Cohort-Specific Peptide Reagents Broaden Depth and Breadth Estimates of the CD8 T Cell Response to HIV-1 Gag Potential T Cell Epitopes.

An effective HIV vaccine will need to stimulate immune responses against the sequence diversity presented in circulating virus strains. In this study, we evaluate breadth and depth estimates of potential T-cell epitopes (PTEs) in transmitted founder virus sequence-derived cohort-specific peptide reagents against reagents representative of consensus and global sequences. CD8 T-cells from twenty-six HIV-1+ PBMC donor samples, obtained at 1-year post estimated date of infection, were evaluated. ELISpot assays compared responses to 15mer consensus (n = 121), multivalent-global (n = 320), and 10mer multivalent cohort-specific (n = 300) PTE peptides, all mapping to the Gag antigen. Responses to 38 consensus, 71 global, and 62 cohort-specific PTEs were confirmed, with sixty percent of common global and cohort-specific PTEs corresponding to consensus sequences. Both global and cohort-specific peptides exhibited broader epitope coverage compared to commonly used consensus reagents, with mean breadth estimates of 3.2 (global), 3.4 (cohort) and 2.2 (consensus) epitopes. Global or cohort peptides each identified unique epitope responses that would not be detected if these peptide pools were used alone. A peptide set designed around specific virologic and immunogenetic characteristics of a target cohort can expand the detection of CD8 T-cell responses to epitopes in circulating viruses, providing a novel way to better define the host response to HIV-1 with implications for vaccine development.

HIV-1 infection and the lack of viral control are associated with greater expression of interleukin-21 receptor on CD8+ T cells.

OBJECTIVES: Interleukin-21 (IL-21) has been linked with the generation of virus-specific memory CD8+ T cells following acute infection with HIV-1 and reduced exhaustion of CD8+ T cells. IL-21 has also been implicated in the promotion of CD8+ T-cell effector functions during viral infection. Little is known about the expression of interleukin-21 receptor (IL-21R) during HIV-1 infection or its role in HIV-1-specific CD8+ T-cell maintenance and subsequent viral control. METHODS: We compared levels of IL-21R expression on total and memory subsets of CD8+ T cells from HIV-1-negative and HIV-1-positive donors. We also measured IL-21R on antigen-specific CD8+ T cells in volunteers who were positive for HIV-1 and had cytomegalovirus-responding T cells. Finally, we quantified plasma IL-21 in treatment-naive HIV-1-positive individuals and compared this with IL-21R expression. RESULTS: IL-21R expression was significantly higher on CD8+ T cells (P = 0.0256), and on central memory (P = 0.0055) and effector memory (P = 0.0487) CD8+ T-cell subsets from HIV-1-positive individuals relative to HIV-1-negative individuals. For those infected with HIV-1, the levels of IL-21R expression on HIV-1-specific CD8+ T cells correlated significantly with visit viral load (r = 0.6667, P = 0.0152, n = 13) and inversely correlated with plasma IL-21 (r = -0.6273, P = 0.0440, n = 11). Lastly, CD8+ T cells from individuals with lower set point viral load who demonstrated better viral control had the lowest levels of IL-21R expression and highest levels of plasma IL-21. CONCLUSION: Our data demonstrates significant associations between IL-21R expression on peripheral CD8+ T cells and viral load, as well as disease trajectory. This suggests that the IL-21 receptor could be a novel marker of CD8+ T-cell dysfunction during HIV-1 infection.

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.

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.

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.

Evaluation of Dried Blood Spots and Oral Fluids as Alternatives to Serum for Human Papillomavirus Antibody Surveillance.

Human papillomavirus (HPV) vaccination elicits high-titer genotype-specific antibody responses that are associated with a reduced risk of cervical disease caused by vaccine-incorporated genotypes. Our objective was to evaluate dried blood spots (DBSs) and oral mucosal transudate (OMT) as alternative samples to serum to confirm HPV vaccine antibody status. A study was carried out to evaluate the feasibility of detecting HPV16 and HPV18 antibodies in OMT, DBSs, and sera among women who self-reported being unvaccinated or fully vaccinated with the HPV vaccine. Serum had the highest sensitivity (100%) for detection of antibodies against both HPV16 and HPV18 but the lowest specificity, due to the detection of natural infection antibodies in 16% of unvaccinated women. Conversely, DBSs and OMT had lower sensitivity (96% and 82%, respectively) but high specificity (98%). We confirmed that these antibodies were functional (i.e., neutralizing) and that their detection was quantitatively reproducible and well correlated between sample types when normalized to IgG content. DBSs and OMT are appropriate alternative sample types for HPV vaccine surveillance. These alternative sample types warrant consideration for the purposes of cervical screening, diagnosis, and management, but more work will be needed to establish the stringent parameters required for such application.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and other anogenital cancers. HPV vaccination, primarily targeted at young girls before the age of sexual debut, is starting to demonstrate population-level declines in HPV infection and early disease associated with vaccine-incorporated genotypes. Monitoring young women for vaccine-specific antibody is important for vaccine surveillance and may be useful as an adjunct test within a cervical screening context. We evaluated serum, dried blood spots, and oral fluid as potential samples for such applications and report robust measures of diagnostic accuracy. This is the first time a direct comparison of alternative sample types has been made between vaccinated and unvaccinated women for the detection and quantitation of HPV antibodies.