Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Dec 1:13:1029029.
doi: 10.3389/fimmu.2022.1029029. eCollection 2022.

Assessment of a diverse panel of transmitted/founder HIV-1 infectious molecular clones in a luciferase based CD8 T-cell mediated viral inhibition assay

Natalia Fernandez  1 , Peter Hayes  1 , Julia Makinde  1 , Jonathan Hare  1   2 , Deborah King  1 , Rui Xu  3 , Ola Rehawi  4 , Allison T Mezzell  4 , Laban Kato  5   6 , Susan Mugaba  5   6 , Jennifer Serwanga  5   6 , James Chemweno  7 , Eunice Nduati  7 , Matt A Price  2   8 , Faith Osier  1 , Christina Ochsenbauer  4 , Ling Yue  3 , Eric Hunter  3 , Jill Gilmour  9 ; IAVI protocol C investigators
Affiliations

Assessment of a diverse panel of transmitted/founder HIV-1 infectious molecular clones in a luciferase based CD8 T-cell mediated viral inhibition assay

Natalia Fernandez et al. Front Immunol. .

Abstract

Introduction: Immunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control.

Methods: The present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects.

Results & discussion: CD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials.

Keywords: CD8 T-cells; HIV; T-cell response; infection; infectious molecular clones; transmitted founder; viral inhibition.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
HIV-1 IMC-LucR replication in expanded CD4 T-cells. Replication in expanded CD4 T-cells from ART naive subjects living with HIV-1 (on the left panel), and HIV-1 uninfected subjects (on the right panel) arranged by IMC HIV-1 clade (A-D) and Recombinant). Replication is measured as RLU of CD4 T-cell cultures at 8 days post-infection. Each data point represents the mean RLU of duplicated values for each subject’s CD4 T-cells infected with a specific HIV-1 IMC-LucR. Bars represent RLU median with interquartile range. The dotted line represents the assay optimal RLU (>1000).
Figure 2
Figure 2
Replication of HIV-1 IMC-LucR in expanded CD4 T-cells from ART naive subjects living with HIV-1 or HIV-1 uninfected subjects. (A) Spearman correlation between replication of HIV-1 IMC-LucR in expanded CD4 T-cells from ART naive subjects living with HIV-1 or HIV-1 uninfected subjects. (B) Scatter plot of the same data with medians of the groups (bar) and p value generated with a Wilcoxon test. Each data point represents the median RLU of an IMC-LucR replicating in expanded CD4 T-cells from ART naive subjects living with HIV-1 or HIV-1 uninfected subjects.
Figure 3
Figure 3
CD8 T-cell-mediated inhibition of HIV-1 replication in autologous CD4 T-cells. Inhibition in T-cell cultures from ART naive subjects living with HIV-1 (on the left panel), and HIV-1 uninfected subjects (on the right panel) arranged by IMC HIV-1 clade (A-D), and Recombinant). Inhibition is measured as log10 reduction RLU of CD4 and CD8 T-cell co-cultures compared with infected CD4 T-cells alone. Each data point represents the mean RLU of duplicated values for each CD4 T-cell infected with a specific HIV-1 IMC-LucR. Bars represent RLU median with interquartile range. The dotted line represents the assay positivity value (>0.8 log10).
Figure 4
Figure 4
Inhibition mediated by CD8 T-cells from ART naive subjects living with HIV-1 and HIV-1 uninfected subjects. (A) Spearman correlation between inhibition of HIV-1 IMC-LucR in ART naive subjects living with HIV-1 or HIV-1 uninfected subjects. (B) Scatter plot of the same data with medians of the groups (bar) and p value generated with a Wilcoxon test. Each data point represents the median log10 inhibition of an IMC-LucR mediated by CD8 T-cells from ART naive subjects living with HIV-1 or HIV-1 uninfected subjects.
Figure 5
Figure 5
Coverage distribution plot for 35 full length HIV-1 Transmitted/founder proteome sequences, representing the cumulative epitope coverage provided against a larger proteome cohort (N=218). Dotted lines reflect cumulative predictive epitope coverage for each potential panel (identified through dashed lines).
Figure 6
Figure 6
Performance of positive control CD4 and CD8 T-cell lines in the IMC-LucR VIA across 3 laboratories. (A) Replication of IMC-LucR in CD4 T-cell line measured by RLU and (B) Inhibition of IMC-LucR replication mediated by an autologous CD8 T-cell line measured by log10 reduction in RLU of CD4 and CD8 T-cell line co-cultures compared with infected CD4 T-cell line alone. Bars represent median values. Triplicate data for laboratories 1 and 2 were analysed using a Mann-Whitney test. The dotted line represents the assay positivity value (>0.8 log10).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. McElrath MJ, Haynes BF. Induction of immunity to human immunodeficiency virus type-1 by vaccination. Immun (2010) 33(4):542–54. doi: 10.1016/j.immuni.2010.09.011 - DOI - PMC - PubMed
    1. Burton DR, Ahmed R, Barouch DH, Butera ST, Crotty S, Godzik A, et al. . A blueprint for HIV vaccine discovery. Cell Host Microbe (2012) 12(4):396–407. doi: 10.1016/j.chom.2012.09.008 - DOI - PMC - PubMed
    1. Arunachalam PS, Charles TP, Joag V, Bollimpelli VS, Scott MKD, Wimmers F, et al. . T Cell-inducing vaccine durably prevents mucosal SHIV infection even with lower neutralizing antibody titers. Nat Med (2020) 26:932–40. doi: 10.1038/s41591-020-0858-8 - DOI - PMC - PubMed
    1. Collins DR, Gaiha GD, Walker BD. CD8(+) T cells in HIV control, cure and prevention. Nat Rev Immunol (2020) 20(8):471–82. doi: 10.1038/s41577-020-0274-9 - DOI - PMC - PubMed
    1. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, et al. . HIV Nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood (2006) 107(12):4781–9. doi: 10.1182/blood-2005-12-4818 - DOI - PMC - PubMed

Publication types