Marion Cornelissen

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

Triple HIV-1 infection.

To the Editor: Dual infection with different strains of HIV type 1 (HIV-1) is reported with increasing frequency, attributed mostly to coinfection at the time of the primary infection. However, some patients were superinfected with a second virus after the original seroconversion,1 which generally accelerated disease progression.2 We encountered a case of serial HIV-1 superinfection resulting in a triple infection in a Dutch patient who was originally infected with a subtype B virus. A 35-year-old homosexual man was found to be HIV-1–seropositive in March 2001 and was referred for follow-up. Early in July 2003, the patient presented with acute onset .xa0.xa0.

Simultaneous introduction of distinct HIV-1 subtypes into different risk groups in Russia Byelorussia and Lithuania.

Objective: To investigate genotypes and serotypes of HIV‐1 variants in Russia, Byelorussia and Lithuania. Patients and methods: Sera from 20 HIV‐1‐infected individuals were tested in an enzyme‐linked immunosorbent assay (ELISA) with 19 V3 synthetic peptides, and serum HIV‐1 V3 RNA was amplified and sequenced. Results: Sequence comparison of the envelope V3 region among specimens tested revealed a 2‐29% range of nucleotide divergence, with a mean of 19%. Phylogenetic analysis clustered the V3 sequences recovered with subtypes A, B, C, D and G. All sequences from the homosexual men were shown to belong to subtype B, and all of the heterosexually infected individuals to subtype C. Sequences from the parenterally infected individuals were more heterogeneous. In the peptide ELISA three reactivity patterns were found. Serum samples from six out of seven homosexual men showed reactivity to peptides p108 or p110 representing V3 amino‐acid sequences found in US/West European HIV‐1 isolates. Serum samples from six out of seven individuals who had acquired HIV‐1 through heterosexual contacts were reactive to peptide p169. Four out of six parenterally infected patients had peak reactivity to p168. Conclusion: Distinct HIV‐1 variants were found in Russia, Byelorussia and Lithuania, which were introduced simultaneously in the mid‐1980s. This diversity was shown to be associated with the route of transmission. Homosexual men appeared to be infected with subtype B and heterosexually infected individuals with subtype C HIV‐1 variants. HIV‐1 subtypes A, C, D and G were found among parenterally infected individuals. AIDS 1995, 9:435‐439

CONSISTENT RISK GROUP-ASSOCIATED DIFFERENCES IN HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 VPR, VPU AND V3 SEQUENCES DESPITE INDEPENDENT EVOLUTION

Human immunodeficiency virus 1 type vpr, vpu and V3 sequences from 15 homosexual men and 19 intravenous drug users in the Amsterdam Cohort studies were analysed. Previously, we reported that V3 domains of viruses from drug users are distinguishable from those of homosexual men on the basis of two silent mutations. Phylogenetic analysis of vpr, vpu and V3 shows that differences in all three regions correlate with risk group. Two positions in both vpr and vpu were found to differ significantly between the risk groups. The distinguishing positions were confirmed for sequences from 11 Scottish and four German samples. The three regions show relatively independent evolution patterns; they resulted in different phylogenies, the only stable clustering being that based on the risk group distinction. Pairwise differences between sequences of the genes were moderately correlated (around 0.30). Surprisingly, when only silent changes are counted, the correlations dropped almost to zero, indicating that the evolution towards independence was more advanced in the silent than in the non-silent positions. This suggests that selection at the amino acid level is not the primary driving force for the independent evolutionary behaviour of the genes. Recombination, combined with restrictions on certain amino acids because of epistatic interactions between the genes, could be an alternative explanation of this phenomenon.

Analysis of human immunodeficiency virus type 1 LTR-LTR junctions in peripheral blood mononuclear cells of infected individuals.

Circularized DNA species containing two long terminal repeat circle junctions were analysed in peripheral blood mononuclear cells of human immunodeficiency virus type 1 (HIV-1)-infected individuals. The circle junction fragments found could be classified into four groups: fragments containing a normal circle junction, fragments with deletions at the circle junction, fragments containing the primer binding site inserted at the circle junction, and fragments containing insertions at the circle junction derived from other regions of the HIV-1 genome.

Virus load in chimpanzees infected with human immunodeficiency virus type 1: effect of pre-exposure vaccination.

Many reports indicate that a long-term asymptomatic state following human immunodeficiency virus type 1 (HIV-1) infection is associated with a low amount of circulating virus. To evaluate the possible effect of stabilizing a low virus load by non-sterilizing pre-exposure vaccination, a quantitative virus isolation method was developed and evaluated in four chronically infected chimpanzees infected with a variety of HIV-1 related isolates. This assay was then used to monitor a group of chimpanzees (n = 6) challenged with HIV-1 following vaccination with gp120 or gp160. Data indicated that of the three vaccinated animals which became infected after challenge, the animal with the lowest neutralizing titre at the time of challenge acquired a virus load similar to the control animals, whereas the two other chimpanzees had reduced numbers of virus producing cells in their peripheral circulation. One animal became virus isolation negative, developed an indeterminant PCR signal on lymph node DNA and subsequently became negative for HIV-1 DNA as determined by PCR on PBMC (peripheral blood mononuclear cells) and bone marrow DNA. Recently, the second animal has also become PCR negative. To confirm observations from quantitative virus isolations, quantification of HIV-1 DNA in PBMC and virus RNA in serum was performed by PCR on serially diluted samples at two different time points. Comparison of virus load as determined by these three methods confirmed that there was an effect of vaccination in reducing virus load and demonstrated a correlation between decreased numbers of virus producing cells, HIV-1 DNA containing cells and virus RNA molecules in serum.

Primary effect of chemotherapy on the transcription profile of AIDS-related Kaposi's sarcoma

BackgroundDrugs & used in anticancer chemotherapy have severe effects upon the cellular transcription and replication machinery. From in vitro studies it has become clear that these drugs can affect specific genes, as well as have an effect upon the total transcriptome.MethodsTotal mRNA from two skin lesions from a single AIDS-KS patient was analyzed with the SAGE (Serial Analysis of Gene Expression) technique to assess changes in the transcriptome induced by chemotherapy. SAGE libraries were constructed from material obtained 24 (KS-24) and 48 (KS-48) hrs after combination therapy with bleomycin, doxorubicin and vincristine. KS-24 and KS-48 were compared to SAGE libraries of untreated AIDS-KS, and to libraries generated from normal skin and from isolated CD4+ T-cells, using the programs USAGE and HTM. SAGE libraries were also compared with the SAGEmap database.ResultsIn order to assess the primary response of AIDS-related Kaposis sarcoma (AIDS-KS) to chemotherapy in vivo, we analyzed the transcriptome of AIDS-KS skin lesions from a HIV-1 seropositive patient at two time points after therapy. The mRNA profile was found to have changed dramatically within 24 hours after drug treatment. There was an almost complete absence of transcripts highly expressed in AIDS-KS, probably due to a transcription block. Analysis of KS-24 suggested that mRNA pool used in its construction originated from poly(A) binding protein (PABP) mRNP complexes, which are probably located in nuclear structures known as interchromatin granule clusters (IGCs). IGCs are known to fuse after transcription inhibition, probably affecting poly(A)+RNA distribution.Forty-eight hours after chemotherapy, mRNA isolated from the lesion was largely derived from infiltrating lymphocytes, confirming the transcriptional block in the AIDS-KS tissue.ConclusionsThese in vivo findings indicate that the effect of anti-cancer drugs is likely to be more global than up- or downregulation of specific genes, at least in this single patient with AIDS-KS. The SAGE results obtained 24 hrs after chemotherapy can be most plausibly explained by the isolation of a fraction of more stable poly(A)+RNA.

Clinical Relevance of HIV-1 Superinfection

Over time a single patient can be infected by multiple intraor intersubtype human immunodeficiency viruses type 1 (HIV-1) strains. These so-called dual infections are divided into co-infections and superinfections. HIV-1 co-infection is described as a second infection taking place before measurable HIV-1 antibody production by the immune system (seroconversion) and HIV-1 superinfection is defined as a second infection occurring after seroconversion. Here the focus lays on superinfections, which have implications for HIV-1 transmission, treatment and vaccine development (Gottlieb et al., 2004). Moreover superinfections can give rise to HIV-1 circulating recombinant forms (CRF); this significantly increases the global epidemiology (Gottlieb et al., 2007). Also due to these recombination events, two different drug resistant HIV-1 viruses could lead to multi drug resistance or even to more pathogenic viruses (Gottlieb et al., 2004, Blackard et al., 2004, Fernandez Larrosa et al., 2006). Another important issue that is not extensively surveyed in literature is the clinical relevance of HIV-1 superinfection, as most descriptions rely on case reports and not on controlled cohort studies. The individual cases differ in severity, as several superinfected patients with rapidly progressive HIV-1 disease have been described since 2002, but superinfection cases were also found by coincidence in long-term non-progressors (LTNPs). These patients were able to control HIV-1 disease before and sometimes also after the superinfection event. So it is not resolved yet how superinfection affects disease progression in general and, if any, specific host or viral factors are involved. In this chapter, first an overview will be given of HIV-1 superinfected patients from several studies, with regard to HIV-1 disease progression. Disease progression is indicated by an increase in the HIV-1 plasma viral load, a decrease in the CD4+ T-cell count, acquired immunodeficiency syndrome (AIDS) related events and/or the start of antiretroviral treatment. These outcomes are compared to values of disease progression for single HIV-1 infected patients, resulting in an indication of the clinical relevance of HIV-1 superinfection.

P.027 No changing incidence in HIV-1 dual infections in Amsterdam, The Netherlands, from 2003–2007

Purpose: The occurrence of HIV-1 dual infections in Amsterdam, The Netherlands, was examined from 2003–2007 to investigate whether or not HIV-1 dual infections are increasing as the number of HIV-1 infected individuals increased here over this time period. Methods: All first HIV-1 genotyping sequences determined between 2003 and 2007 were retrieved and examined for the number of degenerate base codes in the RT fragment. From a total of 73 patients with a degenerate base code count of > 33, a fragment of the V3-V4 region of the env gene was amplified, cloned and sequenced to verify the presence of a HIV-1 dual infection. Dual infections were counted for each year investigated. Results: No significant change in incidence of dual infections was seen in our patient population selected upon the degenerate base count in the HIV-1 genotyping sequence from 2003 till 2007. The frequency of HIV-1 dual infections varied between 1.0%–2.8%/year, with no significant trend in time (p = 0.49). Based upon the number of patients with a high degenerate base code count, an increase in dual infections was expected in 2006 and 2007, but this was not observed. HIV-1 dual infections were similar to HIV-1 single infections in The Netherlands in distribution by risk group, gender and subtype. Conclusion: HIV-1 dual infections in The Netherlands are not increasing from 2003 to 2007 although the HIV-1 infected population is expanding in this period.