Aditya Pattani

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.

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.

Development of a method to quantify the DNA content in cationic peptide-DNA nanoparticles.

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

Intravaginal immunisation using a novel antigen-releasing ring device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses

&NA; The generation of effective levels of antigen‐specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen‐specific immunity, enhance previously existing systemic immunity or re‐target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze‐dried, rod‐shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV‐1 CN54gp140 protein (167 &mgr;g) ± R848 (167 &mgr;g) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed. Sheep received a prime‐boost vaccination regime comprising intramuscular injection of 100 &mgr;g CN54gp140 + 200 &mgr;g R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen‐specific B cells were measured by flow cytometry at necropsy. Vaccine antigen‐specific serum antibody responses were detected in both the intramuscularly‐primed and vaginal mucosally‐primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen‐specific IgG and IgA with the IgA responses 30‐fold greater than systemic levels. Importantly, very high numbers of antigen‐specific B cells were detected in local genital draining lymph nodes. We have elicited local genital antigen‐specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen‐specific mucosal IgA and large numbers of local antigen‐reactive B cells, both likely essential for effective mucosal protection. Graphical abstract Figure. No caption available.

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

The present investigation dealswith development and characterization 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 freezedried 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.

Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended.

Vaccine Delivery Systems: Roles, Challenges and Recent Advances

The overwhelming majority of vaccine antigens are biological macromolecules, such as proteins and polysaccharides, typically with a molecular weight greater than 10,000. As such, they need to be delivered to the body in the correct conformation in order to elicit the desired immune response and to effectively target the immune cells. Currently, most vaccines are administered parenterally via the intradermal, subcutaneous or intramuscular route, the choice largely dependent on whether the antigen is in the adsorbed or nonadsorbed state. However, these routes have major drawbacks, including pain associated with the use of needles, the potential for needle contamination, practicalities of needle disposal and the need for a primary healthcare worker. There is now a particular focus on the development of mucosal vaccines, designed for direct application to mucosal surfaces such as those present in the mouth, nose, vagina and rectum.

Intravaginal immunization using a novel antigen delivery device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses

Background While it is relatively easy to elicit antigen-specific serum antibody it is much more difficult to establish meaningful levels of specific antibody at mucosal surfaces, the major route of viral invasion. We sought to determine if mucosal vaccination using topical vaginal application could initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae.