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Journal articleMcDonald JU, Ekeruche-Makinde J, Ho MM, et al., 2016,
Development of a custom pentaplex sandwich immunoassay using Protein-G coupled beads for the Luminex® xMAP® platform.
, Journal of Immunological Methods, Vol: 433, Pages: 6-16, ISSN: 1872-7905Multiplex bead-based assays have many advantages over ELISA, particularly for the analyses of large quantities of samples and/or precious samples of limited volume. Although many commercial arrays covering multitudes of biologically significant analytes are available, occasionally the development of custom arrays is necessary. Here, the development of a custom pentaplex sandwich immunoassay using Protein G-coupled beads, for analysis using the Luminex® xMAP® platform, is described. This array was required for the measurement of candidate biomarkers of vaccine safety in small volumes of mouse sera. Optimisation of this assay required a stepwise approach: testing cross-reactivity of the antibody pairs, the development of an in-house serum diluent buffer as well as heat-inactivation of serum samples to prevent interference from matrix effects. We then demonstrate the use of this array to analyse inflammatory mediators in mouse serum after immunisation. The work described here exemplifies how Protein G-coupled beads offer a flexible and robust approach to develop custom multiplex immunoassays, which can be applied to a range of analytes from multiple species.
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Journal articleMcKay PF, King DFL, Mann JFS, et al., 2016,
TLR4 and TLR7/8 adjuvant combinations generate different vaccine antigen-specific immune outcomes in minipigs when administered via the ID or IN routes
, PLOS One, Vol: 11, ISSN: 1932-6203The induction of high levels of systemic and mucosal humoral immunity is a key goal for many prophylactic vaccines. However, adjuvant strategies developed in mice have often performed poorly in the clinic. Due to their closer similarity to humans, minipigs may provide a more accurate picture of adjuvant performance. Based on their complementary signalling pathways, we assessed humoral immune responses to model antigens after co-administration with the toll-like receptor 4 (TLR4) stimulator glucopyranosyl lipid adjuvant (GLA-AF) or the TLR7/8 agonist resiquimod (R848) (alone and in combination) via the intradermal (ID), intranasal (IN) or combined routes in the Gottingen minipig animal model. Surprisingly, we discovered that while GLA-AF additively enhanced the adjuvant effect of R848 when injected ID, it abrogated the adjuvant activity of R848 after IN inoculation. We then performed a route comparison study using a CN54 gp140 HIV Envelope model antigen adjuvanted with R848 + GLA-AF (ID) or R848 alone (IN). Animals receiving priming inoculations via one route were then boosted by the alternate route. Although differences were observed in the priming phase (IN or ID), responses converged upon boosting by the alternative route with no observable impact resultant from the order of administration (ID/IN vs IN/ID). Specific IgG responses were measured at a distal mucosal site (vaginal), although there was no evidence of mucosal linkage as these closely reflected serum antibody levels. These data indicate that the complex in vivo cross-talk between innate pathways are likely tissue specific and cannot be predicted by simple in vitro models.
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Journal articleFrancis SC, Hou Y, Baisley K, et al., 2016,
Immune activation in the female genital tract: expression profiles of soluble proteins in women at high risk for HIV infection
, PLOS One, Vol: 11, ISSN: 1932-6203 -
Journal articleVamvaka E, Arcalis E, Ramessar K, et al., 2016,
Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV
, Plant Biotechnology Journal, Vol: 14, Pages: 1427-1437, ISSN: 1467-7652Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of OSGRFT in the best-performing plants was 223 μg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. OSGRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified OSGRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure OSGRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.
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Conference paperHewitt R, Webber J, Farne H, et al., 2016,
Airway Glucose In Virus-Induced COPD Exacerbations
, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X- Author Web Link
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- Citations: 1
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Journal articleVeazey RS, Siddiqui A, Klein K, et al., 2015,
Evaluation of mucosal adjuvants and immunization routes for the induction of systemic and mucosal humoral immune responses in macaques
, Human Vaccines & Immunotherapeutics, Vol: 11, Pages: 2913-2922, ISSN: 2164-5515Delivering vaccine antigens to mucosal surfaces is potentially very attractive, especially as protection from mucosal infections may be mediated by local immune responses. However, to date mucosal immunization has had limited successes, with issues of both safety and poor immunogenicity. One approach to improve immunogenicity is to develop adjuvants that are effective and safe at mucosal surfaces. Differences in immune responses between mice and men have overstated the value of some experimental adjuvants which have subsequently performed poorly in the clinic. Due to their closer similarity, non-human primates can provide a more accurate picture of adjuvant performance. In this study we immunised rhesus macaques (Macaca mulatta) using a unique matrix experimental design that maximised the number of adjuvants screened while reducing the animal usage. Macaques were immunised by the intranasal, sublingual and intrarectal routes with the model protein antigens keyhole limpet haemocyanin (KLH), β-galactosidase (β-Gal) and ovalbumin (OVA) in combination with the experimental adjuvants Poly(I:C), Pam3CSK4, chitosan, Thymic Stromal Lymphopoietin (TSLP), MPLA and R848 (Resiquimod). Of the routes used, only intranasal immunization with KLH and R848 induced a detectable antibody response. When compared to intramuscular immunization, intranasal administration gave slightly lower levels of antigen specific antibody in the plasma, but enhanced local responses. Following intranasal delivery of R848, we observed a mildly inflammatory response, but no difference to the control. From this we conclude that R848 is able to boost antibody responses to mucosally delivered antigen, without causing excess local inflammation.
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Journal articleKing DFL, McKay PF, Mann JFS, et al., 2015,
Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice
, PLOS One, Vol: 10, ISSN: 1932-6203BackgroundAn effective HIV vaccine will likely require induction of both mucosal and systemic cellularand humoral immune responses. We investigated whether intramuscular (IM) delivery ofelectroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal(IN) and IM routes could be combined to induce mucosal and systemic cellular and humoralimmune responses to a model HIV-1 CN54 gp140 antigen in mice.ResultsCo-immunisation of DNA with intranasal protein successfully elicited both serum and vaginalIgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemicor mucosal IgA responses. Cellular IFNγ responses were preserved in coimmunisationprotocols compared to protein-only vaccination groups. The addition of DNAto IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccinationalone. Luminex analysis also revealed that co-immunisation with DNA and IN proteininduced expression of cytokines that promote B-cell function, generation of TFH cells andCCR5 ligands that can reduce HIV infectivity.SignificanceThese data suggest that while IN inoculation alone elicits both cellular and humoralresponses, co-administration with homologous DNA vaccination can tailor these towards amore balanced Th1/Th2 phenotype modulating the cellular cytokine profile while elicitinghigh-levels of antigen-specific antibody. This work provides insights on how to generate differentialimmune responses within the same vaccination visit, and supports co-immunisationwith DNA and protein by a mucosal route as a potential delivery strategy for HIVvaccines.
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Journal articleBadamchi-Zadeh A, McKay PF, Holland MJ, et al., 2015,
Intramuscular Immunisation with Chlamydial Proteins Induces <i>Chlamydia trachomatis</i> Specific Ocular Antibodies
, PLOS ONE, Vol: 10, ISSN: 1932-6203- Author Web Link
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- Citations: 18
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Journal articleMora-Peris B, Winston A, Garvey L, et al., 2015,
HIV-1 CNS in vitro infectivity models based on clinical CSF samples
, Journal of Antimicrobial Chemotherapy, Vol: 71, Pages: 235-243, ISSN: 1460-2091Background The concentration of antiretrovirals in CSF is often utilized as a surrogate for CNS drug exposure. This measurement does not consider pharmacodynamic or combinative effects of ART. We have developed a novel endpoint measurement to assess antiretroviral activity of CSF from subjects on ART.Methods CSF samples were obtained from patients receiving tenofovir/emtricitabine (245/200 mg once daily) with either rilpivirine (25 mg once daily) or lopinavir/ritonavir/maraviroc (400/100/150 mg twice daily) and HIV-uninfected controls. Antiviral activity of ART-containing CSF was assessed in cell cultures using PBMCs and neuro-derived glial (U87) and astrocyte (373) cell lines. Infectivity model half-maximal inhibitory concentration (IMIC50) values were calculated and expressed as −log2IMIC50. Results were correlated with CSF antiretroviral concentrations.Results Compared with controls, CSF from both ART studies demonstrated in vitro antiretroviral activity in all models. CSF antiretroviral activity of patients on lopinavir/ritonavir/maraviroc was significantly greater than that of patients on rilpivirine [−log2IMIC50 (95% CI) 4.82 (4.74–4.89) versus 3.43 (3.33–3.54) in PBMCs, 3.06 (2.98–3.15) versus 2.56 (2.46–2.65) in U87 cells and 6.00 (6.11–5.88) versus 4.90 (5.09–4.72) in 373 cells, respectively]. Positive correlations were observed for individual CSF antiretroviral activity in different cellular models with CSF concentrations of rilpivirine (P = 0.040 in 373 cells) and lopinavir (P = 0.048 in 373 cells), but not maraviroc.Conclusions Antiviral activity of CSF from patients on ART was successfully calculated and was greater with a regimen containing four active drugs compared with three active drugs. The use of neuro-derived cell lines alongside PBMCs to assess the effect of ART on CSF may act as a useful future clinical research tool.
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Journal articleStieh DJ, King DF, Klein K, et al., 2015,
Discrete partitioning of HIV-1 Env forms revealed by viral capture
, RETROVIROLOGY, Vol: 12, ISSN: 1742-4690- Author Web Link
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- Citations: 13
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