Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

Abstract

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Keywords: DNA vaccine; HIV-1; animal models; envelope; neutralizing antibodies.

Figure 1

Immunization regimen and comparing antibody responses in syn.gp140_Bx08 or syn.gp150_Bx08. DNA vaccinated rabbits. (A) Schematic immunization schedule with vertical arrows indicating immunizations. Sera were collected before immunization (w0) and two weeks after last immunization (w14). (B) Average IgG response against recombinant gp120_IIIb (rgp120_IIIb) in immunized rabbits (n = 4). (C) Average neutralizing activity, expressed as IC50, of purified IgG from week 14 rabbit sera against pseudotype virus strains of clade B, C and A (SF162, Bx08, JR-FL, BaL, 92Br025 and 92RW009).

Figure 2

Comparison of the immune responses in animals vaccinated with monovalent or trivalent DNA. Average IgG responses against rgp120_IIIb in immunized. (A) guinea pigs (n = 4) and (C) rabbits (n = 4). Immunization time points are indicated with arrows. Average neutralizing activity, expressed as IC50, of diluted guinea pig serum (B) or purified rabbit IgG (D) from week 14 against pseudotype virus strains of clade A–D and CRF02_AG. Unrelated MLV pseudotype virus was included as non-specific HIV control in the guinea pig setup (red). IgG titers (C) and IC50 values (D) from syn.gp140_Bx08 in the rabbit model are derived from Figure 1B,C.

Figure 3

Schematic representation of HIV-1 envelope DNA constructs and protein expression. DNA constructs encoding gp140. The tissue plasminogen-activator leader sequence (tPA) and the region encoding the variable regions V1 to V5 are indicated (grey boxes). (A) The gp140_ctl21/27 construct with V1-V5 region from ctl21 and ctl27 env flanked by Bx08 env. (B) DNA construct encoding modified gp140 including the SOSIP amino acid substitutions A501C, T605C and I559P (SOSIP), the hexa-arginine cleavage site (R6), the introduced isoleucine-zipper motif (IZ) and the histidine tag (H8). (C) Western blot analysis of protein expression (SDS-PAGE) and oligomerization (Blue-Native PAGE) of Env_Bx08 constructs, encoding gp120, gp140, gp140_SOSIP.R6 and gp140_{SOSIP.R6-IZ-H8}.

Figure 4

Comparison of the immune response in vaccinated guinea pigs (A,B) and rabbits (C,D) with plasmid DNA encoding syn.gp140_mix or syn.gp140_{mix modified.} Average IgG response against recombinant gp120_IIIb (rgp120_IIIb) in immunized (A) guinea pigs (n = 4) and (C) rabbits (n = 4). Immunization time points are indicated with arrows. Asterisk indicates significant difference between the two immunization groups (* p < 0.05, ** p < 0.01, two-way ANOVA). Average neutralizing activity, expressed as IC50, of (B) diluted guinea pig serum or (D) purified rabbit IgG from week 14 animal sera against pseudotype virus strains of clade A–D and CRF02_AG. Amphotropic murine leukemia virus (MLV) pseudotype virus was included as control for the non-specific activity in experiments with guinea pig serum (red). Results from syn.gp140_mix immunizations were derived from Figure 2.

Figure 5

Comparison of immune response in guinea pigs, rabbits and cynomolgus macaques immunized with plasmid DNA encoding syn.gp140_mix. (A) Average IgG response against rgp120_IIIb in immunized animals (n = 4). Immunization time points are indicated with arrows. IgG titers in rabbits and guinea pigs were derived from Figure 2A,C. (B) Average percent neutralization against pseudotype virus strains of clade A–C, by week 14 rabbit IgG or guinea pig sera and week 17 macaque sera. From rabbit sera, IgG was purified and used in neutralization at one fixed concentration (250 or 400 µg/mL). Sera from guinea pigs and macaques were diluted 20 and 30 times, respectively, and used in neutralization. Neutralization results of rabbit and guinea pigs were derived and recalculated from Figure 2B,D. (C and D) Macaque sera was tested for neutralization at 1/30 dilution against SF162 and MW965 viruses with the addition of a final immunization with DNA and protein at w17 (* p < 0.001, One-way ANOVA, Friedman’s test with Dunn’s Multiple Comparison Test).