Skin immunization with third-generation hepatitis B surface antigen using microneedles
Introduction
Hepatitis B is well known as a major global health problem caused by the hepatitis B virus (HBV), which is 7th leading cause of death worldwide. The most effective prevention currently available is immunization with the hepatitis B vaccine [1].
The first generation of HBV vaccines were used beginning in 1980, when the hepatitis B surface antigen (HBsAg) produced from the plasma of chronic HBV infected individuals [2]. The second generation of HBsAg, yeast-derived HBsAg with a small hepatitis B surface protein (S protein), was introduced to prevent the transmission of blood pathogens from human blood products of those treated with the first generation of HBsAg [3]. The S protein of second-generation HBsAg is introduced into virus-like particles (VLPs) that are 22 nm in size, mimicking the structure of the real virus. This second generation of HBsAg is widely used for vaccination of newborns and adults in over 170 countries [4].
Second-generation HBsAg has not produced effective immunogenicity due to factors such as advanced age, renal dysfunction, liver disease, smoking, and immunosuppression [5], [6]. Potential reasons for non-responsiveness are genetically induced by resistance [7] or an increase in S gene mutations [8]. In attempts to improve the effectiveness of vaccination, large- and middle-sized surface proteins (pre S1 and pre S2) were merged into S proteins to induce improved antibody response against HBV [9], [10] and increase the antibody response of S gene mutants [11]. As the demand for enhanced immunogenicity to overcome the non-responsiveness to the S protein of HBsAg has increased, the third-generation HBsAg (with combination of three envelope proteins: S, pre-S1 [L protein] and pre-S2 [M protein]), has been developed and offers promising results for greatly improved vaccine efficacy [12], [13].
An additional approach to improving immunogenicity against HBV is changing the administration route from intramuscular (IM) to intradermal (ID). The epidermal and dermal layers of the skin are rich in antigen-presenting cells such as Langerhans cells and dendritic cells [14]. These colonized cells have high MHC class II expression and are important mediators of antiviral immunity [15], [16]. Thus an ID administration requires a lower dose of vaccine compared to an IM administration [17]. Furthermore, the safety and efficacy of ID administration of the HBV vaccine have been demonstrated by comparing ID to IM administration. [18], [19], [20], However, ID administration requires application into a skin layer at least 1 mm thick and it is not easy to administer precisely. Thus microneedles (MN) have been introduced as an easy-to-use, painless way to control injection depth and delivery of the right dose of antigen into the skin layer. Various antigens have demonstrated improved efficacy with ID administration using MN compared to IM administration [21], [22], [23], [24], [25], [26]. The second-generation hepatitis B vaccine was delivered into the skin layer using MN administration, and MN encapsulating HBsAg showed effective antibody production and improved storage stability [27].
In this study, immunization through the skin using the third generation of the hepatitis vaccine with pre-S1/pre-S2/S (L-HBsAg) was performed with MN for first time, as shown in Fig. 1. Alum was not included in the MN formulation. Stability of the vaccine was observed during the harsh storage conditions and the freeze-thaw cycle. Animal experiments demonstrated immunological efficacy of the vaccine administered intradermally with MN with a stabilizer compared with IM administration.
Section snippets
Animals
Five-week-old female BALB/c mice were purchased from Koatech (Pyungtek, Korea). The animals were housed in standard pathogen-free facilities and maintained with free access to food and water. All studies were approved by Institutional Animal Care and Use Committees (IACUC) at the International Vaccine Institute (2017–001).
Materials
L-HBsAg was obtained from the CHA Vaccine Institute (Seongnam, Korea). PLA was purchased from LACTEL (Birmingham, AL). Sodium carboxymethyl cellulose (CMC), trehalose
Characteristics of L-HBsAg microneedles
L-HBsAg MN were coated with three envelope proteins, L-protein, M-protein, and S-protein, with the amount of the three proteins in the coating formulation measuring about 8%, 17%, and 75%, respectively. L-HBsAg contained virus-like particles with sizes ranging from 20 nm to 40 nm [13].
L-HBsAg has been developed to induce antibodies in non-responding hepatitis B patients and to provide therapeutic treatment for these patients [12]. Formulations consist of a combination of a CMC thickening agent,
Conclusion
The third generation of hepatitis B antigen (L-HBsAg) was formulated without an adjuvant for microneedles, which were delivered into the skin. Microneedle injection (ID administration) induced superior antibody production compared to IM injections of L-HBsAg with an Alum adjuvant. The antigenicity of L-HBsAg-MN was reduced by gamma ray irradiation. When 15% (w/w) of trehalose was added as a stabilizer in a solidified formulation, the antigenicity of L-HBsAg-MN-Tre(F3) did not change even at a
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was funded by grants from Korea Ministry of Trade, Industry & Energy, South Korea (MOTIE, 10067809 (Industrial Strategic Technology Development Program)) and Korea Ministry of Health and Welfare, South Korea (MoHW, HI15C2971 (Technology Development Program of Responding to Infectious Disease)). We appreciate CHA Vaccine Institute to provide L-HBsAg.
References (49)
Hepatitis B vaccination: the key towards elimination and eradication of hepatitis B
J Hepatol
(2009)- et al.
The global impact of vaccination against hepatitis B: a historical overview
Vaccine
(2008) - et al.
Immune response to hepatitis B vaccination in drug using populations: a systematic review and meta-regression analysis
Vaccine
(2014) - et al.
Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications
J Hepatol
(2014) - et al.
Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-S1 and pre-S2 antigens as compared with conventional yeast-derived vaccines
Vaccine
(1994) - et al.
Design and characterisation of a dissolving microneedle patch for intradermal vaccination with heat-inactivated bacteria: a proof of concept study
Int J Pharm
(2018) - et al.
Influenza nucleoprotein DNA vaccination by a skin targeted, dry coated, densely packed microprojection array (Nanopatch) induces potent antibody and CD8+ T cell responses
J Control Release
(2016) - et al.
Effective humoral immune response from a H1N1 DNA vaccine delivered to the skin by microneedles coated with PLGA-based cationic nanoparticles
J Control Release
(2017) - et al.
Diphtheria toxoid and N-trimethyl chitosan layer-by-layer coated pH-sensitive microneedles induce potent immune responses upon dermal vaccination in mice
J Control Release
(2017) - et al.
Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable
Biomaterials
(2017)
Development of novel double-decker microneedle patches for transcutaneous vaccine delivery
Int J Pharm
Stability of influenza vaccine coated onto microneedles
Biomaterials
Bioneedles as alternative delivery system for hepatitis B vaccine
J Control Release
Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity
J Control Release
Storage at −3°C for 24 h alters the immunogenicity of pertussis vaccines
Vaccine
Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles
Vaccine
Mechanism and regulation of Immunoglobulin Isotype Switching54 (1993)
Adv Immunol
Hepatitis B vaccine
N Engl J Med
Human hepatitis B vaccine from recombinant yeast
Nature
Non-responders to hepatitis B vaccination: a review
Commun Dis Public Health
Genetic prediction of nonresponse to hepatitis B vaccine
N Engl J Med
A synthetic peptide vaccine involving the product of the pre-S(2) region of hepatitis B virus DNA: protective efficacy in chimpanzees
Proc Natl Acad Sci USA
Protective efficacy, immunotherapeutic potential, and safety of hepatitis B vaccines
J Med Virol
Enhanced immune response to hepatitis B vaccination through immunization with a Pre-S1/Pre-S2/S vaccine
Med Microbiol Immunol
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These authors equally contributed to this work.