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.

Llama antibody fragments have good potential for application as HIV-1 topical microbicides

There is an urgent global need for preventative strategies against HIV-1 infections. Llama heavy-chain antibody fragments (VHH) are a class of molecules recently described as potent cross-clade HIV-1 entry inhibitors. We studied the potential of a VHH-based microbicide in an application-oriented fashion. We show that VHH can be inexpensively produced in high amounts in the GRAS organism S. cerevisiae, resulting in very pure, and endotoxin free product. VHH are very stable under conditions they might encounter during transport, storage or use by women. We developed active formulations of VHH in aqueous gel and compressed and lyophilized tablets for controlled release from an intra vaginal device. The release profile of the VHH from e.g. a vaginal ring suggests sufficient bioavailability and protective concentration of the molecule at the mucosal site at the moment of the infection. The ex vivo penetration kinetics through human tissues show that the VHH diffuse into the mucosal layer and open the possibility to create a second defense layer either by blocking the HIV receptor binding sites or by blocking the receptors of immune cells in the mucosa. In conclusion, our data show that VHH have

Non-aqueous silicone elastomer gels as a vaginal microbicide delivery system for the HIV-1 entry inhibitor maraviroc

Aqueous semi-solid polymeric gels, such as those based on hydroxyethylcellulose (HEC) and polyacrylic acid (e.g. Carbopol®), have a long history of use in vaginal drug delivery. However, despite their ubiquity, they often provide sub-optimal clinical performance, due to poor mucosal retention and limited solubility for poorly water-soluble actives. These issues are particularly pertinent for vaginal HIV microbicides, since many lead candidates are poorly water-soluble and where a major goal is the development of a coitally independent, once daily gel product. In this study, we report the use of a non-aqueous silicone elastomer gel for vaginal delivery of the HIV-1 entry inhibitor maraviroc. In vitro rheological, syringeability and retention studies demonstrated enhanced performance for silicone gels compared with a conventional aqueous HEC gel, while testing of the gels in the slug model confirmed a lack of mucosal irritancy. Pharmacokinetic studies following single dose vaginal administration of a maraviroc silicone gel in rhesus macaques showed higher and sustained MVC levels in vaginal fluid, vaginal tissue and plasma compared with a HEC gel containing the same maraviroc loading. The results demonstrate that non-aqueous silicone gels have potential as a formulation platform for coitally independent vaginal HIV microbicides.

Binding modes of diketo-acid inhibitors of HIV-1 integrase: A comparative molecular dynamics simulation study

HIV-1 integrase (IN) has become an attractive target since drug resistance against HIV-1 reverse transcriptase (RT) and protease (PR) has appeared. Diketo acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN: however the action mechanism is not well understood. Here, to study the inhibition mechanism of DKAs we performed 10 ns comparative molecular dynamics simulations on HIV-1 IN bound with three most representative DMA inhibitors: Shionogi inhibitor, S-1360 and two Merck inhibitors L-731,988 and L-708,906. Our simulations show that the acidic part of S-1360 formed salt bridge and cation-pi interactions with Lys159. In addition, the catalytic Glu152 in S-1360 was pushed away from the active site to form an ion-pair interaction with Arg199. The Merck inhibitors can maintain either one or both of these ion-pair interaction features. The difference in potencies of the DMA inhibitors is thus attributed to the different binding modes at the catalytic site. Such structural information at atomic level, not only demonstrates the action modes of DMA inhibitors but also provides a novel starting point for structural-based design of HIV-1 IN inhibitors.

Development of liposome-based freeze-dried rods for vaginal vaccine delivery against HIV-1

The present investigation deals with development and characteriza- tion of the liposomes-based freeze-dried rods for the vaginal delivery of gp140 antigen in mice. Positively charged, negatively charged and neutral liposomes were prepared and characterized for various parameters e.g. morphology, size, polydispersity index, zeta potential and antigen encapsulation efficiency. To further improve the efficacy of vaccine delivery, antigen encapsulated liposomes were formulated as polymer gel-based freeze-dried rods, which were then characterized for moisture content. The redispersibility of the liposomes-based freeze- dried rods was determined in simulated vaginal fluid and liposome gel was investigated for mucoadhesion. The developed liposome-based freeze-dried rods systems could offer potential as stable and practical dosage form for the mucosal immunization against HIV-1 infection.

The chemical composition towards the galactic anti-centre .1. Observations and model atmosphere analyses

High resolution spectra of six early B-type main-sequence stars having galactocentric distances of between 10 and 18 kpc are presented. We List the equivalent widths for the metal lines and illustrate their hydrogen and helium line profiles. The stars are analysed using LTE line-blanketed model atmosphere techniques to derive atmospheric parameters and surface chemical compositions. All six stars have similar effective temperatures and surface gravities, allowing a reliable comparison of their metal abundances and distances. Significant variations in the photospheric abundances are evident and are discuss the need for a more detailed line-by-line differential analysis to exactly quantify the differences. This will be presented in a companion paper (Smartt et al. 1996).

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.

Microneedle mediated intradermal delivery of adjuvanted recombinant HIV-1 CN54gp140 effectively primes mucosal boost inoculations

Dissolving polymeric microneedle arrays formulated to contain recombinant CN54 HIVgp140 and the TLR4 agonist adjuvant MPLA were assessed for their ability to elicit antigen-specific immunity. Using this novel microneedle system we successfully primed antigen-specific responses that were further boosted by an intranasal mucosal inoculation to elicit significant antigen-specific immunity. This prime-boost modality generated similar serum and mucosal gp140-specific IgG levels to the adjuvanted and systemic subcutaneous inoculations. While the microneedle primed groups demonstrated a balanced Th1/Th2 profile, strong Th2 polarization was observed in the subcutaneous inoculation group, likely due to the high level of IL-5 secretion from cells in this group. Significantly, the animals that received a microneedle prime and intranasal boost regimen elicited a high level IgA response in both the serum and mucosa, which was greatly enhanced over the subcutaneous group. The splenocytes from this inoculation group secreted moderate levels of IL-5 and IL-10 as well as high amounts of IL-2, cytokines known to act in synergy to induce IgA. This work opens up the possibility for microneedle-based HIV vaccination strategies that, once fully developed, will greatly reduce risk for vaccinators and patients, with those in the developing world set to benefit most.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.