Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection.

A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

Inhibition of immune activation by a novel nuclear factor-kappa B inhibitor in HTLV-I-associated neurologic disease.

The human T-lymphotropic virus type I (HTLV-I) causes a chronic inflammatory disorder of the central nervous system termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-I encodes a protein known to activate several host-signaling pathways involved in inflammation, such as the nuclear factor-κB (NF-κB). The contribution of the NF-κB pathway to the pathogenesis of HAM/TSP, however, has not been fully defined. We show evidence of canonical NF-κB activation in short-term cultures of peripheral blood mononuclear cells (PBMCs) from subjects with HAM/TSP. NF-κB activation was closely linked to HTLV-I viral protein expression. The NF-κB activation in HAM/TSP PBMCs was reversed by a novel small-molecule inhibitor that demonstrates potent and selective NF-κB antagonist activity. Inhibition of NF-κB activation led to a reduction in the expression of lymphocyte activation markers and resulted in reduced cytokine signaling in HAM/TSP PBMCs. Furthermore, NF-κB inhibition led to a reduction in spontaneous lymphoproliferation, a key ex vivo correlate of the immune activation associated with HAM/TSP. These results indicate that NF-κB activation plays a critical upstream role in the immune activation of HAM/TSP, and identify the NF-κB pathway as a potential target for immunomodulation in HAM/TSP.

A single dose investigational subunit vaccine for human use against Nipah virus and Hendra virus.

Nipah and Hendra viruses are highly pathogenic bat-borne paramyxoviruses recently included in the WHO Blueprint priority diseases list. A fully registered horse anti-Hendra virus subunit vaccine has been in use in Australia since 2012. Based on the same immunogen, the Hendra virus attachment glycoprotein ectodomain, a subunit vaccine formulation for use in people is now in a Phase I clinical trial. We report that a single dose vaccination regimen of this human vaccine formulation protects against otherwise lethal challenges of either Hendra or Nipah virus in a nonhuman primate model. The protection against the Nipah Bangladesh strain begins as soon as 7 days post immunization with low dose of 0.1 mg protein subunit. Our data suggest this human vaccine could be utilized as efficient emergency vaccine to disrupt potential spreading of Nipah disease in an outbreak setting.

NF-κB-activating complex engaged in response to EGFR oncogene inhibition drives tumor cell survival and residual disease in lung cancer.

Although oncogene-targeted therapy often elicits profound initial tumor responses in patients, responses are generally incomplete because some tumor cells survive initial therapy as residual disease that enables eventual acquired resistance. The mechanisms underlying tumor cell adaptation and survival during initial therapy are incompletely understood. Here, through the study of EGFR mutant lung adenocarcinoma, we show that NF-κB signaling is rapidly engaged upon initial EGFR inhibitor treatment to promote tumor cell survival and residual disease. EGFR oncogene inhibition induced an EGFR-TRAF2-RIP1-IKK complex that stimulated an NF-κB-mediated transcriptional survival program. The direct NF-κB inhibitor PBS-1086 suppressed this adaptive survival program and increased the magnitude and duration of initial EGFR inhibitor response in multiple NSCLC models, including a patient-derived xenograft. These findings unveil NF-κB activation as a critical adaptive survival mechanism engaged by EGFR oncogene inhibition and provide rationale for EGFR and NF-κB co-inhibition to eliminate residual disease and enhance patient responses.

Dual inhibition of canonical and noncanonical NF-κB pathways demonstrates significant antitumor activities in multiple myeloma.

PURPOSE: NF-κB transcription factor plays a key role in the pathogenesis of multiple myeloma in the context of the bone marrow microenvironment. Both canonical and noncanonical pathways contribute to total NF-κB activity. Recent studies have shown a critical role for the noncanonical pathway: selective inhibitors of the canonical pathway present a limited activity, mutations of the noncanonical pathway are frequent, and bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity. EXPERIMENTAL DESIGN: Multiple myeloma cell lines, primary patient cells, and the human multiple myeloma xenograft murine model were used to examine the biologic impact of dual inhibition of both canonical and noncanonical NF-κB pathways. RESULTS: We show that PBS-1086 induces potent cytotoxicity in multiple myeloma cells but not in peripheral blood mononuclear cells. PBS-1086 overcomes the proliferative and antiapoptotic effects of the bone marrow milieu, associated with inhibition of NF-κB activity. Moreover, PBS-1086 strongly enhances the cytotoxicity of bortezomib in bortezomib-resistant multiple myeloma cell lines and patient multiple myeloma cells. PBS-1086 also inhibits osteoclastogenesis through an inhibition of RANK ligand (RANKL)-induced NF-κB activation. Finally, in a xenograft model of human multiple myeloma in the bone marrow milieu, PBS-1086 shows significant in vivo anti-multiple myeloma activity and prolongs host survival, associated with apoptosis and inhibition of both NF-κB pathways in tumor cells. CONCLUSIONS: Our data show that PBS-1086 is a promising dual inhibitor of the canonical and noncanonical NF-κB pathways. Our preclinical study therefore provides the framework for clinical evaluation of PBS-1086 in combination with bortezomib for the treatment of multiple myeloma and related bone lesions.

Dendrite development regulated by CREST, a calcium-regulated transcriptional activator.

The lasting effects of neuronal activity on brain development involve calcium-dependent gene expression. Using a strategy called transactivator trap, we cloned a calcium-responsive transactivator called CREST (for calcium-responsive transactivator). CREST is a SYT-related nuclear protein that interacts with adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP) and is expressed in the developing brain. Mice that have a targeted disruption of the crest gene are viable but display defects in cortical and hippocampal dendrite development. Cortical neurons from crest mutant mice are compromised in calcium-dependent dendritic growth. Thus, calcium activation of CREST-mediated transcription helps regulate neuronal morphogenesis.

Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques.

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine development. In this study, we evaluated one such structure, expressed as either a gp120-CD4 or a gp140-CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (shCD4) and HIV-1(IIIB) envelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIV(BaL) gp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.