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. 2026 Feb;19(1):1538-1549.
doi: 10.1016/j.mucimm.2025.10.010. Epub 2025 Nov 2.

Protection against reinfection with Mycobacterium tuberculosis extends across heterologous Mtb lineages

Andrew W Simonson  1 , Michael C Chao  2 , Luke E Hood  3 , Rachel A Donlan  4 , Forrest Hopkins  5 , Michael R Chase  6 , Andrew J Vickers  7 , Alanna Callendrello  8 , Edwin Klein  9 , HJacob Borish  10 , Marshall Malin  11 , Pauline Maiello  12 , Charles A Scanga  13 , Philana Ling Lin  14 , Sarah M Fortune  15 , JoAnne L Flynn  16
Affiliations

Protection against reinfection with Mycobacterium tuberculosis extends across heterologous Mtb lineages

Andrew W Simonson et al. Mucosal Immunol. 2026 Feb.

Abstract

Immunological memory elicited either through previous or ongoing M. tuberculosis (Mtb) infection provides a critical mechanism by which hosts protect against re-infection and disease progression upon Mtb re-exposure. Conversely, the uneven competition between distinct Mtb strains suggest certain bacterial clades have enhanced ability to spread across communities and circulate globally, potentially by evading memory responses gained by prior infection with genomically different strains. To address whether memory responses induced by one strain can protect against a genetically distinct strain, we conducted a heterologous reinfection study in cynomolgus macaques involving primary infection by a Lineage 4 Erdman Mtb strain and subsequent re-challenge by a Lineage 2 strain, HT-L2. Recent epidemiologic studies have shown that the clade to which HT-L2 belongs has been spreading successfully over the last decade in Lima, Peru. Here, through microbiologic, PET-CT imaging and sequencing of Mtb genomic barcodes, we show that reinfected animals developed fewer lung lesions and controlled both pulmonary and disseminated forms of infection better than naïve animals without prior exposure to Mtb. Our data support that protection against reinfection is not limited by Mtb lineage, providing optimism that vaccines can be effective across populations and geographic locations.

Keywords: Clinical Mycobacterium tuberculosis strain; Concomitant Immunity; Non-human Primate; Reinfection; Tuberculosis.

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Figures

Fig. 1.
Fig. 1.
Generation and validation of genetically barcoded HT-L2 library. (A) Schematic of barcode design and murine validation model. C57Bl/6 mice (N = 13) were aerosol infected with an isogenic library of HT-L2 Mtb carrying random 18mer barcodes. Animals were sacrificed at 3 (n = 6) and 14 (n = 7) days post-infection and lung homogenates were plated for CFU and for Mtb genomic DNA extraction. The barcodes present in each animal were PCR amplified and quantified by amplicon sequencing. Image prepared using Biorender. (B) Barcodes were PCR amplified from an aliquot of the HT-L2 Mtb barcode master library and subjected to amplicon sequencing. Distinct 18mer barcodes were ordered by abundance (black curve) and library complexity was determined by performing a linear regression of log-transformed counts using a window of 500 adjacent barcodes (red curve). An inflection point, defining approximate library complexity, was defined at barcode rank 36,833 (dotted line). (C) Total lung bacterial burden in mice infected with HT-L2 at 3- and 14-days post-challenge. (D) Quantification of unique barcodes recovered from lungs at 3- and 14-days post-challenge. Only animals that passed QC in the BARTI pipeline were included for panel D. In panels C and D, each symbol represents an animal, lines represent the group median, and p values represent the results of Welch’s unpaired t tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2.
Fig. 2.. Erd-L4 infection in macaques was successfully cleared by antibiotics, although systemic memory responses remained intact.
(A) Schematic of macaque reinfection timeline. Timing of serial PET CT scans indicated by yellow bolts; BAL sampling indicated by blue tube. (B) Quantification of inflammation in lung via serial PET scans throughout primary infection (Erd-L4, blue shading), antibiotic treatment (HRZE, gray shading), and secondary infection (HT-L2, green shading). Thin lines with circles represent individual animals (orange Erd-L4 then HT-L2; purple HT-L2 in naïve animals); thick lines with squares represent group median. (C) Number of granulomas appearing on PET CT scans at 4 and 8 weeks post-primary Erd-L4 infection. Each symbol represents an animal and the p value is the result of a Wilcoxon signed rank test. (D) Estimated thoracic bacterial burden per animal at 8 weeks post-primary Erd-L4 infection based on PET CT scans, as described in Refs. and . Circles represent point estimate; Lines represent extent of 95 % confidence interval. (E) IFN-g release in response to stimulation with ESAT-6 and CFP-10 peptide pools was quantified by ELISpot assay throughout reinfection study. A threshold of 10 spot forming units (SFU) per 2 × 105 PBMCs for positive results was determined by previous studies (Ref. ). Lines connect timepoints for each individual, and each symbol represents and animal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Fig. 3.. Animals previously infected with Erd-L4 were significantly less susceptible to HT-L2 infection.
(A) Number of granulomas found at necropsy that were clinically and microbiologically attributable to HT-L2 infection. (B) Number of unique HT-L2 barcodes recovered from each animal. One animal (10623) is excluded as no sequencing samples passed QC. (C,D) Bacterial burden attributable to HT-L2 in all thoracic (C) and only lung (D) tissues. (E) Percentage of lung bacterial burden obtained from isolated granulomas, rather than in random samples of grossly uninvolved lung tissue, as an indicator of successful granuloma containment. This was calculated by dividing the summed granuloma-attributed CFU by the lung CFU in panel D. (F) Average HT-L2 bacterial burden per granuloma in reinfected and naïve animals. In all plots, each symbol represents an animal and lines represent the group median. Statistics: P-values shown with Mann-Whitney non-parametric ranked sum tests in panels A, B, and E, and Welch’s unpaired t tests for panels C, D and F.
Fig. 4.
Fig. 4.. Granulomas of reinfected animals had fewer cells, driven by a drop in traditionally cytotoxic cell types.
(A) Relative frequency of lymphocyte subsets by flow cytometry. Each bar represents a granuloma analyzed from the animal noted below. (B) Average number of total cells recovered per lesion by animal, as determined by hemocytometer. Lesions with cell counts below limit of detection on hemocytometer (2 × 104 cells) were excluded. (C,D) Average number of cells, broken down by lymphocyte (C) and T cell (D) subsets, in granulomas analyzed by flow cytometry. In panels B-D, each symbol represents an animal, lines represent the group median, and all p values shown are results of Welch’s unpaired t tests.
Fig. 5.
Fig. 5.. T cell populations were more efficient at producing functional responses, with a shift towards granzyme K positive phenotypes.
(A,B) Frequency of CD4 and CD8αβ T cells in lung tissue producing cytotoxic effectors (A) and cytokines (B). p values reported for Mann-Whitney non-parametric ranked sum tests between groups. (C,D) Number of CD4 (C) and CD8αβ (D) T cells per gram of lung tissue producing individual effector molecules. p values reported for Welch’s unpaired t tests, with groups compared for each effector. In panels A-D, each symbol represents an animal and lines represent the group median. (E) Frequency of CD4 T, CD8αβ T and NK cells in lung granulomas producing competent cytotoxic responses, determined by production of at least two of granulysin, perforin, granzyme B and granzyme K. All granulomas were averaged across animal and experimental group.
Fig. 6.
Fig. 6.. Pre-existing immunity from a lineage 4 infection limits dissemination of subsequent lineage 2 infections.
(A) Extent of overall dissemination, shown by the number of tissues sharing a barcode. Each symbol represents a barcode. Reported p value shown for Kolmogorov-Smirnov test. (B) Bacterial burden attributable to HT-L2 infection in lymph node tissues. Reported p value shown for Welch’s unpaired t test. (C) The number of CFU positive lymph nodes per animal. (D) Bacterial burden per CFU + lymph node, shown by animal. Each symbol represents a lymph node, and lines represent the median per animal. (E) Percentage of barcodes recovered from a lung tissue that are shared with a lymph node site of dissemination. In panels B, C and E, each symbol represents an animal and lines represent the group median. In panels C and E, p values are results of Mann-Whitney non-parametric ranked sum test. In panels A and E, one animal (10623) is excluded as no sequencing samples passed QC.
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