Research Article: A panel of correlates predicts vaccine-induced protection of rats against respiratory challenge with virulent Francisella tularensis

Date Published: May 25, 2018

Publisher: Public Library of Science

Author(s): Roberto De Pascalis, Andrew Hahn, Helen M. Brook, Patrik Ryden, Nathaniel Donart, Lara Mittereder, Blake Frey, Terry H. Wu, Karen L. Elkins, Chandra Shekhar Bakshi.


There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat-killed LVS, which were poorly protective. These genes included IFN-γ, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5–7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.

Partial Text

Clinical studies of new vaccines can often be facilitated by taking advantage of correlates of protection, but no correlates have been defined for any intracellular pathogen. Protection against most of these pathogens, such as Francisella tularensis and Mycobacterium tuberculosis, depends heavily on immune responses mediated by T lymphocytes, which can be challenging to measure. In the absence of correlates, clinical trials to evaluate vaccine efficacy require natural exposure of subjects to the infectious agent in regions where the causative agent of the disease is endemic, or through human experimental challenge studies. The low incidence of diseases such as tularemia [1, 2], which is caused by the intracellular bacterium F. tularensis (Ft), makes clinical trials based on natural exposure to microorganisms difficult. On the other hand, human challenge studies are problematic, due to ethical issues and limitations on the number of individuals that can be feasibly enrolled.

Animal models are widely used to evaluate immune responses against infectious diseases, to explore mechanism of protection, and to obtain preliminary assessments of safety and efficacy of new vaccine candidates. Ultimately, vaccine safety and efficacy are usually evaluated in human clinical trials. However, large clinical studies to determine efficacy may be impractical for infectious diseases such as tularemia that have a low incidence rate in nature. In these circumstances, animal models may facilitate selection and evaluation of potential vaccine candidates, particularly if correlates of protection are available to bridge between species and to aid licensure via the FDA “Animal Rule” [3]. The lack of defined correlates for intracellular pathogens impedes development of vaccines against this entire class of microbes, many of which are significant public health concerns.




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