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

285 results found

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

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

Journal article

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

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

Journal article

Jha A, Thwaites RS, Tunstall T, Kon OM, Shattock RJ, Openshaw PJ, Hansel TTet al., 2018, Human Nasal Challenge with TLR7/8 Agonist Resiquimod (R848) Induces Mucosal Interferon-alpha, with Increased Responsiveness in Asthmatic Volunteers, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X

Conference paper

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

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

Journal article

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

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

Journal article

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

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

Journal article

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

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

Journal article

Fischetti L, Zhong Z, Pinder CL, Tregoning JS, Shattock RJet al., 2017, The synergistic effects of combining TLR ligand based adjuvants on the cytokine response are dependent upon p38/JNK signalling., Cytokine, Vol: 99, Pages: 287-296, ISSN: 1043-4666

Toll like receptor (TLR) ligands are important adjuvant candidates, causing antigen presenting cells to release inflammatory mediators, leading to the recruitment and activation of other leukocytes. The aim of this study was to define the response of human blood derived dendritic cells and macrophages to three TLR ligands acting singly or in combination, Poly I:C (TLR3), GLA (TLR4) and R848 (TLR7/8). Combinations of TLR agonists have been shown to have a synergistic effect on individual cytokines, here we look at the global inflammatory response measuring both cytokines and chemokines. Using a custom Luminex assay we saw dose responses in several mediators including CCL3 (MIP1α), IL-1α, IL-1β, IL-12, CXCL10 (IP-10) and IL-6, all of which were significantly increased by the combination of R848 and GLA, even when low dose GLA was added. The synergistic effect was inhibited by specific MAP kinase inhibitors blocking the kinases p38 and JNK but not MEK1. Combining TLR adjuvants also had a synergistic effect on cytokine responses in human mucosal tissue explants. From this we conclude that the combination of R848 and GLA potentiates the inflammatory profile of antigen presenting cells. Since the pattern of inflammatory mediators released can alter the quality and quantity of the adaptive immune response to vaccination, this study informs vaccine adjuvant design.

Journal article

Kratochvil S, McKay PF, Kopycinski JT, Bishop C, Hayes PJ, Muir L, Pinder CL, Cizmeci D, King D, Aldon Y, Wines BD, Hogarth PM, Chung AW, Kent SJ, Held K, Geldmacher C, Dally L, Santos NS, Cole T, Gilmour J, Fidler S, Shattock RJet 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.)

Journal article

Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, Grivel J-C, Shattock RJ, Margolis Let al., 2017, Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions, Scientific Reports, Vol: 7, ISSN: 2045-2322

HIV-1 envelope proteins (Envs) play a critical role in HIV infection. In a correct trimeric conformation, Env mediates virus–cell binding and fusion. Malfunctioning of this machinery renders virions incapable of infecting cells. Each HIV-1 virion carries 10–14 Envs, and therefore a defective Env may not necessarily render a HIV virion non-infectious, since other Env on the same virion may still be functional. Alternatively, it is possible that on a given virion either all the spikes are defective or all are functional. Here, we investigate Env conformations on individual virions using our new nanotechnology, “flow virometry”, and a panel of antibodies that discriminate between various Env conformations. We found that the majority of HIV-1 virions carry either only trimeric (“functional”) or only defective spikes. The relatively small subfraction of virions that carry both functional and nonfunctional Envs contributes little to HIV infection of human lymphoid tissue ex vivo. The observation that the majority of virions exclusively express either functional or nonfunctional forms of Env has important implications for understanding the role of neutralizing and non-neutralizing antibodies in the immune control of HIV infection as well as for the development of effective prophylactic strategies.

Journal article

Aw, McKay, Shattock, Polizzi KMet al., 2017, Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris, AMB Express, Vol: 7, ISSN: 2191-0855

The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 μg mL-1 in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.

Journal article

Joseph S, Quinn K, Greenwood A, Cope A, McKay P, Hayes P, Kopycinski J, Gilmour J, Miller A, Geldmacher C, Nadai Y, Ahmed M, Montefiori D, Dally L, Bouliotis G, Lewis D, Tatoud R, Wagner R, Esteban M, Shattock R, McCormack S, Weber Jet al., 2017, A comparative phase I study of combination, homologous subtype-C DNA, MVA, and Env gp140 protein/adjuvant HIV vaccines in two immunization regimes, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224

There remains an urgent need for a prophylactic HIV vaccine. We compared combined MVA and adjuvanted gp140 to sequential MVA/gp140 after DNA priming. We expected Env-specific CD4+ T-cells after DNA and MVA priming, and Env-binding antibodies in 100% individuals after boosting with gp140 and that combined vaccines would not compromise safety and might augment immunogenicity. Forty volunteers were primed three times with DNA plasmids encoding (CN54) env and (ZM96) gag-pol-nef at 0, 4 and 8 weeks then boosted with MVA-C (CN54 env and gag-pol-nef) and glucopyranosyl lipid adjuvant—aqueous formulation (GLA-AF) adjuvanted CN54gp140. They were randomised to receive them in combination at the same visit at 16 and 20 weeks (accelerated) or sequentially with MVA-C at 16, 20, and GLA-AF/gp140 at 24 and 28 weeks (standard). All vaccinations were intramuscular. Primary outcomes included ≥grade 3 safety events and the titer of CN54gp140-specific binding IgG. Other outcomes included neutralization, binding antibody specificity and T-cell responses. Two participants experienced asymptomatic ≥grade 3 transaminitis leading to discontinuation of vaccinations, and three had grade 3 solicited local or systemic reactions. A total of 100% made anti-CN54gp140 IgG and combining vaccines did not significantly alter the response; geometric mean titer 6424 (accelerated) and 6578 (standard); neutralization of MW965.2 Tier 1 pseudovirus was superior in the standard group (82 versus 45% responders, p = 0.04). T-cell ELISpot responses were CD4+ and Env-dominant; 85 and 82% responding in the accelerated and standard groups, respectively. Vaccine-induced IgG responses targeted multiple regions within gp120 with the V3 region most immunodominant and no differences between groups detected. Combining MVA and gp140 vaccines did not result in increased adverse events and did not significantly impact upon the titer of Env-specific binding antibodies, which were seen in 100% individuals. The ap

Journal article

McKay PF, Mann JFS, Pattani A, Kett V, Aldon Y, King D, Malcolm RK, Shattock Ret al., 2017, Intravaginal immunisation using a novel antigen-releasing ring device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses, Journal of Controlled Release, Vol: 249, Pages: 74-83, ISSN: 1873-4995

The generation of effective levels of antigen-specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze-dried, rod-shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV-1 CN54gp140 protein (167 μg) ± R848 (167 μg) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed.Sheep received a prime-boost vaccination regime comprising intramuscular injection of 100 μg CN54gp140 + 200 μg R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen-specific B cells were measured by flow cytometry at necropsy. Vaccine antigen-specific serum antibody responses were detected in both the intramuscularly-primed and vaginal mucosally-primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen-specific IgG and IgA with the IgA responses 30-fold greater than systemic levels. Importantly, very high numbers of antigen-specific B cells were detected in local genital draining lymph nodes.We have elicited local genital antigen-specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen-specifi

Journal article

Krebs KC, Tian M, Asmal M, Ling B, Nelson K, Henry K, Gibson R, Li Y, Han W, Shattock RJ, Veazey RS, Letvin N, Arts EJ, Gao Yet al., 2016, Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections, AIDS Research and Therapy, Vol: 13, ISSN: 1742-6405

Background:New simian–human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques.Results:Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env’s in the pool, a feature also observed in the HIV establishing new infections in humans.Conclusion:Despite the inability to propagate in primary cells and cell lin

Journal article

Fox J, Tiraboschi JM, Herrera C, Else L, Egan D, Dickinson L, Jackson A, Olejniczak N, Back D, Khoo S, Shattock R, Boffito Met al., 2016, Pharmacokinetic/Pharmacodynamic Investigation of Single-Dose Oral Maraviroc in the Context of HIV-1 Pre-exposure Prophylaxis, JAIDS-Journal of Acquired Immune Deficiency Syndromes, Vol: 73, Pages: 252-257, ISSN: 1525-4135

To investigate the pharmacokinetics/pharmacodynamics ofsingle-dose maraviroc 300 mg in HIV-1 exposure compartments.Maraviroc concentrations in blood, secretions (vaginal, urethral, oral,and rectal), and tissue (vaginal and rectal) were measured, and ex vivochallenge was performed in 54 healthy volunteers to study protectionfrom HIV infection. Maraviroc Cmax occurred within 4 hours inmost compartments. Concentrations from 4 to 72 hours were aboveintracellular (IC) IC90 in all compartments, range 15–8095 ng/mL. MeanAUC0-72 compartment-to-plasma ratios were highest in the rectum (45–819) and urethra (144) compared with the female genital tract (1.6–4.8)and saliva (0.2). No sex differences in AUC0-72 or Cmax were observed.No ex vivo protection from HIV-1BaL occurred in rectal or vaginaltissue. Despite high and sustained concentrations, single-dose maravirocwas not protective against ex vivo challenge of vaginal/rectal tissue.

Journal article

Cheeseman HM, Olejniczak NJ, Rogers PM, Evans AB, King DFL, Ziprin P, Liao H-X, Haynes BF, Shattock RJet al., 2016, Broadly neutralizing antibodies display potential for prevention of HIV-1 infection of mucosal tissue superior to that of nonneutralizing antibodies, Journal of Virology, Vol: 91, ISSN: 1098-5514

Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal

Journal article

Muir L, Mckay PF, Petrova VN, Klymenko OV, Kratochvil S, Kellam P, Shattock RJet al., 2016, Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare subsets in response to effective vs ineffective vaccination, Conference on HIV Research for Prevention (HIV R4P), Publisher: Mary Ann Liebert, Pages: 336-336, ISSN: 0889-2229

Conference paper

Herrera C, Veazey R, Schuetz A, Olejniczak N, Chenine A-L, Phogat S, Sinangil F, Nitayaphan S, Kaewkungwal J, Pitisuttithum P, Rerks-Ngarm S, Michael NL, Robb ML, Excler J-L, O'Connell RJ, Vasan S, Kim JH, Shattock RJet al., 2016, Ex Vivo Evaluation of Mucosal Cytokine Responses to in Vivo Vaccination with ALVAC-HIV/AIDSVAX (R) B/E of Non-human Primates (NHPs) and Humans, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 75-75, ISSN: 0889-2229

Conference paper

Joseph S, Quinn K, Greenwood A, Miller A, Cope AV, Mckay PF, Hayes P, Kopycinski J, Bouliotis G, Gilmour J, McFarlane L, Tatoud R, Shattock RJ, Lewis D, Montefiore D, Esteban M, Wagner R, McCormack S, Weber Jet al., 2016, UK HVC 003: A Phase I Clinical Trial Exploring a Strategy to Maximise HIV Antibody Responses using Subtype C DNA, MVA and GLA Adjuvanted gp140, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 99-99, ISSN: 0889-2229

Conference paper

Herrera C, Harman S, Rogers P, Aldon Y, Armanasco N, Stieh D, Holt J, Nutall J, Shattock RJet al., 2016, Increased Activity of the Entry Inhibitor DS003, a BMS-378806 Analogue, through Binding to the CD4-induced Epitope in HIV-1 gp120, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 233-233, ISSN: 0889-2229

Conference paper

Aldon Y, Kratochvil S, Shattock RJ, McKay PFet al., 2016, Programming T and B cell homing to mucosal sites to induce protective and/or therapeutic vaccination using chemokine-adjuvanted DNA plasmids, Conference on HIV Research for Prevention (HIV R4P), Publisher: Mary Ann Liebert, Pages: 84-84, ISSN: 0889-2229

Conference paper

Herrera C, Kelly C, Shattock RJ, 2016, Preclinical Evaluation of a Reverse Transcriptase and Protease Inhibitory Combination as a Candidate Microbicide, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 233-233, ISSN: 0889-2229

Conference paper

Herrera C, Romas L, Olejniczak N, Plummer F, Shattock RJ, Burgener Aet al., 2016, Preclinical Evaluation of Serpins as Potential Colorectal Microbicides, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 295-295, ISSN: 0889-2229

Conference paper

Cheeseman HM, Rogers P, King DFL, Shattock RJet al., 2016, Peripheral Immune Cells Improve the Inhibitory Activity of Non-neutralising HIV-1-specific Antibodies, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 147-147, ISSN: 0889-2229

Conference paper

Nadai Y, Ahmed MIM, Joseph S, Missanga M, Bauer A, Cope AV, Joachim A, Reimer U, Pollakis G, McCormack S, Tatoud R, Shattock RJ, Robb M, Sandstroem E, Hoelscher M, Bakari M, Maboko L, Kroidl A, Weber J, Geldmacher C, Held Ket al., 2016, Env-specific IgG Responses Induced by Identical and None-identical Immunogen Prime-boost Vaccination Strategies Target Different Antigenic Regions, Conference on HIV Research for Prevention (HIV R4P), Publisher: MARY ANN LIEBERT, INC, Pages: 392-392, ISSN: 0889-2229

Conference paper

Jha A, Progatzky F, Wane M, Thwaites RS, McBrien M, Brimley J, Tunstall T, Shattock RJ, Bugeon L, Openshaw PJM, Dallman MJ, Hansel TTet al., 2016, Human nasal mucosal responses to TLR agonists are mirrored by the zebrafish gill, British Association of Lung Research Summer Congress

Introduction: There are few reliable ways to study respiratory mucosal immune responses to viruses, viral-type toll-like receptor (TLR) agonists and vaccines. To investigate innate immune responses to TLR agonists (TLR3: poly IC/ poly ICLC; TLR7/8: resiquimod), we compared the effects on human nasal mucosa and zebrafish gills in vivo. Methods: Nasal challenge of adult volunteers was performed with saline, poly IC (n=4), poly ICLC (n=4) or resiquimod (n=8; 5 non-atopic, 3 atopic). Nasal mucosal lining fluid (MLF) was obtained by nasosorption at regular intervals up to 24 hours after challenge; nasal obstruction was monitored by peak nasal inspiratory flow (PNIF) and total nasal symptom scores (TNSS). Cytokines and interferons were measured in MLF using electrochemiluminescence on the Meso Scale Discovery (MSD) platform. Adult zebrafish gills were exposed to the same TLR agonists and gene expression was quantified in gill tissue at similar time-points. Results: Nasal challenge with TLR3 agonists failed to elicit any significant responses when compared to saline. In contrast resiquimod (10μg/100μl per nostril) caused a potent induction of cytokines with an early release (1-3 hours) of IFN-α2a, TNF-α and IL-1β and a later release (after 4 hours) of IFN-γ. The 3 volunteers with the highest levels of IFN-α2a were atopic. Six volunteers were asymptomatic and two volunteers had flu-like symptoms. There were no significant changes in clinical correlates of nasal obstruction. After resiquimod administration, but not TLR3 agonists, zebrafish gills showed an immune profile remarkably analogous to human nasal responses. Conclusion: The TLR7/8 agonist resiquimod is a potent mucosal inducer of IFN-α2a, IFN-γ and proinflammatory cytokines, whilst TLR3 agonists failed to stimulate mucosal innate immune responses. Zebrafish gills accurately mimic human nasal mucosal responses following exposure to TLR agonists, offering translational app

Conference paper

Cheeseman HM, Carias AM, Evans AB, Olejniczak NJ, Ziprin P, King DF, Hope TJ, Shattock RJet al., 2016, Expression profile of human Fc receptors in mucosal tissue: implications for antibody-dependent cellular effector functions targeting HIV-1 transmission, PLOS One, Vol: 11, ISSN: 1932-6203

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the ini

Journal article

Cosgrove CA, Lacey CJ, Cope AV, Bartolf A, Morris G, Yan C, Baden S, Cole T, Carter D, Brodnicki E, Shen X, Joseph S, DeRosa SC, Peng L, Yu X, Ferrari G, Seaman M, Montefiori DC, Frahm N, Tomaras GD, Stöhr W, McCormack S, Shattock RJet al., 2016, Comparative immunogenicity of HIV-1 gp140 vaccine delivered by parenteral, and mucosal routes in female volunteers; MUCOVAC2, a randomized two centre study, PLOS One, Vol: 11, ISSN: 1932-6203

BackgroundDefining optimal routes for induction of mucosal immunity represents an important research priority for the HIV-1 vaccine field. In particular, it remains unclear whether mucosal routes of immunization can improve mucosal immune responses.MethodsIn this randomized two center phase I clinical trial we evaluated the systemic and mucosal immune response to a candidate HIV-1 Clade C CN54gp140 envelope glycoprotein vaccine administered by intramuscular (IM), intranasal (IN) and intravaginal (IVAG) routes of administration in HIV negative female volunteers. IM immunizations were co-administered with Glucopyranosyl Lipid Adjuvant (GLA), IN immunizations with 0.5% chitosan and IVAG immunizations were administered in an aqueous gel.ResultsThree IM immunizations of CN54 gp140 at either 20 or 100 μg elicited significantly greater systemic and mucosal antibodies than either IN or IVAG immunizations. Following additional intramuscular boosting we observed an anamnestic antibody response in nasally primed subjects. Modest neutralizing responses were detected against closely matched tier 1 clade C virus in the IM groups. Interestingly, the strongest CD4 T-cell responses were detected after IN and not IM immunization.ConclusionsThese data show that parenteral immunization elicits systemic and mucosal antibodies in women. Interestingly IN immunization was an effective prime for IM boost, while IVAG administration had no detectable impact on systemic or mucosal responses despite IM priming.

Journal article

Herrera C, Armanasco N, García-Pérez J, Ziprin P, Olejniczak N, Alcami J, Nuttall J, Shattock RJet al., 2016, Maraviroc and reverse transcriptase inhibitors combinations as potential pre-exposure prophylaxis candidates, AIDS, Vol: 30, Pages: 1015-1025, ISSN: 0269-9370

Objective: Receptive anal intercourse in both men and women is associated with the highest probability for sexual acquisition of HIV infection. As part of a program to develop an effective prevention strategy, we performed an ex-vivo preclinical evaluation to determine the efficacy of multiple double combinations of maraviroc (MVC) and reverse transcriptase inhibitors (RTIs).Design: The entry inhibitor, MVC, a nucleotide RTI, tenofovir and two non-nucleoside RTIs, UC781 and TMC120 (dapivirine, DPV), were used in double, combinations against a panel of CCR5-using clade B and clade C HIV-1 isolates and against MVC-escape variants. A gel-formulated version of MVC-DPV combination was also tested.Methods: Indicator cells, cocultures of immature dendritic cells with CD4+T cells, and colorectal tissue explants were used to assess antiviral activity of drug combinations.Results: All dual MVC-RTI combinations tested inhibited MVC-sensitive and resistant isolates in cellular and colorectal explants models. All the combinations were positive with no reduction in the activity of MVC. In tissue explants, the combinations against all viral isolates tested produced an increase in the activity of MVC. An initial gel-formulation of MVC-DPV combination showed greater and prolonged antiviral activity of MVC in mucosal tissue explants.Conclusion: This study demonstrates that combinations based on antiretroviral drugs inhibiting HIV transmission at viral entry and reverse transcription have potential as prevention strategies against colorectal transmission of HIV-1 including MVC-resistant isolates. Preclinical evaluation with colorectal tissue explants indicates that a gel-formulation of MVC-DPV is an effective candidate colorectal microbicide.

Journal article

Mann JF, Tregoning JS, Aldon Y, Shattock RJ, McKay PFet al., 2016, CD71 targeting boosts immunogenicity of sublingually delivered influenza haemagglutinin antigen and protects against viral challenge in mice., Journal of Controlled Release, Vol: 232, Pages: 75-82, ISSN: 1873-4995

The delivery of vaccines to the sublingual mucosa is an attractive prospect due to the ease and acceptability of such an approach. However, novel adjuvant and delivery approaches are required to optimally vaccinate at this site. We have previously shown that conjugation of protein antigen to the iron transport molecule, transferrin, can significantly enhance mucosal immune responses. We tested whether conjugating influenza haemagglutinin to transferrin could improve the immune response to sublingually delivered antigen. Transferrin conjugated haemagglutinin induced a significant antibody and T cell response in both naïve animals and previously immunized animals. The immune response generated was able to protect mice against influenza virus challenge. Sublingually administered antigen dispersed more widely through the gastro-intestinal tract than intranasally delivered antigen and transferrin conjugation had a more marked effect on sublingually delivered antigen than intranasal immunisation. From these studies we conclude that transferrin conjugation of antigen is effective at boosting immune responses to sublingually delivered antigen and may be an attractive approach for influenza vaccines, particularly when mass campaigns are required.

Journal article

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