Naturally occurring V1-env region variants mediate simian immunodeficiency virus SIVmac escape from high-titer neutralizing antibodies induced by a protective subunit vaccine

Abstract

Macaques which developed high-titer neutralizing antibodies (htNAb) after immunization with a virion-derived oligomeric envelope glycoprotein subunit vaccine were protected against a homologous simian immunodeficiency virus SIVmac challenge. Here we demonstrate that the htNAb could be overcome by V1-env region variants isolated ex vivo from an SIVmac-infected macaque. The results further suggest that the development of V1-env region neutralization escape mutants is also necessary for survival of the virus in infected macaques. The immunological capacity of a single variable region to induce neutralizing antibodies in vaccinated and infected macaques initiate new ideas for a successful vaccine strategy.

FIG. 1

Construction of V1 chimeras. 1, each V1 region was amplified with the primer pair P5-P6; 5′ and 3′ flanking regions were amplified with primer pairs P1-P2 and P3-P4, respectively. 2, the V1 regions were introduced into the molecular clone SIVmac239 by recombinant PCR. 3, alignment of the V1 regions used for the chimeras, depicted in amino acid single-letter code.

FIG. 2

Production of infectious virus stocks. COS-7 cells were transfected with the different viral clones. Supernatants were harvested over 6 days, and virus production was measured in triplicate by a commercially available p24 antigen ELISA. Here and in subsequent figures, OD₄₅₀ denotes optical density at 450 nm.

FIG. 3

Viral replication in permanent human T-cell lines. Virus-containing supernatants from COS-7 cells were normalized for p27 concentration and used to infect the CD4⁺ cell lines CEMx174 and HUT-78. Virus production was measured in triplicate over 31 days by a commercially available p24 antigen ELISA. Values represent the average and standard deviation from the mean of three experiments.

FIG. 4

Coreceptor-dependent viral replication. Virus-containing supernatants from COS-7 cells were normalized for p27 concentration and used to infect U87.CD4 cells expressing CCR5 or CXCR4. Virus production was measured in triplicate over 17 days by a commercially available p24 antigen ELISA. Values represent the average and standard deviation from the mean of three experiments.

FIG. 5

Neutralization (MT-4 cell) assay. Neutralization was detected with different dilutions of sera from SIVmac-infected macaques Mm1708 and Mm1604 or O-gp130-immunized macaques Mm1698, Mm1701, and Mm1715. Dotted lines indicate the cutoff at 75 and 90% virus neutralization, respectively. Each NAb titer is the mean of three serum samples examined in parallel. In 82% of the assays, the mean deviation was 0; in the remaining assays, the results differed by one dilution step.

FIG. 6

Antibody binding to GST-V1 fusion proteins. GST-V1 fusion proteins were expressed in E. coli, affinity purified, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Fusion proteins were stained with Coomassie blue (A) and detected with sera from SIVmac-infected Mm1604 (B) or SIVmac infected-Mm1708 (C) on the day of infection or 52 wpi, respectively, and with sera from the O-gp130-immunized animal Mm1698, Mm1701, or Mm1715. All sera were diluted 1:200 for Western blot analysis.

FIG. 7

Alignment of predicted proviral V1 amino acid sequences (in single-letter code) from SIVmac-infected rhesus macaque Mm1604 and from SIVmacV1-1708/2, SIVmacV1-1708/4, and SIVmacV1-32H. Comparison was done with the program PILEUP of the Wisconsin Package (Genetics Computer Group, Madison, Wis.). Amino acid identities and deletions are denoted by dashes and periods, respectively.