0, 4, 8, and 16 weeks. 2 weeks later these animals, together with a similar unvaccinated group, were challenged with 10 MID50 (50% monkey infectious doses) of a pool of SIVmac251 previously titrated in vivo. Virus was repeatedly isolated from unvaccinated animals on at least five separate occasions and proviral DNA was detected in circulating lymphocytes by polymerase chain reaction amplification. By contrast, virus and proviral DNA were not found in any of the vaccinated animals. However, the same vaccination regimen used after live virus challenge did not eliminate virus from previously infected macaques.

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.