Amplification of autologous HIV sequences using multiplex PCR.

<p>Panel A. Sequence alignment of multiple HIV isolates, revealed a region of relative conservation with variable residues in positions 7847 and 7848. Primer REVF7830 is perfectly complimentary to consensus sequence B, whereas primers REVF7830.1 and REVF7830.2 encode compensatory mutations in the 3′ region of the primer, indicated in bold. ⃛denotes deletions, -sequence identity, letters indicate alternative bases in the corresponding positions relative to consensus sequence B. Consensus sequences for common HIV clades as well as less frequent isolates are denoted in bold. Panel B. Schematic overview of the Rev RNA amplification strategy. Open bar denotes regions outside of open reading frame of interest, hatched bar denotes RNA region exon 2 Rev, grey bar represent DNA intermediate products durig amplification process. For details on primer design and amplification refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001489#s4" target="_blank">Method</a> section.</p

Selective utilization of primers by RT-PCR from various subjects materials.

<p>The Nef cDNA sequences were analyzed in the regions corresponding to the regions defined by the primers and identity of the primers was identifies by sequence. Total of 15 Nef clones were analyzed for subjects HTM344, HTM 349 and HTM 367. The number in the right three columns represents how many clones contained the identified primer.</p

Composition of primer groups.

<p>Primers were combined according to their position in the genome.</p><p>Bold indicates the primer group name. Non-bold: names of the primers which comprise each primer groups. Sequence of all primers is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001489#pone-0001489-t001" target="_blank">Table 1</a>.</p

Successful clade-independent amplification of HIV RNA encoding for antigens from infectious plasma.

<p>Panel A: Agarose gel electrophoresis analysis of PCR fragment obtained from three diverse plasma. Amplification from subject plasma infected with Clade B sample. M: 100 bp DNA ladder (Invitrogen). Panel B: Amplification from subject plasma infected with Clade C virus. M: 100 bp DNA ladder (Invitrogen). Panel C: Amplification from subject plasma infected with Clade AG virus. M: AmpliSize DNA ladder (BioRad). Analysis of products obtained after the secondary PCR reaction for Gag, Vpr, Rev, and Nef as marked on the top. Panel D. cDNA obtained in preparative secondary PCR reaction corresponding to Gag, Vpr, Rev, and Nef antigens. M: 100 bp DNA ladder (Invitrogen). The molecular weight of representative DNA bands is indicated on the left. Panel E. RNA corresponding to Gag, Vpr, Rev, and Nef antigens obtained by <i>in vitro</i> transcription using amplified PCR products from subjects plasma. M: molecular weight RNA ladder (Promega), representative marker sizes are indicated on the left. G, V, R, N: in vitro transcribed RNAs for Gag, Vpr, Nef and Nef respectively.</p

Panel A: CFSE-low cells expressed as a percentage of total PBMCs.

<p>Mature DCs (CD209: 96%; CD14: 0%; CD80: 100%; CD83: 91%; CD86: 100%; HLA-DR: 96%; and HLA-I: 100%) were electroporated with 4 HIV antigen-encoding RNAs (hatched bar) or eGFP (solid bar) were cultured with CFSE-labeled PBMCs for 6 days. Frequency of CD8+ CFSE-low were cells determined by flow cytometry. Panel B: CD28/CD45RA phenotype of CD8+ cells induced to proliferate (CFSE-low) by DC electroporated with 4 HIV antigen-encoding RNAs (left panel), as compared to the frequency of CD8+ CFSE-low cells induced by eGFP-RNA loaded control DC (right panel), as determined by flow cytometry. Panel C: Frequency of IFN-γ positive cells within the CD8+ CFSE-low subset induced by 4 hr re-stimulation with DC expressing individual HIV antigen-encoding RNAs, or eGFP control RNA, as determined by intracellular staining and flow cytometry. The background response for single HIV RNA stimulators (1ug HIV RNA/10⁶ DC) was calculated at 0.38% from GFP RNA-electroporated DC (1ug GFP RNA/10⁶ DC) and is indicated by the horizontal dashed line.</p

Capture of HIV quasispesies using the developed multiplex RT-PCR approach.

<p>Phylogenetic relationships of nucleotide sequences of isolated full-length Nef clones (Panel A) and amino acid sequences (Panel B). Horizontal scale indicates the number of nucleotide mutations or amino acid substitutions on each clone relative to neighbor clones.</p

Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

Immunization with human (h)AQP4_281-300 leads to an expansion of antigen-specific CD4⁺ T cells <i>in vivo</i>, and an Ig isotype switch in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice.

<p>(A) Following immunization with human (h)AQP4_281-300, an expansion of antigen-specific CD4⁺ T helper cells was detected by tetramer staining of lymph node cells. The fluorescent signal of <i>HLA-DRB1*03</i>:<i>01</i>-loaded tetramers minus the fluorescent signal of empty <i>HLA-DRB1*03</i>:<i>01</i> tetramers is shown. CD4⁺ T helper cells provide soluble mediators that drive B cell differentiation immunoglobulin (Ig) class switching. To determine whether hAQP4_281-300-reactive CD4⁺ T cells are capable of causing IgM to IgG isotype switching in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice, the concentration of Ig against hAQP4_281-300, mAQP4284-299, or with whole-length hAQP4 protein in serum of immunized mice was quantified longitudinally. Since the NMO-IgG is a human IgG1 isotype, both, the murine IgG2a and IgG2b isotype were examined as they have similar properties with regard to complement binding and the Fcγ receptor. A switch from IgM to IgG2b was detected in mice immunized with hAQP4_281-300 peptide with regard to (B) antibody responses against hAQP4_281-300 and (C) whole-length AQP4 protein. An Ig isotype switch from IgM to IgG2b was also detectable in mice immunized with whole-length AQP4 protein with regard to (D) antibody responses against hAQP4_281-300 and (E) whole-length AQP4 protein.</p

Human (h)AQP4_281-300-specific T cells do not cross-react with murine (m) AQP4_281-300.

<p>(A) There is that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290. (B) In lymph node cells of <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 there was a significant proliferation of CD4⁺ T cells when hAQP4_281-300 was used as the recall antigen (* = P-value = 0.01). Only a higher recall antigen dose of 25 μg/ml resulted in a significant increase in proliferation, whereas as a dose of 5 μg/ml did not. (C) There was no proliferative response to mAQP4_281-300 at either dose_. (D) There is a significantly increased frequency of IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 by ELISpot assay when hAQP4_281-300, and hAQP4_281-299 are used as recall antigens. However, we were unable to detect antigen specific IFNγ and IL-17 producing lymph nodes cells when mAQP4_281-300, or the negative control hAQP4_66-79 were used as recall antigens (** = P-value < 0.01). (E) IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with mAQP4_281-300 were undetectable with any of the recall antigens.</p

<i>HLA-DRB1*03</i>:<i>01</i> transgenic mice are disease resistant to active immunization with human aquaporin 4 (hAQP4), and adoptive transfer of hAQP4-specific T cells.

<p>(A) <i>HLA-DRB1*03</i>:<i>01</i> mice were actively immunized with proteolipid protein (PLP)_91-110 (100 μg/100 μl/mouse; positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152720#pone.0152720.ref025" target="_blank">25</a>]), or varying AQP4 antigens*(whole-length hAQP4 protein, hAQP4_281-300, murine (m)AQP4_281-300, hAQP4_281-300 with a Quil-A Incomplete Freund Adjuvant (IFA) booster on day 14 post-immunization, mAQP4_281-300 with a Quil-A IFA booster on day 14 post immunization, and hAQP4_281-300 plus mAQP4_281-300) emulsified in Complete Freund Adjuvant (CFA). Immunization with a positive control proteolipid protein (PLP)_91-110, a dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> led to typical EAE. (B) Lymph node cells taken from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 or mAQP4_281-300 were restimulated for three days and passively transferred into <i>HLA-DRB1*03</i>:<i>01</i> mice. None of these experimental approaches resulted in clinical disease. (C) Paraffin sections were stained with haematoxlin eosin (H&E) and luxol fast blue (LFB). Representative sections of the spinal cords from PLP_91-110 and hAQP4_281-300 immunized mice are shown. On histopathological examination there were no visible signs of cellular infiltration, inflammation, or demyelination within the brain and spinal cord in any experimental paradigms other than in active immunization with PLP_91-110, the dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> that led to typical EAE (spinal cord shown; inflammatory infiltrates and areas of demyelination are indicated by black arrows). (D) Fifteen days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with PLP_91-110 or hAQP4_281-300, pupillary reflex was measured via a mouse pupillometry. Mice actively immunized with hAQP4_281-300 and the control antigen PLP_91-110 did not show altered pupillary responses.</p