Intravaginal immunization using the recombinant HIV-1 clade-C trimeric envelope glycoprotein CN54gp140 formulated within lyophilized solid dosage forms

Vaccine-mediated prevention of primary HIV-1 infection at the heterosexual mucosal portal of entry may be facilitated by highly optimised formulations or drug delivery devices for intravaginal (i.vag) immunization. Previously we described hydroxyethylcellulose (HEC)-based rheologically structured gel vehicles (RSVs) for vaginal immunization of an HIV-1 vaccine candidate, a soluble recombinant trimeric HIV-1 clade-C envelope glycoprotein designated CN54gp140. Here we investigated the efficacy of lyophilized solid dosage formulations (LSDFs) for prolonging antigen stability and as i.vag delivery modalities. LSDFs were designed and developed that upon i.vag administration they would reconstitute with the imbibing of vaginal fluid to mucoadhesive, site-retentive semi-solids. Mice were immunized with lyophilized equivalents of (i) RSVs, (ii) modified versions of the RSVs more suited to lyophilization (sodium carboxymethyl cellulose (NaCMC)-based gels) and (iii) Carbopol® gel, all containing CN54gp140. NaCMC-based LSDFs provided significantly enhanced antigen stability compared to aqueous-based RSVs. Rheological analysis indicated the NaCMC-based LSDFs would offer enhanced vaginal retention in woman compared to more conventional vaginal gel formulations. All LSDFs were well tolerated in the mouse model. Following i.vag administration, all LSDFs boosted systemic CN54gp140-specific antibody responses in sub-cutaneously primed mice. Induction of CN54gp140-specific antibody responses in the female genital tract was evident. Of all the LSDFs the fastest releasing which was lyophilized Carbopol® gel elicited immune responses comparable to buffer instillation of antigen suggesting that rather than slower sustained release, initial high burst release from the LSDFs may suffice. The boosting of specific immune responses upon i.vag administration indicates that LSDFs are viable mucosal vaccine delivery modalities promoting antigen stability and facilitating intimate exposure of CN54gp140 to the mucosal-associated lymphoid tissue of the female genital tract.

Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responses

Rheologically structured vehicle (RSV) gels were developed as delivery systems for vaginal mucosal vaccination with an HIV-1 envelope glycoprotein (CN54gp140). RSVs comprised a mucoadhesive matrix forming and vaginal fluid absorbing polymer. The mucoadhesive and rheological properties of the RSVs were evaluated in vitro, and the distribution, antigenicity and release of CN54gp140 were analysed by ELISA. CN54gp140 was uniformly distributed within the RSVs and continuously released in vitro in an antigenically intact form over 24 h. Vaginal administration to rabbits induced specific serum IgG, and IgG and IgA in genital tract secretions. The RSVs are a viable delivery modality for vaginal immunization.

Development of liposome gel based formulations for intravaginal delivery of the recombinant HIV-1 envelope protein CN54gp140

Mucosally-administered vaccine strategies are widely investigated as a promising means of preventing HIV infection. This study describes the development of liposomal gel formulations, and novel lyophilised variants, comprising HIV-1 envelope glycoprotein, CN54gp140, encapsulated within neutral, positively charged or negatively charged liposomes. The CN54gp140 liposomes were evaluated for mean vesicle diameter, polydispersity, morphology, zeta potential and antigen encapsulation efficiency before being incorporated into hydroxyethyl cellulose (HEC) aqueous gel and subsequently lyophilised to produce a rod-shaped solid dosage form for practical vaginal application. The lyophilised liposome-HEC rods were evaluated for moisture content and redispersibility in simulated vaginal fluid. Since these rods are designed to revert to gel form following intravaginal application, mucoadhesive, mechanical (compressibility and hardness) and rheological properties of the reformed gels were evaluated. The liposomes exhibited good encapsulation efficiency and the gels demonstrated suitable mucoadhesive strength. The freeze-dried liposome-HEC formulations represent a novel formulation strategy that could offer potential as stable and practical dosage form.

Suggested Binding Mechanism of the HIV-gp120 to its CD4 Receptor

The molecular recognition and attachment of the CD4 molecule and the HIV envelope glycoprotein (gp120) might be described as a consecutive three-step molecular recognition process. 1. (a) Long range interaction: electrostatic pre-orientation, 2. (b) short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation) and 3. (c) internal interaction (induced fit): conformational readjustment of the protein molecules. On the basis of the preliminary investigations (X-ray structures of CD4 and biological studies of CD4 and gp120 point mutants) we described a computational model. This approach consists of empirical calculations as well as ab initio level of quantum chemistry. The conformational analysis of the wild type and mutant CD4 molecules was supported by molecular mechanics and dynamics (Amber force field). The latter analysis involves the application of a novel method, the Amino Acid Conformation Assignment of Proteins (ACAP) software, developed for the notation of secondary protein structures. According to the cardinal role of the electrostatic factors during this interaction, several ab initio investigations were performed for better understanding of the recognition process on submolecular level. Using the above mentioned computational model, we could interpret the basic behaviours and predict some additional features of CD4-gp120 interaction, in spite of the missing gp120 X-ray structure.

Computational identification of self-inhibitory peptides from envelope proteins

Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the enveloped (E) proteins function to inhibit the proteinprotein interactions in the membrane fusion step mediated by the viral E protein. Thus, they have the potential to be developed into effective antiviral therapy. Herein, we have developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. The stability of the peptides, which indicates their potential to bind in situ to the E proteins, was evaluated by two different scoring functions, dipolar distance-scaled, finite, ideal-gas reference state and residue-specific all-atom probability discriminatory function. The method was applied to a-helical Class I HIV-1 gp41, beta-sheet Class II Dengue virus (DENV) type 2 E proteins, as well as Class III Herpes Simplex virus-1 (HSV-1) glycoprotein, a E protein with a mixture of a-helix and beta-sheet structural fold. The peptide hits identified are in line with the druggable regions where the self-inhibitory peptide inhibitors for the three classes of viral fusion proteins were derived. Several novel peptides were identified from either the hydrophobic regions or the functionally important regions on Class II DENV-2 E protein and Class III HSV-1 gB. They have potential to disrupt the proteinprotein interaction in the fusion process and may serve as starting points for the development of novel inhibitors for viral E proteins. 

Radiative rates for E1, E2, M1 and M2 transitions in Fe X

Energies of the 54 levels belonging to the (1s(2)2s(2)2p(6)) 3s(2)3p(5), 3s3p(6), 3s(2)3p(4)3d and 3s3p(5)3d configurations of Fe X have been calculated using the GRASP code of Dyall et al. (1989). Additionally, radiative rates, oscillator strengths, and line strengths are calculated for all electric dipole (E1), magnetic dipole (M1), electric quadrupole (E2), and magnetic quadrupole (M2) transitions among these levels. Comparisons are made with results available in the literature, and the accuracy of the data is assessed. Our energy levels are estimated to be accurate to better than 3%, whereas results for other parameters are probably accurate to better than 20%. Additionally, the agreement between measured and calculated lifetimes is better than 10%.

Self-focusing and envelope pulse generation in nonlinear magnetic metamaterials

The self-modulation of waves propagating in nonlinear magnetic metamaterials is investigated. Considering the propagation of a modulated amplitude magnetic field in such a medium, we show that the self-modulation of the carrier wave leads to a spontaneous energy localization via the generation of localized envelope structures (envelope solitons), whose form and properties are discussed. These results are also supported by numerical calculations.

Finite amplitude envelope solitons in pair-ion plasmas

The nonlinear coupling between finite amplitude ion thermal waves (ITWs) and quasistationary density perturbations in a pair-ion plasma is considered. A generalized nonlinear Schrödinger equation is derived for the ITW electric field envelope, accounting for large amplitude quasistationary plasma slow motion describing the ITW ponderomotive force. The present theory accounts for the trapping of ITWs in a large amplitude ion density hole. The small amplitude limit is considered and exact analytical solutions are obtained. Finite amplitude solutions are obtained numerically and their characteristics are discussed.

Characterization of a ligand-attenuated binding site on glycoprotein IIb/IIIa

We describe an epitope on the platelet integrin, GPIIb/IIIa, identified by the monoclonal antibody, 4F8, which is attenuated by small-molecule GPIIb/IIIa ligands. 4F8 did not bind to the ligand binding pocket as it did not compete with a radiolabelled antagonist, H-3-SC-52012. This indicates that the 4F8 epitope behaves as a ligand-attenuated binding site (LABS). Ligand-induced attenuation of 4178 was an active process as it was prevented by pretreating platelets with cytochalasin D and reduced by prostaglandin E-1 or inhibition of protein kinase C. Disappearance of the epitope was required for full platelet activation as 4F8 prevented platelet aggregation without inhibiting fibrinogen binding. These results suggest a model where disappearance of the 4F8 epitope is a secondary event required for full