Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein

Abstract

Identification of optimal antigen(s) and adjuvant combination(s) to elicit potent, protective, and long-lasting immunity has been a major challenge for the development of effective vaccines against chronic viral pathogens, such as HIV-1, for which there are not yet any licensed vaccines. Here we describe the use of a novel adjuvant approach employing Carbopol 971P(®) NF (hereafter referred to as Carbopol971P), a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in-water adjuvant, MF59, as a potentially safe and effective adjuvant to augment humoral immune responses to the HIV-1 envelope glycoprotein (Env). Intramuscular immunization of small animals with recombinant Env glycoprotein (gp140) formulated in Carbopol971P plus MF59 gave significantly higher titers of binding and virus neutralizing antibodies as compared to immunization using gp140 with either MF59 or Carbopol971P alone. In addition, the antibodies generated were of higher avidity. Importantly, the use of Carbopol971P plus MF59 did not cause any serious adverse reactions or any obvious health problems in animals upon intramuscular administration. Hence, the Carbopol971P plus MF59 adjuvant formulation may provide a benefit for future vaccine applications.

Figure 1

Analysis of interaction between SF162 gp140 protein and Carbopol971P. (A) Measurement of particle size of 0.25% (w/v) Carbopol971P suspension and SF162 gp140 protein-Carbopol971P (final concentration 0.25%, w/v) complex using Dynamic Light Scattering (DLS). The increased size of the gp140 protein-Carbopol971P complex (average ~86nm) in comparison to Carbopol971P alone (average ~68nm) indicates the adsorption of the positively-charged protein at pH ≥3 on negatively-charged Carbopol. (B) Direct binding of Carbopol971P suspension to SF162 gp140 protein immobilized on CM5 sensor chip. 5000RU of SF162 gp140 protein was covalently immobilized on sensor chip via amine coupling and 30 µl of 0.1% Carbopol971P suspension was injected at 10 µl/min. The increase in RU (Resonance Unit) (see association phase) indicates binding of polyanionic Carbopol971P at pH ≥3 to immobilized-gp140 (red line). Following injection, as pH alters to 7.4 (running buffer HBS-EP), a bulk-drop in RU occurs (at time = 180s) followed by very slow dissociation of the polymer from the gp140-coated surface (see dissociation phase). The dotted grey line shows the (non-) binding of Carbopol971P suspension to mock-treated control surface. The spikes at the beginning (time = 0s) and end (time = 180s) of injection/association is due to differences in pH and nature of the solvent media.

Figure 2

Stability of SF162 gp140 protein. (A) SDS-PAGE of SF162 gp140 protein upon incubation with 0.5% (w/v) Carbopol971P for 0 h (lane 1, control), 1 h (lane 2), 2 h (lane 3), 3 h (lane 4) and 4 h (lane 5), indicating that the protein is stable in low pH conditions in the presence of Carbopol971P. (B) Binding of CD4IgG2 (surrogate for CD4 receptor) and anti-gp120 mAbs, b12 (CD4BS, conformational), 17b (CD4-induced, CD4i) in the presence or absence of soluble CD4 (sCD4) and F425-B4e8 (V3 variable loop), to SF162 gp140 protein (control) or gp140 protein upon 3 h incubation with 0.5% (w/v) Carbopol971P. A capture ELISA using Ab D3724 was used to analyze the binding of the gp140 protein to CD4IgG2, mAbs b12, 17b (-/+ sCD4) and F425-B4e8 (see Methods section for details). For testing CD4-induced 17b mAb binding, a three-time molar excess of soluble CD4 (sCD4) was added to gp140 protein prior to their addition to the Ab D3724-coated plates.

Figure 2

Stability of SF162 gp140 protein. (A) SDS-PAGE of SF162 gp140 protein upon incubation with 0.5% (w/v) Carbopol971P for 0 h (lane 1, control), 1 h (lane 2), 2 h (lane 3), 3 h (lane 4) and 4 h (lane 5), indicating that the protein is stable in low pH conditions in the presence of Carbopol971P. (B) Binding of CD4IgG2 (surrogate for CD4 receptor) and anti-gp120 mAbs, b12 (CD4BS, conformational), 17b (CD4-induced, CD4i) in the presence or absence of soluble CD4 (sCD4) and F425-B4e8 (V3 variable loop), to SF162 gp140 protein (control) or gp140 protein upon 3 h incubation with 0.5% (w/v) Carbopol971P. A capture ELISA using Ab D3724 was used to analyze the binding of the gp140 protein to CD4IgG2, mAbs b12, 17b (-/+ sCD4) and F425-B4e8 (see Methods section for details). For testing CD4-induced 17b mAb binding, a three-time molar excess of soluble CD4 (sCD4) was added to gp140 protein prior to their addition to the Ab D3724-coated plates.

Figure 3

Total binding gp140 protein-specific antibodies as determined by ELISA following immunization with gp140 protein adjuvanted with MF59, Carbopol971P or MF59 plus Carbopol971P. ELISA mean titers ± SD of SF162 gp140 protein-specific antibodies from animals at various time-points following immunization (2wp2, 2wp3, 2wp4, 4wp4 and 15wp4) to determine the durability of the response upon immunization of gp140 protein adjuvanted with MF59, or Carbopol971P or Carbopol971P plus MF59. The p values for the differences between groups at various time points are indicated.

Figure 4

Avidity of gp140 protein-specific antibodies as determined by ELISA following immunization with gp140 protein adjuvanted with MF59, Carbopol971P or MF59 plus Carbopol971P. Avidity indexes (mean ± SD) of SF162 gp140 protein-specific antibodies from animals at various time-points following immunization (2wp2, 2wp3, 2wp4, 4wp4 and 15wp4) to determine the durability of the response upon immunization of gp140 protein adjuvanted with MF59, or Carbopol971P or Carbopol971P plus MF59. The p values for the differences between groups are indicated.

Figure 5

(A–D): Analysis of breadth by neutralization of HIV-1 pseudoviruses in TZM-bl cells by 2wp3 (p3) and 2wp4 (p4) sera from animals immunized with gp140 protein adjuvanted with MF59, Carbopol971P or Carbopol971P plus MF59. ID50 neutralization titers are shown for the following viral isolates: (A) SF162.LS, MW965.26, (B) Bal.26, .SS1196.1, (C) 6535.3, QH0692.42 and RHPA45259.7. The subtypes of the viruses are highlighted in parenthesis. Neutralization of (D) MLV is shown here as a control.

Figure 6

ID50 titers of sera from animals immunized with gp140 protein adjuvanted with MF59, Carbopol971P, or Carbopol971P plus MF59. ID50 neutralization titers in TZM-bl cells, are indicated against the two Tier 1A (SF162.LS and MW965.26) and one Tier 1B (Bal.26) viruses. The subtypes are indicated in parenthesis. P values for the differences between groups, at 2wp3 (p3) and 2wp4 (p4), in MF59, Carbopol971P or Carbopol971P plus MF59 are listed in the table below.

Figure 7

Body weights of animals following immunization. (A) Changes in percentage (%) body weight of animals immunized with gp140 protein adjuvanted with Carbopol971P plus MF59 to highlight no adverse reactivity or no obvious health problems (NOHP) upon use of Carbopol971P plus MF59 during vaccination. The grey arrows highlight the four immunization time-points. Data for the other two groups, using MF59 only or Carbopol971P only, are similar and show no adverse reactivity and NOHP (not shown). (B) Changes in body weight (in kg) in five animals (#11, 12, 13, 14, 15) monitored before (indicated by black arrow) immunization with gp140 protein adjuvanted with Carbopol971P plus MF59 and two time-points after the immunization. Although subtle drops in body weights were observed, particularly after 3^rd immunization, the differences were not statistically significant (p≥0.05).