Imperial College London

ProfessorRobinShattock

Faculty of MedicineDepartment of Infectious Disease

Chair in Mucosal Infection and Immunity
 
 
 
//

Contact

 

+44 (0)20 7594 5206r.shattock

 
 
//

Location

 

453Wright Fleming WingSt Mary's Campus

//

Summary

 

Publications

Publication Type
Year
to

273 results found

Fu M, Hu K, Hu H, Ni F, Du T, Shattock RJ, Hu Qet al., 2019, Antigenicity and immunogenicity of HIV-1 gp140 with different combinations of glycan mutation and V1/V2 region or V3 crown deletion., Vaccine

The carbohydrate moieties on HIV-1 envelope glycoprotein (Env) act as shields to mask conserved neutralizing epitopes, while the hyperimmunogenic variable regions are immunodominant in inducing non-neutralizing antibodies, representing the major challenge for using Env as a vaccine candidate to induce broadly neutralizing antibodies (bNAbs). In this study, we designed a series of HIV-1 gp140 constructs with the removal of N276/N463 glycans, deletion of the V1/V2 region and the V3 crown, alone or in combination. We first demonstrated that all the constructs had a comparable level of expression and were mainly expressed as trimers. Following purification of gp140s from mammalian cells, we measured their binding to bNAbs and non-NAbs in vitro and capability in inducing bNAbs in vivo. Antibody binding assay showed that removal of N276/N463 glycans together with the deletion of V1/V2 region enhanced the binding of gp140s to CD4-binding site-targeting bNAbs VRC01 and 3BNC117, and CD4-induced epitopes-targeting non-NAbs A32, 17b and F425 A1g8, whereas further deletion of V3 crown in the gp140 mutants demonstrated slightly compromised binding capability to these Abs. Immunogenicity study showed that the above mutations did not lead to the induction of a higher Env-specific IgG response via either DNA-DNA or DNA-protein prime-boost strategies in mice, while neutralization assay did not show an apparent difference between wild type and mutated gp140s. Taken together, our results indicate that removal of glycans at N276/N463 and deletion of the V1/V2 region can expose the CD4-binding site and CD4-induced epitopes, but such exposure alone appears incapable of enhancing the induction of bNAbs in mice, informing that additional modification or/and immunization strategies are needed. In addition, the strategies which we established for producing gp140 proteins and for analyzing the antigenicity and immunogenicity of gp140 provide useful means for further vaccine design and assessm

Journal article

Abraham S, Juel HB, Bang P, Cheeseman HM, Dohn RB, Cole T, Kristiansen MP, Korsholm KS, Lewis D, Olsen AW, McFarlane LR, Day S, Knudsen S, Moen K, Ruhwald M, Kromann I, Andersen P, Shattock RJ, Follmann Fet al., 2019, Safety and immunogenicity of the chlamydia vaccine candidate CTH522 adjuvanted with CAF01 liposomes or aluminium hydroxide: a first-in-human, randomised, double-blind, placebo-controlled, phase 1 trial., Lancet Infectious Diseases, ISSN: 1473-3099

BACKGROUND: Chlamydia is the most common sexually transmitted bacterial infection worldwide. National screening programmes and antibiotic treatment have failed to decrease incidence, and to date no vaccines against genital chlamydia have been tested in clinical trials. We aimed to assess the safety and immunogenicity, in humans, of a novel chlamydia vaccine based on a recombinant protein subunit (CTH522) in a prime-boost immunisation schedule. METHODS: This phase 1, first-in-human, double-blind, parallel, randomised, placebo-controlled trial was done at Hammersmith Hospital in London, UK, in healthy women aged 19-45 years. Participants were randomly assigned (3:3:1) to three groups: CTH522 adjuvanted with CAF01 liposomes (CTH522:CAF01), CTH522 adjuvanted with aluminium hydroxide (CTH522:AH), or placebo (saline). Participants received three intramuscular injections of 85 μg vaccine (with adjuvant) or placebo to the deltoid region of the arm at 0, 1, and 4 months, followed by two intranasal administrations of 30 μg unadjuvanted vaccine or placebo (one in each nostril) at months 4·5 and 5·0. The primary outcome was safety and the secondary outcome was humoral immunogenicity (anti-CTH522 IgG seroconversion). This study is registered with Clinicaltrials.gov, number NCT02787109. FINDINGS: Between Aug 15, 2016, and Feb 13, 2017, 35 women were randomly assigned (15 to CTH522:CAF01, 15 to CTH522:AH, and five to placebo). 32 (91%) received all five vaccinations and all participants were included in the intention-to-treat analyses. No related serious adverse reactions were reported, and the most frequent adverse events were mild local injection-site reactions, which were reported in all (15 [100%] of 15) participants in the two vaccine groups and in three (60%) of five participants in the placebo group (p=0·0526 for both comparisons). Intranasal vaccination was not associated with a higher frequency of related local reactions (reported in seven [47%]

Journal article

Blakney AK, McKay PF, Yus BI, Aldon Y, Shattock RJet al., 2019, Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA, Gene Therapy, Vol: 26, Pages: 363-372, ISSN: 0969-7128

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

Journal article

Kis Z, Shattock R, Shah N, Kontoravdi Cet al., 2019, Correction: Emerging technologies for low‐cost, rapid vaccine manufacture, Biotechnology Journal, Vol: 14, Pages: 1-2, ISSN: 1860-6768

Journal article

Blakney AK, McKay PF, Christensen D, Yus BI, Aldon Y, Follmann F, Shattock RJet al., 2019, Effects of cationic adjuvant formulation particle type, fluidity and immunomodulators on delivery and immunogenicity of saRNA, Journal of Controlled Release, Vol: 304, Pages: 65-74, ISSN: 0168-3659

Self-amplifying RNA (saRNA) is well suited as a vaccine platform against chlamydia, as it is relatively affordable and scalable, has been shown to induce immunity against multivalent antigens, and can result in protein expression for up to 60 days. Cationic adjuvant formulations (CAFs) have been previously investigated as an adjuvant for protein subunit vaccines; here we optimize the CAFs for delivery of saRNA in vivo and observe the immunogenicity profile in the context of both cellular and humoral immunity against the major outer membrane protein (MOMP) of Chlamydia trachomatis. We tested both liposomal and emulsion based CAFs with solid and fluid phase lipids, with or without the TLR agonists R848 and 3M-052, for in vitro transfection efficiency and cytotoxicity. We then optimized the RNA/delivery system ratio for in vivo delivery using saRNA coding for firefly luciferase (fLuc) as a reporter protein in vivo. We observed that while the fluid phase liposome formulations showed the highest in vitro transfection efficiency, the fluid and solid phase liposomes had equivalent luciferase expression in vivo. Incorporation of R848 or 3M-052 into the formulation was not observed to affect the delivery efficiency of saRNA either in vitro or in vivo. MOMP-encoding saRNA complexed with CAFs resulted in both MOMP-specific cellular and humoral immunity, and while there was a slight enhancement of IFN-γ+ T-cell responses when R848 was incorporated into the formulation, the self-adjuvanting effects of RNA appeared to dominate the immune response. These studies establish that CAFs are efficient delivery vehicles for saRNA both for in vitro transfections and in vivo immunogenicity and generate cellular and humoral responses that are proportionate to protein expression.

Journal article

Sliepen K, Han BW, Bontjer I, Mooij P, Garces F, Behrens A-J, Rantalainen K, Kumar S, Sarkar A, Brouwer PJM, Hua Y, Tolazzi M, Schermer E, Torres JL, Ozorowski G, van der Woude P, de la Pena AT, van Breemen MJ, Camacho-Sanchez JM, Burger JA, Medina-Ramirez M, Gonzalez N, Alcami J, LaBranche C, Scarlatti G, van Gils MJ, Crispin M, Montefiori DC, Ward AB, Koopman G, Moore JP, Shattock RJ, Bogers WM, Wilson IA, Sanders RWet al., 2019, Structure and immunogenicity of a stabilized HIV-1 envelope trimer based on a group-M consensus sequence, Nature Communications, Vol: 10, ISSN: 2041-1723

Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses.

Journal article

Blakney AK, McKay PF, Ibarzo Yus B, Hunter JE, Dex EA, Shattock RJet al., 2019, The skin you're in: Design of experiments optimization of lipid nanoparticle self-amplifying RNA formulations in human skin explants, ACS Nano, Vol: 13, ISSN: 1936-0851

Messenger RNA (mRNA) is a promising tool for biotherapeutics, and self-amplifying mRNA (saRNA) is particularly advantageous as it results in abundant protein expression and production is easily scalable. While mRNA therapeutics have been shown to be highly effective in small animals, the outcomes do not scale linearly when these formulations are translated to dose-escalation studies in humans. Here, we utilize a Design of Experiments (DoE) approach to optimize the formulation of saRNA lipid nanoparticles in human skin explants. We first observed that luciferase expression from saRNA peaked after 11 days in human skin. Using DoE inputs of complexing lipid identity, lipid nanoparticle dose, lipid concentration, particle concentration, and ratio of zwitterionic to cationic lipids, we optimized the saRNA-induced luciferase expression in skin explants. Lipid identity and lipid concentration were found to be significant parameters in the DoE model, and the optimized formulation resulted in ~7-fold increase in luciferase expression relative to initial DOTAP formulation. Using flow cytometry, we observed that optimized formulations delivered the saRNA to ~2% of the resident cells in the human skin explants. Although immune cells make up only 7% of the total population of cells in skin, immune cells were found to express ~50% of the RNA. This study demonstrates the powerful combination of using a DoE approach paired with clinically relevant human skin explants to optimize nucleic acid formulations. We expect that this system will be useful for optimizing both formulation and molecular designs of clinically translational nucleic acid vaccines and therapeutics.

Journal article

McKay P, Cizmeci D, Aldon Y, Maertzdorf J, Weiner J, Kaufmann S, Lewis D, van den Berg R, Del Giudice G, Shattock Ret al., 2019, Identification of potential biomarkers of vaccine inflammation in mice, eLife, Vol: 8, ISSN: 2050-084X

Systems vaccinology approaches have been used successfully to define early signatures of the vaccine-induced immune response. However, the possibility that transcriptomics can also identify a correlate or surrogate for vaccine inflammation has not been fully explored. We have compared four licensed vaccines with known safety profiles, as well as three agonists of Toll-like receptors (TLRs) with known inflammatory potential, to elucidate the transcriptomic profile of an acceptable response to vaccination versus that of an inflammatory reaction. In mice, we looked at the transcriptomic changes in muscle at the injection site, the lymph node that drained the muscle, and the peripheral blood mononuclear cells (PBMCs)isolated from the circulating blood from 4 hr after injection and over the next week. A detailed examination and comparative analysis of these transcriptomes revealed a set of novel biomarkers that are reflective of inflammation after vaccination. These biomarkers are readily measurable in the peripheral blood, providing useful surrogates of inflammation, and provide a way to select candidates with acceptable safety profiles.

Journal article

Progatzky F, Jha A, Wane M, Thwaites RS, Makris S, Shattock RJ, Johansson C, Openshaw PJ, Bugeon L, Hansel TT, Dallman MJet al., 2019, Induction of innate cytokine responses by respiratory mucosal challenge with R848 in zebrafish, mice and humans, Journal of Allergy and Clinical Immunology, ISSN: 0091-6749

We compared live zebrafish, mouse and human nasal challenge responses to the TLR7/8 agonist resiquimod (R848). We found remarkably similar induction of mediators in the three species, offering novel mucosal models of innate anti-viral immunity.

Journal article

Kis Z, Shattock R, Shah N, Kontoravdi Ket al., 2019, Emerging technologies for low-cost, rapid vaccine manufacture, Biotechnology Journal, Vol: 14, ISSN: 1860-6768

To stop the spread of future epidemics and meet infant vaccination demands in low‐ and middle‐income countries, flexible, rapid and low‐cost vaccine development and manufacturing technologies are required. Vaccine development platform technologies that can produce a wide range of vaccines are emerging, including: a) humanized, high‐yield yeast recombinant protein vaccines; b) insect cell‐baculovirus ADDomer vaccines; c) Generalized Modules for Membrane Antigens (GMMA) vaccines; d) RNA vaccines. Herein, existing and future platforms are assessed in terms of addressing challenges of scale, cost, and responsiveness. To assess the risk and feasibility of the four emerging platforms, the following six metrics are applied: 1) technology readiness; 2) technological complexity; 3) ease of scale‐up; 4) flexibility for the manufacturing of a wide range of vaccines; 5) thermostability of the vaccine product at tropical ambient temperatures; and 6) speed of response from threat identification to vaccine deployment. The assessment indicated that technologies in the order of increasing feasibility and decreasing risk are the yeast platform, ADDomer platform, followed by RNA and GMMA platforms. The comparative strengths and weaknesses of each technology are discussed in detail, illustrating the associated development and manufacturing needs and priorities.

Journal article

Lopez E, Shattock RJ, Kent SJ, Chung AWet al., 2018, The Multifaceted Nature of Immunoglobulin A and Its Complex Role in HIV, AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol: 34, Pages: 727-738, ISSN: 0889-2229

Journal article

Herrera C, Veazey R, Lemke M, Olejniczak N, Arnold K, Kim JH, Shattock RJet al., 2018, Vaccination with ALVAC-HIV/AIDSVAX (R) B/E of Non-human Primates (NHPs) Elicits Distinct Mucosal and Systemic Responses, HIV Research for Prevention Meeting (HIVR4P) - AIDS Vaccine, Microbicide and ARV-Based Prevention Science, Publisher: MARY ANN LIEBERT, INC, Pages: 306-306, ISSN: 0889-2229

Conference paper

Sliepen K, Han BW, Bontjer I, Garces F, Behrens A-J, Rantalainen K, Brouwer P, Mooij P, Schermer E, van Gils MJ, Alcami J, Scarlatti G, Crispin M, Ward AB, Montefiori DC, Moore JP, Shattock RJ, Bogers W, Wilson I, Sanders RWet al., 2018, Structure and Immunogenicity of a Stabilized HIV-1 Envelope Trimer Based on a Group M Consensus Sequence, HIV Research for Prevention Meeting (HIVR4P) - AIDS Vaccine, Microbicide and ARV-Based Prevention Science, Publisher: MARY ANN LIEBERT, INC, Pages: 335-335, ISSN: 0889-2229

Conference paper

Aldon Y, McKay PF, Allen J, Ozorowski G, Levai RF, Tolazzi M, Rogers P, He L, de Val N, Fabian K, Scarlatti G, Zhu J, Ward AB, Crispin M, Shattock RJet al., 2018, Rational design of DNA-expressed stabilized native-like HIV-1 envelope trimers, Cell Reports, Vol: 24, Pages: 3324-3338.e5, ISSN: 2211-1247

The HIV-1-envelope glycoprotein (Env) is the main target of antigen design for antibody-based prophylactic vaccines. The generation of broadly neutralizing antibodies (bNAb) likely requires the appropriate presentation of stabilized trimers preventing exposure of non-neutralizing antibody (nNAb) epitopes. We designed a series of membrane-bound Envs with increased trimer stability through the introduction of key stabilization mutations. We derived a stabilized HIV-1 trimer, ConSOSL.UFO.750, which displays a dramatic reduction in nNAb binding while maintaining high quaternary and MPER-specific bNAb binding. Its soluble counterpart, ConSOSL.UFO.664, displays similar antigenicity, and its native-like Env structure is confirmed by negative stain-EM and glycosylation profiling of the soluble ConSOSL.UFO.664 trimer. A rabbit immunization study demonstrated that the ConSOSL.UFO.664 can induce autologous tier 2 neutralization. We have successfully designed a stabilized native-like Env trimer amenable to nucleic acid or viral vector-based vaccination strategies.

Journal article

Aw R, McKay P, Shattock R, Polizzi Ket al., 2018, A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization, Protein Expression and Purification, Vol: 149, Pages: 43-50, ISSN: 1046-5928

Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

Journal article

Cheeseman HM, Day S, McFarlane LR, Fleck S, Miller A, Cole T, Sousa-Santos N, Cope A, Cizmeci D, Tolazzi M, Hwekwete E, Hannaman D, Kratochvil S, McKay PF, Chung AW, Kent SJ, Cook A, Scarlatti G, Abraham S, Combadiere B, McCormack S, Lewis DJ, Shattock RJet al., 2018, Combined Skin and Muscle DNA Priming Provides Enhanced Humoral Responses to a Human Immunodeficency Virus Type 1 Clade C Envelope Vaccine, Human Gene Therapy, Vol: 29, Pages: 1011-1028, ISSN: 1043-0342

© Copyright 2018, Mary Ann Liebert, Inc., publishers2018. Intradermal (i.d.) and intramuscular (i.m.) injections when administered with or without electroporation (EP) have the potential to tailor the immune response to DNA vaccination. This Phase I randomized controlled clinical trial in human immunodeficiency virus type 1-negative volunteers investigated whether the site and mode of DNA vaccination influences the quality of induced cellular and humoral immune responses following the DNA priming phase and subsequent protein boost with recombinant clade C CN54 gp140. A strategy of concurrent i.d. and i.m. DNA immunizations administered with or without EP was adopted. Subtle differences were observed in the shaping of vaccine-induced virus-specific CD4+ and CD8+ T cell-mediated immune responses between groups receiving: i.d.EP+ i.m., i.d. + i.m.EP, and i.d.EP+ i.m.EPregimens. The DNA priming phase induced 100% seroconversion in all of the groups. A single, non-adjuvanted protein boost induced a rapid and profound increase in binding antibodies in all groups, with a trend for higher responses in i.d.EP+ i.m.EP. The magnitude of antigen-specific binding immunoglobulin G correlated with neutralization of closely matched clade C 93MW965 virus and Fc-dimer receptor binding (FcγRIIa and FcγRIIIa). These results offer new perspectives on the use of combined skin and muscle DNA immunization in priming humoral and cellular responses to recombinant protein.

Journal article

Vamvaka E, Farre G, Molinos-Albert LM, Evans A, Canela-Xandri A, Twyman RM, Carrillo J, Ordonez RA, Shattock RJ, O'Keefe BR, Clotet B, Blanco J, Khush GS, Christou P, Capell Tet al., 2018, Unexpected synergistic HIV neutralization by a triple microbicide produced in rice endosperm, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 115, Pages: E7854-E7862, ISSN: 0027-8424

Journal article

Blakney A, McKay PF, Shattock R, 2018, Structural components for amplification of positive and negative strand VEEV splitzicons, Frontiers in Molecular Biosciences, Vol: 5, ISSN: 2296-889X

RNA is a promising nucleic acid technology for both vaccines and therapeutics, and replicon RNA has gained traction as a next-generation RNA modality. Replicon RNA self-amplifies using a replicase complex derived from alphaviral non-structural proteins and yields higher protein expression than a similar dose of messenger RNA. Here, we debut RNA splitzicons; a split replicon system wherein the non-structural proteins (NSPs) and the gene of interest are encoded on separate RNA molecules, but still exhibit the self-amplification properties of replicon RNA. We designed both positive and negative strand splitzicons encoding firefly luciferase as a reporter protein to determine which structural components, including the 5′ untranslated region (UTR), a 51-nucleotide conserved sequence element (CSE) from the first nonstructural protein, the subgenomic promoter (SGP) and corresponding untranslated region, and an internal ribosomal entry site (IRES) affect amplification. When paired with a NSP construct derived from the whole, wild type replicon, both the positive and negative strand splitzicons were amplified. The combination of the 51nt CSE, subgenomic promoter and untranslated region were imperative for the positive strand splitzicon, while the negative strand was amplified simply with inclusion of the subgenomic promoter. The splitzicons were amplified by NSPs in multiple cell types and show increasing protein expression with increasing doses of NSP. Furthermore, both the positive and negative strand splitzicons continued to amplify over the course of 72 h, up to >100,000-fold. This work demonstrates a system for screening the components required for amplification from the positive and negative strand intermediates of RNA replicons and presents a new approach to RNA replicon technology.

Journal article

Short CS, Quinlan R, Bennett P, Shattock R, Taylor Get al., 2018, Optimising the collection of female genital tract fluid for cytokine analysis in pregnant women, Journal of Immunological Methods, Vol: 458, Pages: 15-20, ISSN: 0022-1759

Introduction: To better understand the immunology of pregnancy, study of female genital tract fluid (FGF) is desirable. However the optimum method of collection of FGF in pregnant women for immunological methods, specifically cytokine measurement, is unknown.Methods:A prospective study of HIV-uninfected pregnant women comparing two methods of FGF collection: polyvinyl acetal sponge collection of cervical fluid (CF) and menstrual cup collection of cervicovaginal fluid (CVF). Samples were collected at 3 time points across the second and third trimesters: 14-21, 22-25 and 26-31 weeks. Multiplex chemi-luminescent assays were used to measure: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13 and TNF-α. Optimal methodology for cytokine normalisation (sample weight, volume and total protein) was explored. ResultsAll cytokines were measurable in both fluid types. IL-1β, IL-8 and IL-6 were detected at the highest concentrations (ranking order CF > CVF > plasma). CVF collection was simpler, provided the largest volume of sample (median 0.5g) with the potential for undiluted usage, and allowed for self-insertion. CF cytokine concentrations were intrinsically associated with sample weight and protein concentration however CVF cytokines were independent of these. Conclusion:Both methods of collection are robust for measurement of FGF cytokines during pregnancy. We recommend CVF collection using a menstrual cup as a viable option in pregnant women for high dimensional biological techniques.

Journal article

Blakney AK, Yilmaz G, McKay PF, Becer CR, Shattock RJet al., 2018, One size does not fit all: The effect of chain length and charge density of poly(ethylene imine) based copolymers on delivery of pDNA, mRNA, and repRNA polyplexes., Biomacromolecules, Vol: 19, Pages: 2870-2879, ISSN: 1525-7797

Nucleic acid delivery systems are commonly translated between different modalities, such as DNA and RNA of varying length and structure, despite physical differences in these molecules that yield disparate delivery efficiency with the same system. Here, we synthesized a library of poly(2-ethyl-2-oxazoline)/poly(ethylene imine) copolymers with varying molar mass and charge densities in order to probe how pDNA, mRNA, and RepRNA polyplex characteristics affect transfection efficiency. The library was utilized in a full factorial design of experiment (DoE) screening, with outputs of luciferase expression, particle size, surface charge, and particle concentration. The optimal copolymer molar mass and charge density was found as 83 kDa/100%, 72 kDa/100%, and 45 kDa/80% for pDNA, RepRNA, and mRNA, respectively. While 10 of the synthesized copolymers enhanced the transfection efficiency of pDNA and mRNA, only 2 copolymers enhanced RepRNA transfection efficiency, indicating a narrow and more stringent design space for RepRNA. These findings suggest that there is not a "one size fits all" polymer for different nucleic acid species.

Journal article

Aw R, McKay P, Shattock R, Polizzi KMet al., A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization, Protein Expression and Purification, ISSN: 1046-5928

Journal article

Kratochvil S, McKay PF, Chung AW, Kent SJ, Gilmour J, Shattock RJet al., 2018, Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination (vol 8, 1883, 2017), FRONTIERS IN IMMUNOLOGY, Vol: 9, ISSN: 1664-3224

Journal article

Anderson J, Olafsdottir TA, Kratochvil S, McKay PF, östensson M, Persson J, Shattock RJ, Harandi AMet al., 2018, Molecular signatures of a TLR4 agonist-adjuvanted HIV-1 vaccine candidate in humans, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224

Systems biology approaches have recently provided new insights into the mechanisms of action of human vaccines and adjuvants. Here, we investigated early transcriptional signatures induced in whole blood of healthy subjects following vaccination with a recombinant HIV-1 envelope glycoprotein subunit CN54gp140 adjuvanted with the TLR4 agonist glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) and correlated signatures to CN54gp140-specific serum antibody responses. Fourteen healthy volunteers aged 18-45 years were immunized intramuscularly three times at 1-month intervals and whole blood samples were collected at baseline, 6 h, and 1, 3, and 7 days post first immunization. Subtle changes in the transcriptomic profiles were observed following immunization, ranging from over 300 differentially expressed genes (DEGs) at day 1 to nearly 100 DEGs at day 7 following immunization. Functional pathway analysis revealed blood transcription modules (BTMs) related to general cell cycle activation, and innate immune cell activation at early time points, as well as BTMs related to T cells and B cell activation at the later time points post-immunization. Diverse CN54gp140-specific serum antibody responses of the subjects enabled their categorization into high or low responders, at early ( < 1 month) and late (up to 6 months) time points post vaccination. BTM analyses revealed repression of modules enriched in NK cells, and the mitochondrial electron chain, in individuals with high or sustained antigen-specific antibody responses. However, low responders showed an enhancement of BTMs associated with enrichment in myeloid cells and monocytes as well as integrin cell surface interactions. Flow cytometry analysis of peripheral blood mononuclear cells obtained from the subjects revealed an enhanced frequency of CD56 dim NK cells in the majority of vaccines 14 days after vaccination as compared with the baseline. These results emphasize the utility of a systems biology approa

Journal article

Makinde J, Jones C, Bartolf A, Sibeko S, Baden S, Cosgrove C, Shattock RJet al., 2018, Localized cyclical variations in immunoproteins in the female genital tract and the implications on the design and assessment of mucosal infection and therapies, AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Vol: 79, ISSN: 1046-7408

ProblemFluctuating hormones regulate reproductive processes in the female genital tract. Consequent changes in the local immunological environment are likely to affect cellular interaction with infectious agents and the assessment of therapies that target mucosal infections.Method of studyWe compared Softcup and Weck‐Cel sampling protocols and assessed the changes in the concentrations of 39 soluble proteins with menstrual cycle progression in the mucosal and peripheral compartments.ResultsWe demonstrate that the mucosal immunological profile is distinct from serum with inflammatory and migratory signatures that are localized throughout the cycle. The analytes highlighted in the mucosal compartment were generally highest at the follicular phase with a tendency to fall as the cycle progressed through ovulation to the luteal phase.ConclusionOur results underscore the need to consider these localized cyclical differences in studies aimed at assessing the outcome of disease and the efficacy of mucosal vaccines and other therapies.

Journal article

Muir L, McKay PF, Petrova VN, Klymenko OV, Kratochvil S, Pinder CL, Kellam P, Shattock RJet al., 2018, Optimisation ofex vivomemory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; referees: 2 approved], Wellcome Open Research, Vol: 2, Pages: 97-97, ISSN: 2398-502X

Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells. Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry. Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.

Journal article

Pankrac J, Klein K, McKay PF, King DFL, Bain K, Knapp J, Biru T, Wijewardhana CN, Pawa R, Canaday DH, Gao Y, Fidler S, Shattock RJ, Arts EJ, Mann JFSet al., 2018, A heterogeneous human immunodeficiency virus-like particle (VLP) formulation produced by a novel vector system., npj Vaccines, Vol: 3, ISSN: 2059-0105

First identified as the etiological agent behind Acquired Immunodeficiency Syndrome (AIDS) in the early 1980s, HIV-1 has continued to spread into a global pandemic and major public health concern. Despite the success of antiretroviral therapy at reducing HIV-1 viremia and preventing the dramatic CD4+ T-cell collapse, infected individuals remain HIV positive for life. Unfortunately, it is increasingly clear that natural immunity is not, and may never be, protective against this pathogen. Therefore, efficacious vaccine interventions, which can either prevent infection or eradicate the latent viral reservoir and effect cure, are a major medical priority. Here we describe the development of a safe vaccine platform, currently being utilized in on-going prophylactic and therapeutic preclinical studies and consisting of highly heterogeneous virus-like particle formulations that represent the virus diversity within infected individuals. These VLPs contain no 5'LTR, no functional integrase, and have a severely mutated stem loop 1-thereby preventing any potential reverse transcription, integration, and RNA packaging. Furthermore, we demonstrate that these VLPs are morphologically identical to wild-type virus with polyvalent Env in a functional form. Finally, we show that the VLPs are antigenic and capable of generating strong immune recall responses.

Journal article

Kratochvil S, McKay PF, Chung AW, Kent SJ, Gilmour J, Shattock RJet al., 2017, Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224

Antibody subclasses exhibit extensive polymorphisms (allotypes) that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig) G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

Journal article

Pinder CL, kratochvil S, Cizmeci D, Muir L, Guo Y, Shattock R, McKay PFet al., 2017, Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry–Based Ig Capture Assay, Journal of Immunology, ISSN: 1550-6606

We report the development of a novel flow cytometry–based Ig capture assay (ICA) for the identification and sorting of individual Ab-secreting cells based on their Ag reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent Ab analysis, and cloning. We demonstrate the utility of the assay by isolating Ag-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced Ag-specific Abs from these cells and subsequently verified their Ag reactivity via ELISA. Furthermore, we used the ICA to track Ag-specific plasmablast responses in HIV-vaccine recipients over a period of 42 d and performed a head-to-head comparison with a conventional B cell ELISpot. Results were highly comparable, highlighting that this assay is a viable alternative for monitoring Ag-specific plasmablast responses at early time points after infection or vaccination. The ICA provides important added benefits in that phenotypic information can be obtained from the identified Ag-specific cells that can then be captured for downstream applications such as B cell sequencing and/or Ab cloning. We envisage the ICA as being a useful tool in Ab repertoire analysis for future clinical trials.

Journal article

Haidari G, Cope A, Miller A, Venables S, Yan C, Ridgers H, Reijonen K, Hannaman D, Spentzou A, Hayes P, Bouliotis G, Vogt A, Joseph S, Combadiere B, McCormack S, Shattock RJet al., 2017, Combined skin and muscle vaccination differentially impact the quality of effector T cell functions: the CUTHIVAC-001 randomized trial, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322

Targeting of different tissues via transcutaneous (TC), intradermal (ID) and intramuscular (IM) injection has the potential to tailor the immune response to DNA vaccination. In this Phase I randomised controlled clinical trial in HIV-1 negative volunteers we investigate whether the site and mode of DNA vaccination influences the quality of the cellular immune responses. We adopted a strategy of concurrent immunization combining IM injection with either ID or TC administration. As a third arm we assessed the response to IM injection administered with electroporation (EP). The DNA plasmid encoded a MultiHIV B clade fusion protein designed to induce cellular immunity. The vaccine and regimens were well tolerated. We observed differential shaping of vaccine induced virus-specific CD4 + and CD8 + cell-mediated immune responses. DNA given by IM + EP promoted strong IFN-γ responses and potent viral inhibition. ID + IM without EP resulted in a similar pattern of response but of lower magnitude. By contrast TC + IM (without EP) shifted responses towards a more Th-17 dominated phenotype, associated with mucosal and epidermal protection. Whilst preliminary, these results offer new perspectives for differential shaping of desired cellular immunity required to fight the wide range of complex and diverse infectious diseases and cancers.

Journal article

Reuschl AK, Edwards MR, Parker R, Connell DW, Hoang L, Halliday A, Jarvis H, Siddiqui N, Wright C, Bremang S, Newton SM, Beverley P, Shattock R, Kon OM, Lalvani Aet al., 2017, Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways, PLoS Pathogens, Vol: 13, ISSN: 1553-7366

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1β and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myleoid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.

Journal article

This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.

Request URL: http://wlsprd.imperial.ac.uk:80/respub/WEB-INF/jsp/search-html.jsp Request URI: /respub/WEB-INF/jsp/search-html.jsp Query String: respub-action=search.html&id=00698655&limit=30&person=true