Imperial College London

ProfessorRobinShattock

Faculty of MedicineDepartment of Infectious Disease

Chair in Mucosal Infection and Immunity
 
 
 
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Contact

 

+44 (0)20 7594 5206r.shattock

 
 
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Location

 

453Wright Fleming WingSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
to

296 results found

Blakney AK, McKay PF, Hu K, Samnuan K, Jain N, Brown A, Thomas A, Rogers P, Polra K, Sallah H, Yeow J, Zhu Y, Stevens MM, Geall A, Shattock RJet al., 2021, Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines, Journal of Controlled Release, Vol: 338, Pages: 201-210, ISSN: 0168-3659

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Journal article

Sliepen K, Schermer E, Bontjer I, Burger JA, Levai RF, Mundsperger P, Brouwer PJM, Tolazzi M, Farsang A, Katinger D, Moore JP, Scarlatti G, Shattock RJ, Sattentau QJ, Sanders RWet al., 2021, Interplay of diverse adjuvants and nanoparticle presentation of native-like HIV-1 envelope trimers, NPJ VACCINES, Vol: 6

Journal article

Spencer AJ, McKay PF, Belij-Rammerstorfer S, Ulaszewska M, Bissett CD, Hu K, Samnuan K, Blakney AK, Wright D, Sharpe HR, Gilbride C, Truby A, Allen ER, Gilbert SC, Shattock RJ, Lambe Tet al., 2021, Heterologous vaccination regimens with self-amplifying RNA and adenoviral COVID vaccines induce robust immune responses in mice, Nature Communications, Vol: 12, ISSN: 2041-1723

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two-dose heterologous vaccination regimens than single-dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells, which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.

Journal article

van de Berg D, Kis Z, Behmer CF, Samnuan K, Blakney A, Kontoravdi K, Shattock R, Shah Net al., 2021, Quality by design modelling to support rapid RNA vaccine production against emerging infectious diseases, npj Vaccines, Vol: 6, ISSN: 2059-0105

Rapid-response vaccine production platform technologies, including RNA vaccines, are being developed to combat viral epidemics and pandemics. A key enabler of rapid response is having quality-oriented disease-agnostic manufacturing protocols ready ahead of outbreaks. We are the first to apply the Quality by Design (QbD) framework to enhance rapid-response RNA vaccine manufacturing against known and future viral pathogens. This QbD framework aims to support the development and consistent production of safe and efficacious RNA vaccines, integrating a novel qualitative methodology and a quantitative bioprocess model. The qualitative methodology identifies and assesses the direction, magnitude and shape of the impact of critical process parameters (CPPs) on critical quality attributes (CQAs). The mechanistic bioprocess model quantifies and maps the effect of four CPPs on the CQA of effective yield of RNA drug substance. Consequently, the first design space of an RNA vaccine synthesis bioreactor is obtained. The cost-yield optimization together with the probabilistic design space contribute towards automation of rapid-response, high-quality RNA vaccine production.

Journal article

Blakney A, McKay P, Bouton C, Hu K, Samnuan K, Shattock Ret al., 2021, Innate inhibiting proteins enhance expression and immunogenicity of self-amplifying RNA, Molecular Therapy, Vol: 29, Pages: 1174-1185, ISSN: 1525-0016

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. Here, we screen a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determine the effect on protein expression and immunogenicity. We observed that the PIV-5 V and MERS-CoV ORF4a proteins enhance protein expression 100-500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent JAK/STAT inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the RABV-specific IgG titer and neutralization IC50 by ~10-fold in rabbits, but not mice or rats. These experiments provide a proof-of-concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Journal article

Short C-E, Brown R, Quinlan R, Lee Y, Smith A, Marchesi J, Shattock R, Bennett P, Taylor G, MacIntyre Det al., 2021, Lactobacillus-depleted vaginal microbiota in pregnant women living with HIV-1 infection are associated with increased local inflammation and preterm birth, Frontiers in Cellular and Infection Microbiology, Vol: 10, ISSN: 2235-2988

Background: Pregnant women living with HIV-1 infection (PWLWH) have an elevated risk of preterm birth (PTB) of unknown aetiology, which remains after successful suppression of HIV. Women at high risk for HIV have a common bacterial profile which has been associated with poor birth outcomes. We set out to explore factors associated with gestational age at delivery of PWLWH in a UK population.Methods: Prospective study of PWLWH (n = 53) in whom the vaginal microbiota and cervicovaginal cytokine milieu were assessed using metataxonomics and multiplexed immunoassays, respectively. Cross-sectional characterisation of vaginal microbiota in PWLWH were compared with 22 HIV uninfected pregnant women (HUPW) at a similar second trimester timepoint. Within PWLWH the relationships between bacterial composition, inflammatory response, and gestational age at delivery were explored.Findings: There was a high rate of PTB among PWLWH (12%). In the second trimester the vaginal microbiota was more diverse in PWLWH than in HUPW (Inverse Simpson Index, p = 0.0004 and Species Observed, p = 0.009). PWLWH had a lower prevalence of L. crispatus dominant vaginal microbiota group (VMB I, 15 vs 54%) than HUPW and higher prevalence of L. iners dominant (VMB III, 36 vs 9% and VMB IIIB, 15 vs 5%) and mixed anaerobes (VMB IV, 21 vs 0%). Across the second and third trimesters in PWLWH, VMB III/IIIB and IV were associated with PTB and with increased local inflammation [cervicovaginal fluid (CVF) cytokine concentrations in upper quartile]. High bacterial diversity and anaerobic bacterial abundance were also associated with CVF pro-inflammatory cytokines, most notably IL-1β.Interpretation: There is an association between local inflammation, vaginal dysbiosis and PTB in PWLWH. Understanding the potential of antiretroviral therapies to influence this cascade will be important to improve birth outcomes in this population.

Journal article

Spencer AJ, McKay PF, Belij-Rammerstorfer S, Ulaszewska M, Bissett CD, Hu K, Samnuan K, Blakney AK, Wright D, Sharpe HR, Gilbride C, Truby A, Allen ER, Gilbert SC, Shattock RJ, Lambe Tet al., 2021, Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce robust immune responses in mice, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1<jats:sup>+</jats:sup> CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.</jats:p>

Working paper

Samnuan K, Blakney AK, McKay PF, Shattock RJet al., 2021, Design-of-Experiments In Vitro Transcription Yield Optimization of Self-Amplifying RNA

<jats:title>Abstract</jats:title><jats:p>Self-amplifying RNA (saRNA) vaccines are able to induce a higher antigen-specific immune response with a more cost-effective and rapid production process compared to plasmid DNA vaccines. saRNAs are synthesized through in vitro transcription (IVT) however; this process has mainly been optimized for relatively short mRNAs. Here, we optimized the IVT process for long saRNAs, approximately 9.4 kb through a design of experiment (DoE) approach to produce a maximal RNA yield and validated the optimal IVT method on various sizes of RNA. We found that magnesium has the highest impact on RNA yield with acetate ions enabling a higher yield than chloride ions. In addition, the interaction between magnesium and nucleoside triphosphates (NTPs) is highly essential for IVT. Further addition of sodium acetate (NaOAc) during IVT provided no added benefit in RNA yield. Moreover, pyrophosphatase was not essential for productive IVT. The optimal IVT method can be used to synthesize different lengths of RNA. These findings emphasize the ability to synthesize high quality and quantity of saRNA through IVT and that the optimal amount of each component is essential for their interactions to produce a high RNA yield.</jats:p>

Journal article

Sandbrink JB, Shattock RJ, 2020, RNA vaccines: a suitable platform for tackling emerging pandemics?, Frontiers in Immunology, Vol: 11, Pages: 1-9, ISSN: 1664-3224

The COVID-19 pandemic demonstrates the ongoing threat of pandemics caused by novel, previously unrecognized, or mutated pathogens with high transmissibility. Currently, vaccine development is too slow for vaccines to be used in the control of emerging pandemics. RNA-based vaccines might be suitable to meet this challenge. The use of an RNA-based delivery mechanism promises fast vaccine development, clinical approval, and production. The simplicity of in vitro transcription of mRNA suggests potential for fast, scalable, and low-cost manufacture. RNA vaccines are safe in theory and have shown acceptable tolerability in first clinical trials. Immunogenicity of SARS-CoV-2 mRNA vaccines in phase 1 trials looks promising, however induction of cellular immunity needs to be confirmed and optimized. Further optimization of RNA vaccine modification and formulation to this end is needed, which may also enable single injection regimens to be achievable. Self-amplifying RNA vaccines, which show high immunogenicity at low doses, might help to improve potency while keeping manufacturing costs low and speed high. With theoretical properties of RNA vaccines looking promising, their clinical efficacy is the key remaining question with regard to their suitability for tackling emerging pandemics. This question might be answered by ongoing efficacy trials of SARS-CoV-2 mRNA vaccines.

Journal article

Gurnani P, Blakney AK, Yeow J, Bouton CR, Shattock RJ, Stevens MM, Alexander Cet al., 2020, An improved synthesis of poly(amidoamine)s for complexation with self-amplifying RNA and effective transfection, Polymer Chemistry, Vol: 11, Pages: 5861-5869, ISSN: 1759-9954

Cationic polymers are widely used as materials to condense nucleic acids for gene-based therapies. These have been developed to mainly deliver DNA and RNA for cancer therapies but the ongoing COVID-19 pandemic has demonstrated an urgent need for new DNA and RNA vaccines. Given this, suitable manufacturing conditions for such cationic polymers which can protect the nucleic acid in the formulation and delivery stages but release the cargo in the correct cellular compartment effectively and safely are required. A number of polymers based on poly(amidoamine)s fit these criteria but their syntheses can be time-consuming, inefficient and poorly reproducible, precluding their adoption as manufacturable vaccine excipients. Here we report an improved synthesis of poly(cystamine bisacrylamide-co-4-amino-1-butanol), abbreviated as pABOL, via modifications in concentration, reaction time and reaction conditions. Optimisation of monomer contents and stoichiometries, solvents, diluents and temperature, combined with the application of microwaves, enabled the preparation of vaccine candidate pABOL materials in 4 h compared to 48 h reported for previous syntheses. These procedures were highly reproducible in multiple repeat syntheses. Transfection experiments with a model RNA showed that polymers of formulation with appropriate molar masses and mass distributions were as effective in model cell lines as polymers derived from the unoptimised syntheses which have been shown to have high efficacy as RNA vaccine formulation candidates.

Journal article

McKay PF, Hu K, Blakney AK, Samnuan K, Brown JC, Penn R, Zhou J, Bouton CR, Rogers P, Polra K, Lin PJC, Barbosa C, Tam YK, Barclay WS, Shattock RJet al., 2020, Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice, Nature Communications, Vol: 11, Pages: 1-7, ISSN: 2041-1723

The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine. We observe remarkably high and dose-dependent SARS-CoV-2 specific antibody titers in mouse sera, as well as robust neutralization of both a pseudo-virus and wild-type virus. Upon further characterization we find that the neutralization is proportional to the quantity of specific IgG and of higher magnitude than recovered COVID-19 patients. saRNA LNP immunizations induce a Th1-biased response in mice, and there is no antibody-dependent enhancement (ADE) observed. Finally, we observe high cellular responses, as characterized by IFN-γ production, upon re-stimulation with SARS-CoV-2 peptides. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic.

Journal article

Aldon Y, Kratochvil S, Shattock R, McKay Pet al., 2020, Chemokine-adjuvanted plasmid DNA induces homing of antigen-specific and non-Antigen-specific B and T cells to the intestinal and genital mucosae, Journal of Immunology, ISSN: 0022-1767

Plasmid DNA is a promising vaccine platform that together with electroporation can elicit significant systemic antibody responses, however immunity at mucosal sites remains low. Here, we sought to program T and B cells to home to the gastro-intestinal and vaginal mucosa using genetic chemokine adjuvants and assessed their impact on immune homeostasis in various distinct immune compartments. Balb/c mice were immunized intramuscularly with plasmid DNA encoding a model antigen HIV-1 Env gp140 (gp140) and selected chemokines/cytokine and boosted intravaginally with gp140 recombinant protein. Isolated splenocytes, intestinal and genital lymphocytes as well as serum and intestinal luminal contents were assessed for antigen-specific reactivity. In addition, flow cytometric analysis was performed to determine the impact on immune homeostasis at these sites. Different molecular chemokine/cytokine adjuvants effected significant alterations to the recruitment of B and T cells to the spleen, vaginal and intestinal mucosae, for example CCL25 enhanced splenic and vaginal antigen-specific T cell responses while CCL28 increased the levels of specific T cells only in the vaginal mucosa. The levels of antibody could be modulated in the systemic circulation, as well as the vaginal vault and intestinal lumen, with CCL20 playing a central role. Our data demonstrate that the CCL20, CCL25 and CCL28 genetic chemokine adjuvants enhance the vaccine antigen-specific humoral and cellular responses and induce homing to the intestinal and female genital mucosae.

Journal article

Haidari G, Day S, Wood M, Ridgers H, Cope A, Fleck S, Yan C, Reijonen K, Hannaman D, Spentzou A, Hayes P, Vogt A, Combadiere B, Cook A, McCormack S, Shattock RJet al., 2019, The safety and immunogenicity of GTU (R) MultiHIV DNA vaccine delivered by transcutaneous and intramuscular injection with or without electroporation in HIV-1 positive subjects on suppressive ART, Frontiers in Immunology, Vol: 10, Pages: 1-8, ISSN: 1664-3224

Previous studies have shown targeting different tissues via the transcutaneous (TC) and intramuscular injection (IM) with or without electroporation (EP) has the potential to trigger immune responses to DNA vaccination. The CUTHIVTHER 001 Phase I/II randomized controlled clinical trial was designed to determine whether the mode of DNA vaccination delivery (TC+IM or EP+IM) could influence the quality and function of induced cellular immune responses compared to placebo, in an HIV positive clade B cohort on antiretroviral therapy (ART). The GTU®MultiHIV B DNA vaccine DNA vaccine encoded a MultiHIV B clade fusion protein to target the cellular response. Overall the vaccine and regimens were safe and well-tolerated. There were robust pre-vaccination IFN-γ responses with no measurable change following vaccination compared to placebo. However, modest intracellular cytokine staining (ICS) responses were seen in the TC+IM group. A high proportion of individuals demonstrated potent viral inhibition at baseline that was not improved by vaccination. These results show that HIV positive subjects with nadir CD4+ counts ≥250 on suppressive ART display potent levels of cellular immunity and viral inhibition, and that DNA vaccination alone is insufficient to improve such responses. These data suggest that more potent prime-boost vaccination strategies are likely needed to improve pre-existing responses in similar HIV-1 cohorts (This study has been registered at http://ClinicalTrials.gov under registration no. NCT02457689).

Journal article

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, Vol: 37, Pages: 7501-7508, ISSN: 0264-410X

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, Vol: 19, Pages: 1091-1100, 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

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, Vol: 144, Pages: 342-345.e7, 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 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

Nadai Y, Held K, Joseph S, Ahmed MIM, Hoffmann VS, Peterhoff D, Missange M, Bauer A, Joachim A, Reimer U, Zerweck J, McCormack S, Cope A, Tatoud R, Shattock RJ, Robb ML, Sandstroem EG, Hoelscher M, Maboko L, Bakari M, Kroidl A, Wagner R, Weber J, Pollakis G, Geldmacher Cet al., 2019, Envelope-specific recognition patterns of HIV vaccine-induced IgG antibodies are linked to immunogen structure and sequence, Frontiers in Immunology, Vol: 10, Pages: 1-14, ISSN: 1664-3224

Background: A better understanding of the parameters influencing vaccine-induced IgG recognition of individual antigenic regions and their variants within the HIV Envelope protein (Env) can help to improve design of preventive HIV vaccines.Methods: Env-specific IgG responses were mapped in samples of the UKHVC003 Standard Group (UK003SG, n = 11 from UK) and TaMoVac01 (TMV01, n = 17 from Tanzania) HIV vaccine trials. Both trials consisted of three immunizations with DNA, followed by two boosts with recombinant Modified Vaccinia Virus Ankara (MVA), either mediating secretion of gp120 (UK003SG) or the presentation of cell membrane bound gp150 envelopes (TMV01) from infected cells, and an additional two boosts with 5 μg of CN54gp140 protein adjuvanted with glucopyranosyl lipid adjuvant (GLA). Env immunogen sequences in UK003SG were solely based on the clade C isolate CN54, whereas in TMV01 these were based on clades A, C, B, and CRF01AE. The peptide microarray included 8 globally representative Env sequences, CN54gp140 and the MVA-encoded Env immunogens from both trials, as well as additional peptide variants for hot spots of immune recognition.Results: After the second MVA boost, UK003SG vaccinees almost exclusively targeted linear, non-glycosylated antigenic regions located in the inter-gp120 interface. In contrast, TMV01 recipients most strongly targeted the V2 region and an immunodominant region in gp41. The V3 region was frequently targeted in both trials, with a higher recognition magnitude for diverse antigenic variants observed in the UK003SG (p < 0.0001). After boosting with CN54gp140/GLA, the overall response magnitude increased with a more comparable recognition pattern of antigenic regions and variants between the two trials. Recognition of most immunodominant regions within gp120 remained significantly stronger in UK003SG, whereas V2-region recognition was not boosted in either group.Conclusions: IgG recognition of linear antigenic Env regions differe

Journal article

Liu R, Blakney AK, Gokhan Y, Mckay PF, Shattock RJ, Becer Ret al., 2019, Cationic star-shaped glycopolymer brushes for targeted gene delivery, 257th National Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727

Conference paper

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

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

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

McKay PF, Aldon Y, Groepper C, Mottl J, Vogel A, Shattock RJet al., 2018, Development and Pre-clinical Immunogenicity of a Formulated saRNA Replicon Vaccine Expressing Designed Native-like ConSOSL.UFO HIV Env Glycoproteins, HIV Research for Prevention Meeting (HIVR4P) - AIDS Vaccine, Microbicide and ARV-Based Prevention Science, Publisher: MARY ANN LIEBERT, INC, Pages: 333-333, ISSN: 0889-2229

Conference paper

Aldon Y, McKay PF, Blakney A, Shattock RJet al., 2018, Mumps and PIV5 Pseudotyped Virus-like Particles for HIV-1 Env Trimer Display, HIV Research for Prevention Meeting (HIVR4P) - AIDS Vaccine, Microbicide and ARV-Based Prevention Science, Publisher: MARY ANN LIEBERT, INC, Pages: 325-325, ISSN: 0889-2229

Conference paper

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