Elucidating mechanisms of protection conferred by a primary Mycobacterium tuberculosis (Mtb) infection to a secondary Mtb infection in non-human primates

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has lived with man for thousands of years. Yet, TB is still a major global health problem killing more than a million people every year. Bacille Calmette-Guerin was developed almost a hundred years ago and is still the only licensed vaccine for TB. The main hindrance to current vaccine development is the lack of correlates and immune targets of protection. Early published data suggest that individuals with latent TB have a lower risk of developing active TB disease after Mtb re-exposure. A more recent study showed that macaques with an ongoing primary Mtb infection are protected against establishment of granulomas and bacterial growth from a secondary Mtb challenge. The precise immune mechanisms of this protection are largely unknown. The main goal of this dissertation is to investigate the importance of bacterial viability and CD4 T cells in the protection against Mtb reinfection using DNA-barcoded Mtb strains. Eliminating live Mtb bacilli by drug treatment reduced but did not abolish the protection against the establishment of and bacterial growth in granulomas arising from the second infection; although the effect of long term primary Mtb infection against a second infection needs to be further studied. Depletion of CD4 T cells in macaques before reinfection resulted in increased bacterial burden in secondary granulomas, however it only increased the number of secondary granulomas in some macaques showing heterogeneity in these animals. Moreover, we showed that CD4 T cells are important in preventing Mtb dissemination to the lymph nodes. Bacterial burden in secondary granulomas, lungs and lymph nodes in the CD4 T cell-depleted macaques did not reach the level of bacterial burden in the naïve controls suggesting protection is multifactorial. Lastly, we showed that lymph nodes are more than just sites of antigen presentation and immune activation; rather, they are sites of Mtb persistence and growth. Overall, this dissertation provided elements to consider and target in the design and testing of vaccines and therapeutics

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

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

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

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