Pushing the frontiers of T-cell vaccines: accurate measurement of human T-cell responses

Abstract

There is a need for novel approaches to tackle major vaccine challenges such as malaria, tuberculosis and HIV, among others. Success will require vaccines able to induce a cytotoxic T-cell response--a deficiency of most current vaccine approaches. The successful development of T-cell vaccines faces many hurdles, not least being the lack of consensus on a standardized T-cell assay format able to be used as a correlate of vaccine efficacy. Hence, there remains a need for reproducible measures of T-cell immunity proven in human clinical trials to correlate with vaccine protection. The T-cell equivalent of a neutralizing antibody assay would greatly accelerate the development and commercialization of T-cell vaccines. Recent advances have seen a plethora of new T-cell assays become available, including some like cytometry by time-of-flight with extreme multiparameter T-cell phenotyping capability. However, whether it is historic thymidine-based proliferation assays or sophisticated new cytometry assays, each assay has its relative advantages and disadvantages, and relatively few of these assays have yet to be validated in large-scale human vaccine trials. This review examines the current range of T-cell assays and assesses their suitability for use in human vaccine trials. Should one or more of these assays be accepted as an agreed surrogate of T-cell protection by a regulatory agency, this would significantly accelerate the development of T-cell vaccines.

Figure 1. Assays available for measuring the T-cell response to vaccines

The success of a T-cell vaccine requires the induction of specific T-cell memory against the cognate vaccine antigen. A variety of assays are currently available for evaluating the function, immune phenotype and frequency of T-cell responses including: tetramers to quantify total memory T cells, proliferation assays to measure T-cell activation, cytokine-based assays to measure functionality and the profile of the vaccine response and immunophenotyping via flow cytometry-based methods to integrate many of the aforementioned assays. Finally, T-cell effector function assays are critical to show the vaccine-induced T cells are able to kill antigen-expressing target cells. BrdU: 5-bromo-2′-deoxyuridine; CBA: Cytometric bead array; CFSE: Carboxyfluorescein succinimidyl ester; CyTOF: Cytometry by time-of-flight; ELISPOT: Enzyme-linked immunosorbent spot; ICS: Intracellular cytokine staining.

Figure 2. Examples of assays used to assess T-cell responses in a human immunization trial

(A) PBMCs were collected from a clinical trial subject 4 weeks after a first, second or third immunization with hepatitis B surface antigen (HBs) combined with Advax^™, a novel polysaccharide adjuvant. PBMCs were stained with CFSE and cultured in presence of saline control (left column) or HBs antigen (right column) for 5 days then washed and stained with anti-CD4 monoclonal antibody and analyzed by FACS Diva. Increased CD4 T-cell proliferation to HBs is observed 4 weeks after the third immunization as measured by reduction in CFSE intensity (red circle, bottom right figure). (B) Antigen-specific T cells can be identified by stimulating PBMCs with the relevant peptide overnight and performing intracellular cytokine staining for IFN-γ, IL-2 and/or TNF-α. Here, influenza-specific CD4⁺ T cells have been identified using intracellular cytokine staining for IFN-γ and IL-2 following stimulation with relevant vaccine peptide and compared with unstimulated cells. (C) Human antigen-specific T cells can also be detected by cytokine enzyme-linked immunosorbent spot (ELISPOT). Here, fresh or thawed cryopreserved human PBMCs were stimulated in an IFN-γ capture plate with either mitogen mix (pokeweed mitogen and phytohemagglutinin) or inactivated influenza virus (B/Brisbane) or media and then the number of IFN-γ-producing T cells quantified using a cytotoxic T lymphocyte ELISPOT reader. The results confirm that PBMC cryopreservation does not diminish the ability to detect the secretion of IFN-γ production by thawed T cells. CFSE: Carboxyfluorescein succinimidyl ester; PBMC: Peripheral blood mononuclear cell.