83 results found
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
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, 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.
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, 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.
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
McKay PF, Aldon Y, Groepper C, et 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
Aldon Y, McKay PF, Blakney A, et 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
Aldon Y, McKay PF, Allen J, et 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.
Cheeseman HM, Day S, McFarlane LR, et 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.
Aw R, McKay P, Shattock R, et 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.
Petrova V, Muir L, McKay P, et al., 2018, Combined influence of B-cell receptor rearrangement and somatic hypermutation on B-cell class-switch fate in health and in chronic lymphocytic leukaemia, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens.BCRs are generated through genetic recombination and can be diversified throughsomatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoirediversity can vary substantially between different health conditions. We use isotyperesolvedBCR sequencing to compare B-cell evolution and class-switch fate in healthyindividuals and in patients with chronic lymphocytic leukemia (CLL). We show that thepatterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. Weidentify distinct properties of clonal expansion that lead to the generation of antibodiesof different classes in healthy, malignant, and non-malignant CLL BCR repertoires. Wefurther demonstrate that BCR diversity is affected by relationships between antibodyvariable and constant regions leading to isotype-specific signatures of variable geneusage. This study provides powerful insights into the mechanisms underlying the evolutionof the adaptive immune responses in health and their aberration during disease.
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.
Blakney AK, Yilmaz G, McKay PF, et 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.
Gao Y, McKay PF, Mann J, 2018, Advances in HIV-1 vaccine development, Viruses, Vol: 10, ISSN: 1999-4915
An efficacious HIV-1 vaccine is regarded as the best way to halt the ongoing HIV-1 epidemic. However, despite significant efforts to develop a safe and effective vaccine, the modestly protective RV144 trial remains the only efficacy trial to provide some level of protection against HIV-1 acquisition. This review will outline the history of HIV vaccine development, novel technologies being applied to HIV vaccinology and immunogen design, as well as the studies that are ongoing to advance our understanding of vaccine-induced immune correlates of protection.
Aw R, McKay P, Shattock R, et 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, ISSN: 1046-5928
Kratochvil S, McKay PF, Chung AW, et 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
Anderson J, Olafsdottir TA, Kratochvil S, et 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
Muir L, McKay PF, Petrova VN, et 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.
Pankrac J, Klein K, McKay PF, et 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.
Kratochvil S, McKay PF, Chung AW, et 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.
Pinder CL, kratochvil S, Cizmeci D, et 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.
Muir L, McKay P, Petrova V, et al., 2017, Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; peer review: 2 approved], Wellcome Open Research, Vol: 2, 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 cultured ex 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.
Kratochvil S, McKay PF, Kopycinski JT, et al., 2017, A phase 1 human immunodeficiency virus vaccine Trial for cross-profiling the kinetics of serum and mucosal antibody responses to CN54gp140 modulated by two homologous prime-boost vaccine regimens, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224
A key aspect to finding an efficacious human immunodeficiency virus (HIV) vaccine is the optimization of vaccine schedules that can mediate the efficient maturation of protective immune responses. In the present study, we investigated the effect of alternate booster regimens on the immune responses to a candidate HIV-1 clade C CN54gp140 envelope protein, which was coadministered with the TLR4-agonist glucopyranosyl lipid A-aqueous formulation. Twelve study participants received a common three-dose intramuscular priming series followed by a final booster at either 6 or 12 months. The two homologous prime-boost regimens were well tolerated and induced CN54gp140-specific responses that were observed in both the systemic and mucosal compartments. Levels of vaccine-induced IgG-subclass antibodies correlated significantly with FcγR engagement, and both vaccine regimens were associated with strikingly similar patterns in antibody titer and FcγR-binding profiles. In both groups, identical changes in the antigen (Ag)-specific IgG-subclass fingerprint, leading to a decrease in IgG1 and an increase in IgG4 levels, were modulated by booster injections. Here, the dissection of immune profiles further supports the notion that prime-boost strategies are essential for the induction of diverse Ag-specific HIV-1 responses. The results reported here clearly demonstrate that identical responses were effectively and safely induced by both vaccine regimens, indicating that an accelerated 6-month regimen could be employed for the rapid induction of immune responses against CN54gp140 with no apparent impact on the overall quality of the induced immune response. (This study has been registered at http://ClinicalTrials.gov under registration no. NCT01966900.)
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