Abstract
Mycobacterium tuberculosis is the leading cause of death globally due to a single infectious agent. Currently, no licensed vaccine protects against pulmonary tuberculosis, the primary adult disease caused by Mtb infection. CD4 T cells are essential in protection against Mtb infection and inducing a protective CD4 T cell response remains the goal of most Mtb vaccines currently in testing. However, in contrast to other pathogens, most Mtb T cell epitopes do not exhibit sequence variation, suggesting that T cell recognition does not drive selection of escape mutants. We discovered hypothetical Mtb antigens that do exhibit sequence diversity within human T cell epitopes and tested the impact of vaccination with these rare variable antigens (RVMA) using a DNA vaccine platform. We found vaccination with RVMA significantly alters the immune response to Mtb infection in both C57BL/6 and hypersusceptible SP140‒/‒ mice without reducing lung bacterial burdens. RVMA vaccination of hypersusceptible SP140‒/‒ animals prevented necrosis and altered the lesion composition reducing tissue damage and increasing CD4 T cell distribution. Reductions in pathology were associated with increases in RORγt-expressing CD4 T cells and decreases in monocyte-derived cells in the lungs prior to the development of necrotic lesions, and were independent of B cells. These results suggest T cell responses to certain antigens may be involved in preventing pathology without significantly changing bacterial burdens and indicate that these antigens can be efficacious in Prevention of Disease TB vaccines.
1 Introduction
Mycobacterium tuberculosis (Mtb) is the greatest cause of human mortality due to a single infectious agent (). Despite discovering Mtb as the causative agent of tuberculosis (TB) over 140 years ago, we still lack an effective vaccine to prevent pulmonary TB in adults (–). While BCG remains the only approved vaccine against TB, it has variable efficacy in preventing TB disease (–). Recent attempts to develop an effective subunit vaccine have yielded mixed results, with only one candidate, M72A/AS01E, providing approximately 50% efficacy in preventing TB disease (–).
Most subunit TB vaccine candidates aim to generate protective Th1 CD4 T cell responses, and evidence supports the notion that CD4 T cells provide protection against TB (–). However, the optimal antigens to target with a subunit vaccine remain unknown and the discovery of antigens that induce protective immunity is a high priority. Interestingly, Mtb CD4 T cell epitope sequences are uniquely conserved (, ). While other pathogens utilize antigenic variation as a mechanism of immune evasion, Mtb is an exception (). In fact, hyperconservation suggests that purifying selection of predominant Mtb T cell epitopes is due to a beneficial effect of T cell recognition on the bacteria, such as by promoting transmission through induction of T cell-dependent inflammatory lung tissue destruction as in cavitary TB. Therefore, we hypothesized that conserved Mtb T cell antigens may induce suboptimal immunity during infection and that the antigens that exhibit sequence diversity within epitope sequences can be efficacious targets for vaccination. We previously identified a small group of antigens that exhibited sequence diversity within CD4 T cell epitopes, across strains and lineages within the Mycobacterium tuberculosis complex (MTBC) that we hypothesize are under diversifying selection due to T cell responses that are more detrimental to the bacteria than are T cell responses to conserved antigens ().
Lung pathology is an important aspect of TB (). As a human-specific pathogen, Mtb relies on human-to-human transmission via the aerosol route. Humans with more extensive lung pathology shed a higher number of bacteria and are more infectious (–). One form of TB lung pathology termed cavitary TB is associated with enhanced transmission and development of cavitary TB is facilitated by CD4 T cells, since individuals whose CD4 T cells are depleted by HIV infection rarely exhibit cavitary TB (). Moreover, in people infected with HIV that have active TB, the frequency of cavitary TB is lower (, , –). In fact, as CD4 T cell counts decrease the rates of cavitary TB also decrease, which indicates a direct correlation between CD4 T cells and cavitary disease (, ). Together, these results indicate that some CD4 T cell responses can be detrimental in humans with TB.
Given the significance of lung pathology to TB morbidity and transmission, recent vaccine trials have focused on prevention of disease (, ). While many studies have demonstrated the protection afforded by CD4 T cells that make IFNγ, those CD4 T cell responses can be associated with more cavitary TB and higher bacterial shedding (, ). In mice and humans, CD4 T cell responses can be more pathogenic if immunoregulation is lost, such as in the case of anti-PD-1 therapy or genetic deficiency of PD-1 and other immunoregulatory factors (–). Most mouse studies of Mtb infection use C57BL/6 mice, a resistant strain that exhibits limited lung pathology. More highly susceptible strains of mice, such as C3HeB/FeJ mice develop more lung pathology, but genetic differences from the C57BL/6 mice make it less tractable for in-depth immunology studies (). Recently, an isogenic C57BL/6 mouse strain was developed by disrupting the SP140 gene to generate a susceptible mouse model that exhibits severe lung pathology similar to that in C3HeB/FeJ mice (). Using this Sp140-deficient mouse strain, we evaluated the ability of a vaccine targeting Mtb rare variable antigens (RVMA) in preventing severe lung pathology while retaining the ability to evaluate T cell responses in a C57BL/6 genetic background.
In this study, we report on a vaccine containing antigens selected based on increased sequence variation and found that this vaccine reduced necrosis in the hypersusceptible SP140‒/‒ mouse strain without significantly reducing bacterial burdens in the lungs or spleen. We identified differences in the cellular response to infection in vaccinated animals that associated with reduced tissue necrosis. We also characterize the lesion structure and composition in vaccinated animals, demonstrating a shift from necrotic to non-necrotic fibrotic lesions.
2 Materials and methods
2.1 Plasmids
Vaccine cassettes were made by restriction cloning synthetic gene fragments (Genewiz) into the pVAX1 vector backbone (Invitrogen). Sequences were derived from Mtb Erdman strain and mammalian codon optimized. Four variable antigens Rv0990c, Rv0010c, RimJ (Rv0995), and Rv2719c were used to construct the vaccine vector used in these studies, named Cassette 4 (Cas4). NEB 5-alpha (NEB) E. coli was transformed with resulting vaccine plasmids and propagated in LB broth containing 50 µg/mL kanamycin. Vaccine plasmid for immunizations was prepared from 2L of overnight culture by Gigaprep (Zymo Research) with subsequent endotoxin removal. Vaccine plasmid diluted to a concentration of 2 μg/μL in sterile phosphate buffered saline (PBS) and stored at −20 °C.
2.2 Mice
C57BL/6 mice were obtained from Jackson Laboratory. SP140‒/‒ mice were obtained from the laboratory of Russell Vance and bred under specific-pathogen-free conditions after rederivation at the University of California, San Francisco (). Congenic CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ, strain #:002014) were obtained from Jackson Laboratory. Mice aged 8–12 weeks for used for experiments, and mice infected with Mtb were housed in the Animal Biosafety Level 3 facility. For any procedures requiring anesthesia, mice were anesthetized by inhalation of isoflurane using 3%–5% isoflurane in oxygen (1–1.2 L/min). At the appropriate experimental endpoints, animals were euthanized by carbon dioxide inhalation followed by cervical dislocation. All animal protocols used were approved by the University of California, San Francisco, Animal Care and Use Committee.
2.3 Mycobacterial strains, culture, and aerosol infections
Mtb Erdman, obtained from ATCC (ATCC 35801), Mtb H37Rv, and Mtb H37Rv expressing zsGreen, generated previously by our lab, were propagated in Middlebrook 7H9 broth (BD) supplemented with 10% (v/v) ADC (albumin, dextrose, catalase), 0.05% Tween-80, and 0.2% glycerol (). For aerosol infections, a frozen stock of mid-log phase bacteria was thawed and passed through a 25-G needle to ensure a single cell suspension. Bacterial stocks were diluted to an approximate concentration in water to yield an infection with 50–100 CFU per mouse. Mice were infected with Mtb using a Glas-Col inhalation exposure unit as described previously (). Initial infectious doses were confirmed by homogenizing lungs from mice in PBS + 0.05% Tween-80 immediately after the aerosol procedure. Homogenates were plated on 7H11 agar plates containing PANTA (BD) to enumerate CFU ().
2.4 Immunizations and antigen stimulations
Mice were immunized in the right tibialis anterior muscle as described previously (–). Briefly, 50 µL of vaccine plasmid (100 μg total plasmid) was injected using a 28G ½″ 0.5 mL insulin syringe. Mice were immunized three times, three weeks apart. All experiments were performed three weeks after the final vaccination.
For assessing immunogenicity in vaccinated animals, mice were euthanized, and spleens were collected into RPMI-1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 10 mM HEPES, penicillin/streptomycin, and 50 µM 2-mercaptoethanol (complete RPMI). Spleens were mashed through a 70 µm strainer using the back of 3 mL syringe to generate a single cell suspension. Red blood cells (RBCs) were lysed using ACK lysis buffer (Gibco). Spleen cells were cultured in complete RPMI at 37 °C + 5% CO2 in 96-well round bottom plates with 10 µg/mL pooled overlapping peptides (18-mers, overlapping by 11 amino acids) from Genscript or individual peptides where specified. For IFN-γ secretion, cells were incubated for 72 h before supernatants were collected for analysis by IFN-γ ELISA (Invitrogen).
For flow cytometry, cells were incubated for 1 h with or without 10 µg/mL pooled overlapping peptides (18-mers, overlapping by 11 amino acids), prior to the addition of eBioscience protein transport inhibitor cocktail (Invitrogen) followed by an additional 5-h incubation. Stimulated cells were then stained with Live-or-Dye750-777 (Biotium) and anti-CD16/32 (clone 2.4G2; Fc block) for 15 min at 4 °C. After washing, cells were stained for surface markers in FACS buffer (PBS with 2% HI-FBS (v/v) for 30 min at 4 °C containing the following fluorochrome-conjugated antibodies: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD19 (6D5), and CD44 (IM7). After surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences) and stained in BD Perm/Wash buffer containing antibodies for IFNγ (XMG1.2) and TNF (MP6-XT22) for 30 min at 4 °C. Samples were acquired using an Aurora spectral flow cytometer (Cytek Biosciences) and gated as shown in Supplementary Figure S1.
2.5 Tissue harvests and flow cytometry
Where specified, mice were anesthetized by inhalation of isoflurane and retro-orbitally injected with 1 µg of anti-CD45 antibody (clone 30-F11) in 100 µL of PBS, 3 min before euthanasia with CO2 inhalation and cervical dislocation. Lungs were collected into Hanks' Balanced Salt Solution (HBSS) containing 50 µg Liberase TM (Sigma) and 30 µg/mL DNase I (Sigma) and incubated at 37 °C for 30 min, then processed with a gentleMACS dissociator (Miltenyi). To obtain a single cell suspension, the resulting homogenates were passed through a 70 µm strainer. Spleens were also collected into complete RPMI as described for single cell suspensions or PBS + 0.05% (v/v) Tween-80 for enumerating CFU. Aliquots of lung and spleen samples were taken prior to further processing for enumerating CFU by serial dilution and plating on 7H11 agar plates. Lung single cell suspensions were further processed by centrifugation and RBC lysis using ACK lysis buffer (Gibco).
For flow cytometry analysis, lung single cell suspensions were first stained with Live-or-Dye750-777 (Biotium) and Fc block for 15 min at 4 °C. Lung cells were then stained in FACS buffer with two sets of antibodies for analyzing myeloid cell populations as described previously () and CD4 T cell subsets by lineage defining transcription factors. For myeloid cell analysis, cells were stained in FACS buffer containing a cocktail of fluorochrome-conjugated antibodies, as described previously (), for 30 min at 4 °C. For CD4 T cell analysis, cells were stained in FACS buffer for 30 min at 4 °C containing the following fluorochrome-conjugated antibodies: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD19 (6D5), and CD44 (IM7). For CD4 T cell subset analysis, cells were then fixed and permeabilized using the eBioscience FoxP3/Transcription Factor Staining Buffer Set (Invitrogen) prior to being incubated with fluorochrome-conjugated antibodies targeting Tbet (4B10), RORγt (Q31-378), and FoxP3 (MF-14). All cells were analyzed on an Aurora Spectral flow cytometer (Cytek Biosciences). Myeloid cell subsets were identified as described previously (), and CD4 T cell subsets were identified as live > single cells > CD3+CD19‒ > CD4+CD8‒ > CD44+ (Supplementary Figure S1).
2.6 Lung tissue histopathology, cryosectioning, and immunofluorescence
Mice were euthanized, as described above, and lungs were harvested directly into 10% neutral buffered formalin for histopathology. After 48 h, fixed lungs were shipped to Histowiz for processing. All H&E staining and immunohistochemistry was performed by Histowiz. Histopathology was assessed using QuPath to quantitate lymphoid aggregates in lungs from at least 5 mice per group (). Lymphoid aggregates were identified by training a pixel classifier to identify lymphoid-rich regions (dense lymphocyte aggregates) within the lung sections, and B220 IHC confirmed lymphocyte identification by H&E staining. The number of lymphoid aggregates in the lungs was used to calculate the frequency (number/total lung area), and the size of each identified lymphoid aggregate was also measured.
For cryosectioning and immunofluorescence, mice were euthanized, as described above, and lungs were inflated with PBS containing 50% (v/v) OCT compound through the trachea. Lungs were embedded in OCT and flash frozen in liquid nitrogen. Blocks were cut into 6 µm sections by cryostat (Leica Biosystems), and sections were transferred to CFSA 1X slides (Leica Biosystems) using adhesive tape windows (Leica Biosystems). Sections were crosslinked to the slides via UV exposure and fixed in acetone for 15 min. After drying, slides were stored at −20 °C until staining.
For immunofluorescence staining, slides were rehydrated for 10 min in PBS before blocking with PBS + 0.5% BSA for 30 min at room temperature. Sections were then stained in PBS + 0.5% BSA containing fluorochrome-conjugated antibodies against CD4 (GK1.5), CD68 (FA-11), and Ly6G (1A8), or E-Cadherin (DECMA-1) and α-SMA (1A4) for 2 h at room temperature. E-cadherin and α-SMA sections were also stained with a primary unconjugated antibody against Mtb (ab905, Abcam) and subsequently stained with a fluorochrome-conjugated isotype specific secondary antibody for 1 h at room temperature. All sections were washed and stained with DAPI before coverslips were mounted with VECTASHIELD Vibrance Antifade Medium (Vector Laboratories). Images were captured using the 4X and 20X objective of a Nikon Ti inverted microscope with a DS-Qi2 camera. Individual images were stitched together to cover entire areas using NIS Elements software (Nikon).
Lesions were manually identified from whole lung images using the 4X objective and were further imaged using the 20X objective. At least 5 mice per group were imaged. Images were imported into QuPath for analysis (). Lesions identified by Mtb staining or Ly6G-rich regions were sub-divided into core and cuff region by manually annotating the central 80% of the lesion as the core and the outer 20% as the cuff (Supplementary Figures S4A,B). Analysis was performed by utilizing a pixel thresholder in QuPath to mask cells or regions that had positive staining. Frequencies were determined by calculating the total number of identified events and dividing by the total area of the core or cuff regions for CD4 and CD68 staining, as those markers identified discrete cells. For Ly6G, which identified large necrotic areas, the total area masked was used to calculate the relative area of positive staining (positive staining area/total area) due to a lack of discrete objects. Similar analysis was performed for E-cadherin, α-SMA, and Mtb (ab905) staining.
2.7 Sytox Green injection and lung imaging
To quantitate necrosis in lungs from infected mice, we utilized Sytox Green dye injection as described previously (). Briefly, infected mice were anesthetized by isoflurane inhalation and retro-orbitally injected with 100 μL of a 50 μM solution of Sytox Green dye (Invitrogen), a live-cell impermeable dye that stains extracellular DNA, 20 min prior to euthanasia as described above. Lungs were immediately collected into PBS to wash, before being fixed in 10% neutral buffered formalin for 48 h at 4 °C. Fixed whole lungs were imaged using a Leica M205 FCA stereomicroscope and images captured using a Leica K5 monochrome camera. Post acquisition images were processed using LASX software (Leica). Image analysis was performed using ImageJ to quantify Sytox Green MFI of the left lung lobes from at least 5 mice per group. For experiments where Sytox Green injection was performed, the right lung lobes were utilized for flow cytometry and enumeration of bacterial burden by CFU, as described above.
2.8 B cell depletion
To isolate the role of the vaccine-induced T cell response in preventing necrosis in SP140‒/‒ mice, B cells were depleted by anti-CD20 mAb injection (clone MB20-11, Bio X Cell) as described previously (). Briefly, mice were injected intravenously with 10 μg of anti-CD20 antibody in 100 μL of PBS 1 day before the first vaccination with either the pVAX1 empty vector (mock) or Cas4, and again 4 weeks later in the middle of the vaccine regimen, to deplete B cells and prevent a vaccine-specific B cell response. Mice were challenged with Mtb by aerosol after the final vaccination, approximately 5 weeks after the final anti-CD20 antibody injection, when B cell numbers recovered. Each experiment was performed simultaneously along with a separate group of mice that did not receive anti-CD20 antibody. Tissue harvests for bacterial burden enumeration, flow cytometry, and the Sytox Green assay for determining whole-lung necrosis were performed as described above. Statistical comparisons were made between mock- and pCas4-vaccinated groups within the control and B cell depleted mice.
2.9 CD4 T cell adoptive transfers and cell sorting
Congenic B6 CD45.1 mice were vaccinated as described above with RimJ. After vaccination, RimJ-specific CD4 T cells were purified from spleen cells of vaccinated mice by RimJ peptide stimulation and magnetic enrichment using the CD154 enrichment and detection kit (PE, mouse) (Miltenyi Biotec) according to the manufacturer's instructions. Approximately 5 × 105 purified CD154+ cells from vaccinated mice were transferred via retro-orbital injection into recipient SP140‒/‒ mice under isoflurane anesthesia. The day after transferring vaccine CD4 T cells, the mice were infected via aerosol infection with Mtb Erdman expressing zsGreen, as described above. At the indicated time points, lung cells were prepared for cell sorting by staining for T cell flow cytometry as described above with the addition of a fluorochrome-conjugated antibody for CD45.1 (A20). Recipient mice were used for lung sectioning and imaging or antigen stimulation and ICS, as described above.
2.10 Data analysis
Flow cytometry data were processed and analyzed using SpectroFlo 3.3 (Cytek) and FlowJo 10.10.0. For ICS experiments, data is background subtracted where the frequencies in the unstimulated sample were subtracted from the frequencies in the paired stimulated samples. Absolute numbers of cells were calculated using the number of events and volume of sample acquired by the cytometer. GraphPad Prism 10.4.1 (GraphPad) was used for generating plots and performing statistical analysis.
3 Results
3.1 Variable antigens are immunogenic and antigenic in C57BL/6 mice
A DNA cassette consisting of a fusion of 4 RVMA (Rv0010c, RimJ, Rv2719c, and Rv0990c) designed using the sequences in the Mtb Erdman strain () was synthesized and cloned into the commercially available pVAX1 DNA vaccine vector backbone to create pCas4 (Figure 1A). assess immunogenicity of this vaccine plasmid, C57BL/6 mice were vaccinated 3 times, 3 weeks apart and IFNγ responses to the pCas4 antigens were assessed 3 weeks after the final vaccination. Spleen cells from vaccinated animals were stimulated with pooled overlapping peptides spanning the 4 RVMA, and IFNγ was measured after 72 h of stimulation. IFNγ was detected in response to 3 of the 4 RVMA, with RimJ inducing the highest magnitude responses (Figure 1B). Since these antigens were identified in clinical isolates from patients and epitopes were confirmed to produce T cell responses in Mtb-exposed patient samples (, ), we next sought to determine the antigenicity of the 4 RVMA by determining the magnitude of CD4 T cell responses during infection in mice. We performed ICS on lung cells from Mtb H37Rv-infected C57BL/6 mice that were stimulated in vitro with pooled overlapping peptides spanning the pCas4 antigens as well as an Mtb whole cell lysate or the immunodominant antigens Ag85B and ESAT-6. IFNγ-producing CD4 T cell responses were observed for the same 3 antigens that induced a response in vaccinated animals: Rv0010c, Rv2719c, and RimJ (Figure 1C). However, the magnitude of the IFNγ response was lower for all pCas4 antigens compared to the responses to the conserved immunodominant antigens Ag85B and ESAT-6 (Figure 1C). Subdominant antigens have been shown to provide protection when incorporated into vaccines (), indicating that responses to these antigens could still be effective as vaccine antigens. We therefore determined the impact of vaccination on the magnitude of CD4 T cell responses during infection by challenging vaccinated animals with the matched Mtb Erdman strain at 28 days post-infection. Vaccinated animals had a significant increase in the IFNγ+TNF+ CD4 T cell responses to Rv0010c and RimJ, with a trend towards increased responsiveness to Rv2719c, as compared to mock-vaccinated animals (Figure 1D). Vaccination with pCas4 also enhanced the CD4 T cell response to the WCL, indicating that the enhanced T cell response may not be limited to pCas4-specific T cells, or that responses to the WCL were due to the presence of these RVMA (Figure 1D). Similar IFNγ+TNF+ CD4 T cell responses have been associated with reductions in bacterial burden in other Mtb vaccine studies, suggesting pCas4 could be efficacious as well ().
Figure 1
As the antigens in pCas4 were identified by their sequence diversity across strains of Mtb, we assessed protein sequence differences between the two strains of Mtb used in this study, H37Rv and Erdman. Despite both being related lineage 4 strains, we identified multiple amino acid substitutions in the pCas4 RVMA, RimJ. One of these substitutions was located within the predicted core epitope for the mouse H2-IAb allele, RimJ99–116 (Figure 1E). The substitution, Y105C, in the Erdman sequence induced a lower response in spleen cells from H37Rv-infected mice as compared to the H37Rv-matched RimJ99–116 sequence (Figure 1F). In mice that were vaccinated with pCas4, which was designed using the Erdman sequences, the Erdman RimJ99–116 peptide induced greater responses than the H37Rv-matched peptide but did not match the total response induced by the pooled overlapping peptides (Figure 1F). We further determined a secondary epitope in Erdman RimJ, RimJ182–197, that may account for this difference (Supplementary Figure S2). That a naturally occurring substitution in one of the pCas4 antigens alters CD4 T cell responses supports the hypothesis that antigenic variation exists in Mtb Due to the sequence variation of the pCas4 antigens, we used the matched Erdman strain for all further challenge experiments.
3.2 RVMA DNA vaccination reduces lymphoid aggregates during chronic Mtb infection
Recent TB vaccine efforts have expanded to target prevention of disease as well as prevention of infection (). Therefore, we determined the efficacy of pCas4 vaccination in limiting disease burden during chronic infection in C57BL/6 mice. We aimed to measure bacterial burden as well as lung pathology and the immune cell composition of the lungs in mice vaccinated with pCas4 at 12 weeks post infection (Figure 2A). Despite the enhanced T cell responses observed in pCas4 vaccinated mice at 4 weeks post-infection (Figure 1D), we did not observe a reduction in bacterial burdens in either the lungs or spleens in vaccinated mice at 12 weeks post-infection (Figure 2B). However, the gross pathology of lungs from pCas4-vaccinated mice subjectively appeared less than that in mock-vaccinated control animals (data not shown). H&E staining of lung sections from mock- and pCas4-vaccinated mice revealed that there were fewer lymphoid aggregates in pCas4-vaccinated mice (Figure 2C). Quantification of these structures using 2-dimensional image analysis with QuPath confirmed this observation, revealing that lymphoid aggregates were fewer and smaller in pCas4-vaccinated animals (Figures 2D,E) (). Lymphoid aggregates are observed in chronic Mtb infection in mice and are a common feature of some granulomas in humans and non-human primates infected with Mtb (, –48). These lymphoid aggregates mostly consist of B cells, and immunohistochemistry with the B cell marker B220 revealed that these structures were in fact primarily composed of B cells (Figure 2F) (). Consistent with a reduction in these structures, we also observed a significant reduction in the frequency of B cells, as well as T cells, among the lung cells from pCas4-vaccinated animals (Figures 2G,H). These results suggest that pCas4 vaccination limits the immunopathology in mice without affecting bacterial burdens, consistent with the principle of infection tolerance.
Figure 2
3.3 RVMA DNA vaccination reduces necrosis in SP140‒/‒ mice
Having observed reduced immunopathology in RVMA-vaccinated C57BL/6 mice, we investigated the impact of pCas4-vaccination in hypersusceptible SP140‒/‒ mice (). These mice develop more severe and human-like pathology, with large necrotizing granulomas similar to those observed in humans (, ). Impacts to pathology or granuloma architecture without a change in bacterial burden have been observed in other mouse models (). We therefore hypothesized that pCas4-vaccination could reduce the overall pathology in SP140‒/‒ mice without impacting the enhanced bacterial burdens observed in these mice. Since SP140‒/‒ mice are highly susceptible, we restricted our analyses to 5 weeks post-infection. Additionally, we employed intravenous injection of Sytox Green and whole lung fluorescence imaging, as previously described, to more accurately quantify the extent of necrosis as a measure of lung pathology (). SP140‒/‒ mice were vaccinated with pCas4 or the pVAX1 empty vector and challenged with Mtb Erdman to assess the lung immune cell response, bacterial burdens in the lungs and spleens, and lung pathology (Figure 3A). At 5 weeks post-infection we observed no significant differences in the bacterial burden in the lungs or spleens between mock- and pCas4-vaccinated mice (Figure 3B). However, pCas4-vaccinated mice had reduced lung Sytox Green fluorescence, consistent with less necrosis (Figure 3C). Quantification of whole lung fluorescence confirmed these observations showing a significant reduction in Sytox Green fluorescence in pCas4-vaccinated mice (Figure 3E). H&E-stained sections confirmed the presence of large necrotic lesions in mock-vaccinated mice, correlating with the high whole lung Sytox Green fluorescence (Figure 3D). Mice vaccinated with pCas4 had large cellular lesions devoid of necrosis, but also lacking obvious organization (Figure 3D).
Figure 3
The necrosis observed in SP140‒/‒ mice has been largely attributed to neutrophils (, 49). Given the large differences in necrosis and lesion architecture in pCas4-vaccinated mice we predicted that pCas4-vaccination would reduce overall neutrophil numbers in the lungs. However, flow cytometry of lung cells revealed no significant impact of vaccination on frequency or total numbers of neutrophils in pCas4-vaccinated mice compared to mock-vaccinated mice after Mtb challenge (Figures 3F,G). Instead, we observed a reduction in the frequency of MNC2 cells, which we previously demonstrated to be a more restrictive subset of monocyte-derived phagocytes during Mtb infection (), and an increase in the number alveolar macrophages in pCas4-vaccinated mice relative to mock-vaccinated mice (Figures 3F,G). With reduced lung pathology, it is likely that pCas4-vaccination prevents the destruction of healthy lung tissue and sustains higher numbers of alveolar macrophages while reducing the influx or differentiation of inflammatory monocyte-derived macrophages.
Since we identified an enhanced CD4 T cell response to the antigens in Cas4 after vaccination and challenge, we determined whether the lung CD4 T cell population was affected by pCas4-vaccination in SP140‒/‒ mice. Using flow cytometry and staining for lineage defining transcription factors, we determined the frequency and total numbers of Th1 (Tbet+), Th1/17 (Tbet+RORγt+), Th17 (RORγt+Tbet‒), and Treg (FoxP3+) cells (50). Mice that were vaccinated with pCas4 had a reduced frequency of Treg cells, possibly secondary to the reduced tissue damage observed (Figure 3H). Additionally, pCas4 vaccination induced a higher frequency of Tbet+RORγt+ (Th1/17) cells and a trend towards an increase in the total number of Tbet+RORγt+ cells, which have previously been associated with vaccine-mediated protection in mice (Figures 3H,I) (). Although we identified an IL-17A response in the draining lymph nodes of C57BL/6 mice when stimulated with the Cas4 antigen, RimJ, we did not detect antigen-specific cells making IL-17A in the lungs (Supplementary Figure S3). This may be due to a technical limitation of the assay or indicate these cells are not making IL-17A despite expressing RORγt. Overall, we determined that an increased frequency of Th1/17 cells and reduction in a subset of monocyte-derived cells was associated with reduced lung necrosis in SP140‒/‒ mice.
3.4 RVMA vaccination alters immune responses to Mtb infection
To further investigate the impact of pCas4-vaccination on immune responses to Mtb infection, we performed immunofluorescence microscopy of lung sections from DNA vector-only (mock)- and pCas4-vaccinated SP140‒/‒ mice. Since pCas4-vaccinated mice had non-necrotic lesions, we aimed to assess the immune cell composition, as well as the structural and bacterial composition of the lesions. In SP140‒/‒ mice, the lesions that form during Mtb infection resemble human granulomas that consist of a necrotic neutrophil- and macrophage-rich core containing bacteria, surrounded by a lymphocytic cuff (, 49). We used antibodies against CD68, CD4, and Ly6G to identify macrophages, CD4 T cells, and neutrophils, respectively, within the lung lesions and divided each lesion into a core and cuff for analysis. Lesions from mock-vaccinated SP140‒/‒ mice exhibited the expected composition of a large central necrotic core that stained positive for neutrophil and macrophage markers, with no intact cells and predominantly acellular debris (Figure 4A). Macrophages surround the necrotic core, along with CD4 T cells, which were almost entirely absent from the core of the lesion (Figure 4A). In mice that were vaccinated with pCas4, the lesions contained intact neutrophils and pockets of macrophages, with CD4 T cells distributed throughout the lesions (Figure 4B). Quantification of immune cell subsets across lesions from multiple mice revealed a significant reduction in neutrophil staining in pCas4-vaccination mice (Figure 4C). Additionally, we determined that there was a significant increase in the number of CD4 T cells within the core of lesions, consistent with our observation that CD4 T cells were distributed throughout the non-necrotic lesions but absent from the core of necrotic lesions (Figure 4D). No significant differences in the number of macrophages were identified (Figure 4E).
Figure 4
Recent descriptions of granulomas from the Mtb hypersusceptible C3HeB/FeJ mice have revealed that prior Mtb immunity reduced neutrophil-driven pathology and destruction of alveolar epithelium (51). Mice vaccinated with pCas4 had lesions containing fewer neutrophils within the central core, and thus we hypothesized that these lesions would also exhibit less lung parenchymal tissue destruction. To identify lung parenchymal cells we stained lung sections with antibodies against E-cadherin to mark alveolar epithelium and α-smooth muscle actin (α-SMA) to identify fibroblasts, as they have been observed in non-necrotic granulomas in other models (52, 53). Lesions from mock-vaccinated mice exhibited a loss of E-cadherin within the central core, which was expected given the acellular, neutrophil-rich appearance (Figure 4F). Additionally, no fibrosis was observed with little to no α-SMA staining seen (Figure 4F). In pCas4-vaccinated mice, E-cadherin was present throughout the lesions and not lost within the core (Figure 4G). Interestingly, α-SMA staining resembling myofibroblasts associated with lung fibrosis was observed within the core and cuff of lesions from pCas4-vaccinated mice (Figure 4G, Supplementary Figure S4C) (52). Quantification confirmed the observed differences of significant increases in E-cadherin and α-SMA within the core of lesions from pCas4-vaccinated compared with mock-vaccinated mice (Figures 4I,J). Consistent with the lack of difference in bacterial burdens between these two groups, we did not observe a difference in Mtb staining within the core of lesions between mock- and pCas4-vaccinated mice (Figure 4H). Surprisingly, we observed a significant increase in Mtb staining within the cuff of lesions from pCas4-vaccinated mice (Figure 4H). This may represent redistribution of the infected cells, normally contained mostly within the necrotic core, or a less organized lesion with less defined core and cuff regions. Regardless, these data demonstrate that pCas4-vaccination impacts TB lesion structures, in the absence of a significant reduction in bacterial burdens.
To further understand the role of pCas4 vaccination in mediating changes to pathology in SP140‒/‒ mice, we evaluated the immune cell composition in the lungs and the bacterial burden at an earlier time point. As we had only evaluated the impacts of pCas4 vaccination at 5-weeks post-infection, we determined whether differences in bacterial burden preceded changes in pathology and if differences in immune responses could be detected prior to development of lung necrosis in unvaccinated SP140‒/‒ mice (Figure 5A). Consistent with our observations at later time points, we observed no differences in bacterial burdens in either the lungs or spleens between mock- and pCas4-vaccinated mice at this earlier time point (Figure 5B). However, despite a lack of difference in bacterial burdens there were significant differences in the parenchymal immune cell composition in the lungs of pCas4-vaccinated mice. The changes in CD4 T cell subsets observed at 35 days post-infection were more apparent at 21 days post-infection. Specifically, we found a large increase in the frequency of RORγt-expressing CD4 T cells, both Tbet+ (Th1/17) and Tbet- (Th17) (Figures 5C,D) in the pCas4 group. This was accompanied by a decrease in the frequency of Tbet+ (Th1) CD4 T cells (Figures 5C,D). The total number of these CD4 T cells mirrored the frequencies, without differences in the total number of CD4 T cells between the two groups (Figure 5G).
Figure 5
In addition to differences in CD4 T cells, we observed a significant reduction in the frequency of neutrophils and both monocyte-derived macrophage subsets, MNC1 and MNC2, in pCas4-vaccinated mice (Figure 5E). The monocyte-derived cells in pCas4-vaccinated mice also had a reduction in the expression of CD11c (Figure 5F), which may represent a less inflammatory cell state (54, 55). The total number of neutrophils was not significantly different in pCas4-vaccinated mice, but there was a significant reduction in the total number of both monocyte-derived cell subsets in pCas4-vaccinated mice corresponding to a small decrease in the total number of cells isolated from the lungs (Figure 5H). These changes may be leading to the reduction in lung necrosis in pCas4-vaccinated mice. The reduction in neutrophil frequency observed at 21 days post-infection is likely not observed later due to the neutrophil cell death in mock-vaccinated mice reducing the number of neutrophils that are able to be detected by flow cytometry of live cells from digested lung tissue.
3.5 B cells are dispensable for pCas4-mediated protection
B lymphocytes have been shown to be protective in Mtb infection in mice and macaques (56). Indeed, B cells have been shown to be protective against immunopathology, and are associated with the development of tertiary lymphoid structures associated with Mtb granulomas and the regulation of T cell responses (56–59). Therefore, we sought to determine if the effects of pCas4 vaccination on necrosis in SP140‒/‒ mice are mediated by B cells through vaccine-induced antibodies or other mechanisms. To eliminate B cell responses to vaccination without affecting CD4 T cell responses, we used an anti-CD20 antibody to deplete B cells during vaccination (Figure 6A). By depleting B cells during the vaccination regimen, we aimed to eliminate the induction of antigen specific B cells, while allowing B cell numbers to recover to baseline prior to Mtb challenge. At 21 days post-infection, B cell numbers had recovered with only a trend towards reduced parenchymal B cells in anti-CD20 antibody-treated mice (Supplementary Figure S5). Depletion of B cells during vaccination did not impact the reduction in necrosis as measured by Sytox Green fluorescence in pCas4-vaccinated mice (Figures 6B,C). Additionally, no difference in the lung bacterial burdens was observed at either 21- or 28-days post-infection (Figure 6D). A reduction in the frequency of Tbet+ CD4 T cells was observed in pCas4-vaccinated B cell depleted mice similar to untreated pCas4-vaccinated mice (Figures 6E,F). Similar reductions in monocyte-derived cell frequencies were also observed in both the pCas4-vaccinated anti-CD20 antibody treated mice and pCas4-vaccinated control mice (Figures 6G,H). These data indicate that B cells are dispensable for pCas4-mediated protection.
Figure 6
3.6 A vaccine encoding for RimJ alone is sufficient to reduce necrosis in SP140‒/‒ mice
Vaccination with pCas4 induced immune responses to 3 of the 4 RVMA, but RimJ was of special interest due to the amino acid substitution between H37Rv and Erdman Mtb strains. To test whether vaccination with RimJ alone could recapitulate the effects observed after pCas4 vaccination, we vaccinated SP140‒/‒ mice with the pVAX1-RimJ plasmid and challenged with Mtb Erdman (Figure 7A). RimJ vaccination significantly reduced Sytox Green staining in whole lungs of SP140‒/‒ mice compared with mock-vaccination (Figures 7B,C). No significant change in the bacterial burden in lungs or spleens of mock- or RimJ-vaccinated mice was observed, consistent with the observations made using pCas4 (Figure 7D). The immune cell composition was only measured at 28 days post-infection, and most differences were more subtle than what was observed for pCas4-vaccination at 21 days post-infection. Nonetheless, RimJ-vaccinated mice had significantly increased frequencies and numbers of RORγt+ CD4 T cells, as was observed in pCas4-vaccinated mice (Figures 7E,F). However, no significant reduction in monocyte-derived cells was observed at this time point in RimJ-vaccinated animals (Figures 7G,H). Overall, RimJ-vaccination is able to recapitulate most aspects of pCas4-vaccination in SP140‒/‒ mice and thus represents an antigen for further investigation of the differences in T cell responses to this class of antigens and how naturally occurring T cell epitope-disrupting substitutions impact vaccine efficacy.
Figure 7
To further characterize the CD4 T cell response to RimJ, we transferred CD4 T cells from vaccinated congenic mice to allow for phenotyping and tissue localization in infected SP140‒/‒ mice. Spleen cells from vaccinated CD45.1 congenic mice were stimulated with pooled overlapping peptides spanning the entire RimJ protein and selected by magnetic enrichment for antigen-responsive CD154+ cells. CD154+ cells were then transferred into recipient CD45.2 SP140‒/‒ mice one day prior to aerosol infection with Mtb Erdman expressing the fluorescent protein zsGreen. We then assessed the presence, phenotype, and localization of CD45.1+ CD4+ T cells 28 days post-infection using flow cytometry and immunofluorescence (Figures 8A,E). We identified donor CD45.1+CD4+ T cells in the lungs by both modalities (Figures 8A,E). Transcription factor staining revealed a significant reduction in the frequency of Tbet+ cells among the CD45.1+ CD4 T cells compared with endogenous (CD45.1‒) CD4 T cells, similar to what was observed for pCas4 vaccination at 21 days-post infection (Figures 8B,C). RORγt+ cells were also significantly reduced among transferred cells as compared to endogenous CD4 T cells (Figures 8B,C). We confirmed that CD45.1+ CD4 T cells were RimJ-specific by restimulation with RimJ peptides and intracellular cytokine staining for IFNγ, which revealed responses among the CD45.1+ CD4 T cells only to RimJ peptides and not to ESAT-6 peptides (Figure 8D). Additionally, staining for IL-10 revealed no detectable IL-10 expression among the transferred cells, which indicates that RimJ-specific CD4 T cells are not a significant source of this immunosuppressive cytokine (Figure 8D). Immunofluorescence of lung tissue sections revealed large non-necrotic lesions containing CD45.1+CD4+ cells within the lesions near Mtb+ cells (Figure 8E). These data indicate that vaccine-induced RimJ-specific CD4 T cells respond and traffic to the lungs during Mtb infection and likely exhibit a unique phenotype in the context of the bulk CD4 T cell response to Mtb infection.
Figure 8
4 Discussion
We generated DNA vaccines encoding RVMA, which were previously identified by their presence of nonsynonymous mutations within human T cell epitopes suggestive of selection pressure from immunological recognition (, ) and found that vaccination with DNA encoding one or more RVMA prior to Mtb infection limits immune pathology without reducing the bacterial burdens. Vaccination with a DNA vaccine encoding for a fusion of 4 variable antigens reduced lymphoid aggregate formation in resistant C57BL/6 mice and limited formation of necrotic lesions in susceptible SP140‒/‒ mice. Additionally, these non-necrotic lesions in pCas4-vaccinated mice had reduced lung tissue destruction and signs of myofibroblast presence. Corresponding reductions in neutrophils and increased CD4 T cell penetration into the core of lesions in pCas4-vaccinated mice were consistent with other studies in susceptible mice (51). Early differences in immune responses precede these changes in pCas4-vaccinated mice. Reductions in neutrophils as well as monocyte-derived cells, coupled with changes to CD4 T cell subsets, may all contribute to preventing formation of necrotic lesions. B cells were dispensable for this protection in the context of vaccination with pCas4. Further, vaccination with a DNA vaccine encoding for RimJ alone is sufficient to promote reduction in necrosis and changes to the CD4 T cell subsets responding to the infection.
Aside from a higher rate of nonsynonymous mutations within T cell epitope regions of the genes, it is unclear what might be different about these variable antigens as compared to more common vaccine antigens, such as ESAT-6. The most obvious difference is the lack of bacterial secretion of these antigens as compared to the classical immunodominant antigens. This may be the reason we observed a more modest T cell response to RVMA in response to Mtb infection of unvaccinated mice compared to the immunodominant antigens, ESAT-6 and Ag85B. While other work has shown that vaccination with “subdominant” antigens can induce protection, it is unclear whether being subdominant impacts T cell responses to RVMA (). Targeting T cell responses specifically to subdominant epitopes within ESAT-6 in mice was able to improve the quality of CD4 T cell responses to Mtb infection (60). Thus, antigens inducing subdominant CD4 T cell responses may generate CD4 T cell responses that reduce lung pathology while not increasing control of bacterial replication.
Both pCas4 and RimJ vaccination increased RORγt+ CD4 T cells. RORγt+ CD4 T cells are increased in frequency in people who resist TB infection and in vaccinated mice with enhanced restriction of Mtb, and thus may contribute to protective CD4 T cell responses (, 61). While we observed increased frequencies of RORγt+ CD4 T cells in the lungs of pCas4- and RimJ-vaccinated mice, we did not detect IL-17A expression by ICS in the lungs of infected mice after stimulation with RimJ peptides (Supplementary Figure S3B). We did detect IL-17A and IFNγ production in response to RimJ-stimulation in the draining lymph node at early time points post-infection (Supplementary Figure S3A). Thus, CD4 T cell responses to RimJ may represent a natural ex-Th17 response that may be impacting Mtb-induced tissue damage without restricting replication. Indeed, we detected ex-Th17 cells in the lungs of infected mice using an Il17a-cre;Ai14 reporter mouse to fate-map cells that express IL-17A (Supplementary Figure S3C,D), and these cells have been implicated in lung bacterial clearance (62, 63). More recent evidence also implicates ex-Th17 cells in rheumatoid arthritis, and could further indicate ex-Th17 cells play a role in mediating immunopathology during Mtb infection (64). Further studies will be needed to elucidate the impact of ex-Th17 cells in the context of Mtb infection.
Mtb is a unique pathogen that has evolved in humans for at least 70,000 years (65). This timescale, along with the fact that Mtb is an obligate human pathogen with no known reservoirs, has likely favored traits that allow for the maximum persistence and transmission (65). One aspect of immunity to pathogens that is rarely considered in the context of vaccine development is infection or disease tolerance (66). That is the ability of a host to tolerate a pathogen with minimal damage to the host without eliminating the pathogen. While it may seem that this simply represents a failure to resist the infection, it can be beneficial to the host by prioritizing tissue homeostasis over restricting or eliminating the invading pathogen. Indeed, in the context of Mtb infection, prior to the introduction of anti-tuberculosis chemotherapies many infected individuals experienced long-term chronic infections. While the case fatality rate of untreated tuberculosis is high (∼50%), it has also been argued that the pathogen has reached a balance as over 90% of individuals that are exposed to Mtb, do not develop disease (, 67). Additionally, evidence for tolerance to Mtb infection being beneficial to the host is necessity of the checkpoint protein PD-1 to prevent severe tuberculosis in mice, nonhuman primates and humans (, , 68–70). Vaccine development for Mtb infection could benefit from consideration of disease tolerance as a potential end point. The results presented here suggest that T cell responses to RVMAs may represent an immune response that improves infection tolerance in the host. We hypothesize that a vaccine incorporating RVMAs may be more efficacious in a PoD vaccine due to enhanced tolerance preventing lung immunopathology associated with tuberculosis disease and transmission.
Immunopathology in pulmonary TB is associated with enhanced transmission. CD4 T cell counts correlate with rates of cavitary TB, which is known to increase sputum bacillary load and likely transmission (, 71) While TB transmission cannot be modeled in mice, we did identify reduced immunopathology in the hypersusceptible SP140‒/‒ mice that otherwise develop necrotic lesions similar to those observed in humans. Reduced immunopathology and cavitary lesions could coincide with reduced rates of transmission, which further supports the benefits of vaccines that prevent disease and cavitary lesions. However, additional studies are needed to determine whether PoD vaccines will lead to reduced rates of cavitary lesions and transmission. While reducing immunopathology may reduce transmission, it is not clear whether it impacts dissemination of the infection. We did not observe different bacterial burdens in the spleens, which suggests that dissemination was not affected. While lower CD4 counts are associated with reduced cavitary disease, they are also associated with greater dissemination of Mtb in humans (, 72, 73).
A consequence of choosing sequence-variable antigens for these experiments was identifying a naturally occurring substitution in RimJ in the Mtb Erdman strain that was predicted to impact MHCII binding for H2-IAb. We found that the Erdman RimJ peptide variant elicited lower frequencies of CD4 T cells in H37Rv-infected mice and allowed for recognition of a secondary epitope in mice vaccinated with the Erdman variant (Supplementary Figure S2). While this may raise concern regarding the need for multiple sequences in potential RVMA-containing vaccines, it should be noted that there is limited overall sequence diversity in global bacterial populations, and in the case of the RVMA, the number of variants at a given locus is 3 or fewer (, , 65). Thus, polyvalent vaccine design approaches would be sufficient for RVMA-containing vaccines. RimJ vaccination alone was able to recapitulate the effects of pCas4-vaccination, and thus responses to RimJ likely account for many of the effects mediated by pCas4-vaccination. All RVMA may not be equivalent. We began this work with an additional cassette of RVMA, Rv0012 and LldD2, which despite inducing robust immune responses to those antigens, did not impact infection outcomes. We also determined these antigens did not induce any detectable T cell responses in mice with H2-IAb or H2-IAd during Mtb infection, which suggests they are poorly antigenic in mice. RimJ could serve as a model for future studies that aim to investigate the impact of antigenic variation on the CD4 T cell response to Mtb and vaccine efficacy.
There were limitations to our study. We were unable to determine a clear mechanism underlying the reduction in immunopathology observed in mice vaccinated with pCas4. We identified correlations with changes in CD4 T cell subsets between mock- and RVMA-vaccinated mice, but were unable to determine whether these differences were required to mediate the reduction in pathology. We did observe changes to the myeloid compartment in RVMA-vaccinated mice as well, but were unable to determine more functional changes within certain subsets of myeloid cells. It is likely that changes associated with RVMA vaccination involve changes in T cells, and future studies will be needed to characterize those changes. Since B cell depletion did not abrogate the benefit of RVMA vaccination, and since the effects of vaccination were antigen-dependent, it is reasonable to infer that the effects of vaccination were mediated by T cells. We were able to include multiple imaging methods to assess immunopathology. Our use of the Sytox Green method allowed us to evaluate lung necrosis in a more moderate throughput way. Although Sytox Green stains all extracelullar DNA and there is documented evidence of NETosis in the SP140‒/‒ mice, corresponding IHC and immunofluorescence revealed an extent of tissue desctuction and acellularity more consistent with necrosis (, 49, 74). We were also unable to compare the effects of RVMA-vaccination to currently relevant vaccine candidates in our study, such as M72 (). Most vaccines have been selected for their ability to reduce bacterial burdens in preclinical models, and thus differ from our findings using RVMA vaccination. As no vaccine using this metric has been studied in humans, our identification of reduced immunopathology in a hypersusceptible mouse model represents a potentially useful new metric to evaluate vaccine candidates. Additional studies are required to determine how M72, or similar vaccine candidates, compare to RVMA vaccination in a hypersusceptible mouse model.
Immune pathology associated with Mtb infection in mice may not be linked to bacterial burden in all contexts. Indeed, it has been observed in some mouse models, such as mice deficient in the intracellular adhesion molecule 1 (ICAM-1) gene or cyclophilin D (CypD), that changes in pathology can be independent of bacterial burden as described here and even associated with pathogenic T cell responses (75–77). It is even possible to achieve increased pathology with lower bacterial burdens in the context of IFNγ-overexpression (). Identifying a context in which vaccination with specific antigens can create a similar decoupling of pathology and bacterial replication has important consequences for preclinical testing of future Mtb vaccines. Ensuring that bacterial burden is not used as a sole method of ruling out a vaccine may be important for identifying vaccines that achieves substantial efficacy in humans. Additionally, the fact that CD4 T cell responses to Mtb may promote pathology and possibly transmission warrants caution and consideration in early-stage trials. Further studies are needed to determine whether evolutionary pressures are selecting for Mtb that induces CD4 T cell responses to promote transmission, and whether RVMA induce CD4 T cell responses that protect the host from pathology and enhanced transmission.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by University of California, San Francisco Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
ZH: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. AM: Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. WZ: Investigation, Methodology, Validation, Writing – review & editing. JE: Conceptualization, Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was supported by the NIH grants F31 AI172360-01 (ZH) and U01 AI166309 (JE). The funders had no role in study design, data collection or interpretation, or the decision to submit the work for publication.
Acknowledgments
We thank Irina Debnath for early contributions to this work. We also acknowledge the excellent technical assistance provided by I-Chang Chang and Lucas Chen. Microscopy data for this study was acquired at the UCSF Innovation Core at the Weill Institute for Neurosciences and the UCSF Center for Advanced Light Microscopy. Flow cytometry data for this study was generated with assistance from the UCSF Division of Experimental Medicine Core Immunology Lab.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ftubr.2026.1814646/full#supplementary-material
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Summary
Keywords
antigens, CD4 T cells, epitopes, mycobacterium tuberculosis, prevention-of-disease, vaccine
Citation
Howard ZP, Mohapatra A, Zheng W and Ernst JD (2026) A Mycobacterium tuberculosis rare variable antigen vaccine reduces lung pathology in hypersusceptible SP140−/− mice. Front. Tuberc. 4:1814646. doi: 10.3389/ftubr.2026.1814646
Received
20 February 2026
Revised
12 June 2026
Accepted
26 June 2026
Published
15 July 2026
Volume
4 - 2026
Edited by
Mohammad Aqdas, National Institutes of Health (NIH), United States
Reviewed by
Saurabh Chugh, UMR5089 Institut de Pharmacologie et de Biologie Structurale (IPBS), France
Sudhasini Panda, The Scripps Research Institute, United States
Updates
Copyright
© 2026 Howard, Mohapatra, Zheng and Ernst.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Joel D. Ernst joel.ernst@ucsf.edu
Disclaimer
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