Nicole K. T. Back

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV‐1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2′,3′‐dideoxy‐3′‐thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild‐type and 3TC‐resistant HIV‐1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild‐type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC‐resistant RT mutants produce significantly shorter cDNA molecules than the wild‐type enzyme [Met (wt)>Val>Ile], indicating that 3TC‐resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC‐resistant HIV‐1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC‐treated patients that develop drug‐resistant HIV‐1 variants, this may be of considerable clinical benefit.

Transmission networks of HIV-1 among men having sex with men in the Netherlands

Objective: To obtain insight in the HIV-1 transmission networks among men having sex with men (MSM) in the Netherlands. Design: A phylogenetic tree was constructed from polymerase sequences isolated from 2877 HIV-1 subtype B-infected patients monitored as part of the AIDS Therapy Evaluation in the Netherlands (ATHENA) nationwide observational cohort. Methods: For MSM with a known date of infection, the most similar sequences were selected as potential transmission pairs when they clustered with bootstrap value of at least 99%. Time from infection to onward transmission was estimated as the median time between dates of infection for each transmission pair. The source of infections with a resistant strain was traced using the entire phylogenetic tree. Results: Of sequences from 403 MSM with a known date of infection between 1987 and 2007, 175 (43%) formed 63 clusters. Median time to onward transmission was 1.4 years (interquartile range 0.6–2.7). Twenty-four (6%) MSM carried a virus with resistance-related mutations, 13 of these were in eight clusters together with sequences from 28 other patients in the entire phylogenetic tree. Six clusters contained sequences obtained from 29 men all presenting the same resistance-related mutations. Conclusion: From our selection of likely transmission pairs, we conclude that onward transmission of HIV-1 from infected MSM in the Netherlands happens both during and after primary infection. Transmission of resistant strains from the antiretroviral therapy-treated population is limited, but strains with resistance-related mutations have formed subepidemics.

Genomic diversity and antigenic variation of HIV-1: links between pathogenesis, epidemiology and vaccine development.

Recent analysis of primate lentivirus genomes (1) indicates that lentiviruses have infected primates for hundreds of years. The pathogenicity of such viruses may fluctuate due to the high evolution rate of some parts of the viral genome. Fixed nucleic acid substitutions in the gag gene appear to be caused by random fixation of selectively neutral mutants, whereas nonrandom fixation of selectively advantageous mutants, as has been observed for MHC molecules and serine protease inhibitors, appears to be operational for some hypervariable env gene regions. The former is characterized by an excess of silent mutations independent of the rate of change, the latter by an excess of nonsilent mutations. This latter type of selection may especially characterize the third variable region of the external HIV envelope (V3), which contains the principal neutralization domain.—Goudsmit, J.; Back, N. K. T.; Nara, P. L. Genomic diversity and antigenic variation of HIV‐1: links between pathogenesis, epidemiology, and vaccine development. FASEB J. 5: 2427–2436; 1991.

Multicenter Evaluation of the New Abbott RealTime Assays for Quantitative Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA

ABSTRACT The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Phylogenetic reconstruction of transmission events from individuals with acute HIV infection: toward more-rigorous epidemiological definitions.

Phylogenetic reconstructions of transmission events from individuals with acute human immunodeficiency virus (HIV) infection are conducted to illustrate this groups heightened infectivity. Varied definitions of acute infection and assumptions about observed phylogenetic clusters may produce misleading results. We conducted a phylogenetic analysis of HIV pol sequences from 165 European patients with estimated infection dates and calculated the difference between dates within clusters. Nine phylogenetic clusters were observed. Comparison of dates within clusters revealed that only 2 could have been generated during acute infection. Previous analyses may have incorrectly assigned transmission events to the acutely HIV infected when they were more likely to have occurred during chronic infection.

Neutralizing activity of anti-peptide antibodies against the principal neutralization domain of human immunodeficiency virus type 1

Monoclonal antibodies (MAbs) raised against a 15-mer peptide representing the centre of the principal neutralization domain of human immunodeficiency virus type 1 (strain BH10) showed wide variations in neutralizing activity against the homologous strain. The nature of this difference in neutralizing activity was studied by measuring antibody concentration, their affinity for peptide and specificity, by reaction with peptides which differed in the extent of sequence overlap, length and the presence of single amino acid replacements. All MAbs bound to approximately the same region in the principal neutralization domain, within the sequence RIQRGPGRAFV. The peptides with which each antibody was able to react differed by only a few amino acids. The neutralizing activity of each MAb preparation was related to its affinity and concentration; the affinity is related in part to the fine structure of the epitope recognized. MAbs with high affinity for the peptide tended to react only with peptides in which amino acid replacements did not affect the beta-turn potential of the peptide, whereas the reactivity of MABs with low affinity was relatively insensitive to amino acid replacements affecting the beta-turn potential.

Comparison and fine mapping of both high and low neutralizing monoclonal antibodies against the principal neutralization domain of HIV-1.

SummaryMonoclonal antibodies raised against viral lysate of HIV-1 (strain LAV-1) and against recombinant gp 160 of HIV-1 (strain HTLV IIIB) which neutralized HIV-1 in a type specific manner were mapped with the aid of peptides (Pepscan analysis). Each of these monoclonal antibodies bound to peptides located on the principal neutralizing domain (PND) of HIV-1. We found that the antigenic sites of the MAbs described in this paper are represented by linear peptides of at least 10 amino acids long. The affinity of the MAbs is high for these peptides and in the same order of magnitude as for native gp 160. The fine mapping of the epitopes may reflect structural features of the PND, for instance which amino acid side chains are exposed and which are buried in the protein. Furthermore the fine mapping of the epitopes explained the HIV type-specific neutralizing activity of the MAbs. Antibodies that bound to the tip of the loop (amino acids QRGPGRAF) have a higher neutralizing activity than antibodies that bound to amino acids towards the N-terminal side of the loop (amino acids KSIRI). Furthermore, MAbs that bound to virtually the same amino acids on the tip of the loop (amino acids IQRGPGRAF and RGPGRAFV) had different neutralizing activities due to different affinities for native gp 160. These data reveal that neutralizing activity not only is determined by the affinity of an antibody to the neutralizing site but also by its fine binding specificities to the V 3 loop of gp 120.

Routine HIV-1 genotyping as a tool to identify dual infections

Objectives:The incidence of HIV-1 dual infections is generally thought to be low, but as dual infections have been associated with accelerated disease progression, its recognition is clinically important. Methods to identify HIV-1 dual infections are time consuming and are not routinely performed. Design:Genotyping of the HIV-1 protease and reverse transcriptase (prot/RT) genes is commonly performed in the western world to detect drug-resistance mutations in clinical isolates. In our hospital, prot/RT baseline sequencing is part of the patient care for all newly infected patients in the Amsterdam region since 2003. We reasoned that degenerate base codes in this sequence could indicate either extensive viral evolution or infection with multiple HIV-1 strains. Methods:We amplified, cloned and sequenced multiple HIV-1 envelope (env)-V3 and gag sequences from patients with 34 or more (range 34–99) degenerate base codes in the ViroSeq genotyping RT sequence (37 out of 1661 available records) to estimate the number of HIV-1 dual infections in this group. Results:Of the 37 patients included in this study, 16 (43.2%, equal to 1% of the 1661 total records) had an HIV-1 dual infection based on phylogenetic analysis of env-V3/gag sequences. If only sequences with 45 or more degenerate base codes were taken into account, 73.3% of patients showed evidence of a dual infection. Conclusion:We describe an additional use of routinely performed HIV-1 genotyping. In patients with a high number of degenerate bases (≥ 34) in RT it is important to consider the possibility of a dual HIV-1 infection.

Multiple transmissions of a stable human leucocyte antigen-B27 cytotoxic T-cell-escape strain of HIV-1 in The Netherlands.

Objective:The evolution of HIV-1 is largely shaped by the cytotoxic T-cell (CTL) response of the host as encoded by the human leucocyte antigen (HLA) genes. Certain HLA-B alleles can delay disease progression, but it is uncertain whether this protection will sustain or whether the virus is in the process of adaptation. In The Netherlands, HLA-B27 is moderately prevalent (approximately 8–16% of HLA-B alleles). If adaptation to HLA-B alleles is in progress, virus strains carrying escape mutations to HLA-B27 should appear in the epidemic by now. Design:A subtype B HIV-1 strain carrying a HLA-B27 CTL-escape mutation in the main Gag-p24 KK10 epitope, R264G, together with a compensatory mutation outside this epitope, E260D, was detected in four patients from Amsterdam, The Netherlands, by sequence analysis of the gag gene. The patients were a drug user and three men who have sex with men, and were infected with HIV-1 between 2002 and 2008. Methods:Characterization and evolutionary analysis of the HIV-1 CTL-escape strain was done by sequence analysis of serial blood plasma samples. Results:The mutations involved were stable during follow-up and after transmission, also in two individuals lacking HLA-B27. Conclusion:The finding that a stable HLA-B27 CTL-escape strain is circulating in The Netherlands has important implications for the understanding of virus–host interactions and vaccine design alike. Vaccines targeted at inducing a CTL response might easily be circumvented by the virus. Also, patients carrying protective HLA alleles might not be protected anymore from disease progression in the future.

Ten years of HIV testing with fourth generation assays: The Amsterdam experience

Serological HIV assays combining detection of HIV antigen and antibodies are referred to as fourth generation assays. Fourth generation assays were implemented in Europe for routine patient testing about 10 years ago. The Academic Medical Center is one of the main HIV treatment centers in the Netherlands and has now 10 years experience with fourth generation testing, which is summarized here.