Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection . Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, positron emission tomography-computed tomography (PET CT) imaging, flow cytometry, and cytokine profiling, we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination, or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the seven antibiotic-treated animals demonstrated sterilizing immunity against reinfection, while four of the seven non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic-treated macaques.

Antibiotic treatment modestly reduces protection against Mycobacterium tuberculosis reinfection in macaques.

Concomitant immunity is generally defined as an ongoing infection providing protection against reinfection1. Its role in prevention of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is supported by epidemiological evidence in humans as well as experimental evidence in mice and non-human primates (NHPs). Whether the presence of live Mtb, rather than simply persistent antigen, is necessary for concomitant immunity in TB is still unclear. Here, we investigated whether live Mtb plays a measurable role in control of secondary Mtb infection. Using cynomolgus macaques, molecularly barcoded Mtb libraries, PET-CT imaging, flow cytometry and cytokine profiling we evaluated the effect of antibiotic treatment after primary infection on immunological response and bacterial establishment, dissemination, and burden post-secondary infection. Our data provide evidence that, in this experimental model, treatment with antibiotics after primary infection reduced inflammation in the lung but was not associated with a significant change in bacterial establishment, dissemination or burden in the lung or lymph nodes. Nonetheless, treatment of the prior infection with antibiotics did result in a modest reduction in protection against reinfection: none of the 7 antibiotic treated animals demonstrated sterilizing immunity against reinfection while 4 of the 7 non-treated macaques were completely protected against reinfection. These findings support that antibiotic-treated animals were still able to restrict bacterial establishment and dissemination after rechallenge compared to naïve macaques, but not to the full extent of non-antibiotic treated macaques.

CD4+ T cells are homeostatic regulators during Mtb reinfection.

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

Identification and characterization of the T cell receptor (TCR) repertoire of the cynomolgus macaque (Macaca Fascicularis).

BACKGROUND: Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire. RESULT: We identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes. CONCLUSION: The genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Lymph nodes-The neglected battlefield in tuberculosis.

Lymph nodes, particularly thoracic lymph nodes, are among the most common sites of extrapulmonary tuberculosis (TB). However, Mycobacterium tuberculosis (Mtb) infection in these organs is understudied. Aside from being sites of initiation of the adaptive immune system, lymph nodes also serve as niches of Mtb growth and persistence. Mtb infection results in granuloma formation that disrupts and-if it becomes large enough-replaces the normal architecture of the lymph node that is vital to its function. In preclinical models, successful TB vaccines appear to prevent spread of Mtb from the lungs to the lymph nodes. Reactivation of latent TB can start in the lymph nodes resulting in dissemination of the bacteria to the lungs and other organs. Involvement of the lymph nodes may improve Bacille Calmette-Guerin (BCG) vaccine efficacy. Lastly, drug penetration to the lymph nodes is poor compared to blood, lung tissue, and lung granulomas. Future studies on evaluating the efficacy of vaccines and anti-TB drug treatments should include consideration of the effects on thoracic lymph nodes and not just the lungs.

Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.

Whole-genome sequencing and single nucleotide polymorphisms in multidrug-resistant clinical isolates of Mycobacterium tuberculosis from the Philippines.

OBJECTIVES: Thousands of cases of multidrug-resistant tuberculosis (TB) have been observed in the Philippines, but studies on the Mycobacterium tuberculosis (MTB) genotypes that underlie the observed drug resistance profiles are lacking. This study aimed to analyse the whole genomes of clinical MTB isolates representing various resistance profiles to identify single nucleotide polymorphisms (SNPs) in resistance-associated genes. METHODS: The genomes of ten MTB isolates cultured from banked sputum sources were sequenced. Bioinformatics analysis consisted of assembly, annotation and SNP identification in genes reported to be associated with resistance to isoniazid (INH), rifampicin (RIF), ethambutol (ETH), streptomycin, pyrazinamide (PZA) and fluoroquinolones (FQs). RESULTS: The draft assemblies covered an average of 97.08% of the expected genome size. Seven of the ten isolates belonged to the Indo-Oceanic lineage/EA12-Manila clade. Two isolates were classified into the Euro-American lineage, whilst the pre-XDR (pre-extensively drug-resistant) isolate was classified under the East Asian/Beijing clade. The SNPs katG Ser315Thr, rpoB Ser450Leu and embB Met306Val were found in INH- (4/7), RIF- (3/6) and ETH-resistant (2/6) isolates, respectively, but not in susceptible isolates. Mutations in the inhA promoter and in the pncA and gyrA genes known to be involved in resistance to INH, PZA and FQs, respectively, were also identified. CONCLUSIONS: This study represents the first effort to investigate the whole genomes of Philippine clinical strains of MTB exhibiting various multidrug resistance profiles. Whole-genome data can provide valuable insights to the mechanistic and epidemiological qualities of TB in a high-burden setting such as the Philippines.

The changing molecular epidemiology of HIV in the Philippines.

BACKGROUND: The Philippines has one of the fastest-growing HIV epidemics in the world. Possible reasons for this include increased testing, increased local transmission, and possibly more aggressive strains of HIV. This study sought to determine whether local molecular subtypes of HIV have changed. METHODS: Viruses from 81 newly diagnosed, treatment-naive HIV patients were genotyped using protease and reverse transcriptase genes. Demographic characteristics and CD4 count data were collected. RESULTS: The cohort had an average age of 29 years (range 19-51 years), CD4+ count of 255 cells/mm3 (range 2-744 cells/mm3), and self-reported acquisition time of 2.42 years (range 0.17-8.17 years). All were male, including 79 men who have sex with men (MSM). The genotype distribution was 77% CRF01_AE, 22% B, and 1% C. Previous data from 1985-2000 showed that most Philippine HIV infections were caused by subtype B (71%, n=100), followed by subtype CRF01_AE (20%). Comparison with the present cohort showed a significant shift in subtype (p<0.0001). Comparison between CRF01_AE and B showed a lower CD4+ count (230 vs. 350 cells/mm3, p=0.03). Survival data showed highly significant survival associated with antiretroviral (ARV) treatment (p<0.0001), but no significant difference in mortality or CD4 count increase on ARVs between subtypes. CONCLUSIONS: The molecular epidemiology of HIV in the Philippines has changed, with the more aggressive CRF01_AE now being the predominant subtype.

Detection and characterization of mutations of multidrug-resistant tuberculosis isolates of the Philippine General Hospital

Background. Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. Objective. To detect and characterize mutations in rpoB, katG and gyrA. Methods. Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. Results. Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. Conclusion. These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.

Detection and characterization of mutations of multidrug-resistant tuberculosis isolates of the Philippine General Hospital

BACKGROUND: Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. OBJECTIVE: To detect and characterize mutations in rpoB, katG and gyrA. METHODS: Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. RESULTS: Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. CONCLUSION: These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.