Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525.

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity.

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development. Trial registration: ClinicalTrials.gov NCT01994525.

OMIP-064: A 27-Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells.

This 27-color flow cytometry panel was developed in order to assess immunological changes over the course of an immunization and challenge regimen in two experimental malaria vaccine trials. The aim of the study was to find correlates of vaccine-induced protection. Several studies have indicated that protection against malaria appears to involve immune responses at various immunological sites, with liver-resident responses playing an essential role. As it is not feasible to monitor the immune responses within the liver in humans, this panel is developed with the aim to thoroughly characterize the immune responses over time in blood in addition to detecting changes that might reflect what happens in other immunological sites like the liver. The focus of this panel is to detect several innate lymphoid cell populations, including NK cells and their activation status. Moreover, unconventional T cells like mucosal associated invariant T cells and γδ T cells are assessed in the panel. © 2020 International Society for Advancement of Cytometry.

OMIP-056: Evaluation of Human Conventional T Cells, Donor-Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining.

A 26-color staining panel was developed to profile human antigen-specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal-associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross-validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Attenuated IL-2R signaling in CD4 memory T cells of T1D subjects is intrinsic and dependent on activation state.

The IL-2/IL-2R pathway is implicated in type 1 diabetes (T1D). While its role in regulatory T cell (Treg) biology is well characterized, mechanisms that influence IL-2 responses in effector T cells (Teff) are less well understood. We compared IL-2 responses in 95 healthy control and 98 T1D subjects. In T1D, low IL-2 responsiveness was most pronounced in memory Teff. Unlike Treg, CD25 expression did not influence the Teff responses. Reduced IL-2 responses in memory Teff were not rescued by resting, remained lower after activation and proliferation, and were absent in type 2 diabetes. Comparing basal IL-2 responses in resting versus activated cells, memory Teff displayed lower, but more sustained, responses to IL-2 overtime. These results suggest that T1D-associated defects in the Teff compartment are due to intrinsic factors related to activation. Evaluation of both Teff and Treg IL-2R signaling defects in T1D subjects may inform selection of therapies.

A first-in-human germline-targeting HIV nanoparticle vaccine induced broad and publicly targeted helper T cell responses.

The engineered outer domain germline targeting version 8 (eOD-GT8) 60-mer nanoparticle was designed to prime VRC01-class HIV-specific B cells that would need to be matured, through additional heterologous immunizations, into B cells that are able to produce broadly neutralizing antibodies. CD4 T cell help will be critical for the development of such high-affinity neutralizing antibody responses. Thus, we assessed the induction and epitope specificities of the vaccine-specific T cells from the IAVI G001 phase 1 clinical trial that tested immunization with eOD-GT8 60-mer adjuvanted with AS01B. Robust polyfunctional CD4 T cells specific for eOD-GT8 and the lumazine synthase (LumSyn) component of eOD-GT8 60-mer were induced after two vaccinations with either the 20- or 100-microgram dose. Antigen-specific CD4 T helper responses to eOD-GT8 and LumSyn were observed in 84 and 93% of vaccine recipients, respectively. CD4 helper T cell epitope "hotspots" preferentially targeted across participants were identified within both the eOD-GT8 and LumSyn proteins. CD4 T cell responses specific to one of these three LumSyn epitope hotspots were observed in 85% of vaccine recipients. Last, we found that induction of vaccine-specific peripheral CD4 T cells correlated with expansion of eOD-GT8-specific memory B cells. Our findings demonstrate strong human CD4 T cell responses to an HIV vaccine candidate priming immunogen and identify immunodominant CD4 T cell epitopes that might improve human immune responses either to heterologous boost immunogens after this prime vaccination or to other human vaccine immunogens.

Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.

Background: Identifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge. Methods: To identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each. Results: RAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations. Conclusions: This study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.

Achieving intracellular cytokine staining assay concordance on two continents to assess HIV vaccine-induced T-cell responses.

The HIV Vaccine Trials Network (HVTN) conducts clinical trials on 4 continents in pursuit of a safe and effective HIV vaccine. Cellular immune responses to vaccination that define vaccine immunogenicity and/or immune correlates of protection can be measured using multiparameter intracellular cytokine staining (ICS) assays. The HVTN cellular immunology laboratory, located in Seattle, WA, conducts ICS assays for vaccine trials according to Good Clinical Laboratory Practices (GCLP). In 2013, the HVTN established a second GCLP compliant cellular immunology laboratory in Cape Town, South Africa to assess vaccine immunogenicity for HVTN trials conducted on the African continent. To ensure ICS readouts in the 2 laboratories were directly comparable, we conducted concordance testing using PBMC from healthy controls and vaccine trial participants. Despite standardized procedures and instrumentation, shared quality control measures and quality assurance oversight, several factors impacted our ability to obtain close agreement in T-cell responses measured in the 2 laboratories. One of these was the type of fetal bovine serum (FBS) used in the assay, which impacted lymphocyte cell viability and background responses. In addition, the differences in supernatant removal technique also significantly affected our ability to detect positive responses to vaccine antigens. Standardization of these factors allowed us to achieve and maintain ICS assay concordance across the 2 laboratories over multiple years, accelerating our efforts to evaluate HIV vaccines. The insights gained in this process are valuable for assay transfer efforts by groups of investigators that need to directly compare data generated in different laboratories around the globe.

Multiple autoimmune-associated variants confer decreased IL-2R signaling in CD4+ CD25(hi) T cells of type 1 diabetic and multiple sclerosis patients.

IL-2 receptor (IL-2R) signaling is essential for optimal stability and function of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Treg); a cell type that plays an integral role in maintaining tolerance. Thus, we hypothesized that decreased response to IL-2 may be a common phenotype of subjects who have autoimmune diseases associated with variants in the IL2RA locus, including T1D and MS, particularly in cells expressing the high affinity IL-2R alpha chain (IL-2RA or CD25). To examine this question we used phosphorylation of STAT5 (pSTAT5) as a downstream measure of IL-2R signaling, and found a decreased response to IL-2 in CD4(+)CD25(hi) T cells of T1D and MS, but not SLE patients. Since the IL2RArs2104286 haplotype is associated with T1D and MS, we measured pSTAT5 in controls carrying the rs2104286 risk haplotype to test whether this variant contributed to reduced IL-2 responsiveness. Consistent with this, we found decreased pSTAT5 in subjects carrying the rs2104286 risk haplotype. Reduced IL-2R signaling did not result from lower CD25 expression on CD25(hi) cells; instead we detected increased CD25 expression on naive Treg from controls carrying the rs2104286 risk haplotype, and subjects with T1D and MS. However the rs2104286 risk haplotype correlated with increased soluble IL-2RA levels, suggesting that shedding of the IL-2R may account in part for the reduced IL-2R signaling associated with the rs2104286 risk haplotype. In addition to risk variants in IL2RA, we found that the T1D-associated risk variant of PTPN2rs1893217 independently contributed to diminished IL-2R signaling. However, even when holding genotype constant at IL2RA and PTPN2, we still observed a significant signaling defect in T1D and MS patients. Together, these data suggest that multiple mechanisms converge in disease leading to decreased response to IL-2, a phenotype that may eventually lead to loss of tolerance and autoimmunity.

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.