Peptides-Based Vaccine MP3RT Induced Protective Immunity Against Mycobacterium Tuberculosis Infection in a Humanized Mouse Model.

Background: Tuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)'s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines. Methods: In this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine's protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT. Results: Six immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody. Conclusions: Taken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.

Toll like-receptor agonist Pam3Cys modulates the immunogenicity of liposomes containing the tuberculosis vaccine candidate H56.

A major roadblock in the development of novel vaccines is the formulation and delivery of the antigen. Liposomes composed of a dimethyldioctadecylammonium (DDA) backbone and the adjuvant trehalose-6-6-dibehenate (TDB, termed "cationic adjuvant formulation (CAF01)", promote immunogenicity and protective efficacy of vaccines, most notably against infection with Mycobacterium tuberculosis. Specifically, the multicomponent antigen H56 delivered by CAF01 protects against tuberculosis in mice. Here we investigated whether the inclusion of immune-modulatory adjuvants into CAF01 modulates the immunogenicity of H56/CAF01 in vitro and in vivo. Based on our recent findings we selected the active sequence of the mycobacterial 19 kDa lipoprotein, Pam3Cys, which interacts with Toll like receptor 2 to induce an antimicrobial pathway. H56/CAF01-Pam3Cys liposomes were characterized for Pam3Cys incorporation, size, toxicity and activation of primary human macrophages. Macrophages efficiently take up H56/CAF01-Pam3Cys and trigger the release of significantly higher levels of TNF, IL-12 and IL-10 than H56/CAF01 alone. To evaluate the immunogenicity in vivo, we immunized mice with H56/CAF01-Pam3Cys and measured the release of IFN-γ and IL-17A by lymph node cells and spleen cells. While the antigen-specific production of IFN-γ was reduced by inclusion of Pam3Cys into H56/CAF01, the levels of IL-17A remained unchanged. In agreement with this finding, the concentration of the IFN-γ-associated IgG2a antibodies in the serum was lower than in H56/CAF01 immunized animals. These results provide proof of concept that Toll like-receptor agonist can be included into liposomes to modulate immune responses. The discordant results between the in vitro studies with human macrophages and in vivo studies in mice highlight the relevance and complexity of comparing immune responses in different species.

Spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines.

Thermal stability of self-assembled peptide vaccine materials.

The majority of current vaccines depend on a continuous "cold chain" of storage and handling between 2 and 8°C. Vaccines experiencing temperature excursions outside this range can suffer from reduced potency. This thermal sensitivity results in significant losses of vaccine material each year and risks the administration of vaccines with diminished protective ability, issues that are heightened in the developing world. Here, using peptide self-assemblies based on the fibril-forming peptide Q11 and containing the epitopes OVA323-339 from ovalbumin or ESAT651-70 from Mycobacterium tuberculosis, the chemical, conformational, and immunological stability of supramolecular peptide materials were investigated. It was expected that these materials would exhibit advantageous thermal stability owing to their adjuvant-free and fully synthetic construction. Neither chemical nor conformational changes were observed for either peptide when stored at 45°C for 7days. ESAT651-70-Q11 was strongly immunogenic whether it was stored as a dry powder or as aqueous nanofibers, showing undiminished immunogenicity even when stored as long as six months at 45°C. This result was in contrast to ESAT651-70 conjugated to a protein carrier and adjuvanted with alum, which demonstrated marked thermal sensitivity in these conditions. Antibody titers and affinities were undiminished in mice for OVA323-339-Q11 if it was stored as assembled nanofibers, yet some diminishment was observed for material stored as a dry powder. The OVA study was done in a different mouse strain and with a different prime/boost regimen, and so it should not be compared directly with the study for the ESAT epitope. This work indicates that peptide self-assemblies can possess attractive thermal stability properties in the context of vaccine development. STATEMENT OF SIGNIFICANCE: Almost all current vaccines must be maintained within a tight and refrigerated temperature range, usually between 2 and 8°C. This presents significant challenges for their distribution, especially in the developing world. Here we report on the surprisingly robust thermal stability of a self-assembled peptide vaccine. In particular a self-assembled peptide vaccine containing a tuberculosis epitope maintained all of its potency in mice when exposed to an extreme thermal treatment of six months at 45°C. In a different mouse model, we investigated another model epitope and found some storage conditions where potency was diminished. Overall this study illustrates that some self-assembled peptide vaccines can have remarkable thermal stability.

Functional, biochemical and 3D studies of Mycobacterium tuberculosis protein peptides for an effective anti-tuberculosis vaccine.

Tuberculosis (TB) is an air-born, transmissible disease, having an estimated 9.4 million new TB cases worldwide in 2009. Eventual control of this disease by developing a safe and efficient new vaccine able to detain its spread will have an enormous impact on public health policy. Selecting potential antigens to be included in a multi-epitope, minimal subunit-based, chemically-synthesized vaccine containing the minimum sequences needed for blocking mycobacterial interaction with host cells is a complex task due to the multiple mechanisms involved in M. tuberculosis infection and the mycobacterium's immune evasion mechanisms. Our methodology, described here takes into account a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-TB vaccine; it has been based on identifying short mycobacterial protein fragments using synthetic peptides having high affinity interaction with alveolar epithelial cells (A549) and monocyte-derived macrophages (U937) which are able to block the microorganism's entry to target cells in in vitro assays. This manuscript presents a review of the results obtained with some of the MTB H37Rv proteins studied to date, aimed at using these high activity binding peptides (HABPs) as platforms to be included in a minimal subunit-based, multiepitope, chemically-synthesized, antituberculosis vaccine.

Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.

Stabilizing formulations for inhalable powders of an adenovirus 35-vectored tuberculosis (TB) vaccine (AERAS-402).

A powder vaccine intended for aerosol delivery was formulated by spray drying the Ad35-vectored tuberculosis (TB) AERAS-402 vaccine with mannitol-based stabilizers. Thermodynamic properties, water absorption, particle size distribution and morphology of the powders were evaluated. Virus survival during spray drying and storage was determined by medium Tissue Culture Infectious Dose (TCID(50)). A mannitol-based powder (mannitol-cyclodextrin-trehalose-dextran, MCTD) had a narrow size distribution with a median volume diameter around 3.2-3.5microm (suitable for pulmonary vaccination of humans) and good aerosolization characteristics. The spray dry powders generated from mannitol-based formulations were hydrophobic, which benefits virus survival during both production and storage at 4 degrees C, 25 degrees C and 37 degrees C as compared to the hygroscopic formulations (trehalose, sucrose, dextran, PVP, leucine). In conclusion, this study demonstrates that it is possible to produce in a one-step spray drying process a stable dry powder formulation of a TB vaccine suitable for mass vaccination.

Safety and immunogenicity of boosting BCG vaccinated subjects with BCG: comparison with boosting with a new TB vaccine, MVA85A.

OBJECTIVES: To investigate the safety and immunogenicity of a booster BCG vaccination delivered intradermally in healthy, BCG vaccinated subjects and to compare with a previous clinical trial where BCG vaccinated subjects were boosted with a new TB vaccine, MVA85A. DESIGN: Phase I open label observational trial, in the UK. Healthy, HIV-negative, BCG vaccinated adults were recruited and vaccinated with BCG. The primary outcome was safety; the secondary outcome was cellular immune responses to antigen 85, overlapping peptides of antigen 85A and tuberculin purified protein derivative (PPD) detected by ex vivo interferon-gamma (IFN-gamma) ELISpot assay and flow cytometry. RESULTS AND CONCLUSIONS: BCG revaccination (BCG-BCG) was well tolerated, and boosting of pre-existing PPD-specific T cell responses was observed. However, when these results were compared with data from a previous clinical trial, where BCG was boosted with MVA85A (BCG-MVA85A), MVA85A induced significantly higher levels (>2-fold) of antigen 85-specific CD4+ T cells (both antigen and peptide pool responses) than boosting with BCG, up to 52 weeks post-vaccination (p = 0.009). To identify antigen 85A-specific CD8+ T cells that were not detectable by ex vivo ELISpot and flow cytometry, dendritic cells (DC) were used to amplify CD8+ T cells from PBMC samples. We observed low, but detectable levels of antigen 85A-specific CD8+ T cells producing IFNgamma (1.5% of total CD8 population) in the BCG primed subjects after BCG boosting in 1 (20%) of 5 subjects. In contrast, in BCG-MVA85A vaccinated subjects, high levels of antigen 85A-specific CD8+ T cells (up to 14% total CD8 population) were observed after boosting with MVA85A, in 4 (50%) of 8 subjects evaluated. In conclusion, revaccination with BCG resulted in modest boosting of pre-existing immune responses to PPD and antigen 85, but vaccination with BCG-MVA85A induced a significantly higher response to antigen 85 and generated a higher frequency of antigen 85A-specific CD8+ T cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT00654316 NCT00427830.

Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Multicomponent antituberculous subunit vaccine based on immunodominant antigens of Mycobacterium tuberculosis.

The low molecular mass polypeptides of secretory proteome of Mycobacterium tuberculosis are dominant targets for recognition by lymphocytes of human models of immunity to tuberculosis. In the present study, we evaluated the inherent immunogenicity of 102 individual polypeptides purified from low molecular mass region below 40kDa in mouse model of immunization. The aim of this study was to identify molecules relevant for development of subunit vaccine against tuberculosis based on high degree of immunogenecity. Here, we demonstrate that experimental multicomponent subunit vaccine (MSV) consisting of five immunodominant polypeptides with high immunogenicity (CFP-25, CFP-20.5, Ag85B, Ag85A and CPF-32) induced both cellular and humoral immune responses characterized by Th1 and Th2 cytokine induction and imparted significant protection when administered with DDA-MPL adjuvants in C57BL/6J mice. The degree of protection imparted by experimental MSV on the basis of decrease in CFU's from target organs (lungs and spleen) was comparable to BCG and total mycobacterial culture filtrate proteins (CFPs) based vaccines. These results, therefore, suggest the potential of multicomponent subunit vaccination against tuberculosis based on strongly immunogenic proteins of M. tuberculosis.

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein.

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.