Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides

An HIV vaccine capable of inducing high and durable levels of broadly neutralizing antibodies has thus far proven elusive. A promising antigen is the membrane-proximal external region (MPER) from gp41, a segment of the viral envelope recognized by a number of broadly neutralizing antibodies. Though an attractive vaccine target due to the linear nature of the epitope and its highly conserved sequence, MPER peptides are poorly immunogenic and may require display on membranes to achieve a physiological conformation matching the native virus. Here we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. Administration of MPER peptides anchored to the surface of liposomes induced MPER-specific antibodies whereas MPER administered in oil-based emulsion adjuvants or alum did not, even when combined with Toll-like receptor agonists. High-titer IgG responses to liposomal MPER required the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced by incorporating high-Tm lipids in the vesicle bilayer and optimizing the MPER density to a mean distance of ∼10–15 nm between peptides on the liposomes' surfaces. Encapsulation of helper epitopes within the vesicles allowed efficient “intrastructural” T-cell help, which promoted IgG responses to MPER while minimizing competing B-cell responses against the helper sequence. These results define several key properties of liposome formulations that promote durable, high-titer antibody responses against MPER peptides, which will be a prerequisite for a successful MPER-targeting vaccine.Bill & Melinda Gates FoundationNational Institutes of Health (U.S.) (AI091693

Targeting Antibody Responses to the Membrane Proximal External Region of the Envelope Glycoprotein of Human Immunodeficiency Virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Breit neutralisierende Antikörper (bnAk) sind ein wichtiges Ziel der HIV-Impfstoffentwicklung, aber schwer zu induzieren. Drei bnAk (2F5, 4E10, Z13) sind gegen die $\textit {membrane proximal externalregion}$ (MPER) des Transmembranproteins gp41 gerichtet. Um die Antikörperantworten gegen die natürliche Konformation von MPER auszurichten, wurden N-terminal verkürzte Varianten erzeugt, welche in die Zellmembran und in virusähnliche Partikel (VLP) eingebaut werden können. In der Durchflusszytometrie zeigten 2F5 und 4E10 eine erhöhte Bindung an Zellen, welche die MPER-Mutanten anstatt Volllänge gp41 präsentierten. Immunisierung von Mäusen mit VLPs, welche die MPER-Mutanten enthielten, induzierte MPER-spezifische Antikörper. Eine Trimerisierung der MPER und ein DNA $\it {prime}$ konnten deren Titer noch erhöhen. Obwohl 2F5 mit den induzierten Antikörpern um Bindung an die MPER konkurrierte, konnte keine neutralisierende Aktivität detektiert werden. Die Gründe hierfür bedürfen weiterer Untersuchungen

Advances in Immunotherapy for the Treatment of Adult Glioblastoma: Overcoming Chemical and Physical Barriers

Glioblastoma, or glioblastoma multiforme (GBM, WHO Grade IV), is a highly aggressive adult glioma. Despite extensive efforts to improve treatment, the current standard-of-care (SOC) regimen, which consists of maximal resection, radiotherapy, and temozolomide (TMZ), achieves only a 12–15 month survival. The clinical improvements achieved through immunotherapy in several extracranial solid tumors, including non-small-cell lung cancer, melanoma, and non-Hodgkin lymphoma, inspired investigations to pursue various immunotherapeutic interventions in adult glioblastoma patients. Despite some encouraging reports from preclinical and early-stage clinical trials, none of the tested agents have been convincing in Phase III clinical trials. One, but not the only, factor that is accountable for the slow progress is the blood–brain barrier, which prevents most antitumor drugs from reaching the target in appreciable amounts. Herein, we review the current state of immunotherapy in glioblastoma and discuss the significant challenges that prevent advancement. We also provide thoughts on steps that may be taken to remediate these challenges, including the application of ultrasound technologies

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determinedThis work was supported by grants from the European Commission FP6 program (DEC-VAC, LSHP-CT-2005-018685; EUROPRISE network of excellence), the German Research Foundation (TRR60/1) and the Bill & Melinda Gates Foundation (Collaboration for AIDS Vaccine Discovery - CAVD - Grant #OPP38580_01

Antibody response after VLP boost in DNA immunized mice.

<p>(A) Mice were immunized three times with DNA encoding different MPER display mutants and boosted with VLPs containing the different MPER display mutants eight to fifteen weeks after the last DNA immunization. (B) MPER-specific antibody levels in the sera of immunized mice three weeks after the last VLP immunization are presented as log₁₀ values of the relative light units (Log RLU) obtained in an MPER antibody ELISA. Mean and single values for each of the animals are shown.</p

Map of domains and amino acid sequences of the MPER display mutants.

<p>The open reading frames of the MPER display mutants contain a heterologous leader peptide (LP), an isoleucine zipper to promote trimerization, a common tag (Ollas-tag) for easy detection, a flexible linker region (G4S), various regions of gp41 (MPERmut) including an HIV-1 transmembrane region (TM), and the cytoplasmic domain of the VSV-G protein (CT). The amino acid sequence of the different domains is given in the one-letter code. The numbering backward as depicted indicates the number of MPER-derived amino acid residues expressed by each mutant. The target regions for 2F5 and 4E10 monoclonal antibodies are indicated by double arrow-lines.</p

Incorporation of MPER display mutants into VLPs.

<p>VLPs containing MPER display mutants were concentrated from the supernatant of transfected cells and analyzed by Western blot analysis under denaturing (A to C) and non-reducing conditions (D) using the antibodies 2F5 (A), 4E10 (B), anti-Ollas (C, D). Results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038068#pone-0038068-g003" target="_blank">Figures 3</a> A-D are representative of at least three WB analyses using different VLP preparations and different denaturing conditions. ^*MPER47 contains 5 amino acids residue of non-MPER origin at its N-terminus.</p