Fusion intermediates of HIV-1 gp41 as targets for antibody production: design, synthesis, and HR1-HR2 complex purification and characterization of generated antibodies.

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

The effect of human immunodeficiency virus type 1 (HIV-1) gp41 variability on antibody detection.

To determine whether genetic variability influences the ability to detect antibody, nine gp41 ectodomain recombinant proteins from human immunodeficiency virus type 1 (HIV-1) CRF07_BC, CRF01_AE and subtype B' were expressed in a bacterial expression system and purified. An indirect sandwich ELISA was developed with individual purified recombinant proteins. Plasma samples from 26 individuals infected with HIV-1 of different subtypes and four samples from the 1st international antibody reference panel were tested against each recombinant protein by ELISA. Heat-map and two-dimensional hierarchical clustering methods revealed that ELISA reactivity against antigens derived from the same subtypes clustered together. This suggests a similar reactivity pattern among infections of the same subtype, and thus the antigenicity of gp41 recombinant proteins may vary depending on the subtype, and subtype-related serotypes may exist among these antigens. Using association analysis methods, eight signature sites related to the subtype-specific reactivity patterns were identified. This study provides valuable information for the development of diagnostic assays with the ability to detect broadly cross-reactive antibodies induced by infection with different HIV-1 subtypes.

Molecular epidemiology of HIV type 1 subtypes in equatorial guinea.

Equatorial Guinea is endemic for HIV-1. This country borders to the north with Cameroon, where different subtypes belonging to group M, as well as group O strains, are circulating simultaneously. To assess the molecular epidemiology of HIV-1 in Equatorial Guinea we analyzed 76 plasma samples collected throughout 1999 from seropositive individuals. Phylogenetic analysis of the gp41 region revealed that 53 were of subtype A, with 64% of these sequences clustering with CRF02_AG reference strains; 11 were of subtype C; 4 were of subtype D; 2 (closely related to subtype F2) were of subtype F; 3 were of subtype G, two of them forming a separate cluster with the recombinant circulating forms CRF06_cpx; 1 was of subtype H; and 2 were unclassifiable. Although subtype A is predominant, the presence of 14% of subtype C is also noteworthy. This work represents the first HIV-1 subtype distribution study in Equatorial Guinea.

Re-analysis of human immunodeficiency virus type 1 isolates from Cyprus and Greece, initially designated 'subtype I', reveals a unique complex A/G/H/K/? mosaic pattern.

Human immunodeficiency virus type 1 (HIV-1) has been classified into three main groups and 11 distinct subtypes. Moreover, several circulating recombinant forms (CRFs) of HIV-1 have been recently documented to have spread widely causing extensive HIV-1 epidemics. A subtype, initially designated I (CRF04_cpx), was documented in Cyprus and Greece and was found to comprise regions of sequence derived from subtypes A and G as well as regions of unclassified sequence. Re-analysis of the three full-length CRF04_cpx sequences that were available revealed a mosaic genomic organization of unique complexity comprising regions of sequence from at least five distinct subtypes, A, G, H, K and unclassified regions. These strains account for approximately 2% of the total HIV-1-infected population in Greece, thus providing evidence of the great capability of HIV-1 to recombine and produce highly divergent strains which can be spread successfully through different infection routes.