Dae-Goon Yoo

Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic. Methodology/Principal Findings Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro. Conclusions/Significance These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

Enhanced Memory Responses to Seasonal H1N1 Influenza Vaccination of the Skin with the Use of Vaccine-Coated Microneedles

BACKGROUND Morbidity and mortality due to influenza could be reduced by improved vaccination. METHODS To develop a novel skin delivery method that is simple and allows for easy self-administration, we prepared microneedle patches with stabilized influenza vaccine and investigated their protective immune responses. RESULTS Mice vaccinated with a single microneedle dose of trehalose-stabilized influenza vaccine developed strong antibody responses that were long-lived. Compared with traditional intramuscular vaccination, stabilized microneedle vaccination was superior in inducing protective immunity, as was evidenced by efficient clearance of virus from the lung and enhanced humoral and antibody-secreting cell immune responses after 100% survival from lethal challenge. Vaccine stabilization was found to be important, because mice vaccinated with an unstabilized microneedle vaccine elicited a weaker immunoglobulin G 2a antibody response, compared with the stabilized microneedle vaccine, and were only partially protected against viral challenge. Improved trafficking of dendritic cells to regional lymph nodes as a result of microneedle delivery to the skin might play a role in contributing to improved protective immunity. CONCLUSIONS These findings suggest that vaccination of the skin using a microneedle patch can improve protective efficacy and induce long-term sustained immunogenicity and may also provide a simple method of administration to improve influenza vaccination coverage.

Intradermal Vaccination with Influenza Virus-Like Particles by Using Microneedles Induces Protection Superior to That with Intramuscular Immunization

ABSTRACT Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (≤40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.

Improved influenza vaccination in the skin using vaccine coated microneedles

Easy and effective vaccination methods could reduce mortality rates and morbidity due to vaccine-preventable influenza infections. In this study, we examined the use of microneedle patches to increase patient coverage through possible self-administration and enhance vaccine immunogenicity by targeted delivery to skin. We carried out a detailed study of protective immune responses after a single influenza vaccination to the skin of mice with a novel microneedle patch designed to facilitate simple and reliable vaccine delivery. Skin vaccination with inactivated virus-coated microneedles provided superior protection against lethal challenge compared to intramuscular injection as evidenced by effective virus clearance in lungs. Detailed immunologic analysis suggests that induction of virus neutralizing antibodies as well as enhanced anamnestic humoral and cellular responses contributed to improved protection by microneedle vaccination to the skin. These findings suggest that vaccination in the skin using a microneedle patch can improve protective immunity, and simplify delivery of influenza and possibly other vaccines.

Stabilization of Influenza Vaccine Enhances Protection by Microneedle Delivery in the Mouse Skin

Background Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy. Methodology/Principal Findings Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin. However, the stability of the influenza vaccine, as measured by hemagglutination activity, was found to be significantly damaged during microneedle coating. The addition of trehalose to the microneedle coating formulation retained hemagglutination activity, indicating stabilization of the coated influenza vaccine. For both intramuscular and microneedle skin immunization, delivery of un-stabilized vaccine yielded weaker protective immune responses including viral neutralizing antibodies, protective efficacies, and recall immune responses to influenza virus. Immunization using un-stabilized vaccine also shifted the pattern of antibody isotypes compared to the stabilized vaccine. Importantly, a single microneedle-based vaccination using stabilized influenza vaccine was found to be superior to intramuscular immunization in controlling virus replication as well as in inducing rapid recall immune responses post challenge. Conclusions/Significance The functional integrity of hemagglutinin is associated with inducing improved protective immunity against influenza. Simple microneedle influenza vaccination in the skin produced superior protection compared to conventional intramuscular immunization. This approach is likely to be applicable to other vaccines too.

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.

Stability of influenza vaccine coated onto microneedles

A microneedle patch coated with vaccine simplifies vaccination by using a patch-based delivery method and targets vaccination to the skin for superior immunogenicity compared to intramuscular injection. Previous studies of microneedles have demonstrated effective vaccination using freshly prepared microneedles, but the issue of long-term vaccine stability has received only limited attention. Here, we studied the long-term stability of microneedles coated with whole inactivated influenza vaccine guided by the hypothesis that crystallization and phase separation of the microneedle coating matrix damages influenza vaccine coated onto microneedles. In vitro studies showed that the vaccine lost stability as measured by hemagglutination activity in proportion to the degree of coating matrix crystallization and phase separation. Transmission electron microscopy similarly showed damaged morphology of the inactivated virus vaccine associated with crystallization. In vivo assessment of immune response and protective efficacy in mice further showed reduced vaccine immunogenicity after influenza vaccination using microneedles with crystallized or phase-separated coatings. This work shows that crystallization and phase separation of the dried coating matrix are important factors affecting long-term stability of influenza vaccine-coated microneedles.

Microneedle Delivery of H5N1 Influenza Virus-Like Particles to the Skin Induces Long-Lasting B- and T-Cell Responses in Mice

ABSTRACT A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207+ Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.

Kinetics of Immune Responses to Influenza Virus-Like Particles and Dose-Dependence of Protection with a Single Vaccination

ABSTRACT The format of influenza virus-like particles (VLPs) as a nonreplicating particulate vaccine candidate is a promising alternative to conventional egg-based vaccines. In this study, we have investigated the detailed kinetics of immune responses and protective efficacy after a single intranasal immunization with different doses of VLPs alone or in the presence of an Escherichia coli mutant heat-labile enterotoxin [mLT(R192G)] or cholera toxin subunit B as adjuvants. Analysis of immune responses showed differential kinetics in a VLP antigen dose-dependent manner and dynamic changes in the ratios of antibody immunoglobulin G isotypes over the time course. Protection against lethal challenge was observed with a single immunization with influenza VLPs even without adjuvant. The addition of adjuvant showed significant antigen-sparing effects with improved protective efficacy. The protective immune responses, efficacies of protection, and antigen-sparing effects were significantly improved by a second immunization as determined by the levels of neutralizing antibodies, morbidity postchallenge, lung viral titers, and inflammatory cytokines. Our results are informative for a better understanding of the protective immunity induced by a single dose or two doses of influenza VLPs, which is dependent on antigen dosage and the presence of adjuvant, and will provide insights into designing effective vaccines based on VLPs.

Stability of whole inactivated influenza virus vaccine during coating onto metal microneedles

Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating. Vaccine coating in a trehalose-only formulation retained about 40-50% of vaccine activity, which is a significant improvement. The partial viral activity loss observed in the trehalose-only formulation was hypothesized to come from osmotic pressure-induced vaccine destabilization. We found that inclusion of a viscosity enhancer, carboxymethyl cellulose, overcame this effect and retained full vaccine activity on both washed and plasma-cleaned titanium surfaces. The addition of polymeric surfactant, Lutrol® micro 68, to the trehalose formulation generated phase transformations of the vaccine coating, such as crystallization and phase separation, which was correlated to additional vaccine activity loss, especially when coating on hydrophilic, plasma-cleaned titanium. Again, the addition of a viscosity enhancer suppressed the surfactant-induced phase transformations during drying, which was confirmed by in vivo assessment of antibody response and survival rate after immunization in mice. We conclude that trehalose and a viscosity enhancer are beneficial coating excipients, but the inclusion of surfactant is detrimental to vaccine stability.