Tool For Vaccine Development Research Articles

Integrating 16S rRNA profiling and in-silico analysis for an epitope-based vaccine strategy against Achromobacter xylosoxidans infection

Achromobacter xylosoxidans is an aerobic, catalase-positive, non-pigment-forming, Gram-negative, and motile bacterium. It potentially causes a wide range of human infections in cystic fibrosis and non-cystic fibrosis patients. However, developing a safe preventive or therapeutic solution against A. xylosoxidans remains challenging. This study aimed to construct an epitope-based vaccine candidate using immunoinformatic techniques. A. xylosoxidans was isolated from an auto workshop in Lahore, and its identification was confirmed through 16S rRNA amplification and bioinformatic analysis. Two protein targets with GenBank accession numbers AKP90890.1 and AKP90355.1 were selected for the vaccine construct. Both proteins exhibited antigenicity, with scores of 0.757 and 0.580, respectively and the epitopes were selected based on the IC50 value using the ANN 4.0 and NN-align 2.3 epitope prediction method for MHC I and MHC II epitopes respectively and predicted epitopes were analyzed for antigenicity, allergenicity and pathogenicity. The vaccine construct demonstrated structural stability, thermostability, solubility, and hydrophilicity. The vaccine produced 250 B-memory cells per mm3 and approximately 16,000 IgM + IgG counts, indicating an effective immune response against A. xylosoxidans. Moreover, the vaccine candidate interacted stably with toll-like receptor 5, a pattern recognition receptor, with a confidence score of 0.98. These results highlight the potency of the designed vaccine candidate, suggesting its potential to withstand rigorous in vitro and in vivo clinical trials. This epitope-based vaccine could serve as the first preventive immunotherapy against A. xylosoxidans infections, addressing this bacterium’s health and financial burdens. The findings demonstrate the value of employing immunoinformatic tools in vaccine development, paving the way for more precise and tailored approaches to combating microbial threats.

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial

Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

A facile method to prepare non-cationic mRNA lipid-nanoparticles based on frame guided assembly strategy

In recent years, lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) has become a promising tool in vaccine development and protein replacement therapy. However, the cationic or ionizable lipid components in the current LNPs also bring the concerns on toxicity and immunogenicity. In this study, we have developed a novel cationic lipid-independent strategy to prepare mRNA-LNPs through frame-guided assembly (FGA). A general mRNA compacting method has been demonstrated to fold long mRNA strands into compact mRNA particles, which was subsequently served as the amphiphilic frames to guide the assembly of phospholipid through hydrophobic interaction. We have also demonstrated high mRNA encapsulation efficiency, good stability and intracellular delivery capability of our mRNA-LNPs, which is expected as a promising tool in mRNA therapeutics and vaccines.

Computational and Experimental Evaluation of the Immune Response of Neoantigens for Personalized Vaccine Design.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection.

Extracellular vesicles (EVs), produced during viral infections, are of emerging interest in understanding infectious processes and host-pathogen interactions. EVs and exosomes in particular have the natural ability to transport nucleic acids, proteins, and other components of cellular or viral origin. Thus, they participate in intercellular communication, immune responses, and infectious and pathophysiological processes. Some viruses are known to hijack the cell production and content of EVs for their benefit. Here, we investigate whether two pathogenic flaviviruses i.e., Zika Virus (ZIKV) and Dengue virus (DENV2) could have an impact on the features of EVs. The analysis of EVs produced by infected cells allowed us to identify that the non-structural protein 1 (NS1), described as a viral toxin, is associated with exosomes. This observation could be confirmed under conditions of overexpression of recombinant NS1 from each flavivirus. Using different isolation methods (i.e., exosome isolation kit, size exclusion chromatography, Polyethylene Glycol enrichment, and ELISA capture), we showed that NS1 was present as a dimer at the surface of excreted exosomes, and that this association could occur in the extracellular compartment. This finding could be of major importance in a physiological context. Indeed, this capacity of NS1 to address EVs and its implication in the pathophysiology during Dengue or Zika diseases should be explored. Furthermore, exosomes that have demonstrated a natural capacity to vectorize NS1 could serve as useful tools for vaccine development.

Novel and efficient method for the reconstruction of adenoviruses through isothermal assembly and its potential applications.

Adenovirus based vectors are useful tools for vaccine development, gene therapy, and oncolytic virotherapy. Here we describe a novel approach for the genetic engineering of any portion of the adenovirus genome and the reconstruction of either fully replication competent or defective virions. This innovative strategy is rapid, effective and suitable for the manipulation of the entire genome broadening the spectrum of potential applications for the adenovirus system. Our strategy involved insertion of restriction enzyme recognition sequences absent in the native virus into the termini of the adenovirus genome in order to facilitate recovery. These restriction enzyme sites, together with the two inverted terminal repeats and packaging sequences, were synthesized and then subcloned into the pBR322 vector. The remaining internal portion of the adenovirus genome was separated and amplified via PCR into six segments, of which groups of two were joined together by PCR and then subcloned into pBR322 plasmids. During the PCR reaction, an overlap of 30-40 bp was added to the termini of the adjacent fragments, allowing for the subsequent isothermal assembly and correct orientation of all fragments. This approach allows for the genetic modification of each genomic fragment according to the specific research goals, (e.g., deletion, substitution, addition, etc.) To recreate the entire viral genome, the four engineered fragments (each comprised of two adenovirus genomic sections) as well as the pBR322 backbone, were reassembled into a single construct utilizing an isothermal assembly reaction. Finally, the reassembled plasmid containing the entire genome was linearized and transfected into HEK293 cells to recover the complete reconstructed adenoviral vector. Using this approach, we have successfully generated two recombinant reporter adenoviruses, one of human adenovirus serotype 14 and another of serotype 55. The E3 region was replaced by the reporter genes (GFP and Luciferase) to visualize and track the recovery process. Subsequent infection of A549 cells with these reconstructed adenovirus vectors demonstrated that they were replication competent. This method shortens the viral reconstruction process because the one-step isothermal assembly requires less than 4 days, and recombinant adenovirus recovery occurs within 10 days. This new method allows for single or multiple genetic modifications within any portion of the viral genome and can be used to construct or manipulate any adenovirus whose complete genome sequence is known.

Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease.

Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.

Systematic review of reverse vaccinology and immunoinformatics data for non-viral sexually transmitted infections.

Sexually Transmitted Infections (STIs) are a public health burden rising in developed and developing nations. The World Health Organization estimates nearly 374 million new cases of curable STIs yearly. Global efforts to control their spread have been insufficient in fulfilling their objective. As there is no vaccine for many of these infections, these efforts are focused on education and condom distribution. The development of vaccines for STIs is vital for successfully halting their spread. The field of immunoinformatics is a powerful new tool for vaccine development, allowing for the identification of vaccine candidates within a bacterium's genome and allowing for the design of new genome-based vaccine peptides. The goal of this review was to evaluate the usage of immunoinformatics in research focused on non-viral STIs, identifying fields where research efforts are concentrated. Here we describe gaps in applying these techniques, as in the case of Treponema pallidum and Trichomonas vaginalis.

Recent Advances in the Study of Gas Vesicle Proteins and Application of Gas Vesicles in Biomedical Research.

The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein–protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.

Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study.

Current vaccination strategies against influenza focus on generating an antibody response against the viral haemagglutination surface protein, however there is increasing interest in neuraminidase (NA) as a target for vaccine development. A critical tool for development of vaccines that target NA or include an NA component is available validated serology assays for quantifying anti-NA antibodies. Additionally serology assays have a critical role in defining correlates of protection in vaccine development and licensure. Standardisation of these assays is important for consistent and accurate results. In this study we first validated a harmonized enzyme-linked lectin assay (ELLA)- Neuraminidase Inhibition (NI) SOP for N1 influenza antigen and demonstrated the assay was precise, linear, specific and robust within classical acceptance criteria for neutralization assays for vaccine testing. Secondly we tested this SOP with NA from influenza B viruses and showed the assay performed consistently with both influenza A and B antigens. Third, we demonstrated that recombinant NA (rNA) could be used as a source of antigen in ELLA-NI. In addition to validating a harmonized SOP we finally demonstrated a clear improvement in inter-laboratory agreement across several studies by using a calibrator. Importantly we showed that the use of a calibrator significantly improved agreement when using different sources of antigen in ELLA-NI, namely reverse genetics viruses and recombinant NA. We provide a freely available and detailed harmonized SOP for ELLA-NI. Our results add to the growing body of evidence in support of developing biological standards for influenza serology.

A perspective into the Current Global Scenario of Vaccine Development and Approval

An understanding of vaccine epidemiology is essential for global preparedness against disease outbreaks. The gap of knowledge between academic communities, policymakers, and the common public must be narrowed down. With the advent of new technologies, vaccine development and bioproduction are witnessing a new revolution. This review article highlights the critical aspects of vaccine design, development, and approval. The various categories of vaccines have been discussed, highlighting the significant benefits and risks of each type. The bioproduction processes involved in manufacturing vaccines have been described with case studies for the vaccine against Malaria and Haemophilus influenzae type b (Hib) infections. The importance of computational tools in vaccine development has been highlighted as artificial intelligence and medical science collaboratively have highly accelerated the complete process starting from identification of CVV(Candidate vaccine virus) to the approval of a vaccine. The method of vaccine approval in different regions of the world has been elaborated, focusing on the regulatory authorities at the United States, European Union, and India. The article ends with the mention of milestones and insight into how humankind has joined hands to combat the COVID-19 global pandemic.

Vaccine Development in the Time of COVID-19: The Relevance of the Risklick AI to Assist in Risk Assessment and Optimize Performance.

The 2019 coronavirus (COVID-19) pandemic revealed the urgent need for the acceleration of vaccine development worldwide. Rapid vaccine development poses numerous risks for each category of vaccine technology. By using the Risklick artificial intelligence (AI), we estimated the risks associated with all types of COVID-19 vaccine during the early phase of vaccine development. We then performed a postmortem analysis of the probability and the impact matrix calculations by comparing the 2020 prognosis to the contemporary situation. We used the Risklick AI to evaluate the risks and their incidence associated with vaccine development in the early stage of the COVID-19 pandemic. Our analysis revealed the diversity of risks among vaccine technologies currently used by pharmaceutical companies providing vaccines. This analysis highlighted the current and future potential pitfalls connected to vaccine production during the COVID-19 pandemic. Hence, the Risklick AI appears as an essential tool in vaccine development for the treatment of COVID-19 in order to formally anticipate the risks, and increases the overall performance from the production to the distribution of the vaccines. The Risklick AI could, therefore, be extended to other fields of research and development and represent a novel opportunity in the calculation of production-associated risks.

Archaeosomes and Gas Vesicles as Tools for Vaccine Development.

Archaea are prokaryotic organisms that were classified as a new domain in 1990. Archaeal cellular components and metabolites have found various applications in the pharmaceutical industry. Some archaeal lipids can be used to produce archaeosomes, a new family of liposomes that exhibit high stability to temperatures, pH and oxidative conditions. Additionally, archaeosomes can be efficient antigen carriers and adjuvants promoting humoral and cellular immune responses. Some archaea produce gas vesicles, which are nanoparticles released by the archaea that increase the buoyancy of the cells and facilitate an upward flotation in water columns. Purified gas vesicles display a great potential for bioengineering, due to their high stability, immunostimulatory properties and uptake across cell membranes. Both archaeosomes and archaeal gas vesicles are attractive tools for the development of novel drug and vaccine carriers to control various diseases. In this review we discuss the current knowledge on production, preparation methods and potential applications of archaeosomes and gas vesicles as carriers for vaccines. We give an overview of the traditional structures of these carriers and their modifications. A comparative analysis of both vaccine delivery systems, including their advantages and limitations of their use, is provided. Gas vesicle- and archaeosome-based vaccines may be powerful next-generation tools for the prevention and treatment of a wide variety of infectious and non-infectious diseases.

Role of Circulating Immune Complexes in the Pathogenesis of Canine Leishmaniasis: New Players in Vaccine Development.

During canine visceral leishmaniasis (CanL), due to Leishmania infantum (L. infantum), uncontrolled infection leads to a strong humoral immune response. As a consequence of the production of high antibody levels and the prolonged presence of parasite antigens, circulating immune complexes (CIC) are formed, which can be deposited in certain organs and tissues, inducing vasculitis, uveitis, dermatitis and especially glomerulonephritis and renal failure. A method to detect CIC and quantify their levels in serum samples from dogs infected with L. infantum has been recently described. It allowed demonstration of a correlation between CIC levels and disease severity. Thus, CIC measurement may be useful for diagnosis, assessment of disease progression and monitoring response to treatment. This is an interesting finding, considering that there remains an urgent need for identification of novel biomarkers to achieve a correct diagnosis and for optimal disease staging of dogs suffering from Leishmania infection. The objective of the present review is to shed light on the role of CIC in CanL, as well as to highlight their potential use not only as diagnostic and prognostic biomarkers but also as a valuable tool in vaccine development and new immunotherapy strategies to prevent or control disease outcome.

Proteomic Analysis of Exosome-Like Vesicles Isolated From Saliva of the Tick Haemaphysalis longicornis.

Extracellular vesicles (EVs), are considered as vehicles of cellular communication. Parasites usually release EVs in their excretory-secretory products to modulate host environment. However, little is known about the secretion of EVs by ticks. In this study, we show for the first time that the tick Haemaphysalis longicornis secretes EVs in saliva that resembles exosomes. EVs were purified from pilocarpine induced saliva of partially engorged H. longicornis ticks. Electron microscopy analysis revealed the presence of exosome-like vesicles with a size of 100 nm. Proteomic analysis by LC-MS/MS identified a total of 356 proteins in tick-derived EVs. Proteome data of tick-derived EVs was validated by Western blot analysis. Immunodetection of Hsp70 and GAPDH proteins indicated that the proteomics data of tick-derived EVs were highly reliable. Bioinformatics analysis (Gene Ontology) indicated association of certain biological and molecular functions with proteins which may be helpful during tick development. Likewise, KEGG database revealed involvement of vesicular proteins in proton transport, detoxification, ECM-receptor interaction, ribosome, RNA transport, ABC transporters, and oxidative phosphorylation. The results of this study provide evidence that EVs are being secreted in tick saliva and suggest that tick saliva-derived EVs could play important roles in host-parasite relationships. Moreover, EVs could be a useful tool in development of vaccines or therapeutics against ticks.

Development of immunogenic chimeric virus-like particles based on bovine papillomavirus type 6

Virus-like particles (VLPs) are considered useful tools for vaccine development because they induce an immune response and are safe. In addition, VLPs may be useful as a platform for the presentation of foreign antigens to elicit immune responses. In this study, we aimed to produce a chimeric VLP composed of L1 protein of bovine papillomavirus type 6 (BPV6-L1) that can display an entire foreign protein on its surface. Based on prediction of the conformational structure of VLP of BPV6-L1 (BPV6-VLP), candidate insertion sites for the foreign protein into BPV6-VLP were identified. Fusion proteins of BPV6-L1 and EGFP as a model foreign protein were constructed and produced. Only the fusion protein in which EGFP was inserted between amino acids 136 and 137 of BPV6-L1 self-assembled into VLPs and did not exhibit hindrance of the conformation of EGFP. Chimeric BPV6-VLP-immunized mice produced specific IgG against both BPV6 and EGFP. This is the first demonstration of the production of an immunogenic, bivalent, chimeric BPV6-VLP incorporating an entire protein for stable surface display. Thus, immunogenic chimeric BPV6-VLP may constitute a promising vaccine platform.

Novel ssRNA phage VLP platform for displaying foreign epitopes by genetic fusion

Virus-like particles (VLPs) can be used as efficient carriers of various antigens and therefore serve as attractive tools in vaccine development. Although VLPs of different viruses can be used, VLPs of ssRNA phages have convincing advantages due to their unique properties, including efficient protein production in bacterial and yeast expression systems, low production cost and easy and fast purification. Currently, the range of ssRNA phage VLPs is limited. In particular, this is true for VLPs that tolerate insertions at the N- and C-termini of the coat protein. It is therefore necessary to find new alternatives within the known ssRNA phage VLP range. From previous studies, we found approximately 80 new VLPs forming ssRNA phage coat proteins. In the current study, we attached a model peptide to the N- and C-termini of coat proteins. As a model peptide, we used a triple repeat of 23 N-terminal residues of the ectodomain of the influenza M2 protein, used previously in the development of the flu vaccine. Examining 43 novel phage coat proteins for the ability to form chimeric VLPs, we found ten new promising candidates for further vaccine design, five of which were tolerant to insertions at both the N- and C-termini. Furthermore, we demonstrate that most of the chimeric VLPs have good antigenic properties as judged from their reactivity with anti-M2 antibodies.

β-glucan as a new tool in vaccine development.

Vaccination constitutes one of the major breakthroughs in human medicine. At the same time, development of more immunogenic vaccine alternatives to using aluminium-based adjuvants is one of the most important phases of vaccination development. Among different sources of carbohydrate polymers, including plants, microbes and synthetic sources tested, glucans were found to be the most promising vaccine adjuvant, as they alone stimulate various immune reactions including antibody production without any negative side effects. The use of glucan particles as a delivery system is a viable option based on the documented efficient antigen loading and receptor-targeted uptake in antigen-presenting cells. In addition to particles, soluble glucans can be used as novel hydrogels or as direct immunocyte-targeting delivery systems employing novel complexes with oligodeoxynucleotides. This review focuses on recent advances in glucan-based vaccine development from glucan-based conjugates to a glucan-based delivery and adjuvant platform.

A tracer-based method enables tracking of Plasmodium falciparum malaria parasites during human skin infection.

Introduction: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human malaria parasites. To enable in depth analysis of not genetically modified (non-GMO) Plasmodium falciparum (Pf) sporozoite behaviour in human skin, we devised a labelling technology (Cy5M2, targeting the sporozoite mitochondrion) that supports tracking of individual non-GMO sporozoites in human skin.Methods: Sporozoite labelling with Cy5M2 was performed in vitro as well as via the feed of infected Anopheles mosquitos. Labelling was validated using confocal microscopy and flow cytometry and the fitness of labelled sporozoites was determined by analysis of infectivity to human hepatocytes in vitro, and in vivo in a rodent infection model. Using confocal video microscopy and custom software, single-sporozoite tracking studies in human skin-explants were performed.Results: Both in vitro and in mosquito labelling strategies yielded brightly fluorescent sporozoites of three different Plasmodium species. Cy5M2 uptake colocalized with MitoTracker® green and could be blocked using the known Translocator protein (TSPO)-inhibitor PK11195. This method supported the visualization and subsequent quantitative analysis of the migration patterns of individual non-GMO Pf sporozoites in human skin and did not affect the fitness of sporozoites.Conclusions: The ability to label and image non-GMO Plasmodium sporozoites provides the basis for detailed studies on the human skin stage of malaria with potential for in vivo translation. As such, it is an important tool for development of vaccines based on attenuated sporozoites and their route of administration.

Clinical endpoints in the controlled human challenge model for Shigella: A call for standardization and the development of a disease severity score.

BackgroundSince 1946 the controlled human infection model (CHIM) for Shigella has been used to improve understanding of disease pathogenesis, describe clinical and immunologic responses to infection and as a tool for vaccine development. As the frequency and intent for use in vaccine comparisons increases, standardization of the primary endpoint definition is necessary.MethodsSubject-level data were obtained from previously conducted experimental Shigella CHIM studies. Signs and symptoms severity were categorized consistently across all studies. Sign and symptom correlations were estimated and univariate models were utilized to describe the association between stool output and other Shigella-attributable signs and symptoms. Multiple correspondence and hierarchical clustering analyses were performed to describe the co-occurrence of signs and symptoms. A disease score is proposed based on the co-occurrence of these events.ResultsData were obtained on 54 subjects receiving 800 to 2000 colony forming units (cfu) of S. flexneri. The median maximum 24 hour stool output was 514 ml (IQR: 300, 998 ml) with a median frequency of 6 (IQR: 4, 9). Subjects reported abdominal pain or cramps (81.5%), headache (66.7%) and anorexia (64.8%), 50.0% had a fever and 27.8% had gross blood in multiple loose stools. Multiple correspondence analyses highlighted co-occurrence of symptoms based on severity. A 3-parameter disease severity score predicted shigellosis endpoints and better differentiated disease spectrum.ConclusionDichotomous endpoints for Shigella CHIM fail to fully account for disease variability. An ordinal disease score characterizing the breadth of disease severity may enable a better characterization of shigellosis and can decrease sample size requirements. Furthermore, the disease severity score may be a useful tool for portfolio management by enabling prioritization across vaccine candidates with comparable efficacy estimates using dichotomous endpoints.