Patent Application
20250152690
The present invention relates to methods and compositions of matter that are useful for preventing or reducing the possibility of infection caused by Mycobacterium tuberculosis, the agent of tuberculosis, and infection by other pathogenic strains of mycobacteria in humans and/or animals including Mycobacterium bovis and Mycobacterium leprae.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a deadly global disease. It is estimated that one quarter of the world's population has been infected with Mtb, most of whom develop latent TB infection, and that 10 million people develop active TB and 1.5 million people die of TB annually. Mycobacterium bovis Bacillus Calmette-Guérin (BCG), developed more than 100 years ago and the only licensed vaccine against TB, has been used to vaccinate infants and to protect young children against severe forms of TB; however BCG has shown variable efficacy in preventing pulmonary TB in adolescents and adults, the most prevalent form (1). As BCG has been widely used worldwide to vaccinate 88% of infants within the first year of their life (1, 2), booster vaccines that improve upon the efficacy of BCG, even to a small extent, could have a significant impact on the TB pandemic.
Several strategies have been employed to develop booster vaccines for TB, primarily protein/adjuvant vaccines comprising fusion proteins of selected Mtb antigens administered with a strong T-cell stimulating adjuvant and viral-vectored vaccines, wherein viruses including adenovirus, Modified Vaccinia Ankara virus, and cytomegalovirus express recombinant proteins (3-9). In contrast, we have employed a highly attenuated replicating bacterium as a vaccine vector, Lm ΔactA ΔinlB prfA*, a Listeria monocytogenes with deletions in two major virulence genes (actA and inlB) and a single amino acid substitution (G155S) in PrfA (positive regulatory factor A) resulting in constitutive overexpression of PrfA and PrfA-dependent genes, a modification exploited to enhanced vaccine efficacy (10). Listeria monocytogenes is an intracellular bacterium that invades mononuclear phagocytes, resides in a membrane-bound phagosome, and ultimately escapes the phagosome to reside and multiply in the cytoplasm (11). Its intraphagosomal and intracytoplasmic locations favor antigen presentation via both MHC class I and II, respectively, allowing induction of both CD4+ and CD8+ antigen-specific T cells, both important to immunity against TB. A Listeria vector also has other immunologic advantages including the capacity to carry and express a large amount of recombinant protein cargo; the ability to disseminate to organs that are impacted by Mtb, such as the lung and spleen, before being cleared by the immune system, thereby promoting local immunity at sites of Mtb infection; and the fact that pre-existing immunity does not negatively affect efficacy (12, 13). Additional practical advantages of a Listeria vectored vaccine are an established safety profile (14), as the vector has been used safely in cancer vaccines, and low cost of manufacture in simple broth culture, without the need for extensive purification as in the case of protein/adjuvant and viral-vectored vaccines.
A safe and effective vaccine against M. tuberculosis or other species of the genus Mycobacterium that is superior to the currently available vaccines is sorely needed. There is also a need for a M. tuberculosis booster vaccine or a vaccine that can improve the potency of the currently available vaccines by even a small amount. The disclosure provided herein meets this need.
The present disclosure provides a vaccine and method for preventing, reducing the possibility of or treating tuberculosis in humans and animals that is better than the current commercially available vaccines and methods in protecting against pulmonary tuberculosis and dissemination of bacteria to the spleen and other organs. The present disclosure also provides a vaccine and method for preventing, reducing the possibility of or treating leprosy and other mycobacterial diseases. Moreover, the present disclosure provides a vaccine that is easier and cheaper to manufacture than both virus-vectored vaccines, which must be grown in tissue culture cells and then purified, and protein-in-adjuvant vaccines, where the protein needs to be purified. As noted below, the M. tuberculosis vector vaccine described in the present disclosure can simply be grown in broth culture—no purification is necessary.
Listeria monocytogenes is an intracellular bacterium that invades mononuclear phagocytes, resides in a membrane-bound phagosome, and ultimately escapes the phagosome to reside and multiply in the cytoplasm. Its intraphagosomal and intracytoplasmic locations favor antigen presentation via both MHC class I and II, respectively, allowing induction of both CD4+ and CD8+ antigen-specific T cells, both important to immunity against TB. A Listeria vector also has other immunologic advantages including the capacity to carry and express a large amount of recombinant protein cargo; the ability to disseminate to organs that are impacted by M. tuberculosis, such as the lung and spleen, before being cleared by the immune system, thereby promoting local immunity at sites of Mtb infection; and the fact that pre-existing immunity does not negatively affect efficacy. Additional practical advantages of a Listeria vectored vaccine are an established safety profile, as the vector has been used safely in cancer vaccines, and low cost of manufacture in simple broth culture, without the need for extensive purification as in the case of protein/adjuvant and viral-vectored vaccines. Protein/adjuvant vaccines and non-replicating virus-vectored vaccines lack many of these advantages.
Embodiments of the invention include the aforementioned Listeria monocytogenes ΔactA ΔinlB prfA* vector into which antigen cassettes comprising all or parts of various key immunoprotective antigens of M. tuberculosis have been inserted. Each vaccine is administered intradermally or by another route, e.g. subcutaneously, intramuscularly, intranasally, inhaled, or even orally to a mammalian host. The vaccine can be administered as part of a homologous or heterologous prime-boost vaccination strategy. The vaccine induces a strong cell-mediated immune response to pathogen antigens in the vaccine including both CD4 and CD8 T cells.
The invention disclosed herein has a number of embodiments. Embodiments of the invention include compositions of matter comprising at least one fusion protein having antigenic epitopes present in at least five Mycobacterium tuberculosis proteins selected from: immunogenic protein MPT64 (“23.5/Mpt64”), ESAT-6-like protein EsxH (“TB10.4/EsxH”), 6 kDa early secretory antigenic target (“ESAT6/EsxA”), ESAT-6-like protein EsxB (“CFP10/EsxB”), and diacylglycerol acyltransferase/mycolyltransferase Ag85B (“r30/Antigen 85B”); ESAT-6-like protein EsxN (“EsxN”); PPE family immunomodulator PPE68 (“PPE68”); ESX-1 secretion-associated protein EspA (“EspA”) and low molecular weight T-cell antigen TB8.4 (“TB8.4”), wherein the composition comprises live attenuated Listeria monocytogenes expressing the at least one fusion protein. Embodiments of the invention further include polynucleotides encoding the fusion proteins disclosed herein, for example such polynucleotides disposed within a vector.
Typically with the live attenuated Listeria monocytogenes compositions expressing the fusion proteins disclosed herein, when administered to mice as a vaccine, the composition elicits an immune response to Mycobacterium tuberculosis exposure in the mice characterized by an at least 10% reduction in Mycobacterium tuberculosis colony forming units in lungs of mice administered the vaccine as compared to lungs of control mice administered a control composition lacking antigenic epitopes present in Mycobacterium tuberculosis. In illustrative working embodiments of the invention, the fusion protein comprises antigenic epitopes present in the polypeptide sequence:
and/or
In certain embodiments of the invention, the composition comprises at least two fusion proteins. For example, in some embodiments of the invention, the Listeria monocytogenes expresses a first fusion protein encoded by a polynucleotide present in a first locus of the Listeria monocytogenes genome and a second fusion protein encoded by a polynucleotide present in a second locus of the Listeria monocytogenes genome. In certain embodiments of the invention, a first fusion protein comprises epitopes present in at least two Mycobacterium tuberculosis proteins selected from: 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30/Antigen 85B; and a second fusion protein comprises epitopes present in at least two Mycobacterium tuberculosis proteins selected from EsxN; PPE68; EspA and TB8.4.
Embodiments of the invention also include methods of generating an immune response to a Mycobacterium tuberculosis in a mammal (e.g., a mouse, guinea pig or human) comprising administering to the mammal a composition comprising a live attenuated Listeria monocytogenes expressing one or more fusion proteins disclosed herein such that an immune response to Mycobacterium tuberculosis is generated. Typically in such embodiments of the invention, when administered to mice as a vaccine, the composition elicits an immune response to Mycobacterium tuberculosis exposure in the mice characterized by an at least 10%, 25% or 50% (or 0.05, 0.1, or 0.3 log) reduction in Mycobacterium tuberculosis colony forming units in lungs of mice administered the vaccine as compared to lungs of control mice administered a control composition lacking antigenic epitopes present in Mycobacterium tuberculosis.
Typically, the mammal is immunized intranasally, subcutaneously, intradermally, intramuscularly or orally. Certain of these methodological embodiments of the invention include the steps of exposing the mammal to Mycobacterium tuberculosis antigens using a different immunogenic platform administered to the mammal at the same or at a different time, for example methods which comprise immunizing the mammal a composition disclosed herein in combination with Mycobacterium bovis strain Bacille Calmette-Guérin (BCG).
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
Referring now to the drawings and figures:
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
In the following description of the typical embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.
While medical practitioners have been working to generate an effective vaccine for tuberculosis for many years, this goal remains elusive. One of the challenges in this area of technology involves determining which M. tuberculosis protein antigen(s) can be used to create effective vaccines (as the Mycobacterium tuberculosis genome comprises around 4.4 million base pairs, and contains around 4,000 genes). Previously we developed a recombinant Listeria monocytogenes ΔactA ΔinlB prfA* vectored vaccine candidate (rLm30) expressing the Mtb 30-kDa major secretory protein (r30/Ag85B/Rv1886) driven by the hly promoter and leader sequence or the actA promoter and leader sequence to facilitate the expression and secretion of r30 by rLm (see, e.g. PCT International Publication No. WO 2011/159814, the contents of which are incorporated by reference). We found that rLm30 significantly enhances BCG-primed protective efficacy against aerosol challenge with the virulent Mtb Erdman strain in mice and guinea pigs (15). Boosting BCG-primed C57BL/6 mice with rLm30 induces strong antigen-specific T-cell mediated immune responses, including greater frequencies of antigen-specific polyfunctional CD4+ and CD8+ T cells expressing interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and/or Interleukin 2 (IL-2) in the spleens and lungs.
As discussed herein, we have determined that certain constellations of different M. tuberculosis proteins perform unexpectedly well when utilized in the immunogenic Listeria monocytogenes fusion protein compositions disclosed herein (i.e., can elicit significant protection against M. tuberculosis infection). Briefly, in efforts to expand the M. tuberculosis antigen repertoire of our rLm vaccine, we evaluated a number of different M. tuberculosis proteins in addition to r30 for potential inclusion in a multi-antigenic vaccine. We constructed a variety of new rLm multi-antigenic vaccine candidates including vaccines expressing a variety of immunogenic epitopes found on different M. tuberculosis proteins and evaluated them for efficacy against M. tuberculosis aerosol challenge in C57BL/6 and/or BALB/c mice. From these studies, we identified rLm5Ag (30), expressing the fusion protein 23.5 (MPT64)-TB10.4 (EsxH)-ESAT6 (EsxA)-CFP10 (EsxB)-r30 (Ag85B) as one of the most promising vaccine candidates. We then studied the immunogenicity of rLm5Ag (30) in BCG-primed C57BL/6 and BALB/c mice. We found that while the rLm-vectored multi-antigenic vaccine boosts BCG-primed protection in both C57BL/6 and BALB/c mice, this rLm vaccine induces strong T-cell mediated immune responses, evidenced by enhanced antigen-specific frequencies of splenic and lung CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, and/or IL-2, in BCG-primed C57BL/6 mice but not in BALB/c mice, where such responses are markedly limited. Thus, while the rLm vaccines enhance protective immunity in both BCG-immunized mouse strains, they do so via disparate immune responses.
In the studies that led to the invention disclosed herein, we discovered that certain immunogenic epitopes present on selected M. tuberculosis proteins can significantly enhance the protective efficacy of vaccines against M. tuberculosis (e.g., in the Listeria monocytogenes fusion protein platforms disclosed herein). Significantly in these studies, the inventors discovered some M. tuberculosis protein immunogenic epitopes do not function well in the Listeria monocytogenes fusion protein vaccine compositions disclosed herein. For example, immunogenic epitopes found in certain M. tuberculosis proteins (e.g., Hrp1, PE25, HspX, and VapB47) actually compromised the ability of Listeria monocytogenes fusion protein vaccine compositions to prevent or inhibit M. tuberculosis infection (see, e.g,
Embodiments of the invention include composition of matter comprising at least one fusion protein having antigenic epitopes present in at least two (and up to 9) Mycobacterium tuberculosis proteins selected from: 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30/Antigen 85B; EsxN; PPE68; EspA and TB8.4. In certain embodiments of the invention, the composition comprises at least two fusion proteins having antigenic epitopes present in at least two (and up to 9) Mycobacterium tuberculosis proteins selected from: 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30/Antigen 85B; EsxN; PPE68; EspA and TB8.4. In some embodiments of the invention, the at least one fusion protein does not comprise immunogenic epitopes present in at least one Mycobacterium tuberculosis protein selected from Hypoxic response protein 1 (“Hrp1”, UniProt P9WAJ3), PE-PGRS family protein PE25 (“PE25”, UniProt 16X486), Alpha-crystallin (“HSPX” UniProt P9WMK1) and Antitoxin VapB47 “VapB47” UniProt P9WF22). In certain embodiments of the invention, immunogenic epitopes are disposed on the fusion protein such that immunogenic epitopes of 23.5/Mpt64 are N-terminal to other Mycobacterium tuberculosis immunogenic epitopes disposed in the fusion protein. In certain embodiments of the invention, immunogenic epitopes are disposed on the fusion protein such that immunogenic epitopes of r30/Antigen 85B are C-terminal to other Mycobacterium tuberculosis immunogenic epitopes disposed in the fusion protein.
Typically, these compositions comprise Listeria monocytogenes expressing the at least one fusion protein or the at least two fusion proteins (e.g. a strain of Listeria monocytogenes that does not express a functional InlB protein; that does not express a functional actA protein; and/or expresses prfA protein having a G155S substitution mutation). In certain compositions of the invention, the Listeria monocytogenes expresses a first fusion protein encoded by a polynucleotide present in a first locus of the Listeria monocytogenes genome and a second fusion protein encoded by a polynucleotide present in a second locus of the Listeria monocytogenes genome. In one illustrative embodiment of the invention, the first fusion protein comprises epitopes present in at least two Mycobacterium tuberculosis proteins selected from: 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and r30/Antigen 85B; and the second fusion protein comprises epitopes present in at least two Mycobacterium tuberculosis proteins selected from EsxN; PPE68; EspA and TB8.4.
In illustrative working embodiments of the invention, a fusion protein comprises antigenic epitopes present in the polypeptide sequence:
and/or
Embodiments of the invention further include polynucleotides encoding the fusion proteins disclosed herein (e.g. SEQ ID NO: 1 or 2), for example such polynucleotides disposed within a vector
Embodiments of the invention also include methods of generating an immune response to a Mycobacterium tuberculosis comprising immunizing a mammal with a vaccine composition disclosed herein such that an immune response to Mycobacterium tuberculosis is generated. Those of skill in this art understand that the immunization methods disclosed herein can be combined with other methodological steps. For example, certain embodiments of the invention include the step of immunizing the mammal with Mycobacterium bovis strain Bacille Calmette-Guérin (BCG). Optionally in these embodiments, the BCG is used in a primary immunization and the Listeria monocytogenes expressing the at least one fusion protein or the at least two fusion proteins is used in a booster immunization. In such embodiments of the invention, the mammal can be immunized intradermally, intranasally, orally, subcutaneously, percutaneously, intramuscularly, intravenously, or by another conventional route of vaccine delivery. For example, in some of these methods the composition is administered subcutaneously. In certain methods, the composition is administered intradermally.
Further aspects and embodiments of the invention are discussed in the examples below.
Aspects and embodiments of the invention discussed in this example are also discussed in Jia Q, Masleša-Galić S, Nava S, Horwitz M A. mBio. 2022 Jun. 28; 13 (3): e0068722. doi: 10.1128/mbio.00687-22. Epub 2022 Jun. 1, the contents of which are incorporated herein by reference.
Murine (J774A.1, ATCC TIB-67) and human (THP1, ATCC TIB-202) monocytes were differentiated into macrophage-like cells and cultured in Dulbecco's modified Eagle's medium (DMEM) and RMPI 1640 (RPMI) medium, respectively, containing penicillin (100 μg/ml) and streptomycin (100 U/ml) and supplemented with 10% fetal bovine serum (FBS). Mycobacterium bovis BCG Tice was purchased from Organon. M. tuberculosis (Mtb) Erdman strain (ATCC 35801) was harvested from infected outbred guinea pigs to verify virulence, cultured on 7H11 agar, subjected to gentle sonication to obtain a single cell suspension, and frozen at −80° C. for use in animal challenge experiments. All Listeria vector and recombinant Listeria-vectored vaccine stocks were grown to mid log phase in Yeast Extract broth medium and the bacteria collected by centrifugation, resuspended in phosphate buffer saline (PBS), titrated, and stored in 20% glycerol/PBS at −80° C. until use. Six to eight-week-old female C57BL/6 mice were purchased from Harlan (currently Envigo, Livermore, CA, USA) or Jackson Laboratory (Bar Harbor, Maine, USA) and BALB/c mice purchased from Jackson Laboratory.
We constructed Lm-vectored multi-antigenic rLm vaccine candidates using the Lm ΔactA ΔinlB prfA* vector, as we previously described (15). Briefly, to construct rLm vaccine candidates expressing Mtb multi-antigenic proteins, we analyzed the protein sequences of the selected 15 Mtb proteins, removed the predicted signal peptides of TB8.4 (2R-28A), Apa (2H-39A), r30/Ag85B (2Q-43A), and 23.5/Mpt64 (1V-23A) and the internal regions of HspX (1211-128V), PE25 (661-73L), and EspA (111F-193L) that might interfere with protein secretion from the rLm vaccine constructs; we kept the full-length sequences for TB10.4, EsxN, Hrp1/Rv2626c, VapB47/RV3407, EspC, PPE68, CFP-10, and ESAT-6. We optimized the coding sequence for each of the selected proteins for expression in Lm, purchased them from DNA2.0 (Newark, CA), and assembled the optimized DNAs encoding the indicated multi-antigenic proteins with or without a spacer encoding a GGSG (SEQ ID NO: 3) or GSSGGSSG (SEQ ID NO: 4) linker by traditional molecular cloning methods. We cloned the final assembled DNAs into a phage-based Listeria site-specific integration vector derived from pPL1 (kindly provided by P. Lauer) or pPL2e (kindly provided by J. Skoble) (40) downstream of the Lm actA promoter and ligated in-frame to the C-terminus of the ActAN. Subsequently, we integrated the Mtb antigen expression cassette together with the pPL vector into the comK locus or the 3′ the end of the tRNAarg locus on the bacterial chromosome of the recipient Lm vector, as described previously by us (15) and Lauer et al. (10, 16). All molecular plasmid constructs were confirmed by restriction enzyme digestion and nucleotide sequencing. The final rLm strains were verified by PCR using primers, specifically amplifying a unified 548-bp PCR product in strains that contain an integration vector at the bacterial attachment site tRNAarg-attBB′, and using primers, specifically amplifying a PCR product across the inserted gene with various sizes in different strains; the PCR products were further confirmed by nucleotide sequencing.
The growth kinetics of rLm vaccine candidates in broth culture and macrophage-like cells was examined as described by us previously with modifications (15). Glycerol stocks of the Lm vector and rLm vaccine candidates were inoculated into Brain Heart Infusion (BHI) medium supplemented with Streptomycin (200 μg/ml) (the Lm vector is streptomycin resistant) to prevent any contamination and grown overnight under stationary conditions in a 37° C. incubator with 5% CO2. The overnight culture was inoculated into 5 ml fresh BHI with Streptomycin at an initial optical density at 540 nm (OD540) of ˜0.05 and incubated at 37° C. with shaking at 180 rpm. At 0, 3, 5, and 7 hours post inoculation, a 1-ml aliquot of each culture was removed and measured for OD540.
The growth kinetics in macrophage-like cells was assayed by infecting monolayers of murine macrophage-like cells (J774A.1) or phorbol 12-myristate 13-acetate (PMA) differentiated monolayers of human macrophage-like cells (THP-1) with the Lm vector or rLm candidates cultured overnight to stationary phase at a multiplicity of infection of 1:10 for 90 min in DMEM (J774A.1) or RPMI (THP-1) medium supplemented with 10% heat-inactivated FBS (HI-FBS). After 90 min infection, cells were washed three times with PBS supplemented with 2% HI-FBS. The infected cells were cultured for an additional 4.5 hours in DMEM or RPMI supplemented with 10% HI-FBS and gentamycin (10 μg/ml). At 0, 2, 4, and 6 hours post infection, the medium was removed; the monolayers lysed with 0.1% Saponin/PBS; and the cell lysates serially diluted in PBS and plated on BHI agar plates supplemented with streptomycin (200 μg/ml). The plates were incubated at 37° C. for two days and colonies enumerated.
Immunization and Aerosol Challenge of Mice with Virulent M. tuberculosis Erdman Strain
Groups of BALB/c or C57BL/6 mice, 8 or 12/group, were primed with BCG intradermally (i.d.) or intranasally (i.n.). BCG-primed mice were either not boosted or boosted once or twice with 2×106 CFU of a single rLm vaccine or combination of two rLm vaccine candidates expressing multiple Mtb antigens and challenged 3 or 4 weeks later by exposure to aerosolized Mtb Erdman strain generated by a Collison Type-6 Jet Nebulizer (CH Technologies USA, Waltham, MA) from 10 ml of Mtb bacterial suspension (1.6-2.6×105 CFU/ml) for 30 min followed by 5 min to allow for settling of bacteria. The challenge dose was verified by euthanizing two animals and assaying CFUs in their entire lungs at Day 1 post challenge. The mice were euthanized at various times post-challenge, and the spleens and lungs removed and assayed for bacillus burden as described by us previously (15).
To determine the immunogenicity of rLm5Ag (30) expressing the fusion protein of 5 Mtb antigens (23.5-10.4-ESAT6-CFP10-r30) as a booster vaccine, we immunized C57BL/6 and BALB/c mice, 4/group, subcutaneously (s.q.) with BCG at Week 0; boosted them at Weeks 14 and 18 with 2×106 CFU of the Lm vector or rLm5Ag (30); euthanized the mice at 6 days post the last immunization; prepared single cell suspensions of spleen and lung cells; stimulated the single cell suspensions with a single Mtb antigen or pool of multiple Mtb antigens; and assayed T-cell immunity by intracellular cytokine staining (ICS) using methods that we published previously (15, 17) with modifications as described below.
We conducted ICS by using an eight-color flow cytometry panel to simultaneously analyze multiple cytokines at the single-cell level. Specifically, a single cell suspension of 5×105 lung cells per well or 1.0×106 splenocytes per well was seeded in U-bottom 96-well plates and stimulated with medium alone (negative control), 5 μg/ml of recombinant proteins r30/Ag85B (our lab stock, isolated from recombinant Mycobacterium smegmatis), ESAT6/EsxA (BEI Resources), CFP10/EsxB (BEI Resources), TB10.4/EsxH (Aeras), 23.5/Mpt64 (BEI Resources), pool of 5 antigens (5Ag) comprising r30, ESAT6, CFP10, TB10.4, and 23.5, each at 2 μg/ml, or PPD (5 μg/ml) in the presence of anti-CD28 monoclonal antibody (Clone 37.51) for a total of 6 h. Cells stimulated with PMA served as a positive control. Four hours prior to harvest, GolgiPlug (protein transport inhibitor containing Brefeldin A) diluted in T-cell medium was added to all wells; PMA was additionally added to positive control wells. Following in vitro stimulation, cells were harvested, washed with PBS, incubated with Live/Dead Fix Near IR Cell Stain (Invitrogen) for 10 min at room temperature to identify dead cells, and surface stained with antibodies against CD4 (Clone RM4-5, conjugated with Brilliant Violet 510) and CD8 (Clone 53-6.7, conjugated with Brilliant Violet 605). Cells were then fixed/permeabilized with Cytofix/Cytoperm (BD BioSciences) and stained for CD3 (clone 17A2, conjugated with Alexa Fluor 488), IFN-γ (Clone XMG1.2, conjugated with Brilliant Violet 650), IL-2 (Clone JES6-5H4, conjugated with PE), TNF-α (Clone MP6-XT22, conjugated with PerCPCy5.5) and IL-17A (Clone TC11-18H10.1, conjugated with Alexa Fluor 647). Note that due to the internalization of CD3 in responding CD4+ T cells, cells were stained for CD3 after fixing/permeabilization. The Fluorochrome-conjugated antibodies were purchased from BioLegend. For Flow cytometry analysis, a minimum of 100,000 lymphocytes per sample was acquired with an LSRII-HT (BD) flow cytometer. The data were analyzed using FlowJo software. Initial gating of total events included a lymphocyte gate, followed by selection for singlet cells and live CD3+ T cells (Near IR-AF488+); CD4+ and CD8+ T cells were identified by CD4+ (BV510+BV605-) and CD8+ (BV605+BV510-) expression, respectively. The gates for frequencies of antigen-specific IFN-γ, IL-2, TNF-α, and IL-17A producing CD4+ and CD8+ T cells were determined by using the unstimulated cells; Boolean combinations of the four intracellular cytokine gates were used to uniquely discriminate responding cells based on their frequency with respect to cytokine production. Each cytokine-positive cell was assigned to one of the 15 possible combinations. Background frequencies of CD4+ and CD8+ T cells producing cytokines without antigen stimulation were subtracted. Two-way ANOVA with Sidak's multiple comparisons test was performed using GraphPad Prism 8.02 (San Diego, CA) to determine significance in comparisons of mean frequencies of cytokine-producing CD4+ and CD8+ T cells between mice vaccinated with the Lm vector and mice vaccinated with rLm5Ag (30).
One-way or two-way ANOVA with Tukey's multiple comparisons test was performed using GraphPad Prism 9.02 (San Diego, CA) to determine significance in comparisons of mean frequencies of cytokine producing CD4+ and CD8+ T cells and mean organ CFUs in spleens and lungs among mice in vaccinated and control groups.
Construction and verification of new rLm vaccines expressing 1, 3, or 4 recombinant Mtb proteins
Previously we have shown that rLm30, expressing the r30/Ag85B downstream of the Lm actA promoter and ligated to the N-terminal 100 amino acids of ActA (ActAN) as a fusion protein, boosts BCG-primed efficacy against TB (15). To expand the Mtb antigen repertoire of the rLm vaccine platform, we initially constructed 3 new rLm vaccine candidates-rLm23.5, expressing the mature peptide of 23.5/Mpt64A1V-23A); rLm3Ag, expressing the fusion protein of Ag85B (Δ2Q-43A)-RP-TB10.4-GGSG (SEQ ID NO: 3)-ESAT6 [RP, a dipeptide encoded by EagI restriction enzyme site for cloning purposes; GGSG (SEQ ID NO: 3), a flexible fusion protein linker]; and rLm4Ag, expressing the fusion protein of Mpt64 (A1V-23A)-RP-TB10.4-GGSG (SEQ ID NO: 3)-ESAT6-GSSGGSSG (SEQ ID NO: 4)-CFP10 (GSSGGSSG, (SEQ ID NO: 4) a flexible linker) (Table 1). The Mtb antigens in each vaccine construct were expressed as a C-terminal fusion protein to Lm ActAN; the Mtb protein expression cassette was driven by the Lm actA promoter and integrated at the tRNAarg of the Lm ΔactA ΔinlB prfA* chromosome.
a)Isoelectric point
When referring to certain figures as “sFig” in the text in this Example, see the supplemental figures found in Jia Q, Masleša-Galić S, Nava S, Horwitz MA. mBio. 2022 Jun. 28; 13 (3): e0068722. doi: 10.1128/mbio.00687-22. Epub 2022 June, the contents of which are incorporated by reference. We verified the expression of the heterologous protein ActAN-Mpt64 by rLm23.5 as a 31-kDa protein band detected by a polyclonal antibody to a peptide comprising 18 amino acids (A30-K47) of ActAN (AK18) (courtesy of J. Skoble and P. Lauer) (sFIG. 1a); expression of ActAN-Ag85B-TB10.4-ESAT6 by rLm3Ag as a 59-kDa protein band detected by a rabbit polyclonal antibody to r30 (sFIG. 1b, upper panel) or a polyclonal antibody to TB10.4 (sFIG. 1b, lower panel), and expression of ActAN-Mpt64-TB10.4-ESAT6-CFP10 by rLm4Ag as a 63-kDa protein band detected by AK18 (sFIG. 1c).
Boosting BCG-primed C57BL/6 mice with the combined rLm30-10.4-ESAT6 and rLm23.5 vaccines enhances protection against aerosolized Mtb.
To determine the efficacy of these multi-antigenic vaccine candidates as a booster vaccine in protecting BCG-immunized mice against aerosolized Mtb challenge, we immunized C57BL/6 mice, 8/group, i.n. with PBS (sham) or BCG at Week 0 and boosted one group of BCG-immunized mice i.n. with rLm30-10.4-ESAT6 (rLm3Ag)+rLm23.5 at Weeks 7 and 10. The mice were then challenged with aerosolized Mtb Erdman (2.6×105 CFU for 30 min resulting in an average of 21 CFU in the lungs at Day 1 post challenge) at Week 13, euthanized at Week 19, and their lungs and spleens assayed for Mtb CFU. As shown in
Boosting BCG-primed BALB/c mice with the combined rLm23.5-10.4-ESAT6-CFP10 and rLm30 vaccines enhances protection against aerosolized Mtb.
To further verify the immunoprotection against Mtb challenge of multi-antigenic rLm vaccine candidates as a booster vaccine in BCG-immunized mice, we immunized and challenged a different strain of mice, BALB/c mice. We immunized BALB/c mice, 8/group, i.d. with PBS (sham) or BCG at Week 0, did not boost or boosted BCG-immunized mice intramuscularly (i.m.) with Lm vector (LmV) or rLm23.5-TB10.4-ESAT6-CFP10 (rLm4Ag)+rLm30 at Weeks 14 and 18. The mice were then challenged with aerosolized Mtb Erdman (1.6×105 CFU for 30 min resulting in an average of 19 CFU in the lungs at Day 1 post challenge) at Week 22, euthanized at Week 32, and their lungs and spleens assayed for Mtb CFU (
In a companion experiment challenged at the same time as the experiment described in
As shown in
aAs an individual protein
bBlasted using nucleotide blast tool via https://blast.ncbi.nlm.nih.gov/Blast.cgi
c
M. bovis BCG reference strain: Pasteur 1173P2, gene bank access number AM408590.1
Selection of Mtb antigens and construction of 13 new rLm5Ag vaccine candidates
To further expand the Mtb antigen repertoire, we selected 15 Mtb proteins (including r30) as potential vaccine candidates for further investigation (Table 2), including a) Secreted proteins: r30 (3); 23.5/Mpt64/Rv1980c (24, 25, 30); TB8.4/Rv1174c (20); Apa/MPT32/Rv1860 (21); b) ESAT6 and associated proteins secreted by the Esx/Type VII secretion system: ESAT6/EsxA/Rv3875 (30, 32), CFP10/EsxB/Rv3874 (31), TB10.4/EsxH/Rv0288 (19, 33), EspA/Rv3616c (30), EspC/Rv3615c (30), and EsxN/Rv1793; c) Antigenic PE/PPE proteins: PE25/Rv2431c (34) and PPE68/Rv3873 (30, 35); and d) Latency associated proteins: α-crystallin/hspX/Rv2031c (26, 27), Hrpl/Rv2626c (28), and VapB47/Rv3407 (28, 29). Among the 15 selected proteins, all but two (EsxN and PE25) have been shown by us or others to be immunoprotective antigens when incorporated into various vaccines including protein/adjuvant, DNA, Listeria-vectored or virus-vectored vaccines and 4 proteins-23.5/Mpt64, PPE68, CFP-10, and ESAT-6-are absent either from all BCG strains or the modern BCG strain (Mpt64) (36, 37) (Table 2).
We constructed 11 new rLm5Ag vaccine candidates carrying a single copy of an ActAN-Mtb 5 antigen fusion protein expression cassette downstream of the Lm actA promoter integrated at the 3′ end of the tRNAarg locus in the rLm chromosome; in all such cases, the first four proteins in the fusion protein were Mpt64-EsxH-EsxA-EsxB followed by a GSSGGSSG (SEQ ID NO: 4) flexible linker and the fifth protein was one of the 11 other selected Mtb proteins in Table 2. In addition, we constructed 2 rLm vaccine candidates expressing the Mtb 4Ag fusion protein or r30/Ag85B from the comK locus of the rLm chromosome, rLm4Ag (comK) and rLm30 (comK), respectively, to allow a comparison of vaccines expressing proteins at this locus vs. the tRNAarg locus and to explore the possibility of later expressing proteins from both loci in the same vaccine (Table 3).
a) Mpt64 refers to Mpt64(Δ1V-23A)
b) The estimated Mw of each fusion protein is calculated as a secreted form without the signal peptide for ActA (1V-29A, 3.3 kDa).
We examined Mtb fusion protein expression by rLm5Ag vaccine candidates grown in broth medium. As shown in
We also verified the Mtb protein expression cassette integrated at the tRNAarg locus by PCR and nucleotide sequencing of the resultant PCR products. As shown in sFIG. 2a, amplification across the bacterial attachment site tRNAarg-attBB′ with primers NC16 and PL95 resulted in a 548-bp fragment in each selected clone of the rLm candidates (lanes 3-10, 12, 14); with regard to rLm5Ag (VapB47), 2 out of 3 clones selected tested positive (lanes 13, 15, and 16). Amplification with primers 319 and 327 across the antigen expression cassette resulted in various sizes of the PCR product, as shown in sFIG. 2b. Consistent with the PCR result using primers NC16 and PL95, a ˜2154 bp DNA fragment was amplified with primers 319 and 327 from 2 out of 3 selected clones of rLm5Ag (VapB47) (sFIG. 2b, lanes 13, 17 and 18).
We tested the genetic stability of the Mtb 5Ag expression cassette integrated into the rLm chromosome by culturing the vaccine candidates in the presence and absence of erythromycin, a marker used for selection of the rLm constructs. We observed that the Mtb 5Ag antigen expression cassettes were stable after passage in vitro in the absence of antibiotic selection, except for one clone of the rLm5Ag (VapB47), expressing Rv3407 as the fifth protein (sFIG. 3). Rv3407 encodes an antitoxin virulence associated protein B47 that is part of the toxin-antitoxin operon with Rv3408. Expressing Rv3407 independently of Rv3408 may have resulted in instability of this rLm fusion protein. However, when we grew the vaccine candidates carrying the antigen expression cassette including Rv3407 in the presence of antibiotic selection, these vaccines expressed the Mtb fusion proteins abundantly with two major bands of ˜74 (secreted form) and ˜78 kDa (non-secreted form) (
Growth Kinetics of rLm5Ag Vaccine Candidates in Broth Medium and in Infected Murine and Human Macrophages
We examined the growth kinetics in BHI broth of the 11 new rLm5Ag vaccine candidates. As shown in sFIG. 4a-c, all of the rLm5Ag vaccine candidates, except rLm5Ag (VapB47) (this clone was subsequently discarded and replaced by a new clone shown in
To examine the growth kinetics of rLm5Ag vaccine candidates in macrophage-like cells, we infected monolayers of murine J774A.1 cells or monolayers of human THP-1 cells differentiated by PMA with the Lm vector or with the rLm vaccine candidates at a MOI of 10. In general, rLm5Ag vaccine candidates expressing fusion proteins comprising Mtb 4Ag (23.5-10.4-ESAT6-CFP10) ligated with a 5th antigen grew similarly to the Lm vector in both murine (sFIG. 4d-f) and human (sFIG. 4g-i) macrophage-like cells.
Protective Immunity of rLm5Ag Vaccine Candidates Against Aerosol Challenge with Virulent Mtb Erdman Strain in BALB/c Mice.
To screen for optimal Mtb antigens, we examined the protective efficacy against aerosolized Mtb of priming mice i.d. with BCG and boosting them i.m. (this experiment was initiated prior to our obtaining results of the experiment described above that determined that the optimal route was s.q.) with the 11 new rLm5Ag vaccine candidates each expressing the Mtb 4Ag fusion protein (ActAN-Mpt64-10.4-ESAT6-CFP10) ligated at its C-terminus with a new 5th antigen. We immunized BALB/c mice, 8 per group, i.d. with PBS (Sham) or i.d. with 5×105 CFU of BCG at Week 0 and boosted them i.m. once at Week 18 with 2×106 CFU each of the Lm Vector, 11 rLm5Ag candidates, or with one of two rLm vaccine combinations expressing the same 5 Mtb antigens as rLm5Ag (30). At Week 22, we challenged the mice with aerosolized Mtb (average of 19 CFU delivered to the lungs of each animal). At Week 32 (10 weeks post challenge), we euthanized the mice and assayed bacillus burdens in their lungs and spleens (
Overall, of the single vaccines expressing 5 Mtb antigens, we considered rLm5Ag (30) as the most efficacious, as it had the lowest CFU count in the lung, the major site of TB pathology, and the second lowest CFU count in the spleen. Hence, this vaccine was evaluated further for immunogenicity.
Boosting BCG-Primed Mice with rLm5Ag (30) Induces Disparate Antigen-Specific CD4+ and CD8+Immune Responses in C57BL/6 and BALB/c Mice.
To determine the immunogenicity of rLm5Ag (30) as a booster vaccine in BCG-primed mice, we primed C57BL/6 and BALB/c mice, 4/group, i.d. with 5×105 CFU of BCG at Week 0 and boosted them s.q. twice at Weeks 14 and 18 with Lm Vector or rLm5Ag (30). At 6 days post the last immunization, we euthanized the mice, prepared single cell suspensions of spleen and lung cells, seeded the cells in 96-well cell-culture plates, stimulated the cells with various Mtb antigens, and assayed T-cell immunity by ICS.
C57BL/6 mice, but not BALB/c mice, primed-boosted with BCG-rLm5Ag (30) produced a lower frequency of CD4+ T cells, greater frequency of CD8+ T cells, and lower CD4+/CD8+ T cell ratio than mice primed-boosted with BCG-LmVector in their lungs after in vitro stimulation without (Medium control) or with Mtb antigens (sFIG. 5). There are no significant differences in the frequencies of CD4+ and CD8+ T cells in the spleens of C57BL/6 and BALB/c mice (sFIG. 6). With respect to CD4+ T cells in the spleens and lungs of C57BL/6 mice, as shown in
With respect to CD8+ T cells in spleens and lungs of C57BL/6 mice (
In a similar experiment performed in BALB/c mice (
Our study shows that boosting BCG primed C57BL/6 and BALB/c mice with a Lm-vectored multi-antigenic Mtb vaccine candidate expressing combinations of M. tuberculosis proteins, especially rLm5Ag (30), expressing a fusion protein of r30/Ag85B, TB10.4/EsxH, ESAT6/EsxA, CFP10/EsxB, and 23.5/Mpt64, enhances the immunoprotection conferred by BCG against aerosol challenge with virulent M. tuberculosis Erdman strain in both mouse strains. Boosting C57BL/6 mice with rLm5Ag (30) significantly enhances the level of CD8+ T cell expression in the spleens and lungs, the frequency of multifunctional splenic CD4+ T cells expressing IFN-γ, TNF-α, and IL-2 in response to r30/Ag85B, PPD, and the 5Ag pool, and the frequency of splenic and lung CD8+ T cells expressing IFN-γ and TNF-α in response to TB10.4/EsxH and/or ESAT6/EsxA antigens. Although boosting BCG-primed BALB/c mice with rLm5Ag (30) also enhances the frequency of some cytokine-secreting lymphocytes, specifically splenic and lung CD8+ T cells expressing IFN-γ or TNF-α in response to TB10.4/EsxH antigen, the response is much more limited than in BCG-primed C57BL/6 mice.
Of the five recombinant Mtb antigens expressed by rLm5Ag (30), all have previously been demonstrated to be immunoprotective individually as well as in combination with other Mtb antigens. r30/Ag85B has been demonstrated to be highly protective when administered as an adjuvanted recombinant protein (22) or when expressed by recombinant BCG (rBCG30) (3, 23) or an Lm vector (15) in guinea pigs and mice. TB10.4 alone or as part of an Ag85B-TB10.4 fusion protein in adjuvant has been shown to induce protection in mice (19) and guinea pigs (38, 39). ESAT6, alone or in combination with Antigen 85B, administered with the adjuvant monophosphoryl lipid A has been shown to induce protective immunity in mice (32, 33). CFP10 delivered via a DNA vaccine induces protection against aerosolized Mtb Erdman in C3H/HeJ mice (31), and a Salmonella vectored vaccine expressing an ESAT6-CFP10 fusion protein protects C57BL/6 mice against aerosolized Mtb H37Rv (40). Finally, the 23.5/Mpt64 protein expressed by a DNA vaccine (24) or surface expressed by recombinant BCG (25) has been found to induce protective immunity in C57BL/6 mice challenged intravenously with H37Rv (24) or by aerosol with Mtb Erdman (25).
Notably, of the five antigens in rLm5Ag (30), three are absent from BCG entirely (ESAT6/EsxA and CFP10/EsxB) or from modern strains of BCG (23.5/Mpt64). Hence, boosting BCG with rLm5Ag (30) not only enhances the level of immunity to immunoprotective proteins present in BCG, but additionally broadens the immune response to encompass antigens present in M. tuberculosis but absent from BCG.
Also of note, the five proteins comprising rLm5Ag (30) are all secreted or extracellularly released proteins. Such extracellular proteins have been demonstrated to be especially important immunoprotective antigens of intracellular pathogens and hypothesized early on to play a central role in vaccines against such pathogens including Legionella pneumophila and Mycobacterium tuberculosis (33, 41, 42).
Our screen of 11 Listeria vectored vaccines expressing 5 Mtb antigens, all comprising a fusion protein of 23.5-TB10.4-Esat6-CFP10 plus one of 11 additional antigens, revealed several vaccine candidates that induced protection better than BCG and almost comparable to rLm5Ag (30). The most potent alternative “fifth” antigens were EsxN, PPE68, EspA and Tb 8.4. Of these, one antigen, PPE68, is absent from BCG. We have subsequently constructed a 9-antigen Listeria vectored vaccine incorporating these additional four Mtb antigens, and in on-going studies, we are evaluating it for protective efficacy in mice, guinea pigs, and non-human primates.
The rLm5Ag (30) vaccine induced significantly enhanced levels of antigen-specific cytokine-secreting CD4+ and CD8+ T cells in BCG-immunized C57BL/6 but not in BCG-immunized BALB/c mice, where such responses were weak and sporadic, reflecting the well-established Th1 bias of C57BL/6 mice vs. the Th2 bias of BALB/c mice. Similarly, BCG immunization alone has been found to induce a greater Th1 type response in C57BL/6 than BALB/c mice (43, 44), although this has not been observed universally (40). In any case, despite the disparate immune responses induced by the rLm5Ag (30) vaccine in BCG-immunized C57BL/6 and BALB/c mice, rLm vaccines expressing these five antigens boosted protection against Mtb aerosol challenge in both mouse strains. This result mirrors a previous observation that differences in the ability of these two mouse strains to generate Th1 helper cells are not reflected by differences in their ability to resist Mtb infection (45).
A potential major advantage of a Listeria-vectored vaccine, particularly with respect to protein/adjuvant vaccines, is enhanced capacity to induce CD8+ T cells. CD8+ T cells are required to resist Mtb infection, as demonstrated by studies in mice employing antibody-depletion or TAP1 knock-out of CD8+ T cells (46-49). Consistent with these observations, adoptive transfer of CD8+ T cells enhances resistance to TB (50). Of note, CD8+ T cells appear to play a more important role in primates than in rodents (51); hence, efficacy studies in rodents may underestimate the efficacy of Listeria-vectored vaccines in non-human primates and humans. In current studies, we are evaluating the efficacy of a multi-antigenic Listeria-vectored vaccine in non-human primates.
Tuberculosis Vaccine Protects C57BL/6 and BALB/C Mice and Guinea Pigs Against Mycobacterium Tuberculosis Challenge
Aspects and embodiments of the invention discussed in this example are also discussed in Jia Q, Masleša-Galić S, Nava S, Horwitz MA. Commun Biol. 2022 Dec. 20; 5 (1): 1388. doi: 10.1038/s42003-022-04345-1 (termed in this example: “Jia et al.”), the contents of which are incorporated herein by reference.
Embodiments of the TB vaccines disclosed herein comprise a live attenuated replicating Listeria monocytogenes (Lm) bacterium expressing immunoprotective Mtb antigens. The Lm vector was chosen in large part because of its ability to induce robust antigen-specific CD4+ and CD8+ T cell responses to expressed recombinant antigens-both types of T cell immunity are central to immunoprotection against Mtb. The wild-type parent of this vector, a fast growing, Gram-positive, facultative intracellular bacterium that occasionally infects humans and can cause food-borne disease outbreaks, shares important features of its intracellular lifestyle with Mtb. Like Mtb, upon entry into host cells, which include mononuclear phagocytes, Lm initially resides in a phagosome, a site favoring antigen presentation via class II MHC molecules and the induction of antigen-specific CD4+ T cells. Subsequently, also in common with Mtb, Lm escapes the phagosome and multiplies in the host cytosol, a site favoring antigen presentation via class I MHC molecules and the induction of antigen-specific CD8+ T cells. As a result of its capacity to induce long-lived cell-mediated immune responses, genetically attenuated Lm has been developed as a vaccine vector for cancer and infectious diseases24.
The specific Lm vector that we employ, Lm ΔactA ΔinlB prfA* (Lm ΔactA ΔinlB ΔuvrAB prfA*)22,25 has been attenuated from wild-type Lm and rendered more effective as a vaccine vector via several genetic manipulations25. First, actA, a gene encoding the cell surface transmembrane protein ActA, which promotes intracellular motility via actin polymerization, has been deleted. ActA deletional mutants are able to grow within the cytosol of infected cells, but are unable to induce cell-to-cell spread, resulting in ˜1000-fold attenuation in virulence in mouse models24. Second, a deletion of inlB, encoding internalin B, a virulence factor that promotes invasion of various mammalian cells including epithelial cells, endothelial cells and hepatocytes, inhibits Lm uptake into non-phagocytic cells, such as hepatocytes, but not into phagocytic cells, including antigen-presenting cells; hence, in the double deletional ΔactA ΔinlB mutant, off-target toxicity is minimized but not antigen presentation of secreted recombinant antigens in antigen presenting cells26. Third, a point mutation (G155S) in the master virulence regulator PrfA that renders it constitutively active, promotes Lm escape into the host cell cytosol, and as a result of upregulated expression of PrfA and PrfA-dependent genes, shows enhanced expression of downstream recombinant proteins25,27,28.
Using Lm ΔactA ΔinlB prfA* as a vaccine vector, we have developed several recombinant Lm-vectored Mtb vaccines (rLm) including rLm3022, rLmMtb5Ag (rLm5Ag)23, and in this study, rLmMtb9Ag (rLm9Ag). The rLm30 vaccine expresses a single Mtb antigen—the 30-kDa major secretory protein or Antigen 85B (r30 or Ag85B, gene Rv1886); rLm5Ag expresses a fusion protein of 5 Mtb antigens—Mpt64/23.5 (Rv1980c), EsxH/TB10.4 (Rv0288), EsxA/ESAT6 (Rv3875), EsxB/CFP10 (Rv3874) and r30; and rLm9Ag expresses, in addition to the 5 antigens expressed by rLm5Ag, a fusion protein comprising 4 additional Mtb antigens-EspA (Rv3616c), EsxN (Rv1793), PPE68 (Rv3873) and TB8.4 (Rv1174c). These 4 additional proteins were selected on the basis of their capacity, when administered as part of a rLm booster vaccine, to enhance protective immunity against Mtb aerosol challenge in BCG-immunized mice23. In previous studies of rLm30 and rLm5Ag, we have shown that i) immunization of mice with BCG has no significant effect on local replication or systemic dissemination, growth, and clearance of rLm30 administered intradermally 12 or 15 weeks later22,23; ii) boosting BCG-primed mice with rLm30 and rLm5Ag enhances Mtb antigen-specific CD4+ and CD8+ T cell-mediated immune responses22,23; and iii) boosting BCG-primed C57BL/6 and BALB/c mice with rLm30 and rLm5Ag enhances protective immunity against aerosolized Mtb22,23.
Herein, we investigate the immunogenicity and efficacy of rLm5Ag and rLm9Ag as standalone vaccines in three animal models of pulmonary TB-inbred C57BL/6 and BALB/c mice and outbred Hartley guinea pigs. We test them as standalone vaccines—not because we envision them as replacement vaccines for BCG but because, as noted above, most of the people in the world in need of a TB booster vaccine were vaccinated with BCG in infancy; hence, their BCG-induced immunity will have largely waned by the time they would receive a TB booster vaccine many years and often decades later. As testing the potency of a heterologous booster vaccine administered decades after a prime vaccine is not feasible in small animal models, we elected instead to test the vaccines as standalone vaccines so as to mimic the situation in which BCG-induced immunity has completely waned. In so doing, we examined two different strains of mice, C57BL/6 and BALB/c, because these mice display different innate and acquired immune responses to infection, including mycobacterial infection with BCG 29,30 We additionally examined guinea pigs because these animals develop disease more akin to that of humans than do most strains of mice; e.g., they are highly susceptible to clinical disease after low dose infection with M. tuberculosis; they show strong cutaneous delayed-type hypersensitivity to tuberculin; and they display Langhans giant cells in lung lesions and develop caseating granulomas31.
In C57BL/6 and BALB/c mice, we show that homologous priming-boosting with rLm5Ag and rLm9Ag vaccines induces antigen-specific CD4+ and CD8+ T cell immune responses and protective immunity against aerosol challenge with virulent Mtb. In guinea pigs, we show that homologous priming-boosting with rLm9Ag induces Mtb antigen-specific lymphocyte proliferation and elevated frequencies of CD8+ T cells in the lungs and/or spleens, and that immunization with rLm5Ag or rLm9Ag induces significant protective immunity against Mtb aerosol challenge.
Construction and Verification of rLm9Ag Expressing Fusion Proteins of Mtb5Ag and Mtb5AgII from the comK and tRNAarg Loci, Respectively
To construct rLm5Ag (expressing Mtb ActAN-Mpt64-EsxH-EsxA-EsxB-r30)23 (Supplementary Table 1 of Jia et al.) and rLm5AgII (expressing ActA-Mpt64-EsxN-PPE68-EspA-TB8.4) (Supplementary Table 1 of Jia et al.), we integrated the pPL2e-ActAN-Mtb5Ag and pPL2e-ActAN-Mtb5AgII into the tRNAarg locus of the rLm chromosome (
To analyze expression of the heterologous Mtb fusion proteins by rLm growing inside of macrophages, we infected murine macrophage-like cells (J774.A1) with LmVector or rLm expressing various Mtb fusion proteins at a Multiplicity of Infection (MOI) of 10. At 5.5 hours post infection, we harvested the infected cells and analyzed the lysates for protein expression by SDS-PAGE and Western blotting using a polyclonal antibody to a N-terminal peptide comprising 18 amino acids (A30-K47) of ActA (AK18) (courtesy of Justin Skoble and Peter Lauer), which detected the predicted 94-kDa (5Ag) protein band (and non-specific protein bands) from J774A.1 cells infected with rLm5Ag, rLm5AgII, or rLm9Ag clones #1 and 3 (
Genetic Stability and Growth Kinetics of rLm5Ag and rLm9Ag Vaccines
To evaluate the antigen expression cassette stability of rLm grown in broth culture and in infected macrophage-like cells, we examined the growth of rLm vaccine candidates in BHI broth supplemented with various antibiotics and in infected monolayers of the J774A.1 cells with stationary grown rLm vaccines and assayed bacterial replication. As shown in Supplementary
To examine the growth kinetics of rLm vaccine candidates in murine macrophages, we infected monolayers of J774A.1 cells with LmVector or rLm vaccines at an MOI of 10, as described in the legend to Supplementary
rLm5Ag Induces Antigen-Specific T-Cell Mediated Immune Responses in Mice
We examined the capacity of rLm5Ag to induce antigen-specific T cells and cytokine-expressing CD4+ and CD8+ T cells in the lungs and spleens of C57BL/6 mice and BALB/c mice (
With respect to antigen-specific cytokine-expressing T cells, the rLm5Ag-immunized C57BL/6 mice produced significantly greater frequencies of cytokine-producing CD4+ T cells in their lungs and spleens expressing IFN-γ, TNF-α, and/or IL2 and polyfunctional CD4+ T cells expressing two or more cytokines among IFN-γ, TNF-α, and IL-2 in response to in vitro 6 h stimulation with Ag85B (
BALB/c mice immunized three times with rLm5Ag produced substantially greater (˜2-5-fold) frequencies of live lung CD3+ T cells than LmVector-immunized mice after 22h incubation whether incubated with or without antigen (P<0.05-P<0.001) (Supplementary
With respect to antigen-specific cytokine-expressing CD4+ T cells, in the spleens, BALB/c mice immunized with rLm5Ag produced significantly greater amounts of IFN-γ, TNF-α, and/or IL-17A expressing CD4+ T cells in response to 6h in vitro stimulation with Mpt64, EsxH, EsxB, Ag85B, PPD and GI-H37RV than mice immunized with LmVector; the only antigens not inducing a significantly greater response was EsxA and, as expected, PMA (Supplementary
Thus, homologous priming-boosting BALB/c mice with rLm5Ag induces Mtb antigen-specific cytokine-expressing CD4+ and CD8+ T cells, where the CD4+ T cells show specificity to Mpt64, EsxH, EsxB, and Ag85B—all but EsxA—and express predominantly IFN-γ and TNF-α, and CD8+ T cells show specificity to EsxH and EsxA, but express predominantly IL-17A and IFN-γ.
Comparing C57BL/6 and BALB/c mice, immunization with rLm5Ag substantially increases the frequencies of live CD3+ T cells in the lungs of BALB/c mice (Supplementary
rLm5Ag and rLm9Ag Induce Protective Immunity Against Aerosol Challenge with Virulent Mtb Erdman Strain in BALB/c and C57BL/6 Mice
In preliminary studies, we evaluated the protective efficacy in C57BL/6 and BALB/c mice of a combination of two rLm vaccines expressing 5 antigens-r30/Ag85B, 23.5/Mpt64, TB10.4/EsxH, ESAT6/EsxA, and CFP10/EsxB (combination of rLm30+rLm4Ag, designated as rLm5Ag*). First, we explored i.d. and intranasal (i.n.) administration of the composite vaccine rLm5Ag* in C57BL/6 mice. In these experiments, we used BCG as a positive control against which to compare the efficacy of the Lm vaccines, as BCG consistently provides strong efficacy in animal models of TB. We immunized groups of C57BL/6 mice, 8 per group, with PBS or BCG i.d. or i.n. at Week 0, or the rLm5Ag* i.d. or i.n. three times at Weeks 0, 7, and 10, challenged all the mice with aerosolized Mtb (average of 24 CFU of the Mtb Erdman strain delivered to the lungs of each mouse, as assayed on Day 1 post-challenge) at Week 13 and euthanized the mice at Week 23 (
Next, we immunized groups of 8 BALB/c mice with PBS (Sham) or BCG (positive control) i.d. at Week 0, or i.m. with the rLm5Ag* three times at Weeks 10, 14, and 18 or twice at Weeks 14 and 18, then challenged the mice at Week 22 with aerosolized Mtb (average of 30 CFU of the Mtb Erdman strain delivered to the lungs of each mouse, as assayed on Day 1 post-challenge), and euthanized the mice 10 weeks later (Week 32) to assay bacillus burden in their lungs and spleens (
Subsequently, we performed definitive studies comparing the protective efficacy of rLm30, rLm5Ag* (combination of rLm30+rLm4Ag), rLm5Ag (single vaccine expressing the same 5 antigens as rLm5Ag* from the tRNAarg locus), and rLm9Ag (clones #1 and #3) as a standalone vaccine in both C57BL/6 and BALB/c mice immunized three times s.q., a route that was found to be both practical and efficacious against Mtb aerosol challenge in heterologous prime-boost studies involving a BCG prime and an rLm boost. We immunized groups of 8 C57BL/6 and BALB/c mice s.q. three times at Weeks 0, 3, and 6 with rLm30, rLm5Ag*, rLm5Ag, or two individual clones of rLm9Ag (rLm9Ag #1 and rLm9Ag #3); unimmunized (UI) mice or mice immunized i.d. with BCG, or three times s.q. with LmVector served as controls. Four weeks after the last immunization, mice were challenged with aerosolized Mtb (average of 10 CFU of Mtb Erdman strain delivered to the lungs of each mouse, as assayed at Day 1 post challenge). At Week 20, the mice were euthanized and organ bacillus burdens assayed (
As shown in
As shown in
rLm9Ag Induces Antigen Specific T-Cell Proliferation in Guinea Pigs
To evaluate the capacity of the rLm9Ag vaccine to induce T-cell mediated immune responses in guinea pigs, we immunized guinea pigs with rLm9Ag or LmVector three times at Weeks 0, 3, and 6 and 6 days later evaluated responses of spleen and lung lymphocytes, CD4+ T cells, and CD8+ T cells to stimulation with Mtb peptide antigens. As shown in
Thus, rLm9Ag induces significantly increased lung and/or spleen cells in response to 8 of its 9 recombinant Mtb antigens, significantly increased proliferating splenic CD4+ T cells in response to Ag85B and EsxB, and significantly increased proliferating CD8+ T cells in response to 5 of its 9 Mtb antigens.
rLm5Ag and rLm9Ag Induce Protective Immunity Against Aerosol Challenge with Virulent Mtb Erdman Strain in Outbred Guinea Pigs
Finally, we evaluated the capacity of the rLm5Ag and rLm9Ag vaccines to induce protective immunity against Mtb aerosol challenge in the outbred guinea pig model. As shown in
Our studies show that homologous priming-boosting of inbred C57BL/6 and BALB/c mice and outbred guinea pigs with rLm5Ag (expressing a fusion protein of Mtb antigens Mpt64-EsxH-EsxA-EsxB-Ag85B) and rLm9Ag (additionally expressing Mtb antigens Mpt64-EsxN-PPE68-EspA-TB8.4) induces antigen-specific CD4+ and CD8+ T-cell mediated immunity and immunoprotection against aerosol challenge with virulent Mtb Erdman in all three animal models.
Both CD4+ and CD8+ T cells are required to control primary TB infection. CD4+ T cells help CD8+ T cells maintain effector function and prevent exhaustion, and the synergy between CD4+ and CD8+ T cells promotes the survival of mice infected with Mtb 32. Our results show that rLm5Ag induces both CD4+ and CD8+ T cell-mediated immune responses in both C57BL/6 and BALB/c mice. In both mouse models, immunizing with rLm5Ag or rLm9Ag induces elevated frequencies of lung and/or splenic CD8+ T cells, consistent with Lm's reputation as a potent inducer of CD8+ T cells 24. Similarly, in guinea pigs, rLm9Ag induces proliferating antigen-specific CD4+ and CD8+ T cells. CD8+ T cells appear to play a more important role in protection against Mtb in primates than in rodents 33. Hence, studies in rodents may underestimate the efficacy of an rLm vaccine in non-human primates and humans.
In C57BL/6 mice, rLm5Ag induced significantly elevated frequencies of antigen-specific polyfunctional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, and sometimes IL-2 (the CD4+ T cells in response to stimulation with Ag85B, and the CD8+ T cells in response to stimulation with Mpt64 and EsxH); the frequency of IL-17A expressing cells was not significantly elevated. In BALB/c mice, rLm5Ag induced significantly elevated frequencies of CD4+ T cells expressing IFN-γ and TNF-α (in response to stimulation with Mpt64, EsxH, EsxB, and Ag85B), and additionally significantly elevated frequencies of CD4+ and CD8+ T cells secreting IL17A (in response to EsxH and EsxA). Hence, these two mouse strains displayed a somewhat different CD4+ and CD8+ T cell cytokine expression profile after rLm5Ag immunization.
Among the five antigens common to both rLm5Ag and rLm9Ag, all five induced T cell responses in at least one animal model, and four induced T cell responses in multiple animal models. Antigen 85B was an especially dominant antigen, inducing T cell responses in all three animal models-cytokine-expressing CD4+ T cells in both C57BL/6 and BALB/c mice and proliferating CD4+ and CD8+ T cells in guinea pigs. EsxB also stood out, inducing cytokine-expressing CD4+ T cells in BALB/c mice and proliferating CD4+ and CD8+ T cells in guinea pigs. EsxH induced cytokine-expressing CD4+ T cells in both C57BL/6 and BALB/c mice. Finally, Mpt64 induced cytokine-expressing CD4+ T cells in BALB/c mice and CD8+ T cells in C57BL/6 mice. The rLm9 Ag vaccine, containing 4 additional Mtb antigens, was tested for immunogenicity only in guinea pigs. Three of these four new antigens-EsxN, PPE68, and TB8.4-induced proliferating CD8+ T cells in guinea pigs.
An Lm-vectored vaccine has major advantages as a TB vaccine including i) Lm multiplies rapidly intracellularly and secrets foreign antigens into the host cell cytosol, as noted above, and then is rapidly cleared-7 to 10 days post immunization22; ii) Lm-vectored vaccines with ΔactA ΔinlB deletions have an established safety profile in humans; the vaccines were well tolerated in a Phase I study34; iii) pre-existing immunity to Lm35,36 and to BCG does not deleteriously affect immunization with Lm-vectored vaccines 22, in contrast to some virus- and mycobacterium-vectored vaccines; iv) Lm-vectored vaccines have enhanced capacity to induce both CD4+ and CD8+ T cell-mediated immune responses, and is an especially potent inducer of CD8+ T cells, as noted above; and v) Lm-vectored vaccines can be cheaply manufactured in broth medium at large scale without the need for extensive purification as with protein/adjuvant vaccines or virus-vectored vaccines grown in mammalian cells.
Although in this study we tested the Listeria vectored vaccines as standalone vaccines, we envision an rLm vaccine not as replacement vaccine for BCG but as a heterologous booster vaccine for people previously vaccinated with BCG, or in the future with an improved mycobacterial vaccine that eventually replaces BCG. Greater than 5 billion people on earth who have been vaccinated with BCG live in TB endemic areas, and hence might benefit from a heterologous TB booster vaccine. As most of these people, including adolescents and adults, would have been vaccinated with BCG in infancy, their BCG-induced immunity is likely to have largely waned by the time they were to receive such a booster vaccine many years and decades later. For this reason, as noted earlier, we considered it important to test the efficacy of our Listeria vectored vaccines as standalone vaccines. As standalone vaccines, we did not expect the rLm vaccines to be superior to BCG, which shares thousands of antigens with M. tuberculosis. However, that our studies demonstrate that the rLm vaccines are in many cases comparable in potency to BCG is noteworthy and, in our view, strongly supports the continued development of these rLm vaccines as TB booster vaccines. In conclusion, in this study, the protective efficacy of multi-antigenic rLm TB vaccines—including rLm5Ag and rLm9Ag—was demonstrated in three rigorous animal models of pulmonary TB. This follows upon the demonstration in previous studies of the protective efficacy of a single-antigen rLm vaccine (rLm30) and multi-antigenic rLm vaccines—including rLm5Ag—as booster vaccines to enhance the level of immune protection afforded by BCG immunization. Hence, an rLm vaccine expressing multiple Mtb immunoprotective antigens has substantial promise as a new vaccine to combat the TB pandemic.
All animals were maintained in a specific-pathogen-free animal facility and used according to protocols approved by the UCLA Institutional Animal Care and Use committee.
Murine (J774A.1, ATCC TIB-67) monocytes were cultured as we described previously22. M. bovis BCG Tice and Mtb Erdman (ATCC 35801) strains were acquired and stocks prepared as we described previously22. The Listeria vector, Lm ΔactA ΔinlB prfA*22,25, derived from Listeria monocytogenes 10403S strain (phage-cured, DP-L4056)37, and recombinant Lm-vectored vaccines were grown to mid-log phase in Yeast Extract broth medium, collected by centrifugation, resuspended in PBS, titrated, and stored in 20% glycerol at −80° C. until use. Six to eight-week-old female C57BL/6 and BALB/c mice were purchased from Envigo (Indianapolis, IN) or Jackson Laboratory (Bar Harbor, Maine, USA) and three-week-old outbred male Hartley strain guinea pigs were purchased from Charles River Laboratories (Wilmington, MA, USA). The following Mtb protein reagents were obtained through BEI Resources, NIAID, NIH: Ag85B (Gene Rv1886c), Purified Native Protein from Strain H37Rv, NR-14857; ESAT-6, Recombinant Protein Reference Standard, NR-49424; CFP-10, Recombinant Protein Reference Standard, NR-49425; Mpt64, Recombinant Protein Reference Standard, NR-44102; and GI-H37RV, Mtb, Strain H37Rv, Gamma-Irradiated Whole Cells, NR14819. The Mtb protein EsxH/TB10.4 (gene Rv0288) was obtained from Aeras (formerly Rockville, Maryland, United States). Rabbit polyclonal antibody to ActAN (AK18, lot D4698) was obtained courtesy of Justin Skoble and Pete Lauer; rabbit polyclonal antibody to TB10.4 was obtained from Aeras (formerly Rockville, Maryland, United States); monoclonal antibody to Lm P60 (P6007, Lot AG-20A-0022-C100) was purchased from AdipoGen (San Diego, United States); and monoclonal antibody to β-actin (A5441) was purchased from Sigma (St. Louis, United States).
We constructed Lm-vectored multi-antigenic rLm vaccine candidates using the Lm ΔactA ΔinlB prfA* vector25 and two Lm site-specific phage integration vectors, pPL1 and pPL2, through conjugation process, as previously described by us and others22,23,37. The pPL1 conjugation vector (kindly provided by Peter Lauer) utilizes the listeriophage U153 integrase and attachment site for insertion at the comK locus of the rLm chromosome and carries a Gram-positive chloramphenicol acetyltransferase gene; the pPL2e-derived conjugation vector (pBHE666 containing actA promoter and the N-terminal 100 amino acids of ActAN, kindly provided by Justin Skoble) modified from pPL2, utilizes the listeriophage PSA integrase and attachment site for insertion in the 3′ end of the tRNAarg gene of the rLm chromosome and carries an erythromycin resistance gene37. We cloned the genes encoding Mtb proteins, optimized for expression of Mtb proteins in Listeria monocytogenes and purchased from DNA2.0 (currently https://www.atum.bio/) (Newark, CA), into pPL1 and pBHE666 by the restriction enzyme method and in some cases by the Electra Vector System (https://www.atum.bio/) to generate the following plasmids: pPL2e-ActAN-Mtb4Ag (for integration of ActAN-Mtb4Ag expression cassette at the tRNAarg locus to construct rLm4Ag), pPL2e-ActAN-Mtb5Ag (for integration of ActAN-Mtb5Ag expression cassette at the tRNAarg locus to construct rLm5Ag), pPL1-ActAN-Mtb5Ag (for integration of ActAN-Mtb5Ag expression cassette at the comK locus to construct rLm9Ag), and pPL2e-ActAN-Mtb5AgII (for integration of Mtb5AgII expression cassette at the tRNAarg locus to construct rLm9Ag). We have deposited the sequences of these plasmids to Genbank (https://www.ncbi.nlm.nih.gov/genbank/). All molecular plasmid constructs were confirmed by restriction enzyme digestion and nucleotide sequencing. Candidate vaccines rLm30 (expressing ActAN-r30/Ag85B) (Supplementary Table 1 of Jia et al.)22, rLm4Ag (expressing the fusion protein ActAN-Mpt64-EsxH-EsxA-EsxB) (Supplementary Table 1 of Jia et al.), and rLm5Ag (expressing the fusion protein ActAN-Mpt64-EsxH-EsxA-EsxB-r30) (Supplementary Table 1 of Jia et al.) were constructed previously23 [where rLm5Ag is referred to as rLm5Ag (30)]; the Mtb protein expression cassettes in these vaccines were cloned into the pPL2e-derived vector and integrated at the tRNAarg locus of the rLm chromosome. The rLm5AgII vaccine candidate (Supplementary Table 1 of Jia et al.), expressing the Mtb fusion protein of ActAN-Mpt64-EsxN-PPE68-EspA-TB8.4 from the pPL2e vector integrated at the tRNAarg locus as well, was constructed similarly as described previously22,23. The rLm9Ag vaccine candidate (Supplementary Table 1 of Jia et al.) was constructed by integrating the pPL1-ActAN-Mtb5Ag (expressing ActAN-Mpt64-EsxH-EsxA-EsxB-r30) at the comK locus followed by integrating the pPL2e-ActAN-5AgII (expressing ActAN-Mpt64-EsxN-PPE68-EspA-TB8.4) at the tRNAarg locus. The resultant rLm9Ag carries a total of 9 Mtb antigens with Mpt64 being a common antigen located at the N-terminus of both 5Ag and 5AgII fusion proteins. The pPL1-ActAN-Mtb5Ag conjugation vector carries a codon-optimized antigen expression cassette for the fusion protein of Mpt64 (Δ1V-23A)-RP-EsxH-GGSG (SEQ ID NO: 3)-EsxA-GSSGGSSG (SEQ ID NO: 4)-EsxB-GSSGGSSG (SEQ ID NO: 4)-Ag85B (Δ2Q-43A) (abbreviated as Mpt64-EsxH-EsxA-EsxB-r30), in which RP is a dipeptide encoded by an EagI restriction enzyme site, and GSSG (SEQ ID NO: 6) and GSSGGSSG (SEQ ID NO: 4) are flexible fusion protein linkers. The pPL2e-ActAN-Mtb5AgII conjugation vector carries a codon-optimized antigen expression cassette for the fusion protein of Mpt64 (Δ1V-23A)-EsxN-GSSG (SEQ ID NO:)-PPE68-GSSGGSSG (SEQ ID NO: 4)-EspA (Δ111F-193L)-GSSGGSSG (SEQ ID NO: 4)-TB8.4 (Δ2R-28A) (abbreviated as Mpt64-EsxN-PPE68-EspA-TB8.4).
The growth kinetics of rLm in broth and in murine macrophage-like cells were evaluated as described previously by us22,23. To assay the stability of rLm vaccines grown on agar plates, we passaged the Lm vector, rLm5Ag and rLm9Ag daily for 10 consecutive days on BHI agar plates supplemented with streptomycin, and at day 5 and day 10, we transferred 20-25 colonies of each vaccine onto BHI plates supplemented with streptomycin plus erythromycin (marker for antigen expression cassette integrated at the tRNAarg locus) or streptomycin plus chloramphenicol (marker for antigen expression cassette integrated at the comK locus). To assay vaccine stability in macrophage-like cells, we infected monolayers of J774A.1 cells with rLm vaccines in the absence of antibiotic selection for 5.5 hours; lysed the cells; serially diluted the lysates and plated them on BHI agar supplemented with various antibiotics; cultured the plates at 37° C. for 2 days; and counted the colonies.
To determine the immunogenicity of rLm5Ag as a standalone vaccine, we immunized C57BL/6 and BALB/c mice, 4/group, s.q. at Weeks 0, 4, and 8 with 2×106 Colony Forming Units (CFU) of the Lm vector (LmVector) or rLm5Ag (expressing the fusion protein of ActAN-Mpt64-EsxH-EsxA-EsxB-r30 from the tRNAarg locus); euthanized the mice at 6 days post the last immunization; prepared single cell suspensions of spleen and lung cells; stimulated the single cell suspensions with various Mtb antigens for 6 h or 22 h; and assayed T-cell immunity by intracellular cytokine staining (ICS) using an eight-color flow cytometry panel to analyze simultaneously multiple cytokines at the single-cell level as described by us previously22,23.
Immunization and Aerosol Challenge of Mice with Virulent Mtb Erdman Strain.
Groups of BALB/c or C57BL/6 mice, 8/group, were vaccinated intradermally (i.d.), intramuscularly (i.m.), intranasally (i.n.), or s.q. two or three times, 3 or 4 weeks apart, with 106 CFU of LmVector or of multi-antigenic rLm vaccines; challenged 3 or 4 weeks later by exposure to an aerosol generated by a nebulizer from a 10-ml single-cell suspension of Mtb Erdman strain (2.4×104 CFU/ml) for 30 min followed by settling for 5 min; euthanized at 10 weeks post challenge; and spleens and right lungs removed and assayed for bacillus burden as described by us previously22. Control mice were sham vaccinated i.d. with PBS or immunized i.d. or i.n. with 1×106 CFU BCG at Week 0.
Immunization of Guinea Pigs and Lymphocyte Proliferation Assay of their Spleen and Lung Cells
Guinea pigs (male Hartley), 4/group, were immunized i.d. at Weeks 0, 3, and 6 with 106 CFU of the LmVector or rLm9Ag, bled and euthanized 6 days post the last immunization. Spleens and lungs were removed and single cell suspensions of spleen and lung cells prepared and stimulated with or without Mtb antigens. Lymphocyte proliferation using Flow cytometry analysis was assayed as described below.
Briefly, single cell suspensions of 1×107 spleen and lung cells were stained with 1 μM Cell Tracer Violet (CTV, ThermoFisher, labeling cells to trace multiple generations using dye dilution by flow cytometry) for 10 min at 37° C. and washed with Phosphate buffer saline (PBS) supplemented with 5% fetal bovine serum. CTV treated cells were resuspended in T cell medium 22, adjusted to 5×107 cells/ml, seeded in 96-well round-bottom plates (NUNC) (5×106 cells per 0.1 ml per well) and incubated with or without 15-mer peptide pools of each of the 9 Mtb antigens (Ag85B, EspA, EsxA, EsxB, EsxH, EsxN, Mpt64, PPE68, TB8.4) (1 μg/ml per peptide) (PepMix, JPT Peptide Technologies, Berlin, Germany) or PPD (5 μg/ml). Cells incubated with T-cell medium alone served as a negative control and cells incubated with ConA (5 μg/ml) served as a positive control. After 4-days incubation at 37° C. in a CO2 incubator, cells were collected, washed with PBS, and stained with LIVE/DEAD Fixable Near-IR Dead Cell (LD-NIR) (ThermoFisher), followed by staining with cell surface markers of PanT-APC (BioRad), CD4-PE (BioRad), and CD8-FITC (BioRad). Lymphocyte proliferation was analyzed as loss of CTV staining (CTVLow) using an AttuneNxt flow cytometer (ThermoFisher). Data were analyzed using FlowJo software. Initial gating of the events included lymphocytes based on forward scatter vs. side scatter pattern, followed by selection for singlet cells, live PanT+ cells, and subsequently CD4+ and CD8+ T cells. Proliferating live CD4+ and CD8+ T cells were identified by loss of CTV staining (CTVLow) on each of the cell populations. The gates for each cell population were determined by using the cells incubated without addition of antigen and verified by cells incubated with addition of ConA.
Immunization and aerosol challenge of guinea pigs with virulent Mtb Erdman strain.
Groups of guinea pigs (Hartley, male), 10/group, were vaccinated i.d. once with 103 CFU BCG (positive control), or s.q. three times at Weeks 0, 3, and 6 with one dose (106) of LmVector (negative control), or 3 escalating doses (105, 106, and 107) of rLm9Ag; challenged at Week 10 by aerosol with Mtb Erdman strain (2.4×104 CFU/ml); euthanized at Week 20; and spleens and right lungs removed and assayed for bacillus burden as described by us previously 22.
Vaccine Stability after Passaging in Guinea Pigs
Guinea pigs were immunized subcutaneously at the back of the neck area with 1×106 rLm9Ag vaccine diluted in 0.1 ml PBS. At 0, 1, 2, 4, and 8 days post immunization, 2 guinea pigs were euthanized at each time point; the skin at the immunization site (˜1 cm2), spleen, lung and liver of each animal were removed and homogenized in phosphate buffered saline (PBS); and the homogenates were serially diluted in PBS and plated onto BHI agar plates supplemented with streptomycin (200 μg/ml). The plates were incubated for 2 days at 37° C. in a CO2 incubator. Bacterial colonies were recovered from plates of the various tissue homogenates at 0, 1, 2, and 4 days, but not at 8 days post immunization. Recovered colonies were randomly selected and inoculated into 1 ml BHI broth plus streptomycin (200 μg/ml) and grown overnight without agitation. The bacteria were then collected from the overnight culture, lysed in SDS buffer, and subjected to SDS-PAGE and western blotting using a rabbit polyclonal antibody to Lm ActA (AK18).
Two-way ANOVA with Sidak's multiple comparisons test was performed to determine significance in comparisons of mean frequencies of cytokine-producing CD4+ and CD8+ T cells (
Note: In certain embodiments of the invention, the 9Ag fusion proteins are expressed from two fusion protein expression cassettes, 5Ag (ActAN-Mpt64-EsxH-EsxA-EsxB-Ag85B) and 5AgII (ActAN-Mpt64-EsxN-PPE68-EspA-TB8.4). The expression cassette for the 5Ag is integrated at the comK locus and the 5AgII is integrated at the tRNAarg locus; 5Ag and 5AgII have a common antigen of Mpt64 as a leader protein. In both 5Ag and 5AgII fusion proteins, the Listeria monocytogenes ActAN (100 aa) is expressed as a N-terminus fusion to the Mtb proteins.
The vaccines are intended as 1) booster vaccines for hosts initially immunized with BCG or another mycobacterial vaccine or 2) standalone vaccines in hosts not previously immunized with BCG or another mycobacterial vaccine.
Note: The vector for the vaccine, Listeria monocytogenes ΔactA ΔinlB prfA*, was originally provided by Aduro BioTech (evidently now merged into Chinook Therapeutics).
M. tuberculosis polynucleotide and polypeptide sequences are well known in the art as a result of the M. tuberculosis genome project. See, e.g. UniProt Designation “P9WIN9” for Mycobacterium tuberculosis Immunogenic protein MPT64 (“23.5/Mpt64”). See, e.g. UniProt Designation “P9WNK3” for Mycobacterium tuberculosis ESAT-6-like protein EsxH (“TB10.4/EsxH”). See, e.g. UniProt Designation “P9WNK7” for Mycobacterium tuberculosis 6 kDa early secretory antigenic target (“ESAT6/EsxA”). See, e.g. UniProt Designation “P9WNK5” for Mycobacterium tuberculosis ESAT-6-like protein EsxB (“CFP10/EsxB”). See, e.g. UniProt Designation “P9WQP1” for Mycobacterium tuberculosis Diacylglycerol acyltransferase/mycolyltransferase Ag85B (“r30/Antigen 85B”). See, e.g. UniProt Designation “P9WNJ3” for Mycobacterium tuberculosis ESAT-6-like protein EsxN (“EsxN”). See, e.g. UniProt Designation “P9WHW9” for Mycobacterium tuberculosis PPE family immunomodulator PPE68 (“PPE68”). See, e.g. UniProt Designation “P9WJE1” for Mycobacterium tuberculosis ESX-1 secretion-associated protein EspA (“EspA”). See, e.g. UniProt Designation “O50430” for Mycobacterium tuberculosis Low molecular weight T-cell antigen TB8.4 (“TB8.4”).
See also: Cole et al. Nature. Nature 393:537-544, 1998. See also U.S. Pat. Nos. 7,622,107; 7,300,660; 7,002,002; 6,924,118; 6,818,223; 6,761,894; 6,752,993; 6,599,510; 6,471,967; 6,054,133; 6,013,660; and 5,108,745; and U.S. patents application Nos. 20110129492; 20100284963; 20100183547; and 20100092518, the contents of which are incorporated by reference. M. tuberculosis Protein and coding sequences can also be found for example by an online search using the terms: genolist.pasteur.fr/TubercuList/.
It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto. The above specification, examples and data provide a complete description of the manufacture and use of the apparatus and method of the invention. Since many embodiments of the invention can be made without departing from the scope of the invention, the invention resides in the claims hereinafter appended and the equivalents thereto.
References, the contents of which are incorporated by reference.
Those skilled in the art will appreciate that the exemplary discussions of M. tuberculosis that are provided herein are in no way intended to limit the scope of the present invention to the treatment of M. tuberculosis. Similarly, the teachings herein are not limited in any way to the treatment of tubercular infections. On the contrary, this invention may be used to advantageously provide safe and effective vaccines and immunotherapeutic agents against the immunogenic determinants of any pathogenic agent expressing extracellular products and thereby inhibit the infectious transmission of those organisms.
This concludes the description of embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.
Those of skill in this art understand that aspects of this technology can be adapted to form a wide variety of embodiments of the invention. All literature and other references are incorporated herein by reference (e.g. U.S. Pat. Nos. 10,010,595, 7,300,660 and 7,002,002, and PCT Publications WO 01/46473 and WO 02/094848). Literature describing methods and materials that relate to embodiments of the invention includes Brockstedt et al., (2004) Proc Natl Acad Sci USA 101 (38): 13832-7. PMID 15365184; Brockstedt et al., (2005) Nat Med 11 (8): 853-60. PMID 16041382; Colditz et al., (1994).” JAMA 271 (9): 698-702. PMID 8309034; Fine, P. E. (1989) Rev Infect Dis 11 Suppl 2: S353-9. PMID 2652252; Harth et al., (1997) Infect Immun 65 (6): 2321-8. PMID 9169770; Horwitz et al., (1995) Proc Natl Acad Sci USA 92 (5): 1530-4. PMID 7878014; Horwitz et al., (2000) Proc Natl Acad Sci USA 97 (25): 13853-8. PMID 11095745; Horwitz et al., (2005) Infect Immun 73 (8): 4676-83. PMID 16040980; Jia et al., (2009) Vaccine 27 (8): 1216-29. PMID 19126421; Lauer et al., (2008) Infect Immun 76 (8): 3742-53. PMID 18541651; Lee et al., (2006) Infect Immun 74 (7): 4002-13. PMID 16790773; McShane et al., (2004) Nat Med 10 (11): 1240-4. PMID 15502839; Santosuosso et al., (2006) Infect Immun 74 (8): 4634-43. PMID 16861651; Vordermeier et al., (2009) Infect Immun 77 (8): 3364-73. PMID 19487476; Williams et al., (2005) Infect Immun 73 (6): 3814-6. PMID 15908420; Xing et al., (2009) PLOS One 4 (6): e5856. PMID 19516906; and Yan et al., (2008) Infect Immun 76 (8): 3439-50. PMID 18474644.
This application claims the benefit under 35 U.S.C. Section 119 (e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 63/312,289, filed on Feb. 21, 2022, and entitled “NOVEL LIVE MULTI-ANTIGENIC RECOMBINANT VACCINE AGAINST TUBERCULOSIS” which application is incorporated by reference herein. This application is related to PCT International Publication No. WO 2011/159814, the contents of which are incorporated by reference.
This invention was made with government support under Grant Number AI135631, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US23/62733 | 2/16/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63312289 | Feb 2022 | US |
