Immune Modulation through 4-1BB Enhances SIV Vaccine Protection in Non-Human Primates against SIVmac251 Challenge

Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4+ and CD8+ T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8+ T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4+ T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development

Synergistic activity of ixabepilone plus other anticancer agents: preclinical and clinical evidence

Ixabepilone demonstrates marked synergistic activity in combination with capecitabine, which served as the rationale for the evaluation of this combination in the clinic. Ixabepilone plus capecitabine is currently approved for patients with locally advanced or metastatic breast cancer (MBC) progressing after treatment with an anthracycline and a taxane; approval was based on the results of two phase III trials comparing the combination with capecitabine monotherapy. An array of preclinical studies in multiple solid tumor types show that ixabepilone demonstrates therapeutic synergy with targeted therapies including trastuzumab, bevacizumab, brivanib, and cetuximab; with immune-modulating agents such as anti-CTLA-4 antibody; and with other chemotherapy drugs such as irinotecan and epirubicin. Notably, experiments in several xenograft models show that ixabepilone provides greater antitumor synergism when combined with bevacizumab than either paclitaxel or nab-paclitaxel combined with bevacizumab. These preclinical findings provide a foundation for ongoing phase II clinical trials using ixabepilone in combination with trastuzumab or lapatinib in HER2-positive breast cancer; with bevacizumab in breast cancer, endometrial cancer, renal cancer, and non-small cell lung cancer (NSCLC); with cetuximab in breast cancer, NSCLC, and pancreatic cancer; and with brivanib, dasatinib, sorafinib, sunitinib, or vorinostat in MBC. Preliminary results from several of these trials suggest that ixabepilone-based combinations have promising anticancer activity

Polyfunctional profile of CD8⁺ T cells.

<p>PBMCs isolated 2 weeks after the fourth immunization were stimulated <i>in vitro</i> with a SIVpol peptide pool mix for 5 hours. Cells were stained for intracellular production of IFN-γ, TNF-α and IL-2 and degranulation by CD107a. The magnitude of the SIVgag (white), env (grey) and pol (black) responses are shown as stacked means ± SEM for each group (<b>a</b>). The percentage of the total functional response that has a CD28⁻CD95⁺ (black bar) or CD28⁺CD95⁺ (white bar) is shown as group means ± SEM (<b>b</b>). Pie charts show the proportion of antigen-specific CD8⁺ T cells that have 4 functions (purple), 3 functions (yellow), 2 functions (green) or 1 function (light blue) (<b>c</b>). The bar graphs depict the absolute frequency of each of the 15 functional combinations for the DNA (red), 4-1BB (blue), CTLA-4 (orange), Combo (green) and Saline (black) groups in response to SIVgag, env, and pol after background subtraction (<b>d</b>).</p

Study design and immunization schedule.

<p>Study design and immunization schedule.</p

Maintenance of memory T cells.

<p>PBMCs were collected from immunized macaques at 10 months following a fourth immunization to evaluate memory IFN-γ responses. Cells were then stimulated with SIVmac239 gag (white bars), env (grey bars), and pol (black bars) peptide pools in 18-hour IFN-γ ELISpot assays (<b>a</b>). PBMCs were also labelled with CFSE and stimulated with pooled SIVmac239 gag (white bars), env (grey bars), and pol (black bars) peptides for 5 days. Cells were then stained for phenotypic markers and analyzed by flow cytometry to determine the proliferative response to each antigen (<b>b and c</b>). For both ELISpot and proliferation assays, error bars represent mean responses ± SEM.</p

CTLA-4 blockade enhances the proliferative capacity of CD4⁺ and CD8⁺ T cells.

<p>Fresh PBMCs isolated after the second immunization were stained with CFSE and stimulated with SIVgag, env, and pol peptides <i>in vitro</i> for 5 days to determine the proliferative capacity of antigen-specific cells. The proliferative capacity of the CD4⁺(<b>a</b>) and CD8⁺ (<b>b</b>) T cell compartments are shown as stacked group mean responses ± SEM. Statistical differences between groups were determined by a one-way ANOVA with a Tukey post-hoc test.</p

Modulation of Th1/Th2 responses following DNA vaccination with antibody adjuvants.

<p>An IFN-γ ELISpot was used to measure the Th1 response following each immunization (<b>a</b>). The relative contribution of CD4⁺ and CD8⁺ T cells to the IFN-γ response was measured by ELISpot following CD8⁺ T cell depletion of PBMCs isolated after the fourth immunization (<b>b</b>). The Th2 response was assessed by IL-4 production. ELISpot assay (<b>c</b>). Statistical differences between groups was determined by doing pair-wise Mann-Whitney tests with a Bonferroni adjustment with p values less than 0.01 being significant (** = p<0.01). Pair-wise values: CTLA-4 vs. Saline or DNA (p = 0.002, p = 0.009, respectively); Combo vs. Saline, DNA, or 4-1BB (p = 0.002, p = 0.002, p = 0.009, respectively).</p