Development and optimization of a sensitive pseudovirus-based assay for HIV-1 neutralizing antibodies detection using A3R5 cells - PubMed (original) (raw)

Development and optimization of a sensitive pseudovirus-based assay for HIV-1 neutralizing antibodies detection using A3R5 cells

Qingqing Chen et al. Hum Vaccin Immunother. 2018.

Abstract

Sensitive assays for HIV-1 neutralizing antibody detection are urgently needed for vaccine immunogen optimization and identification of protective immune response levels. In this study, we developed an easy-to-use HIV-1 pseudovirus neutralization assay based on a human CD4+ lymphoblastoid cell line A3R5 by employing a high efficient pseudovirus production system. Optimal conditions for cell counts, infection time, virus dose and concentration of DEAE-dextran were tested and identified. For T-cell line-adapted tier 1 virus strains, significantly higher inhibitory efficiency was observed for both monoclonal neutralizing antibody (4 fold) and plasma (2 fold) samples in A3R5 than those in TZM-bl assay. For circulating tier 2 strains, the A3R5 pseudovirus assay showed even much higher sensitivity for both neutralizing antibody (10 fold) and plasma (9 fold) samples. When sequential neutralizing antibody seroconverting samples were tested in both A3R5 and TZM-bl assays, the seroconverting points could be detected earlier for tier 1 (15.7 weeks) and tier 2 (68.3 weeks) strains in A3R5 assay respectively. The high sensitive pseudovirus assay using more physiological target cells could serve as an alternative to the TZM-bl assay for evaluation of vaccine-induced neutralizing antibodies and identification of the correlates of protection.

Keywords: A3R5; HIV-1; TZM-bl; neutralizing antibody; pseudovirus.

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Figures

Figure 1.

Figure 1.

Optimization of cell numbers for the A3R5 pseudovirus assay. (A) Influence of cell numbers on neutralization results when tested against monoclonal neutralizing antibody samples (PG9 and 2F5) with pseudovirus 11036. (B) Influence of cell numbers on neutralization results when tested against serum samples (HB118 and BJ170) with 11036. (C) Effect of the cell density on the fitness of the neutralization curve.

Figure 2.

Figure 2.

Optimization of the pseudovirus input for the A3R5 pseudovirus assay. (A) The neutralization curve under different pseudovirus inoculum. (B) Effect of the pseudovirus input amount on the fitness of the neutralization curve.

Figure 3.

Figure 3.

Optimization of DEAE-dextran concentration for the A3R5 pseudovirus assay. Effect of the DEAE-dextran concentration on infectivity of the pseudovirus (A) and the viability of the A3R5 cells (B).

Figure 4.

Figure 4.

Determination of incubation time for the pseudovirus A3R5 assay. Relationship between the RLU and the incubation time for pseudovirus 39–14 (A) and 11306 (B).

Figure 5.

Figure 5.

Infectivity of pseudoviruses to A3R5 cells. Pseudoviruses were diluted at 5 fold in triplicate and infected A3R5 cell. The average relative luminescent unit (RLU) values are indicated. (A) The infectivity of pseudoviruses from different subtypes. (B) The infectivity of pseudoviruses from different infection phases. (C) The infectivity of pseudoviruses with different neutralization sensitivities.

Figure 6.

Figure 6.

Validation of the pseudovirus A3R5 assay. (A) specificity: 20 HIV-1-negative plasma samples were tested against a panel of seven pseudoviruses (SF162, Bal26 from tier 1 subtype B; the remaining from tier 2 strains, 11317 from subtype C; 11056, 11058 from subtype B; BJ5.11, GX24.8 from CRF01_AE). (B) Reproducibility: two plasma samples (HJ182 and HB188) were employed to test against pseudovirus 11036. Each plasma sample was tested ten times in three independent runs by different operators.

Figure 7.

Figure 7.

Comparison of the pseudovirus A3R5 assay with the TZM-bl assay tested against tier 1 pseudoviruses. Five NAbs (2F5, 4E10, PG16, 2G12 and b12) were tested against two pseudoviruses SF162 (A) and Bal.26 (B). Five plasma samples (HB4, HB118, HB120, BJ182 and TJ208) were tested against the same two pseudoviruses SF162 (C) and Bal.26 (D). The diagonal line depicts x = y (TZM-bl ID50/IC50 = A3R5 ID50/IC50 values). Statistical analyses for NAb (E) and plasma (F) samples were conducted respectively (Wilcoxon matched pairs test, * for p<0.05, and ** for p < 0.01).

Figure 8.

Figure 8.

Comparison of the pseudovirus A3R5 assay with the TZM-bl assay tested against tier 2 pseudoviruses. Five NAbs were tested against four tier 2 pseudoviruses 11036 (A), 11058 (B) and 11317 (C), and 11506 (D). Five plasma samples were tested against the same four pseudoviruses 11036 (E), 11058 (F) and 11317 (G), and 11506 (H). The diagonal line depicts x = y (TZM-bl ID50/IC50 = A3R5 ID50/IC50 values). Statistical analyses for NAb (I) and plasma (J) samples were conducted respectively (Wilcoxon matched pairs test, *** for p<0.001=.

Figure 9.

Figure 9.

Comparison of A3R5 and TZM-bl assay using NAb seroconverting samples against tier 1 pseudoviurs. Serial NAb seroconverting plasma samples collected from 8 CRF01_AE-infected individuals were tested against pseudovirus SF162 in both A3R5 and TZM-bl assays. The dash and full lines indicate the cutoff values for A3R5 and TZM-bl assay respectively.

Figure 10.

Figure 10.

Comparison of A3R5 and TZM-bl assay using NAb seroconverting samples against tier 2 pseudoviurses. Serial NAb seroconverting plasma samples were tested against 3 tier 2 pseudoviruses in both A3R5 and TZM-bl assays. The dash and full lines indicate the cutoff values for A3R5 and TZM-bl assay respectively.

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References

    1. Plotkin SA. Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis. 2008;47(3):401-9. doi:10.1086/589862. PMID:18558875 - DOI - PubMed
    1. Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, Wang SK, Ramos A, Chan-Hui PY, Moyle M, et al.. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011;477(7365):466-70. doi:10.1038/nature10373. PMID:21849977 - DOI - PMC - PubMed
    1. Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, et al.. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326(5950):285-9. doi:10.1126/science.1178746. PMID:19729618 - DOI - PMC - PubMed
    1. Stamatatos L, Morris L, Burton DR, Mascola JR. Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine? Nat Med. 2009;15(8):866-70. PMID:19525964 - PubMed
    1. Ferrantelli F, Rasmussen RA, Buckley KA, Li PL, Wang T, Montefiori DC, Katinger H, Stiegler G, Anderson DC, McClure HM, et al.. Complete protection of neonatal rhesus macaques against oral exposure to pathogenic simian-human immunodeficiency virus by human anti-HIV monoclonal antibodies. J Infect Dis. 2004;189(12):2167-73. doi:10.1086/420833. PMID:15181562 - DOI - PubMed

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