Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

Discovery of Hapten-Specific scFv from a Phage Display Library and Applications for HER2-Positive Tumor Imaging

In this study, an anti-hapten antibody (single chain Fv, scFv) against a hapten probe was developed as a unique reporter system for molecular imaging or therapy. The hapten peptide (histamine-succinyl-GSYK, Him) was synthesized for phage displayed scFv affinity selection and for conjugation with cypate (Cy-Him) for <i>in vivo</i> near-infrared (NIR) optical imaging. Hapten-specific scFvs were affinity selected from the human single fold phage display scFv libraries (Tomlinson I + J) with high specificity and affinity. Utilizing HER2 targeting as a model system, the highest affinity scFv (clone J42) was recombinantly fused to an anti-HER2 affibody (scFv-L-Aff) with no loss of affinity of either protein. The functionality of the hapten-scFv reporter system was tested <i>in vitro</i> with a HER2-positive human breast cancer cell line, SK-BR3, and <i>in vivo</i> with SK-BR3 xenografts. ScFv-L-Aff mediated the binding of the hapten to HER2 on SK-BR3 cells and from tissue from the SK-BR3 xenograft; however, scFv-L-Aff did not mediate uptake of the hapten in the SK-BR3 xenografted tumors, presumably due to rapid internalization of the HER2/scFv-L-Aff complex. Our results suggest that this hapten-peptide and anti-hapten scFv can be a universal reporter system in a wide range of imaging and therapeutic applications

Biological effects of dosing aerobic exercise and neuromuscular electrical stimulation in rats

Aerobic exercise (AE) and non-Aerobic neuromuscular electric stimulation (NMES) are common interventions used in physical therapy. We explored the dose-dependency (low, medium, high) of these interventions on biochemical factors, such as brain derived neurotrophic growth factor (BDNF), vascular endothelial growth factor-A (VEGF-A), insulin-like growth factor-1 (IGF-1) and Klotho, in the blood and brain of normal rats, as well as a treadmill-based maximum capacity test (MCT). A medium dose of AE produced the most improvement in MCT with dose-dependent changes in Klotho in the blood. A dose-dependent increase of BDNF was evident following completion of an NMES protocol, but there was no improvement in MCT performance. Gene expression in the hippocampus was increased after both AE and NMES, with IGF-1 being a signaling molecule that correlated with MCT performance in the AE conditions, but also highly correlated with VEGF-A and Klotho. Blood Klotho levels can serve as a biomarker of therapeutic dosing of AE, whereas IGF-1 is a key molecule coupled to gene expression of other molecules in the hippocampus. This approach provides a translatable paradigm to investigate the mode and mechanism of action of interventions employed in physical therapy that can improve our understanding of how these factors change under pathological conditions

Statistical Analysis of Trim5 haplotype versus outcome.

1<p>2-sided Fisher's Exact Test.</p

Therapeutic DNA vaccination increases mucosal SIV-specific T cell responses in the gut.

<p>(<b>A</b>) The magnitude of the SIV-specific IFN-γ T cell response in the gut was determined by ELISPOT analysis of lamina propria lymphocytes (LPL) isolated from jejunal resections. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033715#s2" target="_blank">Results</a> represent the mean number of IFN-γ spot forming cells (± SEM) for each group from 2 separate experiments performed at 2 time-points post-infection (weeks 42 and 48). (<b>B</b>) Breadth of the T cell response in each macaque in the blood (top panels, PBMC) and gut (lower panels, GALT). SIV-specific T cell responses in PBMC and LPL isolated from the jejunum were measured by IFN-γ ELISPOT assay against 11 separate pools of overlapping peptides (15-mers overlapping by 11 amino acids) comprising the indicated amino acid sequences from SIV Gag, Env, Pol, and Nef. The percent contribution of each peptide-pool specific response to the total response was determined by dividing the mean number of IFN-γ spot forming cells (SFC) measured against each individual peptide pool by the sum of the response against all peptide pools. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033715#s2" target="_blank">Results</a> represent the average of 2 time-points tested at weeks 42 and 48. (<b>C</b>) Mean absolute GALT CD4+ T cell counts measured by flow cytometric analysis of mononuclear cells isolated from the jejunal lamina propria at weeks 12, 42, and 48 post-infection were similar in animals that controlled vs. failed to control viral rebound after ART was stopped. Controllers are animals that contained virus at ≤100 copies for at least 5 months after stopping ART. Non-controllers are animals that exhibited viral rebound within the same timeframe.</p

Therapeutic DNA vaccines increase SIV-specific IFN-γ T cell responses, but not antibody, in the blood.

<p>(<b>A</b>) IFN-γ T cell responses were measured throughout the study by ELISPOT. Shown is the mean (± SEM) SIV-specific IFN-γ T cell response at each time-point minus background. Background levels were <25 SFC/10⁶ PBMC. Differences between the DNA or DNA+LT vaccine groups and controls were significant (P≤0.0063, repeated measures ANOVA). (<b>B</b>) SIV-gp120 specific binding antibody was measured throughout the study by ELISA. Shown are mean (± SEM) endpoint titers. Mean antibody titers in the DNA+LT group were significantly lower (P = 0.0422) than the controls (<b>C</b>) Neutralizing antibody responses were measured against the vaccine strain (SIV/17E-Fr). Shown are mean (± SEM) neutralization titers after the final DNA vaccine dose but before stopping ART (week 40) and 9 weeks after stopping ART (week 54). *P = 0.0292 (<b>D</b>) Neutralizing antibody titers in each animal before (week 40) and after (week 54) stopping ART against the challenge strain (SIV/DeltaB670).</p

LT-adjuvanted DNA vaccine increases T cells with multiple effector functions in the blood.

<p>SIV-specific T cells with 1–4 cytokine (IFN-γ, TNF-α, IL-2) or cytolytic (CD107a) effector functions were measured in PBMC 14 weeks after ART was withdrawn (week 58) by flow cytometry following <i>in vitro</i> stimulation with overlapping peptide pools derived from genes included in the DNA vaccine (RT, Nef, Gag, Env). Boolean gating was performed to identify the total frequency of CD4+ and CD8+ T cells in the blood of ART responders with any one of 1–4 effector functions. (<b>A</b>) Cumulative mean (±SEM) frequency of SIV-specific CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, IL-2 or CD107a. Cumulative mean frequency (± SEM) of SIV-specific (<b>B</b>) CD4+ and (<b>C</b>) CD8+ T cells co-expressing any combination of 2–4 effector functions. Mean (± SEM) frequency of SIV-specific (<b>D</b>) CD4+ and (<b>E</b>) CD8+ T cells expressing the indicated combination of dual effector functions.</p

Therapeutic DNA vaccination reduces viral load and protects from viral rebound after drug is stopped.

<p>Plasma viral RNA load was determined by real time RT-PCR. Boxed areas (ART) indicate period of antiretroviral drug therapy. Arrows indicate DNA vaccine doses. (<b>A</b>) Response to ART (weeks 0–18). Shown are viral loads in 41 SIV-infected macaques before starting ART (weeks 0–6) and during the first 12 weeks of ART only (weeks 6–18). ART responders (black) had declining viral loads during ART only. ART low responders (red) had little or no decline in viral loads during ART only and maintained ≥5×10⁴ viral RNA copies/ml (dotted line). (<b>B</b>) Log-fold reduction in viral load during ART only. The change in viral load during ART in each animal was determined by dividing the mean viral load prior to initiating ART (weeks 4–6) by the mean viral load during the first 12 weeks of ART but prior to initiating the therapeutic vaccinations (weeks 8–18). Shown is the log-fold reduction in mean viral load during ART for each animal. ART low responders had <1 log-fold reduction. ART responders exhibited 1.6–4.9 log-fold reductions in viral load during ART. (<b>C</b>) Mean plasma viral loads in each group prior to initiating ART (week 6, Pre-ART) and after 12 weeks on ART but before starting therapeutic vaccinations (week 18, Pre-vaccine). (<b>D</b>) Mean plasma viral loads during vaccinations and after withdrawing ART. Differences in mean viral loads after withdrawing ART are significant starting at weeks 68 and 48 in the DNA (P = 0.019) and DNA+LT (P = 0.002) groups, respectively, and remained significant thereafter when compared to controls (Wilcoxon test). (<b>E</b>) Viral loads in each animal. Protection from viral rebound was defined as containment of viral load ≤100 viral RNA copies/ml (hashed line) for a period of at least 5 months after drug was withdrawn (weeks 45–66).</p

Relationship between <i>TRIM5</i> haplotype and outcome.

1<p><u>Rebound</u>: Plasma virus load rebounded to >1000 copies/ml within 26 weeks after stopping ART.</p>2<p><u>Elite Controller</u>: plasma virus load is contained post-ART at ≤100 copies/ml for duration of study (44 after stopping ART).</p>3<p><u>Late Rebound</u>: Plasma virus load was contained for at least 26 weeks after stopping ART but rebounded to >1000 copies/ml before end of study.</p>4<p><u>TFP/Q and Q/CypA</u>: associated with intermediate susceptibility to SIVmac251 and SIVsmE660 infection (34, 35).</p>5<p><u>TFP/TFP and TFP/CypA</u>: associated with resistance to SIVmac251 and SIVsmE660 infection (34, 35).</p

Therapeutic DNA vaccination reduces virus production in the lymph nodes and gut mucosa.

<p>(<b>A</b>) Viral load in gut lamina propria (LP), mesenteric lymph nodes (MLN), and axillary lymph nodes (ALN) in each animal 3 weeks before (week 42, solid circles) and 3 weeks after ART was withdrawn (week 48, open circles). Solid lines indicate the median viral load for the group. Hashed line indicates the lower limit of detection for this analysis (3 viral RNA copies). (<b>B</b>) Correlations between mean virus production in the gut lamina propria (left panel), mesenteric lymph nodes (middle panel) and axillary lymph nodes (right panel) vs. mean plasma viral load in each animal post-ART. Each symbol corresponds to mean virus load in the plasma of each animal after stopping ART (weeks 47–88) on the X axis vs. mean virus production measured in tissues at weeks 42 and 48 on the Y axis. Spearman's rank correlation coefficients are shown.</p