Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins

Abstract

Immunization with recombinant serotype 5 adenoviral (rAd5) vectors or a combination of DNA plasmid priming and rAd5 boosting is known to elicit potent immune responses. However, little data exist regarding these immunization strategies and the development of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. We used DNA plasmids and rAd5 vectors encoding the HIV-1 89.6P or chimeric HxB2/BaL envelope glycoprotein to immunize macaque monkeys. A single rAd5 immunization elicited anti-Env antibody responses, but there was little boosting with subsequent rAd5 immunizations. In contrast, rAd5 boosting of DNA-primed monkeys resulted in a rapid rise in antibody titers, including the development of anti-HIV-1 neutralizing antibodies. The potency and breadth of neutralization were evaluated by testing plasma against a panel of 14 clade B primary isolates. Moderate levels of plasma neutralizing activity were detected against about one-third of the viruses tested, and immunoglobulin G fractionation demonstrated that virus neutralization was antibody mediated. After a challenge with a chimeric simian-human immunodeficiency virus (SHIV89.6P), an anamnestic neutralizing antibody response was observed, although the breadth of the response was limited to the subset of viruses that were neutralized after the primary immunization. These data are the first detailed description of the anti-HIV-1 neutralizing antibody response in nonhuman primates elicited by DNA and rAd5 immunization. In addition to the well-established ability of DNA priming and rAd5 boosting to elicit potent anti-HIV-1 cellular immune responses, this immunization strategy elicits anti-HIV-1 neutralizing antibodies and therefore can be used to study novel Env immunogens designed to elicit more potent neutralizing antibodies.

FIG. 1.

End-point anti-Env antibody titers in plasma against 89.6 gp140 were measured during the course of immunizations. (A) Data for two animals immunized with rAd5 encoding SIVmac239 Gag-Pol and HIV89.6P Env at 0, 8, and 26 weeks. rAd5 SIVmac239 and HIV89.6P Env were each administered at a dose of 10¹² particle units. (B) Data for two animals immunized with DNA plasmids at 0, 4, and 8 weeks and boosted with rAd5 at week 26. DNA plasmids encoded SIVmac239 Gag-Pol-Nef and HIV89.6P Env. Each plasmid was administered at a dose of 4.5 mg; the rAd5 constructs were administered as described above.

FIG. 2.

Neutralization of HIV89.6 mediated by a 1:5 dilution of plasma. The y axis shows the percent neutralization value for each plasma sample at a 1:5 dilution (based on a comparison to the corresponding preimmune plasma). The value above each bar is the reciprocal plasma titer that produced 50% neutralization (i.e., the IC₅₀ value). For each of four animals, the bars show neutralization at weeks 28 and 32, which were 2 and 6 weeks, respectively, after the last immunization.

FIG. 3.

(A) Anti-Env plasma antibody ELISA performed with BaL gp120 and 89.6P gp120. The immunization groups, immunized with either 89.6P Env or HxB2/BaL Env, are indicated within each graph. Data for all six animals in each group are shown. Low levels of anti-Env antibodies were detected after DNA plasmid immunization; a substantial boost was observed after rAd5 immunization. (B) Kinetics of neutralizing antibody responses in six monkeys that received the HxB2/BaL Env immunogen. Plasma samples from weeks 10, 27, 28, 32, and 38 were tested at a 1:5 dilution against SF162. The week 10 time point was 2 weeks after the third DNA immunization; subsequent time points were after the rAd5 booster immunization that occurred at week 26.

FIG. 4.

Plasma neutralization at week 28 of a panel of 15 clade B HIV-1 isolates. Plasma samples were tested at a 1:5 dilution, and the percent neutralization was calculated by a comparison to the corresponding preimmune plasma sample for each animal. HxB2 is a T-cell-adapted isolate, and all other HIV-1 strains are primary isolates. Each bar shows the neutralization value for one of six animals. (A) Animals that received the HxB2/BaL Env immunogen; (B) animals that received the 89.6P Env immunogen. Plasma samples demonstrating virus neutralization were retested in two to four independent neutralization experiments. For these cases, the means and SEM are shown. In some cases, little or no neutralization was observed in the first set of assays, and the assay was not repeated. An arbitrary horizontal line was drawn to show 50% neutralization.

FIG. 5.

Antibody-mediated neutralization of HIV-1 BaL and JRCSF. IgG was purified from the week 28 plasma samples of two macaques that received the HxB2/BaL Env immunogen (closed symbols) and of one animal that received the mock Env immunogen (open symbols). The Hx/BaL Env-immunized macaques were the same two animals represented by the second and third bars in Fig. 4A. Note that the starting IgG concentration of 2 mg/ml is roughly equivalent to a 1:5 plasma dilution.

FIG. 6.

Neutralizing antibodies measured after SHIV89.6P challenge. (A) IC₅₀ values (50% neutralization titers) in plasma against the SHIV89.6P challenge virus. Values are means ± SEM for six animals in each group. (B) Percent neutralization of HIV BaL mediated by a 1:5 dilution of plasma. Values are means ± SEM for six animals in each group. (C) A 1:5 dilution of plasma from week 6 postchallenge was tested against a panel of eight HIV-1 strains. The four immunization groups are shown on the x axis; each bar represents one of the eight viruses. The bars show the mean percentages of neutralization ± SEM for six animals per group against each virus. An arbitrary horizontal line was drawn to show 50% neutralization.

FIG. 6.

Neutralizing antibodies measured after SHIV89.6P challenge. (A) IC₅₀ values (50% neutralization titers) in plasma against the SHIV89.6P challenge virus. Values are means ± SEM for six animals in each group. (B) Percent neutralization of HIV BaL mediated by a 1:5 dilution of plasma. Values are means ± SEM for six animals in each group. (C) A 1:5 dilution of plasma from week 6 postchallenge was tested against a panel of eight HIV-1 strains. The four immunization groups are shown on the x axis; each bar represents one of the eight viruses. The bars show the mean percentages of neutralization ± SEM for six animals per group against each virus. An arbitrary horizontal line was drawn to show 50% neutralization.