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Most human T cell lymphotropic virus type (HTLV)-1-infected individuals mount a strong and persistently activated cytotoxic T lymphocyte (CTL) response to the virus, which implies that there is abundant chronic transcription of HTLV-1 genes. On the other hand, several observations suggest that HTLV-1 might be latent in vivo and therefore not detectable by CTLs. To clarify these discrepancies, we quantified the frequency of provirus-positive peripheral blood mononuclear cells (PBMCs) that were capable of expressing the HTLV-1 Tax protein, which is known to be the immunodominant target antigen recognized by HTLV-1-specific CTLs. The analysis showed that a significant proportion of HTLV-1-infected cells (from 14 to 100%) starts to express the Tax protein within a few hours of culture ex vivo. Phenotypic analysis confirmed that the main cell subpopulation expressing the Tax protein is CD4 positive. Frequent Tax expression in CD4(+) T lymphocytes in vivo might account for the chronic activation of the cytotoxic immune response observed in the majority of HTLV-1-infected patients and might contribute to the pathogenesis of HTLV-1-associated diseases.

Infection and gene expression by the human T lymphotropic virus type I (HTLV-I) in vivo have been thought to be confined to CD4(+) T lymphocytes. We show here that, in natural HTLV-I infection, a significant proportion of CD8(+) T lymphocytes are infected by HTLV-I. Interestingly, HTLV-I-specific but not Epstein-Barr virus-specific CD8(+) T lymphocytes were shown to be infected. Furthermore, HTLV-I protein expression in naturally infected CD8(+) T lymphocytes renders them susceptible to fratricide mediated by autologous HTLV-I-specific CD8(+) T lymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-I and possibly other lymphotropic viruses.

Phylogenetic analysis of nucleotide sequences of gag and env genes of type 1 human immunodeficiency virus (HIV-1) variants isolated from individuals infected through sexual intercourse or nosocomially (by injections with nonsterile syringes) showed that 5 of 27 (18.5%) isolated strains were recombinants. Two viruses found in the Russian Far East had gagAenvE genotype, three other recombinants had envG genotype; gag gene of one isolate belonged to subtype A and gag genes of two others belonged to subtype D. Detection of new recombinant variants in addition to the A/B recombinant described previously shows that these viruses can contribute to the HIV-1 genetic variability in Russia.

This study compares the performance of a line probe assay (LiPA) for the detection of the major mutations associated with reduced sensitivity to nucleoside analogues with a well characterised point mutation assay (PMA). Plasma samples obtained from patients in a trial of four reverse transcriptase inhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutations in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from plasma by the Boom method and amplified by RT-PCR prior to being tested by LiPA or PMA. Assay discrepancies were further investigated by sequencing of the RT gene. Of 275 samples available from 98 trial subjects, 246 samples were successfully amplified by PCR and analysed by LiPA and PMA for six mutations. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a result for 32 codons from 22 samples and PMA failed with 58 codons from 38 samples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequencing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PMA result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are genotypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases.

Objective: To assess the prevalence of human T cell leukaemia/lymphoma virus (HTLV) infection in pregnant women in the United Kingdom., Design: Population study., Subjects: Guthrie card samples from babies born in 1997-8. Samples were linked to data on mother's age and ethnic status and parents' country of birth and then anonymised., Setting: North Thames Regional Health Authority., Main Outcome Measures: Presence of antibodies against HTLV in eluates tested by gelatin particle agglutination assay and results confirmed by immunoblot., Results: Of 126 010 samples tested, 67 had confirmed antibodies to HTLV (59 HTLV-I, 2 HTLV-II, 6 untyped) and six had indeterminate results. Seroprevalence was 17.0 per 1000 (95% confidence interval 9.2 to 28.3) in infants whose mothers were born in the Caribbean, 3.2/1000 (1.5 to 5.9) with mothers born in west and central Africa, and 6.8/1000 (3.1 to 12.9) in infants of black Caribbean mothers born in non-endemic regions. In infants with no known risk (both parents born in non-endemic regions and mother not black Caribbean) seroprevalence was 0.06-0.12 per 1000. Mother's country of birth, father's country of birth, and mother's ethnic status were all independently associated with neonatal seroprevalence. An estimated 223 (95% confidence interval 110 to 350) of the 720 000 pregnant women each year in the United Kingdom are infected with HTLV., Conclusions: The prevalence of HTLV and HIV infections in pregnant women in the United Kingdom are comparable. The cost effectiveness of antenatal HTLV screening should be evaluated, and screening of blood donations should be considered.

Apoptosis in peripheral blood lymphocyte populations in HTLV-I-infected people in vivo was examined, to study the lymphocyte dynamics in HTLV-I infection. Freshly isolated lymphocytes from 10 non-infected healthy people, eight asymptomatic HTLV-I carriers and 15 patients with HAM/TSP were stained with FITC-labelled annexin V to detect phosphatidylserine (PS) residue exposure at the outer plasma membrane leaflet as an early marker of apoptosis. There was no significant difference in annexin V positivity in CD4+ and CD8+ lymphocytes between non-infected subjects, asymptomatic carriers and HAM/TSP patients, but there was a greatly increased exposure of PS on CD19+ lymphocytes (B cells) detected by FITC-annexin V in 12 out of 15 (80%) HAM/TSP patients, while only two out of eight (25%) asymptomatic carriers and none of the non-infected healthy people showed this aberrant PS exposure on B cells. The intensity of annexin V staining of B cells in HAM/TSP was intermediate, as distinct from the high annexin V staining on advanced apoptotic cells. However, annexin V positivity was decreased when the cells were stained after 24 h of culture, suggesting that the intermediate PS exposure on the B cell in HAM/TSP is not a consequence of an apoptotic process, but rather reflects reversible membrane damage. B cells with PS exposure in vivo might provide a site for coagulation and inflammation, and so contribute to the pathogenesis of HAM/TSP and its complications.

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax(11-19)-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo. (Blood. 2000;95:1386-1392)

New independent states of the former Soviet Union are facing a rapidly growing epidemic of HIV-1 among injecting drug users (IDUs). This epidemic is caused by three HIV-1 populations, one belonging to HIV-1 subtype A (IDU-A), another to subtype B (IDU-B), and the third being a recombinant of the IDU-A and IDU-B viruses (IDU-A/B, gagA/envB). Each of these populations is characterized by a high level of genetic homogeneity. We identified a unique synonymous nucleotide substitution in the first isoleucine codon at the IHIGPGR motif (ATT), which was observed in the env subtype B V3 sequences derived from IDUs in Russia and the Ukraine. This substitution was observed in none of 179 sequences obtained from IDUs in western Europe, northern America, and Asia. Molecular epidemiological analysis of HIV-1 strains based on this sequence pattern could be useful for tracing the origin and spread of the IDU-B viruses to other countries and risk groups.

We determined the relationship between the presence of Mycoplasma fermentans and Mycoplasma penetrans and the rate of progression of HIV-associated disease in a nested case-control study based on a cohort of 159 HIV-infected patients with different rates of disease progression. Study participants were divided into 3 progression groups: non-progressors who had been HIV-1 seropositive for at least 9 years and had remained asymptomatic with a CD4 cell count of > 500/mm3; slow progressors who had been HIV-1 seropositive for at least 9 years and whose CD4 cell count had fallen below 500 cells, and who had developed symptomatic disease or AIDS; and rapid progressors who had developed AIDS within 5 years of HIV infection. Peripheral blood mononuclear cells (PBMCs) were collected at enrollment and examined by mycoplasma polymerase chain reaction (PCR) assays. Three (7%) of 46 non-progressors, 3 (3%) of 86 slow progressors, and 2 (7%) of 27 rapid progressors were M. fermentans positive. The PBMCs from 91 subjects were tested for M. penetrans DNA and none was positive. The small proportion of M. fermentans-positive patients indicates that the mycoplasma cannot be important in the development of AIDS in the large majority of patients. Furthermore, no association was found between its presence and more rapid HIV disease progression.

Objective: To assess the safety of imiquimod, an immune response modifier, in the topical treatment of external anogenital warts in HIV-infected patients., Setting: Clinical sites in the United Kingdom (eight) and the United States (five)., Design: A prospective, randomized, double-blind, vehicle-controlled study of imiquimod 5% cream or vehicle applied for 8+/-2 h three times per week for a maximum of 16 weeks in HIV-seropositive males (n = 97) and females (n = 3) aged 18 years or more with clinically diagnosed external anogenital warts, CD4 T lymphocyte count of > or = 100 x 10(6) cells/l and Karnofsky score > or = 70., Main Outcome Measures: Safety was assessed through the incidence and severity of local skin reactions and other adverse events, and through clinical laboratory tests. Wart clearance was documented by two-dimensional measurements of warts and by photography., Results: Among the patients treated with imiquimod (n = 65) and vehicle (n = 35), the most common local skin reaction was erythema, (41.9 and 26.7%, respectively) and the incidence of patients reporting at least one adverse event was 69.2 and 65.7%, respectively. No clinically meaningful differences or changes in laboratory values were observed between treatment groups, nor were drug-related adverse effects observed in regard to HIV disease. While there was no significant difference between treatment groups in the number of patients who totally cleared their baseline warts (imiquimod 11% versus vehicle 6%, P = 0.488), more imiquimod-treated patients experienced a > or = 50% reduction in baseline wart area (38% versus 14%, P = 0.013)., Conclusion: Most local skin reactions were mild and no adverse effects on HIV disease were observed. Topically applied imiquimod 5% cream reduced wart area and may have clinical utility in treating external anogenital warts in some HIV-infected patients.

Patients with human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) typically have a high HTLV-1 proviral load in peripheral blood mononuclear cells and abundant, activated HTLV-1-specific cytotoxic T lymphocytes (CTLs). No effective treatment for HAM/TSP has been described so far. We report a 10-fold reduction in viral DNA for five patients with HAM/TSP during treatment with the reverse transcriptase inhibitor lamivudine. In one patient with recent-onset HAM/TSP, the reduction in viral DNA was associated with a fall in the frequency of CTLs specific to two peptides in the immunodominant viral antigen Tax. The half-life of peripheral blood mononuclear cell populations was estimated from changes in viral DNA copy number, CTL frequency, reduction in CD25 expression, and the loss of dicentric chromosomes following radiation-induced damage. Each of these four different techniques indicated a cellular half-life of approximately 3 days consistent with continuous lymphocyte replication and destruction. These results indicate that viral replication through reverse transcription significantly contributes to the maintenance of HTLV-1 viral DNA load. The relative contribution of proliferation versus replication may vary between infected people.

The prevention of mother to child transmission of HIV-1 by zidovudine monotherapy is well known, but increasingly combination anti-retroviral therapy is prescribed during pregnancy. In this prospective study, 19 pregnant women with human immunodeficiency virus-1 (HIV-1) infection who elected to take anti-retroviral therapy during the second and third trimesters were treated with zidovudine or zidovudine plus lamivudine. Fourteen women treated with zidovudine monotherapy had a mean 0.3 log(10) reduction in viral load and a mean 52 x 10(6)/L (17%) increase in CD4+ lymphocytes at delivery compared with pre-treatment samples. Genotypic mutations associated with decreased susceptibility to zidovudine were detected in 2 of 10 women at delivery. Five women with more advanced HIV-1 infection were treated with zidovudine plus lamivudine and a mean 1.5 log(10) reduction in viral load together with a mean 30 x 10(6)/L (33%) increase in CD4+ lymphocytes was observed in this group. However, four of five women in the dual therapy arm had the M184V mutation in the reverse transcriptase gene associated with decreased susceptibility to lamivudine at delivery. We conclude that zidovudine plus lamivudine reduced HIV-1 plasma viraemia to low levels in pregnant women with advanced HIV-1 disease but the rapid development of genotypic resistance to lamivudine indicates that additional therapy is required both for the long-term benefit of the mothers and to prevent the development of resistant virus that may be transmitted to the infant.

A prospective clinical study of 20 initially asymptomatic HTLV-I-seropositive carriers was commenced in 1991 to determine the natural history of the infection in relation to HTLV-I proviral load, immune responses, and lymphocyte phenotype. Proviral load varied widely between carriers but was relatively constant within an individual over time. The lymphocyte phenotype and prevalence of activated lymphocytes were not predictive of disease and the magnitude of the cytotoxic T-lymphocyte response to HTLV-I was independent of proviral load. Incident conditions, some related to HTLV-I infection, including a case of HTLV-I-associated myelopathy (HAM), were documented in 9 carriers. Development of myelopathy and uveitis was associated with high peripheral blood HTLV-I proviral load that predated symptoms. Persistently high proviral load appears to predate the development of HTLV-I-associated inflammation in neuro-ophthalmic tissue.

Thirteen protease inhibitor-naive patients with HIV-1 infection, and 12 patients with a median of 58 months prior treatment with saquinavir (SQV) monotherapy, were treated with SQV (400 mg twice daily) and ritonavir (RIT, 500 mg twice daily) in a study designed to assess the effect of prior treatment with SQV monotherapy on the antiretroviral activity of RIT-SQV combination therapy. Median baseline viral load and CD4+ cell counts were 155,000 and 262,000 copies/ml and 333 and 225 cells/mm3 in the naive and experienced groups, respectively. Mean viral load changes at 24 weeks were -1.63 and -0.27 log copies/ml in the naive and SQV-experienced groups, respectively (intent-to-treat analysis). Baseline genotype by point mutation assay and sequencing in the SQV-experienced group was highly predictive of virological response. Eight of 11 SQV-experienced patients had evidence of phenotypic resistance to RIT at baseline, despite previous treatment with SQV only. There was strong correlation between phenotypic resistance to RIT and the presence of the L90M mutation. We conclude that prolonged prior treatment with saquinavir monotherapy may produce cross-resistance to ritonavir and reduce the subsequent response to ritonavir-saquinavir in combination. In this study, both phenotypic resistance to ritonavir and presence of the L90M mutation predicted the viral load response to ritonavir-saquinavir.

The significance of the maternal humoral immune response in relation to vertical transmission of HIV-1 was investigated in 123 mothers infected with subtype E from Thailand. Antibody binding titers to HIV-1 env domains (monomeric gp120, the CD4/gp120 binding site [BS], V3 loop, and gp41) and antibody-mediated neutralization of primary and T-cell line-adapted (TCLA) subtypes B and E HIV-1 isolates were investigated. No correlation between maternal anti HIV-1 antibodies at delivery and vertical transmission of HIV-1 subtype E was found. However, a trend to higher titer antibody-mediated cross-neutralization of a heterologous subtype B TCLA isolate, HIV-1MN, was observed in nontransmitting mothers postpartum. The HIV-1-specific antibody titers in these infected mothers increased significantly from delivery to 6 months postpartum (p < .05), but this was only partially attributable to hemodilution and an additional factor or factors appear to affect humoral immunity to HIV-1 during late pregnancy.

This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.

The risk of disease associated with persistent virus infections such as HIV-I, hepatitis B and C, and human T-lymphotropic virus-I (HTLV-I) is strongly determined by the virus load. However, it is not known whether a persistent class I HLA-restricted antiviral cytotoxic T lymphocyte (CTL) response reduces viral load and is therefore beneficial or causes tissue damage and contributes to disease pathogenesis. HTLV-I-associated myelopathy (HAM/TSP) patients have a high virus load compared with asymptomatic HTLV-I carriers. We hypothesized that HLA alleles control HTLV-I provirus load and thus influence susceptibility to HAM/TSP. Here we show that, after infection with HTLV-I, the class I allele HLA-A*02 halves the odds of HAM/TSP (P < 0.0001), preventing 28% of potential cases of HAM/TSP. Furthermore, HLA-A*02(+) healthy HTLV-I carriers have a proviral load one-third that (P = 0.014) of HLA-A*02(-) HTLV-I carriers. An association of HLA-DRB1*0101 with disease susceptibility also was identified, which doubled the odds of HAM/TSP in the absence of the protective effect of HLA-A*02. These data have implications for other persistent virus infections in which virus load is associated with prognosis and imply that an efficient antiviral CTL response can reduce virus load and so prevent disease in persistent virus infections.

This study set out to investigate whether plasma mannose-binding protein (MBP) deficiency caused by mutations in the MBP gene associates with pyogenic or opportunistic infections in HIV-infected patients. Plasma samples were selected randomly from 131 HIV-infected patients followed prospectively for a period not exceeding 12 months or until death. Plasma MBP concentrations were measured by an ELISA and genotyping was determined by amplification of exon 1 of the MBP gene by polymerase chain reaction (PCR) technology, followed by restriction enzyme analysis and Southern blotting using sequence-specific oligonucleotide probes. Neither MBP concentration nor genotype was found to associate with disease progression or opportunistic infection rate. There was an unexpected increased bacterial infection rate in patients with MBP levels greater than 100 ng/ml and wild type genotype. Thus, MBP does not appear to play a role in HIV infection. MBP is an acute phase reactant and this may explain the higher levels in those with more frequent pyogenic infections.

A nested PCR was designed using primers from the pol and tax genes of human T-cell leukaemia virus type I (HTLV-I). The assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1 x 10(5) Peripheral blood mononuclear cells (PBMCs). Using serial dilutions of sample DNA, the assay was applied prospectively to study proviral load in patients with HTLV-associated disease and carriers. The median proviral load expressed as number of copies/100 PBMCs was found to be 14.0 copies in patients with HAM and 1.55 copies in initially asymptomatic carriers. The assay was used to test for low proviral load in subjects who may have HTLV-I infection, and to monitor response to therapy.

The effect on normal vaginal flora of three intravaginal microbicides potentially active against human immunodeficiency virus type 1 was examined. Volunteers received dextrin sulfate (D2S), nonoxynol-9 (N-9), or docusate sodium in separate placebo-controlled studies. High vaginal swabs were obtained for bacterial culture before and after microbicide application. D2S did not affect the vaginal flora. However, lactobacilli decreased by > or = 10(2) cfu/mL in 9 (56%) of 16 women given N-9 and in 5 (63%) of 8 women given docusate sodium. Women using N-9 were also significantly more likely to become colonized abnormally (usually with aerobic gram-negative rods) than were those using placebo, as were women using docusate sodium. Women with reduced lactobacilli were less likely to regain normal flora than were those whose lactobacilli were unaffected. However, coliform colonization occurred whether lactobacilli produced H2O2 or not. Continuous use of N-9 could induce susceptibility to urinary and gynecological infection. It is essential that potential microbicides are examined for activity against normal vaginal flora.

The majority of vertical infections with human immunodeficiency virus type 1 (HIV-1) occur at or near delivery, strongly suggesting a mucosal route of transmission. The frequency and level of intrapartum mucosal exposure to HIV-1 of 22 infants born to infected mothers was investigated. Maternal plasma HIV-1 RNA and CD4 cell count were measured at delivery. Infant oropharyngeal aspirates obtained at birth were examined for HIV-1 RNA by reverse transcription-polymerase chain reaction and qualitative nucleic acid sequence-based amplification. Nine infants (41%) had detectable levels of HIV-1 RNA, 3 of which were quantifiable (mean, 3000 copies/mL). This mucosal exposure to HIV-1 during delivery did not lead to infection of any infant. Cesarian delivery did not reduce mucosal exposure to HIV-1. Mucosal exposure did not correlate with maternal CD4 cell count but did correlate with maternal plasma virus load and was reduced by antiretroviral therapy.

Objectives: To examine the change in uptake of interventions to reduce transmission of HIV from mothers to infants from January 1994 to July 1997., Design: Review of mother-infant pairs who presented for infant diagnosis of HIV infection., Setting: Central London hospital with facilities for diagnosis of infant HIV infection., Subjects: 57 consecutive mother-infant pairs, mainly from central London but also referred from surrounding hospitals., Interventions: Data were collected on mother's country of origin; CD4 count at delivery; plasma HIV RNA copies/ml; mode of delivery; antiretroviral therapy; infant feeding; and HIV infection in infants., Main Outcome Measures: HIV infection of infants., Results: The vertical transmission rate was 12% (7 pairs; 95% confidence interval 3% to 22%). All mothers chose not to breast feed. The caesarean section rate was 53% (30/57). Antiretroviral therapy was taken by 68.5% (39/57) of mother-infant pairs. With antiretroviral therapy or caesarean section, or both, transmission occurred in 6% (0% to 13%) of pairs (3/50). During the 24 months of 1994 and 1995, 21% (4/19) of infants were infected with HIV; 7.9% (3/38) were infected over the 19 months January 1996 to July 1997. The caesarean section rate did not change over these periods. Use of antiretroviral therapy increased from 31.5% (6/19) to 86.8% (33/38) (P < 0.0001)., Conclusion: Women with a diagnosis of HIV infection acted to reduce the risk of transmission to their infants. Uptake of antiretroviral therapy increased significantly over time, and the caesarean section rate was persistently high.

The aim of this phase II study was to evaluate the safety, immunogenicity and tolerability of the yeast-derived virus-like particle immunogen, Ty.p24.VLP (p24-VLP), in HIV-antibody-positive asymptomatic volunteers. Fifteen informed and consented volunteers, with p24 Antibody titres >1/100, p24 Antigen <20 pg/l, and CD4>350 x 10(9)/l were enrolled. Five were immunized with aluminium hydroxide placebo, five with 25 microg, and five with 100 microg p24-VLP in Alum adjuvant at weeks 0 and 4 by the intramuscular route. Patients were followed for 16 weeks post vaccination and the main outcome assessments were CD4 and CD8 lymphocyte counts, p24 antigen and antibody, Ty antibody and quantitative viral cultures. No serious adverse events were observed in any of the groups. There were increases in CD4 counts in the treated groups but not in the controls, although these changes were not statistically significant. There were no significant intrasubject or intergroup changes in the other parameters, such as p24 antigen and antibody. No pattern of change in plasma viraemia was detected, and most cultures were negative. Therefore we conclude that p24-VLP immunizations of 25 microg and 100 microg are well tolerated, and the CD4 changes are encouraging, but higher doses and larger numbers are required to see if there are significant humoral or cellular responses, and extended phase II studies are now in progress.

Ninety male patients with acute nongonococcal urethritis (NGU) who presented for follow-up 10-92 days after initiation of treatment were evaluated. A polymerase chain reaction assay and direct fluorescence antibody test were used to detect Chlamydia trachomatis at presentation and during follow-up. Chlamydial heat-shock protein 60 kD (hsp60) serology with an enzyme-linked immunosorbent assay and C. trachomatis serology with a microimmunofluoresence test were undertaken. In 62 (69%) of the men, evidence of chronic urethritis was noted during follow-up. C. trachomatis was detected in only two patients during follow-up. Chlamydial hsp60 antibody was associated with the development of chronic urethritis between 10 and 92 days after treatment began (P < .04), that is, at 10-29 days (P < .02) and at 30-92 days (P < .008). These results are consistent with the theory that immune response to hsp60 is important in the development of this chronic disease. The results also suggest that chronic NGU is not a consequence of continued production of hsp60 by C. trachomatis.

Objectives: To evaluate the effect of sequence variation within the gp120 V3 loop on CD4 T-cell recognition., Design: CD4 T-cell clones were generated using synthetic peptides to circumvent the difficulties of using polyclonal T-cell responses. Peptides based on other HIV isolates were then used to determined the influence of single and multiple sequence differences., Results: Three of the panels of T-lymphocyte clones (TLC), which were all specific to diverse HIV-1 clade B gp120 V3-loop peptides differing in a limited number of residues, had heterogeneous patterns of response to peptides differing in length and sequence indicating that they recognized distinct but overlapping epitopes. The panels of TLC also differed in the extent to which they tolerated sequence differences between cell-culture-adapted or primary HIV-1 isolates. One panel responded to peptides based on several clade B and one clade D isolate. In contrast, two panels, generated from two different donors using the same peptide, only responded to a limited number of clade B isolates, whereas another only recognized HIV-1BRU. Two of the panels were also stimulated by peptides based on clinical isolates from one patient with some sequence changes enhancing T-cell recognition., Conclusions: These data are consistent with highly diverse CD4 T-cell recognition of the HIV-1 gp120 V3 loop, which is influenced by the sequence differences within the T-cell epitopic region and has implications for the pathogenesis and design of vaccines against HIV-1.

The first human foamy virus (HFV) to be described was isolated from nasopharyngeal carcinoma tissue from a Kenyan patient. Early seroepidemiology concluded that there was a significant infection rate, particularly among Africans. Awareness of foamy viruses as potential vectors has stimulated interest in the natural seroprevalence of HFV infection. We, therefore, investigated the prevalence of HFV infection in more than 5000 human sera collected from diverse populations. To maximize the chances of including the major antigenic epitopes, recombinant proteins derived from the HFV gag and env genes divided into three (the 5' amino terminal, the 3' carboxy terminal, and an internal overlapping region) were used as antigens in an ELISA. In contrast to most other seroepidemiological investigations of HFV infection, highly reactive sera identified by ELISA were subjected to further analysis by additional serological assays and, where PBMCs were available, PCR. None of the serum samples were confirmed as positive. It is worth noting that with our ELISA, the highest level of serum reactivity to HFV was found in subjects from Pacific islands (17%), and in Central Africa (34% in Malawi), areas previously cited as having a high level of HFV infection. Taken together with sequence analysis endorsing the phylogenetic closeness of HFV to SFV-6/7, these data strongly suggest that HFV is not naturally found in the human population.

In studies of the natural history of human immunodeficiency virus type 1 (HIV-1) infection, it has been repeatedly shown that higher-titer antibody responses to the HIV gag p24 protein correlate with less rapid disease progression. In HIV-negative persons, immunization with HIV-1 p17/p24:Ty virus-like particles (p24-VLP) induced humoral and cellular immune responses to p24. This construct was therefore studied as a potential immunotherapeutic agent with the objective of augmenting the immune response to p24 in a double-blind placebo-controlled trial involving 74 p24 antibody-positive, asymptomatic HIV-1-infected subjects with CD4 cell counts > 350/mm3. Immunization with p24-VLP was generally well tolerated. Immunization with p24-VLP did not increase p24 antibody levels and had no effect on CD4 cell counts or virus load. The failure to increase p24 antibody titers cannot entirely be explained by the subjects' immunodeficiency because most generated an antibody response to Ty, a yeast component of the immunogen.

Aims: To develop an indirect ELISA using the heat shock protein (hsp60) of Chlamydia trachomatis as antigen., Methods: The hsp60 gene was amplified by PCR, expressed in the vector pDEV-107 and transformed into Escherichia coli. The recombinant protein, expressed as a beta-galactosidase fusion product, was captured onto a solid phase using a monoclonal antibody directed against beta-galactosidase. Following incubation with goat anti-human antibody conjugated to peroxidase and colour development on addition of peroxidase substrate, antibody recognition of antigen was quantified by optical density at 492 nm., Results: A sensitive and relatively specific ELISA to detect hsp60 has been produced, which can be exploited to determine the antibody response to C trachomatis hsp60., Conclusions: This assay will permit the future investigation of the immunopathogenesis of persistent inflammation following C trachomatis infection.

A retrospective review of all 248 liver biopsies performed in patients with HIV infection at two referral centres in London over a 12 year period revealed five cases of major bleeding following biopsy, with four deaths. The risk of major bleeding was 2.0%, and mortality was 1.6% following liver biopsy. The risk of bleeding as much higher than in published series of biopsies done in patients without HIV infection, owing in part to the high prevalence of thrombocytopaenia and clotting abnormalities in patients with HIV infection. HIV infection per se may also increase the risk of bleeding following liver biopsy.

We evaluated saquinavir, an orally active, selective inhibitor of HIV proteinase, in a randomised, double-blind, dose-ranging study in 49 zidovudine-naive HIV-positive patients with few or no symptoms and CD4 cell counts of 500 or less. The study was designed to assess the antiviral activity and tolerability of saquinavir. Patients were randomised to receive 25, 75, 200, or 600 mg of saquinavir three times daily for 16 weeks. No serious adverse events occurred. CD4 cell counts showed a trend indicative of a dose response in favour of the 600 mg dosage, the maximum increase being seen around week 4. In none of the 8 patients with positive plasma viraemia at baseline did cultures become negative after treatment; peripheral blood mononuclear cell and plasma-viral load by culture and DNA and RNA PCR all showed a trend towards reduction at higher doses of saquinovir. Saquinavir was well tolerated in this group of previously untreated patients with few or no symptoms; this study shows that an HIV-proteinase inhibitor is active in HIV-infected patients.

1. Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1. Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested. 2. In saturation binding studies, [3H]-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells. It bound to other human T-cell lines in a similar manner. 3. There was very little binding of [3H]-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of [3H]-D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of [3H]-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells. The Kd of binding did not change significantly during the course of these experiments.4. [3H]-D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h.5. These results suggest that there is a relationship between the expression of the [3H]-D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV- 1.

A simple and rapid assay for the quantification of infectious HIV-1 in plasma was developed using short-term culture and DNA PCR. This method, called culture PCR, allows detection and quantification of infectious HIV-1 viraemia within 48 hours, and measures the number of infectious cell-free HIV-1 particles, expressed as culture PCR infectious doses (CPID/ml). 42 HIV infected subjects were assessed by this method. The titres obtained by CPID closely correlated with CD4+ count and clinical status. CPID titres had significant correlation with infectious virus titre determined by conventional limiting dilution tissue-culture methods. This culture-PCR technique permits rapid assessment of infectious plasma viraemia, and is comparable to longer culture based assay methods.

Objective: To investigate the HIV-1 V3 sequence diversity in the former Soviet Union in 30 subjects infected with HIV-1 via different modes of transmission., Patients: A cohort of children infected after exposure to nonsterile needles during the epidemic in 1988-1989 in southern Russia (Elista, n = 12 and Rostov-on-Don, n = 10), and eight HIV-seropositive subjects from Belarus (Minsk), infected via sexual (n = 7) and parenteral (n = 1) infection., Methods: The HIV-1 V3 encoding region was amplified by nested polymerase chain reaction on DNA of primary peripheral blood mononuclear cells collected from the study subjects and then cloned and sequenced., Results: The alignment of 127 V3 sequences from 22 patients in the cohort group demonstrated common consensus sequences in both the Elista and Rostov samples. The average means of interperson variation were 5.9 and 6.6% in Elista and Rostov subjects, respectively, and comparable to the mean intraperson variation. The average mean interperson variation between nucleotide sequences of HIV patients infected through sexual transmission was considerably higher (14.9%)., Conclusion: V3 sequence analysis confirms the epidemiologic data which support the transmission of HIV-1 in children from a single source, and suggests the infection of a mother from her parenterally infected child. Furthermore, the genetic variability of HIV-1 V3 in the noncohort group was particularly divergent indicating the heterogeneity of the virus circulating in the former Soviet Union.

Objective: To describe patterns of attendance for follow-up among HIV infected women in Parkside, UK and their correlates., Design: Retrospective cohort study., Subjects: 103 HIV infected women., Main Outcome Measures: Whether patients attended for follow-up between three and 18 months., Results: 31% of women were married and 46% had children. Women born in sub-Saharan Africa were significantly less likely to attend for follow-up after three months (56%) than women born in other areas who had acquired HIV either heterosexually (82%) or through injecting drug use (81%). This pattern persisted on multivariate analysis controlling for whether women were symptomatic, had had a previous positive test, were married or had children., Conclusions: HIV positive sub-Saharan African women are less likely to reattend for follow-up than women with heterosexually acquired HIV from other areas or those who acquired infection through intravenous drug use. Further studies are needed to identify barriers to follow-up for women and to shape the development of more appropriate and accessible services for HIV infected women, especially those of sub-Saharan African origin.

Serologic V3 loop peptide-binding assays have been used to predict divergent human immunodeficiency virus type 1 (HIV-1) strains from the Commonwealth of Independent States (former Soviet Union) that have been subsequently confirmed by sequencing of the V3 region. Initial screening was done by MN V3 peptide binding; 12 parenterally infected HIV-1-positive subjects from Elista and Rostov (group 1) with low-titer MN binding and 6 heterosexually infected HIV-1-positive adults from Byelorussia (group 2) with high-titer MN binding were selected. A consensus sequence from the Elista and Rostov areas was generated; a corresponding 14-mer peptide was synthesized and used in an indirect ELISA to screen sera from 392 individuals from diverse geographic areas. Reactivity to the consensus peptide was 82% in subjects from the homologous areas and 11%-38% in other areas. Antibody binding to a panel of synthetic V3 peptides may be used to predict the presence of diverse strains of HIV-1 within virally heterogenous populations.

Objectives: To determine the relationship between infectious virus titre and proviral copy number in peripheral blood mononuclear cells (PBMC) of infected subjects and to ascertain which, if either, is most closely related to CD4+ cell loss and disease progression., Design and Methods: Cellular HIV-1 viraemia was quantified in 45 infected subjects who had not received antiretroviral therapy using limiting dilution tissue culture infective dose (PBMC TCID) and quantitative polymerase chain reaction (PCR) techniques., Results: Proviral DNA was detected in 44 (98%) and infectious virus in 38 (82%) of the 45 subjects. Viraemia as measured by both culture and PCR was inversely correlated with patient CD4+ cell count and associated with disease status. Measurement using both techniques correlated with each other (Spearman's rank correlation rho = 0.52; P = 0.0006). The ratio of proviral copies to PBMC TCID ranged from 1:1 1000:1. to > 1000:1., Conclusions: The ratio of provirus:PBMC TCID was highest when the PBMC TCID was low, and approached unity when PBMC TCID was high. This ratio could be influenced by a variety of factors but did not correlate significantly with patient disease status or CD4+ cell count.

Mycoplasmas have been suggested as a co-factor to explain various puzzling features of infection by human immunodeficiency virus 1 (HIV-1). We sought Mycoplasma fermentans by means of a semi-nested polymerase chain reaction (PCR) in samples of peripheral-blood mononuclear cells (PBMC), throat swabs, and urine samples from 117 HIV-seropositive patients (of whom 114 were homosexual men). M fermentans was detected in 12 (10%) PBMC samples, 15 (23%) of 65 throat samples, and 4 (8%) of 55 urine samples from the seropositive subjects. The organism was detected in similar proportions among 73 HIV-seronegative patients recruited from a sexually transmitted diseases clinic (9%, 20%, and 6%, respectively); again, most of the men (40 of 50) in this group were homosexual. We found no association between infection by the mycoplasma and stage of disease, CD4 count, or HIV-1 load. These findings do not, however, eliminate the possibility that the mycoplasmal infection could affect the speed of disease progression.

Objective: To study the induction of group-specific (gs) neutralizing antibodies to HIV-1 after seroconversion., Design and Methods: Serum samples taken sequentially from seven Dutch homosexual men and four British haemophiliacs (anonymous sample, therefore sex not known) before and after seroconversion were tested for neutralizing antibodies effective against five diverse HIV-1 strains. Strains of HIV-1 tested included isolates from the United States, Europe and Africa., Results: The gs neutralizing antibody response varied between individuals. Only five of the 11 individuals studied produced detectable neutralizing antibodies to laboratory-adapted HIV-1 strains (for example, IIIB) within 32 weeks of seroconversion. Most individuals initially produced antibodies effective against US/European isolates; the response then generally broadened to include the more diverse strains, i.e., African., Conclusions: These results suggest that the gs neutralizing target for HIV-1 is poorly immunogenic in vivo and is probably not highly conserved among diverse HIV-1 strains.

A cohort of homosexual men at risk for human immunodeficiency virus (HIV) infection were studied prospectively over a 7-year period (1982/88) to assess trends in sexual behaviour and amyl nitrite intake. During the period, there were dramatic declines in the proportion of HIV seropositive and seronegative subjects reporting multiple casual partners for anal intercourse, unprotected anal intercourse and recreational use of amyl nitrite. Reported rates of orogenital intercourse remained the same during the period, whilst the total number of seroconversions fell from 17 for the period 1982-84 to 8 for 1985-88. High-risk sexual and related social behaviour among homosexual men, as assessed by patterns of anal intercourse behaviour and nitrite intake, changed over the 7-year period, with the greatest changes apparent before the widespread availability of HIV antibody testing and public education campaigns. This highlights the effectiveness of peer-group and community-based programmes in modifying the sexual behaviour of their members.