Autologous neutralizing antibodies to the transmitted/founder viruses emerge late after simian immunodeficiency virus SIVmac251 infection of rhesus monkeys

Abstract

While the simian immunodeficiency virus (SIV)-infected rhesus monkey is an important animal model for human immunodeficiency virus type 1 (HIV-1) infection of humans, much remains to be learned about the evolution of the humoral immune response in this model. In HIV-1 infection, autologous neutralizing antibodies emerge 2 to 3 months after infection. However, the ontogeny of the SIV-specific neutralizing antibody response in mucosally infected animals has not been defined. We characterized the kinetics of the autologous neutralizing antibody response to the transmitted/founder SIVmac251 using a pseudovirion-based TZM-bl cell assay and monitored env sequence evolution using single-genome amplification in four rhesus animals that were infected via intrarectal inoculations. We show that the SIVmac251 founder viruses induced neutralizing antibodies at 5 to 8 months after infection. Despite their slow emergence and low titers, these neutralizing antibodies selected for escape mutants that harbored substitutions and deletions in variable region 1 (V1), V2, and V4 of Env. The neutralizing antibody response was initially focused on V4 at 5 to 8 months after infection and then targeted V1/V2 and V4 by 16 months. These findings reveal a striking delay in the development of neutralizing antibodies in SIVmac-infected animals, thus raising questions concerning the suitability of SIVmac251 as a challenge strain to screen AIDS vaccines that elicit neutralizing antibodies as a means to prevent virus acquisition. They also illustrate the capacity of the SIVmac quasispecies to modify antigenic determinants in response to very modest titers of neutralizing antibodies.

FIG. 1.

Autologous neutralizing antibodies after SIVmac251 infection. Plasmas from 4 SIVmac251-infected animals, PBE (A), CP1W (B), CR2A (C), and CR53 (D), were tested at multiple time points p.i. for neutralizing antibodies against the founder env-pseudotyped virus isolated from matched animals. Percent neutralization is plotted along the y axes, and the reciprocal log₁₀ plasma dilution (from 10¹ to 10⁸) is plotted along the x axes. The dotted lines indicate 50% neutralization. The gray symbols and lines indicate time points when the reduction in neutralization was below the 50% neutralization level (ID₅₀ titers < 10), and the black symbols and lines indicate time points when the reduction in neutralization scored above 50%. ID₅₀ titers are indicated in parentheses.

FIG. 2.

Phylogenetic analysis of env quasispecies from the 4 monkeys infected with SIVmac251. The maximum-likelihood tree is rooted on the consensus of the inoculum (blue circles). Sequences from each monkey clustered together and are represented by similarly colored symbols, as indicated. The transmitted viruses at peak viremia in each monkey are represented by open circles, viruses from 5 to 8 months (Mo) p.i. by open triangles, and quasispecies from 16 months p.i. by open squares and 22 months p.i. by closed squares. The scale bar indicates a genetic distance of 0.001 (1 nucleotide difference per 1,000 sites) in the phylogram.

FIG. 3.

Accumulation of env nucleotide sequence changes in replicating viruses after intrarectal SIVmac251 infection. (A) Highlighter alignment of env genes that were transmitted from the inoculum (black rectangle at top right of each alignment), at peak viremia (dark blue rectangles), or 5 or 8 (green rectangles), 16 (light blue rectangles), and 22 (yellow rectangles) months p.i. in animals PBE and CP1W. Each horizontal line on the vertical axis depicts individual Env variants sequenced at various time points postinfection. Identical sequences were removed for clarity. The red tic marks indicate differences that are nonsynonymous in the env coding frame. Deletions are indicated by gray tics. The horizontal axis indicates the amino acid position in the Env alignment. (B) Identification of putative sites of positive selection using three independent tests. The horizontal axis indicates the nucleotide positions in env, aligned to SIVmac239 (nt 6604 to 9108), and the vertical axis indicates the statistical significance for each of the three tests. The tests for enriched mutations and clustered mutations (turquoise and purple bars, respectively) used sliding windows of 45 and 27 nt, respectively. The FEL test from HYPHY is a tree-based statistic that identifies single codons under positive selection (green bars). For reference, the black boxes depict the locations of V1 to V5 in gp120, and the horizontal lines depict the locations of gp41, tat exon 2, rev exon 2, and nef (in reading frames 1, 3, 2, and 3, respectively). The asterisks denote Env variants from each animal that were cloned for further neutralization testing.

FIG. 4.

Accumulation of env nucleotide sequence changes in replicating viruses after infection. (A) Highlighter alignment of env genes from animals CR2A and CR53. The time points p.i. when env genes were sequenced are indicated. The red tic marks represent nonsynonymous mutations, while deletions are indicated by gray tics. (B) Identification of putative sites of positive selection using nonrandom-mutation and clustered-mutation statistics and the FEL test. The asterisks denote Env variants from each animal that were cloned for further neutralization testing.

FIG. 5.

Comparison of the amino acid sequences of the variable regions of SIVmac251 gp120. The sequences of representative Env clones obtained throughout infection are aligned to the Env that was transmitted to each animal from the SIVmac251 inoculum, which is shown at the top. The first amino acid residue in the alignment represents residue number 101 in the SIVmac239 Env. The name of each Env sequence identifies the monkey and the time point when the variant was isolated. The dots represent amino acid identity and the dashes represent deletions. Variable regions V1 through V5 are boxed. The amino acid residue number is on the right of each row. Yellow highlights amino acids that represent a consensus sequence for PNGs that have been shifted, deleted, or added. PNGs in variable regions that remained unchanged throughout infection are highlighted in gray. A potential O-linked glycan attachment site in the V1 region is underlined. These Env variants were cloned for further neutralization testing.

FIG. 6.

Neutralizing antibodies against autologous viral variants. The panels show the ability of autologous plasma sampled over time following infection to neutralize the founder virus and mutant viruses in animals PBE (A), CP1W (B), CR2A (C), and CR53 (D). The x axes indicate the times when plasma samples were collected, and ID₅₀ titers are shown on the y axes. The ID₅₀ titers against the autologous founder Envs over the course of infection are represented by solid lines and filled circles, while the neutralization titers against the mutant Env are depicted by dotted lines and open symbols. The time points p.i. when viral variants were isolated and the mutations/deletions these mutant viruses contain are indicated in parentheses.