Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination
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{"title"=>"Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination", "type"=>"journal", "authors"=>[{"first_name"=>"Danushka K.", "last_name"=>"Wijesundara", "scopus_author_id"=>"36452497500"}, {"first_name"=>"Charani", "last_name"=>"Ranasinghe", "scopus_author_id"=>"6603109978"}, {"first_name"=>"Ronald J.", "last_name"=>"Jackson", "scopus_author_id"=>"56224698400"}, {"first_name"=>"Brett A.", "last_name"=>"Lidbury", "scopus_author_id"=>"6701446320"}, {"first_name"=>"Christopher R.", "last_name"=>"Parish", "scopus_author_id"=>"35515377100"}, {"first_name"=>"Benjamin J.C.", "last_name"=>"Quah", "scopus_author_id"=>"9238617500"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"25170620", "sgr"=>"84907536539", "doi"=>"10.1371/journal.pone.0105366", "scopus"=>"2-s2.0-84907536539", "pui"=>"373874973", "issn"=>"19326203"}, "id"=>"2724973c-793e-394f-b8ba-4ab5ce15dc62", "abstract"=>"Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.", "link"=>"http://www.mendeley.com/research/vivo-fta-assay-assess-magnitude-functional-avidity-epitope-variant-crossreactivity-t-cell-responses", "reader_count"=>9, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>2, "Student > Postgraduate"=>1, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>1, "Student > Ph. D. Student"=>2, "Student > Postgraduate"=>1, "Student > Master"=>2, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Medicine and Dentistry"=>1, "Agricultural and Biological Sciences"=>3, "Social Sciences"=>2, "Immunology and Microbiology"=>2, "Economics, Econometrics and Finance"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Social Sciences"=>{"Social Sciences"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1655030"], "description"=>"<p>Six BALB/c mice were vaccinated with 5×10<sup>6</sup> PFU VV Western Reserve i.p. A FTA was constructed using mouse splenocytes and comprised of fluorescent target cells pulsed with 6 different concentrations of the MHC-I binding peptides F2L, F2L mut, A52R, and HIV neg (as a negative control). FTA target cells were injected i.v. into infected mice 6 days post vaccination and after 18 hr <i>in vivo</i> % specific killing calculated for FTA target cells from harvested spleens. a) <i>In vivo</i> killing responses from six infected animals. b) Summary of responses from all mice with means of % specific killing and standard error of mean. c) Mean area under curve (AUC) measurements from % specific killing response curves and associated standard error of means. d) Mean effective concentration of peptides used to pulse target cells that generated half maximal responses (EC<sub>50</sub>) and associated standard error of means and P values.</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155585, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.g002", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_FTA_assay_can_measure_magnitude_functional_avidity_and_epitope_variant_cross_reactivity_of_CTL_responses_in_vivo_/1155585", "title"=>"The FTA assay can measure magnitude, functional avidity and epitope variant cross-reactivity of CTL responses <i>in vivo</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655035"], "description"=>"<p>Mice were vaccinated with 24 different vaccine regimes based on all combinations of FPV-HIV and VV-HIV administered either i.n. or i.m. in a prime-boost strategy as outlined in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105366#pone-0105366-t001\" target=\"_blank\">Table 1</a>. Mice were vaccinated with 5×10<sup>6</sup> PFU of each vaccine and booster vaccinations were given 2 weeks after the priming vaccination. T cell responses were assessed using a 252-parameter FTA comprised of fluorescent target cells pulsed with 6 concentrations of the MHC-I binding peptides F2L, F2L mut, HIV Gag, HIV Gag mut, HIV Pol and HIV Env, and the MHC-II binding peptide Gag T<sub>H</sub>. FTA target cells were injected i.v. into vaccinated mice 6 days post vaccination and responses assessed after 18 hr <i>in vivo</i> using harvested spleens. % specific killing and T<sub>H</sub> cell activity was calculated as described in the Materials and Methods. a) Cumulative magnitude of T cell responses as AUC was plotted as a heat map depicting the range of highest (darkest colour) to lowest (lightest colour) T<sub>H</sub> cell (upper panel) or CTL (lower panels) responses. b) AUC heat maps of responses to the three HIV Gag epitopes. c) EC<sub>50</sub> values of responses to the three HIV Gag epitopes. Values are only shown where EC<sub>50</sub> values were calculable as described in the Materials and Methods. AUC and EC<sub>50</sub> values are depicted as means from 6 intra-animal replicates. The results are representative of three independent experiments</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155589, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.g003", "stats"=>{"downloads"=>1, "page_views"=>31, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_High_throughput_screening_of_HIV_1_poxvirus_vaccination_regimes_for_high_magnitude_high_functional_avidity_and_high_epitope_variant_cross_reactive_T_cell_responses_in_vivo_/1155589", "title"=>"High throughput screening of HIV-1 poxvirus vaccination regimes for high magnitude, high functional avidity and high epitope variant cross-reactive T cell responses <i>in vivo</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655039"], "description"=>"<p>Mice were vaccinated i.n. with FPV-HIV and/or i.m. VV-HIV. Mice were vaccinated with 5×10<sup>6</sup> PFU of each pox virus vaccine. Booster vaccinations were given 2 weeks post the previous vaccination. T-cell responses were assessed using 252-parameter FTAs as in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105366#pone-0105366-g003\" target=\"_blank\">Figure 3</a>. a) % specific killing of FTA cells <i>in vivo</i> by CTL and b) T<sub>H</sub> cell activity induced by prime, boost, and prime-boost vaccination regimes, showing all 6 intra-animal replicate responses (upper panels) and means and standard error of means (lower panels) to the various CTL epitopes. b) Mean and standard error of means from a). AUC and EC<sub>50</sub> values of: c) CTL responses and: d) T<sub>H</sub> cell responses. Values are only shown where EC<sub>50</sub> values were calculable as described in the Methods. AUC and EC<sub>50</sub> values are depicted as means from 5 intra-animal replicates. The results are representative of seven independent experiments.</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155594, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.g004", "stats"=>{"downloads"=>0, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_n_FPV_HIV_i_m_VV_HIV_prime_boost_vaccination_improves_the_magnitude_functional_avidity_and_epitope_variant_cross_reactivity_of_T_cell_responses_compared_to_prime_or_boost_vaccinations_alone_/1155594", "title"=>"i.n.FPV-HIV/i.m.VV-HIV prime-boost vaccination improves the magnitude, functional avidity and epitope variant cross-reactivity of T-cell responses compared to prime or boost vaccinations alone.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655040"], "description"=>"<p>24 different vaccination regimes were included based on all combinations of FPV-HIV and VV-HIV administered either i.n. or i.m. in a prime-boost format. A control naïve animal receiving no vaccination (Nil) was also included.</p><p>HIV-1 pox virus vaccination regimes used in this study.</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155595, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.t001", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_HIV_1_pox_virus_vaccination_regimes_used_in_this_study_/1155595", "title"=>"HIV-1 pox virus vaccination regimes used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655041"], "description"=>"<p>The number of animals required for <i>in vivo</i> T cell assays to screen the 25 vaccine strategies (including a naïve control and 24 vaccinations) depicted in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105366#pone-0105366-g003\" target=\"_blank\">Figure 3</a> using a conventional 2-parameter <i>in vivo</i> assay or a 252-parameter FTA <i>in vivo</i> assay.</p><p>Animal number requirements for screening assays.</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155596, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.t002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Animal_number_requirements_for_screening_assays_/1155596", "title"=>"Animal number requirements for screening assays.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655043"], "description"=>"<div><p>Qualitative characteristics of cytotoxic CD8<sup>+</sup> T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically <i>in vivo</i> are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time <i>in vivo</i>. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses <i>in vivo</i>. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an <i>in vivo</i> setting.</p></div>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155598, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Use_of_an_In_Vivo_FTA_Assay_to_Assess_the_Magnitude_Functional_Avidity_and_Epitope_Variant_Cross_Reactivity_of_T_Cell_Responses_Following_HIV_1_Recombinant_Poxvirus_Vaccination/1155598", "title"=>"Use of an <i>In Vivo</i> FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-08-29 02:47:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1655029"], "description"=>"<p>Splenocytes from mice were labeled with combinations of CTV (0 nM, 350 nM, 1295 nM, 4792 nM, 17729 nM and 65595 nM), CFSE (0 nM, 79 nM, 315 nM, 1106 nM 3859nm, 13505nm and 47269 nM) and CPD (0 nM, 106 nM, 690 nM, 2738 nM, 10262 nM and 38506 nM) to generate 252 discernable cell clusters. Cell clusters were pulsed with MHC-binding peptides (as outlined in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105366#pone-0105366-g003\" target=\"_blank\">Figure 3</a>) to generate a panel of 42 peptide pulsed clusters and this repeated 6 times to generate 6 intra-animal repeats (i.e., 252 target cell clusters in total). Target cells were also labeled with DiI for discrimination from host splenocytes (not shown). (a) FTA cells were injected i.v. into host mice that had 6 days earlier been vaccinated with VV-HIV or left unvaccinated as a control. 18 hr after FTA injection, splenocytes were collected and target cells delineated from host splenocytes by DiI label using flow cytometry (not shown). (b) 2D plots of the fluorescence intensities of a panel of one replicate of the 42 peptide-pulsed clusters from the unvaccinated and vaccinated animals and an associated histogram analysis of clusters pulsed with titrations of the F2L CTL epitope, revealing killing in vaccinated animals. (c) An example of histogram analysis of the FTA T helper assay, with B220<sup>+</sup> FTA cells pulsed with the Gag Th T<sub>H</sub> cell epitope being assessed for CD69 up-regulation from the vaccinated animal compared to those from the unvaccinated animal.</p>", "links"=>[], "tags"=>["Recombinant Poxvirus Vaccination", "FTA assay", "T cell responses", "HIV epitope variants", "Vivo FTA Assay", "poxvirus vaccination strategies", "vv", "infection", "avidity", "vivo", "quality CTL responses", "fpv", "screening tool"], "article_id"=>1155583, "categories"=>["Biological Sciences"], "users"=>["Danushka K. Wijesundara", "Charani Ranasinghe", "Ronald J. Jackson", "Brett A. Lidbury", "Christopher R. Parish", "Benjamin J. C. Quah"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0105366.g001", "stats"=>{"downloads"=>2, "page_views"=>28, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_a_252_parameter_FTA_assay_/1155583", "title"=>"Schematic representation of a 252-parameter FTA assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-29 02:47:29"}

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Immunology", "average_usage"=>[293]}, {"subject_area"=>"/Biology and life sciences/Organisms", "average_usage"=>[310]}, {"subject_area"=>"/Medicine and health sciences/Pathology and laboratory medicine", "average_usage"=>[287]}, {"subject_area"=>"/Medicine and health sciences/Public and occupational health", "average_usage"=>[327, 510]}]}
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