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Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.

We have recently shown that 'alloimmune sera' derived from polytransfused patients (PTP sera) are able to recognise and neutralise HIV in vitro. In this study we try to identify the protein(s), which are recognised by the PTP sera and elucidate mechanisms responsible for the neutralising capacity of these sera. The PTP sera allowed immunoprecipitation (IP) of HLA class II molecules on HIV-infected cells. To detect a potential cross-reactivity of alloreactive antibodies (Ab) with the HIV envelope protein gp160 or its subunits gp120/gp41 and HLA proteins, ELISA and FACS analyses were performed. The lack of reactivity of the PTP sera against rsgp160 in ELISA or FACS analysis indicated that recognition of cells was independent of HIV infection. To clarify whether interaction of the PTP sera with target cells has any effect on the infection process, virus neutralisation assays were performed. Inhibition of HIV infection was observed only when virus was pre-incubated with the PTP sera. Complement enhanced neutralisation of HIV-1 significantly. This enhancement was not due to complement-mediated lysis, because pre-incubation of the target cells with PTP sera did not inhibit HIV replication. Therefore, the neutralising effect of the Ab was due to blocking of the viral attachment/fusion process and not to negative signalling after infection. Since steric hindrance is possible only when HLA and gp120/gp41 are in close vicinity, isolation of rafts and IP assays were performed. These experiments revealed that gp120 and MHC class II molecules are indeed co-localised. The close physical association of gp120/gp41 and HLA strongly supports a mechanism for neutralisation of HIV by anti-HLA-Ab based on steric hindrance., (Copyright 2003 S. Karger AG, Basel)

After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC.

Objective: To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5., Design and Methods: Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5., Results: Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5., Conclusion: Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.