EA040435B1 - SKIN TESTING FOR TUBERCULOSIS IN IMMUNE COMPROMISED PEOPLE - Google Patents

Description

The field of technology to which the present invention relates

The present invention relates to the use of Mycobacterium tuberculosis antigens to provide a specific skin test composition with improved diagnostic performance for TB in humans co-infected with M. tuberculosis and HIV. The present invention provides a composition containing Mycobacterium tuberculosis (Mtb) antigens for in vivo diagnosis of Mtb infection in immunocompromised or HIV co-infected individuals, wherein the reagent contains a mixture of two ESAT6 and CFP-10 antigens.

Prior Art

Overview of TB/PPD.

Since its discovery in 1908 by Robert Koch, purified tuberculin protein derivative (PPD) has been used as a diagnostic test for latent Mtb infection.

The use of a diagnostic test for latent Mtb infection to guide prophylactic treatment has a significant impact on the further development of TB disease. People infected with Mtb are asymptomatic and require a milder treatment protocol, whereas patients with active TB disease have severe disease, high mortality, and require longer and more complex treatment. It is estimated that 1/3 of the world population is infected with Mtb, 8 million people develop active tuberculosis each year, of which 1.3 million die.

When used as a diagnostic test, a small volume of 0.1 ml of PPD is injected into the top layer of the skin, and after 2-3 days a positive reaction can be determined by measuring skin compaction using a simple ruler. The immunological basis of the skin reaction to PPD is a type IV delayed hypersensitivity reaction, a reaction mediated primarily by PPD-specific Th1 T cells.

PPD, when used as described by Mendel and Mantoux, has been approved by the World Health Organization since 1955, and the PPD-based tuberculin skin test (TST) remains one of the most widely used diagnostic tests, with over 50 million tests performed annually.

PPD is a complex culture filtrate from M. tuberculosis, including, in addition to lipids and carbohydrates, more than 1000 protein antigens. This makes PPD widely recognized in Mtb-infected individuals and is highly immunogenic, stimulating a strong Th1 T cell response in the skin. However, the crude and complex formulation of PPD also causes problems with PPD TST. Importantly, multiple antigens in PPD are also recognized by regulatory T cells, CD-1 restricted T cells, and Th2 cells, which can selectively inhibit the type IV response. In addition, multiple antigens in PPD are also expressed by environmental mycobacteria and Bacillus Calmette-Guerin (BCG) used for tuberculosis vaccination, making the PPD test non-specific.

HIV and immunocompromised people.

Another major concern with the use of TDP PPD is its unreliability in immunocompromised individuals. In particular, in people infected with the human immunodeficiency virus (HIV), the PPD TST often becomes non-responsive and false negative, or may be false positive due to the use of too low a threshold.

The immunological basis for the effect of HIV on the ability of PPD to induce a type IV skin reaction is complex, but the underlying cause has been identified as the selective loss of CD4 T cells due to HIV infection. Therefore, immunodeficiency caused by loss of CD4 T cells is a fundamental factor leading to disease from opportunistic pathogenic viruses and bacteria (a hallmark of HIV/AIDS) as well as failure to respond to PPD TST.

The consequences of this dilemma can be deadly. Often, HIV infection (or other immunodeficiency) is not diagnosed during PPD TST testing. Therefore, the responsible physician often erroneously relies on the PPD TST result. This false sense of security often leads to misdiagnosis and lack of adequate treatment for Mtb infection, leading to the further development of active TB disease. In fact, Mtb-infected HIV/AIDS patients have a 10% annual risk of developing TB, and TB is the disease that kills most HIV/AIDS patients.

Solving the problem of cross-reactivity to BCG.

As stated above, the main limitation for PPD TST is cross-reactivity with BCG and environmental mycobacteria. Cross-reactivity reduces specificity and potentially leads to overtreatment due to false positive responses. The problem of specificity can be solved to some extent, but not completely, by increasing the threshold for TST from 5 mm thickening to 10 mm or even 15 mm, however, increasing the threshold reduces the sensitivity of the test.

If adequate treatment for Mtb infection has not been offered, further desensitization can be fatal, especially for HIV-infected and other large cohorts of patients with

- 1 040435 high risk of developing TB. Thus, for optimal PPD TST performance, testing for HIV would be required prior to PPD TST testing, for example, in BCG vaccinated individuals. However, due to limited resources and inhibitions among both healthcare providers and patients, HIV testing is often not offered or accepted by the person who needs PPD TST testing.

The era of specific testing.

The discovery of Mtb-specific immunodominant antigens has led to a significant new way to diagnose Mtb infection. Previous work has shown the possibility of replacing the PPD TST with a test that assays in vitro production of interferon-gamma (IFN-γ) by T cells in response to Mtb antigens. Around the same time, an important advance was the discovery of highly immunogenic antigens, early secretory antigenic target-6 (ESAT-6) and culture filtrate protein 10 (CFP-10), which significantly improved specificity. These antigens are encoded in the gene 1 (RD1) difference region of the pathogen and, therefore, are absent in all vaccine strains of Bacille Calmette Guerin (BCG) bacteria and most non-tuberculous mycobacteria (with the exception of Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium szulgai).

The basis for the detection of Mtb infection in two commercially available IFN-γ secretion assays (IGRAs): QuantiFERON-TB Gold (QIAGEN, Germany) and T-SPOT.TB (Oxford Immunotec, Oxford, UK) are IFN-γ responses to overlapping peptides encoded RD antigens ESAT-6 and CFP10.

QuantiFERON-TB Gold (QFT, QIAGEN, Germany), an enzyme-linked whole blood immune assay (ELISA) method, has a European mark of conformity and US Food and Drug Administration (FDA) approval for the detection of both latent TB infection and and diseases.

T-SPOT.TB (Oxford Immunotec, Oxford, UK), an enzyme-linked immunosorbent assay (ELISPOT) that uses peripheral blood mononuclear cells, has the European Conformity Mark and FDA approval.

IGR restrictions.

However, despite the apparent superiority found for IGRA over TST, IGRA has several limitations.

IGRA tests based on blood samples require blood sampling, sophisticated laboratory equipment, and trained personnel to administer it. These limitations make the test nearly impossible to implement in resource constrained environments where such tests are most in demand. IGRA tests are also prone to problems in the pre-analytical stage, such as false negative responses due to the delayed start of the incubation stage.

Compared to PPD TST, the diagnostic performance of IGRA tests also requires that the person being tested be able to mount a robust immune response. Just as with PPD TST, IGRA sensitivity may be impaired in immunosuppressed individuals. In particular, patients with HIV infection and low CD4 T cell counts or patients taking immunosuppressive treatment often experience false negative or equivocal responses.

A likely underlying reason for the low performance of IGRA tests in immunosuppressed is that they are based on the detection of a very weak IFN-γ signal, considered positive (17.5 pg/ml or 6 reactive T cells per 200,000 PBMCs). These low responses border on the detection limits of the most sensitive assays used in clinical medicine.

Thus, the skin test, in which an immune response can be elicited by potentially any specific T cell available in the body, appears to be much more reliable per se compared to the in vitro diagnostic test, in which the number of T cells available is limited. a small number of cells contained in the sample.

A meta-analysis by Cattamanchi and colleagues (JAIDS 2011, 56(3) 230-238) showed that for HIV-infected people with active TB (a surrogate reference standard for latent TB infection) in low- and middle-income countries, pooled sensitivity estimates were heterogeneous, but higher for the T-SPOT.TB assay (72%; 95% CI, 62-81%) than for the QFT assay (61%; 95% CI, 47-75%). However, in a head-to-head comparative study, none of the IGRAs was more sensitive than the PPD TST. The authors concluded that IGRA may be less affected by the degree of HIV-induced immunosuppression compared to TST PPD, but results vary geographically.

The potentially life-threatening consequences of false-negative PPD TST and IGRA results have been described by Sester et al. (Am J Respir Crit Care Med. 2014 Nov 15; 190(10):1168-76. doi: 10.1164/rccm.201405-0967OC). In this large prospective study of 768 patients with HIV infection, eight patients with HIV infection developed active TB disease during follow-up. Five out of 8 patients with an established IGRA test result at screening

- 2 040435 had a false negative IGRA test and were not offered prophylactic treatment. In another 3 HIV-infected patients who developed TB, the IGRA test result was questionable. PPD TST was performed in all 11 who developed TB. In this group, only 4 out of 11 tested were positive using the HIV-optimized cut-off of 5 mm.

Diaskintest is a recombinant CFP10-ESAT6 fusion protein produced by E. coli BL21 (DE3)/pCF-ESAT. The protein is expressed as a 6x histidine-labeled protein, and Generium is produced in the Russian Federation. The dose is 0.2 mcg / 0.1 ml. Any size of the lump is considered to be indicative of infection with Mtb. The histidine label causes the risk of neoepitopes. Diaskintest is reported with acceptable specificity, but with a high incidence (4-14%) of vesicular necrotic changes, lymphangitis and lymphadenitis (Kiselev, VI; Probl. Tuberk. Bolezn. Legk. 2009; 2:11-6). Repeated injections of high doses of antigen may pose a risk of sensitization, which may lead to false positive reactions, in addition to causing a risk of adverse reactions. Diaskintest is the only product in clinical use. Several studies have examined the effectiveness of diaskintest in HIV-infected people and found a very strong negative effect of HIV infection on the effectiveness of diaskintest. Pankratova found 42.9% sensitivity in HIV-positive subjects compared to 79.7% in subjects without HIV infection (Pankratova L et al, European Respiratory Journal 2012, vol 40, Suppl 56/P431), similar significant results were obtained Litvinov; 43.5% in HIV-infected versus 89.7% in HIV-negative cases (Litvinov et al, Am J Respir Crit Care Med 185;2012:A4703).

Thus, the PPD TST is an attractive and simple test for latent Mtb infection, however, the PPD antigen component is non-specific, and PPD TST results in HIV-infected individuals are often false negative. The IGRA test is based on the specific Mtb ESAT-6 and CFP10 antigens and addresses issues related to the specificity of the PPD TST test, however, these tests are difficult to implement and perform, and are often false negative in HIV-infected people. Diaskintest provides a skin test with the same specificity as IGRA, however, the effectiveness of diaskintest in HIV-infected people is significantly impaired, and in HIV-infected people the sensitivity is reduced by half compared to those not infected with HIV. Thus, there is a need for a new diagnostic skin test for Mtb infection with a specificity superior to the TST PPD and increased sensitivity in HIV-infected individuals.

Brief summary of the present invention

The present invention discloses an in vivo diagnostic method (skin testing) and diagnostic agents for diagnosing Mtb in immunocompromised or HIV co-infected individuals, individuals who would not otherwise be recognized as M. tuberculosis infection due to the lack of sensitivity of existing tests.

The result is achieved by using a mixture of M. tuberculosis ESAT6 and CFP10 antigens to produce a skin test agent consisting of a mixture containing these antigens. In a preferred embodiment, a mixture of rdESAT-6 and rCFP-10 antigens is used. The mixture of CTb antigens is highly functional in HIV infection, showing an overall reduction in sensitivity of only 33% compared to 45% for Quantiferon. Unlike other testing methods, C-Tb appears to be affected only by HIV infection in the most severe cases with CD4+ T cell counts <100 CD4+ T cells/µL.

Detailed disclosure of the present invention

The present invention discloses a composition containing Mycobacterium tuberculosis antigens for use in determining in vivo presence of Mtb infection in immunocompromised or HIV co-infected individuals, wherein the agent contains a mixture of two antigens ESAT6 and CFP10.

The present invention discloses the use of Mycobacterium tuberculosis antigens to provide a skin testing diagnostic tool (skin test tool) for reliably assessing the presence of Mtb infection in a human, the subject being an immunocompromised human or a human co-infected with HIV.

The present invention also discloses a method for diagnosing Mtb infection in immunocompromised or HIV-coinfected individuals. The diagnostic method is based on measuring the intensity of the response in the skin generated in response to stimulation of specific immune cells with specific Mtb antigens.

The two antigens are preferably cloned, fermented and purified in a suitable organism, eg from Lactococcus lactis, rESAT6 and rCFP10, and most preferably double ESAT-6 (rdESAT6, two ESAT-6 molecules fused together). rdESAT-6 and rCFP10 are preferably mixed in a 1:1 (m/m) ratio. The composition contains two antigens mixed in a vehicle, the most preferred vehicle being phosphate buffered saline (PBS) with 0.01% polysorbate 20 and 0.5% phenol.

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Definitions.

The term tuberculosis refers to the clinical condition of tuberculosis caused by various strains of mycobacteria, usually Mycobacterium tuberculosis.

The term latent M. tuberculosis infection refers to subclinical or latent M. tuberculosis infection, which is defined by the presence of an immune response to M. tuberculosis without signs or symptoms of active disease.

The term ESAT-6 in the present invention refers to a 6 kDa early secretory antigen target produced by Mycobacterium tuberculosis (esxA) and is a secretory protein and potent antigen for T cells, locus tag Rv3875.

The terms CFP10 in the present invention refers to the secreted 10 kDa antigen from Mycobacterium tuberculosis, also known as the ESAT-6-like protein esxB, or the secreted antigenic protein MTSA-10, or the 10 kDa culture filtrate antigen, Rv3874.

The terms cocktail and antigenic cocktail in the present invention refer to at least two proteins together in a vehicle or solution.

The term immunodeficiency or immunosuppression or immunocompromised refers to a condition in which the ability of the immune system to fight an infectious disease or mount an immune response is compromised or completely absent.

The term C-Tb refers to a formulation of the composition of rdESAT-6 and rCFP-10.

The term HIV refers to the human immunodeficiency lentivirus that causes HIV infection and, over time, can lead to acquired immunodeficiency syndrome (AIDS).

The term fusion protein is understood to mean a random order of two or more immunogenic polypeptides from M. tuberculosis or their analogues, fused together with an amino acid linker/spacer (amino acid linkers/spacers) or without an amino acid linker/spacer (amino acid linkers/spacers) of arbitrary length and sequence. To avoid protein aggregation in further production, all cysteines in the fusion protein can be replaced by any amino acid, but serine is the preferred substituent due to its high structural similarity to cysteine.

The surprisingly improved diagnostic sensitivity in HIV-infected people is likely achieved through a combination of mechanisms. The C-Tb skin test reagent consists of a mixture of antigens, compared to, for example, Diaxintest, which is a hybrid construct containing two proteins connected by a linker. The design of the C-Tb mixture offers several potential advantages, including better exposure of epitopes without the interference of linker proteins. In addition, the skin test format offers advantages over the IGRA format. The long incubation period allows the recruitment of the same population of effector T cells that is monitored by IGRA, but also the recruitment of a population of less differentiated cells. Wider T cell recruitment, combined with the skin test mix format, likely resulted in superior sensitivity.

The skin test diagnostic contains two antigens ESAT6 and CFP10. In a preferred embodiment of the skin test, the reagent consists of a mixture of recombinant versions of the two antigens. In the most preferred embodiment, the two antigens are double ESAT-6 (rdESAT-6; two ESAT-6 molecules fused together) and rCFP-10. The two antigens are cloned, fermented and purified in a suitable organism, for example from Lactococcus lactis or E. coli. The antigens are mixed in a vehicle or pharmaceutically acceptable carrier. In the most preferred embodiment, rdESAT-6 and CFP10 are mixed in a 1:1 (m/m) ratio in phosphate buffered saline (PBS) vehicle with 0.01% Polysorbate 20® (and 0.5% phenol). The dose of the antigen is in the range of 0.25-2.0 μg/ml of each antigen. The preferred amount is 0.1 μg/0.1 ml, corresponding to a total concentration of 1 μg antigens/ml, corresponding to 0.5 μg/ml each. This specific preparation of rdESAT-6 and rCFP-10 is referred to as C-Tb. The reagent is not limited to these antigens, but may additionally include other antigens such as Rv3615.

Performing a skin test.

Skin testing is performed by injecting a small volume of C-Tb intradermally, eg 0.1 ml, into the inner surface of the forearm.

The injection should be done with a tuberculin syringe with the needle pointing up. However, other means of intradermal injection may also be used. In the correct area, C-Tb injection should cause a pale skin elevation (papule) 6 to 10 mm in diameter.

The skin test reaction should be read between 48 and 72 hours after administration. A patient who does not return within 72 hours should be rescheduled for another skin test.

The magnitude of the skin reaction can be determined by measuring the induration in millimeters (the diameter of the palpable, elevated area of induration or swelling). The diameter of the compacted area should be measured across the forearm (perpendicular to the long axis of the forearm).

A compaction of 5 mm or more, according to the illustrative material, is the preferred threshold, without prejudice to the fact that another threshold can be used, including 10, 4, 3, 2, 1, or even any response above 0 mm.

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The concept of C-Tb is to combine the well-known Mantoux technique using specific antigens from IGRA with a specific skin test using a single universal threshold of 5 mm induration, regardless of BCG vaccination and HIV status, read after 2-3 days to identify individuals infected Mtb delivered and interpreted by medical or nursing staff at the point of care.

Diagnostics.

Testing the skin with a specific reagent is especially helpful for people with immunosuppression. HIV represents a key risk population in which Mtb infection can rapidly progress to active disease. HIV-infected individuals have compromised CD4 T cell function, so skin testing with reagent-specific targets, Th1 T cells rather than Th2 or regulatory T cells, may be superior.

The target population for C-Tb includes people exposed to Mtb or people showing signs or symptoms of TB, with particular attention to groups at increased risk of developing TB after infection. These groups include, but are not limited to, newly infected cases identified through contact tracing, children under 5 years of age, and HIV-infected individuals.

The present invention provides a skin test reagent for reliably assessing the presence of Mtb infection in people infected with HIV. As clearly shown in the examples, the specific skin test example (C-Tb) has excellent performance for both PPD TST and IGRA.

rdESAT-6 and CFP10 are mixed in a 1:1 (m/m) ratio in vehicle, although in a preferred embodiment the ratios are other than 1:1 m/m ratios including 1:5, 1:4, 1:3, 1 :2; 2:1, 3:1, 4:1, 5:1, and other ratios covering 1:20 and 20:1 are expected to be useful.

The dose value of the antigen is in the range of 0.25-20.0 μg/ml of each antigen. The preferred amount is 0.1 μg/0.1 ml, corresponding to a total concentration of 1 μg of antigens/ml, corresponding to 0.5 μg/ml each. More antigen is expected to result in a stronger skin tightening reaction, but increases the risk of adverse or overly strong reactions. Depending on the clinical situation, genetic or ethnic composition, the degree of immunosuppression, or other characteristics of the test population, the dose of antigen can be selected in the range of 0.25-20.0 0.25-20.0 μg/ml of each antigen.

Although the present invention describes a specific preparation of rdESAT-6 and rCFP-10, the agent is not limited to these antigens, but may additionally include other antigens such as espC (Rv3615) or other specific antigens expressed by M. tuberculosis. Some antigens known to be specifically expressed by infectious agents in the M. tuberculosis complex may be a suitable addition or replacement for ESAT-6 and CFP10 in C-Tb. These antigens include IS1 restricted antigens, IS1 associated antigens, and antigens with immunodiagnostic potential such as Rv2564, Rv3865, Rv3877, Rv2348, Rv3614, Rv3616 and others known to those skilled in the art.

In a preferred embodiment of the C-Tb test, intradermal injection is provided. It should be understood that injection using a syringe and needle is only one of several methods of administration. Alternative methods include the Giff pistol (Sternidle) and various other methods such as, but not limited to, jet injection, bullet delivery using powder and gold particles (i.e., gene gun), hard microneedle patches (metal or dissolving microneedles), topical addition of C-Tb after pre-treatment with microneedles, tattoo guns, or techniques designed to increase skin permeability in combination with a delivery patch, such as abrasion, ultrasound, electroporation, laserporation, chemical enhancers, and thermal skin ablation.

Description of figures

Fig. 1 - frequency of positive C-Tb (black; cut-off 5 mm) and QFT (white; cut-off 0.35 IU/ml) among 534 HIV-negative and 277 HIV-positive suspected TB. The error value shows 95% CI.

Fig. 2 is the frequency of positive C-Tb (black) and QFT (white) tests, stratified according to CD4 count among 292 suspected TB. The error value shows 95% CI.

Examples

Diagnostic C-Tb was obtained by cloning, fermentation and purification of recombinant versions of the two antigens rdESAT-6 and rCFP-10 from Lactococcus lactis. Antigens were mixed in equimolar amounts of ESAT-6 and CFP-10 corresponding to a 1:1 m/m ratio of rdESAT-6 and rCFP10 in phosphate buffered saline (PBS) vehicle with 0.01% Polysorbate 20® (and 0.5% phenol).

The diagnostic performance of C-Tb, PPD and an IGRA called QuantiFERON®-TB Gold In Tube (QFT) was compared in a stage III trial in infants and children under 5 years of age with suspected Mtb infection, and in older children and adults with suspected TB disease. The trial of 1190 participants included 299 HIV positive and 730 HIV negative. Blood for testing

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Claims (17)

QFT was collected before the skin test. C-Tb and PPD were injected in a double-blind manner in different groups with a randomized design with a separation of participants. Available results from volunteers with paired results were included. As can be seen from FIG. 1, C-Tb and QFT showed a similar positivity rate among HIV-negatives, 292/534 (54.7%) were positive with C-Tb and 285/534 (53.4%) were positive with QFT. In total, 277 HIV-infected people were tested in both tests. Surprisingly, C-Tb detected significantly more infected Mtb than the QFT test, 102/277 (36.8%) were positive with C-Tb and only 80/277 (28.9%) were positive with QFT (table, McNemar ; P=0.009). HIV positive + QFT S+ 59 43 102 l H - 21 154 175 ΰ _____ s 80 197 277 P = 0.009 When stratifying the rest of the results by CD4 T cell counts (Figure 2), it becomes clear that the higher sensitivity to C-Tb is due to a significantly increased resistance not only in the low strata of CD4 T cells, but across the spectrum of CD4 T-cell counts. cells. CLAIM 1. Use of a composition containing a mixture of two Mtb ESAT6 and CFP-10 antigens for in vivo skin test diagnosis of latent Mycobacterium tuberculosis (Mtb) infection in immunocompromised or HIV co-infected individuals. 2. Use according to claim 1, wherein said two antigens are cloned, produced and purified in a suitable organism, for example from Lactococcus lactis. 3. Use according to any one of the preceding claims, wherein the composition consists of a mixture of two antigens of recombinant ESAT6 dual and recombinant CFP-10. 4. Use according to any one of the preceding claims, wherein said antigens are mixed in the vehicle. 5. Use according to claim 4, wherein the recombinant double ESAT6 and recombinant CFP10 are mixed in a 1:1 (m/m) ratio. 6. Use according to claim 4, wherein the recombinant double ESAT6 and recombinant CFP10 are mixed in ratios between 1:20 (m/m) and 20:1 (m/m). 7. Use according to any one of the preceding claims, wherein the amount of said antigens is between 0.25 and 2.0 μg/ml of each antigen. 8. Use according to any one of claims 4 to 7, wherein the vehicle comprises phosphate buffered saline (PBS) with 0.01% polysorbate 20 and 0.5% phenol. 9. The use according to any one of the preceding claims, wherein the diagnosis is based on measuring the intensity of the response in the skin generated in response to stimulation of immune cells by said composition administered by intradermal injection. 10. Use according to claim 9, wherein the intensity of the reaction is measured between 48 and 72 hours after administration of said composition. 11. Use according to any one of the preceding claims, wherein the composition contains one or more additional Mycobacterium tuberculosis antigens. 12. Use according to claim 10, wherein the additional antigens are selected from the group consisting of RD1 restricted antigens, RD1-α associated antigens, and Rv2564, Rv3865, Rv3877, Rv2348, Rv3614, Rv3615 and Rv3616. 13. Use according to any one of the preceding claims, wherein the subject is a child. 14. Use according to any one of the preceding claims, wherein the subject is an adult. 15. Use according to any one of the preceding claims, wherein the subject is suspected of having Mtb infection. 16. Use according to any one of the preceding claims, wherein the subject is suspected of having a TB disease. 17. Use according to any one of the preceding claims, wherein the subject has no signs and/or symptoms of active TB disease. -