Understanding the interactions between host and pathogen is important for the development and assessment of medical countermeasures to infectious agents, including potential biodefence pathogens such as
Understanding host-pathogen interactions is important for the development and assessment of medical countermeasures to infectious agents. The advent of new imaging and “omic” technologies has provided the ability to follow these interactions from whole animal to cellular and molecular levels, enabling a greater understanding of the mechanisms involved; this facilitates the development and refinement of new and existing vaccines and therapeutics. For example, advances in bioimaging provide a noninvasive means of identifying the internal systemic spread of infection in animal models and the impact of a prophylaxis or a therapy on the disease process. This can be combined with the analysis of responses at a cellular level using flow cytometry and microscopy techniques. The use of microarrays has also enhanced our understanding of the host response to infection and provides supportive information to help elucidate the innate and adaptive immune mechanisms essential for protection against pathogens, as well as the virulence mechanisms deployed by the pathogen. Although in its infancy, next generation sequencing also holds great potential for defining host-pathogen interactions. This review will assess the impact of these technologies on the ability to assess the host response and how this has been applied to help progress the development of vaccines and immunotherapies against biodefence agents described in the Centers for Disease Control and prevention (CDC) Select Agent list (
Traditionally, many immunological studies have focused on examining single immune parameters, such as cytokines, using techniques like ELISA and ELISpot. This approach does not highlight interconnecting pathways that control the immune response when the host encounters an infectious agent. With the emergence of transcriptomic technologies, such as microarray and next-generation sequencing, thousands of parameters of the immune system can be measured at the same time at a genome-wide scale. This allows a systematic, unbiased approach to understand how transcript changes correlate with diverse states of the immune system [
A DNA microarray consists of a solid surface, usually a glass microscope slide onto which DNA molecules (probes), in picomolar concentrations, are chemically bonded. The purpose of a microarray is to detect the presence and abundance of labelled nucleic acids (targets) in a biological sample, which will hybridise to the DNA on the array. The level of binding between a probe and its target is quantified by measuring the fluorescence emitted by the hybridized targets when scanned. In the majority of microarray experiments the labelled nucleic acids are derived from the mRNA of a sample or tissue, and so the microarray measures gene expression [
Most microarrays are prepared so that they cover the whole genome of a species; however, in the absence of a fully sequenced organism, researchers have used smaller focused arrays designed from publically available gene sequences [
DNA microarrays have revolutionized our understanding of the host gene expression changes in response to infection with various pathogens. This information has largely been obtained from
Microarray studies performed with various Biodefence Agents.
Pathogen | Purpose of study | Arrays used | Material tested | Reference |
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Response to infection | ||||
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Profile human antibody responses in healthy and recovered patients. | Protein array containing 154 |
Human plasma from healthy and recovered melioidosis patients. | [ |
|
Gene expression changes following intravenous infection with bacteria in BALB/c mice. | Sentrix MouseRef-8 cDNA array (Illumina). | Liver and spleen from BALB/c mice. | [ |
|
Differences in gene expression after 2 hour exposure to |
GeneChip human genome U133 (Affymetrix). | A549 human lung epithelial cells. | [ |
|
Gene expression changes in cells exposed to Edema toxin. | GeneChip murine genome (Affymetrix). | RAW 264.7 murine macrophages. | [ |
|
Gene expression changes in cells exposed to lethal toxin. | GeneChip human genome U133 plus 2.0 (Affymetrix). | Human monocytes from the blood of naïve volunteers. | [ |
|
Murine macrophage gene expression changes following exposure to protective antigen and lethal factor from |
PCR product DNA array. | RAW 264.7 murine macrophages. | [ |
|
Gene expression profiling of human macrophages following infection |
GeneChip human genome U133 Plus 2.0 (Affymetrix). | Human alveolar macrophages following bronchoscopy. | [ |
|
Gene expression analysis of mucosal epithelial cells following infection |
10K human ESTs microarray (Microarray centre, Ontario, Canada). | Epithelial-like human HeLa cell line. | [ |
|
Kinetics of human antibody responses to acute and chronic brucellosis. |
|
Sera from brucellosis patients. | [ |
|
Investigate host gene changes |
Custom-made 13K bovine 70 mer oligo array. | Infected Peyer’s patch from calf ligated ileal loop. | [ |
|
Full proteome-wide serological analysis of |
Protein microarray containing 3046 proteins from |
Sera from brucellosis patients. | [ |
|
Profile humoral immune response of naïve and acute Q-fever patients. | Protein microarray containing 84% of |
Human sera from Q-fever patients. | [ |
|
Comparison of the antibody profiles from acute and chronic Q-fever patients. | Protein microarray containing 93% of |
Human sera from Q-fever patients. | [ |
|
Define the humoral immune profile using Q-fever patient sera. | Custom-made protein microarray containing 19 proteins from |
Human sera from Q-fever patients. | [ |
Ebola and Marburg viruses | Gene signatures following infection |
Human cDNA array (Agilent). | Human hepatoblastoma (Huh7) cells. | [ |
Ebola virus | Entry into human macrophages. Infection studies |
GeneChip human genome HG-U95Av2 array (Affymetrix). | Primary human macrophages. | [ |
|
Human neutrophil gene expression, |
GeneChip human genome U133 plus 2.0 (Affymetrix). | Polymorphonuclear leukocytes (PMNs) from human blood. | [ |
|
|
Human gene array (Affymetrix). | Human peripheral blood mononuclear cells (PBMCs). | [ |
|
Gene expression following inhalation of |
Mouse array covering 1500 genes. (Ocimumbio). | Lung tissue taken from infected BALB/c mice. | [ |
|
Gene expression following aerosol exposure with |
Custom-made mouse cDNA array. | Lung tissue taken from infected C57BL/6 mice. | [ |
|
Gene expression of human monocytes infected |
GeneChip human genome U133 plus 2 (Affymetrix). | Naïve human peripheral blood monocytes. | [ |
|
Comparison of mouse global transcriptional responses to |
Mouse whole genome 44K arrays (Agilent). | Lung tissue from infected BALB/c mice. | [ |
Monkeypox and Vaccinia virus | Comparison of gene expression profiles following infection |
Human cDNA arrays with 406 Variola and Vaccinia virus genes. | Primary human macrophages, primary human fibroblasts and HeLa cells. | [ |
Monkeypox and Vaccinia virus | Comparison of gene expression profiles, |
Whole human genome oligo microarray (Agilent). | HeLa cells. | [ |
Monkeypox virus | Gene expression changes |
Rhesus macaque genome microarrays (Affymetrix). |
|
[ |
Monkeypox virus | Comparison of antibody responses to monkeypox virus infection and human smallpox vaccination. | Protein array covering 92–95% of representative proteins from Monkeypox and Vaccinia virus. | Blood from humans with smallpox vaccination and cyno macaques infected with Monkeypox virus. | [ |
Variola virus | Host gene expression changes in Variola virus infected cynomolgus macaques. | Human cDNA microarrays. | PBMC’s sampled from infected monkeys. | [ |
|
Gene expression changes following infection |
Human nylon blots (1185 cDNA spots) (Clontech). | Primary human monocytes and/or mixed with lymphocytes (PBMCs). | [ |
Venezuelan equine encephalitis virus (VEEV) | Gene expression of VEEV infected mice. | Oligo array mouse 70 mer. (Operon) & GEArray, focused mouse Toll-like receptor signaling microarray. | VEEV infected mouse brain CD-1 mice. | [ |
|
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Vaccines | ||||
Anthrax vaccine adjuvant CpG ODN | Measure gene expression changes in mice and splenocytes treated with CpG ODN. | Murine oligonucleotide array (custom-made). | Spleens and splenocytes from various breds of mice. | [ |
|
Assess the memory response of PBMCs taken from LVS vaccinated and naïve humans. | GeneChip human genome U133 (Affymetrix). | Re-stimulated PBMCs from LVS vaccinated and naïve humans. | [ |
Killed |
Define antibody profiles of vaccinated mice. | Whole proteome microarray custom-made. | BALB/c mice vaccinated with LVS. | [ |
Q-fever vaccine | Assess antibody immune profiles of Q-Vax vaccinated humans. |
|
Sera from vaccinated humans. | [ |
Smallpox vaccines | Assess antibody profiles generated to MVA, Acam2000 and/or Dryvax smallpox vaccines. | Protein array containing Vaccinia virus proteins [ |
Mouse, rabbit, macaque, black-tailed prairie dog and human sera. | [ |
|
||||
Therapies | ||||
Anti-coagulant treatments for Ebola virus infection | Comparison of NHP host genome responses responding to candidate therapeutics following infection with Ebola virus. | Human genome cDNA microarray. | PBMC’s from rhesus macaques infected with ZEBOV and treated shortly after exposure with rNAPc2 or rhAPC. | [ |
Omission from this table does not constitute absence of data.
DNA microarray analysis has been used to improve our understanding of the host response following exposure to the bacterium [
Despite the ready availability of DNA microarrays for use with different animal species, relatively few
Some microarray studies have been performed using nonhuman primates (NHPs) infected with Ebola virus [
Protein microarray is a more recent technology, providing a platform for high-throughput proteomics. Construction is similar to DNA microarrays, except that the immobilised species is a protein or a peptide, and the array aims to represent partially or wholly the entire proteome [
One of the most powerful applications of protein microarrays is in the study of the humoral immune response to infection. Arrays have been used to assess host antibody profiles (or “immunosignature”) in response to infection with
Microarray technology has been used to help understand the cell-mediated and humoral immune responses following infection with infectious agents; furthermore it has also improved our understanding of the mechanism of action of therapeutics and biodefence vaccines. For instance a transcriptomic approach, using DNA microarrays, was used to assess the host response to treatment with therapeutic agents (rNAPc2 or rhAPC) designed to block the coagulation pathway during Ebola virus infection in NHPs [
A limited number of studies have been performed using DNA microarrays to understand the underlying protective mechanisms of licensed or novel biodefence vaccines. DNA arrays have been used to examine the immunostimulatory properties of CpG motifs [
The antibody profile evoked by smallpox vaccines has been examined in detail following the development of a Vaccinia proteome microarray by Davies et al. in 2005 [
Protein arrays have also been used to examine the immunosignature of mice vaccinated with killed
Advances in “omic” technology have also assisted with the identification of candidate T-cell antigens. An ORFeome flexible cloning approach was developed by Jing and colleagues whilst analysing the CD4 T-cell response to vaccinia virus using PBMCs from Smallpox vaccinated individuals in 2009 [
Next generation sequencing (NGS; also known as high-throughput, short-read, or deep sequencing) has revolutionised sequence-based analyses over the last decade. The underlying principle is that it uses micro-/nanotechnologies to run millions of parallel sequencing reactions, generating millions or billions of bases per run (which is up to 6 logs greater than the output using the Sanger method). Read lengths are typically comparatively short, a result of which is that any particular base is sequenced many times (known as coverage or read depth). There are a number of competing platforms, with Illumina, ABI SOLiD, Roche 454, and Ion Torrent technologies being widely used, each having different characteristics with regard to average read length, total bases sequenced per run, and cost-per-base [
NGS technology can be applied to both DNA (DNA-seq), and RNA (after conversion to cDNA-RNA-seq). RNA-seq analysis aims to identify the transcriptome (the complete set of transcripts of the cell, which includes mRNA, noncoding RNAs, and small RNAs). RNA-seq is increasingly being used as an alternative to microarray as a method of measuring gene expression [
Upon infection of a host with a pathogen, changes in the expression of both organisms occur. These are usually investigated separately due to the low pathogen:host transcript ratio (up to 200-fold); thus enrichment of the pathogen transcripts is often required [
RNA-seq has been used to investigate the host response to different virulent strains of
The human host response to Dengue virus infection has also been reported using RNA-Seq [
Ideally it would be preferable to monitor the gene expression profiles of the pathogen and host simultaneously. This “dual RNA-seq” approach is technically and bioinformatically more challenging [
Dual RNA-seq has also been used to evaluate the immune response following smallpox vaccination. PBMCs taken from Dryvax vaccinated individuals were either stimulated with or without live Vaccinia virus for 8 hours [
Imaging infection using optical sources relies on the detection of specific targets using fluorescence or bioluminescence. Fluorescent light is emitted with a characteristic emission spectrum following excitation at specific wavelengths. Fluorescent molecules may be used to tag specific molecules of interest. Very often the molecule of interest will be an antibody which in turn will be directed to specific targets (e.g., surface receptors on host cells). Alternatively, endogenous proteins can be made to fluoresce, for example, in genetically modified animals or pathogens, or fluorescent dyes can be used to label pathogens or cells. Bioluminescence is produced by the reaction of a luciferase enzyme with its substrate and requires energy and oxygen to occur. Unlike fluorescence imaging, where the signal is still detectable for some hours after the host has died, bioluminescent imaging requires living cells. This section aims to review how our understanding of biodefence pathogens, vaccines, and immunotherapies and their interactions with the host has been greatly aided by imaging techniques such as flow cytometry, fluorescence microscopy and real time
This technique is routinely used as an important tool for assessing cellular responses to infection and vaccination in both human patients and animal models of infection. It is used for cellular phenotyping and functional assays including fluorescence-based proliferation assays. Bead-based assays are also available to assess levels of soluble factors including cytokines in samples from
Flow cytometry has highlighted a key role for various cell types in murine infection models of
Neutrophil inflammatory responses have been characterised following infection in mouse models with both
Using flow cytometry to understand host-pathogen interactions has the potential to enable an association between host immune markers with protective effects following treatment with therapeutics and vaccines. A number of studies have assessed the immune response to immunotherapeutic approaches for treatment of infection to further understand potentially protective immune responses in
Flow cytometry bead-based assays have the potential to aid our understanding of potential protective mechanisms of novel immunotherapeutics by assessing cytokine responses. These assays have been used in studies investigating the effects of IFN-
Immune responses elicited
In addition to examining lymphocyte responses, the role of antigen-presenting cells in generating protective immunity during vaccination to
Fluorescent-activated cell sorting and cDNA technologies have recently been used together to generate antigen-specific monoclonal antibodies [
A number of recent advances in fluorescent microscopy techniques such as confocal microscopy, intravital 2-photon microscopy, dynamic live cell imaging, and super resolution microscopy have been used to interrogate host-pathogen interactions, based on detection of specific fluorescent signals to provide detailed images of pathogens colocalised with or within host cells. Fluorescence microscopy is also being investigated for its utility in the diagnosis of infections including
Fluorescence microscopy has enabled us to understand the intracellular nature and niches of pathogens and their ability to evade immune pathways to enable their survival within host cells. For example, identifying the lysosomal escape mechanism of
Visualising the infectious disease process as it occurs inside a living animal is of major benefit to the development of medical countermeasures. This can be achieved using biphotonic imaging (BPI), a sensitive and noninvasive method of detecting light emitted either as a bioluminescent (BL) or fluorescent (FL) signal, using photon detectors such as those based on a charge coupled device (CCD) camera. BPI has enabled new insights into pathogen dissemination, host responses to infection, interactions between host and pathogen, and the effects of antimicrobials and vaccines. This technique can also be used to refine animal experiments with each animal acting as its own control, therefore, increasing the power of these studies. The creation of BL or FL strains of pathogenic organisms has enabled this field to progress and description of the processes involved in BPI and creation of these strains are comprehensively reviewed in Andreu et al. [
Using BPI different patterns of pathogen dissemination can be readily observed allowing discrimination of the growth and spread of different forms of the pathogen or target organs depending on route of infection. For example, BL expressing variants of
BPI studies showed that dissemination of
The pattern of dissemination of BL
These pathogen dissemination models have subsequently been used to assess the effect of antibiotics and immunotherapies. A substantial reduction of bacterial signal was found in
The effects of immunisation with protective antigen (PA) vaccine demonstrated that, in immunised mice, dissemination of BL
Recent developments in flow cytometry include the development of imaging flow cytometers including the ImagestreamX. Imaging flow cytometry adds another dimension to flow cytometric applications with images of each cell being produced in addition to fluorescence readouts and has the ability to further advance our understanding of host-pathogen interactions. It is particularly suitable for assessing the colocalisation of pathogens within host cells and for examining cellular processes for uptake and processing of pathogens, for example, phagocytosis, autophagy, and apoptosis. The number of publications which incorporate use of ImagestreamX for investigating host-pathogen interactions is growing each year with a limited number of publications on a wide range of public health related pathogens including
ImageStreamX Mk1 imaging depicting intracellular infection of
Ultrasound magnetic resonance imaging (MRI) and radiography are all technologies which have been used clinically in the diagnosis of infectious diseases including anthrax and tuberculosis. Some of these technologies have been used in biodefence research, for example, positron emission tomography (PET)/computer tomography (CT) imaging was used to examine inflammation patterns in Monkeypox virus infection of primates [
Biodefence agents are dangerous pathogens that pose unique challenges for researchers. Human cases of these diseases are relatively rare and therefore animal models play a key role in helping to understand pathogenesis. Imaging and “omic” technologies have greatly aided our ability to study the host response during the course of an infection and have thus provided important insights. Also since it is neither ethical nor feasible to conduct conventional phase III efficacy trials, using biodefence agents in human volunteers, these key technologies can play an important role in the evaluation of vaccines and therapies. They provide evidence to support the concepts defined by the Food and Drug Agency (FDA) Animal Rule [
Currently the majority of studies using the “omic” and imaging techniques, described in this review, have examined the host response independently from pathogen virulence. In the future, however, due to rapid advances in NGS platform technologies and imaging technologies, it is anticipated that examining pathogen virulence whilst simultaneously interrogating host responses will be achieved. Overall, this should reduce and refine animal experiments and thus allow the identification of both host and pathogen markers during infection at the same time. This will further enhance our knowledge of host-pathogen interactions and aid in the development of vaccines and therapeutics for these dangerous pathogens.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to acknowledge Dominic Jenner (Dstl) for his contribution to this review.