The Need for New Surrogate Markers for Immune Monitoring and Efficacy Assessment

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Current Paradigm Needs ChangeIt is of concern of all in pharma and biotech research fields that the number of vaccines and drugs that get to the market is very low compared to the number that enter phase I clinical trial testing.  For this to change, among other improvements, new surrogate markers must be identified early on for the purpose of patient stratification, immune monitoring and/or therapeutic or prophylactic efficacy assessment.

Until the investment is made to identify these surrogates, the marketplace will be condemned to relive the current paradigm of high failure rates, very high overall costs associated with vaccine and drug development, few therapies reaching patients with needs, and very slow changes in prevention, morbidity and mortality rates.  Identification of a few surrogate markers in this field would have a profound effect on all of the above and would be a wise investment.

As the search continues for surrogates to determine vaccine efficacy and therapeutic response,   the utility of cell mediated immunity (CMI) assays in the assessment of immune response or immunogenicity is increasing significantly.

Once critical assay reproducibility and robustness of data has been established, what needs to be done in relevant clinical trial settings to identify immune markers that can be used as surrogates of efficacy?

ELISPOT, ICS assays or both to determine CMI?

There are several advantages to using Enzyme-linked Immunosorbent Spot (ELISPOT) assays to interrogate cell functionality.  ELISPOT assays do require fewer cells compared to intracellular cytokine staining (ICS) assays and there have been studies that have shown comparability of fresh to frozen PBMCs in ELISPOT validation studies. Both these parameters are critical for the logistics of running clinical trials. ELISPOT is also an easier assay to run compared to ICS (ELISA with cells), however, there continues to be issues with precision in both assays being run manually with acceptable CVs being as high as 70%.  (Ref: Clin Vaccine Immunol. 2009 February; 16(2): 147–155 Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents (CVs of 30% or less but some as high as 70% for positivity 50spots/10 6 cells), and J Immunol Methods. 2011 Jan 5;363(2):143-57. Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing. (CVs of less than 35% for 0.2% and higher positivity).

A key disadvantage of ELISPOT is the inability for polyspectral immunophenotyping using lineage-specific markers to identify the responding cell populations. Given the publications in the past five years on the association of long-term non-progression in HIV positive individuals with higher polyfunctionality of the cellular response, it is anticipated that these are the kinds of studies that will need to be conducted in large scale clinical trials to determine if vaccine and therapeutic strategies elicit similar responses. (Ref: HIV nonprogressors preferentially maintain highly functional HIV-specific CD8 T cells; Blood 2006;107:4781-4789)

Recent publications in the field have identified biomarkers of tolerance in transplant patients that have required a holistic systems biology approach (combining gene expression-secreted protein profiling, polyfunctional flow cytometry evaluations as well as ELISPOT evaluations). (Ref: J Clin Invest. 2010 Jun 1;120(6):1848-61, Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans)

Can CMI assays be performed in a more cost conscious manner?

Both ELISPOT and polyspectral flow cytometry (PSFC) assays are more complex than most of the assays used in current clinical trial settings.  First, both assay formats are configured to assess function and not just presence of cell types.  And, although we would like to identify a single marker as a surrogate, many studies now predict that there will be a mosaic signature, whether it is a cellular or gene expression assay.  In some initial studies with clinical trial organizations, ImmunoSite Technologies (IST) used up to 60 different five-color combinations as screening tools to dissect the immune response and to find the candidate markers of choice.

Once the candidate marker cocktails have been identified and validated, the cost associated with testing can be significantly reduced.  Plus, the automation of these assays has not only reduced associated labor costs, but has improved reproducibility and has significantly reduced the cost of retesting of samples.  All of these progressive steps have reduced sample testing costs while at the same time improved assay results.

Can the paradigm be changed?

Given critical assay reproducibility and robustness of data, would not a holistic approach be best to identify immune markers that can be used as surrogates of efficacy in relevant clinical trial settings?  And given that the right screening can identify the immune response and find candidate surrogate markers of efficacy, should not investment be made to identify these markers?

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