Journal: Journal of Virology

Article Title: Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIV mac251 at Challenge Exposure

doi: 10.1128/JVI.02863-12

Figure Lengend Snippet: Effect of vaccination on SIV replication and CD4+ T cells. (A) Immunization regimen. All macaques were immunized with DNA and ALVAC SIVgpe at the time indicated by arrows. The alum-adjuvanted monomeric native SIVmac251 gp120 protein was given at weeks 40 and 57/61. Twenty control animals received alum only. Eight vaccinated and eight control macaques were exposed to a single dose of 6,100 TCID50 of SIVmac251 at week 61. Twelve vaccinated and 12 control macaques were exposed weekly to 470 TCID50 of SIVmac251 until all control animals had detectable plasma virus. Plasma SIV-RNA values are depicted as means ± standard errors for vaccinated (green) and control (blue) animals exposed to one dose of 6,100 TCID50 (B) or two doses of 470 TCID50 (C) of SIVmac251. The arrows indicate the time of exposure, and the P values indicate differences between the vaccinated and controls within groups (Wilcoxon rank sum test). Changes in the CD4+ T-cell count in blood expressed as means ± standard errors of the percentage of change with respect to the prechallenge level in vaccinated and control macaques exposed to 6,100 TCID50 (D) or to 470 TCID50 (E) of SIVmac251. CD4+ T-cell numbers were reconstituted significantly in vaccinated macaques exposed to 470 TCID50 of SIVmac251 (weeks 16 to 30, P < 0.005, by the Wilcoxon rank sum test). The SIV-exposed uninfected animals were excluded from the analysis.

Article Snippet: Briefly, the primary antibodies used included monoclonal anti-CD4 + T-cell mouse serum antibody (clone IF6; Vector, Burlingame, CA).

Techniques: Cell Counting

Journal: Journal of Virology

Article Title: Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIV mac251 at Challenge Exposure

doi: 10.1128/JVI.02863-12

Figure Lengend Snippet: Virus levels in vaccinated and control macaques exposed to 6,100 and 470 TCID 50

Article Snippet: Briefly, the primary antibodies used included monoclonal anti-CD4 + T-cell mouse serum antibody (clone IF6; Vector, Burlingame, CA).

Techniques: Infection, Variant Assay

Journal: Journal of Virology

Article Title: Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIV mac251 at Challenge Exposure

doi: 10.1128/JVI.02863-12

Figure Lengend Snippet: Vaccination affects the number of virus variants transmitted and virus level. (A) Estimated number of transmitted viral lineages at 6,100 TCID50 and 470 TCID50 in vaccinated (green and red bars) and control animals (blue bars). (B) Phylogenetic tree of env gene from the inoculum stock (black ovals), and 3 vaccinated animals challenged with 470 TCID50. Sequences from P013 (orange ovals) and M890 (green ovals) cluster together in 1 or 2 lineages, which demonstrates evidence of 1 and 2 variants establishing systemic infection. Sequences from P001 (purple ovals) are found dispersed throughout the tree, providing evidence of multiple founder viruses (>10). Sequences were obtained from blood plasma with the exception of jejunum (^) or rectal (*) biopsy specimen sequences obtained at week 2 postinfection. (C) Log-transformed viral DNA copies in snap-frozen rectal or jejunal biopsy specimens collected at week 2 after infection in vaccinated and control macaques exposed to either 6,100 TCID50 (left) or 470 TCID50 (right) of SIVmac251. Vaccinated animals are in green or red and the respective controls in blue. P values are calculated using the Wilcoxon rank sum test, excluding the three vaccinated but uninfected animals, P006, P018, and P010. The frequency of CD4+ T cells per mm2 in the rectal mucosa of vaccinated and control macaques was enumerated by immunohistochemistry prior to infection, and at weeks 3 and 11, following the challenge exposure of vaccinated and control macaques to a single dose of 6,100 TCID50 (D) or two doses of 470 TCID50 (E) of SIVmac251. P values are calculated by repeated-measure analysis of variance, and the vaccinated uninfected animals P006, P018, and P010 are excluded from this analysis.

Article Snippet: Briefly, the primary antibodies used included monoclonal anti-CD4 + T-cell mouse serum antibody (clone IF6; Vector, Burlingame, CA).

Techniques: Infection, Transformation Assay, Immunohistochemistry

Journal: Journal of Virology

Article Title: Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIV mac251 at Challenge Exposure

doi: 10.1128/JVI.02863-12

Figure Lengend Snippet: Immune responses that correlate with protection from high plasma virus levels. For panels A to E, single dots represent the frequency in blood of cytokine-producing memory (CD95+) CD4+ or CD8+ T cells from all vaccinated macaques at 3 weeks before SIVmac251 exposure; the background measured from nonstimulated cells has already been subtracted. Vaccinated animals exposed to 470 TCID50 are depicted in red; vaccinated animals exposed to 6,100 TCID50 are depicted in green, controls are in blue, and the protected animals P010, P006, and P018 are in black. The frequency of memory CD8+ T cells producing IFN-γ after stimulation with Gag (A) and Env (B) peptides is shown in the left panel for all vaccinated animals. The correlations between the values of vaccinated macaques exposed to 6,100 TCID50 and the peak viral loads and the median of viral loads measured from weeks 7 to 31 are depicted in the left panels. The frequency of envelope-specific memory CD4+ T cells producing IFN-γ (C) or TNF-α (D) is depicted in the left panel for all vaccinated animals. The correlations between these values and the peak viral loads for vaccinated macaques exposed to 470 TCID50 are depicted in the left panel. The values plotted for the peak virus level correspond to the highest RNA SIV levels measured after viral exposure (week 2 or 3) for each animal. The values plotted for the peak viral load correspond to the highest RNA SIV levels measured in the plasma after viral exposure (week 2) for each animal. (E) Frequency of Gag CD107+ TNF-α+-producing memory (CD95+) CD8+ T cells measured in mononuclear cells from lymph nodes in all macaques (left) and correlation between these values and the median of viral loads measured from weeks 8 to 34 in macaques exposed to the 470 TCID50 dose (right).

Article Snippet: Briefly, the primary antibodies used included monoclonal anti-CD4 + T-cell mouse serum antibody (clone IF6; Vector, Burlingame, CA).

Techniques:

Journal: Journal of Virology

Article Title: Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIV mac251 at Challenge Exposure

doi: 10.1128/JVI.02863-12

Figure Lengend Snippet: Correlates of protection from a high level of SIV replication in vaccinated macaque

Article Snippet: Briefly, the primary antibodies used included monoclonal anti-CD4 + T-cell mouse serum antibody (clone IF6; Vector, Burlingame, CA).

Techniques: Infection

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Potential mechanisms for infected cell resistance to CTL-mediated lysis. CD4 + T cells exhibit stable differences in susceptibility to lysis (A and B). If resistance is associated with properties that allow preferential infection, then infected cells may show greater resistance than uninfected cells (A). Alternatively, intrinsically susceptible cells may be eliminated over time, with death-resistant HIV-1-harboring cells remaining (B). A third possibility (C) is that HIV-1 induces phenotypic changes that increase cell death resistance of infected cells (HIV-induced resistance).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Infection, Lysis

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Experimental method to assess resistance to CTL-mediated killing. (A) Co-culture setup: primary CD4 + T cells from PLWH are pulsed with mutant p53 peptide and cultured with autologous, pre-expanded CD8 + T cells and p53-specific scDb. IPDA is performed on the viable CD4 + T cell population isolated before and after the co-culture to assess for enrichment of infected cells, indicative of resistance. (B) scDb mechanism of action: scDbs bind to cognate peptide:MHC complexes on the target cell surface and also to the CD3ε subunit of the TCR complex to induce localized cytotoxicity. (C) Flow cytometry gating strategy to determine frequency of viable target cells. (D) Representative histograms of co-cultures with and without scDbs. The reduction in the viable CD4 + T cell count (marked region) was used to determine the amount of CTL-mediated lysis. I Representative viability data for study participant OM5418 for the experimental condition and seven control conditions.

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Isolation, Infection, Flow Cytometry, Cell Characterization, Lysis, Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Ex vivo co-cultures with cells from PLWH do not show enrichment for infected cells in 10/12 participants. (A) Representative ddPCR plots of IPDA performed on CD4 + T cells from donor CIRC0226 before and after co-culture. Each dot represents one droplet out of at least 10,000 droplets measured per assay. Measured frequencies were corrected for cell count and DNA shearing as described previously . (B) Pre- and post-culture frequency of intact provirus per 1x10 6 CD4 + T cells. Each point represents the calculated frequency of intact provirus for one assay for the indicated study participant. (C) Violin plot of enrichment for total study cohort. Each dot represents the average enrichment score for an individual study participant. Colors and symbols coding as in (B). Dashed line marks no change in frequency; horizontal lines mark the median and first and third quartile. (D) Correlation between extent of killing and enrichment for intact proviruses. (E) Correlation between time on ART and enrichment of intact provirus. (F) Comparison of enrichment of intact provirus between study participants with less than 10 years of continuous ART (short-term) and over 13 years of continuous ART (long-term).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Ex Vivo, Infection, Co-Culture Assay, Cell Characterization, Comparison

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: pMHC-specific latency reversal by the p53-specific scDb. (A) Frequencies of CD69 + CD4 + T cells in individual co-cultures without and with scDb across at least two co-cultures for all twelve study participants. Co-cultures were set up as described in . Error bars represent standard deviation. (B) Induction of viral RNA by the p53-specific scDb. Primary CD4 + T cells from HIV-1-positive study participants were pulsed with the p53 R175H peptide (n = 3) or an irrelevant control peptide (HLA-A2 signal peptide, VMAPRTLVL; n = 1) and cultured for 18 hours with or without p53-specific scDb. RNA was isolated and reverse transcribed. Digital PCR was performed with probes binding to poly-adenylated HIV-1 RNA (poly-A), the integrase region of HIV-1 pol (int) or the splice junction for vpu-env mRNA. (C-E) pMHC-specific increase in HIV-1 transcripts induced by the p53-specific scDb. Measurements were averaged over two technical replicates for each of three identical culture wells per condition, and normalized to transcript level in the no treatment (NT) control. PMA/ionomycin was included as positive control. Colors represent treatment conditions: black: no treatment; red: p53-specific scDb; green: PMA/I. NC: non-cognate peptide (HLA-A2 signal peptide SP-2A, VMAPRTLVL), C: cognate peptide (p53 R175H , HMTEVVRHC).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Standard Deviation, Control, Cell Culture, Isolation, Reverse Transcription, Digital PCR, Binding Assay, Positive Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Effect of active viral expression on cell death resistance. (A) Experimental setup: Healthy donor CD4 + T cells were activated for 72 hours with PHA and infected with different HIV-1 GFP-reporter constructs. Cells were then pulsed with exogenous p53 R175H peptide and co-cultured with pre-expanded autologous CD8 + T cells and p53-specific scDb or irrelevant control scDb. Viability and GFP-expression were analyzed through flow cytometry. (B) Genome maps of the HIV-1 reporter constructs used to infect target cells. Each row represents one reading frame. Striped segments indicate gene segments that are no longer translated due to insertion of a STOP-codon. (C) Representative flow plot showing viable uninfected (GFP-negative) and HIV-1-expressing (GFP-positive) cell populations. (D) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-A2:p53 R175H -restricted scDb. Each dot shows the average of three technical replicates per condition. Symbols represent individual donors; assay was performed 2 independent times for each of the n=5 healthy donors, except for the full-length condition, where one donor was only tested once for technical reasons. A p-value less than 0.05 was considered significant. Abbreviations: PHA, phytohemagglutinin; LTR, long terminal repeat; IRES, internal ribosome entry site. ns: no significance (p > 0.05).

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Expressing, Infection, Construct, Cell Culture, Control, Flow Cytometry, Co-Culture Assay

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Modified assay targeting stably expressed HLA-E. (A) Cell surface levels of classical HLA-A2 and non-classical HLA-E molecules on untreated, uninfected and HIV-1-expressing cells. Healthy donor CD4 + T cells were either left untreated (NT) or activated with PHA over 72 hours and infected with indicated reporter virus construct. Cells were then sorted by GFP-expression and stained with PE-labeled monoclonal antibodies for HLA-A2 or HLA-E. Each bar represents average measurements across n=3 healthy donors. (B) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-E-restricted RLP-13 scDb. Each dot shows the average of three technical replicates per condition. Symbols represent individual donors; assay was performed 2 independent times for each of the n=5 healthy donors. Bars indicate average viability and standard deviation.

Article Snippet: To quantify the frequency of intact provirus before and after co-cultures, viable CD4 + T cells were isolated using the Milteniy Dead Cell Removal kit (Cat. No# 130-090-101) and the StemCell EasySep Human CD4 + T cell Enrichment kit (Cat. No# 19052), and genomic DNA was isolated with the Qiagen QIAamp DNA Micro kit (Cat. No# 56304) and used in the IPDA as previously described ( ).

Techniques: Modification, Stable Transfection, Expressing, Infection, Virus, Construct, Staining, Labeling, Bioprocessing, Co-Culture Assay, Standard Deviation

Journal: Viral Immunology

Article Title: Intraepithelial T-Cell Cytotoxicity, Induced Bronchus-Associated Lymphoid Tissue, and Proliferation of Pneumocytes in Experimental Mouse Models of Influenza

doi: 10.1089/vim.2014.0077

Figure Lengend Snippet: Primary Antibodies Used to Label Tissue Sections

Article Snippet: Staining sections with CD4 and CD8 antibodies was attempted using several different antibodies/clones and all listed antigen retrieval methods, without success. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Target Source Clone Host Concentration Antigen retrieval CD3 T-cells Abcam ab5690 Polyclonal Rabbit 1:25 Tris HCl buffer CD3 a T-cells AbD Serotec MCA1477 CD3-12 Rat 1:25 Tris HCl buffer F4/80 a Tissue macrophages, including Kupffer and Langerhans cells, some dendritic cells, and microglia but not macrophages in T-cell areas of spleen and lymph node AbD Serotec MCA497G CLA3-1 Rat 1:10,000 b Proteinase K digestion Pax5 a B–cells Abcam ab109443 3852-1 Rabbit 1:20,000 b Citrate buffer Prosurfactant protein C a Type II pneumocytes, airway epithelial cells Abcam ab40879 Polyclonal Rabbit 1:2,000 Tris HCl buffer CD4 T-cell subset BD Pharmingen no. 550280 RM4-5 Rat 1:20 Multiple 1:20,000 b CD4 T-cell subset eBioscience no. 14-0042 RM4-5 Rat 1:20 Multiple 1:20,000 b CD8a T-cell subset AbD Serotec MCA609B KT15 Rat 1:10 Multiple 1:20,000 b CD8a T-cell subset BD Pharmingen no. 550 281 53-6.7 Rat 1:10 Multiple 1:20,000 b CD8b T-cell subset eBioscience no. 14-0083 H35-17.2 Rat 1:10 Multiple 1:20,000 b CD45R B- cells, LAK, NK AbD Serotec MCA 1258 RA3-6B2 Rat 1:400 Citrate buffer CD68 Blood monocytes, tissue macrophages.

Techniques: Concentration Assay

Journal: Viral Immunology

Article Title: Intraepithelial T-Cell Cytotoxicity, Induced Bronchus-Associated Lymphoid Tissue, and Proliferation of Pneumocytes in Experimental Mouse Models of Influenza

doi: 10.1089/vim.2014.0077

Figure Lengend Snippet: Summary of Experimental Models and Results

Article Snippet: Staining sections with CD4 and CD8 antibodies was attempted using several different antibodies/clones and all listed antigen retrieval methods, without success. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Target Source Clone Host Concentration Antigen retrieval CD3 T-cells Abcam ab5690 Polyclonal Rabbit 1:25 Tris HCl buffer CD3 a T-cells AbD Serotec MCA1477 CD3-12 Rat 1:25 Tris HCl buffer F4/80 a Tissue macrophages, including Kupffer and Langerhans cells, some dendritic cells, and microglia but not macrophages in T-cell areas of spleen and lymph node AbD Serotec MCA497G CLA3-1 Rat 1:10,000 b Proteinase K digestion Pax5 a B–cells Abcam ab109443 3852-1 Rabbit 1:20,000 b Citrate buffer Prosurfactant protein C a Type II pneumocytes, airway epithelial cells Abcam ab40879 Polyclonal Rabbit 1:2,000 Tris HCl buffer CD4 T-cell subset BD Pharmingen no. 550280 RM4-5 Rat 1:20 Multiple 1:20,000 b CD4 T-cell subset eBioscience no. 14-0042 RM4-5 Rat 1:20 Multiple 1:20,000 b CD8a T-cell subset AbD Serotec MCA609B KT15 Rat 1:10 Multiple 1:20,000 b CD8a T-cell subset BD Pharmingen no. 550 281 53-6.7 Rat 1:10 Multiple 1:20,000 b CD8b T-cell subset eBioscience no. 14-0083 H35-17.2 Rat 1:10 Multiple 1:20,000 b CD45R B- cells, LAK, NK AbD Serotec MCA 1258 RA3-6B2 Rat 1:400 Citrate buffer CD68 Blood monocytes, tissue macrophages.

Techniques: Knock-Out

Journal: Viral Immunology

Article Title: Intraepithelial T-Cell Cytotoxicity, Induced Bronchus-Associated Lymphoid Tissue, and Proliferation of Pneumocytes in Experimental Mouse Models of Influenza

doi: 10.1089/vim.2014.0077

Figure Lengend Snippet: Lungs of WT mice receiving naïve or memory CD4 T-cells at 2 days after intranasal challenge with 10,000 EID50 PR8 influenza virus. (A) normal (10×), (B) Naïve (10×), (C) memory (10×). (D, E, F) memory CD3 (20×, 20×, 40×), (G) H&E (20×), (H) CD3 (20×), (I) Pax5 (20×). There is mild (grade 1) mononuclear cell perivascular (D, E) and peribronchial inflammation (F) after flu challenge to mice receiving naïve cells (B). There is much greater infiltrate (grade 4) in challenged mice after transfer of memory cells (C). In addition, in (C), there is infiltration of the vascular wall of some arteries as well as necrosis of bronchial epithelium with luminal exudate (G–I). Most of the perivascular and peribronchial cells stain with CD3 (T-cells; D–F), with a few cells staining with Pax5 (B-cells, arrow in I, below bronchus) or F4/80 (not shown). At this time point, there is little invasion of the bronchial epithelium by T-cells. However, some sections show invasion and loss of the basement membrane (large arrows in F–H). WT, wild type.

Article Snippet: Staining sections with CD4 and CD8 antibodies was attempted using several different antibodies/clones and all listed antigen retrieval methods, without success. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Target Source Clone Host Concentration Antigen retrieval CD3 T-cells Abcam ab5690 Polyclonal Rabbit 1:25 Tris HCl buffer CD3 a T-cells AbD Serotec MCA1477 CD3-12 Rat 1:25 Tris HCl buffer F4/80 a Tissue macrophages, including Kupffer and Langerhans cells, some dendritic cells, and microglia but not macrophages in T-cell areas of spleen and lymph node AbD Serotec MCA497G CLA3-1 Rat 1:10,000 b Proteinase K digestion Pax5 a B–cells Abcam ab109443 3852-1 Rabbit 1:20,000 b Citrate buffer Prosurfactant protein C a Type II pneumocytes, airway epithelial cells Abcam ab40879 Polyclonal Rabbit 1:2,000 Tris HCl buffer CD4 T-cell subset BD Pharmingen no. 550280 RM4-5 Rat 1:20 Multiple 1:20,000 b CD4 T-cell subset eBioscience no. 14-0042 RM4-5 Rat 1:20 Multiple 1:20,000 b CD8a T-cell subset AbD Serotec MCA609B KT15 Rat 1:10 Multiple 1:20,000 b CD8a T-cell subset BD Pharmingen no. 550 281 53-6.7 Rat 1:10 Multiple 1:20,000 b CD8b T-cell subset eBioscience no. 14-0083 H35-17.2 Rat 1:10 Multiple 1:20,000 b CD45R B- cells, LAK, NK AbD Serotec MCA 1258 RA3-6B2 Rat 1:400 Citrate buffer CD68 Blood monocytes, tissue macrophages.

Techniques: Staining