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.

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.

In addition to its surface glycoprotein (GP1,2), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP1,2 and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP12 antibodies, but only from mice that have been immunized by sGP. We term this phenomenon “antigenic subversion”, and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP1,2, thereby allowing it to absorb anti-GP1,2 antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP1,2 response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.

Author Summary

The function of the Ebola virus (EBOV) secreted glycoprotein (sGP) has been long debated, and the fact that sGP production is conserved among all known EBOV species strongly indicates an important role in the viral life cycle. Furthermore, the recent finding that EBOV mutates to a predominantly non-sGP-forming phenotype in cell culture, while the mutant virus reverts to an sGP-forming phenotype in vivo, suggests that sGP is critical for EBOV to survive in its infected host. Here we demonstrate that sGP can function to absorb anti-GP antibodies. More importantly, instead of simply passively absorbing host antibodies, sGP actively subverts the host immune response to induce cross-reactivity with epitopes it shares with membrane-bound GP1,2. Immune subversion by sGP represents a distinct mechanism from the use of secreted antigens as antibody decoys, an immune evasion tactic previously proposed for other viruses, and should be an important consideration for future EBOV vaccine design efforts since vaccines may need to be specifically tailored to avoid subversion.

Killed and live attenuated influenza virus vaccines are effective in preventing and curbing the spread of influenza epidemics when the strains present in the vaccines are closely matched with the predicted epidemic strains. These vaccines are primarily targeted to induce immunity to the variable major target antigen, hemagglutinin (HA) of influenza virus. However, current vaccines are not effective in preventing the emergence of new pandemic or highly virulent viruses. New approaches are being investigated to develop universal influenza virus vaccines as well as to apply more effective vaccine delivery methods. Conserved vaccine targets including the influenza M2 ion channel protein and HA stalk domains are being developed using recombinant technologies to improve the level of cross protection. In addition, recent studies provide evidence that vaccine supplements can provide avenues to further improve current vaccination.

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.

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.

Background. A major goal in influenza vaccine development is induction of serological memory and cellular responses to confer long-term protection and limit virus spread after infection. Here, we investigate induction of long-lived immunity against the 2009 H1N1 virus after skin vaccination.

Methods. BALB/c mice received a single dose of 5 μg inactivated A/California/04/09 virus via coated metal microneedles (MN) applied to skin or via subcutaneous injection.

Results. MN or subcutaneous vaccination elicited similar serum IgG and hemagglutination inhibition titers and 100% protection against lethal viral challenge 6 weeks after vaccination. Six months after vaccination, the subcutaneous group exhibited a 60% decrease in functional antibody titers and extensive lung inflammation after challenge with 10 × LD50 of homologous virus. In contrast, the MN group maintained high functional antibody titers and IFN-γ levels, inhibition of viral replication, and no signs of lung inflammation after challenge. MN vaccination conferred complete protection against lethal challenge, whereas subcutaneous vaccination induced only partial protection. These findings were further supported by high numbers of bone marrow plasma cells and spleen antibody-secreting cells detected in the MN group.

Conclusions. A single skin vaccination with MN induced potent long-lived immunity and improved protection against the 2009 H1N1 influenza virus, compared with subcutaneous injection.

Low pH treatment of influenza virus hemagglutinin (HA) exposes its relatively conserved stalk domain, suggesting a potential immunogen with capability to induce broader immune responses. Here, we describe characterization, immunogenicity, antigenicity, and protective immunity induced by low pH treated inactivated whole viral vaccine in comparison with the untreated vaccine. The acidic pH treated viral vaccine showed high susceptibility to proteolytic cleavage and low hemagglutination activity indicating conformational changes. Immunization of mice with low pH treated viral vaccine induced lower levels of homologous or heterologous virus-specific binding and neutralizing antibodies compared to the untreated vaccine. Also, low pH treated influenza viral antigen showed lower antigenicity compared to the untreated influenza viral antigen. Lower efficacy of cross-protection against heterosubtypic virus was observed in the low-pH treated vaccine group. The results provide evidence that there is a correlation between protective efficacy and the stability of vaccines.

Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite of their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of non-influenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.

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.

Virus-like particles (VLPs) have a number of features that make them attractive influenza vaccine candidates. Microneedle (MN) devices are being developed for the convenient and pain-free delivery of vaccines across the skin barrier layer. Whilst MN-based vaccines have demonstrated proof-of-concept in mice, it is vital to understand how MN targeting of VLPs to the skin epidermis affects activation and migration of Langerhans cells (LCs) in the real human skin environment. MNs coated with vaccine reproducibly penetrated freshly excised human skin, depositing 80% of the coating within 60 seconds of insertion. Human skin experiments showed that H1 (A/PR/8/34) and H5 (A/Viet Nam/1203/04) VLPs, delivered via MN, stimulated LCs resulting in changes in cell morphology and a reduction in cell number in epidermal sheets. LC response was significantly more pronounced in skin treated with H1 VLPs, compared with H5 VLPs. Our data provides strong evidence that MN-facilitated delivery of influenza VLP vaccines initiates a stimulatory response in LCs in human skin. The results support and validate animal data, suggesting that dendritic cells (DCs) targeted through deposition of the vaccine in skin generate immune response. The study also demonstrates the value of using human skin alongside animal studies for preclinical testing of intradermal (ID) vaccines.

Immunization of the world population before an influenza pandemic such as the 2009 H1N1 virus spreads globally is not possible with current vaccine production platforms. New influenza vaccine technologies, such as virus-like-particles (VLPs), offer a promising alternative. Here, we tested the immunogenicity and protective efficacy of a VLP vaccine containing hemagglutinin (HA) and M1 from the 2009 pandemic H1N1 influenza virus (H1N1pdm) in ferrets and compared intramuscular (i.m.) and intranasal (i.n.) routes of immunization. Vaccination of ferrets with VLPs containing the M1 and HA proteins from A/California/04/2009 (H1N1pdm) induced high antibody titers and conferred significant protection against virus challenge. VLP-vaccinated animals lost less weight, shed less virus in nasal washes, and had markedly lower virus titers in all organs tested than naïve controls. A single dose of VLPs, either i.m. or i.n., induced higher levels of antibody than did two doses of commercial split vaccine. Ferrets vaccinated with split vaccine were incompletely protected against challenge; these animals had lower virus titers in olfactory bulbs, tonsils, and intestines, but lost weight and shed virus in nasal washes to a similar extent as naïve controls. Challenge with heterologous A/Brisbane/59/07 (H1N1) virus revealed that the VLPs conferred minimal cross-protection to heterologous infection, as revealed by the lack of reduction in nasal wash and lung virus titers and slightly higher weight loss relative to controls. In summary, these experiments demonstrate the strong immunogenicity and protective efficacy of VLPs compared to the split vaccine and show that i.n. vaccination with VLPs has the potential for highly efficacious vaccination against influenza.

We investigated the roles of MyD88, an innate adaptor signaling molecule, in inducing protective humoral immunity after vaccination with influenza virus-like particles (VLPs). MyD88 knockout C57BL/6 mice (MyD88−/− mice) vaccinated with influenza VLPs showed significant defects in inducing IgG2a/c isotype antibodies and in generating splenic recall memory B cell responses and antibody-secreting plasma cells in the bone marrow. The protective efficacy of influenza VLP vaccination was lower in MyD88−/− mice than in the wild-type mice. Our findings indicate that MyD88-mediated innate signaling pathways are important for effectively inducing primary and boost immune responses, T helper type 1 isotype-switched antibodies, and gamma interferon (IFN-γ)-secreting T cell responses. In particular, the results in this study demonstrated for the first time that MyD88-mediated immune activation is likely an essential pathway for effective generation of long-lived antibody-secreting plasma cells and highly protective immunity after vaccination with influenza VLPs. This study provides insight into mechanisms by which recombinant viral vaccines induce protective immunity via the MyD88-mediated innate immune signaling pathway.

Influenza infection represents a major socio-economic burden worldwide. Novel delivery methods can render influenza vaccination easier and more acceptable by the public, and importantly confer protection equal or superior to that induced by conventional systemic administration. An attractive target for vaccine delivery is the skin. Recent studies have demonstrated improved immune responses after transdermal delivery of inactivated influenza virus with microneedle patches. Here we show that immunization with a licensed influenza subunit vaccine coated on metal microneedles can activate both humoral and cellular arms of the immune response and confer improved long-term protection in the mouse model when compared to the conventional systemic route of delivery. These results demonstrate the promising potential of microneedle delivery of licensed influenza subunit vaccines, that could be beneficial in increasing vaccine coverage and protection and reducing influenza-related mortality worldwide.

Ginseng polysaccharide has been known to have multiple immunomodulatory effects. In this study, we investigated whether Panax ginseng polysaccharide (GP) would have a preventive effect on influenza infection. Administration of mice with GP prior to infection was found to confer a survival benefit against infection with H1N1 (A/PR/8/34) and H3N2 (A/Philippines/82) influenza viruses. Mice infected with the 2009 H1N1 virus suspended in GP solution showed moderately enhanced survival rates and lower levels of lung viral titers and the inflammatory cytokine (IL-6). Daily treatment of vaccinated mice with GP improved their survival against heterosubtypic lethal challenge. This study demonstrates the first evidence that GP can be used as a remedy against influenza viral infection.

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.

The rapid spread of human immunodeficiency virus (HIV) worldwide makes it a high priority to develop an effective vaccine. Since live attenuated or inactivated HIV are not likely to be approved as a vaccine due to safety concerns, HIV virus like particles (VLPs) offer an attractive alternative because they are safe due to the lack of a viral genome. Although HIV VLPs have been shown to induce humoral and cellular immune responses, it is important to understand the mechanisms by which they induce such responses and to improve their immunogenicity. We generated HIV VLPs, and VLPs containing Flt3 ligand (FL), a dendritic cell growth factor, to target VLPs to dendritic cells, and investigated the roles of these VLPs in the initiation of adaptive immune responses in vitro and in vivo. We found that HIV-1 VLPs induced maturation of dendritic cells and monocyte/macrophage populations in vitro and in vivo, with enhanced expression of maturation markers and cytokines. Dendritic cells pulsed with VLPs induced activation of splenocytes resulting in increased production of cytokines. VLPs containing FL were found to increase dendritic cells and monocyte/macrophage populations in the spleen when administered to mice. Administration of VLPs induced acute activation of multiple types of cells including T and B cells as indicated by enhanced expression of the early activation marker CD69 and down-regulation of the homing receptor CD62L. VLPs containing FL were an effective form of antigen in activating immune cells via dendritic cells, and immunization with HIV VLPs containing FL resulted in enhanced T helper type 2-like immune responses.

Purpose

This study sought to determine the effects of microneedle coating formulation, drying time and storage time on antigen stability and in vivo immunogenicity of influenza microneedle vaccines.

Methods

The stability of inactivated influenza virus vaccine was monitored by hemagglutination (HA) activity and virus particle aggregation as a function of storage time and temperature with or without trehalose. In vivo immunogenicity of inactivated influenza vaccines coated onto microneedles was determined in mice by virus-specific antibody titers and survival rates.

Results

In the absence of trehalose, HA activity decreased below 10% and to almost zero after 1 h and 1 month of drying, respectively. Addition of trehalose maintained HA activity above 60% after drying and above 20% after 1 month storage at 25°C. Loss of HA activity generally correlated with increased virus particle aggregation. Administration of microneedles coated with trehalose-stabilized influenza vaccine yielded high serum IgG antibody titers even after 1 month storage, and all animals survived with minimal weight loss after lethal challenge infection.

Conclusions

Inactivated influenza virus vaccine coated on microneedles with trehalose significantly improved the HA activity as well as in vivo immunogenicity of the vaccine after an extended time of storage.

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.

To address limitations of conventional influenza vaccine manufacturing and delivery, this study investigated administration of virus-like particle (VLP) influenza vaccine using a microneedle patch. The goal was to determine if skin immunization with influenza VLP vaccine using microneedles enables dose sparing. We found that low-dose influenza (A/PR/8/34 H1N1) VLP vaccination using microneedles was more immunogenic than low-dose intramuscular (IM) vaccination and similarly immunogenic as high-dose IM vaccination in a mouse model. With 1 µg dose of vaccine, both routes showed similar immune responses and protective efficacy, with microneedle vaccination being more effective in inducing recall antibody responses in lungs and antibody secreting cells in bone marrow. With a low dose of vaccine (0.3 µg), microneedle vaccination induced significantly superior protective immunity, which included binding and functional antibodies as well as complete protection against a high dose of lethal infection with A/PR/8/34 virus, whereas IM immunization provided only partial (40%) protection. Therefore, this study demonstrates that microneedle vaccination in the skin confers more effective protective immunity at a lower dose, thus providing vaccine dose sparing effects.

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.

The emergence of the swine-origin 2009 influenza pandemic illustrates the need for improved vaccine production and delivery strategies. Skin-based immunization represents an attractive alternative to traditional hypodermic needle vaccination routes. Microneedles (MNs) can deliver vaccine to the epidermis and dermis, which are rich in antigen-presenting cells (APC) such as Langerhans cells and dermal dendritic cells. Previous studies using coated or dissolvable microneedles emphasized the use of inactivated influenza virus or virus-like particles as skin-based vaccines. However, most currently available influenza vaccines consist of solubilized viral protein antigens. Here we test the hypothesis that a recombinant subunit influenza vaccine can be delivered to the skin by coated microneedles and can induce protective immunity. We found that mice vaccinated via MN delivery with a stabilized recombinant trimeric soluble hemagglutinin (sHA) derived from A/Aichi/2/68 (H3) virus had significantly higher immune responses than did mice vaccinated with unmodified sHA. These mice were fully protected against a lethal challenge with influenza virus. Analysis of postchallenge lung titers showed that MN-immunized mice had completely cleared the virus from their lungs, in contrast to mice given the same vaccine by a standard subcutaneous route. In addition, we observed a higher ratio of antigen-specific Th1 cells in trimeric sHA-vaccinated mice and a greater mucosal antibody response. Our data therefore demonstrate the improved efficacy of a skin-based recombinant subunit influenza vaccine and emphasize the advantage of this route of vaccination for a protein subunit vaccine.

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.

Mortality due to seasonal and pandemic influenza could be reduced by increasing the speed of influenza vaccine production and distribution. We propose that vaccination can be expedited by (1) immunizing with influenza virus-like particle (VLP) vaccines, which are simpler and faster to manufacture than conventional egg-based inactivated virus vaccines, and (2) administering vaccines using microneedle patches, which should simplify vaccine distribution due to their small package size and possible self-administration. In this study, we coated microneedle patches with influenza VLP vaccine, which was released into skin by dissolution within minutes. Optimizing the coating formulation required balancing factors affecting the coating dose and vaccine antigen stability. Vaccine stability, as measured by an in vitro hemagglutination assay, was increased by formulation with increased concentration of trehalose or other stabilizing carbohydrate compounds and decreased concentration of carboxymethylcellulose (CMC) or other viscosity-enhancing compounds. Coating dose was increased by formulation with increased VLP concentration, increased CMC concentration, and decreased trehalose concentration, as well as increased number of dip coating cycles. Finally, vaccination of mice using microneedles stabilized by trehalose generated strong antibody responses and provided full protection against high-dose lethal challenge infection. In summary, this study provides detailed analysis to guide formulation of microneedle patches coated with influenza VLP vaccine and demonstrates effective vaccination in vivo using this system.

Hepatic natural killer (NK) cells mediate antigen (Ag)-specific contact hypersensitivity (CHS) in T-cell and B-cell deficient mice. We now report that hepatic, but not splenic or naïve NK cells also develop specific memory to vaccines containing Ags from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus-1 (HIV). Adoptive transfer of virus-sensitized NK cells to naïve recipients enhanced the animals' survival upon lethal challenge with the sensitizing virus, but not a different virus. NK cell memory to haptens and viruses depended upon CXCR6, a chemokine receptor on hepatic NK cells that was required for memory NK cell persistence but not for Ag recognition. Hence, hepatic NK-cells can develop adaptive immunity to structurally diverse Ags, an activity that requires NK-cell-expressed CXCR6.