Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition

Glioblastoma multiforme (GBM) are resistant to TNFα-induced apoptosis and blockade of TNFα-induced NF-κB activation sensitizes glioma cells to apoptosis. As Casein kinase-2 (CK2) induces aberrant NF-κB activation and as we observed elevated CK2 levels in GBM tumors, we investigated the potential of CK2 inhibitors (CK2-Is) - DRB and Apigenin in sensitizing glioma cells to TNFα-induced apoptosis. CK2-Is and CK2 small interfering RNA (siRNA) reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation, and sensitized cell to TNFα-induced apoptosis. Importantly, CK2-Is activated p53 function in wild-type but not in p53 mutant cells. Activation of p53 function involved its increased transcriptional activation, DNA-binding ability, increased expression of p53 target genes associated with cell cycle progression and apoptosis. Moreover, CK2-Is decreased telomerase activity and increased senescence in a p53-dependent manner. Apoptotic gene profiling indicated that CK2-Is differentially affect p53 and TNFα targets in p53 wild-type and mutant glioma cells. CK2-I decreased MDM2-p53 association and p53 ubiquitination to enhance p53 levels. Interestingly, CK2-Is downregulated SIRT1 activity and over-expression of SIRT1 decreased p53 transcriptional activity and rescued cells from CK2-I-induced apoptosis. This ability of CK2-Is to sensitize glioma to TNFα-induced death via multiple mechanisms involving abrogation of NF-κB activation, reactivation of wild-type p53 function and SIRT1 inhibition warrants investigation

Immunization with Cocktail of HIV-Derived Peptides in Montanide ISA-51 Is Immunogenic, but Causes Sterile Abscesses and Unacceptable Reactogenicity

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886

Dual Neonate Vaccine Platform against HIV-1 and M. tuberculosis

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the world's most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborn's mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA222. mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA222–mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life

The role of neutralizing antibodies in prevention of HIV-1 infection: what can we learn from the mother-to-child transmission context?

International audienceIn most viral infections, protection through existing vaccines is linked to the presence of vaccine-induced neutralizing antibodies (NAbs). However, more than 30 years after the identification of AIDS, the design of an immunogen able to induce antibodies that would neutralize the highly diverse HIV-1 variants remains one of the most puzzling challenges of the human microbiology. The role of antibodies in protection against HIV-1 can be studied in a natural situation that is the mother-to-child transmission (MTCT) context. Indeed, at least at the end of pregnancy, maternal antibodies of the IgG class are passively transferred to the fetus protecting the neonate from new infections during the first weeks or months of life. During the last few years, strong data, presented in this review, have suggested that some NAbs might confer protection toward neonatal HIV-1 infection. In cases of transmission, it has been shown that the viral population that is transmitted from the mother to the infant is usually homogeneous, genetically restricted and resistant to the maternal HIV-1-specific antibodies. Although the breath of neutralization was not associated with protection, it has not been excluded that NAbs toward specific HIV-1 strains might be associated with a lower rate of MTCT. A better identification of the antibody specificities that could mediate protection toward MTCT of HIV-1 would provide important insights into the antibody responses that would be useful for vaccine development. The most convincing data suggesting that NAbs migh confer protection against HIV-1 infection have been obtained by experiments of passive immunization of newborn macaques with the first generation of human monoclonal broadly neutralizing antibodies (HuMoNAbs). However, these studies, which included only a few selected subtype B challenge viruses, provide data limited to protection against a very restricted number of isolates and therefore have limitations in addressing the hypervariability of HIV-1. The recent identification of highly potent second-generation cross-clade HuMoNAbs provides a new opportunity to evaluate the efficacy of passive immunization to prevent MTCT of HIV-1

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1–4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection