GB2581470A - Method and associated kit and uses for assessing and/or monitoring non-human primate health - Google Patents

Abstract

A method for assessing and/or monitoring the health status of a non-human primate comprising evaluating the expression of a non-human primate cellular marker antigenically equivalent to Human Leukocyte Antigen-DR (HLA-DR) in a biological sample, in particular the method incorporating the steps of measuring the expression level in a first and second biological sample and comparing the relative expression levels. The HLA-DR equivalent may be expressed on neutrophils. The invention also relates to an associated kit and uses of the invention in particular for assessing the health status of a non-human primate infected by Burkholderia pseudomallei, Francisella tularensis or Coxiella burnetti.

Description

METHOD AND ASSOCIATED KIT AND USES FOR ASSESSING AND/OR MONITORING NON-HUMAN PRIMATE HEALTH

Technical Field of the Invention

The invention relates to a method for assessing and/or monitoring the health status of a non-human primate comprising evaluating the expression of a non-human primate cellular marker antigenically equivalent to Human Leukocyte Antigen-DR in a biological sample, in particular the method incorporating the steps of measuring the expression level of a non-human primate cellular marker antigenically equivalent to Human Leukocyte Antigen-DR in a first and second biological sample and comparing the relative expression levels. The invention also relates to an associated kit and uses of the invention.

is Background to the Invention

The need to monitor the health status of populations of non-human primates covers a variety of scenarios. In one example, zoos and other forms of animal sanctuaries may house non-human primates, in particular rare breeds or species under threat of extinction, wherein maintaining animal numbers is of a primary concern. Preserving the health of such populations, and isolating any sick animals, can ensure that disease does not spread and lead to a significant loss of animals.

As a contrasting, and possibly more emotive, example, the use of non-human primates in medical animal experimentation (for example for drug and vaccine trials) can require careful ethical and animal welfare practises. Ensuring healthy populations of non-human primates are provided for experimental research is of key importance, as the incorporation of animals with underlying conditions or infection in studies may impact on the outcomes of experiments, preventing valid conclusions being drawn from experimental research. Ensuring that healthy animals are provided for such studies can negate the need for any undesired repeat experiments, as a consequence of erroneous results, therefore preventing the need for unnecessary animal usage. Furthermore, certain types of non-human primates (e.g. New World monkeys such as marmosets) are prone to diseases such as colitis, heightening the need for a simple, robust and effective means for screening the health status of nonhuman primate populations during animal studies.

In addition, non-human primates may act as vectors for certain zoonotic diseases (e.g. Herpes B virus). Thus, assessing and/or monitoring the health status of a nonhuman primate population in a natural geographical location, for example in low-tomiddle-income countries, may assist in early detection of non-human primate colonies carrying diseases capable of spreading to other animal populations such as humans.

Existing screening tests for monkeys include the use of polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) testing. However, such diagnostic tools typically identify the present of an epitope associated with a specific agent e.g. simian retrovirus (SRV), simian immunodeficiency virus (Sly), and hepatitis A, B and C, and therefore likely will not identify the presence of alternative infective agents in a biological sample isolated from a non-human primate. Furthermore, antibody-based testing (as exemplified by ELISAs) may have a turnaround time in the region of hours to days and may not be suitable for a relatively rapid health surveillance screening assay.

Therefore, there is a need for an efficient, low-cost and rapid screening tool for assessing and/or monitoring the health status of non-human primate populations.

Summary of the Invention

According to a first aspect, the invention provides a method for assessing and/or monitoring the health status of a non-human primate comprising evaluating the expression of HLA-DR in a biological sample.

The term 'FILA-DR' herein refers to a non-human primate protein antigenically equivalent to the human form of Human Leukocyte Antigen-DR isotype. HLA-DR is one of the so-called classical molecules of the MHC class II (also including HLA-DP and HLA-DQ). In humans, Human Leukocyte Antigen-DR is represented by transmembrane glycoproteins composed of two polypeptide chains, alpha (a) and beta (3), found on antigen presenting cells (for example dendritic cells, macrophages, B-cells, neutrophils, lymphocytes) that function by binding specific (non-host) peptides that can be presented to CD4+ T-cells. Thus, by acting as a ligand for the T-cell receptor, Human Leukocyte Antigen-DR can initiate an adaptive immune response against foreign antigens, such as those from bacteria or virus. The Human Leukocyte Antigen-DR a-chain is encoded by the HLA-DRA locus, while the Human Leukocyte Antigen-DR 3-chain is encoded by a combination of genes associated with a corresponding genetic locus (HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLADRB5).

Preferably, the invention provides a method comprising the steps of: taking a first biological sample and a second biological sample from the non-human primate; employing a means for measuring an expression level of HLA-DR in the first biological sample and the second biological sample; and comparing the expression level of HLA-DR in the second biological sample relative to the first biological sample, wherein a respective HLA-DR expression level reduced in the second biological sample, relative to the first biological sample, is an indicator of reduced health status of the non-human primate.

The term 'assessing and/or monitoring' includes undertaking an analytical measurement in a biological sample, in particular a first and second biological sample respectively, to a) evaluate a specific parameter (i.e. assessing), and/or b) evaluate any trends in changes over time in a specific parameter (i.e. monitoring), in order to make a determination with respect to the health status of a non-human primate. In the case of a biological sample, such evaluation may be undertaken by comparing the parameter measurement with a known reference range. In the case of a first and second biological sample, the first sample may acts as a reference relative to the second biological sample in terms of the parameter being measured. The parameter measurement with respect to the first and second biological samples may be compared with a known reference range. With respect to the invention, a reduction in the expression of HLA-DR, as identified by the assessing and/or monitoring, reflects a reduced health status of a non-human primate. In particular, the change in health status may be due to infection by a pathogenic, or potentially pathogenic, agent (e.g. bacteria, virus, fungi). However, other conditions may be envisaged e.g. immune disease.

The term 'non-human primate' would be understood by the skilled person to include any non-human primate, for example: New World monkeys such as the common marmoset (Caffithrix jacchus); and Old World monkeys such as the Rhesus macaque (Macaca mulatta), Cynomolgus macaque (macaca fascicularis), and the African Green monkey (Chlorocebus sabaeus).

The definition of HLA-DR' extends to cover any type of non-human primate gene product (for example also compassing transcribed molecules, such as RNA e.g. mRNA, or those molecules that can be derivative from transcribed molecules e.g. cDNA, as well as translated molecules such as protein) of at least one non-human primate gene, which can show cross-reactivity with a recognition element (for example a complementary nucleic acid sequence, peptide, protein such as a primer, aptamer or antibody respectively, or an alternative molecule or substrate) capable of reacting specifically with a gene product from at least one human gene associated with encoding Human Leukocyte Antigen-DR protein/peptide(s).

The specificity of such recognition elements ensures a lack of cross-reactivity with unrelated gene products, or indeed other members of e.g. the MHC class II. Example of such recognition elements include an antibody which only recognises an Human Leukocyte Antigen-DR protein epitope, such as an epitope on HLA-DRa on account of the correct folding of the HLA-DR a/13 heterodimer, and which does not cross-react with an epitope on other MHC class II members e.g. HLA-DP or HLA-DO.

An equivalent protein (i.e. antigenically equivalent) variant of Human Leukocyte Antigen-DR has been described in non-human primates. For example, Caja-DR is expressed in the common marmoset via the Caja-DRB region. Encoding for the DR beta chain appears to consist of three genes per chromosome/haplotype: a nearly monomorphic DRBW*12 gene, a DRB1*03 locus which is variable but mostly a pseudogene and a polymorphic DRBW*16 gene (Antunes et al. 1998. The Common marmoset: A new world primate species with limited Mhc class II variability. Proc. Natl. Acad. Sci:95, 11745-11750) Thus, in the case of common marmoset, a biological sample, in particular a first and second biological sample, would be taken from an animal and the expression of an individual gene, or combination of genes, from the Caja-DRB region, would be determined. Such determination may be achieved by measuring the level of gene product(s) in the biological sample, potentially with comparison to a known reference range, or by measuring the relative level of gene product(s) in the first and second biological samples, reflected by the quantity of transcribed RNA and/or translated protein from the Caja-DRB region in the sample(s). Observing a reduction in CajaDRB region gene product(s) expression would be indicative of a decrease in health status of a common marmoset under investigation. However, it will be understood by the skilled person that equivalent investigations could be undertaken in different nonhuman primates (e.g. based on bioinformatic data identifying Human Leukocyte Antigen-DR equivalent proteins/peptide(s) and/or associated gene(s) for the nonhuman primate in question).

The term 'biological sample' as understood by the skilled person may refer to a clinical sample taken (i.e. extracted) from a subject, in this case the subject being limited to non-human primates, with such samples including, but not exclusively, cerebrospinal fluid (CSF), blood or samples derived thereof (for example whole blood, plasma, serum, cell-free serum, cell-free plasma), tissues, cells, saliva, transpired secretion, urine, faeces, stomach fluid, digestive fluid, nasal fluid, cytosolic fluid or other biological tissue or fluid sample recognised in the art. The term biological sample includes a sample(s) taken from a non-human primate pre-and/or post-treatment e.g. pre-and/or post-treatment with an antimicrobial agent (antibiotics, antivirals).

It is to be understood that the first biological sample and the second biological sample are taken consecutively from the subject. For example, the first biological sample may serve as a control or baseline sample, in particular with respect to HLADR expression by cells (i.e. immune cells). The second biological sample provides a sample for which HLA-DR expression levels are compared against the corresponding levels determined for the first sample (i.e. assessing). There may be biological samples taken in the period between the first and second samples. Furthermore, the samples may represent continued 'test' samples relative to a prior control or baseline samples, wherein the trend for HLA-DR expression is being investigated (i.e. monitoring). The time period between taking the first and second biological samples may be measured by durations that include: hours e.g. at least 1 hour, at least 5 hours, at least 12 hours at least 18 hours; days e.g. at least 1 day (24 hours), at least 1.5 days (36 hours), at least 2 days (48 hours), at least 3 days; weeks e.g. at least 1 week, at least 2 weeks, at least 3 weeks; months. e.g. at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 10 months; and years e.g. at least 1 year, at least 2 years, at least 3 years, at least 5 years, at least 10 years.

It is to be understood that additional target testing may be required e.g. use of housekeeping gene or genes that are constitutively expressed by cells also 25 expressing HLA-DR, to enable a standardisation of HLA-DR expression.

Preferably, the biological sample(s) taken for the method of the invention is blood. This ensures testing can be performed on a sample that is relatively easy and quick to acquire from a subject i.e. relative to samples which require a more invasive extraction such as tissue or CSF.

Wu et al. (Changes of monocyte human leukocyte antigen-DR expression as a reliable predicator of morality in severe sepsis. Critical Care. 2011, 15:R220) describes the utility of monitoring changes in Human Leukocyte Antigen-DR expression as a predictor of morality. However, this document is specific to measuring Human Leukocyte Antigen-DR only in relation to predicting mortality in human cases in where severe sepsis was already identified.

In contrast, the inventors have shown that HLA-DR is a surprisingly accurate indicator of disease of varying levels of severity in non-human primates, ranging from non-lethal to lethal models. The method of the invention has shown to determine with a high accuracy the development of infection in non-human primates e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and even as high as 100%. Furthermore, the invention has shown success in animals with no overt signs of disease (i.e. asymptomatic), thus having utility as a post-exposure, pre-is symptomatic diagnostic method.

HLA-DR expression following Coxiefia burnetti infection was assessed in a non-lethal marmoset model. For this particular model, animals display a fever for several days that spontaneously resolves with no other observable clinical signs. The results obtained by the inventors show a reduction in HLA-DR expression by day 7, in the event of mild disease where the animals have an increase in body temperature but no other clinical signs. However, by day 14, HLA-DR expression had returned to pre-infection levels, highlighting the utility of HLA-DR in non-lethal, non-human primate infection models.

In the case of lethal models of disease, a correlation of decreasing levels of HLA-DR expression was also observed in infected animals. For example, significant reduction in HLA-DR expression in blood was shown to occur in terminal Francis° tularensis disease in marmosets (i.e. a different bacterial agents and model of disease with increased severity, relative to C. bumetti). Similarly, HLA-DR expression was significantly reduced, relative to pre-infection levels, in 100% of animals challenged with a lethal dose of Burkholderia pseudornallei.

Particularly advantageously, this method provides for a pre-symptomatic test to identify non-human primates with a reduced health status. For example, it has been shown that for aerosol-challenged animals, HLA-DR expression in spleen and blood samples was dramatically reduced by the earliest time point of testing (12 hours post-challenge), despite bacteria at this time point being localised to the lung and the animals being pre-symptomatic.

Furthermore, fluctuation in HLA-DR expression during treatment regimens enables this particular immune biomarker to provide an indication of the effectiveness of medical countermeasures in ameliorating disease. For example, treatment of B. pseudornallei-infected marmosets with antibiotics, after development of fever, saw levels of HLA-DR expression return to re-challenge levels, indicating treatment was successful in controlling and/or eradicating infection.

It is further noted that the trend in HLD-DR expression was observed for different routes of infection e.g. inhalational or subcutaneous administration of an infective agent in non-human primates. For example, in B. pseudo/mai/chi-infected marmosets, infection via the aerosol or subcutaneous route conferred a reduction in HLA-DR expression.

Preferably, the HLA-DR is neutrophil-expressed HLA-DR.

Wu et al. (Changes of monocyte human leukocyte antigen-DR expression as a reliable predicator of morality in severe sepsis. Critical Care. 2011, 15:R220) is specific to measuring Human Leukocyte Antigen-DR expression on human monocytes. Furthermore, other studies have indicated that human-acquired neutrophils have no, or very little, constitutive expression of MHC-II required for antigen presentation, with up-regulation of MHC-II only obtained in the presence of both antigen and antigen-specific memory CD4+ T-cells or supernatant from activated T-cells (Vono et al. 2017. Neutrophils acquire the capacity for antigen presentation to memory CD4+ T cells in vitro and ex vivo. Blood:129(14): 1991- 2001). This situation is in contrast to other human APCs (e.g. dendritic cells) that persistently express MCH-II. Indeed, Vono et al. discloses that even when conditions lead to up-regulation of Human Leukocyte Antigen-DR on human neutrophils, substantially lower expression levels were observed when compared to dendritic cells and monocytes. Based on this evidence, neutrophils would not be considered suitable immune cells for assessing and/or monitoring the expression levels of Human Leukocyte Antigen-DR, and by extension H LA-DR in non-human primates, in particular as expression of Human Leukocyte Antigen-DR marker is not normally associated with human neutrophils (thus for example hampering establishing a baseline or comparative HLA-DR expression level in a subject).

In contrast, the inventors have shown that neutrophil-associated HLA-DR in nonhuman primates is an accurate, timely and reliable single marker for use as a guide to a non-human primate's health. The inventors have shown that neutrophil percentage in the blood of non-human primates can increase during the course of disease, thus providing a source of immune cells particularly relevant to the invention. In this study, the reduction of HLA-DR expression on neutrophils was highly significant and linked to disease, and observed on all neutrophils regardless of location. For example, HLA-DR expression on neutrophils in the spleen and blood of B. pseudomallei-infected non-human primates was dramatically reduced by the earliest time point (12 hours) despite bacteria at this time being localised to the lung and the animals being pre-symptomatic. The suitability of HLA-DR as an accurate immune marker for reduced health status was recorded for all the types of infectious agents investigated (B. pseudomallei, F. tularensis, C. bumetti). Furthermore, upon administering treatment of infection, for example in the case of B. pseudomallei infection of marmosets, the expression of HLA-DR on neutrophils was shown to recover to pre-challenge levels. Therefore, the method of the invention in particular enables assessing and/or monitoring the effectiveness of disease treatment in nonhuman primates via assessing the expression of HLA-DR on neutrophils.

Preferably, neutrophils are isolated from a biological sample(s) prior to measuring neutrophil-expressed HLA-DR. This offers a more concentrated, purer sample in terms of neutrophil content. As understood by the skilled person, techniques to physically separate neutrophils from a biological sample(s) extracted from a nonhuman primate include, but are not limited to, density gradient separation, affinity flow fractionation or other immune-based methodologies for obtaining a neutrophil-only (or as uniform as possible) cell population.

Preferably, the level of HLA-DR expression reduces by at least 5% in the second sample, relative to the first sample, to indicate a reduced health status of the nonhuman primate. For example, the level of HLA-DR expression reduces by at least 5% in the biological sample, relative to a known reference range. In the case of a comparing a first and second biological sample, the level of HLA-DR expression reduces by at least 5% in the second sample, relative to the first sample. Given the high correlation and sensitivity between a decrease in HLA-DR expression in nonhuman primate and a reduced health status, it is submitted that a reduction in HLA-DR expression by at least 5% may be capable of indicating adverse health, such as the presence of an infection. Furthermore preferably, the level of HLA-DR expression reduces by at least 10% to indicate a reduced health status of the non-human primate. Most preferably, the level of HLA-DR expression reduces by at least 20% to indicate a reduced health status of the non-human primate. For example, in marmosets challenged with a lethal aerosol of B. pseudomallei and tested before clinical signs were apparent, in comparing all of the matched blood samples (pre-challenge to post-challenge, regardless of the time after challenge) a reduction of 20% in HLA-DR positively stained neutrophils would have correctly identified 86% as requiring treatment (46/53) and 20% (1/5) of the healthy animals.

Preferably, the means for measuring the expression level of HLD-DR in the biological sample(s) is antibody-based. This approach provides the advantage of potentially incorporating already available, commercial-off-the-shelf antibodies for use in the method, to enable relative levels of HLA-DR to be determined for each sample. Such antibodies may be labelled e.g. with a fluorophore, magnetic particle, isotype or coloured particle (e.g. latex, gold) to enable relative quantification. As understood by the skilled person, such antibodies could be any antibody type e.g. IgG, in particular non-human primate IgG or a recombinant form of IgG.

Preferably, the means incorporates two antibodies: a first antibody specific for neutrophils to enable this cell type to be rapidly isolated by the method; and a second antibody to react with HLA-DR to enable quantification of the relative level of expression of this particular immune biomarker in each sample. A third antibody could be incorporated to provide expression levels of a housekeeping gene.

Preferably, the antibody-based means is incorporated in a lateral flow assay. This approach enables a rapid, simple and easily portable technology for a visual assessment of the relative level of HLA-DR in biological samples.

Preferably, the antibody-based means is incorporated in a flow cytometry assay e.g. fluorescence-activated cell sorting (FACS). This approach enables accurate measurement of HLA-DR expression by a population of cells from a sample(s), in particular using gating during analysis to select for a specific cell population (in particular neutrophils) to interrogate for the level of HLA-DR expression in a biological sample, or relative level of HLA-DR expression across different biological samples. Associated controls as understood by the skilled person could be applied to such an assay.

Alternatively, the means for measuring the expression level of HLD-DR in the biological sample(s) is nucleic acid-based. This provides a highly accurate and scalable approach for rapid diagnostic assessment and/or monitoring of non-human primate health status.

Preferably, the nucleic acid-based means is incorporated in a Reverse Transcription Polymerase Chain Reaction (RT-PCR) or microarray reaction. As understood by the skilled person, such approaches may utilise nucleic acid primers or immobilised probes respectively, directed towards a gene or genes associated with HLA-DR expression, to obtain accurate quantified data of HLA-DR expression in a biological sample(s), extracted from a non-human primate, to assess and/or monitor the nonhuman primate's health status. In the case of RT-PCR, such approaches can allow for real time assessment of transcription rates of genes associated with HLA-DR expression in non-human primates. The expression level(s) of a housekeeping gene may be provided to ensure a reliable assessment of relative HLA-DR expression in different samples.

According to a second aspect, the invention provides use of a bioactive substance capable of binding to a gene product of a gene or genes encoding HLA-DR as a means for assessing and/or monitoring the health status of a non-human primate.

Examples of the bioactive substance includes a nucleic acid molecule e.g. primer, immobilised probe, and/or peptide/protein e.g. aptamer, antibody, or alternative small molecule. Preferably such bioactive substance are labelled to enable detection/quantification e.g. via a fluorophore, magnetic particle, isotype or coloured particle (e.g. latex, gold) According to a third aspect, the invention provides a kit for performing the method of the first aspect, the kit comprising a means to specifically detect HLA-DR.

According to a fourth aspect, the invention provides use of a kit according to the third aspect as a means for assessing and/or monitoring the health status of a non-human primate.

The kit of the invention provides the means for detecting levels of a gene product from the gene(s) associated with expression of HLA-DR in non-human primates. Any suitable nucleic acid-based and/or protein-based technique can be applied for measuring the expression level of HLA-DR e.g. use of a recognition element(s) and/ or immobilised probe(s), for example nucleic acid elements such as primers in the case of nucleic acid detection, or antibodies in the case of peptide/protein detection.

Such kits may incorporate a means for detecting a nucleic acid, in particular RNA such as mRNA, or derivative molecular e.g. cDNA. In particular, the kit may comprise the reagents suitable for performing amplification of a gene product(s) for a gene(s) associated with HLA-DR, in particular a quantifiable amplification reaction such as RT-PCR. The kit may comprise primers for amplification of a gene or genes. The kits may further comprise labels (fluorescent labels and/or oligonucleotide probes) to allow the amplification reaction to be monitored in real-time using known assays e.g. TagMan®, LUX, SYBRO Green etc. The kits may also contain reagents such as buffers, enzymes and salts e.g. MgCI, which are required for performed a nucleic acid amplification reaction. Alternatively, the kit may comprise immobilised nucleic acid probes, for example in the form of a microarray.

In the case of detecting a protein or peptide, the kit may be an HLA-DR-specific lateral flow assay.

Preferably, the kit provides a means for assessing and/or monitoring the health 15 status of a non-human primate infected by Burkholderia pseudomallei, Franc/se/la tularensis or Coxiella bumetti.

Any feature in one aspect of the invention may be applied to any other aspects of the invention, in any appropriate combination. In particular, method aspects may be applied to use and/or kit aspects and vice versa. The invention extends to a method, use or kit substantially as herein described, with reference to the Examples.

In all aspects, the invention may comprise, consist essentially of, or consist of any feature or combination of features.

The present invention will now be described, with reference to the following non-limiting examples and Figures in which:

Brief Description of the Figures

Figure 1 shows graphs of granulocytes, macrophages and lymphocytes from B. pseudomallei aerosol-infected animals over time; Figure 2 shows graphs of expression of neutrophil activation markers in samples from post challenge marmosets; Figure 3 shows a graph of changes in expression of HLA-DR on neutrophils in blood samples following two different challenge routes of B. pseudomallei; Figure 4 shows a graph of changes in expression of HLA-DR on neutrophils, in pre-challenge samples to 12 h post challenge samples, prior to the onset of clinical symptoms, in aerosol infection of B. pseudomallei; Figure 5 shows a graph of changes in expression of HLA-DR on neutrophils in blood samples, taken at the point of fever, following B. pseudomallei infection; Figure 6 shows a graph of recovering HLA-DR expression following antibiotic treatment for B. pseudomallei infection, Figure 7 shows graphs of the discriminatory capacity of three canonical functions generated by Principle Component Analysis to differentiate animals based on the timing of blood sample extraction; Figure 8 shows a graph of changes in expression of HLA-DR following F. tularensis infection with and without treatment; and Figure 9 shows activation status of neutrophils in the blood of marmosets at different times following inhalational challenge with C. bumetti.

Detailed Description

Methods Bacteria/strains B. pseudomallei K96243 or HPUB10303a were grown in Luria broth at 37°C on a rotary platform for aerosol challenges and enumerated on L-Agar plates.

Glycerol stocks of F. tularensis strain SCHU S4 were obtained from ATTC. Bacteria were recovered from the vial onto blood cysteine glucose agar (BCGA) plates and incubated at 37 °C for 24 h, prior to recovery into PBS (pH 7.3). The 0D590 of the suspension was adjusted to 0.1, equivalent to approximately 1x108 colony forming units (CFU) m1-1, 1 ml of which was used to inoculate 100 ml modified cysteine partial hydrolysate broth. The broth was shaken at 180 r.p.m. for 48 h at 37 °C. Prior to challenge, the 0D590 of the culture was adjusted to 0.1 and serially diluted in PBS to the appropriate concentration for challenge. Viable counts were performed by subculture onto BCGA retrospectively. Plates were incubated at 37 °C for 72 h prior to enumeration.

C. bumetti Nine Milne phase I (NMI; RSA493) was cultured axenically in ACMM2 broth in a vented conical flask within a sealed contained using a GENbox microaer gas generator (BioMeriuex). The flasks were incubated at 37 °C at 75 rpm for 6 days. The bacteria were pelleted by centrifugation and stored frozen at -80 °C in PBS until required. On the day of challenge, a frozen aliquot was removed and diluted to the required concentration in PBS.

All bacteriological procedures were carried out under UK Advisory Committee on Dangerous Pathogens containment level 3 conditions in Class 3 microbiological safety cabinets compliant with British Standard BS5726.

Marmosets Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony. Animals included in these studies were between 18 months and 5 years old and weighed between 320g to 500g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.

Animal studies Challenge by the inhalation route was performed using an AeroMP platform and a modified Henderson apparatus. Animals were removed from the home cage and sedated with 10 mg of ketamine hydrochloride by the intramuscular route. Marmosets were placed in a head-only exposure chamber (plethysmograph tube) and exposed for 10 min to a dynamic aerosol generated by a Collison nebulizer which generates aerosol particles of approximately 1-3 pm. The total accumulated tidal volume for each animal during challenge was determined by whole-body real-time plethysmography with a Fleisch pneumotachograph (EMMS). The concentration of the aerosol cloud was quantified after sampling from a sample port into an all-glass impinger (AGI-30; SKU) by serial dilution and plating onto suitable media.

For B. pseudomallei studies, animals were challenged via the inhalational route with B. pseudomallei strains K96243 or HPUB10303a and either euthanised at various time points post-challenge or when they had reached a humane end point. Two weeks prior to challenge animals had blood collected to determine baseline immunological parameters.

A total of 40 animals were infected by the inhalational route with a retained dose of 40.8 (1-201) CFU which gave an average time to death (humane endpoint) of 71.6 hours (81.7-53.4). Of the 16 marmosets that received less than 10 CFU, 5 animals survived 7 days (or more) without a measured temperature increase and 2 of these animals had no culturable B. pseudomallei bacteria in organs post-modem. All other animals had fever at 24 hours and reached a fever plateau by 36 hours. Samples were available also from a further 32 animals from different studies, which were infected with a retained dose of 47.9 (16-123) CFU and culled in groups of 8 at 4 time points post infection: 12 hours before onset of fever or signs; 24 hours low grade fever apparent in most animals; 36 hours fever in all animals, most having high grade fever; and 48 hours high fever and other mild clinical signs. Bacterial counts were determined in key organs at each time point. Additionally, three animals of similar age were used to provide naïve samples.

Additionally, 60 animals were challenged with lethal doses of B. pseudomallei strain K96243 by either the inhalational, ingestion or subcutaneous route. For ingestion administration, 100 j_d_ of the 1 x 108 CFU/mL suspension of bacteria (neat OD-adjusted culture) was added to 1 mL of banana-favoured Nesquik® powder dissolved in water. The liquid was presented in a syringe to pre-conditioned animals to accept. For subcutaneous challenge, 100 ht,L of the 1 x 103 CFU/mL suspension of bacteria 113 (10-s dilution of the neat OD-adjusted culture) was injected into a pinch of skin fold in the inner thigh of the animals. 39 of the animals received antibiotic, starting at either 6 hours post-challenge or at the onset of fever.

For F. tularensis studies, animals were challenged via the inhalational route with F. 15 tularensis strain Schu S4 and euthanised when they had reached a humane end point. Half of the animals received antibiotic starting 24 hours post-challenge.

For Coxiella studies, marmosets were challenged in a non-lethal manner with target doses of C. bumetti or sham (PBS) by the inhalational route. The animals were observed at least three times per day after challenge, for 21 days post-challenge.

Clinical signs were scored during physical entry into the room and/or temperature and remote camera observations were recorded during silent hours. Animals were also weighed daily.

All animals challenged with C. burnetti exhibited a febrile response, except two animals that received a low dose of C. bumetti (less than 10 CFU). The latter animals did not get a fever and maintained a normal diurnal rhythm for the duration of the study. All remaining animals became febrile between 4.25 and 5.25 days post-challenge (mean time of 4.75 days). Onset of fever (defined as greater than 40 °C) was statistically correlated to the dose the animals received (Pearson's Correlation R2 = 0.60, p = 0.0081). However, the duration of fever varied from 2.5 to 7.75 days and was not associated with the dose the animals received.

Flow cytometry on leukocyte populations Tissue samples were homogenised to provide single cell suspensions. Red blood cells were lysed, and the mixed leukocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and HLA-DR (MCHII) (L243) (BD Bioscience, BioLegend, AbD serotec).

Whole cells were detected by nuclear staining allowing the area of interest to be defined by forward and side scatter. Forward and side scatter were also used to gate areas for detection of lymphocytes (T-and B-cells), natural killer (NK) cells, macrophages (MO) and neutrophils.

is Statistics Statistical analysis, Kruskal Wallis tests and ANOVAs were performed using Graphpad PRISM V6.0 The statistical package SPSS V21.0 was used for principal component analysis using the discriminant analysis module. For this analysis, missing data points were substituted with group means (8 of 264 data points).

Results 5. pseudomallei The proportion of intact identifiable neutrophils changed during the course of disease The proportions of the major cell types, granulocytes (neutrophils), macrophages/monocytes and lymphocytes (expressed as a percentage of total cells) were determined in blood and from homogenates of both the lung and the spleen at 12 hour time points during the disease course (Fig. 1; error bars show median and interquartile ranges. Mean level of bacteraemia or bacterial load per gram of associated sample shown with presence of fever (-indicates absence, + indicates presence). The average bacterial count found in the samples is also given. There was a decline in both the percentage of neutrophils and macrophages identifiable in lung tissue from the earliest time point until 48 hours post infection. There was a steady rise in the neutrophil percentage in the blood during the course of disease and a marked loss of these cells in the terminal samples. There was a corresponding change in the proportion of lymphocytes in these samples, which was predominantly caused by changes in T-cells, with the proportion of B-cells remaining constant. The cell profiles from the terminal samples were most similar to naïve samples.

There was an immediate change in the markers expressed on the intact neutrophils in both tissue samples and blood in response to infection There was an initial decrease in the proportion of neutrophils in the lungs immediately after challenge, followed at 36 hours by an increase in both tissues and blood (Fig. 1). The level of activation markers expressed on these cells changed significantly suggesting influx of immature cells, in particular HLA-DR (MHCII) which is highly expressed on normal functional marmoset neutrophils. This marker was reduced, as disease progressed, on all neutrophils in all tissues (Fig. 2; significance difference from baseline (naïve sample) using Dunn's post-tests is marked (P<0.001 = ***, P<0.01 = ** and P<0.05 = *)). The decrease coincided with loss of activation markers CD16 (neutrophil maturation marker) and CD54 (adhesion/migration marker) in the lung. This trend was not as apparent in either the spleen homogenates or the blood. HLA-DR expression on neutrophils in the spleen and blood was dramatically reduced by the earliest time point suggesting that there was a systemic response to disease despite bacteria at this time being localised to the lung and the animals being pre-symptomatic.

HLA-DR expression was compared on neutrophils in blood samples, taken across time source studies, from animal challenged via the inhalation/aerosol and subcutaneous routes respectively (Fig 3; samples were a) before challenge (pre) b) at 12 hour time point intervals after challenge and c) when the animals succumbed to disease (term). Routes of infection were inhalational (aero) and sub-cutaneous (SC) left hand side = inhalation/aerosol challenge, right hand side = subcutaneous challenge). Both routes of infection conferred a reduction in HLA-DR expression, with greater significance observed in subcutaneous disease.

Significant reduction in HLA-DR expression was consistently observed at 12 hours post-challenge compared to baseline levels in 6/8 animals (P=0.004 by paired t-test) (Fig. 4). These animals were challenged with a lethal aerosol of B. pseudomallei and tested before clinical signs were apparent. Comparing all of the matched blood samples (pre-challenge to post-challenge, regardless of the time after challenge), a reduction of 20% in HLA-DR positively stained neutrophils would have correctly identified 86% as requiring treatment (46/53) and 20% (1/5) of the healthy animals. In a further experiment, during the course of severe disease, F-ILA-DR expression was significantly reduced, compared to pre-infection levels, in 10/10 animals at the point of fever (Fig. 5). These animals were challenged with a lethal dose of B. pseudomelfel (by inhalation, ingestion or subcutaneously) and a femoral blood sample taken at the point at which the animals started to display symptoms (in this case at 2 °C above normal body temperature, which was typically observed just over 24 hours after challenge). Reduction of HLA-DR was always observed in terminal animals.

It was observed that upon treatment of animals with antibiotics, after development of fever, levels of HLA-DR expression returned to pre-challenge levels (Figure 6; levels of HLA-DR in: pre-challenge animals Clare HLA-DR N'); animals displaying mild fever following infection (lever HLA-DR N') and surviving animals at 14 days after the cessation of treatment (late HLA-DR Ni). Thus, upon treatment of infection, the expression of HLA-DR on neutrophils recovers to pre-challenge levels.

Key characteristics from blood samples were able to discriminate between health and apparently healthy 12 hours post-challenge animals In order to explore whether collected data might be used to discriminate samples with regards to their infection status, Principal Component Analysis (PCA) was performed. The analysis used eight variables; four variables relating to the proportions of neutrophils and their activation (HLA-DR, CD14, CD16 and CD54 expression), and a further four variables consisting of key inflammatory cytokines (IFN-y, TNF-a, IL-13 and IL-6). The model was able to discriminate between matched blood samples from animals pre-and post-challenge with 100% accuracy (Table 1). For surviving animals, 1 out of 5 marmosets was sorted into the 12 hr post infection group. This animal and two others had low counts of cultivable B. pseudomallei found only in their lungs. It was observed that this animal was the only animal to have a reduced (outside normal pre-bleed range) expression of circulating neutrophil HLA-DR. The basis of discrimination was largely based on one canonical function, which used predominantly the expression of HLA-DR, CD14 and CD16 on neutrophils and IFN-y concentrations (Fig. 7; biomarkers broken down into the square of the Fisher's standardisation coefficients to show which data contributed to each function).

Actual membership Predicted Group Membership Total 12 48 Pre- Survivors hours hours infection 12 hours 8 0 0 0 8 48 hours 1 7 0 0 8 Pre-infection 0 0 21 2 23 Survivors 1 0 0 4 5 Table 1: The discriminatory capability of a PCA model to differentiate animals regarding on when blood samples were taken based on four activation states of neutrophils and four inflammatory cytokines.

F. tularensis Significant reduction in blood neutrophil HLA-DR expression has been shown to also occur in terminal F. tularensis disease in marmosets (Figure 8), wherein test animals were challenged with a lethal inhalation challenge of F. tularensis and culled at the human end point or 2 weeks after antibiotic treatment. As shown in the left-hand side of the graph, 7 out of 8 healthy surviving treated animals had no significant reduction in HLA-DR expression. In contrast, 8 out of 8 samples taken from terminal animals had a significant reduction in HLA-DR expression.

C. bumetti HLA-DR expression by neutrophils following C. bumetti infection was assessed in a non-lethal marmoset model. For this particular model, animals display a slightly fever for several days that spontaneously resolves with no other observable clinical signs.

The results obtained show a reduction in HLA-DR expression by day 7, in the event of mild disease where the animals have an increase in body temperature but no other clinical signs (Figure 9; data presented as median with the interquartile range). However, by days 14 and 21, HLA-DR expression had returned to pre-infection levels.

Various modifications to the invention can be made as will be apparent to those skilled in the art. For example, the invention may incorporate any method understood by the skilled person to be capable of detecting an amount, or expression levels of, HLA-DR from a biological sample. For example, the method may be based on: a chromatography method, such as high performance liquid chromatography (HPLC); a spectrometry method, such as mass spectrometry (MS); a combination of chromatography and spectrometry methods, for example liquid chromatography-mass spectrometry (LC-MS); other methods for identifying/quantifying a specific protein in a sample (either alternatively or in combination with the methods of the invention, such as enzyme-linked immunosorbent assay (EL ISA), protein immunoprecipitation, immunoelectrophoresis, Western blot, protein immunostaining; or other forms of protein assay, such as Bradford assay or ninhydrin assay.

It will be understood that the present invention has been described above purely by 25 way of example, and modification of detail can be made within the scope of the invention. Each feature disclosed in the description, and (where appropriate) the claims and may be provided independently or in any appropriate combination.

Moreover, the invention has been described with specific reference to a method and associated uses and kit, and more specifically with reference to assessing and/or monitoring the health status of non-human primates. Additional applications of the invention will occur to the skilled person.

Claims (15)

1. CLAIMS1. A method for assessing and/or monitoring the health status of a non-human primate comprising evaluating the expression of HLA-DR in a biological sample.
2. 2. A method according to claim 1 comprising the steps of: taking a first biological sample and a second biological sample from the non-human primate; employing a means for measuring an expression level of HLA-DR in the first biological sample and the second biological sample; and comparing the expression level of HLA-DR in the second biological sample in relative to the first biological sample, wherein a respective HLA-DR expression level reduced in the second biological sample, relative to the first biological sample, is an indicator of reduced health status of the non-human primate.
3. 3. A method according to claim 1 to claim 2 wherein the biological sample(s) is 15 blood.
4. 4. A method according to any preceding claim wherein the HLA-DR is neutrophilexpressed H LA-DR.
5. 5. A method according to claim 4 wherein the neutrophils are isolated from the biological sample(s) prior to measuring neutrophil-expressed HLA-DR.
6. 6. A method according to any preceding claim wherein the level of HLA-DR expression reduces by at least 5% to indicate a reduced health status of the nonhuman primate
7. 7. A method according to any preceding claim wherein the means for measuring the expression level of HLA-DR in the biological sample(s) is antibody-based.
8. 8. A method according to claim 7 wherein the antibody-based means system is incorporated in a lateral flow assay.
9. 9. A method according to claim 7 wherein the antibody-based means is incorporated in a flow cytometry assay.
10. 10. A method according to claim 1 to claim 6 wherein the means for measuring the expression level of HLA-DR in the biological sample(s) is nucleic acid-based.
11. 11. A method according to claim 10 wherein the nucleic acid-based means is incorporated in a RT-PCR or microarray reaction.
12. 12. Use of a bioactive substance capable of binding to a gene product of a gene or genes encoding HLA-DR as a means for assessing and/or monitoring the health status of a non-human primate.in
13. 13. A kit for performing the method of claim 1 to claim 11 comprising a means to specifically detect HLA-DR.
14. 14. Use of a kit according to claim 13 as a means for assessing and/or monitoring the health status of a non-human primate.
15. 15. Use of a kit according to claim 14 as a means for assessing and/or monitoring 15 the health status of a non-human primate infected by Burkholdene pseudomallei, Francisella tularensis or Coxiella burnetti.