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1.  Memory T Cells Generated by Prior Exposure to Influenza Cross React with the Novel H7N9 Influenza Virus and Confer Protective Heterosubtypic Immunity 
PLoS ONE  2015;10(2):e0115725.
Influenza virus is a source of significant health and economic burden from yearly epidemics and sporadic pandemics. Given the potential for the emerging H7N9 influenza virus to cause severe respiratory infections and the lack of exposure to H7 and N9 influenza viruses in the human population, we aimed to quantify the H7N9 cross-reactive memory T cell reservoir in humans and mice previously exposed to common circulating influenza viruses. We identified significant cross-reactive T cell populations in humans and mice; we also found that cross-reactive memory T cells afforded heterosubtypic protection by reducing morbidity and mortality upon lethal H7N9 challenge. In context with our observation that PR8-primed mice have limited humoral cross-reactivity with H7N9, our data suggest protection from H7N9 challenge is indeed mediated by cross-reactive T cell populations established upon previous priming with another influenza virus. Thus, pre-existing cross-reactive memory T cells may limit disease severity in the event of an H7N9 influenza virus pandemic.
doi:10.1371/journal.pone.0115725
PMCID: PMC4324938  PMID: 25671696
2.  Cross-protection by co-immunization with influenza hemagglutinin DNA and inactivated virus vaccine using coated microneedles 
The need for annual revaccination against influenza is a burden on the healthcare system, leads to low vaccination rates and makes timely vaccination difficult against pandemic strains, such as during the 2009 H1N1 influenza pandemic. In an effort toward achieving a broadly protective vaccine that provides cross-protection against multiple strains of influenza, this study developed a microneedle patch to co-immunize with A/PR8 influenza hemagglutinin DNA and A/PR8 inactivated virus vaccine. We hypothesize that this dual component vaccination strategy administered to the skin using microneedles will provide cross-protection against other strains of influenza. To test this hypothesis, we developed a novel coating formulation that did not require additional excipients to increase coating solution viscosity by using the DNA vaccine itself to increase viscosity and thereby enable thick coatings of DNA vaccine and inactivated virus vaccine on metal microneedles. Co-immunization in this way not only generated robust antibody responses against A/PR8 influenza but also generated robust heterologous antibody responses against pandemic 2009 H1N1 influenza in mice. Challenge studies showed complete cross-protection against lethal challenge with live pandemic 2009 H1N1 virus. Control experiments using A/PR8 inactivated influenza virus vaccine with placebo DNA coated onto microneedles produced lower antibody titers and provided incomplete protection against challenge. Overall, this is the first study showing DNA solution as a microneedle coating agent and demonstrating cross-protection by co-immunization with inactivated virus and DNA vaccine using coated microneedles.
doi:10.1016/j.jconrel.2013.04.016
PMCID: PMC3815987  PMID: 23643528
Microneedle; DNA vaccine; Influenza virus; Coating; Cross-protection
3.  Mucosal Adjuvants for Influenza Virus-Like Particle Vaccine 
Viral Immunology  2013;26(6):385-395.
Abstract
To find an effective mucosal adjuvant for influenza virus-like particles (VLPs), we compared the effects of known adjuvants Alum, CpG DNA, monophosphoryl lipid A (MPL), poly IC, gardiquimod, and cholera toxin (CT). Mice that were intranasally immunized with Alum, CpG, MPL, and CT adjuvanted VLPs showed higher levels of antibodies in both sera and mucosa. Hemagglutination inhibition and virus neutralizing activities were enhanced in groups adjuvanted with Alum, MPL, or CT. Influenza virus specific long-lived cells secreting IgG and IgA antibodies were found at high levels both in bone marrow and spleen in the Alum, CpG and CT adjuvanted groups. A similar level of protection was observed among different adjuvanted groups, except the CT adjuvant that showed a higher efficacy in lowering lung viral loads after challenge. Alum and CT adjuvants differentially increased influenza VLP-mediated activation of dendritic cells and splenocytes in vitro, supporting the in vivo pattern of antibody isotypes and cytokine production. These results suggest that Alum, MPL, or CpG adjuvants, which have been tested clinically, can be developed as an effective mucosal adjuvant for influenza VLP vaccines.
doi:10.1089/vim.2013.0013
PMCID: PMC3868302  PMID: 24236855
4.  Protocatechuic Acid, a Novel Active Substance against Avian Influenza Virus H9N2 Infection 
PLoS ONE  2014;9(10):e111004.
Influenza virus H9N2 subtype has triggered co-infection with other infectious agents, resulting in huge economical losses in the poultry industry. Our current study aims to evaluate the antiviral activity of protocatechuic acid (PCA) against a virulent H9N2 strain in a mouse model. 120 BALB/c mice were divided into one control group, one untreated group, one 50 mg/kg amantadine hydrochloride-treated group and three PCA groups treated 12 hours post-inoculation with 40, 20 or 10 mg/kg PCA for 7 days. All the infected animals were inoculated intranasally with 0.2 ml of a A/Chicken/Hebei/4/2008(H9N2) inoculum. A significant body weight loss was found in the 20 mg/kg and 40 mg/kg PCA-treated and amantadine groups as compared to the control group. The 14 day survivals were 94.4%, 100% and 95% in the PCA-treated groups and 94.4% in the amantadine hydrochloride group, compared to less than 60% in the untreated group. Virus loads were less in the PCA-treated groups compared to the amantadine-treated or the untreated groups. Neutrophil cells in BALF were significantly decreased while IFN-γ, IL-2, TNF-α and IL-6 decreased significantly at days 7 in the PCA-treated groups compared to the untreated group. Furthermore, a significantly decreased CD4+/CD8+ ratio and an increased proportion of CD19 cells were observed in the PCA-treated groups and amantadine-treated group compared to the untreated group. Mice administered with PCA exhibited a higher survival rate and greater viral clearance associated with an inhibition of inflammatory cytokines and activation of CD8+ T cell subsets. PCA is a promising novel agent against bird flu infection in the poultry industry.
doi:10.1371/journal.pone.0111004
PMCID: PMC4206475  PMID: 25337912
5.  Cutaneous immunization: an evolving paradigm in influenza vaccines 
Expert opinion on drug delivery  2014;11(4):615-627.
Introduction
Most vaccines are administered by intramuscular injection using a hypodermic needle and syringe. Some limitations of this procedure include reluctance to be immunized because of fear of needlesticks, and concerns associated with the safe disposal of needles after their use. Skin delivery is an alternate route of vaccination that has potential to be painless and could even lead to dose reduction of vaccines. Recently, microneedles have emerged as a novel painless approach for delivery of influenza vaccines via the skin.
Areas covered
In this review, we briefly summarize the approaches and devices used for skin vaccination, and then focus on studies of skin immunization with influenza vaccines using microneedles. We discuss both the functional immune response and the nature of this immune response following vaccination with microneedles.
Expert opinion
The cutaneous administration of influenza vaccines using microneedles offers several advantages: it is painless, elicits stronger immune responses in preclinical studies and could improve responses in high-risk populations. These dry formulations of vaccines provide enhanced stability, a property of high importance in enabling their rapid global distribution in response to possible outbreaks of pandemic influenza and newly emerging infectious diseases.
doi:10.1517/17425247.2014.885947
PMCID: PMC4009492  PMID: 24521050
cutaneous; immunization; influenza; review
6.  Multiple heterologous M2 extracellular domains presented on virus-like particles confer broader and stronger M2 immunity than live influenza A virus infection 
Antiviral research  2013;99(3):328-335.
The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza virus at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine.
doi:10.1016/j.antiviral.2013.06.010
PMCID: PMC3788098  PMID: 23811283
M2e VLPs; M2e cross-reactivity; cross protection
7.  A protective role of murine langerin+ cells in immune responses to cutaneous vaccination with microneedle patches 
Scientific Reports  2014;4:6094.
Cutaneous vaccination with microneedle patches offers several advantages over more frequently used approaches for vaccine delivery, including improved protective immunity. However, the involvement of specific APC subsets and their contribution to the induction of immunity following cutaneous vaccine delivery is not well understood. A better understanding of the functions of individual APC subsets in the skin will allow us to target specific skin cell populations in order to further enhance vaccine efficacy. Here we use a Langerin-EGFP-DTR knock-in mouse model to determine the contribution of langerin+ subsets of skin APCs in the induction of adaptive immune responses following cutaneous microneedle delivery of influenza vaccine. Depletion of langerin+ cells prior to vaccination resulted in substantial impairment of both Th1 and Th2 responses, and decreased post-challenge survival rates, in mice vaccinated cutaneously but not in those vaccinated via the intramuscular route or in non-depleted control mice. Our results indicate that langerin+ cells contribute significantly to the induction of protective immune responses following cutaneous vaccination with a subunit influenza vaccine.
doi:10.1038/srep06094
PMCID: PMC4135340  PMID: 25130187
8.  Virus-Like Particles Containing the Tetrameric Ectodomain of Influenza Matrix Protein 2 and Flagellin Induce Heterosubtypic Protection in Mice 
BioMed Research International  2013;2013:686549.
The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation.
doi:10.1155/2013/686549
PMCID: PMC3745920  PMID: 23984396
9.  Enhanced Influenza Virus-Like Particle Vaccines Containing the Extracellular Domain of Matrix Protein 2 and a Toll-Like Receptor Ligand 
The extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region of Salmonella enterica serovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.
doi:10.1128/CVI.00153-12
PMCID: PMC3416094  PMID: 22647270
10.  Host responses in human skin after conventional intradermal injection or microneedle administration of virus-like-particle influenza vaccine 
Advanced healthcare materials  2013;2(10):1401-1410.
Miniaturized microneedle devices are being developed for painlessly targeting vaccines to the immune cell populations in skin. As skin immunization studies are generally restricted to animal models however, where skin architecture and immunity is greatly different to human, surprisingly little is known about the local human response to intradermal (ID) vaccines. Here we use surgically excised human skin to explore for the first time the complex molecular and cellular host responses to a candidate influenza vaccine comprising nanoparticulate virus-like-particles (VLPs), administered via conventional hypodermic injection or reduced scale microneedles. Responses at the molecular level are determined by microarray analysis (47,296 discrete transcripts) and validated by quantitative PCR (96 genes). Cellular response is probed through monitoring migration of dendritic cells in viable skin tissue. Gene expression mapping, ontological analysis and qPCR reveal up-regulation of a host of genes responsible for key immunomodulatory processes and host viral response, including cell recruitment, activation, migration and T cell interaction following both ID and microneedle injection of VLPs; the response from the microneedles being more subtle. Significant morphological and migratory changes to skin dendritic cells are also apparent following microneedle VLP delivery. This is the first study displaying the global, multifaceted immunological events that occur at the site of vaccine deposition in human skin and will subsequently influence the degree and nature of innate and adaptive immune responses. An increased understanding of the detailed similarities and differences in response against antigen administered via different delivery modalities will inform the development of improved vaccines and vaccine delivery systems.
doi:10.1002/adhm.201300006
PMCID: PMC4009496  PMID: 23564440
Intradermal; Microneedle; Human skin; Gene expression; Influenza; Virus-like-particles
11.  Universal Influenza Vaccines, a Dream to Be Realized Soon 
Viruses  2014;6(5):1974-1991.
Due to frequent viral antigenic change, current influenza vaccines need to be re-formulated annually to match the circulating strains for battling seasonal influenza epidemics. These vaccines are also ineffective in preventing occasional outbreaks of new influenza pandemic viruses. All these challenges call for the development of universal influenza vaccines capable of conferring broad cross-protection against multiple subtypes of influenza A viruses. Facilitated by the advancement in modern molecular biology, delicate antigen design becomes one of the most effective factors for fulfilling such goals. Conserved epitopes residing in virus surface proteins including influenza matrix protein 2 and the stalk domain of the hemagglutinin draw general interest for improved antigen design. The present review summarizes the recent progress in such endeavors and also covers the encouraging progress in integrated antigen/adjuvant delivery and controlled release technology that facilitate the development of an affordable universal influenza vaccine.
doi:10.3390/v6051974
PMCID: PMC4036552  PMID: 24784572
influenza; cross protection; universal influenza vaccine
12.  Effects of Stabilization of the gp41 Cytoplasmic Domain on Fusion Activity and Infectivity of SIVmac239 
AIDS Research and Human Retroviruses  2011;27(11):1213-1222.
Abstract
We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell–cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.
doi:10.1089/aid.2010.0321
PMCID: PMC3206742  PMID: 21434848
13.  Viruslike Particle Vaccine Induces Protection Against Respiratory Syncytial Virus Infection in Mice 
The Journal of Infectious Diseases  2011;204(7):987-995.
Background. Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Despite decades of research with traditional or subunit vaccine approaches, there are no approved RSV vaccines. New approaches are therefore urgently needed to develop effective RSV vaccines.
Methods. We developed viruslike particles (VLPs) consisting of an influenza virus matrix (M1) protein core and RSV-F or -G on the surface. We tested the immunogenicity and vaccine efficacy of these VLPs (RSV-F, RSV-G) in a mouse model.
Results. Intramuscular vaccination with RSV-F or RSV-G VLPs elicited IgG2a dominant RSV-specific immunoglobulin G (IgG) antibody responses against RSV-A2 viruses in both serum and lung extract. Mice immunized with VLPs (RSV-F or RSV-G) showed higher viral neutralizing antibodies in vitro and significantly decreased lung virus loads in vivo after live RSV-A2 challenge. RSV-G VLPs showed better protective efficacy than RSV-F VLPs as determined by the levels of lung virus loads and morbidity postchallenge.
Conclusions. This study demonstrates that VLP vaccination provides effective protection against RSV infection. VLPs containing RSV-F and/or RSV-G are potential vaccine candidates against RSV.
doi:10.1093/infdis/jir474
PMCID: PMC3164432  PMID: 21881112
14.  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.
doi:10.1016/j.jconrel.2012.12.002
PMCID: PMC3578180  PMID: 23246470
Coated microneedle patch; Skin vaccination; Influenza vaccine stability; Coating formulation; Crystallization; Phase separation; Osmotic pressure
15.  Long-Term Protective Immunity from an Influenza Virus-Like Particle Vaccine Administered with a Microneedle Patch 
Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown to induce protection similar to or better than that induced by intramuscular immunization. In this study, we examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 virus 14 months after a single vaccine dose. Influenza virus-specific total IgG response and hemagglutination inhibition (HAI) titers were maintained at high levels for over 1 year after microneedle vaccination. Microneedle vaccination also induced substantial levels of lung IgG and IgA antibody responses, and antibody-secreting plasma cells from spleen and bone marrow, as well as conferring effective control of lung viral loads, resulting in complete protection 14 months after vaccination. These strong and long-lasting immune responses were enabled in part by stabilization of the vaccine by formulation with trehalose during microneedle patch fabrication. Administration of the stabilized vaccine using microneedles was especially effective at enabling strong recall responses measured 4 days after lethal virus challenge, including increased HAI and antibody-secreting cells in the spleen and reduced viral titer and inflammatory response in the lung. The results in this study indicate that skin vaccination with VLP vaccine using a microneedle patch provides long-term protection against influenza in mice.
doi:10.1128/CVI.00251-13
PMCID: PMC3889580  PMID: 23863506
17.  Local Response to Microneedle-Based Influenza Immunization in the Skin 
mBio  2012;3(2):e00012-12.
ABSTRACT
Microneedle patches (MN) provide a novel method of vaccine delivery to the skin with the objective of targeting the large network of resident antigen-presenting cells to induce an efficient immune response. Our previous reports demonstrated that cutaneous delivery of inactivated influenza virus-coated MN to mice protects against lethal infection. Protection is correlated with sustained levels of anti-influenza virus serum antibodies, hemagglutination inhibition titers, and robust cellular responses that are often stronger than those generated by intramuscular vaccination. Here we dissect the early events occurring in murine skin after microneedle delivery of inactivated influenza virus. We demonstrate correlation of immunization against influenza virus with a local increase of cytokines important for recruitment of neutrophils, monocytes and dendritic cells at the site of immunization. We also observed prolonged antigen deposition, and migration of matured dendritic cells bearing influenza virus antigen from the skin.
IMPORTANCE
The immunological mechanisms by which MN vaccination confers protective immunity are not well understood. The present study provides a first analysis of the early immune events after microneedle-based vaccination.
doi:10.1128/mBio.00012-12
PMCID: PMC3302568  PMID: 22396479
18.  Improved protection against avian influenza H5N1 virus by a single vaccination with virus-like particles in skin using microneedles 
Antiviral research  2010;88(2):244-247.
Summary
To develop a more effective vaccination method against H5N1 virus, we investigated the immunogenicity and protective efficacy after skin vaccination using microneedles coated with influenza virus-like particles containing hemagglutinin derived from A/Vietnam/1203/04 H5N1 virus (H5 VLPs). A single microneedle vaccination of mice with H5 VLPs induced increased levels of antibodies and provided complete protection against lethal challenge without apparent disease symptoms. In contrast, intramuscular injection with the same vaccine dose showed low levels of antibodies and provided only partial protection accompanied by severe body weight loss. Post-challenge analysis suggested that improved protection was associated with lower lung viral titers and enhanced generation of recall antibody secreting cells by microneedle vaccination. Thus, this study provides evidence that skin delivery of H5 VLP vaccines using microneedles designed for self-administration induces improved protection compared to conventional intramuscular immunization.
doi:10.1016/j.antiviral.2010.09.001
PMCID: PMC2980851  PMID: 20851715
H5N1; pandemic vaccine; single dose; skin vaccination; microneedles
19.  Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine 
Vaccine  2009;28(24):4103-4112.
Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in E. coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.
doi:10.1016/j.vaccine.2009.07.058
PMCID: PMC3187848  PMID: 19654062
20.  Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles 
Virology  2010;405(1):165-175.
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 5 fold high 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.
doi:10.1016/j.virol.2010.05.034
PMCID: PMC2925114  PMID: 20580392
Virus like particles; subunit vaccine; influenza virus; H5N1; protection; memory immune responses
22.  Enhanced Mucosal Immune Responses to HIV Virus-Like Particles Containing a Membrane-Anchored Adjuvant 
mBio  2011;2(1):e00328-10.
Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine.
IMPORTANCE
A prophylactic vaccine is urgently needed to control the spread of HIV/AIDS. Antigens inducing strong systemic and mucosal immune responses are promising as vaccines for this mucosally transmitted disease. We found that novel HIV virus-like particles (VLPs) presenting a high level of Env in its native membrane-bound form and coincorporating an innate immune-signaling adjuvant in the same particles were effective in inducing enhanced systemic and mucosal immunity. As new HIV vaccine candidates, these VLPs bridge the gaps of the innate and adaptive, as well as systemic and mucosal, immune responses, providing a new approach for HIV vaccine development.
doi:10.1128/mBio.00328-10
PMCID: PMC3039440  PMID: 21325038
23.  Closely Related Influenza Viruses Induce Contrasting Respiratory Tract Immunopathology 
PLoS ONE  2013;8(9):e76708.
The swine-origin H1N1 virus which emerged in 2009 resulted in the first influenza pandemic of the 21st century. Although the majority of infections were moderate, a significant proportion of infections were severe and characterized by acute respiratory distress syndrome and pulmonary edema. We compared two isolates from the 2009 H1N1 pandemic; A/California/07/09 (CA/07) and A/Netherlands/602/09 (NL/602) viruses that share greater than 99% sequence identity. Though genetically similar, these viruses exhibit contrasting pathological effects. Mice that were infected with 800 plaque forming unit (PFU) of CA/07 virus rapidly lost weight, which was concurrent with detection of high pulmonary concentrations of MCP-1, MIG, IP-10 and TIMP-1. Initially, severe bronchiolar epithelial necrosis and acute respiratory distress was observed, followed by marked bronchiolar epithelial hyperplasia. Mononuclear cell infiltration was initially localized to perivascular and peribronchiolar interstitium and then spread to adjacent alveoli. Infiltrating cells were phenotypically CD11bhi, F4/80lo. In contrast, when mice were infected with 800 PFU of NL/602 virus, minimal weight loss was observed, and concentrations of cytokines in the lung were significantly lower. Inflammation was primarily restricted to the bronchioles and perivascular interstitium with minimal spread to alveoli. Infiltrating cells include foamy macrophages and surface markers were characterized as CD11blo/-, F4/80hi. These two genetically similar viruses can be useful strains with which to investigate immune-regulatory determinants of pathogenesis of influenza virus.
doi:10.1371/journal.pone.0076708
PMCID: PMC3784437  PMID: 24086762
24.  Enhanced memory responses to H1N1 influenza vaccination in the skin using vaccine coated-microneedles 
The Journal of infectious diseases  2010;201(2):190-198.
Background
Morbidity and mortality due to influenza could be reduced by improved vaccination.
Methods
To develop a novel skin delivery method for simple and self administration, we prepared microneedle patches with stabilized influenza vaccine and investigated their protective immune responses.
Results
Mice immunized by a single microneedle dose of trehalose-stabilized influenza vaccine developed strong antibody responses that were long-lived. Compared to traditional intramuscular immunization, stabilized microneedle vaccination was superior in inducing protective immunity as evidenced by efficient lung viral clearance and enhanced humoral and antibody secreting cell immune responses after lethal challenge. Vaccine stabilization was found to be important, because mice immunized with an unstabilized microneedle vaccine elicited weaker IgG2a antibody response and were only partially protected against viral challenge. Improved trafficking of dendritic cells to regional lymph nodes by microneedle delivery to the skin might play a role in contributing to improved protective immunity.
Conclusions
These findings suggest that vaccination in the skin using a microneedle patch can improve protective efficacy, induce long-term sustained immunogenicity, and may provide a simple method of administration to improve influenza vaccination.
doi:10.1086/649228
PMCID: PMC2798016  PMID: 20017632
influenza virus; vaccine; microneedle; memory response; intradermal immunization
25.  Influenza vaccines based on virus-like particles 
Virus research  2009;143(2):140-146.
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.
doi:10.1016/j.virusres.2009.04.005
PMCID: PMC2753524  PMID: 19374929

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