Selected abstracts from Track A presentations at Toronto

9 September 2006. Related: Conference reports, Basic science and immunology, World AIDS 16 Toronto 2006.

Svilen Konov, HIV i-Base

The conference included several important posters and presentations in the basic science Track A, particularly related to viral entry and genetic responses to treatment.

On the first day, Goto and colleagues from the Kyoto and Osaka Universities presented a detailed structural analysis of the attachment of HIV to the cell membrane at the nanoscale level. [1]

In order to clarify the process of attachment, they used automated electron microscopic tomography. HIV was inoculated into culture lymphocytes, and after a designated time the cells were harvested, fixed, and embedded for microscopy. The images were recorded automatically with a CCD (charged-coupled device) camera and the data was 3-D constructed for tomography. The results revealed that at the first contact point, the distance between the virus and the cell membrane corresponds closely to the length of the extended gp41 molecule. Further stages of attachment are currently under investigation.

Bosch and colleagues from Fundacio IrsiCaixa (Spain) and LaboRetro (France) investigated the cell-to-cell HIV-1 transmission and reported that it probably occurs through a caveolin independent but clathrin-dependent endocytic process, that results in the formation of endosomal vesicles containing complete HIV-1 particles. [2]

They localised internalised virus particles in large intracellular vesicles in cocultures of primary CD4+ T-cells with T-cells continually infected by either one of three HIV-1 isolates (NL4-3, Bal, and CI-1-SI-clinical). The non-stimulated CD4+ cells expressed only residual levels of the early endosomal marker EEA-1 or the late endosomal marker CD63 (as they have a very low metabolism). The HIV-1 infected cells, on the other hand, were strongly CD63 positive. Coculture of infected and uninfected cells produced a translocation into T CD4+ cells of both the HIV-1 Gag antigen and the CD63 marker (seen using immuno-confocal microscopy). Internalised virus was colocalised with clathrin-mediated endocytosis EEA-1 marker, but not with the caveolin-1 marker. The CD63 staining was observed by flow-cytometry after cell-to-cell contacts and viral transmission. Importantly, CD63 was present in the transferred viral particles, which explains the presence of a late endosomal marker in EEA-1 positive, Gag positive endosomes. The dependence on CD4 and the transmission of complete viral particles was confirmed by an analysis of CD4 and envelope subcellular localisation, which revealed their colocalisation with Gag in the polarised phenotype. Even though this finding does not have an immediate clinical impact, it may be important in the identification of new targets for novel compounds.

Pugach and colleagues from Cornell University, USA, studied how HIV-1 escapes from small molecule CCR5 inhibitors in vitro, by creating resistant viruses and examining their properties. [3]

Their results showed that the drug-resistant virus acquired the ability to utilise CCR5 in its inhibitor-bound form. Consequently, the resistant viruses are sensitive to inhibition by PSC-RANTES, but they are considerably resistant to this chemokine derivative when a small molecule inhibitor is also present. This finding may have implications on the research and possible future clinical use of maraviroc and vicriviroc.

Maeda and colleagues reported that CXCR4 antagonist induced co-receptor switching from X4 to R5 phenotype in vitro was determined by a single amino acid substitution in the V3 region of HIV-1 gp120. [4]

An R5/X4 variant (89.6 strain) was passaged in the presence of a CXCR4 antagonist T140 using a cell line highly susceptible to R5 variants, in order to select coreceptor switch mutants. The mutant harboured an amino acid substitution in the V3 region of the Env (Arginine 308 to Serine, R308S). The substitution conferred total resistance to CXCR4 antagonists when luciferase-reporter HIV-1 pseudotyped with the mutant Env. At the same time sensitivity to the CCR5 antagonist TAK-779 increased in both CCR5 and CXCR4-expressing cells. The analysis demonstrated that the virus with the mutation largely utilised CCR5 while retaining CXCR4 usage.

Humbert from Georg-Speyer-Haus, Molecular Virology, Frankfurt, Germany and colleagues successfully applied phage display technology to identify HIV-1 specific mimotopes for neutralising antibodies that are expected to have protective role in the long-term non-progressors (LTNP). [5]

Sera of LTNP and a control group of regular progressors were analysed and in the former, the titers of the neutralising bodies against HIV-1 were logically significantly higher. The team selected more than 1400 phage clones with LTNP IgG (analysed by ELISA) and more than 700 phage inserts were sequenced. They identified motifs related to the immunodominant epitopes in HIV-1 Env, but also conformational epitopes overlapping with receptor binding sites on the surface of gp120. Sera from mice immunised with ceratin phage groups showed neutralising activity against HIV in vitro, proving that the phage mimotopes indeed mimic epitopes for neutralising antibodies. These mimotopes may represent candidates for derivation of vaccine-relevant immunogens.

Several very interesting small studies dealing with polymorphisms were presented as posters. De la Tribonnierre and colleagues reported that a polymorphism in MDR-1 alleles that is associated with virological efficacy in HIV-positive naive patients treated with non-boosted PI-containing HAART, regimens but not in those treated with boosted PI-containing regimens. [6]

The MDR-1 genetic single nucleotide polymorphism (SNP) in exon 26 (C3435T) regulates P-gp expression. The study assessed the influence of the above-mentioned SNP on the virological responses to first-line PI-containing regimens. They included 182 HIV-infected subjects who received HAART from 1997 to 2002 and were followed through December 2004. The proportion of subjects with MDR-1 exon 26 genotypes CC, CT, and TT were 37%, 44%, and 19% respectively. Female patients and patients from sub-Saharan Africa had more frequently exon 26 genotype CC (p<0.05). A multivariate Cox model showed that time to first undetectable viral load when the patients were on an unboosted PI regimen was shorter in the subgroup with the CT genotype (p=0.01) and not those with TT genotype [HR=0.69; 95% CI, 0.41-1.17 and p=0.17] and CC genotype. When comparing the subgroups on boosted PIs, however, the difference disappeared. The difference may be a result of the higher concentration achieved with the boosted PIs.

Two other polymorphisms studies were performed in geographically specific regions and in particular populations. Pavia-Ruz and colleagues analysed the T303A CCR5 gene polymorphism in Mayan and Mestizo populations of Yucatan, Mexico. [7]

Other CCR5 gene polymorphisms that cause premature termination of translation apart from T303A are the CCR5 delta-32 and 893delC. T303A, however, poses particular interest as it occurs in variable allele frequencies (e.g in 0.014 of Afro-Americans, 0.07 in French populations and is completely absent in three Brazilian ethnic groups). To determine the frequency of T303A in Mayan and Mestizo populations in Mexico, 100 samples were analysed (50 of each ethnic group). DNA was extracted from whole blood. Surprisingly, T303A (wildtype genotype) was present in all Mayan individuals, but only in 90% of the Mestizo samples. The heterozygous genotype was present in 10% of the Mestizo participants but in none of the Mayan population.

Tumanov and colleagues studied the distribution of CCR5-delta-32 and CCR2-64I alleles among the basic ethnic groups of Mongolian population. [8] 253 samples were analysed by PCR-restriction fragment length polymorphism assay. No subjects homozygous for CCR5-delta-32 genotype were found, while the percentage of the heterozygous genotype was 2.4% (6/253). The CCR2-64I allele frequency was 0.246. Both these results match the currently available data about the population in the East Asian region.

References:

Unless stated otherwise, all references are to the Programme and abstracts from the XVI International AIDS Conference, Toronto, Canada, 13-18 August 2006.

1. Goto T, Hasegawa T, Kajimura N et al. Attachment in HIV entry process by electronic microscopic tomography. Oral abstract MOAA0101.
2. Bosch B, Grigorov B, Senserrich J et al. Cell-to-cell HIV-1 transmission through a clathrin-dependent endocytic pathway. Oral abstract MOAA0102.
3. Pugach P, Kuhmann S, Ketas T et al. The mechanism of HIV-1 escape from small molecule CCR5 antagonists. Oral abstract MOAA0105.
4. Maeda Y, Yusa K, Harada S. CXCR4 antagonist-induced coreceptor switch from X4 to R5 phenotype in vitro determined by a single amino acid substitution in the V3 region of human immunodeficiency virus type 1 gp120. Oral abstract MOAA0104.
5. Humbert M, Antoni S, Landersz M et al. Vaccine relevant mimotopes selected with neutralising IgG present in plasma from long-term non –progressors (LTNP) by phage display. Oral abstract MOAA0204.
6. Tribonniere X De la, Broly F, Burban Deuffic S et al. Polymorphism in MDR-1 alleles associated with virological efficacy in naive HIV-infected patientstreated with non-boosted PI-containing HAART regimensbut not in those treated with boosted PI-containing regimens. MOPE0012.
7. Pavia-Ruz N, Quintal-Ortiz I, Valadez-Gonzalez N et al. Analysis of the CCR5 gene T303A polymorphism in Mayan and Mestizo populations of Yukatan, Mexico. Poster abstract MOPE0007.
8. Tumanov A, Amarjagal Y, Munkhtuvshin N et al. Polymorphism of CCR5 and CCR2 genes associated with HIV-1 resistance in Mongolia. Poster abstract MOPE0005.