Sensitivity of fetal RHD screening for safe guidance of targeted anti-D immunoglobulin prophylaxis: prospective cohort study of a nationwide programme in the Netherlands.

OBJECTIVE:  To determine the accuracy of non-invasive fetal testing for the RHD gene in week 27 of pregnancy as part of an antenatal screening programme to restrict anti-D immunoglobulin use to women carrying a child positive for RHD DESIGN:  Prospectively monitoring of fetal RHD testing accuracy compared with serological cord blood typing on introduction of the test. Fetal RHD testing was performed with a duplex real time quantitative polymerase chain reaction, with cell-free fetal DNA isolated from 1 mL of maternal plasma The study period was between 4 July 2011 and 7 October 2012. The proportion of women participating in screening was determined. SETTING:  Nationwide screening programme, the Netherlands. Tests are performed in a centralised setting. PARTICIPANTS:  25 789 RhD negative pregnant women. MAIN OUTCOME MEASURES:  Sensitivity, specificity, false negative rate, and false positive rate of fetal RHD testing compared with serological cord blood typing; proportion of technical failures; and compliance to the screening programme. RESULTS:  A fetal RHD test result and serological cord blood result were available for 25 789 pregnancies. Sensitivity for detection of fetal RHD was 99.94% (95% confidence interval 99.89% to 99.97%) and specificity was 97.74% (97.43% to 98.02%). Nine false negative results for fetal RHD testing were registered (0.03%, 95% confidence interval 0.01% to 0.06%). In two cases these were due to technical failures. False positive fetal RHD testing results were registered for 225 samples (0.87%, 0.76% to 0.99%). Weak RhD expression was shown in 22 of these cases, justifying anti-D immunoglobulin use. The negative and positive predictive values were 99.91% (95% confidence interval 99.82% to 99.95%) and 98.60% (98.40% to 98.77%), respectively. More than 98% of the women participated in the screening programme. CONCLUSIONS:  Fetal RHD testing in week 27 of pregnancy as part of a national antenatal screening programme is highly reliable and can be used to target both antenatal and postnatal anti-D immunoglobulin use.

[Foetal Rhesus-D typing added to antenatal screening for infectious diseases and erythrocyte immunisation]. / Foetale resus-D-typering toegevoegd aan prenatale screening op infectieziekten en erytrocytenimmunisatie.

OBJECTIVE: Investigation of the accuracy and national implementation of foetal Rhesus-D typing (fRhD), introduced in 2011 for RhD-negative pregnant women. DESIGN: Descriptive, national study. METHOD: Results of fRhD in pregnant women in the first year after implementation were compared to results from cord blood RhD typing of the child. RESULTS: 0.05% of the fRhD results was false negative (9/18.383; 95% CI: 0.02-0.09), and 0.85% was false positive (157/18.383; 95% CI: 0.73-1.00). fRhD was incorrectly omitted in fewer than 1% of pregnant women. In 96.1% of the pregnant women, antenatal administration of anti-D prophylaxis was recorded. Recording of postnatal anti-D administration turned out to be lower (92%), but locally recorded data showed that postnatal anti-D was omitted in fewer than 2% of cases. CONCLUSION: The percentage of false negative fRhD was statistically significantly lower (p < 0.05) than the critical limit of 0.25% determined beforehand by the Programme Committee of the Dutch Antenatal Screening Programme for Infectious Diseases and Erythrocyte Immunisation. The percentage of false positive fRhD was considered acceptable, and implementation of fRhD was more or less complete. Routine RhD-typing on cord blood was therefore omitted from January 2013. Antenatal and postnatal anti-D administration is now specifically indicated for RhD-negative pregnant women when fRhD is positive, thus saving about 10,000 unnecessary antenatal anti-D administrations per year. RhD-typing on cord blood is now performed only if the RhD blood type of the child of a RhD-negative mother is unknown at delivery, if a positive fRhD was found for multiple births, and in exceptional situations, e.g. if fRhD typing is not possible due to a rare genetic variation.

Animal models for cutaneous vaccine delivery.

Main challenges in skin vaccination are overcoming the stratum corneum (SC) barrier and targeting the antigen presenting cells (APC) in the epidermis and the dermis. For this purpose many delivery techniques are being developed. In vivo immunogenicity and safety studies in animals are mandatory before moving to clinical trials. However, the results obtained in animals may or may not be predictive for humans. Knowledge about differences and similarities in skin architecture and immunology within a species and between species is crucial. In this review, we discuss variables, including skin morphology, skin barrier function, mechanical properties, site of application and immunology, which should be taken into account when designing animal studies for vaccination via the skin in order to support the translation to clinical trial outcomes.

Phase 1 clinical study with Bioneedles, a delivery platform for biopharmaceuticals.

Vaccination campaigns using syringes, needles, vials and refrigeration have a heavy logistic burden. A vaccination platform that circumvents these problems would improve the quality of vaccination campaigns by faster and safer vaccination of populations anywhere. A clinical phase I study in eighteen volunteers has been carried out, using biodegradable mini-implants (Bioneedles™), made of a polymer based on starch, allowing for high speed vaccination of thermostable vaccines and omitting the use of syringes, needles, vials and refrigeration. Clinical and histological assessment demonstrated excellent local tissue tolerance for the Bioneedle polymer. Histological findings were those of normal tissue restitution without evidence for infection, allergic or granulomatous response, or scar formation. The Bioneedle polymer was mostly degraded within hours, remnants of it being cleared by macrophages. The technique of subcutaneous insertion of Bioneedles with a kinetic energy of 0.33 Joule and higher is 100% effective and pain free. All volunteers graded global tolerance of Bioneedles as "very good" or "good". Bioneedles offer a safe, effective and very fast alternative vaccination platform.

Minipigs as an animal model for dermal vaccine delivery.

Appropriate animal models for intradermal vaccine delivery are scarce. Given the high similarity of their skin anatomy to that of humans, minipigs may be a suitable model for dermal vaccine delivery. Here we describe the immunization of Göttingen minipigs by using intradermal and intramuscular delivery of hepatitis B surface antigen (HBsAg). Intradermal vaccine delivery by needle and syringe and by needle-free jet injection induced humoral antiHBsAg responses. Priming immunization by using the disposable syringe jet injector (DSJI) resulted in a higher antibody titer than did conventional intradermal immunization and a titer comparable to that after intramuscular vaccination with HBsAg and Al(OH)3 adjuvant. This study highlights the utility of the minipig model in vaccine studies assessing the efficacy of conventional and novel methods of dermal delivery. Moreover, we include suggestions regarding working with minipigs during dermal vaccine delivery studies, thereby fostering future work in this area of vaccinology.

A combined approach of vesicle formulations and microneedle arrays for transcutaneous immunization against hepatitis B virus.

In the search for an optimal approach for the transcutaneous immunization (TCI) of hepatitis B surface antigen (HBsAg), two vesicle formulations, L595 vesicles (composed of sucrose-laurate ester and octaoxyethylene-laurate ester) and sPC vesicles (composed of soybean-phosphatidylcholine and Span-80) were prepared and characterized in vitro and in vivo. HBsAg was associated to the vesicles, resulting in sPC-HBsAg vesicles (±170nm) with 79% HBsAg association and L595-HBsAg vesicles (±75nm) with only 29% HBsAg association. The vesicles induced in mice via TCI an antibody response only when the skin was pretreated with microneedles. This response was improved by the adjuvant cholera toxin. The sPC-HBsAg vesicle formulations showed to be the most immunogenic for TCI, which was related to the higher degree of HBsAg association.

Bioneedles as alternative delivery system for hepatitis B vaccine.

An alternative vaccine delivery system for needle injections is the Bioneedle. Hepatitis B surface antigen (HBsAg) was formulated with Bioneedles. Three formulations were used: plain antigen, HBsAg adjuvated with aluminum hydroxide and HBsAg with LPS-derived lpxL1. Bioneedles with HBsAg-lpxL1 were the most stable and the most immunogenic formulations. The conventional liquid alum adjuvated vaccine lost 40% of its antigenicity after 1week at 50°C whereas the HBsAg-lpxL1 Bioneedles showed no significant decrease after 3 weeks at 50°C. In vivo studies revealed that the HBsAg-lpxL1 Bioneedle formulations induced comparable IgG titers as conventional liquid formulations after 2 immunizations, but higher IgG2a titers were found already after 1 immunization. The in vivo and in vitro studies showed that the Bioneedle is an attractive alternative for needle injections of HBsAg vaccines.

Preparation and characterization of diphtheria toxoid-loaded elastic vesicles for transcutaneous immunization.

The objective was to develop surfactant-based vesicle formulations containing diphtheria toxoid (DT) for transcutaneous immunization. Formulation variables were molar ratio of the surfactants (sucrose-laurate ester, octaoxyethylene-laurate ester, and sodium bistridecyl sulfosuccinate), DT concentration, pH, and ionic strength. The formulations were characterized by visual inspection, dynamic light scattering and zeta potential measurements. DT loading efficiency and capacity of the vesicle formulations were studied by protein assay and enzyme-linked immunosorbent assay. DT-containing vesicle formulations were optimized for colloidal stability. The pH had a dramatic effect on DT-vesicle association: at pH 4.5 > 70% of the protein was associated with the vesicles, vs. < 20% at pH 5.0. By varying the ionic strength of the buffer, we revealed that hydrophobic interactions play an important role in the association. DT-vesicles formulated at pH 4.5 were stable and showed high association of DT with preserved antigenicity, and are therefore excellent candidates for future in vivo studies.

Bioneedles as vaccine carriers.

Bioneedles are small hollow mini implants fabricated from biodegradable polymers which can be filled with antigen. Bioneedles can be used for vaccination without syringes and needles. Formulations have been prepared containing tetanus toxoid with and without aluminum phosphate. Stability and immunogenicity of Bioneedles were compared with liquid formulations. The antigen, when formulated in Bioneedles, retained fully its antigenicity up to 60 degrees C for 1 week whereas the antigen, in its liquid form, lost all activity at 60 degrees C after 1 week. After 3 weeks at 60 degrees C, a recovery of 60% was still found in the Bioneedles. Mice injected with Bioneedles with adjuvanted tetanus toxoid showed a comparable functional antibody response to the group receiving conventional liquid injections. This response was achieved with a four times lower antigen concentration when using the Bioneedles compared to the regular injections. We conclude that Bioneedles are good alternatives for injections using needles and syringes.

Needle-free vaccine delivery.

The need for minimally invasive delivery methods is urgent. As the number of registered vaccines increases, so does the number of injections. The use of sharps can be unsafe and needle immunisation is less suitable for mass immunisations during emergencies such as pandemics or bioterrorist attacks. The approach of combining vaccines has limitations due to high development costs, risk of pharmaceutical or immunological interference and economic risks. Advancements in the development of alternatives to injection with syringes and needles are discussed in this paper, and include: mucosal vaccination, injection without needles and vaccine delivery via the skin.

Diphtheria toxoid-containing microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs.

In this study, the potential of N-Trimethyl chitosan (TMC, degree of quaternization 50%) and dextran microparticles for pulmonary delivery of diphtheria toxoid (DT) was investigated. The antigen-containing microparticles were prepared by drying of an aqueous solution of polymer and DT through a supercritical fluid (SCF) spraying process. The median volume diameter of the dry particles, as determined by laser diffraction analysis, was between 2 and 3 microm and the fine particle mass fractions smaller than 5 microm, as determined by cascade impactor analysis, were 35 and 56% for the dextran and TMC formulations, respectively. The water content of the particles as measured by Karl-Fischer titration was 2-3% (w/w). Pulmonary immunization with DT-TMC microparticles containing 2 or 10 Lf of DT resulted in a strong immunological response as reflected by the induction of IgM, IgG, IgG subclasses (IgG1 and IgG2) antibodies as well as neutralizing antibody titers comparable to or significantly higher than those achieved after subcutaneous (SC) administration of alum-adsorbed DT (2 Lf). Moreover, the IgG2/IgG1 ratio after pulmonary immunization with DT-TMC microparticles was substantially higher as compared to SC administered alum-adsorbed DT. In contrast, pulmonarily administered DT-dextran particles were poorly immunogenic. Among the tested formulations only pulmonarily administered DT-containing TMC microparticles induced detectable pulmonary secretory IgA levels. In conclusion, in this paper it is demonstrated that TMC microparticles are a potent new delivery system for pulmonary administered DT antigen.

Antigen delivery systems.

Many vaccine candidates are highly purified, sometimes monomeric antigens and as a result, not very immunogenic. Antigen delivery systems optimize the presentation of antigens. They also play a major role in solving the problem of there being an increasing number of vaccines but limited opportunities in which to include these vaccines in immunization programs. The number of injections is restricted and combining vaccines may lead to immunological and physicochemical incompatibility. In this review, the current status with respect to parenteral and mucosal delivery systems is discussed. These include lipid-based systems such as liposomes and immunostimulating complexes, as well as polymeric microspheres. In addition, developments in needle-free, dermal delivery devices such as jet injectors, microneedles and patches are presented.