Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

A Stronger Innate Immune Response During Hyperacute Human Immunodeficiency Virus Type 1 (HIV-1) Infection Is Associated With Acute Retroviral Syndrome.

BACKGROUND: Acute retroviral syndrome (ARS) is associated with human immunodeficiency virus type 1 (HIV-1) subtype and disease progression, but the underlying immunopathological pathways are poorly understood. We aimed to elucidate associations between innate immune responses during hyperacute HIV-1 infection (hAHI) and ARS. METHODS: Plasma samples obtained from volunteers (≥18.0 years) before and during hAHI, defined as HIV-1 antibody negative and RNA or p24 antigen positive, from Kenya, Rwanda, Uganda, Zambia, and Sweden were analyzed. Forty soluble innate immune markers were measured using multiplexed assays. Immune responses were differentiated into volunteers with stronger and comparatively weaker responses using principal component analysis. Presence or absence of ARS was defined based on 11 symptoms using latent class analysis. Logistic regression was used to determine associations between immune responses and ARS. RESULTS: Of 55 volunteers, 31 (56%) had ARS. Volunteers with stronger immune responses (n = 36 [65%]) had increased odds of ARS which was independent of HIV-1 subtype, age, and risk group (adjusted odds ratio, 7.1 [95% confidence interval {CI}: 1.7-28.8], P = .003). Interferon gamma-induced protein (IP)-10 was 14-fold higher during hAHI, elevated in 7 of the 11 symptoms and independently associated with ARS. IP-10 threshold >466.0 pg/mL differentiated stronger immune responses with a sensitivity of 84.2% (95% CI: 60.4-96.6) and specificity of 100.0% (95% CI]: 90.3-100.0). CONCLUSIONS: A stronger innate immune response during hAHI was associated with ARS. Plasma IP-10 may be a candidate biomarker of stronger innate immunity. Our findings provide further insights on innate immune responses in regulating ARS and may inform the design of vaccine candidates harnessing innate immunity.

Treating severe paediatric asthma with mepolizumab or omalizumab: a protocol for the TREAT randomised non-inferiority trial.

INTRODUCTION: A minority of school-aged children with asthma have persistent poor control and experience frequent asthma attacks despite maximal prescribed maintenance therapy. These children have higher morbidity and risk of death. The first add-on biologic therapy, omalizumab, a monoclonal antibody that blocks immunoglobulin (Ig)E, was licensed for children with severe asthma in 2005. While omalizumab is an effective treatment, non-response is common. A second biologic, mepolizumab which blocks interleukin 5 and targets eosinophilic inflammation, was licensed in 2018, but the licence was granted by extrapolation of adult clinical trial data to children. This non-inferiority (NI) trial will determine whether mepolizumab is as efficacious as omalizumab in reducing asthma attacks in children with severe therapy resistant asthma (STRA) and refractory difficult asthma (DA). METHODS AND ANALYSIS: This is an ongoing multicentre 1:1 randomised NI open-label trial of mepolizumab and omalizumab. Up to 150 children and young people (CYP) aged 6-17 years with severe asthma will be recruited from specialist paediatric severe asthma centres in the UK. Prior to randomisation, children will be monitored for medication adherence for up to 16 weeks to determine STRA and refractory DA diagnoses. Current prescribing recommendations of serum IgE and blood eosinophils will not influence eligibility or enrolment. The primary outcome is the 52-week asthma attack rate. Bayesian analysis using clinician-elicited prior distributions will be used to calculate the posterior probability that mepolizumab is not inferior to omalizumab. Secondary outcomes include Composite Asthma Severity Index, Paediatric Asthma Quality of Life Questionnaire, lung function measures (forced expiratory volume in one second (FEV1), bronchodilator reversibility), fractional exhaled nitric oxide, Asthma Control Test (ACT), health outcomes EuroQol 5 Dimension (EQ-5D) and optimal serum IgE and blood eosinophil levels that may predict a response to therapy. These outcomes will be analysed in a frequentist framework using longitudinal models. ETHICS AND DISSEMINATION: The study has been approved by the South Central-Berkshire Research Ethics Committee REC Number 19/SC/0634 and had Clinical Trials Authorisation from the Medicines and Healthcare Products Regulatory Agency (MHRA) (EudraCT 2019-004085-17). All parents/legal guardians will give informed consent for their child to participate in the trial, and CYP will give assent to participate. The results will be published in peer-reviewed journals, presented at international conferences and disseminated via our patient and public involvement partners. TRIAL REGISTRATION NUMBER: ISRCTN12109108; EudraCT Number: 2019-004085-17.

Exposure to common infections may shape basal immunity and potentially HIV-1 acquisition amongst a high-risk population in Coastal Kenya.

Introduction: The impact of exposure to endemic infections on basal immunity and susceptibility to HIV-1 acquisition remains uncertain. We hypothesized that exposure to infections such as cytomegalovirus (CMV), malaria and sexually transmitted infections (STIs) in high-risk individuals may modulate immunity and subsequently increase susceptibility to HIV-1 acquisition. Methods: A case-control study nested in an HIV-1 negative high-risk cohort from Coastal Kenya was used. Cases were defined as volunteers who tested HIV-1 positive during follow-up and had a plasma sample collected 3 ± 2 months prior to the estimated date of HIV-1 infection. Controls were individuals who remained HIV-1 negative during the follow-up and were matched 2:1 to cases by sex, age, risk group and follow-up time. STI screening was performed using microscopic and serologic tests. HIV-1 pre-infection plasma samples were used to determined exposure to CMV and malaria using enzyme-linked immunosorbent assays and to quantify forty-one cytokines and soluble factors using multiplexing assays. Multiplexing data were analyzed using principal component analysis. Associations between cytokines and soluble factors with subsequent HIV-1 acquisition were determined using conditional logistic regression models. Results and discussion: Overall, samples from 47 cases and 94 controls were analyzed. While exposure to malaria (p=0.675) and CMV (p=0.470) were not associated with HIV-1 acquisition, exposure to STIs was (48% [95% CI, 33.3 - 63] vs. 26% [95% CI, 17.3 - 35.9]. Ten analytes were significantly altered in cases compared to controls and were clustered into four principal components: PC1 (VEGF, MIP-1ß, VEGF-C and IL-4), PC2 (MCP-1, IL-2 and IL-12p70), PC3 (VEGF-D) and PC4 (Eotaxin-3). PC1, which is suggestive of a Th2-modulatory pathway, was significantly associated with HIV-1 acquisition after controlling for STIs (adjusted odds ratio, (95% CI), p-value: 1.51 [1.14 - 2.00], p=0.004). Elevation of Th2-associated pathways may dampen responses involved in viral immunity, leading to enhanced susceptibility to HIV-1 acquisition. Immunomodulatory interventions aimed at inhibiting activation of Th2-associated pathways may be an additional strategy to STI control for HIV-1 prevention and may reduce dampening of immune responses to vaccination.

Optimization and validation of an ELISA assay for the determination of antibody responses to CN54gp140 and AIDSVAX BE for use in the Phase IIb PrEPVacc vaccine trial.

PrEPVacc is an international, multi-centre, double-blind vaccine study comparing experimental combination vaccine regimens including DNA/AIDSVAX BE and DNA/CN54gp140 with placebo control. Simultaneously, daily oral PrEP is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites. An important clinical trial outcome is the accurate measurement of in vivo antibody titer induced through vaccination. Here we report the validation of two ELISAs for CN54gp140 and AIDSVAX BE at Uganda Virus Research Institute that demonstrates precision, specificity, and robustness for assessing the reciprocal antibody end point titer in human serum. This is a critical endpoint for determining whether vaccination can provide any protection against HIV in populations at risk of acquiring HIV.

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries.

The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.

A Novel Sample Selection Approach to Aid the Identification of Factors That Correlate With the Control of HIV-1 Infection.

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.

Utilizing Computational Machine Learning Tools to Understand Immunogenic Breadth in the Context of a CD8 T-Cell Mediated HIV Response.

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage.

Involvement of hypoxia-inducible factor-1 HiF(1alpha) in IgE-mediated primary human basophil responses.

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the alpha-subunit of hypoxia-inducible factor 1alpha (HIF-1alpha), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1alpha, although the latter crucially affected degranulation. Abrogating HIF-1alpha expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1alpha protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4.

The evolution of accelerated long-term forgetting: Evidence from the TIME study.

OBJECTIVE: Accelerated long-term forgetting (ALF) occurs when newly learned information decays faster than normal over extended delays. It has been recognised most frequently in temporal lobe epilepsy, including Transient Epileptic Amnesia (TEA), but can also be drug-induced. Little is known about the evolution of ALF over time and its impacts upon other memory functions, such as autobiographical memory (ABM). Here we investigate the long-term outcome of ALF and ABM in a group of patients with TEA and a single case of baclofen-induced ALF. METHODS: Study 1 involved a longitudinal follow-up of 14 patients with TEA over a 10-year period. Patients repeated a neuropsychological battery, three ALF measures (with free recall probed at 30-min and 1-week), and a modified Autobiographical Memory Interview (MAMI). Performance was compared with a group of healthy age-matched controls. In Study 2, patient CS, who previously experienced baclofen-induced ALF, was followed over 4 years, and re-tested now, 18 months after ceasing baclofen. CS repeated a neuropsychological battery, three ALF experimental tasks (each probed after 30 min and 1 week), and a modified autobiographical interview (AI). Her performance was compared with healthy age-matched controls. RESULTS: On ALF measures, the TEA group performed significantly below controls, but when analysed individually, 4 of the 7 patients who originally showed ALF no longer did so. In two, this was accompanied by improvements in ABM for recent but not remote memory. Patient CS no longer demonstrated ALF on standard lab-based tests and now appeared to retain new episodic autobiographical events with a similar degree of episodic richness as controls. CONCLUSION: Long-term follow up suggests that ALF can resolve, with improvements translating to recent ABM in some cases.

Adeno-associated virus vectored immunoprophylaxis to prevent HIV in healthy adults: a phase 1 randomised controlled trial.

BACKGROUND: A preventive vaccine for HIV is a crucial public health need; adeno-associated virus (AAV)-mediated antibody gene delivery could be an alternative to immunisation to induce sustained expression of neutralising antibodies to prevent HIV. We assessed safety and tolerability of rAAV1-PG9DP, a recombinant AAV1 vector encoding the gene for PG9, a broadly neutralising antibody against HIV. METHODS: This first-in-human, proof-of-concept, double-blind, phase 1, randomised, placebo-controlled, dose-escalation trial was done at one clinical research centre in the UK. Healthy men aged 18-45 years without HIV infection were randomly assigned to receive intramuscular injection with rAAV1-PG9DP or placebo in the deltoid or quadriceps in one of four dose-escalating cohorts (group A, 4 × 1012 vector genomes; group B, 4 × 1013 vector genomes; group C, 8 × 1013 vector genomes; and group D, 1·2 × 1014 vector genomes). Volunteers were followed up for 48 weeks. The primary objective was to assess safety and tolerability. A secondary objective was to assess PG9 expression in serum and related HIV neutralisation activity. All volunteers were included in primary and safety analyses. The trial is complete and is registered with ClinicalTrials.gov, number NCT01937455. FINDINGS: Between Jan 30, 2014, and Feb 28, 2017, 111 volunteers were screened for eligibility. 21 volunteers were eligible and provided consent, and all 21 completed 48 weeks of follow-up. Reactogenicity was generally mild or moderate and resolved without intervention. No probably or definitely related adverse events or serious adverse events were recorded. We detected PG9 by HIV neutralisation in the serum of four volunteers, and by RT-PCR in muscle biopsy samples from four volunteers. We did not detect PG9 by ELISA in serum. PG9 anti-drug antibody was present in ten volunteers in the higher dose groups. Both anti-AAV1 antibodies and AAV1-specific T-cell responses were detected. INTERPRETATION: Future studies should explore higher doses of AAV, alternative AAV serotypes and gene expression cassettes, or other broadly neutralising HIV antibodies. FUNDING: International AIDS Vaccine Initiative, United States Agency for International Development, Bill & Melinda Gates Foundation, US National Institutes of Health.

The GABAB receptor agonist, baclofen, contributes to three distinct varieties of amnesia in the human brain - A detailed case report.

We describe a patient in whom long-term, therapeutic infusion of the selective gamma-amino-butyric acid type B (GABAB) receptor agonist, baclofen, into the cerebrospinal fluid (CSF) gave rise to three distinct varieties of memory impairment: i) repeated, short periods of severe global amnesia, ii) accelerated long-term forgetting (ALF), evident over intervals of days and iii) a loss of established autobiographical memories. This pattern of impairment has been reported in patients with temporal lobe epilepsy (TLE), in particular the subtype of Transient Epileptic Amnesia (TEA). The amnesic episodes and accelerated forgetting remitted on withdrawal of baclofen, while the autobiographical amnesia (AbA) persisted. This exceptional case highlights the occurrence of 'non-standard' forms of human amnesia, reflecting the biological complexity of memory processes. It suggests a role for GABAB signalling in the modulation of human memory over multiple time-scales and hints at its involvement in 'epileptic amnesia'.

Involvement of hypoxia-inducible factor-1 in the inflammatory responses of human LAD2 mast cells and basophils.

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli.

Basophils as critical orchestrators of Th2-type immune responses.

Basophils are relatively rare leukocytes that potentially play a role in both systemic anaphylaxis and, owing to their ability to migrate from the blood into various other tissues, in more localized aspects of allergic inflammation. Given their greater sensitivities to allergen provocation compared with their tissue-fixed mast cell counterparts, and by virtue of their capacity to more readily generate Th2-type cytokines, basophils have been considered to play more than a bystander role in initiating and maintaining allergic disorders. However, only very recently has clearer evidence shed light on the abilities of this cell type to orchestrate chronic allergic inflammation and promote Th2 immunity in the early induction stages of allergy. This review summarizes these recent advances in understanding the role of basophils in orchestrating and maintaining allergic responses.