To understand the link between high titers of Ad5 specific Nabs and HIV infection we investigated the effects of Ad5 specific Nabs on various immune cell types. In particular, we hypothesized that dendritic cells (DCs) may play a pivotal role. Previous studies in our lab showed that adenovirus are potent vaccine vectors and have a good capacity to activate dendritic cells. We noticed that infection of dendritic cells (DC) by Ad5 vector alone led to a different DC phenotype than infection by Ad5 vector opsonized with plasma containing Ad5-Nabs (Ad5 immune complexes or Ad5-IC). We showed that in the presence Ad5-IC, DCs became strongly activated (up regulation of co-stimulatory molecules and secretion of pro-inflammatory cytokines). This activation was mediated through FCgRII internalization followed by TLR9 engagement.
We proposed that Ad5-IC strongly activated DCs and provides conditions favoring HIV infection by binding and bringing HIV in close contact with activated Ad5 specific CD4+ T-cells, highly susceptible to HIV infection. One likely scenario is that subjects with Ad5 pre-existing immunity generated a strong cellular immune response following Ad5-immunization leading to an increased level of Ad5-specific CD4+T-cell activation in particular anatomic compartments such as mucosal tissues, which are one of the main portals of entry for HIV. We therefore evaluated the homing capacity of Ad-specific CD4+T-cells isolated from peripheral blood of Ad-naturally infected volunteers versus Ad5-vaccinee. We found that Ad5-specific CD4 T-cells isolated from peripheral blood of Ad5-naturally infected individuals were endowed with mucosal homing capacity. Additionally, we showed that Ad-specific CD4 T-cells residing in gut mucosal tissues of Ad-naturally infected volunteers, express HIV co-receptors CCR5 and 47 integrin and were susceptible to HIV infection in vitro.
We then explored which Ad5 component (proteins, virus-associated RNA, or the double-stranded DNA genome) is involved in Ad IC-mediated DC maturation. We found that the genome of Ad5 vector harbors critical signals leading to DC activation and maturation through stimulation of TLR9. We identified the presence of the GTCGTT hexamer motif within common and rare Ad-derived vector genomes (i.e. Ad5, Ad6, Ad26, Ad35, Ad36 and Ad41 vectors), a motif known to strongly stimulate human TLR9-expressing cells. The number of GTCGTT motifs was found to vary among different Ad vector serotypes. Interestingly, the number of GTCGTT motifs present in each Ad vector genome correlated with the potency of Ad-IC-mediated DC maturation. We could also show that ICs generated with rare Ad vector serotypes such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation. Their poor ability to induce DC maturation strongly correlated with the low number of TLR9-agonist motifs present in their genome.
In this project we aim to characterize the profile of Ad5-neutralizing antibodies (quantitatively and qualitatively). We showed that Ad5-neutralizing antibodies targeting fiber or hexon proteins of the virus have different abilities to induce DC maturation. We will investigate if antibody responses following Ad-5 natural infection or Ad5 immunization have the same characteristics in terms of quantities and specificity. We are also interested in comparing the antibody profiles of Ad5-Nabs in naturally infected people prior and after vaccination.
The different antibody profiles that may arise through natural infection or/and vaccination, may lead to diverse pattern of DC maturation. We therefore will compare the ability of the different antibody profiles (naturally infected versus vaccinated versus naturally infected and vaccinated) to form Ad5-immuno complexes and measure their ability to induce DC maturation. We are also investigating if Ad5-ICs formed with sera from HIV-1 infected patients or sera from non-infected individuals have the same ability to induce DC maturation.
Interestingly the ability to activate DCs depends on the stability of the adenovirus particle once inside the cell as well as the way through which the adenovirus accesses the inside of the cell. We are currently investigating the role of adenoviruses specific antibodies on the stability of the adenovirus virus particle. We are exploring the mechanisms by which opsonizing and neutralizing Ad5-specific antibodies lead to DC maturation.