Determination of Depth-Dependent Intradermal Immunogenicity of Adjuvanted Inactivated Polio Vaccine Delivered by Microinjections via Hollow Microneedles
TL;DR: CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intrader mal microinjection by HMN.
Abstract: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT). A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization. The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50–900 μm), -volumes (1–100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1–2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold). Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.
Summary (4 min read)
- Poliomyelitis can be prevented through vaccination by either oral polio vaccine (OPV) or inactivated polio vaccine (IPV).
- It may cause outbreaks of vaccine-derived polioviruses (1).
- Therefore, the World Health Organization aims to eliminate the use of OPV and substitute it by IPV in its goal towards worldwide polio eradication (2).
- IPV vaccination is more costly because of higher production costs and a higher dose requirement in comparison to OPV.
3 Intravacc (Institute for Translational
- Vaccinology), Bilthoven, The Netherlands several strategies for dose sparing to reduce the cost of IPV vaccination have been proposed, including intradermal immunization and IPV adjuvantation (3).
- Another novel strategy for intradermal injection is microneedles, which are micron-sized needles (7).
- Langerhans cells (LCs) reside in the epidermis (topmost skin layer) and dermal dendritic cells (DDCs) reside in the dermis (lower layer) (14).
- This applicator only allowed a maximum flow rate of 2 μL/min without leakage in the microinjection system.
- Besides establishing the potential effect of intradermal injection depth on the immune response, the use of adjuvants can improve IPV immunization efficiency and therefore may result in IPV dose sparing and thus cost reduction (3,15).
- Polyimide coated fused silica capillary (375 μm outer diameter, 100 and 20 μm inner diameter) was obtained from Polymicro, Phoenix, USA.
- CapTite™ connections were obtained from Labsmith, USA.
- Parafilm was purchased from Bemis, Monceau-sur-Sambre, Belgium.
- Tissue-Tek O.C.T. compound was ordered at Sakura Finetek, Alphen aan den Rijn, the Netherlands.
- Horseradish peroxidase-conjugated goat-antirat IgG was obtained from Southern Biotech, Birmingham, AL, USA.
Fabrication of HMN
- HMN were produced by an in-house process as described previously (8).
- These silicone oil-filled capillaries were subsequently wet etched into HMN by immersing the ends in a container with hydrofluoric acid (49% w/w) for 4 h.
- To expose the microneedle tips, the polyimide coating at the etched ends of the capillaries was removed by immersing them in concentrated sulfuric acid (95–98%) at 250°C for 5 min.
Visualization of Depth-Controlled Microinjections
- To obtain evidence that skin layers at different preselected depths were targeted, microinjections of a trypan blue solution were performed on ex-vivo rat skin, which was then photographed at both sides (Fig. 3a).
- Whereas the trypan blue spot of the shallowermicroinjection at a skin injection depth of 250 μm was more clearly visible at the outer side of the skin, the trypan blue spot of the deeper injection at 550 μm was hardly visible.
- Contrarily, the trypan blue spot of the deeper microinjection at a skin injection depth of 550 μm was more clearly visible at the inner side of the skin than the trypan blue spot of the shallower injection (250 μm).
- Moreover, as shown in Fig. 3b, visualization of the skin injection depth in crosssectioned skin indicated that the microinjections were indeed performed at different pre-defined skin injection depths.
Investigation of Depth-Dependent Intradermal Immunogenicity
- Two immunization studies were performed under the guidelines and regulations enforced by the animal ethic committee of the Nether lands , and were approved by the BDierexperimentencommissie Universiteit Leiden (UDEC)^ under number 12084.
- The animals were housed in groups of 5 and were assigned to different immunization groups (10 rats per immunization group).
- Afterwards, threefold serial dilutions of serum samples in assay buffer were added at 100 μL/ well and subsequently incubated at 37°C for 2 h.
- Subsequently, plates were thoroughly washed before adding TMB substrate solution (100 μL/well) which consisted of 1.1 M sodium acetate, 100 mg/mL TMB and 0.006% (v/v) hydrogen peroxide.
- The endpoint titer was defined as the reciprocal of the serum dilution producing a 450 nm absorbance equal to that of the mean 450 nm absorbance with addition of three times the standard deviation of eight samples of IPV1-specific-antibody negative serum samples.
- In addition to the determination of the IPV1-specific IgG antibody responses by ELISA, protectivity of these antibodies against wildtype poliovirus was measured in a wildtype poliovirus-neutralizing antibody assay.
- Some slight differences in mean VN titer per immunization group were observed.
- Moreover, this VN titer was similar to the VN titers of intradermal and intramuscular IPV1 immunization adjuvanted with CpG, however lower than the VN titer of intradermal IPV1 immunization adjuvanted with CT, which had the highest VN titer.
- Both during and immediately after intradermal immunization with HMN, a small bleb was observed on the skin.
- This bleb disappeared within 5 min after injection.
- Statistics were performed using GraphPad Prism (v.6.00, GraphPad Software, LaJolla, CA, USA).
- Kruskall-Wallis tests with Dunn’s post-hoc tests were performed as IgG titers were non-normally distributed and considered significant at p< 0.05.
HMN Applicator Optimization and Performance
- To allow for increased injection rates and thereby short injection times, connections in the fluidics system of the HMN applicator were optimized by applying high-pressure resistant CapTite™ connectors and high-pressure resistant polyimide fused silica capillaries, as shown in Fig. 1a and b.
- Furthermore, the dead volume in the fluidics system was kept to a minimum to maximize injection volume accuracy and minimize the loss of vaccine formulation.
- The hydrofluoric acid etching procedure of polyimide coated fused silica capillaries resulted in sharp HMN (Fig. 1c).
- This volume loss was observed on the skin surface at the microinjection sites.
- At all investigated injection depths, all microinjections resulted in ±1–2% volume loss.
Injection Depth-Dependent Intradermal Immunogenicity
- To assess whether injection of IPV1 at different injection depths in the skin affects IPV1-specific antibody responses, two immunization studies in rats were conducted.
- Intradermal immunization was performed using the HMN applicator in a depth-controlled manner and this was compared to intramuscular immunization with a conventional 26G hypodermic needle.
- IPV1-specific IgG titers obtained after intradermal immunization with non-adjuvanted IPV1 at injection depths ranging between 50 and 550 μmare shown in Fig.
- Three weeks after prime immunization (Fig. 4a) and boost immunization (Fig. 4b), no significant differences in IPV1-specific IgG titers were observed at different injection depths.
- No IPV1-specific antibody responses were observed in the mock treated group.
Immunogenicity Enhancing Effects of Adjuvants CpG and CT
- To assess the potential increase in antibody responses and the applicability for use as intradermal adjuvants, CpG and CT were used as adjuvants in intradermal and intramuscular IPV immunization (Fig. 4a and b).
- After boost immunization there were no significant differences for intramuscular IPV1 immunization adjuvanted with CT.
- Some differences were observed between CpG and CT, when they were compared against non-adjuvanted groups.
- After prime intramuscular immunization, CpG and CT adjuvanted IPV1 immunization resulted in 2.7 and 12.1 fold increased IgG titers, respectively, in comparison to the non-adjuvanted intramuscular IPV1 group.
- Thus, the IgG titer enhancing effect was more pronounced in prime immunization than in booster immunizat ion.
- The relationship between injection depth and intradermal immunization has not been studied before, because this requires accurate, precise and reproducible depth-controlled intradermal injections with small injection volumes.
- The redesigned applicator allowed for a 30-fold increased injection rate (60 μL/min) compared to previously achieved rates (2 μL/ min) (8).
- It is not known whether the distribution of the injected volume in the skin influences the targeting of intradermal injection depths.
- This behavior, chemotaxis and chemoattraction of other DCs and the complex interplay between different DC classes, may explain the absence of differences in immune response as function of the injection depth.
VN antibody titers (log2)
- This may put a smaller burden on the design specifications of microneedle applicators.
- For polio prime immunization in newborn infants, intradermal 20% IPV dose resulted in similar (4,5) or inferior (31–33) seroconversion rates compared to a full intramuscular dose.
- These findings were seemingly not dependent on the intradermal immunization method used, because jet injectors (5,6,31,32,34,35), the Mantoux technique (4,35) or HMN arrays (13,33) evenly resulted in either similar or inferior results to full intramuscular dose.
- Adjuvants may be used as another strategy for IPV dose sparing (3).
- In the present study, immuneenhancing effects on IPV1-specific IgG responses by the adjuvants CpG and CT were significant, which indicates that CpG and CT might be potential adjuvant candidates for intradermal IPV1 immunization and may lead to dose sparing.
- An unique dose sparing strategy for IPV1 was investigated by intradermal microinjections of small volumes of IPV1 formulation at dif ferent predetermined depths in rat skin in vivo.
- To enable this, a HMN applicator controllable in injection rate, −volume and –depth was developed that allowed for intradermal microinjections.
- Results indicated however, that intradermal immunogenicity was not dependent on the injection depth.
- Nonetheless, IPV1 immunization by minimally-invasive intradermal microinjections resulted in similar IPV1-specific antibody responses in comparison to IPV1 immunization by more invasive and painful intramuscular injections.
- Moreover, intradermal IPV1 immunization antibody responses were significantly increased (7-fold) by adjuvants CpG and CT, such that a single minimally-invasive intradermal immunization by HMN resulted in comparable antibody responses to two intramuscular immunizations.
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Cites background from "Determination of Depth-Dependent In..."
...On the other hand, the variation of injection depth with hollowMNs did not affect the immune response (109)....
...However, while a controlled antigen dose was released at different skin depth by hollow microneedles (109), not clear is the antigen dose released into...
"Determination of Depth-Dependent In..." refers background in this paper
...arrays, as in arrays all needles need to be penetrated and unclogged to ensure an accurate injection and prevent leakage (28,29)....
"Determination of Depth-Dependent In..." refers background in this paper
...Although OPV is an inexpensive and easy to administer vaccine, it may cause outbreaks of vaccine-derived polioviruses (1)....
...The microneedle insertion speed (1 to 3 m/s) is controlled by an electromagnet (1)....
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Frequently Asked Questions (2)
Q1. What contributions have the authors mentioned in the paper "Immunogenicity of adjuvanted inactivated polio vaccine delivered by microinjections via hollow microneedles" ?
The aim of this study was to investigate the depthdependent intradermal immunogenicity of inactivated polio vaccine ( IPV ) delivered by depth-controlled microinjections via hollow microneedles ( HMN ) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 ( CpG ) or cholera toxin ( CT ).
Q2. What are the future works mentioned in the paper "Immunogenicity of adjuvanted inactivated polio vaccine delivered by microinjections via hollow microneedles" ?
This allows to study LC function in intradermal immunity in the future. Furthermore, results presented in this study are important in microneedle patch design for intradermal IPV immunization as the independence of IPV1 immunization on skin depth suggests that microneedle length will not affect the immune response and thus precise dosing at a certain skin depth is not