Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

ABSTRACTIn order to identify immunodominant antigens ofMycobacterium tuberculosisthat may be used in the serodiagnosis of active tuberculosis (TB), we designed anM. tuberculosisfusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed inEscherichia coliBL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.

Download Full-text

Differential Live Mycobacterium tuberculosis-, M. bovis BCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00091-09 ◽

2009 ◽

Vol 16 (7) ◽

pp. 991-998 ◽

Cited By ~ 15

Author(s):

Zahra Hasan ◽

Bushra Jamil ◽

Mussarat Ashraf ◽

Muniba Islam ◽

Maqboola Dojki ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture Filtrate ◽

Mononuclear Cells ◽

Interleukin 10 ◽

Peripheral Blood Mononuclear ◽

High Prevalence ◽

Ifn Γ ◽

Mycobacterial Antigens ◽

Induced Immunity ◽

Culture Filtrate Protein

ABSTRACT The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST−; induration, <10 mm) and TST-positive (TST+) donors were studied separately, both TST− and TST+ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST+ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.

Download Full-text

Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6

Infection and Immunity ◽

10.1128/iai.72.4.2170-2176.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2170-2176 ◽

Cited By ~ 62

Author(s):

Caroline Demangel ◽

Priscille Brodin ◽

Paul J. Cockle ◽

Roland Brosch ◽

Laleh Majlessi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture Filtrate ◽

Envelope Protein ◽

Protective Efficacy ◽

Cell Envelope ◽

Genomic Region ◽

Low Molecular Weight ◽

Ifn Γ ◽

Culture Filtrate Protein ◽

Low Molecular Weight Proteins

ABSTRACT The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6.

Download Full-text

Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00482-12 ◽

2012 ◽

Vol 19 (12) ◽

pp. 1907-1915 ◽

Cited By ~ 39

Author(s):

Desta Kassa ◽

Leonie Ran ◽

Wudneh Geberemeskel ◽

Mekashaw Tebeje ◽

Amelewerk Alemu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Pulmonary Tuberculosis ◽

Immune Responses ◽

Expression Profiles ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Active Pulmonary Tuberculosis ◽

Tnf Α ◽

Wide Range ◽

Ifn Γ

ABSTRACTCharacterizing host immune responses to molecular targets ofMycobacterium tuberculosisis essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series ofM. tuberculosisantigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n= 34) was stimulatedin vitrowithM. tuberculosisantigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classicalM. tuberculosisantigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to severalM. tuberculosisantigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenicM. tuberculosisantigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets ofM. tuberculosisantigens may be promising for the development of immunodiagnostics.

Download Full-text

Mycobacterium tuberculosis Culture Filtrate Protein 10-Specific Effector/Memory CD4+and CD8+T Cells in Tubercular Pleural Fluid, with Biased Usage of T Cell Receptor Vβ Chains

Infection and Immunity ◽

10.1128/iai.00014-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3358-3365 ◽

Cited By ~ 12

Author(s):

Dan Qiao ◽

Li Li ◽

Jian Guo ◽

Suihua Lao ◽

Xianlan Zhang ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Pleural Fluid ◽

Culture Filtrate ◽

T Cell Subsets ◽

Effector Memory ◽

Content Type ◽

Culture Filtrate Protein

ABSTRACTT cell-mediated immunity is critical for the control ofMycobacterium tuberculosisinfection. Identifying the precise immune mechanisms that lead to control of initialM. tuberculosisinfection and preventing reactivation of latent infection are crucial for combating tuberculosis. However, a detailed understanding of the role of T cells in the immune response to infection has been hindered. In addition, there are few flow cytometry studies characterizing the Vβ repertoires of T cell receptors (TCRs) at local sites ofM. tuberculosisinfection in adult tuberculosis. In this study, we used culture filtrate protein 10 (CFP-10) fromM. tuberculosisto characterize T cells at local sites of infection. We simultaneously analyzed the correlation of the production of cytokines with TCR Vβ repertoires in CFP-10-specific CD4+and CD8+T cell subsets. For the first time, we demonstrate that CFP-10-specific CD4+or CD8+T cells from tubercular pleural fluid can produce high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and upregulate the expression of CD107a/b on the cell surface. The CFP-10-specific cells were effector/memory cells with a CD45RO+CD62L−CCR7−CD27−expression profile. In addition, we found CFP-10-specific CD4+and CD8+T cells in tubercular pleural fluid, with biased usage of TCR Vβ9, Vβ12, or Vβ7.2. Our findings of CFP-10-specific CD4+and CD8+T cells in tubercular pleural fluid are critical for understanding the mechanisms of the local cellular immune response and developing more effective therapeutic interventions in cases ofM. tuberculosisinfection.

Download Full-text

Vaccine-Elicited 10-Kilodalton Culture Filtrate Protein-Specific CD8+ T Cells Are Sufficient To Mediate Protection against Mycobacterium tuberculosis Infection

Infection and Immunity ◽

10.1128/iai.00024-08 ◽

2008 ◽

Vol 76 (5) ◽

pp. 2249-2255 ◽

Cited By ~ 39

Author(s):

Ying Wu ◽

Joshua S. Woodworth ◽

Daniel S. Shin ◽

Sheldon Morris ◽

Samuel M. Behar

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Culture Filtrate ◽

Protective Immunity ◽

Protective Antigen ◽

T Cell Response ◽

Rapid Expansion ◽

Mycobacterium Tuberculosis Infection ◽

Infected People ◽

Culture Filtrate Protein

ABSTRACT The 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigen of T cells (ESAT-6) are secreted in abundance by Mycobacterium tuberculosis and are frequently recognized by T cells from infected people. The genes encoding these proteins have been deleted from the genome of the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG), and it is hypothesized that these proteins are important targets of protective immunity. Indeed, vaccination with ESAT-6 elicits protective CD4+ T cells in C57BL/6 mice. We have previously shown that M. tuberculosis infection of C3H mice elicits CFP-10-specific CD8+ and CD4+ T cells. Here we demonstrate that immunization with a CFP-10 DNA vaccine stimulates a specific T-cell response only to the H-2Kk-restricted epitope CFP-1032-39. These CFP-1032-39-specific CD8+ cells undergo a rapid expansion and accumulate in the lung following challenge of immunized mice with aerosolized M. tuberculosis. Protective immunity is induced by CFP-10 DNA vaccination as measured by a CFU reduction in the lung and spleen 4 and 8 weeks after challenge with M. tuberculosis. These data demonstrate that CFP-10 is a protective antigen and that CFP-1032-39-specific CD8+ T cells elicited by vaccination are sufficient to mediate protection against tuberculosis.

Download Full-text

Comparative Analysis of B- and T-Cell Epitopes of Mycobacterium leprae and Mycobacterium tuberculosis Culture Filtrate Protein 10

Infection and Immunity ◽

10.1128/iai.72.6.3161-3170.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3161-3170 ◽

Cited By ~ 30

Author(s):

John S. Spencer ◽

Hee Jin Kim ◽

Angela M. Marques ◽

Mercedes Gonzalez-Juarerro ◽

Monica C. B. S. Lima ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

T Cell ◽

Culture Filtrate ◽

Mycobacterium Leprae ◽

Cross Reactivity ◽

Interferon Response ◽

Positive Staining ◽

T Cell Epitopes ◽

Peptide Epitopes ◽

Culture Filtrate Protein

ABSTRACT Culture filtrate protein 10 (CFP-10) from Mycobacterium tuberculosis is a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells from M. tuberculosis-infected mice and humans. The sequence of the Mycobacterium leprae homologue of CFP-10 shows only 40% identity (60% homology) at the protein level with M. tuberculosis CFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinant M. leprae and M. tuberculosis CFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for either M. leprae or M. tuberculosis CFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against either M. leprae or M. tuberculosis CFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections of M. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting that M. leprae CFP-10, like its homologue in M. tuberculosis, is a secreted protein.

Download Full-text

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00219-17 ◽

2017 ◽

Vol 24 (11) ◽

Cited By ~ 4

Author(s):

Ahreum Kim ◽

Yun-Gyoung Hur ◽

Sunwha Gu ◽

Sang-Nae Cho

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Vaccine Development ◽

Protective Efficacy ◽

Interleukin 2 ◽

T Cell Epitopes ◽

Content Type ◽

Complete Form ◽

Ifn Γ

ABSTRACT The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

Download Full-text

Evaluation of a lateral flow assay for rapid and simple detection of IFN-γ for the diagnosis of Mycobacterium tuberculosis infection

10.21203/rs.3.rs-20838/v1 ◽

2020 ◽

Author(s):

Jeong-Ran Kim ◽

Hae Yeong Kang ◽

Su-Bin Seong ◽

Nari Kim ◽

Tae Sun Shim ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Tuberculosis Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Mycobacterium Tuberculosis Infection ◽

Blood Samples ◽

Simple Detection ◽

Laboratory Workers ◽

Ifn Γ

Abstract Background: Interferon-gamma (IFN-γ) release assays (IGRAs) are useful for the diagnosis of Mycobacterium tuberculosis infection. Current IGRAs use either enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay, which require complex procedures and techniques to determine IFN-γ secretion. We aimed to compare the usefulness of the easy-to-use lateral flow assay (LFA) with that of the QuantiFERON-TB Gold In-Tube (QFT-GIT) or QuantiFERON-TB Gold Plus (QFT-plus) ELISAs for detecting IFN-γ, produced by the blood T cells stimulated by tuberculosis (TB) antigen. Methods: Following informed consent, 176 participants, including health care workers such as TB laboratory workers and radiologists, were enrolled for the study from June 2017 to June 2018. Blood samples were collected and tested using QFT-GIT and QFT-plus. The secreted IFN-γ was quantified by LFA, which took approximately 15 min, and ELISA, which took approximately 3 h. Results: A total of 176 blood samples were screened. The positive rates of QFT-GIT and QFT-plus were 34.1% and 37.5%, respectively. Overall agreement between QFT-GIT and QFT-plus was 93.1% ( κ = 0.86). The positive rates of LFA with QFT-GIT tube and QFT-plus tube were 25.6% and 31.3%, respectively, overall agreement of LFA being 90.3% ( κ = 0.78) and 89.2% ( κ = 0.77), respectively, compared to the QFT-GIT and QFT-plus ELISA. Conclusion: The ability of LFA to measure IFN-γ was similar to that of ELISA. The current findings suggested that the new LFA could be more conveniently utilized for diagnosing TB infection.

Download Full-text

Serodiagnostic Potential of Culture Filtrate Antigens of Mycobacterium tuberculosis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.7.4.662-668.2000 ◽

2000 ◽

Vol 7 (4) ◽

pp. 662-668 ◽

Cited By ~ 43

Author(s):

K. M. Samanich ◽

M. A. Keen ◽

V. D. Vissa ◽

J. D. Harder ◽

J. S. Spencer ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture Filtrate ◽

Bacterial Load ◽

Enzyme Linked Immunosorbent Assay ◽

E Coli ◽

Immunodeficiency Virus ◽

Antigen Profile ◽

Smear Negative ◽

Humoral Responses ◽

Hiv Negative

ABSTRACT Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens ofMycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534–1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49–56, 1997), in which all sera are preadsorbed againstEscherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies.

Download Full-text

Enhanced Influenza Virus-Like Particle Vaccines Containing the Extracellular Domain of Matrix Protein 2 and a Toll-Like Receptor Ligand

Clinical and Vaccine Immunology ◽

10.1128/cvi.00153-12 ◽

2012 ◽

Vol 19 (8) ◽

pp. 1119-1125 ◽

Cited By ~ 37

Author(s):

Bao-Zhong Wang ◽

Harvinder S. Gill ◽

Sang-Moo Kang ◽

Li Wang ◽

Ying-Chun Wang ◽

...

Keyword(s):

Influenza Virus ◽

Fusion Protein ◽

Influenza A ◽

Matrix Protein ◽

Protective Efficacy ◽

Extracellular Domain ◽

Toll Like Receptor ◽

Influenza A Viruses ◽

Live Virus ◽

Content Type

ABSTRACTThe extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region ofSalmonella entericaserovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.

Download Full-text