Aggregate complexes of HIV-1 induced by multimeric antibodies

Abstract

Background: Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry.

Results: The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA.

Conclusions: These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.

Figure 1

Size-exclusion chromatography (SEC) and dynamic light scattering in tandem enable more accurate characterization of the complexes formed with 2F5 IgA. Virus incubated with 2F5 dIgA was separated over a size exclusion column and followed by particle detection by (A) infrared, (B) ultraviolet, and (C) DLS detection. Size was determined for each peak eluted from the SEC system and the % of signal intensity corresponding to complex size is shown. Three peaks were detected with dIgA preparations, eluted at (D) 19, (E) 21, and (F) 23 minutes, measuring 450 nm, 298 nm and 180 nm, respectively. Experiments were performed in triplicate. Results shown are from one representative experiment.

Figure 2

Size-exclusion chromatography and dynamic light scattering in tandem allow more accurate characterization of viral complexes formed with b12 IgA. Virus incubated with b12 dIgA was separated over a size exclusion column and followed by particle detection by (A) infrared, (B) ultraviolet, and (C) DLS detection. Size was determined for each peak eluted from the SEC system and the % of signal intensity corresponding to complex size is shown. Two peaks were detected with dIgA preparations, eluted at (D) 21.5 and (E) 23 minutes, measuring 310 nm and 180 nm, respectively. Experiments were performed in triplicate. Results shown are from one representative experiment.

Figure 3

Sizing measurements of pre-immunization compared to post-immunization samples for RV144 serum IgA. (A) Scatter plots and (B) paired size measurements for pre and post immunization IgA samples incubated with HIV-1_A244. Purified virions and IgA samples were incubated for 1 hour and measured using dynamic light scattering. Each circle shown represents the average of 3 measurements, with experiments performed three times for each sample. Horizontal dashed line indicates the size of untreated viral preparations. Error bars indicate the standard deviation within the measurements for each patient at each visit.

Figure 4

RV144 Vaccine Induced IgA mediated Infectious HIV-1 Virion Capture. Purified plasma IgA at visit 1 (pre-vaccination) and visit 8 (2 weeks post last vaccination) from 22 (CM244 or WITO) and 29 (NL4-3) vaccinees with detectable HIV-1 Env binding antibody responses were selected and measured in infectious HIV-1 capture assays using micro-plate based assay [29]. Relative light units (RLU) produced by infected target cells quantify the amount of infectious virus bound. Dashed line (RLU ≥1000) indicates positive samples. The percentage of tested vaccinees with virion capture capacity are 34.5% (10/29) NL4-3, and 60.9% (14/23) for lab adapted strain NL4-3 and CM244, respectively. The transmitted/founder WITO was captured by 31.8% (7/22) vaccinees. Dashed line indicates positivity threshold, defined by the background levels of capture (≥1000).