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. 2016 Jun 24;90(14):6276-6290.
doi: 10.1128/JVI.00557-16. Print 2016 Jul 15.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization

Sumire Iseda  1   2 , Naofumi Takahashi  1 , Hugo Poplimont  1   2 , Takushi Nomura  1 , Sayuri Seki  1 , Taku Nakane  1   2 , Midori Nakamura  1 , Shoi Shi  1   2 , Hiroshi Ishii  1 , Shota Furukawa  1 , Shigeyoshi Harada  1 , Taeko K Naruse  3 , Akinori Kimura  3 , Tetsuro Matano  1   2 , Hiroyuki Yamamoto  4
Affiliations

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization

Sumire Iseda et al. J Virol. .

Abstract

Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control.

Importance: In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

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Figures

FIG 1
FIG 1
Sustained SIV replication control by acute-phase NAb passive immunization. (A) Long-term follow-up of plasma viral loads (pVL) after SIVmac239 challenge (SIV gag RNA copies/ml plasma) in macaques that had been infused with 300 mg of anti-SIV NAbs at week 1 postchallenge (red), showing sustained control in animals possessing four different MHC-I haplotypes. pVL in MHC-I haplotype-matched, NAb-uninfused animals are shown in gray. Viral loads were determined as described previously (25). The lower detection limit was approximately 4 × 102 copies/ml. R06-019 and R06-038 had been infused with 300 mg of nonspecific control IgG at week 1. The dotted lines show geometric mean viral loads for the previously reported haplotype-possessing naive infected animals (n ≥ 5) (19, 20). (B) Comparison of peak (weeks 1 and 2), set point (week 12), and chronic-phase (geometric [geo] mean after year 1) pVL in NAb-uninfused and infused animals (paired t tests). Viral loads below the detection limits were calculated as the lower limit of detection (4 × 102 copies/ml). A significant difference was observed in the chronic phase. (C) NAb titers during viremia control. Titers were examined as 100% SIVmac239-neutralizing endpoints by a 10-TCID50 killing assay on MT4-R5 cells (3, 17, 18). The lower detection limit was 1:2; no de novo NAb induction was observed in NAb-infused controllers. For macaques R06-038 and R03-013, euthanized at weeks 95 and 90 postinfection, the week 95 and 90 samples were used for the data around year 2. N.D., not determined. (D) Virus-binding IgG responses assessed by immunoblotting. Antibody responses were detected using a commercial Western blotting system (ZeptoMetrix). Levels of Gag/Pol and Env antigen binding by plasma samples are shown, separated by slashes. ++, bands detected for more than two antigens; +, bands detected for one or two antigens; −, no bands detected. A week 5 plasma sample for R06-038 was unavailable.
FIG 2
FIG 2
Viral mutation patterns before SIV control. Amino acid sequences of plasma SIVmac239 vRNA in NAb-infused SIV controllers at week 8 postchallenge (direct sequencing) are shown. Pink stripes, dominant amino acid mutations; black stripes, subdominant amino acid mutations. The red and blue triangles beneath the sequences show the first-line and second-line, respectively, immunodominant CTL epitopes aligned. Mutations selected within the epitopes are shown in corresponding colored stripes (unified for dominant and subdominant selection). *, CTL-unrelated viral-fitness mutations (58); Δ, residue deletions.
FIG 3
FIG 3
Viral mutation patterns within CTL epitopes in NAb-uninfused and infused animals. (A) Amino acid sequences encoded by plasma vRNA from NAb-uninfused macaques during persistent infection at week 8 and years 1.5 to 2 postchallenge. (B) Amino acid sequences encoded by plasma vRNA from SIV controller R03-013 before and during viremia rebound. (C) Immunodominant CTL epitope amino acid sequences encoded by proviral DNA from CD4+ T cells of controller PBMCs around year 2. For R03-013, the data at week 90 are shown. (A and B) Dominant substitutions are colored corresponding to the CTL epitope colors in Fig. 2A. Subdominant substitutions are in black.
FIG 4
FIG 4
SIV-specific T-cell responses during NAb-induced SIV control. (A) SIV Gag, Pol, Vif, Vpx, Vpr, Tat, Rev, Env, and Nef proteome-specific IFN-γ+ CD8+ T-cell responses in PBMCs of NAb-uninfused and infused animals at around year 2 post-SIV challenge. A+, D+, E+, and H+ represent 90-120-Ia+, 90-010-Id+, 90-010-Ie+, and 90-030-Ih+, respectively, comprising immunodominant epitope-specific CTLs in each MHC-I haplotype. These proteins are referred to as “immunodominant.” (B) Comparison of total SIV-specific CD8+ T-cell responses and immunodominant CD8+ T-cell targeting frequencies (paired t tests; MHC-I paired) in NAb-uninfused and infused animals around year 2 postinfection. The latter was calculated as follows: percentages of responses against immunodominant proteins, including the identified CTL epitopes, divided by total SIV-specific CD8+ T-cell responses. (C) SIV proteome-specific IFN-γ+ CD8+ T-cell responses at week 16. (D) Temporal curve of SIV-specific CD8+ T-cell subdominance (percentages within total SIV protein-specific CD8+ T-cell responses) (left) and comparison of subdominance deviation (percentages) from week 16 to around year 2 in NAb-uninfused and infused animals (paired t test; MHC-I paired) (right). (E) SIV proteome-specific IFN-γ+ CD4+ T-cell responses at around year 2 in NAb-uninfused and infused animals. (F) Comparison of total SIV-specific CD4+ T-cell responses in NAb-uninfused and infused animals around year 2 postinfection (paired t test; MHC-I paired). For macaques R06-038, R04-013, and R03-013, euthanized before 2 years postinfection, samples at years 1.5 to 2 were used for the data around year 2.
FIG 5
FIG 5
NAb-mediated enhancement of DC antigen cross-presentation to CD8+ T cells in vitro. (Left) Specific T-cell responses in effector PBMCs (SIV controller R07-006; week 48) (23) cocultured with VSV-pseudotyped SIV-infected autologous B-LCLs used as surrogate target cells. (Right) NAb- and FcγRI-dependent facilitation of stimulation-specific MIP-1β induction in the same effector under coculture with autologous MDDCs pulsed with the indicated antigens. Activation of CD8+ T cells upon encounter with antigen-loaded DCs is observed when an effector molecule (here, MIP-1β) different from IFN-γ is assessed. The data are representative of two (left) or four (right) experiments with similar results performed in duplicate. The error bars indicate standard error of the mean (SEM).
FIG 6
FIG 6
CTL escape mutant SIV suppression in vitro by early CD8+ cells in SIV controllers. (A) CD8+ cell-mediated suppression of replication of SIVs carrying single CTL escape mutations. CD8+ cells at week 5 postchallenge were assessed in NAb-uninfused and infused animal pairs. Fold reductions of SIV p27 in culture supernatants post-CD8+ effector/SIV-preinfected CD8− target cell coculture versus the levels in CD8− target cell cultures at day 8 are shown. Mean values of triplicate CD8+ cell-CD8− target cell cocultures are shown for each CTL escape mutant virus in the corresponding MHC-I-matched animal pairs. (B) Comparison of CTL escape mutant SIV suppression levels at week 5 (epitopes were pooled; paired t test; data were paired for evaluated mutant SIVs).
FIG 7
FIG 7
pERK-pAMPK patterns depicting epitope-specific CTLs in sustained SIV control. (A) Immunodominant epitope-specific CTL levels in PBMCs in three NAb-uninfused and infused pairs at around year 2 (y axes, log10 scale). Samples around weeks 87 to 123 are shown as “around yr 2.” (B) Comparison of pooled epitope-specific CTL levels around year 2 (paired t test). (C) Representative gating of epitope-specific IFN-γ+ CD8+ T cells and their intracellular expression of pERK, pS6RP, PTEN, and pAMPK (R03-011; week 90; Nef121–129 specific). (D) Comparison of pAMPK expression levels within total pERKhi CD28− epitope-specific CTLs between NAb-uninfused viremic animals R06-019 (Vif114–124-specific; week 95), R06-034 (Nef121–129-specific; week 96), and R06-038 (Rev7–21-specific; week 87) versus NAb-infused SIV controllers R06-003 (Gag206–216, Gag241–249, and Nef193–203 specific; week 123), R06-023 (Nef35–43 and Nef121–129 specific; week 102), and R03-011 (Nef45–53 and Nef121–129 specific; week 90). (E) Comparison of pAMPKlo pERKhi subpopulations in epitope-specific CD28− (left) and total IFN-γ+ CD8+ T cells (right) (unpaired t tests). Bars indicate mean values in individual groups.
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