T-Cell Immune Responses Against Env from CRF12_BF and Subtype B HIV-1 Show High Clade-Specificity that Can Be Overridden by Multiclade Immunizations

BACKGROUND: The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors. METHODOLOGY/PRINCIPAL FINDINGS: As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence. Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B. CONCLUSIONS/SIGNIFICANCE: This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate

TMIC-69. MITOCHONDRIAL TRANSFER FROM ASTROCYTES ENHANCES METABOLISM AND DRIVES PROLIFERATION OF GLIOBLASTOMA

Abstract Mitochondrial transfer occurs both in stroke (central nervous system) and inflammatory pain (peripheral nerves). However, its role in glioblastoma (GBM) remains poorly understood. We hypothesized that mitochondrial transfer from non-malignant to GBM cells supports tumor metabolism and growth. Using transgenic mice expressing fluorophore-tagged mitochondria, we found that ~50% of orthotopically-implanted mouse GBM cells acquire mitochondria. Brain-resident cells, especially astrocytes, were the primary mitochondrial donors in vitro and in vivo. Mitochondrial transfer also occurred from immortalized human astrocytes to patient-derived xenograft (PDX) models in vitro at rates of 15-35%. GBM cells that acquired mitochondria expressed higher levels of the ATP-synthase subunit ATP5A and produced more ATP, while metabolomics revealed multiple upregulated pathways in recipient cells. These data point to increased metabolic activity in recipient cells. In vivo, mouse GBM cells that acquired mitochondria were more likely to be in S/G2/M cell cycle phases. We observed a similar effect in PDX that acquired astrocyte mitochondria in vitro, suggesting that transfer drives GBM proliferation. Using sorted mouse and human GBM cells with/without in vitro astrocyte mitochondrial acquisition, we found that mitochondrial transfer promoted in vitro self-renewal and in vivo tumorigenicity, leading to significant reduction in survival and increased penetrance in orthotopic GBM models. Transfer in mouse and human systems was contact-dependent and was abrogated by physical separation of donor and recipient cells by transwell inserts. Pharmacologic inhibition of cytoskeleton and gap junctions did not affect transfer rate, while blocking growth-associated protein 43 (GAP43) function by c-Jun N-terminus kinase inhibition decreased transfer rate by 15-30%, suggesting a potential role of GAP43. Taken together, mitochondrial transfer comprises a fundamental, protumorigenic mechanism of GBM, enhancing metabolic activity and driving tumor cell proliferation. Elucidating the molecular machinery regulating astrocyte mitochondrial transfer and its downstream protumorigenic effects will lead to therapeutic opportunities targeting this understudied tumor microenvironment interaction

Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant

Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4(+) T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205(+) DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4(+) immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen

CNSC-15. MITOCHONDRIA TRANSFER VIA GLIOMA-ASTROCYTE NETWORK MICROTUBES REPROGRAMS TUMOR CELLS FOR ENHANCED TUMORIGENICITY

Abstract Glioblastoma (GBM) interaction with neural cells is critical to its pathobiology. Emerging evidence suggests that GBM cells form an interconnected network with astrocytes, facilitating tumor persistence. Given reports of intercellular transfer of mitochondria in ischemic stroke and other pathologic disease states outside the CNS, we hypothesized that this network facilitates mitochondria transfer from astrocytes to GBM with protumorigenic sequelae. Employing transgenic mice and intracranial viral vector transductions in rats, we found that mitochondria transfer from the TME to GBM occurs in intracranial mouse and patient-derived xenograft models (in nude rats) of GBM. Mitochondria transfer from bone marrow-derived immune cells was minimal in bone marrow chimera mouse models of orthotopic GBM, suggesting that neural cells were the primary mitochondria donors. We confirmed this in vitro, where mouse astrocytes were the major mitochondria donors, followed by microglia and to a much smaller extent bone marrow-derived macrophages. Immortalized human astrocytes transduced with mitochondria-localized mCherry (mito-mCherry) also transferred their mitochondria to numerous patient-derived glioma stem cell (GSC) models at rates of ~5-20%, assessed by flow cytometry and confocal microscopy. Mitochondria were visualized along intercellular actin bridges, structurally resembling tumor microtubes. Blocking actin polymerization or knocking down GAP43 (previously linked to microtube formation) decreased mitochondria transfer from astrocytes to GBM in vitro. Functionally, sorted mito-mCherry+ patient-derived GSCs displayed higher mitochondrial respiration, metabolomic reprogramming and proliferation-promoting phospho-signaling. Mito-mCherry+ GBM cells were more likely to be in the proliferative G2/M phases of the cell cycle, and when sorted from co-cultures had high self-renewal (in vitro) and tumor-initiating capacity (in vivo xenograft mouse model). In ongoing work, we are investigating the role of retrograde GBM to astrocyte transfer of mitochondria by dual-color labeling of the organelle, as well as further delineating the protein machinery involved in this fundamental protumorigenic process, with the goal of identifying novel therapeutic targets

GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development