Tuberculosis vaccines: Rising opportunities.

Johan Vekemans, Katherine O'Brien, and Jeremy Farrar discuss recent breakthroughs in the search for a highly effective tuberculosis vaccine.

Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells.

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.

In silico design of Mycobacterium tuberculosis epitope ensemble vaccines.

Effective control of Mycobacterium tuberculosis is a global necessity. In 2015, tuberculosis (TB) caused more deaths than HIV. Considering the increasing prevalence of multi-drug resistant forms of M. tuberculosis, the need for effective TB vaccines becomes imperative. Currently, the only licensed TB vaccine is Bacillus Calmette-Guérin (BCG). Yet, BCG has many drawbacks limiting its efficacy and applicability. We applied advanced computational procedures to derive a universal TB vaccine and one targeting East Africa. Our approach selects an optimal set of highly conserved, experimentally validated epitopes, with high projected population coverage (PPC). Through rigorous data analysis, five different potential vaccine combinations were selected each with PPC above 80% for East Africa and above 90% for the World. Two potential vaccines only contained CD8+ epitopes, while the others included both CD4+ and CD8+ epitopes. Our prime vaccine candidate was a putative seven-epitope ensemble comprising: SRGWSLIKSVRLGNA, KPRIITLTMNPALDI, AAHKGLMNIALAISA, FPAGGSTGSL, MLLAVTVSL, QSSFYSDW and KMRCGAPRY, with a 97.4% global PPC and a 92.7% East African PPC.

Lyophilization of an Adjuvanted Mycobacterium tuberculosis Vaccine in a Single-Chamber Pharmaceutical Cartridge.

Although substantial effort has been made in the development of next-generation recombinant vaccine systems, maintenance of a cold chain is still typically required and remains a critical challenge in effective vaccine distribution. The ability to engineer alternative containment systems that improve distribution and administration represents potentially significant enhancements to vaccination strategies. In this work, we evaluate the ability to successfully lyophilize a previously demonstrated thermostable tuberculosis vaccine formulation (ID93 + GLA-SE) in a cartridge format compared to a traditional vial container format. Due to differences in the shape of the container formats, a novel apparatus was developed to facilitate lyophilization in a cartridge. Following lyophilization, the lyophilizate was assessed visually, by determining residual moisture content, and by collecting melting profiles. Reconstituted formulations were assayed for particle size, protein presence, and GLA content. Based on assessment of the lyophilizate, the multicomponent vaccine was successfully lyophilized in both formats. Also, the physicochemical properties of the major components in the formulation, including antigen and adjuvant, were retained after lyophilization in either format. Ultimately, this study demonstrates that complex formulations can be lyophilized in alternative container formats to the standard pharmaceutical glass vial, potentially helping to increase the distribution of vaccines.

Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

Understanding and overcoming the barriers to T cell-mediated immunity against tuberculosis.

Despite the overwhelming success of immunization in reducing, and even eliminating, the global threats posed by a wide spectrum of infectious diseases, attempts to do the same for tuberculosis (TB) have failed to date. While most effective vaccines act by eliciting neutralizing antibodies, T cells are the primary mediators of adaptive immunity against TB. Unfortunately, the onset of the T cell response after aerosol infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes TB, is exceedingly slow, and systemically administered vaccines only modestly accelerate the recruitment of effector T cells to the lungs. This delay seems to be orchestrated by Mtb itself to prolong the period of unrestricted bacterial replication in the lung that characterizes the innate phase of the response. When T cells finally arrive at the site of infection, multiple layers of regulation have been established that limit the ability of T cells to control or eradicate Mtb. From this understanding, emerges a strategy for achieving immunity. Lung resident memory T cells may recognize Mtb-infected cells shortly after infection and confer protection before regulatory networks are allowed to develop. Early studies using vaccines that elicit lung resident T cells by targeting the lung mucosa have been promising, but many questions remain. Due to the fundamental nature of these questions, and the need to understand and manipulate the early events in the lung after aerosol infection, only coordinated approaches that utilize tractable animal models to inform human TB vaccine trials will move the field toward its goal.

The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines.

Given the urgent need for designing a new antituberculosis vaccine conferring total protection on patients of all ages, following the line of research adopted by our institute, this work has identified Mycobacterium tuberculosis (Mtb) Rv3166c protein high-activity binding peptides (HABPs) which are able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The presence and transcription of the rv3166c gene in the Mtb species complex was confirmed by polymerase chain reaction (PCR) and reverse transcriptase-PCR; Rv3166c expression was evaluated by western blot and cellular localisation confirmed by immunoelectron microscopy. Its presence was mainly determined on cell surface. Sixteen peptides covering its entire length were chemically synthesised and tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. The four HABPs found inhibited Mtb entry by 15.07-94.06%. These results led us to including Rv3166c HABPs as candidates for further studies contributing towards the search for a multiepitope, chemically synthesised, subunit-based antituberculosis vaccine.

The importance of adjuvant formulation in the development of a tuberculosis vaccine.

An effective protein-based vaccine for tuberculosis will require a safe and effective adjuvant. There are few adjuvants in approved human vaccines, including alum and the oil-in-water-based emulsions MF59 (Novartis Vaccines and Diagnostics), AS03 and AS04 (GlaxoSmithKline Biologics), AF03 (Sanofi), and liposomes (Crucell). When used with pure, defined proteins, both alum and emulsion adjuvants are effective at inducing primarily humoral responses. One of the newest adjuvants in approved products is AS04, which combines monophosphoryl lipid A, a TLR-4 agonist, with alum. In this study, we compared two adjuvants: a stable oil-in-water emulsion (SE) and a stable oil-in-water emulsion incorporating glucopyranosyl lipid adjuvant, a synthetic TLR-4 agonist (GLA-SE), each together with a recombinant protein, ID93. Both the emulsion SE and GLA-SE adjuvants induce potent cellular responses in combination with ID93 in mice. ID93/SE induced Th2-biased immune responses, whereas ID93/GLA-SE induced multifunctional CD4(+) Th1 cell responses (IFN-γ, TNF-α, and IL-2). The ID93/GLA-SE vaccine candidate induced significant protection in mice and guinea pigs, whereas no protection was observed with ID93/SE, as assessed by reductions in bacterial burden, survival, and pathology. These results highlight the importance of properly formulating subunit vaccines with effective adjuvants for use against tuberculosis.

Dual neonate vaccine platform against HIV-1 and M. tuberculosis.

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the world's most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborn's mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA(222). mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA(222)-mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life.

Rational design of multiple TB antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis.

PURPOSE: Rational design of recombinant antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis. The main purpose is to obtain a large quantity of soluble antigens. METHODS: Recombinant antigens were cloned in frame with the N-terminal thioredoxin and expressed in E. coli. The thioredoxin tag was removed by TEV protease. Nickel-affinity and size-exclusion chromatography were used to purify antigens to homogeneity. Antigen stability at different pH levels was studied by photon correlation spectrometry. Circular dichroism was used to probe antigen secondary structure and thermal stability. RESULTS: N-terminal thioredoxin fusion dramatically increased antigen solubility. Soluble TB10.4 and TB10.4-Ag85B were purified to homogeneity and obtained in milligram quantity. Co-expression of bacteria chaperons increased the yield of TB10.4-Ag85B. Soluble TB10.4 and TB10.4-Ag85B purified from the inclusion body showed a reversible structure change. However, Ag85B and soluble TB10.4-Ag85B showed a clear melting temperature, above which the secondary structure was lost dramatically. CONCLUSION: Soluble TB10.4 and TB10.4-Ag85B were purified from the E. coli in significant quantities. The methods to purify and characterize these recombinant antigens were established, which paved the way for further vaccine development based on these antigens.

Tuberculosis subunit vaccine design: the conflict of antigenicity and immunogenicity.

The attempts to find an effective antituberculous subunit vaccine are based on the assumption that it must drive a Th1 response. In the absence of effective correlates of protection, a vast array of mycobacterial components are being evaluated worldwide either on the basis of their ability to be recognized by T lymphocytes in in vitro assays during early stage of animal or human infection (antigenicity) or their capacity to induce T cell response following immunization in animal models (immunogenicity). The putative vaccine candidates selected using either of these strategies are then subjected to challenge studies in different animal models to evaluate the protective efficacy. Here we review the outcome of this current scheme of selection of vaccine candidates using an 'antigenicity' or 'immunogenicity' criterion on the actual protective efficacy observed in experimental animal models. The possible implications for the success of some of the leading vaccine candidates in clinical trials will also be discussed.