AMS Professorship Award for Professor Christine Goffinet, Liverpool School of Tropical Medicine

The HIV-1 pandemic has been one of the most devastating health crises of modern times. An estimated 38 million people are currently living with HIV-1 worldwide. The burden has been particularly severe in sub-Saharan Africa.

While combinatory antiretroviral therapy (cART) has dramatically improved the life quality and expectancy of people living with HIV-1 (PLHIV), its current implementation alone is insufficient to end the HIV-1 pandemic. Many regions, particularly in low- and middle-income countries, face barriers to obtaining and affording treatment.

Limited healthcare infrastructure, inadequate resources, high drug costs and resistance development of cART pose substantial challenges. Finally, cART is not curative.

The challenge in establishing an HIV-1 cure approach lies in the virus persisting in reservoirs even during cART, predominantly in long-lived resting CD4+ memory T-cells.

The hallmark of persistent, latent HIV-1 infection is integrated viral DNA in infected cells´ genome with limited, if at all, expression of viral RNA and proteins.

The majority of latently infected cells is thus immunologically invisible and unsusceptible to cytolytic killing. “Shock-and-kill” is one of the most conceptually advanced strategies towards HIV-1 cure.

Treatment of PLHIV with latency-reverting agents (LRAs), combined with cART, reactivates HIV-1 transcription in vivo, however, fails to reduce the size of the viral reservoir, indicating its insufficiency in terms of elimination of latently infected cells.

While elements of cell-intrinsic innate immunity clearly limit virus spread during acute infection, our understanding of their multifactorial impact on HIV-1 latency regulation is limited.

Previous work from the group established that specific LRAs dramatically suppress the cell-intrinsic antiviral state, which is beneficial in terms of efficient viral reactivation, but disadvantageous in terms of elimination of reactivated cells.

In this project conducted at the LSTM, we plan to identify, characterize and modulate key cellular factors and parameters involved in the regulation of HIV-1 latency. The project structure encompasses three parallel arms with partially complementing methodology: Aim 1.

Using targeted and unbiased approaches in CD4+ T-cells from PLHIV and diverse cellular models of HIV-1 latency, we will identify and validate cellular factors that facilitate reversal of proviral quiescence and increase the susceptibility of HIV-1-reactivating CD4+ T-cells to immune cell-mediated cytotoxicity.

We recently established a list of human genes being differentially expressed in HIV-1 RNA-positive CD4+ T-cells from aviremic PLHIV, as opposed to their T-cells with undetectable HIV-1 RNA. Furthermore, expression of some genes correlated with intracellular HIV-1 RNA abundance, suggesting co-regulation.

These genes represent potential biomarkers of the transcriptionally competent reservoir.

In a parallel, unbiased approach, we will conduct a genome-wide CRISPR-Cas9 knock-out screen to identify genes that regulate HIV-1 latency and/or predict the HIV-1 latency status.

The role of candidate genes in HIV-1 latency establishment, maintenance and reversal will be probed in functional (knock-out, heterologous expression, CRISPa-induced expression) and mechanistic (mutations, subcellular localization, druggability) assays. Aim 2.

Reversal of HIV-1 quiescence is almost exclusively studied in the context of pharmacological reactivation via addition of LRAs, which however induce a plethora of gene expression changes per se.

Using single-cell omics, we will identify perturbations of the intra-T-cellular milieu induced specifically upon reactivation of transcription from the HIV-1 LTR promoter.

This attempt will likely lead to identification of virus-mediated strategies towards evasion from immune recognition, which vice versa could be pharmacologically targeted in order to improve elimination of reactivating cells in an HIV-1 cure approach.

To this end, we will develop a CRISPRa-based approach using HIV-1 LTR-targeting guides to directly stimulate HIV-1 transcription and monitor resulting cellular chromatin accessibility and gene expression changes, in relation to the degree and quality of HIV-1 reactivation. Aim 3.

Finally, we will rank existing and newly identified “shock-and-kill” strategies in a multidimensional cellular latency model that takes into account all consecutive steps of HIV-1 reactivation.

We will capitalize on our recently established experimental platform, consisting of 70 latently HIV-1-infected Jurkat T-cell clones each with a unique proviral integration site of a recombinant HIV-1 which expresses an EGFP OPT in HIV-1 Env.

This fully infectious virus allows identifying latency reversal by high-throughput EGFP OPT FACS and, simultaneously, sensitive quantification of cell surface expression of HIV-1 Env EGFP OPT via immunostaining with anti-GFP antibodies.

We will merge cellular and viral transcriptomic data with corresponding Env EGFPOPT surface levels at the single-cell level using the feature barcoding approach.

In addition, antibody-dependent cytotoxicity experiments will define clone- and LRA-specific susceptibility to immune elimination.

This work has the potential to uncover so far unappreciated pitfalls of currently applied shock-and-kill-regimen, which might include LRA-increased resistance to immune cytotoxicity despite efficient reactivation.

Furthermore, it will generate several hypotheses and work models how to improve and advance The added value of an AMS Professorship will be manifold.

First, since I am starting a Professor position in a (for me) new academic system and I am able to transfer only a limited number of active grants to LSTM, I highly appreciate the welcoming character of this call being focused on researchers moving from overseas to the UK.

Therefore, I assume that AMS Professorship Scheme awardees are curious researchers, who look forward to move out of their comfort zone, are keen to explore new environments and widen their scientific and personal horizon.

Funding will provide me a financial security to smoothly start one my research projects which is closest to my heart, namely the interplay of innate immunity and HIV-1 infection and latency. That would not be possible given the limited in-house budget associated to the position. Furthermore, it will increase my competitiveness regarding future acquisition of both intramural and external funding.

As a non-UK scientist, I highly value the possibility to access additional, non-monetary support, including careers support, and benefit from the Academy’s networks across academia, industry and healthcare.

Most importantly, the methodological elements and approaches that I plan to develop and use in the proposed work will nourish other important research projects, for which I will seek separate funding sources.

Therefore, acquisition of funding within the AMS Professorship Scheme will allow me to establish an excellent basis for the development of my future research activities.