Functional studies on the interaction of immunoglobulins with HIV-2 envelope

Abstract

Infection with HIV-2 has a much more limited geographic distribution than HIV-1, being less readily transmitted and is generally less pathogenic. The role of immunoglobulins in defence against pathogens in different body compartments is known, where IgG is the principal isotype in the blood and extracellular fluid while lgA predominates in the mucosal compartment. The aim of this thesis was to study the interaction of lgA and IgG with the HIV-2 envelope in an attempt to understand the role of the humoral immune response in controlling HIV-2 infection.

Since HIV is a sexually transmitted disease, lgA may play a role in controlling transmission of HIV2 across mucosal surfaces. lgA was purified from the sera of HIV-2 infected individuals. The antigenic sites of HIV-2 envelope important for binding lgA were studied using recombinant gp105 and gp36, as well as peptides spanning the whole envelope. Prominent lgA binding was defined to the central region of gp36, corresponding to residues 644658. The purified lgA demonstrated neutralizing activity against HIV-2 in 59% of the sera tested, suggesting a role for IgA in preventing HIV-2 transmission.

The V3 region expressed on the HIV envelope has been identified as a main neutralization target for both HIV- 1 and HIV-2. However, studies have indicated that this site is not exposed in HIV- 1 primary isolates. To study the exposure of the V3 region, both functionally and structurally, the envelope gene from a primary CD4-independent HIV-2 isolate was cloned, and two constructs of HIV-2 gp125 were designed as previously described for HIV-1 gp120, where in the gp125deltaV1V2 construct the V1/V2 region was excluded. These gp125 constructs were expressed in CHOlec cells as glycosylated proteins with minimal glycosylation heterogeneity, and were characterized using circular dichroism and mass spectrometry analyses.

Two V3-specific murine monoclonal antibodies (7C8 & 3C4) were used to study the exposure of the V3 region on gp125, 7C8 being linear site-specific, while 3C4 is conformationally sensitive. Using different lectin affinity chromatography techniques, both monomeric and oligomeric forms of the gp125 proteins were purified. These monomeric and oligomeric gp125 proteins were recognized by 7C8, while only gp125 oligomers were recognized by 3C4. Previous studies have demonstrated that HIV-1 binding to CD4 causes conformational changes in the envelope, where the V1 and V2 loops reorient and expose the V3 region. Surface plasmon resonance analysis of CD4 binding to gp125 and gpl20 glycoproteins demonstrated that CD4 binds to gp125 at a lower affinity and with different kinetics compared to gpl20. Molecular modeling of gp125 revealed a residue that fills the hydrophobic cavity implicated in CD4 binding which may be responsible for the CD4-induced conformation of gp125. Furthermore, the accessibility of the V3 region in polymeric gp125 suggests that gp125 does not require CD4 to reorient the position of the V 1 and V2 loops in order to expose the V3 region.

To understand the role of the gp125 V3 region in neutralization of HIV-2, the murine antibody responses induced by immunization with recombinant gp125 proteins, and the neutralization capacity of V3-specific murine mAbs were studied. Antibodies at titres of 8000 to 128000 recognizing gp125 or gp125deltaV1V2 were induced in the sera of the immunized mice with either recombinant gp125 protein. However, none of the sera neutralized HIV-2 or bound a peptide spanning the center and C-terminus of the V3 region. While neither 7C8 nor 3C4 mAbs neutralized HIV-2, their respective Fab fragments blocked infection.

In conclusion, the results from this thesis suggest a role for lgA and V3-specific IgG in controlling infection by HIV2. The analysis of the conformation of gp125 and the exposure of the V3 region presented in this work could provide an explanation underlying the CD4-independence observed for most HIV-2 isolates. However, while the V3 region may be exposed on HIV-2, smaller Fab fragments are required to neutralize HIV-2. The specific conformation of gp125, exposing the V3 region, and V3-specific small molecules may provide clues for the design of vaccines against HIV.