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Increased susceptibility to influenza virus infection during pregnancy has been attributed to immunological changes occurring before and during gestation in order to "tolerate" the developing fetus. These systemic changes are most often characterized by a suppression of cell-mediated immunity and elevation of humoral immune responses referred to as the Th1-Th2 shift. However, the underlying mechanisms which increase pregnant mothers' risk following influenza virus infection have not been fully elucidated. We used pregnant BALB/c mice during mid- to late gestation to determine the impact of a sub-lethal infection with A/Brisbane/59/07 H1N1 seasonal influenza virus on completion of gestation. Maternal and fetal health status was closely monitored and compared to infected non-pregnant mice. Severity of infection during pregnancy was correlated with premature rupture of amniotic membranes (PROM), fetal survival and body weight at birth, lung viral load and degree of systemic and tissue inflammation mediated by innate and adaptive immune responses. Here we report that influenza virus infection resulted in dysregulation of inflammatory responses that led to pre-term labor, impairment of fetal growth, increased fetal mortality and maternal morbidity. We observed significant compartment-specific immune responses correlated with changes in hormonal synthesis and regulation. Dysregulation of progesterone, COX-2, PGE2 and PGF2α expression in infected pregnant mice was accompanied by significant remodeling of placental architecture and upregulation of MMP-9 early after infection. Collectively these findings demonstrate the potential of a seasonal influenza virus to initiate a powerful pro-abortive mechanism with adverse outcomes in fetal health.

Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 - 6×10-4 cm s-1) and high Arrhenius activation energy (Ea = 15.0 kcal mol-1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.

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.

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.

The swine-origin H1N1 virus which emerged in 2009 resulted in the first influenza pandemic of the 21(st) 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 CD11b(hi), F4/80(lo). 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 CD11b(lo/-), F4/80(hi). These two genetically similar viruses can be useful strains with which to investigate immune-regulatory determinants of pathogenesis of influenza virus.

In addition to its surface glycoprotein (GP(1,2)), 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 GP(1,2) and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP(12) 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 GP(1,2), thereby allowing it to absorb anti-GP(1,2) antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP(1,2) 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.

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.

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 µg of monovalent H1N1 subunit vaccine alone or with 1 µg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.

Background: Many pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies., Methodology/principal Findings: Here we report a novel recombinant, attenuated Sendai virus vector (GP42-H1) in which the hemagglutinin (HA) gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu) of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, bronchoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD(50) (1250 pfu) of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected., Conclusions: These results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.

Background: Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health., Methodology/principal Findings: To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms., Conclusions/significance: These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.

We demonstrated previously that the incorporation of a membrane-anchored form of flagellin into influenza virus-like particles (VLPs) improved the immunogenicity of VLPs significantly, inducing partially protective heterosubtypic immunity by intramuscular immunization. Because the efficacy of mucosal vaccination is highly dependent on an adjuvant, and is particularly effective for preventing mucosal infections such as influenza, we determined whether the membrane-anchored flagellin is an efficient adjuvant for VLP vaccines by a mucosal immunization route. We compared the adjuvant effect of membrane-anchored and soluble flagellins for immunization with influenza A/PR8 (H1N1) VLPs by the intranasal route in a mouse model. The results demonstrate that membrane-anchored flagellin is an effective adjuvant for intranasal (IN) immunization, inducing enhanced systemic and mucosal antibody responses. High cellular responses were also observed as shown by cytokine production in splenocyte cultures when stimulated with viral antigens. All mice immunized with flagellin-containing VLPs survived challenge with a high lethal dose of homologous virus as well as a high dose heterosubtypic virus challenge (40 LD(50) of A/Philippines/82, H3N2). In contrast, no protection was observed with a standard HA/M1 VLP group upon heterosubtypic challenge. Soluble flagellin exhibited a moderate adjuvant effect when co-administered with VLPs by the mucosal route, as indicated by enhanced systemic and mucosal responses and partial heterosubtypic protection. The membrane-anchored form of flagellin incorporated together with antigen into influenza VLPs is effective as an adjuvant by the mucosal route and unlike standard VLPs, immunization with such chimeric VLPs elicits protective immunity to challenge with a distantly related influenza A virus.

Background: The recent swine-origin H1N1 pandemic illustrates the need to develop improved procedures for rapid production of influenza vaccines. One alternative to the current egg-based manufacture of influenza vaccine is to produce a hemagglutinin (HA) subunit vaccine using a recombinant expression system with the potential for high protein yields, ease of cloning new antigenic variants, and an established safety record in humans., Methodology/principal Findings: We generated a soluble HA (sHA), derived from the H3N2 virus A/Aichi/2/68, modified at the C-terminus with a GCN4pII trimerization repeat to stabilize the native trimeric structure of HA. When expressed in the baculovirus system, the modified sHA formed native trimers. In contrast, the unmodified sHA was found to present epitopes recognized by a low-pH conformation specific monoclonal antibody. We found that mice primed and boosted with 3 microg of trimeric sHA in the absence of adjuvants had significantly higher IgG and HAI titers than mice that received the unmodified sHA. This correlated with an increased survival and reduced body weight loss following lethal challenge with mouse-adapted A/Aichi/2/68 virus. In addition, mice receiving a single vaccination of the trimeric sHA in the absence of adjuvants had improved survival and body weight loss compared to mice vaccinated with the unmodified sHA., Conclusions/significance: Our data indicate that the recombinant trimeric sHA presents native trimeric epitopes while the unmodified sHA presents epitopes not exposed in the native HA molecule. The epitopes presented in the unmodified sHA constitute a "silent face" which may skew the antibody response to epitopes not accessible in live virus at neutral pH. The results demonstrate that the trimeric sHA is a more effective influenza vaccine candidate and emphasize the importance of structure-based antigen design in improving recombinant HA vaccines.

There is a significant gap in our fundamental understanding of early morphological and migratory changes in human Langerhans cells (LCs) in response to vaccine stimulation. As the vast majority of LCs studies are conducted in small animal models, substantial interspecies variation in skin architecture and immunity must be considered when extrapolating the results to humans. This study aims to determine whether excised human skin, maintained viable in organ culture, provides a useful human model for measuring and understanding early immune response to intradermally delivered vaccine candidates. Excised human breast skin was maintained viable in air-liquid-interface organ culture. This model was used for the first time to show morphological changes in human LCs stimulated with influenza virus-like particle (VLP) vaccines delivered via intradermal injection. Immunohistochemistry of epidermal sheets and skin sections showed that LCs in VLP treated skin lost their typical dendritic morphology. The cells were more dispersed throughout the epidermis, often in close proximity to the basement membrane, and appeared vertically elongated. Our data provides for increased understanding of the complex morphological, spatial and temporal changes that occur to permit LC migration through the densely packed keratinocytes of the epidermis following exposure to vaccine. Significantly, the data not only supports previous animal data but also provides new and essential evidence of host response to this vaccination strategy in the real human skin environment.

Background: The respiratory illnesses caused by influenza virus can be dramatically reduced by vaccination. The current trivalent inactivated influenza vaccine is effective in eliciting systemic virus-specific antibodies sufficient to control viral replication. However, influenza protection generated after parenteral immunization could be improved by the induction of mucosal immune responses., Methodology/principal Findings: Transcutaneous immunization, a non-invasive vaccine delivery method, was used to investigate the quality, duration and effectiveness of the immune responses induced in the presence of inactivated influenza virus co-administered with retinoic acid or oleic acid. We observed an increased migration of dendritic cells to the draining lymph nodes after dermal vaccination. Here we demonstrate that this route of vaccine delivery in combination with certain immunomodulators can induce potent immune responses that result in long-term protective immunity. Additionally, mice vaccinated with inactivated virus in combination with retinoic acid show an enhanced sIgA antibody response, increased number of antibody secreting cells in the mucosal tissues, and protection from a higher influenza lethal dose., Conclusions/significance: The present study demonstrates that transdermal administration of inactivated virus in combination with immunomodulators stimulates dendritic cell migration, results in long-lived systemic and mucosal responses that confer effective protective immunity.

Background: The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines., Methodology/principal Findings: We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection., Conclusion/significance: This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.

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.

Background: Influenza is a contagious disease caused by a pathogenic virus, with outbreaks all over the world and thousands of hospitalizations and deaths every year. Due to virus antigenic drift and short-lived immune responses, annual vaccination is required. However, vaccine coverage is incomplete, and improvement in immunization is needed. The objective of this study is to investigate a novel method for transdermal delivery using metal microneedle arrays (MN) coated with inactivated influenza virus to determine whether this route is a simpler and safer approach than the conventional immunization, capable to induce robust immune responses and confer protection against lethal virus challenge., Methodology/principal Findings: Inactivated A/Aichi/2/68 (H3N2) influenza virus was coated on metal microneedle arrays and applied to mice as a vaccine in the caudal dorsal skin area. Substantial antibody titers with hemagglutination inhibition activity were detected in sera collected two and four weeks after a single vaccine dose. Challenge studies in mice with 5 x LD(50) of mouse adapted Aichi virus demonstrated complete protection. Microneedle vaccination induced a broad spectrum of immune responses including CD4+ and CD8+ responses in the spleen and draining lymph node, a high frequency of antigen-secreting cells in the lung and induction of virus-specific memory B-cells. In addition, the use of MN showed a dose-sparing effect and a strong Th2 bias when compared to an intramuscular (IM) reference immunization., Conclusions/significance: The present results show that delivery of inactivated influenza virus through the skin using metal microneedle arrays induced strong humoral and cellular immune responses capable of conferring protection against virus challenge as efficiently as intramuscular immunization, which is the standard vaccination route. In view of the convenience of delivery and the potential for self-administration, vaccine-coated metal microneedles may provide a novel and highly effective immunization method.

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.