Dr Anita Milicic

Research Area: Immunology
Technology Exchange: Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease
Keywords: Vaccine development, Trained Immunity, Vaccine dose reduction, Vaccine adjuvants and Encapsulation

I studied Molecular Biology and Physiology at the University of Belgrade, Serbia, before completing an MSc in Genetics and DPhil in Immunology at the University of Oxford, UK. This was followed by Post-doc positions at Oxford, studying cellular immunity to HIV, CD8+ T cell activation mechanisms, and currently, vaccine development. 

Over the last few years my research has focused on developing a new vaccine encapsulation technology, with the aim of reducing the number of immunisations required for full protection against a disease. Almost all current vaccines are administered in two or three doses at specific time intervals. Inadequate immunisation coverage remains a major hurdle in the successful implementation of immunisation schedules worldwide, due to missed or mistimed visits. Our goal is to avoid the need for booster vaccinations by providing the booster dose, encapsulated into microcapsules for a later in vivo release, together with the priming vaccine. This would reduce multiple dose immunisation regimens, with the capsule serving either to generate long lasting persistence of the vaccine in the body (continuous release), or by releasing discrete burst(s) of the booster vaccine (pulsatile release). Through a close collaboration with the Institute of Biomedical Engineering at Oxford, we are developing vaccine encapsulation using microfluidics and electro-spraying methods. These technologies use different approaches to precisely tune particle synthesis parameters such as size and antigen loading, and minimise the production of heterogeneous emulsions, which will facilitate the in vivo assessment of the immunogenicity and efficacy of encapsulated formulations.

In parallel, I manage the Jenner Institute Adjuvant Facility and collaborate on vaccine adjuvant research and development with external partners. I am on the Management Committee of an EU COST (Cooperation in Science and Technology) Action Programme ENOVA (European Network of Vaccine Adjuvants) and Working Group leader for Prophylactic Vaccine Adjuvants www.enova-adjuvant.eu

My long-term interest are mechanisms of immune activation, in particular the interplay between the innate and the adaptive immunity. I have recently started looking at different aspects of this interaction in the context of enhancing the protective efficacy of a viral vectored vaccine against malaria, and am looking to broaden my observations to vaccines against other complex intracellular pathogens.

I am also the Jenner Institute lead for Public Engagement with Research (twitter.com/JennerInstitute, facebook.com/jennerinstitute).

Name Department Institution Country
Professor Adrian VS Hill Jenner Institute Oxford University, Old Road Campus Research Building United Kingdom
Professor Eleanor Stride Engineering Science University of Oxford United Kingdom
Dr Jelena Bezbradica Mirkovic University of Oxford United Kingdom
Associate Professor Christine S Rollier Jenner Institute Oxford University, Centre for Clinical Vaccinology and Tropical Medicine United Kingdom
Dr David Wyllie Jenner Institute Oxford University, Henry Wellcome Building for Molecular Physiology United Kingdom
Professor Arturo Reyes-Sandoval Jenner Institute Oxford University, Henry Wellcome Building for Molecular Physiology United Kingdom
Coughlan L, Sridhar S, Payne R, Edmans M, Milicic A, Venkatraman N, Lugonja B, Clifton L, Qi C, Folegatti PM et al. 2018. Corrigendum to "Heterologous Two-dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-lasting Cellular Immunity to Influenza Virus A in Healthy Adults" [EBioMedicine 29 (2018) 146-154]. EBioMedicine, 31 pp. 321. | Show Abstract | Read more

© 2018 The Authors The authors wish to point out that L. Coughlan and S. Sridhar were both at the Jenner Institute, University of Oxford, OX3 7DQ, UK, when the work for the paper was completed.

Coughlan L, Sridhar S, Payne R, Edmans M, Milicic A, Venkatraman N, Lugonja B, Clifton L, Qi C, Folegatti PM et al. 2018. Heterologous Two-Dose Vaccination with Simian Adenovirus and Poxvirus Vectors Elicits Long-Lasting Cellular Immunity to Influenza Virus A in Healthy Adults. EBioMedicine, 29 pp. 146-154. | Show Abstract | Read more

BACKGROUND: T-cell responses against highly conserved influenza antigens have been previously associated with protection. However, these immune responses are poorly maintained following recovery from influenza infection and are not boosted by inactivated influenza vaccines. We have previously demonstrated the safety and immunogenicity of two viral vectored vaccines, modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAdOx1 expressing conserved influenza virus antigens, nucleoprotein (NP) and matrix protein-1 (M1). We now report on the safety and long-term immunogenicity of multiple combination regimes of these vaccines in young and older adults. METHODS: We conducted a Phase I open-label, randomized, multi-center study in 49 subjects aged 18-46years and 24 subjects aged 50years or over. Following vaccination, adverse events were recorded and the kinetics of the T cell response determined at multiple time points for up to 18months. FINDINGS: Both vaccines were well tolerated. A two dose heterologous vaccination regimen significantly increased the magnitude of pre-existing T-cell responses to NP and M1 after both doses in young and older adults. The fold-increase and peak immune responses after a single MVA-NP+M1 vaccination was significantly higher compared to ChAdOx1 NP+M1. In a mixed regression model, T-cell responses over 18months were significantly higher following the two dose vaccination regimen of MVA/ChAdOx1 NP+M1. INTERPRETATION: A two dose heterologous vaccination regimen of MVA/ChAdOx1 NP+M1 was safe and immunogenic in young and older adults, offering a promising vaccination strategy for inducing long-term broadly cross-reactive protection against influenza A. FUNDING SOURCE: Medical Research Council UK, NIHR BMRC Oxford.

Flaxman A, van Diemen PM, Yamaguchi Y, Allen E, Lindemann C, Rollier CS, Milicic A, Wyllie DH. 2017. Development of persistent gastrointestinal S. aureus carriage in mice. Sci Rep, 7 (1), pp. 12415. | Show Abstract | Read more

One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.

Milicic A, S Rollier C, Tang CK, Longley R, Hill AVS, Reyes-Sandoval A. 2017. Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria. Sci Rep, 7 (1), pp. 7284. | Show Abstract | Read more

The majority of routinely given vaccines require two or three immunisations for full protective efficacy. Single dose vaccination has long been considered a key solution to improving the global immunisation coverage. Recent infectious disease outbreaks have further highlighted the need for vaccines that can achieve full efficacy after a single administration. Viral vectors are a potent immunisation platform, benefiting from intrinsic immuno-stimulatory features while retaining excellent safety profile through the use of non-replicating viruses. We investigated the scope for enhancing the protective efficacy of a single dose adenovirus-vectored malaria vaccine in a mouse model of malaria by co-administering it with vaccine adjuvants. Out of 11 adjuvants, only two, Abisco®-100 and CoVaccineHTTM, enhanced vaccine efficacy and sterile protection following malaria challenge. The CoVaccineHTTM adjuvanted vaccine induced significantly higher proportion of antigen specific central memory CD8+ cells, and both adjuvants resulted in increased proportion of CD8+ T cells expressing the CD107a degranulation marker in the absence of IFNγ, TNFα and IL2 production. Our results show that the efficacy of vaccines designed to induce protective T cell responses can be positively modulated with chemical adjuvants and open the possibility of achieving full protection with a single dose immunisation.

Diemen PMV, Leneghan DB, Brian IJ, Miura K, Long CA, Milicic A, Biswas S, Rollier CS, Wyllie DH. 2017. The S. aureus 4-oxalocrotonate tautomerase SAR1376 enhances immune responses when fused to several antigens. Sci Rep, 7 (1), pp. 1745. | Show Abstract | Read more

A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them.

Walters AA, Krastev C, Hill AVS, Milicic A. 2015. Next generation vaccines: single-dose encapsulated vaccines for improved global immunisation coverage and efficacy. J Pharm Pharmacol, 67 (3), pp. 400-408. | Show Abstract | Read more

OBJECTIVES: Vaccination is considered the most successful health intervention; yet incomplete immunisation coverage continues to risk outbreaks of vaccine preventable diseases worldwide. Vaccination coverage improvement through a single-dose prime-boost technology would revolutionise modern vaccinology, impacting on disease prevalence, significantly benefiting health care and lowering economic burden of disease. KEY FINDINGS: Over the past 30 years, there have been efforts to develop a single-dose delayed release vaccine technology that could replace the repeated prime-boost immunisations required for many current vaccines. Biocompatible polymers have been employed to encapsulate model vaccines for delayed delivery in vivo, using either continuous or pulsed release. Biomaterial considerations, safety aspects, particle characteristics and immunological aspects of this approach are discussed in detail. SUMMARY: Despite many studies showing the feasibility of vaccine encapsulation for single-dose prime-boost administration, none have been translated into convincing utility in animal models or human trials. Further development of the encapsulation technology, through optimising the particle composition, formulation, antigen loading efficacy and stability, could lead to the application of this important approach in vaccine deployment. If successful, this would provide a solution to better global vaccination coverage through a reduction in the number of immunisations needed to achieve protection against infectious diseases. This review provides an overview of single-dose vaccination in the context of today's vaccine needs and is derived from a body of literature that has not been reviewed for over a decade.

Sadeyen J-R, Wu Z, Davies H, van Diemen PM, Milicic A, La Ragione RM, Kaiser P, Stevens MP, Dziva F. 2015. Immune responses associated with homologous protection conferred by commercial vaccines for control of avian pathogenic Escherichia coli in turkeys. Vet Res, 46 (1), pp. 5. | Show Abstract | Read more

Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.

Warimwe GM, Lorenzo G, Lopez-Gil E, Reyes-Sandoval A, Cottingham MG, Spencer AJ, Collins KA, Dicks MDJ, Milicic A, Lall A et al. 2013. Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice. Virol J, 10 (1), pp. 349. | Show Abstract | Read more

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. METHODS: Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. RESULTS: A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. CONCLUSIONS: Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

Antrobus RD, Lillie PJ, Berthoud TK, Spencer AJ, McLaren JE, Ladell K, Lambe T, Milicic A, Price DA, Hill AVS, Gilbert SC. 2012. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years. PLoS One, 7 (10), pp. e48322. | Show Abstract | Read more

BACKGROUND: Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults. METHODS: Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach. RESULTS: Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination. CONCLUSIONS: MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT00942071.

Milicic A, Kaur R, Reyes-Sandoval A, Tang C-K, Honeycutt J, Perrie Y, Hill AVS. 2012. Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses. PLoS One, 7 (3), pp. e34255. | Show Abstract | Read more

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFNγ responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang C-K, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AVS. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Mol Ther, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

Cited:

30

Scopus

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang CK, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AVS. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity Molecular Therapy, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (AdMVA) enhanced CD8++T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same AdMVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8++T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the AdMVA mixture led to consistent boosting of the transgene-specific CD8++response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens. © The American Society of Gene & Cell Therapy.

de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P, Chauhan VS, Chitnis CE, Gilbert SC, Hill AVS, Draper SJ. 2011. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus. J Immunol, 187 (5), pp. 2602-2616. | Show Abstract | Read more

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Cited:

79

Scopus

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AVS. 2011. CD8<sup>+</sup>T effector memory cells protect against liver-stage malaria Journal of Immunology, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines.We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8+ T cells into effector, effector memory (TEM), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and TEM response that requires long intervals for an efficient boost. A preferential TEM phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8+ TEM cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Agspecific TEM cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 TEMpopulations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design. Copyright © 2011 by The American Association of Immunologists, Inc.

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AVS. 2011. CD8+ T effector memory cells protect against liver-stage malaria. J Immunol, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

Cited:

187

European Pubmed Central

Berthoud TK, Hamill M, Lillie PJ, Hwenda L, Collins KA, Ewer KJ, Milicic A, Poyntz HC, Lambe T, Fletcher HA et al. 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis, 52 (1), pp. 1-7. | Show Abstract | Read more

BACKGROUND: Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes. METHODS: We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA-NP+M1) and conducted a phase I clinical trial in healthy adults. RESULTS: The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8(+). CONCLUSIONS: We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

Kaur R, Milicic A, Reyes-Sandoval A, Hill A, Perrie Y. 2010. Addition of immunostimulatory components to DDA-TDB adjuvant delivery system JOURNAL OF PHARMACY AND PHARMACOLOGY, 62 (10), pp. 1245-1246.

Choudhuri K, Parker M, Milicic A, Cole DK, Shaw MK, Sewell AK, Stewart-Jones G, Dong T, Gould KG, van der Merwe PA. 2009. Peptide-major histocompatibility complex dimensions control proximal kinase-phosphatase balance during T cell activation. J Biol Chem, 284 (38), pp. 26096-26105. | Show Abstract | Read more

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Varela-Rohena A, Molloy PE, Dunn SM, Li Y, Suhoski MM, Carroll RG, Milicic A, Mahon T, Sutton DH, Laugel B et al. 2008. Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor. Nat Med, 14 (12), pp. 1390-1395. | Show Abstract | Read more

HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.

Milicic A, Harrison L-A, Goodlad RA, Hardy RG, Nicholson AM, Presz M, Sieber O, Santander S, Pringle JH, Mandir N et al. 2008. Ectopic expression of P-cadherin correlates with promoter hypomethylation early in colorectal carcinogenesis and enhanced intestinal crypt fission in vivo. Cancer Res, 68 (19), pp. 7760-7768. | Show Abstract | Read more

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

Laugel B, van den Berg HA, Gostick E, Cole DK, Wooldridge L, Boulter J, Milicic A, Price DA, Sewell AK. 2007. Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties. J Biol Chem, 282 (33), pp. 23799-23810. | Show Abstract | Read more

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.

Purbhoo MA, Li Y, Sutton DH, Brewer JE, Gostick E, Bossi G, Laugel B, Moysey R, Baston E, Liddy N et al. 2007. The HLA A*0201-restricted hTERT(540-548) peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor. Mol Cancer Ther, 6 (7), pp. 2081-2091. | Show Abstract | Read more

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540-548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT(540-548) peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT(540-548) on tumor cells: (a) a CD8(+) CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT(540-548) complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT(540-548) peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications.

Laugel B, Price DA, Milicic A, Sewell AK. 2007. CD8 exerts differential effects on the deployment of cytotoxic T lymphocyte effector functions. Eur J Immunol, 37 (4), pp. 905-913. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) are equipped with a range of effector functions that contribute both to the control of intracellular pathogens and dysregulated cellular proliferation and to the development of certain immunopathologies such as autoimmune disease. Qualitative analyses of various CTL responses have revealed substantial heterogeneity in the diversity of functions that are mobilized in response to antigen. Here, we studied the influence of the CD8 co-receptor, which is known to enhance antigen recognition by CTL, on the secretion of eight different cytokines and chemokines by human CTL clones using flow cytometric bead array. Our results show that abrogation of MHC class I/CD8 interactions exerts a differential influence on the distinct individual effector functions that are elicited in response to agonist ligands. The magnitude of this co-receptor blockade inhibitory effect was clearly related to the hierarchy of cytokine secretion in terms of activation threshold because those functions requiring the highest amounts of antigen were most affected. Thus, modulation of CD8 activity can effectively tune not only the sensitivity but also the qualitative profile of CTL responses.

Zimbwa P, Milicic A, Frater J, Scriba TJ, Willis A, Goulder PJR, Pillay T, Gunthard H, Weber JN, Zhang H-T, Phillips RE. 2007. Precise identification of a human immunodeficiency virus type 1 antigen processing mutant. J Virol, 81 (4), pp. 2031-2038. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.

Frater AJ, Edwards CTT, McCarthy N, Fox J, Brown H, Milicic A, Mackie N, Pillay T, Drijfhout JW, Dustan S et al. 2006. Passive sexual transmission of human immunodeficiency virus type 1 variants and adaptation in new hosts. J Virol, 80 (14), pp. 7226-7234. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences.

Wooldridge L, Scriba TJ, Milicic A, Laugel B, Gostick E, Price DA, Phillips RE, Sewell AK. 2006. Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers. Eur J Immunol, 36 (7), pp. 1847-1855. | Show Abstract | Read more

The T cell coreceptors CD8 and CD4 bind to invariable regions of peptide-MHC class I (pMHCI) and class II (pMHCII) molecules, respectively, and facilitate antigen recognition by a number of mechanisms. It is established that some antibodies (Ab) specific for the CD8 molecule, which stabilizes TCR/pMHCI interactions, can alter the binding of pMHCI tetramers to cell surface TCR. In contrast, the extremely weak pMHCII/CD4 interaction does not stabilize TCR/pMHCII interactions or contribute to cognate tetramer binding; consequently, it is assumed that anti-CD4 Ab do not affect pMHCII binding. Here, we used a panel of point-mutated HLA A2 molecules with a range of affinities for CD8 spanning over three orders of magnitude to demonstrate that anti-CD8 Ab-mediated inhibition of pMHCI tetramer binding and cognate T cell activation correlates directly with the strength of the pMHCI/CD8 interaction. Further, some anti-CD4 Ab were found to block pMHCII tetramer binding; these effects were also paralleled in T cell activation assays. In sum, these data challenge the assertion that anti-coreceptor Ab exert their effects on T cell activation and pMHC binding solely by blocking pMHC/coreceptor interactions.

Milicic A, Edwards CTT, Hué S, Fox J, Brown H, Pillay T, Drijfhout JW, Weber JN, Holmes EC, Fidler SJ et al. 2005. Sexual transmission of single human immunodeficiency virus type 1 virions encoding highly polymorphic multisite cytotoxic T-lymphocyte escape variants. J Virol, 79 (22), pp. 13953-13962. | Show Abstract | Read more

Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral "challenge" which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the "donor" and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic T lymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.

Milicic A, Price DA, Zimbwa P, Booth BL, Brown HL, Easterbrook PJ, Olsen K, Robinson N, Gileadi U, Sewell AK et al. 2005. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef. J Immunol, 175 (7), pp. 4618-4626. | Show Abstract | Read more

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.

Wooldridge L, van den Berg HA, Glick M, Gostick E, Laugel B, Hutchinson SL, Milicic A, Brenchley JM, Douek DC, Price DA, Sewell AK. 2005. Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface. J Biol Chem, 280 (30), pp. 27491-27501. | Show Abstract | Read more

The off-rate (k(off)) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a "stabilization factor" that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.

Steer S, Fisher SA, Fife MS, Lad B, Grumley J, Milicic A, Wordsworth P, Worthington J, Lewis CM, Repository ARCN. 2004. REFINEMENT OF THE CRH HAPLOTYPE IN RHEUMATOID ARTHRITIS RHEUMATOLOGY, 43 pp. 35-35.

Milicic A, Misra R, Agrawal S, Aggarwal A, Brown MA, Wordsworth BP. 2003. The F158V polymorphism in Fc gamma RIIIA shows disparate associations with rheumatoid arthritis in two genetically distinct populations (vol 61, pg 1021, 2002) ANNALS OF THE RHEUMATIC DISEASES, 62 (1), pp. 96-96.

Newton J, Brown MA, Milicic A, Ackerman H, Darke C, Wilson JN, Wordsworth BP, Kwiatkowski D. 2003. The effect of HLA-DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin alpha-tumor necrosis factor haplotype. Arthritis Rheum, 48 (1), pp. 90-96. | Show Abstract | Read more

OBJECTIVE: HLA-DRB1, a major genetic determinant of susceptibility to rheumatoid arthritis (RA), is located within 1,000 kb of the gene encoding tumor necrosis factor (TNF). Because certain HLA-DRB1*04 subtypes increase susceptibility to RA, investigation of the role of the TNF gene is complicated by linkage disequilibrium (LD) between TNF and DRB1 alleles. By adequately controlling for this LD, we aimed to investigate the presence of additional major histocompatibility complex (MHC) susceptibility genes. METHODS: We identified 274 HLA-DRB1*04-positive cases of RA and 271 HLA-DRB1*04-positive population controls. Each subject was typed for 6 single-nucleotide polymorphisms within a 4.5-kb region encompassing TNF and lymphotoxin alpha (LTA). LTA-TNF haplotypes in these unrelated individuals were determined using a combination of family data and the PHASE software program. RESULTS: Significant differences in LTA-TNF haplotype frequencies were observed between different subtypes of HLA-DRB1*04. The LTA-TNF haplotypes observed were very restricted, with only 4 haplotypes constituting 81% of all haplotypes present. Among individuals carrying DRB1*0401, the LTA-TNF 2 haplotype was significantly underrepresented in cases compared with controls (odds ratio 0.5 [95% confidence interval 0.3-0.8], P = 0.007), while in those with DRB1*0404, the opposite effect was observed (P = 0.007). CONCLUSION: These findings suggest that the MHC contains genetic elements outside the LTA-TNF region that modify the effect of HLA-DRB1 on susceptibility to RA.

MacKay K, Milicic A, Lee D, Tikly M, Laval S, Shatford J, Wordsworth P. 2003. Rheumatoid arthritis susceptibility and interleukin 10: a study of two ethnically diverse populations. Rheumatology (Oxford), 42 (1), pp. 149-153. | Show Abstract | Read more

INTRODUCTION: IL-10 is an immunoregulatory cytokine which may modulate disease expression in rheumatoid arthritis (RA). The IL-10 gene is highly polymorphic with a number of single nucleotide polymorphisms in the promoter region and two microsatellite loci, IL10.R and IL10.G, 4 kb and 1.1 kb 5' of the transcription initiation site. It has been reported that allele 2 of the IL10.R microsatellite (IL10.R2) is associated with increased IL-10 secretion and IL10.R3 with reduced secretion. Subsequently, over-representation of IL10.R2 and under-representation of IL10.R3 in three independent RA groups has been reported. The aim of the current study is to determine whether there is an association between the IL10.R2 allele and RA in two ethnically distinct populations. METHODS: IL10.R genotypes were determined by semi-automated DNA sequencing technology in 186 UK Caucasians and 138 South Africans of Zulu or Sotho origin, fulfilling the 1987 American College of Rheumatology (ACR) criteria for RA. The Caucasian patients had relatively severe disease and comprised 75 patients with RA vasculitis, 22 with Felty's syndrome and 89 who had undergone a joint replacement (hip or knee) within 15 years of the onset of disease. Allele frequencies were compared with 296 Caucasians and/or 73 South Africans. RESULTS: The frequency of the IL10.R2 allele was significantly greater in the South Africans (RA and controls) than in the Caucasians (0.78 vs 0.66, P=1 x 10(-6)), while the frequency of IL10.R3 was less common (0.16 vs 0.3, P=1 x 10(-8)). No differences were observed in either IL10.R2 or IL10.R3 frequencies between patients and controls in either population. CONCLUSIONS: We were unable to confirm any association between IL10.R alleles and RA in this study. However, significant differences were demonstrated in the frequency of IL10.R2 and IL10.R3 between the two ethnic groups. The relatively high frequency of IL10.R2 in the South African population (0.78) would have reduced the power to detect an association with RA.

Harney S, Newton J, Milicic A, Brown MA, Wordsworth BP. 2003. Non-inherited maternal HLA alleles are associated with rheumatoid arthritis. Rheumatology (Oxford), 42 (1), pp. 171-174. | Show Abstract | Read more

BACKGROUND: Rheumatoid arthritis (RA) is strongly associated with a series of HLA-DRB1 alleles that encode a conserved sequence of amino acids ((70)Q/R K/R R A A(74)) in the DRbeta1 chain, known as the shared epitope (SE). However 30% of patients are negative for DRB1*04 and 15% are SE-negative. Exposure to these alleles as non-inherited maternal antigens (NIMA) might explain this discrepancy. We undertook a family study to investigate the role of NIMA in RA. METHODS: One hundred families, including the RA proband and both parents, were recruited. HLA-DRB1 genotyping was performed using an allele-specific polymerase chain reaction by standard methods. The frequencies of NIMA and non-inherited paternal antigens (NIPA) were compared using contingency tables and a two-tailed P test. We then reviewed four previously published studies of NIMA in RA and conducted an analysis of the combined data RESULTS: We identified 36 families in which the proband was DRB1*04-negative and 13 in which the proband lacked the SE. There was an excess of DRB1*04 and SE NIMA (P=0.05) compared with NIPA. Combined analysis with previous studies showed that 53/231 mothers (23%) versus 25/205 fathers (12%) had a non-inherited DRB1*04 (P=0.003) and 30/99 mothers versus 18/101 fathers had a non-inherited SE allele (P=0.03). CONCLUSION: A role for HLA NIMA in RA is suggested by these results.

Milicic A, Misra R, Agrawal S, Aggarwal A, Brown MA, Wordsworth BP. 2002. The F158V polymorphism in FcgammaRIIIA shows disparate associations with rheumatoid arthritis in two genetically distinct populations. Ann Rheum Dis, 61 (11), pp. 1021-1023. | Show Abstract | Read more

OBJECTIVES: To investigate the association of the FcgammaRIIIA gene with rheumatoid arthritis (RA) in two genetically distinct groups: a white group from the United Kingdom and a northern Indian group. METHODS: The distributions of the two alleles of the FcgammaRIIIA F158V polymorphism were determined in 398 white patients from the United Kingdom and 63 Indian patients with RA and compared with those from 289 United Kingdom and 93 Indian healthy controls, respectively. RESULTS: Among the Indian patients, the frequency of the rare 158V allele and the proportion of 158VV homozygotes were reduced (relative risk (RR)=0.3, 95% confidence interval (95% CI) 0.1 to 1.1, p<0.06), reaching statistical significance for carrying the 158VV phenotype relative to 158FV or FF (RR=0.2, 95% CI 0.05-0.9, p<0.02). Conversely, no significant deviation in allelic frequencies was noted between the patients and controls from the United Kingdom. CONCLUSIONS: The 158VV phenotype showed a weak protective effect against developing RA in the Indian group. However, this sample was small (resulting in a low power for statistical analysis) and no independent confirmation was found in the larger white United Kingdom group. Thus the FcgammaRIIIA locus is unlikely to be of major importance in causing RA.

Milicic A, Lee D, Brown MA, Darke C, Wordsworth BP. 2002. HLA-DR/DQ haplotype in rheumatoid arthritis: novel allelic associations in UK Caucasians. J Rheumatol, 29 (9), pp. 1821-1826. | Show Abstract

OBJECTIVE: To elucidate the relative importance of the HLA-DR and HLA-DQ loci in conferring genetic predisposition to rheumatoid arthritis (RA). METHODS: HLA-DRB1 and HLA-DQB1 alleles were typed in a set of 685 patients with RA using sequence-specific polymerase chain reaction. Allele and phenotype frequencies were compared with those in 2 large sets of historical, ethnically matched healthy controls, using the relative predispositional effect method. RESULTS: Positive association was confirmed with the shared epitope positive HLA-DRB1 alleles associated with RA in Caucasians. A significant susceptibility effect was observed with HLA-DRB1*09, described in other ethnically diverse populations but not in Caucasians. A significant underrepresentation of the HLA-DRB1*0103 variant was noted among the RA cases, supporting the proposed protective role of the DERAA motif at residues 70-74 of the DRbeta molecule. No HLA-DRB1 independent association of the HLA-DQB1 alleles, implicated in predisposing to RA, was evident. CONCLUSION: These data corroborate the shared epitope hypothesis of susceptibility to RA and provide strong evidence for the DRB1 locus as the primary RA susceptibility factor in the HLA region.

MacKay K, Eyre S, Myerscough A, Milicic A, Barton A, Laval S, Barrett J, Lee D, White S, John S et al. 2002. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom (vol 46,pg 632, 2002) ARTHRITIS AND RHEUMATISM, 46 (5), pp. 1406-1406. | Read more

Harney S, Newton J, Milicic A, Wordsworth BP. 2002. NON-INHERITED MATERNAL ANTIGENS INFLUENCE SUSCEPTIBILITY TO RHEUMATOID ARTHRITIS RHEUMATOLOGY, 41 pp. 128-128.

Newton J, Brown MA, Milicic A, Ackerman H, Darke C, Wilson J, Wordsworth BP, Kwiatkowski D. 2002. LTA-TNF HAPLOTYPE STUDIES IDENTIFY ADDITIONAL RHEUMATOID ARTHRITIS SUSCEPTIBILITY REGIONS IN THE MHC RHEUMATOLOGY, 41 pp. 13-13.

MacKay K, Eyre S, Myerscough A, Milicic A, Barton A, Laval S, Barrett J, Lee D, White S, John S et al. 2002. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum, 46 (3), pp. 632-639. | Show Abstract | Read more

OBJECTIVE: To undertake a systematic whole-genome screen to identify regions exhibiting genetic linkage to rheumatoid arthritis (RA). METHODS: Two hundred fifty-two RA-affected sibling pairs from 182 UK families were genotyped using 365 highly informative microsatellite markers. Microsatellite genotyping was performed using fluorescent polymerase chain reaction primers and semiautomated DNA sequencing technology. Linkage analysis was undertaken using MAPMAKER/SIBS for single-point and multipoint analysis. RESULTS: Significant linkage (maximum logarithm of odds score 4.7 [P = 0.000003] at marker D6S276, 1 cM from HLA-DRB1) was identified around the major histocompatibility complex (MHC) region on chromosome 6. Suggestive linkage (P < 7.4 x 10(-4)) was identified on chromosome 6q by single- and multipoint analysis. Ten other sites of nominal linkage (P < 0.05) were identified on chromosomes 3p, 4q, 7p, 2 regions of 10q, 2 regions of 14q, 16p, 21q, and Xq by single-point analysis and on 3 sites (1q, 14q, and 14q) by multipoint analysis. CONCLUSION: Linkage to the MHC region was confirmed. Eleven non-HLA regions demonstrated evidence of suggestive or nominal linkage, but none reached the genome-wide threshold for significant linkage (P = 2.2 x 10(-5)). Results of previous genome screens have suggested that 6 of these regions may be involved in RA susceptibility.

Steer S, Fife MS, Fisher SA, Shah CJ, Newton J, McKay K, Milicic A, Wordsworth P, Polley A, Rosenthal A et al. 2001. Identification of a CRH genomic region haplotype in RA. ARTHRITIS AND RHEUMATISM, 44 (9), pp. S101-S101.

Milicic A, Brown MA, Wordsworth BP. 2001. Polymorphism in codon 17 of the CTLA-4 gene (+49 A/G) is not associated with susceptibility to rheumatoid arthritis in British Caucasians. Tissue Antigens, 58 (1), pp. 50-54. | Show Abstract | Read more

The role of the CTLA-4 antigen in the development of autoimmune diseases is well documented, with several autoimmune disorders showing association or linkage with the CTLA-4 locus. Its role in the aetiology of rheumatoid arthritis (RA) however, remains unclear, as the functional studies of the B7-CTLA-4 pathway in mouse models of RA and genetic studies in humans have given contrasting results. We have studied the single nucleotide polymorphism at position +49 (A/G) of the CTLA-4 gene, in a cohort of 421 RA cases and 452 healthy controls from the UK. Despite the high statistical power to detect even a weak susceptibility effect, no significant association was found. We also analysed the distribution of the allele and genotype frequencies with respect to the presence of the shared epitope (a known RA susceptibility factor) and found no statistically significant differences. We conclude that, although the importance of the B7-CTLA-4 interaction in the development of RA can not be excluded, the CTLA-4 gene is unlikely to be a predisposing factor to this disease.

Fife MS, Steer S, Shah CJ, Newton J, Wordsworth P, Milicic A, Polley A, Rosenthal A, Thoren G, Dix K et al. 2001. Genomic sequence and variability in the RA-associated CRH region (8q13) RHEUMATOLOGY, 40 pp. 84-84.

Laval SH, Timms A, Edwards S, Bradbury L, Brophy S, Milicic A, Rubin L, Siminovitch KA, Weeks DE, Calin A et al. 2001. Whole-genome screening in ankylosing spondylitis: evidence of non-MHC genetic-susceptibility loci. Am J Hum Genet, 68 (4), pp. 918-926. | Show Abstract | Read more

Ankylosing spondylitis (AS) is a common inflammatory arthritis predominantly affecting the axial skeleton. Susceptibility to the disease is thought to be oligogenic. To identify the genes involved, we have performed a genomewide scan in 185 families containing 255 affected sibling pairs. Two-point and multipoint nonparametric linkage analysis was performed. Regions were identified showing "suggestive" or stronger linkage with the disease on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q. The MHC locus was identified as encoding the greatest component of susceptibility, with an overall LOD score of 15.6. The strongest non-MHC linkage lies on chromosome 16q (overall LOD score 4.7). These results strongly support the presence of non-MHC genetic-susceptibility factors in AS and point to their likely locations.

Milicic A, Brown MA, Wordsworth BP. 2001. The Fc gamma RIIIa 158F/V variant does not predispose to RA RHEUMATOLOGY, 40 pp. 15-15.

Steer S, Fife MS, Fisher SA, Shah CJ, Newton J, McKay K, Milicic A, Polley A, Rosenthal A, Worthington J et al. 2001. A CRH genomic region haplotype in familial and sporadic rheumatoid arthritis RHEUMATOLOGY, 40 pp. 63-63.

Milicic A, Misra R, Brown MA, Wordsworth BP. 2001. Disparate associations of the FcgammaRIIIa 158/V variant with RA in two diverse populations ARTHRITIS RESEARCH & THERAPY, 3 (S2), | Read more

Milicic A, Lindheimer F, Laval S, Rudwaleit M, Ackerman H, Wordsworth P, Hohler T, Brown MA. 2000. Interethnic studies of TNF polymorphisms confirm the likely presence of a second MHC susceptibility locus in ankylosing spondylitis. Genes Immun, 1 (7), pp. 418-422. | Show Abstract | Read more

The objective of this study was to investigate TNF promoter region polymorphisms for association with susceptibility to ankylosing spondylitis (AS). The TNF -238 and -308 polymorphisms were genotyped in 306 English AS cases and 204 ethnically matched healthy B27-positive controls, and 96 southern German AS cases, 58 B27-positive and 251 B27-negative ethnically matched controls. Additionally, the TNF -376 polymorphism was genotyped in the southern German cases and controls. In the southern German AS patients a significant reduction in TNF -308.2 alleles was seen, compared with B27 positive controls (odds ratio 0.4, P = 0.03, 95% confidence interval 0.2-0.9), but no difference in allele frequencies was observed at TNF -238. Significant association between AS and both TNF -238 and TNF -308 was excluded in the English cases. These results confirm previous observations in the southern German population of association between TNF promoter region polymorphisms and AS, but the lack of association in the English population suggests that these polymorphisms themselves are unlikely to be directly involved. More likely, a second, non-HLA-B, MHC locus is involved in susceptibility to AS in these two populations.

Milicic A, Lee D, Brown M, Darke C, Mackay K, Wordsworth B. 2000. A protective association of the HLA-DR*0103 polymorphism with rheumatoid arthritis. AMERICAN JOURNAL OF HUMAN GENETICS, 67 (4), pp. 335-335.

MacKay KR, Brown MA, Milicic A, Laval S, Lee D, Wordsworth BP. 2000. Interleukin 10 (IL-10) haplotypes are associated with rheumatoid arthritis (RA). ARTHRITIS AND RHEUMATISM, 43 (9), pp. S270-S270.

MacKay KR, Eyre S, Barrett J, Laval S, Myerscough A, Milicic A, Barton A, Lee D, John S, Edwards S et al. 2000. Linkage analysis of potential rheumatoid arthritis non-HLA susceptibility loci. ARTHRITIS AND RHEUMATISM, 43 (9), pp. S69-S69.

MacKay K, Lee D, Milicic A, Laval S, Shatford J, Wordsworth P. 1999. The role of IL-10 in rheumatoid arthritis (RA) in two ethnically diverse populations. ARTHRITIS AND RHEUMATISM, 42 (9), pp. S247-S247.

Flaxman A, van Diemen PM, Yamaguchi Y, Allen E, Lindemann C, Rollier CS, Milicic A, Wyllie DH. 2017. Development of persistent gastrointestinal S. aureus carriage in mice. Sci Rep, 7 (1), pp. 12415. | Show Abstract | Read more

One fifth to one quarter of the human population is asymptomatically, naturally and persistently colonised by Staphylococcus aureus. Observational human studies indicate that although the whole population is intermittently exposed, some individuals lose S. aureus rapidly. Others become persistent carriers, as assessed by nasal cultures, with many individuals colonised for decades. Current animal models of S. aureus colonisation are expensive and normally require antibiotics. Importantly, these animal models have not yet contributed to our poor understanding of the dichotomy in human colonisation status. Here, we identify a single strain of S. aureus found to be persistently colonising the gastrointestinal tract of BALB/c mice. Phylogenetic analyses suggest it diverged from a human ST15 lineage in the recent past. We show that murine carriage of this organism occurs in the bowel and nares, is acquired early in life, and can persist for months. Importantly, we observe the development of persistent and non-persistent gastrointestinal carriage states in genetically identical mice. We developed a needle- and antibiotic-free model in which we readily induced S. aureus colonisation of the gastrointestinal tract experimentally by environmental exposure. Using our experimental model, impact of adaptive immunity on S. aureus colonisation could be assessed. Vaccine efficacy to eliminate colonisation could also be investigated using this model.

Milicic A, S Rollier C, Tang CK, Longley R, Hill AVS, Reyes-Sandoval A. 2017. Adjuvanting a viral vectored vaccine against pre-erythrocytic malaria. Sci Rep, 7 (1), pp. 7284. | Show Abstract | Read more

The majority of routinely given vaccines require two or three immunisations for full protective efficacy. Single dose vaccination has long been considered a key solution to improving the global immunisation coverage. Recent infectious disease outbreaks have further highlighted the need for vaccines that can achieve full efficacy after a single administration. Viral vectors are a potent immunisation platform, benefiting from intrinsic immuno-stimulatory features while retaining excellent safety profile through the use of non-replicating viruses. We investigated the scope for enhancing the protective efficacy of a single dose adenovirus-vectored malaria vaccine in a mouse model of malaria by co-administering it with vaccine adjuvants. Out of 11 adjuvants, only two, Abisco®-100 and CoVaccineHTTM, enhanced vaccine efficacy and sterile protection following malaria challenge. The CoVaccineHTTM adjuvanted vaccine induced significantly higher proportion of antigen specific central memory CD8+ cells, and both adjuvants resulted in increased proportion of CD8+ T cells expressing the CD107a degranulation marker in the absence of IFNγ, TNFα and IL2 production. Our results show that the efficacy of vaccines designed to induce protective T cell responses can be positively modulated with chemical adjuvants and open the possibility of achieving full protection with a single dose immunisation.

Walters AA, Krastev C, Hill AVS, Milicic A. 2015. Next generation vaccines: single-dose encapsulated vaccines for improved global immunisation coverage and efficacy. J Pharm Pharmacol, 67 (3), pp. 400-408. | Show Abstract | Read more

OBJECTIVES: Vaccination is considered the most successful health intervention; yet incomplete immunisation coverage continues to risk outbreaks of vaccine preventable diseases worldwide. Vaccination coverage improvement through a single-dose prime-boost technology would revolutionise modern vaccinology, impacting on disease prevalence, significantly benefiting health care and lowering economic burden of disease. KEY FINDINGS: Over the past 30 years, there have been efforts to develop a single-dose delayed release vaccine technology that could replace the repeated prime-boost immunisations required for many current vaccines. Biocompatible polymers have been employed to encapsulate model vaccines for delayed delivery in vivo, using either continuous or pulsed release. Biomaterial considerations, safety aspects, particle characteristics and immunological aspects of this approach are discussed in detail. SUMMARY: Despite many studies showing the feasibility of vaccine encapsulation for single-dose prime-boost administration, none have been translated into convincing utility in animal models or human trials. Further development of the encapsulation technology, through optimising the particle composition, formulation, antigen loading efficacy and stability, could lead to the application of this important approach in vaccine deployment. If successful, this would provide a solution to better global vaccination coverage through a reduction in the number of immunisations needed to achieve protection against infectious diseases. This review provides an overview of single-dose vaccination in the context of today's vaccine needs and is derived from a body of literature that has not been reviewed for over a decade.

Reyes-Sandoval A, Rollier CS, Milicic A, Bauza K, Cottingham MG, Tang C-K, Dicks MD, Wang D, Longley RJ, Wyllie DH, Hill AVS. 2012. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Mol Ther, 20 (8), pp. 1633-1647. | Show Abstract | Read more

Substantial protection can be provided against the pre-erythrocytic stages of malaria by vaccination first with an adenoviral and then with an modified vaccinia virus Ankara (MVA) poxviral vector encoding the same ME.TRAP transgene. We investigated whether the two vaccine components adenovirus (Ad) and MVA could be coinjected as a mixture to enhance protection against malaria. A single-shot mixture at specific ratios of Ad and MVA (Ad+MVA) enhanced CD8(+) T cell-dependant protection of mice against challenge with Plasmodium berghei. Moreover, the degree of protection could be enhanced after homologous boosting with the same Ad+MVA mixture to levels comparable with classic heterologous Ad prime-MVA boost regimes. The mixture increased transgene-specific responses while decreasing the CD8(+) T cell antivector immunity compared to each vector used alone, particularly against the MVA backbone. Mixed vector immunization led to increased early circulating interferon-γ (IFN-γ) response levels and altered transcriptional microarray profiles. Furthermore, we found that sequential immunizations with the Ad+MVA mixture led to consistent boosting of the transgene-specific CD8(+) response for up to three mixture immunizations, whereas each vector used alone elicited progressively lower responses. Our findings offer the possibility of simplifying the deployment of viral vectors as a single mixture product rather than in heterologous prime-boost regimens.

de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P, Chauhan VS, Chitnis CE, Gilbert SC, Hill AVS, Draper SJ. 2011. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus. J Immunol, 187 (5), pp. 2602-2616. | Show Abstract | Read more

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Reyes-Sandoval A, Wyllie DH, Bauza K, Milicic A, Forbes EK, Rollier CS, Hill AVS. 2011. CD8+ T effector memory cells protect against liver-stage malaria. J Immunol, 187 (3), pp. 1347-1357. | Show Abstract | Read more

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.

Kaur R, Milicic A, Reyes-Sandoval A, Hill A, Perrie Y. 2010. Addition of immunostimulatory components to DDA-TDB adjuvant delivery system JOURNAL OF PHARMACY AND PHARMACOLOGY, 62 (10), pp. 1245-1246.

Varela-Rohena A, Molloy PE, Dunn SM, Li Y, Suhoski MM, Carroll RG, Milicic A, Mahon T, Sutton DH, Laugel B et al. 2008. Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor. Nat Med, 14 (12), pp. 1390-1395. | Show Abstract | Read more

HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.

Laugel B, van den Berg HA, Gostick E, Cole DK, Wooldridge L, Boulter J, Milicic A, Price DA, Sewell AK. 2007. Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties. J Biol Chem, 282 (33), pp. 23799-23810. | Show Abstract | Read more

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.

Laugel B, Price DA, Milicic A, Sewell AK. 2007. CD8 exerts differential effects on the deployment of cytotoxic T lymphocyte effector functions. Eur J Immunol, 37 (4), pp. 905-913. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTL) are equipped with a range of effector functions that contribute both to the control of intracellular pathogens and dysregulated cellular proliferation and to the development of certain immunopathologies such as autoimmune disease. Qualitative analyses of various CTL responses have revealed substantial heterogeneity in the diversity of functions that are mobilized in response to antigen. Here, we studied the influence of the CD8 co-receptor, which is known to enhance antigen recognition by CTL, on the secretion of eight different cytokines and chemokines by human CTL clones using flow cytometric bead array. Our results show that abrogation of MHC class I/CD8 interactions exerts a differential influence on the distinct individual effector functions that are elicited in response to agonist ligands. The magnitude of this co-receptor blockade inhibitory effect was clearly related to the hierarchy of cytokine secretion in terms of activation threshold because those functions requiring the highest amounts of antigen were most affected. Thus, modulation of CD8 activity can effectively tune not only the sensitivity but also the qualitative profile of CTL responses.

Milicic A, Price DA, Zimbwa P, Booth BL, Brown HL, Easterbrook PJ, Olsen K, Robinson N, Gileadi U, Sewell AK et al. 2005. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef. J Immunol, 175 (7), pp. 4618-4626. | Show Abstract | Read more

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.

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