Publications by authors named "Gustavo Cabral-Miranda"

20 Publications

  • Page 1 of 1

The relationship between cytokine and neutrophil gene network distinguishes SARS-CoV-2-infected patients by sex and age.

JCI Insight 2021 05 24;6(10). Epub 2021 May 24.

Department of Immunology, Institute of Biomedical Sciences, and.

The fact that the COVID-19 fatality rate varies by sex and age is poorly understood. Notably, the outcome of SARS-CoV-2 infections mostly depends on the control of cytokine storm and the increasingly recognized pathological role of uncontrolled neutrophil activation. Here, we used an integrative approach with publicly available RNA-Seq data sets of nasopharyngeal swabs and peripheral blood leukocytes from patients with SARS-CoV-2, according to sex and age. Female and young patients infected by SARS-CoV-2 exhibited a larger number of differentially expressed genes (DEGs) compared with male and elderly patients, indicating a stronger immune modulation. Among them, we found an association between upregulated cytokine/chemokine- and downregulated neutrophil-related DEGs. This was correlated with a closer relationship between female and young subjects, while the relationship between male and elderly patients was closer still. The association between these cytokine/chemokines and neutrophil DEGs is marked by a strongly correlated interferome network. Here, female patients exhibited reduced transcriptional levels of key proinflammatory/neutrophil-related genes, such as CXCL8 receptors (CXCR1 and CXCR2), IL-1β, S100A9, ITGAM, and DBNL, compared with male patients. These genes are well known to be protective against inflammatory damage. Therefore, our work suggests specific immune-regulatory pathways associated with sex and age of patients infected with SARS-CoV-2 and provides a possible association between inverse modulation of cytokine/chemokine and neutrophil transcriptional signatures.
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http://dx.doi.org/10.1172/jci.insight.147535DOI Listing
May 2021

Discovery of four new B-cell protective epitopes for malaria using Q beta virus-like particle as platform.

NPJ Vaccines 2020 8;5:92. Epub 2020 Oct 8.

The Jenner Institute, University of Oxford, Oxford, UK.

Malaria remains one of the world's most urgent global health problems, with almost half a million deaths and hundreds of millions of clinical cases each year. Existing interventions by themselves will not be enough to tackle infection in high-transmission areas. The best new intervention would be an effective vaccine; but the leading and vaccine candidates, RTS,S and VMP001, show only modest to low field efficacy. New antigens and improved ways for screening antigens for protective efficacy will be required. This study exploits the potential of Virus-Like Particles (VLP) to enhance immune responses to antigens, the ease of coupling peptides to the Q beta (Qβ) VLP and the existing murine malaria challenge to screen B-cell epitopes for protective efficacy. We screened TRAP (PvTRAP) immune sera against individual 20-mer PvTRAP peptides. The most immunogenic peptides associated with protection were loaded onto Qβ VLPs to assess protective efficacy in a malaria sporozoite challenge. A second approach focused on identifying conserved regions within known sporozoite invasion proteins and assessing them as part of the Qβ. Using this VLP as a peptide scaffold, four new protective B-cell epitopes were discovered: three from the disordered region of PvTRAP and one from Thrombospondin-related sporozoite protein (TRSP). Antigenic interference between these and other B-cell epitopes was also explored using the virus-like particle/peptide platform. This approach demonstrates the utility of VLPs to help identifying new B-cell epitopes for inclusion in next-generation malaria vaccines.
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http://dx.doi.org/10.1038/s41541-020-00242-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546618PMC
October 2020

A Single Monoclonal Antibody against the Peanut Allergen Ara h 2 Protects against Systemic and Local Peanut Allergy.

Int Arch Allergy Immunol 2020 10;181(5):334-341. Epub 2020 Mar 10.

Immunology, RIA, Inselspital, University of Bern, Bern, Switzerland.

Background: Peanut allergy is the most prevalent and dangerous food allergy. Peanuts consist of a large number of different allergens and peanut-allergic patients are frequently sensitized to multiple allergens. Hence, conventional desensitization approaches aim at targeting as many allergens as possible.

Methods: The monoclonal anti-Ara h 2 antibody (mAb) was produced by hybridoma cells derived from WT BALB/c mice after immunization with a vaccine based on virus-like particles coupled to Ara h 2. BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum and mAbs were applied i.v. Challenge was performed the next day with the whole peanut extract intravenously and via skin prick test.

Results: Here we show in peanut-allergic mice that a single high-affinity mAb specific for Ara h 2 is able to block systemic and local allergic reactions induced by the complex peanut extract. We confirm in vitro binding of the mAb to the inhibitory low-affinity FcγRIIb receptor using a sensitive biosensor and demonstrate in vivo that protection was dependent on FcγRIIb.

Conclusion: A single mAb specific for Ara h 2 is able to improve local and systemic allergic symptoms induced by the whole allergen mixture.
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http://dx.doi.org/10.1159/000505917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265771PMC
November 2020

Vaccination Against Amyloidogenic Aggregates in Pancreatic Islets Prevents Development of Type 2 Diabetes Mellitus.

Vaccines (Basel) 2020 Mar 2;8(1). Epub 2020 Mar 2.

Department of Rheumatology, Immunology and Allergology (RIA), University Hospital, University of Bern, 3010 Bern, Switzerland.

Type 2 diabetes mellitus (T2DM) is a chronic progressive disease characterized by insulin resistance and insufficient insulin secretion to maintain normoglycemia. The majority of T2DM patients bear amyloid deposits mainly composed of islet amyloid polypeptide (IAPP) in their pancreatic islets. These-originally β-cell secretory products-extracellular aggregates are cytotoxic for insulin-producing β-cells and are associated with β-cell loss and inflammation in T2DM advanced stages. Due to the absence of T2DM preventive medicaments and the presence of only symptomatic drugs acting towards increasing hormone secretion and action, we aimed at establishing a novel disease-modifying therapy targeting the cytotoxic IAPP deposits in order to prevent the development of T2DM. We generated a vaccine based on virus-like particles (VLPs), devoid of genomic material, coupled to IAPP peptides inducing specific antibodies against aggregated, but not monomeric IAPP. Using a mouse model of islet amyloidosis, we demonstrate in vivo that our vaccine induced a potent antibody response against aggregated, but not soluble IAPP, strikingly preventing IAPP depositions, delaying onset of hyperglycemia and the induction of the associated pro-inflammatory cytokine Interleukin 1β (IL-1β). We offer the first cost-effective and safe disease-modifying approach targeting islet dysfunction in T2DM, preventing pathogenic aggregates without disturbing physiological IAPP function.
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http://dx.doi.org/10.3390/vaccines8010116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157615PMC
March 2020

Correction: -Derived E-DIII Protein Displayed on Immunologically Optimized VLPs Induces Neutralizing Antibodies without Causing Enhancement of Infection. 2019, , 72.

Vaccines (Basel) 2020 Feb 20;8(1). Epub 2020 Feb 20.

The Jenner Institute, Nuffield Department of Medicine, Centre for Cellular and Molecular Physiology (CCMP), University of Oxford, Oxford OX1 2JD, UK.

The authors wish to make the following correction to their paper [...].
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http://dx.doi.org/10.3390/vaccines8010094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157487PMC
February 2020

Early Transcriptional Signature in Dendritic Cells and the Induction of Protective T Cell Responses Upon Immunization With VLPs Containing TLR Ligands-A Role for CCL2.

Front Immunol 2019 2;10:1679. Epub 2019 Aug 2.

The Jenner Institute, Oxford University, Oxford, United Kingdom.

Inducing T cell responses by therapeutic vaccination requires appropriate activation of antigen presenting cells (APCs). The use of virus-like particles (VLPs) containing Toll-like receptor (TLR) ligands has demonstrated remarkable potential in activating APCs and modulating the immune response both for prophylactic vaccines as well as immunotherapy. Here, we employed VLPs associated to TLR ligands as tools to modulate cytotoxic response mediated by CD8 T cells and provide further insight in the development of T cell-based immunotherapy. We have investigated the transcriptional signature in dendritic cells (DCs) from mice immunized with VLPs containing distinct classes of nucleic acid and correlated the expression patterns with the efficiency of induced T cell responses. We identified key pathways activated in DCs that are involved in the appropriated induction of T cell responses and show evidence for the modulatory effect of CCL2 in CD8 T cells responses. These insights shed light on immune networks that are pivotal for the induction of potent cytotoxic T cell responses and identify key genes for appropriate DC activation and subsequent modulation of the adaptive immune response.
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http://dx.doi.org/10.3389/fimmu.2019.01679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687836PMC
October 2020

-Derived E-DIII Protein Displayed on Immunologically Optimized VLPs Induces Neutralizing Antibodies without Causing Enhancement of Infection.

Vaccines (Basel) 2019 Jul 23;7(3). Epub 2019 Jul 23.

The Jenner Institute, Nuffield Department of Medicine, Centre for Cellular and Molecular Physiology (CCMP), University of Oxford, Oxford OX1 2JD, UK.

(ZIKV) is a similar to (DENV) in terms of transmission and clinical manifestations, and usually both viruses are found to co-circulate. ZIKV is usually transmitted by mosquitoes bites, but may also be transmitted by blood transfusion, via the maternal-foetal route, and sexually. After 2015, when the most extensive outbreak of ZIKV had occurred in Brazil and subsequently spread throughout the rest of South America, it became evident that ZIKV infection during the first trimester of pregnancy was associated with microcephaly and other neurological complications in newborns. As a result, the development of a vaccine against ZIKV became an urgent goal. A major issue with DENV vaccines, and therefore likely also with ZIKV vaccines, is the induction of antibodies that fail to neutralize the virus properly and cause antibody-dependent enhancement (ADE) of the infection instead. It has previously been shown that antibodies against the third domain of the envelope protein (EDIII) induces optimally neutralizing antibodies with no evidence for ADE for other viral strains. Therefore, we generated a ZIKV vaccine based on the EDIII domain displayed on the immunologically optimized (CuMVtt) derived virus-like particles (VLPs) formulated in dioleoyl phosphatidylserine (DOPS) as adjuvant. The vaccine induced high levels of specific IgG after a single injection. The antibodies were able to neutralise ZIKV without enhancing infection by DENV in vitro. Thus, the here described vaccine based on EDIII displayed on VLPs was able to stimulate production of antibodies specifically neutralizing ZIKV without potentially enhancing disease caused by DENV.
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http://dx.doi.org/10.3390/vaccines7030072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789886PMC
July 2019

Targeting Mutated Plus Germline Epitopes Confers Pre-clinical Efficacy of an Instantly Formulated Cancer Nano-Vaccine.

Front Immunol 2019 15;10:1015. Epub 2019 May 15.

Nuffield Department of Medicine, Jenner Institute, University of Oxford, Oxford, United Kingdom.

Personalized cancer vaccines hold promises for future cancer therapy. Targeting neoantigens is perceived as more beneficial compared to germline, non-mutated antigens. However, it is a practical challenge to identify and vaccinate patients with neoantigens. Here we asked whether two neoantigens are sufficient, and whether the addition of germline antigens would enhance the therapeutic efficacy. We developed and used a personalized cancer nano-vaccine platform based on virus-like particles loaded with toll-like receptor ligands. We generated three sets of multi-target vaccines (MTV) to immunize against the aggressive B16F10 murine melanoma: one set based on germline epitopes (GL-MTV) identified by immunopeptidomics, another set based on mutated epitopes (Mutated-MTV) predicted by whole exome sequencing and a last set combines both germline and mutated epitopes (Mix-MTV). Our results demonstrate that both germline and mutated epitopes induced protection but the best therapeutic effect was achieved with the combination of both. Our platform is based on Cu-free click chemistry used for peptide-VLP coupling, thus enabling bedside production of a personalized cancer vaccine, ready for clinical translation.
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http://dx.doi.org/10.3389/fimmu.2019.01015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532571PMC
September 2020

Correction to: Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

J Immunother Cancer 2019 May 23;7(1):137. Epub 2019 May 23.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Following publication of the original article [1], the author reported an author's family name has been misspelled. Paul Engroff should be replace Paul Engeroff.Furthermore, there are two mistake in two affiliations: 9) Department of dermatology, University of Zurich, Bern, Switzerland and 10) Department of Oncology, University of Lausanne, Bern,Switzerland should be replace with 9) Department of dermatology, University of Zurich, Zurich, Switzerland.10) Department of Oncology, University of Lausanne, Lausanne, Switzerland.
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http://dx.doi.org/10.1186/s40425-019-0616-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532182PMC
May 2019

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

J Immunother Cancer 2019 04 26;7(1):114. Epub 2019 Apr 26.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot.

Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMV-VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMV-VLPs using bio-orthogonal Cu-free click chemistry. The CuMV-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum.

Results: Our results showed that CuMV-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMV-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8, specific p33 T cell response or tumour protection were assessed.

Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic.
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http://dx.doi.org/10.1186/s40425-019-0587-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485085PMC
April 2019

DOPS Adjuvant Confers Enhanced Protection against Malaria for VLP-TRAP Based Vaccines.

Diseases 2018 Nov 21;6(4). Epub 2018 Nov 21.

The Jenner Institute, Nuffield Department of Medicine, Centre for Cellular and Molecular Physiology (CCMP), Roosevelt Drive, Oxford OX3 7BN, UK.

Vaccination remains the most effective and essential prophylactic tool against infectious diseases. Enormous efforts have been made to develop effective vaccines against malaria but successes remain so far limited. Novel adjuvants may offer a significant advantage in the development of malaria vaccines, in particular if combined with inherently immunogenic platforms, such as virus-like particles (VLP). Dioleoyl phosphatidylserine (DOPS), which is expressed on the outer surface of apoptotic cells, represents a novel adjuvant candidate that may confer significant advantage over existing adjuvants, such as alum. In the current study we assessed the potential of DOPS to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using thrombospondin-related adhesive protein (TRAP) as a target antigen. TRAP was chemically coupled to VLPs derived from the cucumber mosaic virus fused to a universal T cell epitope of tetanus toxin (CuMVtt). Mice were immunized with TRAP alone or formulated in alum or DOPS and compared to TRAP coupled to CuMVtt formulated in PBS or DOPS. Induced immune responses, in particular T cell responses, were assessed as the major protective effector cell population induced by TRAP. The protective capacity of the various formulations was assessed using a transgenic expressing PfTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral and T cell immunogenicity for PfTRAP compared to the antigen alone. Display on VLPs, in particular if formulated with DOPS, induced the strongest and most protective immune response. Thus, the combination of VLP with DOPS may harness properties of both immunogenic components and optimally enhance induction of protective immune responses.
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http://dx.doi.org/10.3390/diseases6040107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313579PMC
November 2018

Biosensor-based selective detection of Zika virus specific antibodies in infected individuals.

Biosens Bioelectron 2018 Aug 1;113:101-107. Epub 2018 May 1.

Centre for Cellular and Molecular Physiology (CCMP), The Jenner Institute, University of Oxford, Oxford, UK; Immunology, RIA, Inselspital, University of Bern, Switzerland. Electronic address:

Zika virus (ZIKV) recently emerged as a global threat subsequent to its global spread because it induces microencephaly and other brain damages in infants born to infected mothers. Epidemiological monitoring of infection has been hampered by the absence of reliable serological tests capable to distinguish between ZIKV and other Flavivirus infections, in particular Dengue virus (DENV). As both viruses are transmitted by the same mosquito-species, their distributions largely overlap and reliable serological distinction between the viruses is essential. Here we develop a novel biosensor which is based on recombinant forms of ZIKV non-structural protein 1 (NS1) and the domain III of the envelope protein (EDIII). Using electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), we demonstrate that in addition to extremely sensitive detection of ZIKV-specific antibodies in serum and saliva, the biosensor promptly distinguished ZIKV and DENV-specific antibodies. Hence, this novel biosensor allows assessing ZIKV antibodies in blood and saliva and results are unaffected by presence of DENV virus-specific antibodies.
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http://dx.doi.org/10.1016/j.bios.2018.04.058DOI Listing
August 2018

Microcrystalline Tyrosine (MCT): A Depot Adjuvant in Licensed Allergy Immunotherapy Offers New Opportunities in Malaria.

Vaccines (Basel) 2017 Sep 27;5(4). Epub 2017 Sep 27.

Nuffield Department of Medicine, Centre for Cellular and Molecular Physiology (CCMP), The Jenner Institute, University of Oxford, Oxford OX3 7BN, UK.

Microcrystalline Tyrosine (MCT) is a widely used proprietary depot excipient in specific immunotherapy for allergy. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria. To this end, we formulated the circumsporozoite protein (CSP) of in MCT and compared the induced immune responses to CSP formulated in PBS or Alum. Both MCT and Alum strongly increased immunogenicity of CSP compared to PBS in both C57BL/6 and BALB/c mice. Challenge studies in mice using a chimeric expressing CSP of demonstrated clinically improved symptoms of malaria with CSP formulated in both MCT and Alum; protection was, however, more pronounced if CSP was formulated in MCT. Hence, MCT may be an attractive biodegradable adjuvant useful for the development of novel prophylactic vaccines.
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http://dx.doi.org/10.3390/vaccines5040032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748599PMC
September 2017

Major findings and recent advances in virus-like particle (VLP)-based vaccines.

Semin Immunol 2017 12 5;34:123-132. Epub 2017 Sep 5.

Jenner Institute, University of Oxford, Roosevelt Dr, Oxford OX3 7BN, UK; Inselspital, Universitatsklinik RIA, Immunologie, Sahlihaus 1, 3010 Bern, Switzerland. Electronic address:

Virus-like particles (VLPs) have made giant strides in the field of vaccinology over the last three decades. VLPs constitute versatile tools in vaccine development due to their favourable immunological characteristics such as their size, repetitive surface geometry, ability to induce both innate and adaptive immune responses as well as being safe templates with favourable economics. Several VLP-based vaccines are commercially available including vaccines against Human Papilloma Virus (HPV) such as Cervarix, Gardasil & Gardasil9 and Hepatitis B Virus (HBV) including the 3rd generation Sci-B-Vac™. In addition, the first licensed malaria-VLP-based vaccine Mosquirix™ has been recently approved by the European regulators. Several other VLP-based vaccines are currently undergoing preclinical and clinical development. This review summarizes some of the major findings and recent advances in VLP-based vaccine development and technologies and outlines general principles that may be harnessed for induction of targeted immune responses.
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http://dx.doi.org/10.1016/j.smim.2017.08.014DOI Listing
December 2017

Virus-Like Particle (VLP) Plus Microcrystalline Tyrosine (MCT) Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax.

Vaccines (Basel) 2017 May 2;5(2). Epub 2017 May 2.

The Jenner Institute, Nuffield Department of Medicine, Centre for Cellular and Molecular Physiology (CCMP), University of Oxford, Oxford OX3 7BN, UK.

Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT) is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs), which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using thrombospondin-related adhesive protein (TRAP) as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt), formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant . Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.
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http://dx.doi.org/10.3390/vaccines5020010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492007PMC
May 2017

Adjusted Particle Size Eliminates the Need of Linkage of Antigen and Adjuvants for Appropriated T Cell Responses in Virus-Like Particle-Based Vaccines.

Front Immunol 2017 6;8:226. Epub 2017 Mar 6.

The Jenner Institute, Oxford University, Oxford, UK; Immunology, Inselspital, Bern, Switzerland.

Since the discovery of the first virus-like particle (VLP) derived from hepatitis B virus in 1980 (1), the field has expanded substantially. Besides successful use of VLPs as safe autologous virus-targeting vaccines, the powerful immunogenicity of VLPs has been also harnessed to generate immune response against heterologous and even self-antigens (2-4). Linking adjuvants to VLPs displaying heterologous antigen ensures simultaneous delivery of all vaccine components to the same antigen-presenting cells. As a consequence, antigen-presenting cells, such as dendritic cells, will process and present the antigen displayed on VLPs while receiving costimulatory signals by the VLP-incorporated adjuvant. Similarly, antigen-specific B cells recognizing the antigen linked to the VLP are simultaneously exposed to the adjuvant. Here, we demonstrate in mice that physical association of antigen, carrier (VLPs), and adjuvant is more critical for B than T cell responses. As a model system, we used the E7 protein from human papilloma virus, which spontaneously forms oligomers with molecular weight ranging from 158 kDa to 10 MDa at an average size of 50 nm. E7 oligomers were either chemically linked or simply mixed with VLPs loaded with DNA rich in non-methylated CG motifs (CpGs), a ligand for toll-like receptor 9. E7-specific IgG responses were strongly enhanced if the antigen was linked to the VLPs. In contrast, both CD4 and CD8 T cell responses as well as T cell-mediated protection against tumor growth were comparable for linked and mixed antigen formulations. Therefore, our data show that B cell but not T cell responses require antigen-linkage to the carrier and adjuvant for optimal vaccination outcome.
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http://dx.doi.org/10.3389/fimmu.2017.00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337491PMC
March 2017

Delivering adjuvants and antigens in separate nanoparticles eliminates the need of physical linkage for effective vaccination.

J Control Release 2017 04 28;251:92-100. Epub 2017 Feb 28.

University of Oxford, Roosevelt Dr, Oxford OX3 7BN, UK; Inselspital, Universitatsklinik RIA, Immunologie, Sahlihaus 1, 3010 Bern, Switzerland.

DNA rich in unmethylated CG motifs (CpGs) engage Toll-Like Receptor 9 (TLR-9) in endosomes and are well described stimulators of the innate and adaptive immune system. CpGs therefore can efficiently improve vaccines' immunogenicity. Packaging CpGs into nanoparticles, in particular into virus-like particles (VLPs), improves the pharmacological characteristics of CpGs as the protein shell protects them from DNAse activity and delivers the oligomers to the endosomal compartments of professional antigen presenting cells (APCs). The current consensus in packaging and delivering CpGs in VLP-based vaccines is that both adjuvants and antigens should be kept in close proximity (i.e. physically linked) to ensure delivery of antigens and adjuvants to the same APCs. In the current study, we harness the draining properties of the lymphatic system and show that also non-linked VLPs are efficiently co-delivered to the same APCs in lymph nodes. Specifically, we have shown that CpGs can be packaged in one VLP and mixed with another VLP displaying the antigen prior to administration in vivo. Both VLPs efficiently reached the same draining lymph node where they were taken up and processed by the same APCs, namely dendritic cells and macrophages. This resulted in induction of specific CTLs producing cytokines and killing target cells in vivo at levels seen when using VLPs containing both CpGs and chemically conjugated antigen. Thus, delivery of antigens and adjuvants in separate nanoparticles eliminates the need of physical conjugation and thus can be beneficial when designing precision medicine VLP-based vaccines or help to re-formulate existing VLP vaccines not naturally carrying immunostimulatory sequences.
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http://dx.doi.org/10.1016/j.jconrel.2017.02.031DOI Listing
April 2017

Detecting circulating antibodies by controlled surface modification with specific target proteins: Application to malaria.

Biosens Bioelectron 2017 May 15;91:833-841. Epub 2017 Jan 15.

BioMark/CINTESIS-ISEP, School of Engineering of the Polytechnique School of Porto, Portugal. Electronic address:

Sensitive detection of specific antibodies by biosensors has become of major importance for monitoring and controlling epidemics. Here we report a development of a biosensor able to specifically measure antibodies in a drop of unmodified blood serum. Within minutes, the detection system measures presence of antibodies against Plasmodium vivax, a causing agent for malaria. The biosensor consists of a layer of carbon nanotubes (CNTs) which were casted on a carbon working electrode area of a three-electrode system and oxidized. An amine layer was produced next by modifying the surface with EDAC/NHS followed by reaction with a diamine compound. Finally, the protein fragments derived from P. vivax containing well-known antigen sequences were casted on this layer and bound through electrostatic interactions, involving hydrogen and ionic bonding. All these chemical changes occurring at the carbon surface along the biosensor assembly were followed and confirmed by Fourier Transformed Infrared s pectrometry (FTIR) and Raman spectroscopy. The presence of antibodies in serum was detected by monitoring the electrical properties of the layer, making use of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), against a standard iron probe. Overall, the charge-transfer resistance decreased after antibody binding, because there was an additional amount of protein bound to the surface. This hindered the access of the iron redox probe to the conductive support at the electrode surface. Electrical changes could be measured at antibody concentration as low as ~6-50pg/L (concentrations in the range of 10-15M) and as high as ~70μg/L. Specific measurement with low background was even possible in undiluted serum. Hence, this novel biosensor allows assessing serum antibody levels in real time and in un-manipulated serum samples on-site where needed.
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http://dx.doi.org/10.1016/j.bios.2017.01.031DOI Listing
May 2017

Backside-surface imprinting as a new strategy to generate specific plastic antibody materials.

J Mater Chem B 2014 May 14;2(20):3087-3095. Epub 2014 Apr 14.

BioMark/ISEP, School of Engineering, Polytechnic Institute of Porto, António Bernardino de Almeida, 431, 4200-072 Porto, Portugal.

A backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular "tape". The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL) versus charged tranfer resistance (R, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL (R = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest.
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http://dx.doi.org/10.1039/c3tb21740jDOI Listing
May 2014

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies.

J Mater Chem B 2014 Feb 10;2(5):477-484. Epub 2013 Dec 10.

Laboratory of Immunophysiopathology, Department of Immunology at the Institute of Biomedical Sciences, University of Sao Paulo, Brazil.

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by self-assembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 μg mL. The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.
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http://dx.doi.org/10.1039/c3tb21048kDOI Listing
February 2014