Response to COVID-19 vaccination imaged by PD-L1 PET scanning.

BACKGROUND: During a phase 0 clinical trial of an investigational programmed cell death ligand-1 (PD-L1) PET tracer in patients with non-small cell lung cancer (NSCLC), three patients received booster doses of COVID-19 vaccines before PD-L1 imaging. METHODS: Five patients underwent whole-body PET/CT imaging with a novel PD-L1 tracer, constructed by attaching 89Zr to the anti PD-L1 antibody durvalumab. Intramuscular (deltoid) booster doses of mRNA BNT162b2 COVID-19 mRNA vaccine were coincidentally given to three patients in the month before PD-L1 tracer injection. RESULTS: Two recently-vaccinated patients, in remission of NSCLC and receiving non-immunosuppressive cancer therapies (immunotherapy and tyrosine kinase inhibitor respectively), showed increasing PD-L1 tracer uptake in ipsilateral axillary lymph nodes. No asymmetric nodal uptake was seen in a third recently-vaccinated patient who was receiving immunosuppressive chemotherapy, or in two patients not recently-vaccinated. CONCLUSION: Immune response to mRNA BNT162b2 vaccination may involve regulation by PD-L1 positive immune cells in local draining lymph nodes in immunocompetent patients. TRIAL REGISTRATION: This trial was registered with the Australian New Zealand Clinical Trials Registry. Registration number ACTRN12621000171819. Date of Trial Registration 8/2/2021. Date of enrolment of 1st patient 11/4/2021. URL of trial registry record: https://www.australianclinicaltrials.gov.au/anzctr/trial/ACTRN12621000171819 .

The roles of the kynurenine pathway in COVID-19 neuropathogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the highly contagious respiratory disease Corona Virus Disease 2019 (COVID-19) that may lead to various neurological and psychological disorders that can be acute, lasting days to weeks or months and possibly longer. The latter is known as long-COVID or more recently post-acute sequelae of COVID (PASC). During acute COVID-19 infection, a strong inflammatory response, known as the cytokine storm, occurs in some patients. The levels of interferon-γ (IFN-γ), interferon-ß (IFN-ß), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) are particularly increased. These cytokines are known to activate the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), catalysing the first step of tryptophan (Trp) catabolism through the kynurenine pathway (KP) leading to the production of several neurotoxic and immunosuppressive metabolites. There is already data showing elevation in KP metabolites both acutely and in PASC, especially regarding cognitive impairment. Thus, it is likely that KP involvement is significant in SARS-CoV-2 pathogenesis especially neurologically.

CRISPR-Cas13b-mediated suppression of hepatitis B virus replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches are urgently required that target multiple aspects of the hepatitis B virus (HBV) replication cycle to improve rates of functional cure. HBV RNA represents a novel therapeutic target. Here, we programmed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13b endonuclease, to specifically target the HBV pregenomic RNA (pgRNA) and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pgRNA. Mammalian cells with replication competent wildtype HBV DNA of different genotypes, a HBV stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-blue fluorescent protein (BFP) and crRNAs plasmids and the impact on HBV replication and protein expression was measured. WT HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and sera HBsAg was measured. PspCas13b mRNA and crRNA were also delivered by lipid nanoparticles (LNP) in a HBsAg-expressing stable cell line and the impact on secreted HBsAg determined. RESULTS: Our HBV targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p<0.0001). HBV protein expression was also reduced in an HBV stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p<0.0001) in vivo. LNP-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p=0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: There is an urgent need for new treatments that target multiple aspects of the HBV replication cycle. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.

Predictors of liver disease progression in people living with HIV-HBV co-infection on antiretroviral therapy.

BACKGROUND: In people living with HIV-HBV, liver fibrosis progression can occur even with suppressive antiretroviral therapy (ART). We investigated the relationship between liver fibrosis and biomarkers of inflammation, apoptosis, and microbial translocation. METHODS: In this observational cohort study adults living with HIV-HBV already on effective ART were recruited in Australia and Thailand and followed for 3 years including 6 monthly clinical review and blood tests and annual transient elastography. Differences in clinical and laboratory predictors of liver fibrosis progression were tested followed by regression analysis adjusted for CD4+ T-cells at study entry. A linear mixed model was fitted to longitudinal data to explore changes over time. FINDINGS: 67 participants (85% male, median age 49 y) were followed for 175 person-years. Median duration of ART was 10 years (interquartile range (IQR) 8-16 years). We found 11/59 (19%) participants during 3-years follow-up (6/100 person-years) met the primary endpoint of liver disease progression, defined as increased Metavir stage from baseline to final scan. In regression analysis, progressors compared to non-progressors had higher levels of high mobility group box 1 protein (HGMB1), (median (IQR) 3.7 (2.6-5.0) and 2.4 ng/mL (1.5-3.4) respectively, adjusted relative risk 1.47, 95% CI [1.00, 2.17]) and lower nadir CD4+ T-cell percentage (median 4% (IQR 2-8) and 11% (4-15) respectively (relative risk 0.93, 95% CI [0.88, 0.98]). INTERPRETATION: Progression in liver fibrosis occurs in people with HIV-HBV on suppressive ART. Fibrosis progression was associated with higher HMGB1 and lower percentage nadir CD4+ T-cell count, highlighting the importance of early initiation of HBV-active ART. FUNDING: This work was supported by NHMRC project grant 1101836; NHMRC practitioner fellowship 1138581 and NHMRC program grant 1149990. The funder had no role in study design, data collection, data analysis, interpretation, writing of this manuscript or decision to submit for publication.

Interim results from a phase I randomized, placebo-controlled trial of novel SARS-CoV-2 beta variant receptor-binding domain recombinant protein and mRNA vaccines as a 4th dose booster.

BACKGROUND: SARS-CoV-2 booster vaccination should ideally enhance protection against variants and minimise immune imprinting. This Phase I trial evaluated two vaccines targeting SARS-CoV-2 beta-variant receptor-binding domain (RBD): a recombinant dimeric RBD-human IgG1 Fc-fusion protein, and an mRNA encoding a membrane-anchored RBD. METHODS: 76 healthy adults aged 18-64 y, previously triple vaccinated with licensed SARS-CoV-2 vaccines, were randomised to receive a 4th dose of either an adjuvanted (MF59®, CSL Seqirus) protein vaccine (5, 15 or 45 µg, N = 32), mRNA vaccine (10, 20, or 50 µg, N = 32), or placebo (saline, N = 12) at least 90 days after a 3rd boost vaccination or SARS-CoV-2 infection. Bleeds occurred on days 1 (prior to vaccination), 8, and 29. CLINICALTRIALS: govNCT05272605. FINDINGS: No vaccine-related serious or medically-attended adverse events occurred. The protein vaccine reactogenicity was mild, whereas the mRNA vaccine was moderately reactogenic at higher dose levels. Best anti-RBD antibody responses resulted from the higher doses of each vaccine. A similar pattern was seen with live virus neutralisation and surrogate, and pseudovirus neutralisation assays. Breadth of immune response was demonstrated against BA.5 and more recent omicron subvariants (XBB, XBB.1.5 and BQ.1.1). Binding antibody titres for both vaccines were comparable to those of a licensed bivalent mRNA vaccine. Both vaccines enhanced CD4+ and CD8+ T cell activation. INTERPRETATION: There were no safety concerns and the reactogenicity profile was mild and similar to licensed SARS-CoV-2 vaccines. Both vaccines showed strong immune boosting against beta, ancestral and omicron strains. FUNDING: Australian Government Medical Research Future Fund, and philanthropies Jack Ma Foundation and IFM investors.

GITR activation ex vivo impairs CD8 T cell function in people with HIV on antiretroviral therapy.

Glucocorticoid-induced tumor necrosis factor related protein (GITR) is a co-stimulatory immune checkpoint molecule constitutively expressed on regulatory T cells (Tregs) and on activated T conventional cells (Tconv). In blood collected from PWH on suppressive ART, GITR expression was reduced in multiple activated CD4 and CD8 T cell subsets but was increased in Tregs. HIV specific CD8 T cells expressed higher levels of GITR and programmed cell death protein 1 (PD-1) compared to total CD8 T cells. Following stimulation with HIV peptides and GITR-ligand (L), we demonstrated a significant decrease in killing by HIV specific CD8 T cells and an increased exhausted profile. T cell receptor co-stimulation with GITR-L abrogated Treg suppression and induced expansion of CD4 Tconv. We conclude that GITR activation is an additional factor contributing to an impaired HIV immune response in PWH on ART and that GITR agonist antibodies should not be pursued for HIV cure strategies.

Characterisation of HIV-1 reservoirs in paediatric populations: protocol for a systematic review and meta-analysis.

INTRODUCTION: The success of antiretroviral therapy (ART) has changed HIV from a deadly to a chronic infection, thus increasing the transitioning from infancy toward adulthood. However, the virostatic nature of antiretrovirals maintains viruses in sanctuaries, with reactivation potentials. Because current ARTs are very limited for children, the emergence of new HIV epidemics driven by HIV drug-resistance mutations is favoured. Our systematic review aims to estimate the global burden of archived drug-resistance mutations (ADRMs) and the size of reservoir (HIV-1 DNA load), and their associated factors in children and adolescents. METHODS AND ANALYSIS: Papers from the PubMed/MEDLINE, Google Scholar, ScienceDirect, African Journals Online and Academic Medical Education Databases will be systematically identified using the keywords: "HIV-1 reservoirs", "viral reservoirs", "HIV-1 DNA", infants, adolescents, child and children, linked by the following Boolean operators: 'OR' and 'AND'. Randomised and non-randomised trials, cohort studies and cross-sectional studies published in French or English from January 2002 will be included, while case reports, letters, comments, reviews, systematic reviews and meta-analyses, and editorials will be excluded. All studies describing data on ADRMs, HIV-1 DNA load and/or immunological markers among children/adolescents will be eligible. A random-effects model will be used to calculate the pooled prevalence of ADRMs. Data will be reported according to type of viral reservoir (peripheral blood mononuclear cells, CD4 cells), geographical location (country/continent), ethnicity/race, age (infants vs adolescents), gender, HIV-1 clades, ART exposure (naïve vs treated, drug class, type of regimen, age at ART initiation and treatment duration), WHO clinical staging (I, II, III, IV), immune status (immune compromised vs immune competent) and virological response (viraemic vs non-viraemic). Multivariate logistic regression will be performed to determine predictors of HIV reservoir profile in paediatric populations. The primary outcome will be to assess the genotypical and quantitative profile of HIV reservoirs, while the secondary outcomes will be to identify factors associated with ADRMs and reservoir size in paediatric populations. ETHICS AND DISSEMINATION: Ethical approval is not applicable for this study as it will be based on published data. Results will be disseminated via a peer-reviewed scientific journal and relevant conferences. PROSPERO REGISTRATION NUMBER: CRD42022327625.

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial.

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756 .

Design and analysis considerations for early phase clinical trials in hepatitis B (HBV) cure research: the ACTG A5394 study in persons with both HIV and HBV.

With growing interest and efforts to achieve a hepatitis B (HBV) cure, HBV therapeutics have increasingly entered the clinical testing phase. In designing an early phase clinical trial aimed at HBV cure, the heterogeneity in participants and the choice of a biomarker endpoint that signals a cure requires careful consideration. We describe the key elements to consider during the development of HBV clinical trials aimed at a functional cure, and how we have addressed them in the design of a phase II AIDS Clinical Trials Group (ACTG) study, A5394 (NCT05551273). The trial we present is for persons with both HIV and HBV, a unique population that has much to gain from an HBV cure. Our decisions on the design elements are specific to the study agent and the targeted population, but our deliberations may be informative in the emerging field of early phase HBV trials aimed at cure.

Effect of high dose vitamin D3 on the HIV-1 reservoir: A pilot randomised controlled trial.

Introduction: Antiretroviral therapy for people living with HIV-1 must be taken lifelong due to the persistence of latent virus in long-lived and proliferating CD4+ T cells. Vitamin D3 is a steroidal gene transcription regulator which exerts diverse effects on immune and epithelial cells including reductions in CD4+ T cell proliferation and improvement in gut barrier integrity. We hypothesised that a high dose of vitamin D3 would reduce the size of the HIV-1 reservoir by reducing CD4+ T cell proliferation. Methods: We performed a randomised placebo-controlled trial evaluating the effect of 24 weeks of vitamin D3 (10,000 international units per day) on the HIV-1 reservoir and immunologic parameters in 30 adults on antiretroviral therapy; participants were followed for 12 weeks post-treatment. The primary endpoint was the effect on total HIV-1 DNA at week 24. Parameters were assessed using mixed-effects models. Results: We found no effect of vitamin D3 on the change in total HIV-1 DNA from week 0 to week 24 relative to placebo. There were also no changes in integrated HIV-1 DNA, 2-long-terminal repeat (2-LTR) circles or cell-associated HIV-1 RNA. Vitamin D3 induced a significant increase in the proportion of central memory CD4+ and CD8+ T cells, a reduction in the proportion of senescent CD8+ T cells and a reduction in the natural killer cell frequency at all time points including week 36, 12 weeks after the study drug cessation. At week 36, there was a significant reduction in total HIV-1 DNA relative to placebo and persistently elevated 25-hydroxyvitamin D levels. No significant safety issues were identified. Conclusions: Vitamin D3 administration had a significant impact on the T cell differentiation but overall effects on the HIV-1 reservoir were limited and a reduction in HIV-1 DNA was only seen following cessation of the study drug. Additional studies are required to determine whether the dose and duration of vitamin D3 can be optimised to promote a continued depletion of the HIV-1 reservoir over time. Trial registration: ClinicalTrials.gov NCT03426592.

Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice.

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4+ T cells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4+ T cells from PLWH ex vivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.

HIV infection.

The AIDS epidemic has been a global public health issue for more than 40 years and has resulted in ~40 million deaths. AIDS is caused by the retrovirus, HIV-1, which is transmitted via body fluids and secretions. After infection, the virus invades host cells by attaching to CD4 receptors and thereafter one of two major chemokine coreceptors, CCR5 or CXCR4, destroying the host cell, most often a T lymphocyte, as it replicates. If unchecked this can lead to an immune-deficient state and demise over a period of ~2-10 years. The discovery and global roll-out of rapid diagnostics and effective antiretroviral therapy led to a large reduction in mortality and morbidity and to an expanding group of individuals requiring lifelong viral suppressive therapy. Viral suppression eliminates sexual transmission of the virus and greatly improves health outcomes. HIV infection, although still stigmatized, is now a chronic and manageable condition. Ultimate epidemic control will require prevention and treatment to be made available, affordable and accessible for all. Furthermore, the focus should be heavily oriented towards long-term well-being, care for multimorbidity and good quality of life. Intense research efforts continue for therapeutic and/or preventive vaccines, novel immunotherapies and a cure.

Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform.

Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted 'intact proviral DNA assay' (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5' defective and 3' defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3' defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5' defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses.

Regional Analysis of Intact and Defective HIV Proviruses in the Brain of Viremic and Virally Suppressed People with HIV.

Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.

Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy.

Introduction: In people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function. Methods: We used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles. Results: Soluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART. Discussion: Plasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.