Divergence of primary cognate B- and T-cell proliferative responses to subcutaneous and intravenous immunization with virus-like particles

Abstract

A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL) on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes.

Figure 1

Design, production and characterization of HIV-virus-like particles (VLPs) containing an HA epitope. (A) Schematic representation of the Hgpsyn-HA and Hgsyn-HA expression cassettes. CMV, immediate early promoter of human cytomegalovirus; MA, matrix; CA, capsid; Pol, polymerase. HA_110-120, influenza virus hemagglutinin epitope. (B) HEK293T cells were transfected with Hgpsyn, Hgpsyn-HA, Hgsyn or Hgsyn-HA. VLPs were recovered from the supernatants of transfected cells by ultracentrifugation through a sucrose cushion and analyzed by western blot with an antibody against capsid. (C) Naive untouched CD4⁺ T-cells were isolated from TCR-HA and BALB/c mice, labeled with CFSE and co-cultured with splenic DC at a 10:1 ratio for 64 h at 37 °C in the presence of the indicated VLPs (50 ng of capsid/mL). VLPs were prepared by co-transfection of an Env expression plasmid with one of the Gag or GapPol expression plasmids, as described in Table 1. After incubation, the cells were stained with anti-CD3 and anti-CD4 antibodies, and the CFSE fluorescence intensity of the CD3⁺ CD4⁺ cells is shown.

Figure 2

Proliferative response of naive cognate CD4⁺ T-cells after s.c. and i.v. immunization. Naive untouched CD4⁺ T-cells were isolated from TCR-HA mice labeled with CFSE and adoptively transferred into recipient BALB/c mice (2.5 × 10⁶ cells per mouse). Four hours later, the recipient mice were immunized with GagPol-VLP-HA (s.c. or i.v.) or with Inflexal^® V s.c. (A) Proliferation of the transferred CD4⁺ T-cells from the spleen and draining lymph nodes (LNs) was analyzed 72 h after immunization. The CFSE fluorescence intensity of CD3⁺ CD4⁺ is shown. (B) Expansion of transferred T-cells is evident by the increase in the percentage of CD4⁺ CFSE⁺ T-cells from total CD4⁺ lymphocytes in the spleen and lymph nodes. The histogram gives the mean of one experiment ± the standard deviation (n = 3). * Significant difference for the group in comparison to the other groups (p < 0.05; Student’s t-test). The experiment was repeated two more times with Gag-VLP-HA with similar results.

Figure 3

Proliferative response and expansion of naive cognate B-cells after s.c. and i.v. immunization. Naive untouched B-cells were isolated from SW-HEL mice, labeled with CFSE and adoptively transferred into recipient BL6 mice (5 × 10⁶ cells per mouse). Four hours later, the recipient mice were immunized with HEL-VLP (s.c. or i.v.). (A) Three days later, spleen and LN cells were stained with Alexa647-conjugated HEL and anti-B220 antibodies. The CFSE fluorescence intensity of HEL⁺ B220⁺ cells is shown. (B) The expansion of HEL⁺ B220⁺ CFSE⁺ B-cells in draining LN on Day 3 is evident by the increase in their percentage of the total B220⁺ cells. The histogram gives the mean ± the standard deviation of one representative experiment (n = 4). Two independent experiments with three to four mice were performed. * Significant difference for the group comparison to the other groups (p < 0.05; Student’s t-test). (C, D) For the analyses of B-cell expansion on Day 7, non-labeled SW-HEL B-cells were transferred. Seven days after VLP injection, cells were stained with Alexa647-conjugated HEL and anti-B220 antibodies, and the expansion of HEL⁺ B220⁺ cells is presented as the percentage of the total B220⁺ cells (C) and as their absolute numbers in the cell preparations from spleen and lymph nodes (D). The histograms represent the mean within one experiment ± the standard deviation (n = 4). *** Significantly different from the other groups (p < 0.01; Student’s t-test). ^# Significantly different from the control group (p < 0.01; Student’s t-test).

Figure 4

Transfer of particulate and soluble HEL by DC to cognate B-cells in vitro. Naive untouched B2-cells were isolated from (A) SW-HEL and (B) BL6 mice and labeled with CFSE. Freshly isolated splenic DC or B-cells were separately incubated for 2 h in the presence of either HEL-VLP or mHEL. After extensive washing, DC and B-cells either exposed to VLPs/mHEL as marked by the “+” sign or non-exposed as marked by the “-” sign were mixed together and co-cultured for three days. The experiment was performed three times independently of each other, and one representative experiment is shown.