Tuberculosis In Nonhuman Primates Research Articles

Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates

Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. Macaques that developed LTBI (n=36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n=21; p<0.01, q<0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p<0.05) and increasing airway IgG responses to some proteins over time. Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Surveillance of Daughter Micronodule Formation Is a Key Factor for Vaccine Evaluation Using Experimental Infection Models of Tuberculosis in Macaques

Tuberculosis (TB) is still a major worldwide health problem and models using non-human primates (NHP) provide the most relevant approach for vaccine testing. In this study, we analysed CT images collected from cynomolgus and rhesus macaques following exposure to ultra-low dose Mycobacterium tuberculosis (Mtb) aerosols, and monitored them for 16 weeks to evaluate the impact of prior intradermal or inhaled BCG vaccination on the progression of lung disease. All lesions found (2553) were classified according to their size and we subclassified small micronodules (<4.4 mm) as 'isolated', or as 'daughter', when they were in contact with consolidation (described as lesions ≥ 4.5 mm). Our data link the higher capacity to contain Mtb infection in cynomolgus with the reduced incidence of daughter micronodules, thus avoiding the development of consolidated lesions and their consequent enlargement and evolution to cavitation. In the case of rhesus, intradermal vaccination has a higher capacity to reduce the formation of daughter micronodules. This study supports the 'Bubble Model' defined with the C3HBe/FeJ mice and proposes a new method to evaluate outcomes in experimental models of TB in NHP based on CT images, which would fit a future machine learning approach to evaluate new vaccines.

Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates.

Simple SummaryTuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is the most lethal infectious disease from a single pathogen for which there is no effective vaccine available. Rhesus macaques are extremely susceptible to MTB and therefore represent relevant models to study the pathogenesis of TB and assess the potential of TB drugs and vaccines. However, there are no diagnostic tools currently available that allow rapid, user-friendly detection of TB for either TB research purposes or monitoring nonhuman primate colonies. To develop a rapid diagnostic test, we investigated whether low complexity lateral flow assays (LFAs) that we recently developed for rapid and quantitative detection of human serum proteins are applicable to detect and monitor active pulmonary TB in NHPs. We found that serum levels of SAA1, IP-10, and IL-6 detected by LFAs were significantly increased after MTB infection in rhesus macaques. Moreover, levels of these biomarkers correlated with disease severity as determined by pathology scoring and allowed detection of the effect of vaccination and drug treatment in experimentally MTB infected macaques. These UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be used to assess new therapeutic and prophylactic treatment methods in macaques to tackle TB.Nonhuman primates (NHPs) are relevant models to study the pathogenesis of tuberculosis (TB) and evaluate the potential of TB therapies, but rapid tools allowing diagnosis of active pulmonary TB in NHPs are lacking. This study investigates whether low complexity lateral flow assays utilizing upconverting reporter particles (UCP-LFAs) developed for rapid detection of human serum proteins can be applied to detect and monitor active pulmonary TB in NHPs. UCP-LFAs were used to assess serum proteins levels and changes in relation to the MTB challenge dosage, lung pathology, treatment, and disease outcome in experimentally MTB-infected macaques. Serum levels of SAA1, IP-10, and IL-6 showed a significant increase after MTB infection in rhesus macaques and correlated with disease severity as determined by pathology scoring. Moreover, these biomarkers could sensitively detect the reduction of bacterial levels in the lungs of macaques due to BCG vaccination or drug treatment. Quantitative measurements by rapid UCP-LFAs specific for SAA1, IP-10, and IL-6 in serum can be utilized to detect active progressive pulmonary TB in macaques. The UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be applied in studies on TB vaccine and drug development involving macaques.

Defining the role of CD8 T cell subsets in tuberculosis in non-human primates

Abstract Tuberculosis (TB) is the number one cause of death by a single infectious agent with no protective vaccine available. Challenges in vaccine development are partially attributed to a deficiency in knowledge regarding specific immune cells’ contribution to protection. CD8 T cells comprise ~30–40% of T cells in the lung granuloma, an organized collection of immune cells that is a hallmark of Mycobacterium tuberculosis (Mtb) infection. However, few studies have explored the diversity, function and contribution to protection of CD8 subsets during TB. We investigated the roles of conventional CD8αβ T cells and unconventional CD8αα T cells during early Mtb infection. Cynomolgus macaques were CD8-depleted using anti-CD8α or anti-CD8β antibodies prior to and during Mtb infection and necropsied at 6 weeks post-infection. CD8α depleted NHPs (CD8αα and CD8αβ depletion) had higher bacterial burdens, worse pathology and overall poorer disease outcome compared to IgG controls, while CD8β depleted (CD8αβ only depletion) displayed an intermediate phenotype. This provides the first evidence for an important early role for non-conventional CD8 T cells in TB. Using spectral flow cytometry we identified multiple CD8 subsets and assessed their functions in granulomas for the first time in NHPs. After CD8α depletion, the repertoire of CD8 subsets dramatically changed in granulomas. This was associated with functional alterations in other immune cell populations which we validated and expanded upon using single-cell RNA sequencing. These studies provide a functional immune landscape of early events in Mtb granulomas as well as the effects of CD8 depletion, shedding light on the protective potential of CD8 T cell subsets during primary Mtb infection.

Characterization of a potential host-directed therapy against TB in non-human primates

Abstract An estimated 558,000 cases of drug-resistant tuberculosis (TB) occurred in 2017, emphasizing a need for new treatment strategies. We assessed a host-directed therapy (HDT) for TB using two FDA-approved drugs. Linezolid (LZD) is a potent antibiotic for antibiotic-resistant Gram-positive infections and is an effective treatment for TB. However, lengthy antibiotic regiments (&amp;gt;4 weeks) may lead to LZD-associated host toxicities, most commonly bone marrow (BM) suppression. LZD toxicities are mediated by IL-1, a pathway important for early immunity during Mtb infection that later contributes to pathological TB disease. We hypothesized LZD efficacy could be enhanced by addition of Anakinra, a recombinant IL-1R antagonist (rIL-1RA), by reducing BM toxicity and TB-associated inflammation. We tested this HDT in a non-human primate (NHP) model which emulates the spectrum of human TB, combined with PET/CT to monitor disease progression. NHPs with active TB were treated with LZD or LZD+rIL-1RA and tracked by PET/CT to monitor granuloma formation and inflammation. Analysis of BM erythroid progenitor cells determined little evidence of BM suppression associated with short-term high dose LZD. However, we observed sterilization of the majority of granulomas and a trend of enhanced bacterial clearance with rIL-1RA. Addition of rIL-1RA decreased granuloma inflammation (measured by PET/CT) compared to LZD alone, however few differences in immune cell populations were observed. Together, these data indicate LZD as a promising addition to treatment of drug resistant TB and the potential of HDTs in improving therapy.

Immunization of Vγ2Vδ2 T cells programs sustained effector memory responses that control tuberculosis in nonhuman primates

Tuberculosis (TB) remains a leading killer among infectious diseases, and a better TB vaccine is urgently needed. The critical components and mechanisms of vaccine-induced protection against Mycobacterium tuberculosis (Mtb) remain incompletely defined. Our previous studies demonstrate that Vγ2Vδ2 T cells specific for (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) phosphoantigen are unique in primates as multifunctional effectors of immune protection against TB infection. Here, we selectively immunized Vγ2Vδ2 T cells and assessed the effect on infection in a rhesus TB model. A single respiratory vaccination of macaques with an HMBPP-producing attenuated Listeria monocytogenes (Lm ΔactA prfA*) caused prolonged expansion of HMBPP-specific Vγ2Vδ2 T cells in circulating and pulmonary compartments. This did not occur in animals similarly immunized with an Lm ΔgcpE strain, which did not produce HMBPP. Lm ΔactA prfA* vaccination elicited increases in Th1-like Vγ2Vδ2 T cells in the airway, and induced containment of TB infection after pulmonary challenge. The selective immunization of Vγ2Vδ2 T cells reduced lung pathology and mycobacterial dissemination to extrapulmonary organs. Vaccine effects coincided with the fast-acting memory-like response of Th1-like Vγ2Vδ2 T cells and tissue-resident Vγ2Vδ2 effector T cells that produced both IFN-γ and perforin and inhibited intracellular Mtb growth. Furthermore, selective immunization of Vγ2Vδ2 T cells enabled CD4+ and CD8+ T cells to mount earlier pulmonary Th1 responses to TB challenge. Our findings show that selective immunization of Vγ2Vδ2 T cells can elicit fast-acting and durable memory-like responses that amplify responses of other T cell subsets, and provide an approach to creating more effective TB vaccines.

Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques.

Although recent studies in mice have shown that components of B cell and humoral immunity can modulate the immune responses against Mycobacterium tuberculosis, the roles of these components in human and nonhuman primate infections are unknown. The cynomolgus macaque (Macaca fascicularis) model of M. tuberculosis infection closely mirrors the infection outcomes and pathology in human tuberculosis (TB). The present study used rituximab, an anti-CD20 antibody, to deplete B cells in M. tuberculosis-infected macaques to examine the contribution of B cells and humoral immunity to the control of TB in nonhuman primates during the acute phase of infection. While there was no difference in the overall pathology, disease profession, and clinical outcome between the rituximab-treated and untreated macaques in acute infection, analyzing individual granulomas revealed that B cell depletion resulted in altered local T cell and cytokine responses, increased bacterial burden, and lower levels of inflammation. There were elevated frequencies of T cells producing interleukin-2 (IL-2), IL-10, and IL-17 and decreased IL-6 and IL-10 levels within granulomas from B cell-depleted animals. The effects of B cell depletion varied among granulomas in an individual animal, as well as among animals, underscoring the previously reported heterogeneity of local immunologic characteristics of tuberculous granulomas in nonhuman primates. Taken together, our data clearly showed that B cells can modulate the local granulomatous response in M. tuberculosis-infected macaques during acute infection. The impact of these alterations on disease progression and outcome in the chronic phase remains to be determined.

Modeling tuberculosis in nonhuman primates.

Nonhuman primates have emerged as an excellent model of human tuberculosis, in large part because they recapitulate the full spectrum of infection outcome and pathology seen in humans. Several variables inherent to the nonhuman primate models of tuberculosis are discussed in this review, including the monkey species, Mycobacterium tuberculosis strains, and routes of infection, all of which can influence the model to be chosen for various studies. New technologies for studying the microbiology, immunology, and pathogenesis of tuberculosis in nonhuman primates have greatly expanded the capabilities of this model for basic and translational studies, including the development and testing of new treatment and prevention strategies for tuberculosis.

Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.

&lt;i&gt;Mycobacterium Tuberculosis&lt;/i&gt; Infection in Rhesus Monkeys (&lt;i&gt;Macaca mulatta&lt;/i&gt;) and Evaluation of ESAT-6 and CFP10 as Immunodiagnostic Antigens

Tuberculosis (TB) in nonhuman primates is a serious menace to the welfare of the animals and human who come into contact with them, while the rapid, accurate, and robust diagnosis is challenging. In this study, we first sought to establish an appropriate primate TB model resembling natural TB in nonhuman primates. Four rhesus monkeys (Macaca mulatta) of Chinese origin were infected intratracheally with two low doses of M. tuberculosis H37Rv. Regardless of the infectious doses, all monkeys were demonstrated to be successfully infected by clinical assessments, tuberculin skin test conversions, peripheral immune responses, gross observations, histopathology analysis, and M. tuberculosis burdens. Furthermore, we extended the usefulness of this model for assessing the following immunodiagnostic antigens: CFP10, ESAT-6, CFP10-ESAT-6, and an antigen cocktail of CFP10 and ESAT-6. The data showed that CFP10 was an M. tuberculosis-specific, “early” antigen used for serodiagnosis of TB in nonhuman primates. In conclusion, we established a useful primate TB model depending on low doses of M .tuberculosis and affording new opportunities for studies of M. tuberculosis disease and diagnostics.

Guidelines for the prevention and control of tuberculosis in non‐human primates: recommendations of the European Primate Veterinary Association Working Group on Tuberculosis

Effective tuberculosis (TB) control requires accurate diagnostic methods but the tuberculin skin test has serious limitations. Both false-negative and false-positive reactions are common, resulting in the spread of the infection and devastating TB outbreaks. Results of questionnaire surveys concerning TB testing practices in primate housing facilities showed great differences in testing practices. Although there was some uniformity regarding the sites of application, the amounts of tuberculin used and the time intervals for retesting, a great deal of variety was revealed considering the types of tuberculin preparations, the interpretation of tests and the susceptibility of animals. Here, we summarize the most common practices as regards TB control and prevention for non-human primates, and attempt to establish a uniform guideline based upon our experience with primate husbandry and care programmes as well as recent developments in the literature. The present guideline represents a consensus recommendation intending to harmonize the existing protocols.

Immunological concepts in tuberculosis diagnostics for non‐human primates: a review

Accurate diagnosis of tuberculosis in non-human primates is of critical importance. As with natural human infection with Mycobacterium tuberculosis, infected primates develop a broad spectrum of disease, including subclinical (latent) infection, chronic primary tuberculosis, rapidly progressing fulminant disease, and reactivation tuberculosis. In a primate colony, clinical suspicion is the key to diagnosis. The course of action should be based on careful and thorough clinical assessments in conjunction with screening and microbiologic methods. Diagnostic modalities can be categorized into pathogen identification and immunologic host response techniques. While the classic tuberculin skin test is the standard screening tool, it has limited sensitivity and specificity. Other tools such as interferon gamma releasing assays have similar accuracy results but use different immunologic mechanisms and may be helpful as an additional screening tool. Advantages and disadvantages to these and other assays (e.g., lymphocyte proliferation assay, antibody detection) are also discussed. Surrogates to sputum sample (e.g., gastric aspirate, stool samples, respiratory sample via bronchoscopy) should be obtained for microbiologic identification, as acid-fast smear and culture are critical to pathogen identification for optimal sensitivity and specificity. Interpretation of these immunologic screening tools should be performed cautiously and must be correlated with level of suspicion. While the identification of M. tuberculosis or M. bovis confirms the diagnosis of tuberculosis, negative results do not exclude the diagnosis. Without pathogen detection to confirm diagnosis, thorough gross and microscopic pathological review at necropsy may be required to make a definitive diagnosis. Lastly, the risk and benefits to the primate colony, staff and resources must be carefully weighed when deciding to euthanize monkeys to make the diagnosis of tuberculosis.

New Approaches to Tuberculosis Surveillance in Nonhuman Primates

Despite significant progress in reducing the incidence of tuberculosis in nonhuman primates (NHPs) maintained in captivity, outbreaks continue to occur in established colonies, with potential serious consequences in human exposures, animal losses, disruption of research, and costs related to disease control efforts. The intradermal tuberculin skin test (TST) using mammalian old tuberculin (MOT) has been the mainstay of NHP tuberculosis surveillance and antemortem diagnosis for more than 60 years. But limitations of the TST, particularly its inability to reliably identify animals with latent TB infections, make it unsuitable for use as a single, standalone test for TB surveillance in nonhuman primates in the 21st century. Advances in technology and the availability of Mycobacterium spp. genomic sequence data have facilitated the development and evaluation of new immune-based screening assays as possible adjuncts and alternatives to the TST, including in vitro whole blood assays that measure the release of interferon gamma in response to stimulation with tuberculin or specific mycobacterial antigens, and assays that detect antibodies to highly immunogenic secreted proteins unique to M. tuberculosis, M. bovis, and other species belonging to the M. tuberculosis complex. It is becoming apparent that no single screening test will meet all the requirements for surveillance and diagnosis of tuberculosis in nonhuman primates. Instead, the use of several tests in combination can increase the overall sensitivity and specificity of screening and surveillance programs and likely represents the future of TB testing in nonhuman primates. In this article we describe the characteristics of these newer screening tests and discuss their potential contributions to NHP tuberculosis surveillance programs.

PrimaTB STAT-PAK assay, a novel, rapid lateral-flow test for tuberculosis in nonhuman primates.

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

Public health implications of exotic pet mycobacteriosis

The most common species of mycobacteria occurring in avian and exotic pet animals are as follows: Mycobacterium genavense and M avium-complex (MAC) in birds; MAC, M marinum, M chelonae, M fortuitum, M smegmatis , and M phlei in reptiles; M tuberculosis in nonhuman primates; and M fortuitum, M xenopi and M marinum in amphibians. Spontaneous mycobacteriosis in rabbits, gerbils, mice, hamsters, and guinea pigs is very rare and not associated with a specific mycobacterial species. M tuberculosis or M bovis , both known as zoonotic agents, play no important role in exotic pets. The public health significance of the other mycobacteria remains to be elucidated. Newly developed laboratory techniques such as media for fastidious mycobacteria, gene probes, and nucleic acid amplification methods will allow the identification and closer characterization of agents found in this group of animals.

Mycobacterium bovis infection in primates in Dublin Zoo: epidemiological aspects and implications for management.

An outbreak of tuberculosis in non-human primates was successfully contained in the isolation area of Dublin Zoo. A Mayotte lemur, a lion-tailed macaque, a Patas monkey and a Siamang gibbon developed tuberculosis, and Mycobacterium bovis was isolated from all but the lemur, from which lesions were not cultured. Procedures for the prevention of tuberculosis in primate collections and for the management of outbreaks are discussed. The need for typing of the isolated pathogen is emphasized.