103 results found
Gallinaro A, Pirillo MF, Aldon Y, et al., 2022, Persistent immunogenicity of integrase defective lentiviral vectors delivering membrane-tethered native-like HIV-1 envelope trimers, NPJ VACCINES, Vol: 7
Pollock KM, Cheeseman HM, Szubert AJ, et al., 2022, Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial, EClinicalMedicine, Vol: 44, ISSN: 2589-5370
Background: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is a novel technology formulated as a low dose vaccine against COVID-19. Methods: A phase I first-in-human dose-ranging trial of a saRNA COVID-19 vaccine candidate LNP-nCoVsaRNA, was conducted at Imperial Clinical Research Facility, and participating centres in London, UK, between 19th June to 28th October 2020. Participants received two intramuscular (IM) injections of LNP-nCoVsaRNA at six different dose levels, 0.1-10.0μg, given four weeks apart. An open-label dose escalation was followed by a dose evaluation. Solicited adverse events (AEs) were collected for one week from enrolment, with follow-up at regular intervals (1-8 weeks). The binding and neutralisation capacity of anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, immunoblot, SARS-CoV-2 pseudoneutralisation and wild type neutralisation assays. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20). Findings: 192 healthy individuals with no history or serological evidence of COVID-19, aged 18-45 years were enrolled. The vaccine was well tolerated with no serious adverse events related to vaccination. Seroconversion at week six whether measured by ELISA or immunoblot was related to dose (both p<0.001), ranging from 8% (3/39; 0.1μg) to 61% (14/23; 10.0μg) in ELISA and 46% (18/39; 0.3μg) to 87% (20/23; 5.0μg and 10.0μg) in a post-hoc immunoblot assay. Geometric mean (GM) anti-S IgG concentrations ranged from 74 (95% CI, 45-119) at 0.1μg to 1023 (468-2236) ng/mL at 5.0μg (p<0.001) and was not higher at 10.0μg. Neutralisation of SARS-CoV-2 by participant sera was measurable in 15% (6/39; 0.1μg) to 48% (11/23; 5.0μg) depending on dose level received. Interpretation: Encapsulated saRNA is safe for clinical development, is immunogenic at low dose levels but failed to induce 100% seroconversion. Modifications to optimis
Blakney AK, McKay PF, Hu K, et 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.
Peter AS, Roth E, Schulz SR, et al., 2021, A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model, EUROPEAN JOURNAL OF IMMUNOLOGY, ISSN: 0014-2980
Aldon Y, McKay PF, Herrero JM, et al., 2021, Immunogenicity of stabilized HIV-1 Env trimers delivered by self-amplifying mRNA, Molecular Therapy - Nucleic Acids, Vol: 25, Pages: 483-493, ISSN: 2162-2531
Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLX), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an IgG2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.
Zhou J, Peacock T, Brown J, et al., 2021, Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.
<jats:title>Abstract</jats:title> <jats:p>SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.</jats:p>
Zhou J, Peacock TP, Brown JC, et al., 2021, Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway
<jats:title>Abstract</jats:title><jats:p>SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.</jats:p>
McKay P, Murray S, 2021, Chlamydia trachomatis: cell biology, immunology and vaccination, Vaccine, Vol: 39, Pages: 2965-2975, ISSN: 0264-410X
Chlamydia trachomatis is the causative agent of a highly prevalent sexually transmitted bacterial disease and is associated with a number of severe disease complications. Current therapy options are successful at treating disease, but patients are left without protective immunity and do not benefit the majority asymptomatic patients who do not seek treatment. As such, there is a clear need for a broad acting, protective vaccine that can prevent transmission and protect against symptomatic disease presentation. There are three key elements that underlie successful vaccine development: 1) Chlamydia biology and immune-evasion adaptations, 2) the correlates of protection that prevent disease in natural and experimental infection, 3) reflection upon the evidence provided by previous vaccine attempts. In this review, we give an overview of the unique intra-cellular biology of C. trachomatis and give insight into the dynamic combination of adaptations that allow Chlamydia to subvert host immunity and survive within the cell. We explore the current understanding of chlamydial immunity in animal models and in humans and characterise the key immune correlates of protection against infection. We discuss in detail the specific immune interactions involved in protection, with relevance placed on the CD4+ T lymphocyte and B lymphocyte responses that are key to pathogen clearance. Finally, we provide a timeline of C. trachomatis vaccine research to date and evaluate the successes and failures in development so far. With insight from these three key elements of research, we suggest potential solutions for chlamydial vaccine development and promising avenues for further exploration.
Spencer AJ, McKay PF, Belij-Rammerstorfer S, et 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.
Blakney A, McKay P, Bouton C, et 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.
Brown JC, Goldhill DH, Zhou J, et al., 2021, Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape
<jats:title>Abstract</jats:title><jats:p>Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.</jats:p>
Blakney AK, McKay PF, 2021, Next-generation COVID-19 vaccines: here come the proteins Comment, LANCET, Vol: 397, Pages: 643-645, ISSN: 0140-6736
Blakney A, Deletic P, McKay P, et al., 2021, Effect of complexing lipids on cellular uptake and expression of messengerRNA in human skin explants, Journal of Controlled Release, Vol: 330, Pages: 1250-1261, ISSN: 0168-3659
Messenger RNA (mRNA) represents a promising next-generation approach for both treatment and vaccination. Lipid based particles are one of the most investigated delivery systems for mRNA formulations. Here we explore how the complexing lipid affects uptake and translation of lipoplex-delivered RNA in resident cells in human skin explants and, we explore a more modular delivery system that utilizes mRNA added to pre-formed nanoparticles prior to dosing. We prepared formulations of lipoplexes with ionizable, cationic or zwitterionic lipids, externally complexed these with mRNA, and observed which cells internalized and/or expressed the mRNA over 72 h after intradermal injections into primary, human, skin explants. Using a flow cytometry panel to assess cellular phenotypes, mRNA uptake and mRNA expression, we found that, unlike other cell types, adipocytes expressed mRNA efficiently at 4 and 24 h after mRNA-lipoplex injection and contributed the greatest proportion of total RNA-encoded protein expression, despite being the lowest frequency cell type. Other cell types (epithelial cells, fibroblasts, T cells, B cells, dendritic cells, monocytes, NK cells, Langerhans cells, and leukocytes) had increasing mRNA expression over the course of 72 h, irrespective of lipoplex formulation. We observed that overall charge of the particle, but not the complexing lipid classification, was predictive for the pattern of mRNA uptake and expression among resident cell types in this model.This study provides insight into maximizing protein expression, using modular mRNA lipoplexes that are more compatible with product development, in a clinically relevant, human skin explant model.
Spencer AJ, McKay PF, Belij-Rammerstorfer S, et 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>
Samnuan K, Blakney A, McKay P, et al., 2021, Design-of-experiments in vitro transcription yield optimization of self-amplifying RNA, Publisher: Cold Spring Harbor Laboratory
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.
Pinder CL, McKay PF, Shattock RJ, 2021, Use of chlamydial elementary bodies as probes to isolate pathogen-specific human monoclonal antibodies., Methods in Molecular Biology, Vol: 2183, Pages: 19-28, ISSN: 1064-3745
Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.
Siris S, Gladstone CA, Guo Y, et al., 2021, Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs) Using Bacterial Whole Cells as Molecular Probes., Methods Mol Biol, Vol: 2183, Pages: 9-18
The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.
J C, Najer A, Blakney A, et al., 2020, Neutrophils enable local and non-invasive liposome delivery to inflamed skeletal muscle and ischemic heart, Advanced Materials, Vol: 32, Pages: 1-10, ISSN: 0935-9648
Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non‐invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil‐mediated delivery system able to transport drug‐loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome‐loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug‐loaded liposomes to an inflamed skeletal muscle in mice. A single low‐dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome‐loaded neutrophils in a human‐disease‐relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier‐loaded neutrophils as a universal platform to deliver anti‐inflammatory drugs to promote tissue regeneration in inflammatory diseases.
Gurnani P, Blakney AK, Terracciano R, et al., 2020, The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA, BIOMACROMOLECULES, Vol: 21, Pages: 3242-3253, ISSN: 1525-7797
Mann JFS, Pankrac J, Klein K, et al., 2020, A targeted reactivation of latent HIV-1 using an activator vector in patient samples from acute infection., EBioMedicine, Vol: 59, Pages: 1-15, ISSN: 2352-3964
BACKGROUND: During combined anti-retroviral treatment, a latent HIV reservoir persists within resting memory CD4 T cells that initiates viral recrudescence upon treatment interruption. Strategies for HIV-1 cure have largely focused on latency reversing agents (LRAs) capable of reactivating and eliminating this viral reservoir. Previously investigated LRAs have largely failed to achieve a robust latency reversal sufficient for reduction of latent HIV pool or the potential of virus-free remission in the absence of treatment. METHODS: We utilize a polyvalent virus-like particle (VLP) formulation called Activator Vector (ACT-VEC) to 'shock' provirus into transcriptional activity. Ex vivo co-culture experiments were used to evaluate the efficacy of ACT-VEC in relation to other LRAs in individuals diagnosed and treated during the acute stage of infection. IFN-γ ELISpot, qRT-PCR and Illumina MiSeq were used to evaluate antigenicity, latency reversal, and diversity of induced virus respectively. FINDINGS: Using samples from HIV+ patients diagnosed and treated at acute/early infection, we demonstrate that ACT-VEC can reverse latency in HIV infected CD4 T cells to a greater extent than other major recall antigens as stimuli or even mitogens such as PMA/Iono. Furthermore, ACT-VEC activates more latent HIV-1 than clinically tested HDAC inhibitors or protein kinase C agonists. INTERPRETATION: Taken together, these results show that ACT-VEC can induce HIV reactivation from latently infected CD4 T cells collected from participants on first line combined antiretroviral therapy for at least two years after being diagnosed and treated at acute/early stage of infection. These findings could provide guidance to possible targeted cure strategies and treatments. FUNDING: NIH and CIHR.
McKay PF, Hu K, Blakney AK, et 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.
Blakney AK, Liu R, Yilmaz G, et al., 2020, Precisely targeted gene delivery in human skin using supramolecular cationic glycopolymers, POLYMER CHEMISTRY, Vol: 11, Pages: 3768-3774, ISSN: 1759-9954
Blakney AK, Abdouni Y, Yilmaz G, et al., 2020, Mannosylated Poly(ethylene imine) Copolymers Enhance saRNA Uptake and Expression in Human Skin Explants, BIOMACROMOLECULES, Vol: 21, Pages: 2482-2492, ISSN: 1525-7797
Blakney AK, Zhu Y, McKay PF, et al., 2020, Big is beautiful: enhanced saRNA delivery and immunogenicity by a higher molecular weight, bioreducible, cationic polymer, ACS Nano, Vol: 14, Pages: 5711-5727, ISSN: 1936-0851
Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to mRNA (mRNA), thus minimizing the required dose. However, previous delivery strategies were optimized for siRNA or mRNA and do not necessarily deliver saRNA efficiently due to structural differences of these RNAs, thus motivating the development of saRNA delivery platforms. Here, we engineer a bioreducible, linear, cationic polymer called “pABOL” for saRNA delivery and show that increasing its molecular weight enhances delivery both in vitro and in vivo. We demonstrate that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics.
Aldon Y, Kratochvil S, Shattock R, et 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.
Blakney AK, McKay PF, Yus BI, et 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.
Blakney AK, McKay PF, Christensen D, et 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.
Blakney AK, McKay PF, Ibarzo Yus B, et 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.
McKay P, Cizmeci D, Aldon Y, et 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.
Liu R, Blakney AK, Gokhan Y, et 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
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