Immune subdominant antigens as vaccine candidates against Mycobacterium tuberculosis.

Unlike most pathogens, many of the immunodominant epitopes from Mycobacterium tuberculosis are under purifying selection. This startling finding suggests that M. tuberculosis may gain an evolutionary advantage by focusing the human immune response against selected proteins. Although the implications of this to vaccine development are incompletely understood, it has been suggested that inducing strong Th1 responses against Ags that are only weakly recognized during natural infection may circumvent this evasion strategy and increase vaccine efficacy. To test the hypothesis that subdominant and/or weak M. tuberculosis Ags are viable vaccine candidates and to avoid complications because of differential immunodominance hierarchies in humans and experimental animals, we defined the immunodominance hierarchy of 84 recombinant M. tuberculosis proteins in experimentally infected mice. We then combined a subset of these dominant or subdominant Ags with a Th1 augmenting adjuvant, glucopyranosyl lipid adjuvant in stable emulsion, to assess their immunogenicity in M. tuberculosis-naive animals and protective efficacy as measured by a reduction in lung M. tuberculosis burden of infected animals after prophylactic vaccination. We observed little correlation between immunodominance during primary M. tuberculosis infection and vaccine efficacy, confirming the hypothesis that subdominant and weakly antigenic M. tuberculosis proteins are viable vaccine candidates. Finally, we developed two fusion proteins based on strongly protective subdominant fusion proteins. When paired with the glucopyranosyl lipid adjuvant in stable emulsion, these fusion proteins elicited robust Th1 responses and limited pulmonary M. tuberculosis for at least 6 wk postinfection with a single immunization. These findings expand the potential pool of M. tuberculosis proteins that can be considered as vaccine Ag candidates.

Clinical research and development of tuberculosis diagnostics: moving from silos to synergy.

The development, evaluation, and implementation of new and improved diagnostics have been identified as critical needs by human immunodeficiency virus (HIV) and tuberculosis researchers and clinicians alike. These needs exist in international and domestic settings and in adult and pediatric populations. Experts in tuberculosis and HIV care, researchers, healthcare providers, public health experts, and industry representatives, as well as representatives of pertinent US federal agencies (Centers for Disease Control and Prevention, Food and Drug Administration, National Institutes of Health, United States Agency for International Development) assembled at a workshop proposed by the Diagnostics Working Group of the Federal Tuberculosis Taskforce to review the state of tuberculosis diagnostics development in adult and pediatric populations.

Synthetic and natural TLR4 agonists as safe and effective vaccine adjuvants.

Natural derivatives and synthetic analogues of lipopolysaccharide are potent stimulators of the mammalian immune system. Retained adjuvant activity with reduced toxicity was obtained by the development of monophosphoryl lipid A (MPL((R))), which is approved for use in several vaccine products. Ongoing research and development of synthetic TLR4 agonists may offer increased purity and biological activity with reduced cost. Extensive research has elucidated the mechanism of action of TLR4 agonists and structure-function relationships. Moreover, the formulation of TLR4 agonists has been shown to significantly affect the type and magnitude of elicited immune response. TLR4 agonists comprise a promising class of adjuvants for safe and effective vaccines.

Identification of human T cell antigens for the development of vaccines against Mycobacterium tuberculosis.

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-gamma recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-gamma, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.

Identification of Novel Antigens Recognized by Serum Antibodies in Bovine Tuberculosis.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (∼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy.

Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.

The structure of Escherichia coli cytosine deaminase.

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent.

The ID93 tuberculosis vaccine candidate does not induce sensitivity to purified protein derivative.

The tuberculin skin test (TST) is a simple and inexpensive test to determine whether individuals have been exposed to Mycobacterium tuberculosis. This test is not always reliable, however, in people previously immunized with BCG and/or who have been exposed to environmental mycobacterial species due to a reaction to purified protein derivative (PPD) used in the skin test. An issue with BCG, therefore, is that the resulting sensitization to PPD in some individuals compromises the diagnostic use of the skin test. The ability to induce protective immune responses without sensitizing to the tuberculin skin test will be important properties of next-generation tuberculosis (TB) vaccine candidates. We show here that guinea pigs immunized with the candidate TB vaccine ID93/GLA-SE, currently in clinical trials, do not react to intradermal PPD administration. In contrast, positive DTH responses to both ID93 and components thereof were induced in ID93/GLA-SE-immunized animals, indicating robust but specific cellular responses were present in the immunized animals. Noninterference with the TST is an important factor for consideration in the development of a vaccine against M. tuberculosis.

Therapeutic vaccination against relevant high virulence clinical isolates of Mycobacterium tuberculosis.

The purpose of this study was to attempt to develop therapeutic or post-exposure vaccines that could slow progressive disease in guinea pigs infected by low dose aerosol with very high virulence Beijing isolates of Mycobacterium tuberculosis, currently classified as Category C biodefense pathogens by the NIH and CDC. After screening several candidates we focused on the use of three candidates; these were a pool of bacterial iron acquisition proteins, a pool of antigens recognized by T cells from chronically infected mice thought to represent proteins made by the bacillus in response to decreases in local oxygen tension, and a bacterial lipoprotein that is a potent TLR2 agonist. When delivered in a potent GLA-based adjuvant [targeting TLR4 and TLR9], in most cases we were unable to reduce the bacterial load in the lungs. However, the pathologic appearance of lungs from these animals showed that, while primary lesions were most unaffected and had become necrotic, the development of large, lung consolidating secondary lesions seemed to have been mostly prevented. In animals given both a priming vaccination and a boost the effects were prominent, and almost certainly contributed to significantly prolonged survival in these animals. In a biodefense situation, this prolonged survival would be potentially long enough to allow for the organism to be identified and a drug susceptibility profile determined.

Comparison of point-of-care tests for the rapid diagnosis of visceral leishmaniasis in East African patients.

The development of rK39-based rapid diagnostic tests (RDTs) has greatly aided the diagnosis of visceral leishmaniasis, especially in the Indian subcontinent and Brazil, by offering high sensitivity and specificity. However, these tests have been less sensitive and less specific in sub-Saharan Africa. To improve upon the performance of rK39 in Africa, we engineered the fusion molecule rK28, which retained some of the rK39 repeats and combined them with repeat sequences from two additional Leishmania genes. This polyprotein was used in the development of several prototype RDTs by different commercial manufacturers with the goal of assessing relative performance in inexpensive formats. Here, we report field studies showing that the rK28 antigen could be readily adapted to a variety of RDT formats to achieve high sensitivity, generally > 90%, and adequate specificity to aid in the diagnosis of human visceral leishmaniasis in East Africa, Asia, and South America.

Adjuvants containing natural and synthetic Toll-like receptor 4 ligands.

The last decade has seen an increased focus on the development of adjuvants for vaccines, and several novel adjuvants are now in licensed products or in late-stage clinical development. These advancements have been aided by the discovery of receptors and signaling pathways of the innate immune system and an increased understanding of how these innate responses influence the adaptive immune response. Successful vaccine development relies on knowledge of which adjuvants to use and the proper formulation of adjuvants and antigens to achieve safe, stable and immunogenic vaccines. In this review, the authors focus on the current use of natural and synthetic lipopolysaccharide analogues that retain their adjuvant properties with reduced toxicity compared with the parent compound for use in emerging vaccines. The authors review how these compounds initiate signal transduction through Toll-like receptor 4, insights from structure-function studies and how formulation parameters can influence their effectiveness as vaccine adjuvants.

Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle.

In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1ß and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.

A synthetic TLR4 agonist formulated in an emulsion enhances humoral and Type 1 cellular immune responses against GMZ2--a GLURP-MSP3 fusion protein malaria vaccine candidate.

GMZ2 adjuvanted by aluminum hydroxide is a candidate malaria vaccine that has successfully passed phase 1 clinical testing in adult German and Gabonese volunteers and Gabonese children under five. Here we report a preclinical study screening a series of adjuvant vehicles and Toll-like receptor (TLR) agonists in CB6F1 mice to identify an improved formulation of GMZ2 suitable for further human clinical studies. GMZ2 formulated in an oil-in-water emulsion plus the synthetic TLR4 agonist GLA elicits the highest (a) vaccine-specific IgG2a and total IgG titers, (b) parasite-specific IFA titers, (c) levels of Type 1 cytokine responses (IFN-γ), and (d) number of long-lived-plasma cells (LLPC) secreting antibodies against both the GMZ2 fusion and its two components. Thus, GLA helps to elicit a vaccine-specific Type 1 antibody profile together with high levels of LLPC, both of which are thought to be essential for the development of long-term protective immunity against clinical malaria.

Identification of Mycobacterium tuberculosis antigens of high serodiagnostic value.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, with several million new cases detected each year. Current methods of diagnosis are time-consuming and/or expensive or have a low level of accuracy. Therefore, new diagnostics are urgently needed to address the global tuberculosis burden and to improve control programs. Serological assays remain attractive for use in resource-limited settings because they are simple, rapid, and inexpensive and offer the possibility of detecting cases often missed by routine sputum smear microscopy. The aim of this study was to identify M. tuberculosis seroreactive antigens from a panel of 103 recombinant proteins selected as diagnostic candidates. Initial library screening by protein array analysis and enzyme-linked immunosorbent assay (ELISA) identified 42 antigens with serodiagnostic potential. Among these, 25 were novel proteins. The reactive antigens demonstrated various individual sensitivities, ranging from 12% to 78% (specificities, 76 to 100%). When the antigens were analyzed in combinations, up to 93% of antibody responders could be identified among the TB patients. Selected seroreactive proteins were used to design 3 new polyepitope fusion proteins. Characterization of these antigens by multiantigen print immunoassay (MAPIA) revealed that the vast majority of the TB patients (90%) produced antibody responses. The results confirmed that due to the remarkable variation in immune recognition patterns, an optimal multiantigen cocktail should be designed to cover the heterogeneity of antibody responses and thus achieve the highest possible test sensitivity.

A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis.

Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.

Intradermal immunization improves protective efficacy of a novel TB vaccine candidate.

We have developed the Mycobacterium tuberculosis (Mtb) fusion protein (ID83), which contains the three Mtb proteins Rv1813, Rv3620 and Rv2608. We evaluated the immunogenicity and protective efficacy of ID83 in combination with several emulsion-formulated toll-like receptor agonists. The ID83 subunit vaccines containing synthetic TLR4 or TLR9 agonists generated a T helper-1 immune response and protected mice against challenge with Mtb regardless of route. The ID83 vaccine formulated with gardiquimod (a TLR7 agonist) also resulted in a protective response when administered intradermally, whereas the same vaccine given subcutaneously failed to provide protection. This highlights the need to explore different routes of immunization based on the adjuvant formulations used.

Microseed matrix screening to improve crystals of yeast cytosine deaminase.

A crystallization strategy termed 'microseed matrix screening' is described where the optimal conditions for nucleation versus extended lattice growth are not compatible. This method is an extension of conventional seeding techniques in which microseeds from the nucleation step are systematically seeded into new conditions where all components of the drop are allowed to vary to screen for subsequent growth of well ordered specimens. The structure of a crystal form of yeast cytosine deaminase produced by streak-seeding using a single condition for both nucleation and growth is compared with the structure of a related crystal form produced by separating nucleation and growth conditions. The resulting structural comparison demonstrates that differential chelation patterns of cations by acidic surface residues of proteins within crystal lattice contacts is a critical parameter of crystal nucleation and growth.

Design of a novel globular protein fold with atomic-level accuracy.

A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.