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. 2001 Jul;75(13):5879-90.
doi: 10.1128/JVI.75.13.5879-5890.2001.

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques

D C Montefiori  1 , J T Safrit, S L Lydy, A P Barry, M Bilska, H T Vo, M Klein, J Tartaglia, H L Robinson, B Rovinski
Affiliations

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques

D C Montefiori et al. J Virol. 2001 Jul.

Abstract

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.

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Figures

FIG. 1
FIG. 1
Western blot analysis of HIV-1 antigen-specific antibodies. Serum samples were assayed at a 1:100 dilution. Panels: A, sera obtained at week 6; B, sera obtained at week 46. wks, weeks; HIV−, HIV negative; HIV+, weakly HIV positive; HIV++, strongly HIV positive.
FIG. 2
FIG. 2
Neutralization of HIV-1Bx08 by sera from immunized animals. Bar height represents the percent reduction in p24 relative to the amount of p24 produced in the presence of the corresponding preimmunization serum from each animal as assayed at a 1:2 dilution. Values above the bars are the highest serum dilutions at which p24 production was reduced by 80%. Panels: A, animal groups with detectable neutralizing antibodies; B, animal groups with no detectable neutralizing antibodies.
FIG. 3
FIG. 3
Bx08-V3 peptide-binding antibodies. Serum samples obtained 2 weeks after the fourth immunization (week 46) were tested at a 1:50 dilution for antibodies reactive with a peptide corresponding to the V3 loop of HIV-1Bx08. Bar height represents the average A450 of duplicate tests. All duplicate values agreed within 5% of the average. The value obtained with serum from an HIV-1-naive macaque is shown by a dashed line. Values greater than twice that of the negative control were considered to be positive. Serum samples that neutralized HIV-1Bx08 are indicated by asterisks above the bars. OD, optical density; grp, group.
FIG. 4
FIG. 4
Inability to detect neutralizing antibodies specific for the HIV-1Bx08 V3 loop. Two serum samples that contained antibodies reactive with a Bx08 V3 peptide and that neutralized HIV-1Bx08 were tested for neutralizing activity in the presence and absence of the Bx08 V3 peptide. Samples were preincubated with either peptide (50 μg/ml) or an equal volume of sterile PBS for 1 h at 37°C and assayed at a 1:3 dilution. Both postimmunization serum samples were obtained at week 46 (2 weeks after final boosting). Dark-shaded bars, no V3 peptide; light-shaded bars, with V3 peptide; pre, preimmunization serum; post, postimmunization serum. Each error bar represents the standard deviation of the average of triplicate values.
FIG. 5
FIG. 5
Number of HIV-1 Gag-specific IFN-γ-producing cells detected by ELISpot assay with PBMC from individual animals. Solid bars represent cells stimulated with peptide pool 1, covering the amino-terminal half of Gag. Hatched bars represent cells stimulated with peptide pool 2, covering the carboxyl-terminal half of Gag. Values on the y axis are numbers of IFN-γ-producing cells per million cells. ND, not done.
FIG. 6
FIG. 6
Average number of IFN-γ-producing cells in each group of animals. The values below the x axis are the fractions of animals that were positive for Bx08-specific neutralizing antibodies at weeks 26 and 46.
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