HIV Envelope gp120 Activates LFA-1 on CD4 T-Lymphocytes and Increases Cell Susceptibility to LFA-1-Targeting Leukotoxin (LtxA)

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals

Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents

Longitudinal Study of Primary HIV-1 Isolates in Drug-Naïve Individuals Reveals the Emergence of Variants Sensitive to Anti-HIV-1 Monoclonal Antibodies

To study how virus evolution affects neutralization sensitivity and to determine changes that occur in and around epitopes, we tested the ability of 13 anti-HIV-1 gp120 (anti-V2, anti-V3, anti-CD4bd and anti-carbohydrate) human monoclonal antibodies (mAbs) to neutralize sequential viruses obtained from five HIV-1 chronically infected drug naïve individuals. Overall, primary viruses collected from patients at first visit were resistant to neutralization by all anti-HIV-1 mAbs with the exception of one virus sensitive to IgG1b12. Four of the five patients' viruses evolved increased sensitivity to neutralization by anti-V3 mAbs. Virus collected from a patient obtained 31 months later, evolved increased sensitivity to anti-V2, anti-V3, and anti-CD4bd mAbs. Furthermore, the anti-V2 and anti-CD4bd mAbs also exhibited increased neutralization capacities against virus collected from a patient 29 months later. Of the seven anti-V3 mAbs, five showed increased potency to neutralize the evolved virus from a patient collected after 11 months, and three exhibited increased potency against viruses from two patients collected 29 and 36 months later. Anti-V3 mAbs exhibited the most breadth and potency in neutralizing the evolving viruses. Sequence analysis of the envelope regions revealed amino acid conservation within the V3 loop, while most of the changes identified occurred outside the core epitopes and in particular within the C3 region; these may account for increased neutralization sensitivity. These studies demonstrate that in vivo, HIV-1 can evolve increased neutralization sensitivity to mAbs and that the spectrum of neutralization capacities by mAbs can be broader when studied in longitudinal analysis

Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis

HIV-1 Infection of DC: Evidence for the Acquisition of Virus Particles from Infected T Cells by Antigen Uptake Mechanism

Dendritic cells (DC) play a pivotal role in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection trap virus particles via DC-SIGN and transfer the virus to the interacting naïve T cells. This prompted us to ask the question whether DC could acquire virus from infected T cells during DC-T cell interaction. To address this, we investigated the likely transfer of virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results indicate that DC acquire virus from infected T cells via antigen uptake mechanism and this results in infection of DC with expression of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in infection of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data show that DC infection is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate virus