Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Abstract

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.

FIG. 1.

Schematic representation of the experimental protocol for immunization of rhesus macaques with rBCG Env V3 and challenge with either SHIV-MN or SHIV-89.6PD. A total of 24 macaques were assigned to either the rBCG Env V3 vaccine or rBCG vector control group. The animals each received a single subcutaneous injection and were then split into three groups prior to challenge with either low-dose SHIV-MN, high-dose SHIV-MN, or SHIV-98.6PD.

FIG. 2.

Serum anti-V3 antibody titers determined by peptide-based ELISA. Preimmune and immune sera from macaques inoculated with rBCG Env V3 were collected and stored at −80°C until they were used. Sera from naïve macaques were used as controls. Data using preimmune sera were within the control levels (data not shown). The results are expressed as the means ± SD of four independent assays.

FIG. 3.

HIV-1-specific neutralization antibody responses in macaques vaccinated with rBCG Env V3. Analysis of in vitro neutralization of SHIV-MN by anti-rBCG-HIV-1 antibodies using M8166 cell-based virus neutralization assays. Serum IgG was purified from preimmune or immune sera of macaques inoculated with rBCG Env V3 at the indicated times. The results are expressed as the means ± SD of four independent assays.

FIG. 4.

Neutralization of HIV-1_BZ167 and HIV-1_SF2 in GHOST-X4 cells by immune sera from macaques vaccinated with rBCG Env V3. Dilutions of immune sera (closed symbols) and preimmune sera (open symbols) were tested in duplicate, and the percent neutralization was calculated using the mean value. The dose-response curves represent the means of three independent assays.

FIG. 5.

Alignment of the amino acid sequences of HIV-1 Env V3 from primary and laboratory isolates. The spaces indicate amino acid deletions; dashes indicate homology. The V3 motif of a neutralization-sensitive HIV-1 strain is enclosed in the shaded rectangle (37).

FIG. 6.

Comparison of infection kinetics following high-dose (200 TCID₅₀) inoculation of SHIV-MN in macaques vaccinated with either rBCG Env V3 or rBCG vector control. (A) Viral RNA copy number per milliliter of serum. (B) CD4⁺-T-cell count as a percentage of total lymphocytes. The results in individual animals are expressed.

FIG. 7.

Comparison of infection kinetics following challenge with pathogenic SHIV-89.6PD in macaques vaccinated with either rBCG Env V3 or rBCG vector control. (A) Plasma viral-RNA copy numbers per milliliter. (B) CD4⁺-T-cell count as a percentage of total lymphocytes. The results in individual animals are expressed.