Vaccination Method Affects Immune Response and Bacterial Growth but Not Protection in the Salmonella Typhimurium Animal Model of Typhoid

Understanding immune responses elicited by vaccines, together with immune responses required for protection, is fundamental to designing effective vaccines and immunisation programs. This study examines the effects of the route of administration of a live attenuated vaccine on its interactions with, and stimulation of, the murine immune system as well as its ability to increase survival and provide protection from colonisation by a virulent challenge strain. We assess the effect of administration method using the murine model for typhoid, where animals are infected with S. Typhimurium. Mice were vaccinated either intravenously or orally with the same live attenuated S. Typhimurium strain and data were collected on vaccine strain growth, shedding and stimulation of antibodies and cytokines. Following vaccination, mice were challenged with a virulent strain of S. Typhimurium and the protection conferred by the different vaccination routes was measured in terms of challenge suppression and animal survival. The main difference in immune stimulation found in this study was the development of a secretory IgA response in orally-vaccinated mice, which was absent in IV vaccinated mice. While both strains showed similar protection in terms of challenge suppression in systemic organs (spleen and liver) as well as survival, they differed in terms of challenge suppression of virulent pathogens in gut-associated organs. This difference in gut colonisation presents important questions around the ability of vaccines to prevent shedding and transmission. These findings demonstrate that while protection conferred by two vaccines can appear to be the same, the mechanisms controlling the protection can differ and have important implications for infection dynamics within a population.     

Microneedle Vaccination Elicits Superior Protection and Antibody Response over Intranasal Vaccination against Swine-Origin Influenza A (H1N1) in Mice

Influenza is one of the critical infectious diseases globally and vaccination has been considered as the best way to prevent. In this study, immunogenicity and protection efficacy between intranasal (IN) and microneedle (MN) vaccination was compared using inactivated swine-origin influenza A/H1N1 virus vaccine. Mice were vaccinated by MN or IN administration with 1 μg of inactivated H1N1 virus vaccine. Antigen-specific antibody responses and hemagglutination-inhibition (HI) titers were measured in all immunized sera after immunization. Five weeks after an immunization, a lethal challenge was performed to evaluate the protective efficacy. Furthermore, mice were vaccinated by IN administration with higher dosages (> 1 μg), analyzed in the same manner, and compared with 1 μg-vaccine-coated MN. Significantly higher antigen-specific antibody responses and HI titer were measured in sera in MN group than those in IN group. While 100% protection, slight weight loss, and reduced viral replication were observed in MN group, 0% survival rate were observed in IN group. As vaccine dose for IN vaccination increased, MN-immunized sera showed much higher antigen-specific antibody responses and HI titer than other IN groups. In addition, protective immunity of 1 μg-MN group was similar to those of 20- and 40 μg-IN groups. We conclude that MN vaccination showed more potential immune response and protection than IN vaccination at the same vaccine dosage.     

Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4⁺IL-17A⁺TNFα⁺). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development     

Approaches towards DNA Vaccination against a Skin Ciliate Parasite in Fish

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.     

Specific in vitro lymphocyte transformation with Venezuelan equine encephalitis virus.

Specific lymphocyte transformation to viral antigen was detected in individuals vaccinated with live, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain TC-83. Suspensions of purified, inactivated virus were used an antigen in 250-μ1 lymphocyte cultures, and under optimal conditions the assay demonstrated 10-fold greater incorporation of ¹⁴C-thymidine by lymphocytes from immune than from nonimmune people. The rise in lymphocyte transformation response occurred 1 week after vaccination. The magnitude and range of the lymphocyte transformation response to the VEE viral antigen were similar to the responses seen using antigens derived from five other microbial sources: Francisella tularensis, Coccidioides immitis, Mycobacterium tuberculosis, Streptococcus, and parainfluenza virus. Autologous plasma containing antibody exerts an inhibitory effect on cultures from immune individuals. The onset, magnitude of response, and specificity of this in vitro assay are correlated with the clinical and pathological events of VEE virus infection.     

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.     

Oral Vaccination with Lipid-Formulated BCG Induces a Long-lived,Multifunctional CD4+ T Cell Memory Immune Response

Oral delivery of BCG in a lipid formulation (Liporale™-BCG) targets delivery of viable bacilli to the mesenteric lymph nodes and confers protection against an aerosol Mycobacterium tuberculosis challenge. The magnitude, quality and duration of the effector and memory immune response induced by Liporale™-BCG vaccination is unknown. Therefore, we compared the effector and memory CD4⁺ T cell response in the spleen and lungs of mice vaccinated with Liporale™-BCG to the response induced by subcutaneous BCG vaccination. Liporale™-BCG vaccination induced a long-lived CD4⁺ T cell response, evident by the detection of effector CD4⁺ T cells in the lungs and a significant increase in the number of Ag85B tetramer-specific CD4⁺ T cells in the spleen up to 30 weeks post vaccination. Moreover, following polyclonal stimulation, Liporale™-BCG vaccination, but not s.c. BCG vaccination, induced a significant increase in both the percentage of CD4⁺ T cells in the lungs capable of producing IFNγ and the number of multifunctional CD4⁺ T cells in the lungs at 30 weeks post vaccination. These results demonstrate that orally delivered Liporale™-BCG vaccine induces a long-lived multifunctional immune response, and could therefore represent a practical and effective means of delivering novel BCG-based TB vaccines.     

Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit

Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.     

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.     

Removing the Age Restrictions for Rotavirus Vaccination: A Benefit-Risk Modeling Analysis

Background

To minimize potential risk of intussusception, the World Health Organization (WHO) recommended in 2009 that rotavirus immunization should be initiated by age 15 weeks and completed before 32 weeks. These restrictions could adversely impact vaccination coverage and thereby its health impact, particularly in developing countries where delays in vaccination often occur.

Methods and Findings

We conducted a modeling study to estimate the number of rotavirus deaths prevented and the number of intussusception deaths caused by vaccination when administered on the restricted schedule versus an unrestricted schedule whereby rotavirus vaccine would be administered with DTP vaccine up to age 3 years. Countries were grouped on the basis of child mortality rates, using WHO data. Inputs were estimates of WHO rotavirus mortality by week of age from a recent study, intussusception mortality based on a literature review, predicted vaccination rates by week of age from USAID Demographic and Health Surveys, the United Nations Children''s Fund (UNICEF) Multiple Indicator Cluster Surveys (MICS), and WHO-UNICEF 2010 country-specific coverage estimates, and published estimates of vaccine efficacy and vaccine-associated intussusception risk. On the basis of the error estimates and distributions for model inputs, we conducted 2,000 simulations to obtain median estimates of deaths averted and caused as well as the uncertainty ranges, defined as the 5th–95th percentile, to provide an indication of the uncertainty in the estimates.We estimated that in low and low-middle income countries a restricted schedule would prevent 155,800 rotavirus deaths (5th–95th centiles, 83,300–217,700) while causing potentially 253 intussusception deaths (76–689). In contrast, vaccination without age restrictions would prevent 203,000 rotavirus deaths (102,000–281,500) while potentially causing 547 intussusception deaths (237–1,160). Thus, removing the age restrictions would avert an additional 47,200 rotavirus deaths (18,700–63,700) and cause an additional 294 (161–471) intussusception deaths, for an incremental benefit-risk ratio of 154 deaths averted for every death caused by vaccine. These extra deaths prevented under an unrestricted schedule reflect vaccination of an additional 21%–25% children, beyond the 63%–73% of the children who would be vaccinated under the restricted schedule. Importantly, these estimates err on the side of safety in that they assume high vaccine-associated risk of intussusception and do not account for potential herd immunity or non-fatal outcomes.

Conclusions

Our analysis suggests that in low- and middle-income countries the additional lives saved by removing age restrictions for rotavirus vaccination would far outnumber the potential excess vaccine-associated intussusception deaths. Please see later in the article for the Editors'' Summary     

Vaccination of School Children With Live Mumps Virus Vaccine

Live, attenuated mumps virus vaccine (Mumpsvax) was administered to 146 school children 6 to 9 years of age. One child developed clinical mumps nine days after vaccination; epidemiological and serological data strongly suggest that this child had become infected before vaccination. Apart from this single instance there were no apparent clinical reactions that could be ascribed to the administration of the vaccine. Sixty-three of the 146 children with no clinical history of mumps had an initial serum neutralizing antibody titre of less than 1:2. Specific antibodies to mumps virus were detected in 93.5% of the sera of the susceptible children 28 days after vaccination, and the geometric mean antibody titre of these sera was low (1:6). Of the 80 initially seropositive children 21 (26.2%) showed a significant antibody response to the vaccine and this was influenced by the pre-existing antibody level. These data have further demonstrated the safety and efficacy of the live mumps vaccine in children.     

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

Background

Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-?? (chIFN-??) and chicken interleukin-18 (chIL-18) as natural immunomodulators.

Results

Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-?? and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-?? or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-?? and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens.

Conclusions

Our results indicate the value of combined administration of chIFN-?? and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.     

DNA Vaccination Affords Significant Protection against Feline Immunodeficiency Virus Infection without Inducing Detectable Antiviral Antibodies

To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVΔRT). In a first experiment, FIVΔRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-γ) DNA. The DNA was administered in four 100-μg doses at 0, 10, and 23 weeks. Immunization with FIVΔRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVΔRT vaccinates than in the controls. Immunization with FIVΔRT in conjunction with IFN-γ gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVΔRT plus IFN-γ and IFN-γ alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines.     

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte?Cmacrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40?mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4?mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2?days after paclitaxel; and (c) administration of 4?mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8⁺ T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.     

Phase I trial of a recombinant yeast-CEA vaccine (GI-6207) in adults with metastatic CEA-expressing carcinoma

Yeast-CEA (GI-6207) is a therapeutic cancer vaccine genetically modified to express recombinant carcinoembryonic antigen (CEA) protein, using heat-killed yeast (Saccharomyces cerevisiae) as a vector. In preclinical studies, yeast-CEA induced a strong immune response to CEA and antitumor responses. Patients received subcutaneous vaccines every 2 weeks for 3 months and then monthly. Patients were enrolled at 3 sequential dose levels: 4, 16, and 40 yeast units (10⁷ yeast particles/unit). Eligible patients were required to have serum CEA > 5 ng/mL or > 20 % CEA⁺ tumor block, ECOG PS 0–2, and no history of autoimmunity. Restaging scans were performed at 3 months and then bimonthly. Peripheral blood was collected for the analysis of immune response (e.g., by ELISPOT assay). Twenty-five patients with metastatic CEA-expressing carcinomas were enrolled. Median patient age was 52 (range 39–81). A total of 135 vaccines were administered. The vaccine was well tolerated, and the most common adverse event was grade 1/2 injection-site reaction. Five patients had stable disease beyond 3 months (range 3.5–18 months), and each had CEA stabilization while on-study. Some patients showed evidence post-vaccination of increases in antigen-specific CD8⁺ T cells and CD4⁺ T lymphocytes and decreases in regulatory T cells. Of note, a patient with medullary thyroid cancer had substantial T cell responses and a vigorous inflammatory reaction at sites of metastatic disease. Yeast-CEA vaccination had minimal toxicity and induced some antigen-specific T cell responses and CEA stabilization in a heterogeneous, heavily pre-treated patient population. Further studies are required to determine the clinical benefit of yeast-CEA vaccination.     

Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.     

Endocine?, N3OA and N3OASq; Three Mucosal Adjuvants That Enhance the Immune Response to Nasal Influenza Vaccination

Annual outbreaks of seasonal influenza are controlled or prevented through vaccination in many countries. The seasonal vaccines used are either inactivated, currently administered parenterally, or live-attenuated given intranasally. In this study three mucosal adjuvants were examined for the influence on the humoral (mucosal and systemic) and cellular influenza A-specific immune responses induced by a nasally administered vaccine. We investigated in detail how the anionic Endocine™ and the cationic adjuvants N3OA and N3OASq mixed with a split inactivated influenza vaccine induced influenza A-specific immune responses as compared to the vaccine alone after intranasal immunization. The study showed that nasal administration of a split virus vaccine together with Endocine™ or N3OA induced significantly higher humoral and cell-mediated immune responses than the non-adjuvanted vaccine. N3OASq only significantly increased the cell-mediated immune response. Furthermore, nasal administration of the influenza vaccine in combination with any of the adjuvants; Endocine™, N3OA or N3OASq, significantly enhanced the mucosal immunity against influenza HA protein. Thus the addition of these mucosal adjuvants leads to enhanced immunity in the most relevant tissues, the upper respiratory tract and the systemic circulation. Nasal influenza vaccination with an inactivated split vaccine can therefore provide an important mucosal immune response, which is often low or absent after traditional parenteral vaccination.