Enzymatic removal of mannose moieties can increase the immune response to HIV-1 gp120 in vivo

Abstract

The Env glycoproteins gp120 and gp41 are used in humoral immunity-based vaccines against human immunodeficiency virus (HIV-1) infection. One among many obstacles to such a vaccine is the structural defenses of Env glycoproteins that limit their immunogenicity. For example, gp120 mannose residues can induce immunosuppressive responses in vitro, including IL-10 expression, via mannose C-type lectin receptors on antigen-presenting cells. Here, we have investigated whether mannose removal alters gp120 immunogenicity in mice. Administering demannosylated gp120 (D-gp120) in the T(H)2-skewing adjuvant Alum induced approximately 50-fold higher titers of anti-gp120 IgG, compared to unmodified gp120. While the IgG subclass profile was predominantly T(H)2-associated IgG1, Abs of the T(H)1-associated IgG2a and IgG3 subclasses were also detectable in D-gp120 recipients. Immunizing with D-gp120 also improved T-cell responses. Giving an IL-10 receptor blocking MAb together with unmodified gp120 in Alum increased the anti-gp120 IgG titer, implicating IL-10 as a possible mediator of auto-suppressive responses to gp120.

Figure 1. Biochemical properties of demannosylated gp120

Preparations of gp120 were assessed for (A) m.wt. changes, (B) the presence of α(1–2,3,6)-mannosidase using a non-reducing 4–12% Bis-Tris NuPage gel. The D-gp120 preparation shown in (A) is from batch #2 after CHT column purification.

Figure 2. Antigenic properties of demannosylated gp120

Unmodified gp120, mannosidase-treated gp120 (D-gp120, batch #1) or gp120 processed in the absence of mannosidase (M-gp120) were captured onto ELIS plates via Ab D7324, then reacted with serial 3-fold dilutions of the antibodies indicated on each panel. The OD₄₉₀ values displayed are the means from duplicate wells. The test MAbs recognize terminal mannoses (2G12); a discontinuous C1–C4 epitope (A32); a CD4-induced (CD4i) epitope (17b ± sCD4); V3 epitopes (447-52D, F425-B4e8); or epitopes overlapping the CD4 binding site (CD4BS) (b6, b12, 15e). CD4IgG2 binds the CD4Bs and HIVIG is a polyclonal antibody preparation derived from the sera of HIV-1-infected people.

Figure 3. Comparative immunogenicity of D-gp120 in BALB/c mice

Mice (5 per group) were immunized with gp120, M-gp120 or D-gp120 (batch #1) in Alum (A) or Quil A (B) adjuvants, as indicated. Anti-gp120 IgG titers were determined using a gp120 capture ELISA. Each symbol represents the mean (± SEM) reciprocal endpoint anti-gp120 titer for each group of mice (n=5). Titers <100 (grey shaded area) are considered negative. The mean reciprocal end-point titer values for prebleed samples were all < 1.0 × 10² (data not shown).

Figure 4. Comparative immunogenicity of D-gp120 in C57BL/6 mice

Mice were immunized with M-gp120 (grey symbols) or D-gp120 batch #1 (black symbols) at the times indicated by the arrows. Main figure: Alum adjuvant; inset, Quil A adjuvant. Each symbol represents the mean (± SEM) reciprocal endpoint anti-gp120 titer for each group of mice (n=5 initially, but n=4 for M-gp120/Alum from week 7 due to the death of one mouse from an unrelated cause). Titers <100 (grey shaded area) are considered negative.

Figure 5. The increased immunogenicity of D-gp120 is not due to contaminant mannosidase

Mice (C57BL/6, 5 per group) were immunized with M-gp120 (grey symbols) or D-gp120 (batch #2; enyzme free; black symbols) using alum adjuvant, at the times indicated by the arrows. Each symbol represents the mean (± SEM) reciprocal endpoint anti-gp120 titer for each group of mice. Titers <100 (grey shaded area) are considered negative.

Figure 6. Blocking IL-10 activity during immunization with gp120 in Alum adjuvant, but not Quil A, increases the anti-gp120 titer in C57BL/6 mice

Mice were immunized with gp120 in the presence of an anti-mIL-10R MAb (black symbols) or an isotype control MAb (white symbols) at the times indicated by the arrows (black, gp120; grey, MAbs). Main figure: Alum adjuvant; inset, Quil A adjuvant. Each symbol represents the mean (± SEM) reciprocal endpoint anti-gp120 titer for each group of mice (n=5 initially, but n=4 for gp120 + control MAb in Alum from week 2 due to the death of one mouse from an unrelated cause). Titers <100 (grey shaded area) are considered negative.

Figure 7. gp120-specific IgG subclass responses in C57BL/6 mice immunized with M-gp120 or D-gp120

Serum IgG subclasses were determined 7 weeks after primary immunization. Shown are reciprocal endpoint titers for each individual mouse (horizontal bar indicates median values) that received M-gp120 (grey symbols) or D-gp120 batch #1 (black symbols) in either Alum (panel A) or Quil A (panel B) adjuvants. Negative responders (titer < 100) are shown in the grey boxed area.

Figure 8. gp120-specific IgG subclass responses in C57BL/6 mice immunized with gp120 in the presence or absence of an IL-10 receptor Mab

Serum IgG subclasses were determined 7 weeks after primary immunization. Shown are reciprocal endpoint titers for each individual mouse (horizontal bar indicates median values) that received gp120 together with an anti-IL-10 receptor MAb (grey symbols) or an isotype control MAb (black symbols) in Alum adjuvant. Negative responders (titer < 100) are shown in the grey boxed area.

Figure 9. D-gp120 immunization improves antigen specific T cell responses

Unfractionated splenocytes (2.5 × 10⁵ cells/well) derived from the indicated groups of C57BL/6 mice were cultured for 72h with 10μg/ml of recombinant gp120 (left panel), 2μg/ml of anti-CD3 (center panel) or media (right panel). Cytokine levels in supernatants from triplicate wells were measured by ELISA and are expressed in pg/ml as the mean value ± SD above the respective assay detection limits. The mice were immunized with M-gp120 (white bars) or D-gp120 batch #1 (black bars) in the adjuvant indicated at the foot of the figure. * p < 0.05 (one-tailed Student’s t test).

Figure 10. Antigen-specific T cell responses from gp120-immunized C57BL/6 mice

Unfractionated splenocytes (2.5 × 10⁵ cells/well) derived from the indicated groups of C57BL/6 mice were cultured for 72h with 10μg/ml of recombinant gp120 (left panel), 2μg/ml of anti-CD3 (center panel) or media (right panel). Cytokine levels in supernatants from triplicate wells were measured by ELISA and are expressed in pg/ml as the mean value ± SD (the y-axis starts at the manufacturer specified detection limit for each cytokine). The mice were immunized with gp120 in the adjuvant indicated at the foot of the figure, and in the presence of an isotype control MAb (white bars) or the anti-mIL-10R MAb (black bars). * p < 0.05 (one-tailed Student’s t test).