Fu-Shi Quan

Virus-Like Particle Vaccine Induces Protective Immunity against Homologous and Heterologous Strains of Influenza Virus

ABSTRACT Recurrent outbreaks of highly pathogenic avian influenza virus pose the threat of pandemic spread of lethal disease and make it a priority to develop safe and effective vaccines. Influenza virus-like particles (VLPs) have been suggested to be a promising vaccine approach. However, VLP-induced immune responses, and their roles in inducing memory immune responses and cross-protective immunity have not been investigated. In this study, we developed VLPs containing influenza virus A/PR8/34 (H1N1) hemagglutinin (HA) and matrix (M1) proteins and investigated their immunogenicity, long-term cross-protective efficacy, and effects on lung proinflammatory cytokines in mice. Intranasal immunization with VLPs containing HA induced high serum and mucosal antibody titers and neutralizing activity against PR8 and A/WSN/33 (H1N1) viruses. Mice immunized with VLPs containing HA showed little or no proinflammatory lung cytokines and were protected from a lethal challenge with mouse-adapted PR8 or WSN viruses even 5 months postimmunization. Influenza VLPs induced mucosal immunoglobulin G and cellular immune responses, which were reactivated rapidly upon virus challenge. Long-lived antibody-secreting cells were detected in the bone marrow of immunized mice. Immune sera administered intranasally were able to confer 100% protection from a lethal challenge with PR8 or WSN, which provides further evidence that anti-HA antibodies are primarily responsible for preventing infection. Taken together, these results indicate that nonreplicating influenza VLPs represent a promising strategy for the development of a safe and effective vaccine to control the spread of lethal influenza viruses.

Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity

Microneedle patches coated with solid-state influenza vaccine have been developed to improve vaccine efficacy and patient coverage. However, dip coating microneedles with influenza vaccine can reduce antigen activity. In this study, we sought to determine the experimental factors and mechanistic pathways by which inactivated influenza vaccine can lose activity, as well as develop and assess improved microneedle coating formulations that protect the antigen from activity loss. After coating microneedles using a standard vaccine formulation, the stability of influenza vaccine was reduced to just 2%, as measured by hemagglutination activity. The presence of carboxymethylcellulose, which was added to increase viscosity of the coating formulation, was shown to contribute to vaccine activity loss. After screening a panel of candidate stabilizers, the addition of trehalose to the coating formulation was found to protect the antigen and retain 48-82% antigen activity for all three major strains of seasonal influenza: H1N1, H3N2 and B. Influenza vaccine coated in this way also exhibited thermal stability, such that activity loss was independent of temperature over the range of 4-37 degrees C for 24h. Dynamic light scattering measurements showed that antigen activity loss was associated with virus particle aggregation, and that stabilization using trehalose largely blocked this aggregation. Finally, microneedles using an optimized vaccine coating formulation were applied to the skin to vaccinate mice. Microneedle vaccination induced robust systemic and functional antibodies and provided complete protection against lethal challenge infection similar to conventional intramuscular injection. Overall, these results show that antigen activity loss during microneedle coating can be largely prevented through optimized formulation and that stabilized microneedle patches can be used for effective vaccination.

Influenza vaccines based on virus-like particles

The simultaneous expression of structural proteins of virus can produce virus-like particles (VLPs) by a self-assembly process in a viral life cycle even in the absence of genomic material. Taking an advantage of structural and morphological similarities of VLPs to native virions, VLPs have been suggested as a promising platform for new viral vaccines. In the light of a pandemic threat, influenza VLPs have been recently developed as a new generation of non-egg based cell culture-derived vaccine candidates against influenza infection. Animals vaccinated with VLPs containing hemagglutinin (HA) or HA and neuraminidase (NA) were protected from morbidity and mortality resulting from lethal influenza infections. Influenza VLPs serve as an excellent model system of an enveloped virus for understanding the properties of VLPs in inducing protective immunity. In this review, we briefly describe the characteristics of influenza VLPs assembled with a lipid bilayer containing glycoproteins, and summarize the current progress on influenza VLPs as an alternative vaccine candidate against seasonal as well as pandemic influenza viruses. In addition, the protective immune correlates induced by vaccination with influenza VLPs are discussed.

Induction of Heterosubtypic Immunity to Influenza Virus by Intranasal Immunization

ABSTRACT Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4+ or CD8+ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.

Incorporation of Membrane-Anchored Flagellin into Influenza Virus-Like Particles Enhances the Breadth of Immune Responses

ABSTRACT We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H1N1) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H3N2) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.

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.