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. 2012 Aug;86(16):8835-47.
doi: 10.1128/JVI.00923-12. Epub 2012 Jun 13.

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Fan Wu  1 , Ilnour Ourmanov, Takeo Kuwata, Robert Goeken, Charles R Brown, Alicia Buckler-White, Ranjini Iyengar, Ronald Plishka, Scott T Aoki, Vanessa M Hirsch
Affiliations

Sequential evolution and escape from neutralization of simian immunodeficiency virus SIVsmE660 clones in rhesus macaques

Fan Wu et al. J Virol. 2012 Aug.

Abstract

Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.

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Figures

Fig 1
Fig 1
Peripheral blood mononuclear cell (PBMC) infection with SIVsmE660 clones. PBMCs were stimulated with 10% IL-2 for 3 days and infected with SIVsmE660 clones at a multiplicity of infection (MOI) of 0.001. Noninfected PBMCs were used as negative controls, and PBMCs infected with SIVsmE660 stock virus were used as positive controls. Virus production was quantified and shown as RT values in supernatants collected at 3-day intervals.
Fig 2
Fig 2
Replication of SIVsmE660 clones in rhesus macaques. (A) Viral loads were quantified and shown as numbers of RNA copies per ml in plasma samples. (B) SIV RNA-positive cells (black spots) were detected by ISH staining from lymph nodes collected at day 10 from macaque RH805. (C to E) Peripheral CD4+ T cells (C), peripheral naïve CD4+ T cells (CD4+ CD28+ CD95−) (D), and peripheral memory CD4+ T cells (CD4+ CD28+ CD95+) (E) were quantified by FACS and shown as the absolute numbers per microliter of blood. (F) Percentages of CD4+ cells in BAL fluid were quantified by FACS.
Fig 3
Fig 3
ID50 titers of sera against parental SIVsmE660 clones. Serum samples were collected at 0, 4, 8, 16, and 24 w.p.i and tested against the parental SIVsmE660 clones FL6 and FL14 by a TZM-bl neutralization assay. (A) Serum samples collected from Rh805 were tested against SIVsmE660-FL6. ID50 titers were calculated with nonlinear regression by PRISM5 and expressed as the reciprocals of the highest dilutions of sera which mount a 50% reduction in RLU from that of the virus control. (B) ID50 titers of sera from infected macaques against the inoculated parental clones.
Fig 4
Fig 4
Diversity of Env sequences. Env sequences from Rh805 (A) and Rh807 (B) at 2, 4, 8, 16, and 24 w.p.i. were aligned with Env sequences from SIVsmE660-FL6 or SIVsmE660-FL14. Phylogenetic trees (right) were constructed by maximum likelihood methods using the Hasegawa-Kishino-Yano plus gamma with invariant sites (HKY+G+I) model. The left panels show nucleotide mutations in Env regions as silent or synonymous changes (green), nonsynonymous or nonsilent changes (red), and deletions (gray) in a Highlighter plot.
Fig 5
Fig 5
Alignment of Env gp120 amino acid sequences. The gp120 amino acids of typical Env variants from Rh805 and Rh806 (A) and Rh807 and Rh808 (B) were aligned to parental SIVsmE660-FL6 (A) and SIVsmE660-FL14 (B). Identical amino acids are shown as dots (.), and deletions are shown as dashes (-) and highlighted with gray. Amino acid substitutions are shown and highlighted with yellow. Potential glycosylation sites are indicated with asterisks (*) in SIVsmE660-FL6 and -FL14, and mutations resulting in a change of potential glycosylation sites are highlighted with red in the Env variants.
Fig 5
Fig 5
Alignment of Env gp120 amino acid sequences. The gp120 amino acids of typical Env variants from Rh805 and Rh806 (A) and Rh807 and Rh808 (B) were aligned to parental SIVsmE660-FL6 (A) and SIVsmE660-FL14 (B). Identical amino acids are shown as dots (.), and deletions are shown as dashes (-) and highlighted with gray. Amino acid substitutions are shown and highlighted with yellow. Potential glycosylation sites are indicated with asterisks (*) in SIVsmE660-FL6 and -FL14, and mutations resulting in a change of potential glycosylation sites are highlighted with red in the Env variants.
Fig 6
Fig 6
Neutralization of sera against clones from different time points. Serum samples from Rh805 (A) and Rh807 (B) were tested against selected clones from the same macaque at different time points by the TZM-bl neutralization assay. ID50 titers were calculated and are shown. Viruses tested are identified in the legend by the rhesus ID, the plasma sample that was the source of the envelope clone, and a clone number (e.g., 805 24w-2).
Fig 7
Fig 7
Neutralization of late-stage sera against Env escape variants. Serum samples from Rh805, Rh806, Rh807, and Rh808 at a late stage were tested against selected escape Env variants. ID50 titers were calculated and are shown. Viruses tested are identified in the legend by the rhesus ID, the plasma sample that was the source of the envelope clone, and a clone number (e.g., 805 24w-2).
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