F. de Wolf

Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection : a composite of redistribution and proliferation

The origin of CD4+ T cells reappearing in the blood following antiretroviral therapy in human immunodeficiency virus type-1 (HIV-1) infection is still controversial. Here we show, using mathematical modeling, that redistribution of T cells to the blood can explain the striking correlation between the initial CD4+ and CD8+ memory T-cell repopulation and the observation that 3 weeks after the start of treatment memory CD4+ T-cell numbers reach a plateau. The increase in CD4+ T cells following therapy most likely is a composite of initial redistribution, accompanied by a continuous slow repopulation with newly produced naive T cells.

Immunological abnormalities in human immunodeficiency virus (HIV)-infected asymptomatic homosexual men. HIV affects the immune system before CD4+ T helper cell depletion occurs.

To investigate the effect of persistent HIV infection on the immune system, we studied leukocyte functions in 14 asymptomatic homosexual men (CDC group II/III) who were at least two years seropositive, but who still had normal numbers of circulating CD4+ T cells. Compared with age-matched heterosexual men and HIV-negative homosexual men, the CD4+ and CD8+ T cells from seropositive men showed decreased proliferation to anti-CD3 monoclonal antibody and decreased CD4+ T-helper activity on PWM-driven differentiation of normal donor B cells. Monocytes of HIV-infected homosexual men showed decreased accessory function on normal T cell proliferation induced by CD3 monoclonal antibody. The most striking defect in leukocyte functional activities was observed in the B cells of HIV-infected men. B cells of 13 out of 14 seropositive men failed to produce Ig in response to PWM in the presence of adequate allogeneic T-helper activity. These findings suggest that HIV induces severe immunological abnormalities in T cells, B cells, and antigen-presenting cells early in infection before CD4+ T cell numbers start to decline. Impaired immunological function in subclinically HIV-infected patients may have clinical implications for vaccination strategies, in particular the use of live vaccines in groups with a high prevalence of HIV seropositivity.

ASSOCIATION BETWEEN BIOLOGICAL PROPERTIES OF HUMAN IMMUNODEFICIENCY VIRUS VARIANTS AND RISK FOR AIDS AND AIDS MORTALITY

49 individuals seropositive for human immunodeficiency virus (HIV) antibody were studied longitudinally for the relation between in-vitro properties of their sequential HIV isolates and clinical course before and after the development of AIDS. They were classified into three groups according to the syncytium-inducing capacity, replication rate, and host range of their HIV isolates. The most rapid progression to AIDS (median 15 months) and the lowest survival rate following AIDS diagnosis (median survival 12.5 months) were observed in individuals with high-replicating, syncytium-inducing HIV isolates, followed by individuals with high-replicating, non-syncytium-inducing isolates. In contrast, most individuals with low-replicating, non-syncytium-inducing HIV isolates remained symptom-free during the study period (median follow-up until AIDS diagnosis greater than 42 months), and the few individuals from this group in whom AIDS developed were still alive at the end of the study period (median survival greater than 34 months). In addition, AIDS patients from the three groups differed with respect to their symptoms. Zidovudine treatment in the symptom-free period seemed to delay the onset of AIDS in all risk groups, although stabilisation of CD4+ cell numbers was observed only in individuals with non-syncytium-inducing HIV variants.

Persistent HIV antigenaemia and decline of HIV core antibodies associated with transition to AIDS.

Sequential serum samples from 13 homosexual men who seroconverted for antibodies to human immunodeficiency virus (HIV) were tested for HIV antigen. In one of these men, who developed the acquired immune deficiency syndrome (AIDS), HIV antigenaemia preceded the onset of AIDS by more than a year and persisted throughout the course of the disease. This antigenaemia was accompanied by the disappearance of IgG antibody reactivity to the major HIV core protein p24. In none of the 12 others, who all remained without serious disease, were serum concentrations of HIV antigen detected, except on one occasion in one man. All their serum samples showed strong IgG antibody reactivity to p24. Nine children who were infected with HIV in 1981 by plasma transfusion from a single donor were also followed up for HIV antigenaemia. HIV antigen was almost constantly present in the serum (26 of 28 samples) of five children who developed AIDS related complex or AIDS and less often in the serum (four of 10 samples) of four children who remained free of symptoms. The two children who developed AIDS showed a virtual absence of antibody reactivity to p24. These results indicate that increased HIV gene expression is a contributing factor to the development of AIDS and also provide evidence for a switch from latent to active HIV infection.

Association between CCR5 genotype and the clinical course of HIV-1 infection.

Viral, immune, and host genetic factors may influence the clinical course of HIV-1 infection. High viral load [1, 2], presence of syncytium-inducing HIV-1 [3-5], low T-lymphocyte function [6], and certain HLA types [7, 8] have been associated with rapid disease progression [9]. Several coreceptors for HIV-1 have recently been identified. Syncytium-inducing, T-cell line-adapted HIV-1 variants use the C-X-C chemokine receptor 4, macrophagetropic variants use the C-C chemokine receptor 5 (CCR5), and primary syncytium-inducing viruses can use both [10-16]. Persons who have been exposed to HIV-1 on multiple occasions but remain uninfected seem to be homozygous for a 32-nucleotide deletion (delta32) in the CCR5 gene [17, 18]; this concurs with the idea that macrophage-tropic HIV-1 variants establish new infections [19, 20]. In vitro, HIV-1 replication in cells that were heterozygous for CCR5 delta32 was reduced compared with the level of HIV-1 replication in wild-type cells [18]. Several cohort studies [17, 21-24] have shown a substantial correlation between CCR5 delta32 heterozygosity and delayed disease progression. To further substantiate this finding and to examine the biological principle underlying the protection offered by CCR5 delta32 heterozygosity, we analyzed the role of CCR5 genotype alone and in relation to established progression markers in the clinical course of HIV-1 infection in participants from the Amsterdam Cohort Studies. Methods Study Sample Between October 1984 and March 1986, 961 asymptomatic men who were living in the Amsterdam area and who reported having had at least two homosexual contacts in the preceding 6 months were enrolled in a prospective study on the prevalence and incidence of HIV-1 infection and risk factors for AIDS [25]. In the first serum sample taken, 728 men tested negative for HIV-1 antibodies; 131 of these men underwent seroconversion during the study. The remaining 238 men were positive for HIV antibodies; 5 of these men refused to participate further. Enrollment of seropositive persons was stopped after 6 months (in April 1985). Epidemiologic studies on the incidence of HIV-1 infection [26] showed that infection in seroprevalent homosexual men must have occurred an average of 1.5 years before entry into the Amsterdam Cohort Studies. Therefore, the time of seroconversion for seroprevalent men was set at 1.5 years before study entry. No differences in AIDS-free survival were found between persons who underwent seroconversion during the study and seroprevalent persons by using Kaplan-Meier (P > 0.2) and Cox proportional-hazard analyses in which the development of AIDS was the end point criterion (relative hazard, 1.17 for persons who had seroconversion compared with seroprevalent persons [95% CI, 0.84 to 1.63]). This result suggests a good estimation of the seroconversion date in the latter group. When we restricted our analyses to persons who had seroconversion, relative hazards were similar but less precise than estimates for the group as a whole. Therefore, we used 131 persons who had seroconversion and 233 seroprevalent persons as one study sample. Every 3 months, clinical and epidemiologic data were collected and serum and peripheral blood mononuclear cells were cryopreserved. Most seropositive men (n = 242 [66%]) did not receive early treatment. The remaining 122 men (34%) received zidovudine (70 [19%]), didanosine 10 [3%]), or other antiretroviral therapy (42 [12%]) before AIDS was diagnosed. None of the men received a combination of more than two antiretroviral drugs during our study. The mean age of participants at the time of seroconversion was 34.5 years (range, 19.5 to 57.7 years). By 1 January 1996 (the censor date), 189 men had developed AIDS according to the 1987 definition of AIDS [27] (median follow-up, 5.9 years [range, 0.6 to 12.3 years]), 94 men had not developed AIDS (median follow-up, 10.1 years [range, 0.3 to 13.7 years]), and 81 men were lost to follow-up (median follow-up, 2.0 years [range, 0.6 to 12.5 years]). A nested casecontrol study done using the same group of participants from the Amsterdam Cohort Studies was designed to identify factors that may be correlated with long-term survival. Long-term survivors (n = 23) remained free of clinical diseases for at least 9 years, with a mean CD4+ T-lymphocyte count of more than 400 cells/mm3 in the eighth and ninth year of HIV-1-positive follow-up (median follow-up, 10.8 years [range, 9.1 to 11.1 years]; mean CD4+ T-lymphocyte counts in the ninth year of follow-up, 534 cells/mm3 [range, 408 to 953 cells/mm3]). Each long-term survivor was matched with two progressors (men who developed AIDS after 2 to 7 years of HIV-1-positive follow-up). Matching was based on mean CD4+ T-lymphocyte count ( 250 cells/mm3) in year 2 of HIV-positive follow-up, HIV-1 serostatus at entry in the cohort study, and age ( 10 years). Use of Polymerase Chain Reaction for CCR5 Genotyping Samples of DNA were available for CCR5 genotyping for 343 of 364 men (94%). Genomic DNA was isolated from cryopreserved peripheral blood mononuclear cells (Qiagen blood kit, Qiagen, Hilden, Germany) and 100 mg of DNA was analyzed by using polymerase chain reaction (PCR) with primers (sense, position 612 to 635 in CCR5, 5-GATAGGTACCTGGCTGTCGTCCAT-3; antisense, position 829 to 850 in CCR5, 5-AGATAGTCATCTTGGGGCTGGT-3) flanking the described 32-nucleotide deletion in the CCR5 gene [17, 18]. Samples were amplified with 1 unit of Taq polymerase (Promega, Madison, Wisconsin) in the provided buffer with a final MgCl2 concentration of 3 mmol/L. Conditions of PCR comprised 5 minutes of denaturation at 95C; 30 cycles of 1 minute at 95C, 1 minute at 56C, and 2 minutes at 72C; and 5 minutes of elongation at 72C in a Perkin Elmer Cetus DNA thermal cycler 480 (Perkin Elmer, Foster City, California). Products of PCR were analyzed by using 2% agarose gel electrophoresis and ethidium bromide staining. Five randomly chosen samples with a reduced product size revealed the described 32-base pair deletion on automatic DNA sequencing (data not shown) [17, 18]. Virologic Assays Cocultivation of HIV-1-positive peripheral blood mononuclear cells with MT2 cells was performed every 3 months to detect syncytium-inducing HIV-1 variants [28, 29]. Serum viral load was measured by using a quantitative HIV-1 RNA nucleic acid-based sequence amplification (Organon Teknika, Boxtel, the Netherlands) with electrochemiluminescent labeled probes [30]. Serum samples obtained approximately 2 years after seroconversion (1 year after seroconversion; mean time point, 2.3 years [range, 1.5 to 3.0 years]) were available for measurement of HIV-1 RNA viral load for 335 of 364 participants (92%). Serum levels of HIV-1 RNA were analyzed after log10 transformation. Numbers of RNA copies that were below the test threshold of quantification were arbitrarily set at 10 (3).0 copies/mL. Immunologic Assays Antibodies to HIV-1 were detected in serum by using a commercial recombinant HIV-1/-2 enzyme immunoassay (Abbott, Chicago, Illinois) and were confirmed with an HIV-1 Western blot IgG assay (version 1.2, Diagnostic Biotechnology Ltd., Singapore, Thailand). Enumeration of CD4+ and CD8+ T lymphocytes was done by using flow cytofluorometry. For seroprevalent persons for whom we estimated the time of seroconversion to have been 18 months before entry into the cohort study, CD4+ T-lymphocyte count was first measured 18 months after the estimated time of seroconversion. Beginning in January 1988, reactivity of T lymphocytes in response to stimulation with CD3 monoclonal antibodies in vitro was routinely determined in whole-blood cultures [31]. The proliferative response measured after 4 days of culture by incorporation of [3H] thymidine was expressed as a percentage of the median values of the responses measured in two to five healthy controls tested on the same day. Statistical Analysis The Fisher exact test was used to compare HIV-1-seronegative participants with HIV-1-seropositive participants for CCR5 genotype distributions. In the casecontrol study, conditional logistic regression was performed to estimate the chance that a CCR5 delta32 heterozygote would be a long-term survivor. The Mann-Whitney U test was used to compare CCR5 delta32 heterozygotes and CCR5 wild-type homozygotes. For each participant, the slope of the decrease in CD4+ T lymphocytes was determined separately by fitting a simple regression line to his CD4+ T-lymphocyte count. At least three CD4+ T-lymphocyte counts had to be available for analysis; this was the case for 66 (97%) of the 68 CCR5 delta32 heterozygotes and 250 (91%) of the 275 CCR5 wild-type homozygotes. A Kaplan-Meier analysis was used to estimate the cumulative incidence of conversion to syncytium-inducing HIV-1 variants in relation to CCR5 genotype. We also estimated the duration of AIDS-free survival in relation to CCR5 genotype for the period during which only non-syncytium-inducing variants were present (conversion to syncytium-inducing HIV-1 was used as a censor criterion) or for the period after conversion to syncytium-inducing HIV-1 variants. A Kaplan-Meier analysis and a Cox proportional-hazards analysis were used to study the predictive value of CCR5 genotype alone or in combination with serum viral RNA load, CD4+ T-lymphocyte count, T-lymphocyte function, and syncytium-inducing phenotype. We evaluated the predictive value of the markers by fitting separate Cox models at 2, 4, 6, and 8 years after seroconversion. Participants were at risk from each specific time point; this method excluded participants who had previously developed AIDS. Because data on HIV-1 RNA load were available approximately 2 years after seroconversion only, data on viral load were not included in the models at 4, 6, and 8 years after seroconversion. All markers were also analyzed as time-dependent covariates. Participants who did not have AIDS were censored at 1 January 1996. Significance in

Late presentation of HIV infection: a consensus definition

Objectives Across Europe, almost a third of individuals infected with HIV do not enter health care until late in the course of their infection. Surveillance to identify the extent to which late presentation occurs remains inadequate across Europe and is further complicated by the lack of a common clinical definition of late presentation. The objective of this article is to present a consensus definition of late presentation of HIV infection.

Human immunodeficiency virus type 1 Rev- and Tat-specific cytotoxic T lymphocyte frequencies inversely correlate with rapid progression to AIDS

Immunological correlates of AIDS-free survival after human immunodeficiency virus type 1 (HIV-1) infection are largely unknown. Cytotoxic T lymphocyte (CTL) responses are generally believed to be a major component of protective immunity against viral infections. However, the relationship between HIV-1-specific CTL responses and disease progression rate is presently unclear. Here we show in twelve HIV-1-infected individuals that detection of Rev-specific CTL precursors (CTLp) early in the asymptomatic stage, as well as detection of Rev- and Tat-specific CTLp later during follow-up, inversely correlate with rapid disease progression. No such correlation was found for detection of CTLp against Gag, RT or Nef. Further studies are required to determine whether a protective mechanism is indeed the basis of the observed correlation. The data presented are in agreement with the hypothesis that CTL against proteins that are important for early viral transcription and translation are of particular importance in protection from rapid disease progression.

HIV treatment response and prognosis in Europe and North America in the first decade of highly active antiretroviral therapy: a collaborative analysis.

BACKGROUND Highly active antiretroviral therapy (HAART) for the treatment of HIV infection was introduced a decade ago. We aimed to examine trends in the characteristics of patients starting HAART in Europe and North America, and their treatment response and short-term prognosis. METHODS We analysed data from 22,217 treatment-naive HIV-1-infected adults who had started HAART and were followed up in one of 12 cohort studies. The probability of reaching 500 or less HIV-1 RNA copies per mL by 6 months, and the change in CD4 cell counts, were analysed for patients starting HAART in 1995-96, 1997, 1998, 1999, 2000, 2001, and 2002-03. The primary endpoints were the hazard ratios for AIDS and for death from all causes in the first year of HAART, which were estimated using Cox regression. RESULTS The proportion of heterosexually infected patients increased from 20% in 1995-96 to 47% in 2002-03, and the proportion of women from 16% to 32%. The median CD4 cell count when starting HAART increased from 170 cells per muL in 1995-96 to 269 cells per muL in 1998 but then decreased to around 200 cells per muL. In 1995-96, 58% achieved HIV-1 RNA of 500 copies per mL or less by 6 months compared with 83% in 2002-03. Compared with 1998, adjusted hazard ratios for AIDS were 1.07 (95% CI 0.84-1.36) in 1995-96 and 1.35 (1.06-1.71) in 2002-03. Corresponding figures for death were 0.87 (0.56-1.36) and 0.96 (0.61-1.51). INTERPRETATION Virological response after starting HAART improved over calendar years, but such improvement has not translated into a decrease in mortality.

Decrease of HIV-1 RNA levels in lymphoid tissue and peripheral blood during treatment with ritonavir, lamivudine and zidovudine

Objectives: Triple combination treatment of HIV-1 infection using two reverse transcriptase inhibitors and a protease inhibitor can result in significant and sustained decreases in the quantity of viral RNA in peripheral blood. Lymphoid tissue, however, constitutes the major reservoir of HIV in infected patients. Study of the viral burden in these tissues has provided additional insight in the efficacy of antiretroviral treatment. Design: Patients were randomized into two groups in order to study differences in the development of resistance to reverse transcriptase inhibitors. Group I started treatment with all three drugs simultaneously. Group II started with ritonavir monotherapy, aiming at initial reduction in virus production before the addition of lamivudine and zidovudine 3 weeks later. Methods: Changes in the amount of HIV in plasma and tonsillar lymphoid tissue during 24 weeks of treatment with ritonavir, lamivudine and zidovudine were studied by reverse transcriptase polymerase chain reaction. Results: Thirty-three antiretroviral-naive HIV-infected patients were included for analysis. After 24 weeks, median CD4+ cell count increased by 152 × 106/l and median plasma viral RNA levels decreased by at least 2.87 log10 copies/ml. In 88% of the patients remaining on treatment, plasma RNA levels were below the quantification limit of the assay used (mean, 2.4 log10 copies/ml). The lymphoid tissue viral burden, ranging from 9.16 to 8.52 log10 copies/g at baseline, was markedly reduced with at least 2.1 log10 copies/g by week 24 in the five patients analysed. Eight patients (24%) withdrew because of side-effects. In one patient in group II, ritonavir and lamivudine resistance-associated mutations developed. Conclusions: Treatment with this triple antiretroviral drug combination produced a durable and strong decrease of HIV-1 RNA burden in both plasma and lymphoid tissue.

Improvement of chronic diarrhoea in patients with advanced HIV-1 infection during potent antiretroviral therapy

Background:A substantial number of patients with advanced HIV infection suffer from intractable diarrhoea. The aim of this study was to evaluate whether potent antiretroviral therapy could alleviate such diarrhoea. Methods:In an open randomized study the effect of the HIV protease inhibitor indinavir in combination with nucleoside analogue reverse transcriptase inhibitors on chronic HIV-related diarrhoea was investigated in 14 late-stage (CD4+ lymphocyte count ≤ 50 × 106 cells/l) HIV-infected patients. Data concerning stool frequency, stool consistency and antidiarrhoeal drug use were collected in daily diaries over a 24-week period. Endpoints of the study were reduction of stool frequency, improvement of stool consistency, weight gain, and in case of diarrhoea due to Enterocytozoon bieneusi or Cryptosporidium sp. disappearance of these parasites from stool. Results:Thirteen patients started the study drug indinavir. One patient died after 1 week and one patient withdrew prematurely after 18 weeks. Median stool frequency declined from 5.8 daily at baseline to 2.3 daily after 24 weeks (P = 0.04). Stool consistency improved considerably over the study period: before treatment 56% of stools were watery and 0% were formed; at week 24 these figures were 0 and 35%, respectively. Body weight increased significantly with a median increment of 6.6 kg at week 24 (P = 0.0006). In two out of six patients with microsporidiosis and both patients with cryptosporidiosis, stools were free of parasites at week 24. Five out of six patients who used non-specific antidiarrhoeal medication on a regular basis prior to the study had ceased to do so at the end. Conclusion:The use of potent antiretroviral therapy in patients with advanced HIV infection can improve chronic HIV-related diarrhoea and in some cases lead to disappearance of E. bieneusi and Cryptosporidium sp. from the stools.