Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides

Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins' which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311-320) or hepatitis C virus (HCV) NS5 (P17, residues 2423-2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs.     

Recombinant pseudorabies virus carrying a plasmodium gene: herpesvirus as a new live viral vector for inducing T- and B-cell immunity.

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ T lymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10(7) plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281-296) from the P. yoelii CS protein designated PYCTL1. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8+ T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.     

Development of Th1-mediated CD8+ effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes

There have been many studies for tumor therapy mediated by cytotoxic T lymphocytes (CTL) that recognize tumor-associated antigen. It is generally accepted that CTL responses are induced when antigen is delivered into the cytosol. The pH-sensitive liposomes as vehicles are well known for their capacity to deliver the antigen into the cytosol. In this work, immunization of mice with CTL epitope peptides from Hantaan nucleocapsid protein (M6) or human papilloma virus E7 encapsulated in pH-sensitive liposomes induced effective antigen-specific CTL responses. The CTL responses induced by M6 peptide encapsulated in pH-sensitive liposomes blocked the formation of tumor mass from Hantaan NP transfected B16 melanoma cells in C57BL/6 mice and delayed the growth of preinoculated melanoma cells. During the blockade of the tumor growth, the CTL response was maintained for at least approximately 6 weeks, and the mice secreted Th1 type cytokines such as IL-2 and IFN-gamma. These results suggested that the pH-sensitive liposomes might provide an effective peptide delivery system for CTL-mediated tumor therapy.     

Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization

Hepatitis C Virus (HCV) causes most cases of posttransfusion hepatitis. Chronic HCV infection is highly related to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Current therapies are only minimally effective and no vaccine has been developed. DNA-based immunization could be of prophylactic and therapeutic value for HCV infection. By intramuscular inoculation in BALB/c mice with an HCV recombinant plasmid pCI-HCV-C, we found significant levels of IgM antibody, but no significant IgG rise. After boost the immunized mice with recombinant HCV-core protein (cp1-10; 1-164aa), the anticore IgG, verified by Western-blotting, rose rapidly, which was two weeks earlier than that with control plasmid. Spleen cells from pCI-HCV-C immunized mice gave higher proliferation index (PI) than control (P < 0.05). The PI of cp1-10 boosted mice was even higher. Proliferation blocking assay with mAb proved the responding cell to be of CD4+ CD8- phenotype, supporting specific priming of T helper cells. A 51Cr-releasing CTL assay specific for HCV-core was developed, and a specific CTL response against HCV-core was demonstrated in both pCI-HCV-C immunized mice and mice boosted with cp1-10. Strong cytotoxic activity against peptide-pulsed p815 cells (H-2d), but not EL-4 cells (H-2b), suggested MHC class I restriction of the CTL activity. Blocking of CTL with mAb proved the effector cells to be of CD4- CD8+. Three CTL epitopes in HCV-core protein were demonstrated. We failed to detect CTL when immunized only with core protein. The results suggested that vaccination with HCV-core derived DNA sequences could be an effective method to induce humoral and cellular immune responses to HCV.     

Effects of diet composition on liver antioxidant defense and detoxification enzymes in mice with murine AIDS

Liver antioxidant defense and detoxification enzymes in murine AIDS-infected mice fed cereal based-diet or purified diet were studied with 32 C57BL/6 female mice in a 2×2×2 (diet × virus × period) treatment design. The mice were divided into two groups and fed Purina mice chow or a liquid diet. One week later, half of the mice in each diet group were injected intraperitoneally with LPBM5 murine retrovirus (MAIDS) stock. Two and 4 weeks after infection, half of the mice in each of the 4 treatment groups were killed, and the livers were excised for biochemical analysis. The results showed that MAIDS virus infection significantly decreased activities of glutathione peroxidase (GP) at 2 weeks postinfection in the liquid diet group, superoxide dismutase (SOD) at both periods in the liquid diet group, and glutathione-S-transferase (GST) toward 1,2-dichloro-4-nitrobenzene (DCNB) at both periods in both diet groups when compared to the control groups. MAIDS virus infection did not affect reduced glutathione (GSH) levels, or activities of catalase and glutathione reductase (GR). GSH levels and activities of catalase and GSTs were significantly lower in the mice fed the liquid diet than in those fed mice chow. Virus-mediated decline in antioxidant defense and detoxification capability of MAIDS infected mice may contribute to further development of the disease. The results suggest that chow diet provides a higher antioxidant defense capability than the liquid diet.     

Self HSP60 peptide serves as an immunogenic carrier for a CTL epitope against persistence of murine cytomegalovirus in the salivary gland

Murine cytomegalovirus (MCMV) infection is associated with persistence of virus in the salivary glands, despite relatively rapid clearance of virus from the spleen. An effective immunization against MCMV should prevent such viral persistence. We previously reported that a peptide (p458) from the sequence of the 60 kDa heat shock protein (HSP60) molecule in a conjugate vaccine can provide T cell help for the induction of protecting antibody against bacterial capsular polysaccharides. We now report that the p458 peptide as a carrier peptide can also enhance the immunogenicity of a dominant CTL epitope of the MCMV pp89 antigen-89pep. We synthesized a linear combined peptide: chimeric p458-89pep. We immunized young BALB/c mice and challenged them with MCMV. We found that the p458-89pep chimeric peptide was more effective than the 89pep in inducing 89pep-specific IFN(gamma) secretion and specific CTL activity. Moreover, the p458-89pep chimeric peptide induced sustained IFN(gamma) secretion in the salivary gland specific to 89pep and only this immunization was associated with clearance of virus from the salivary gland. These results suggest that a peptide epitope of HSP60 may be advantageous as a T cell carrier peptide in the induction of specific T cell immunity against infectious agents.     

Role of T cell competition in the induction of cytotoxic T lymphocyte activity during viral vector-based immunization regimens

T cell competition between antigen- and vector-specific T cells may determine the outcome of viral vector-based immunization regimens, as we previously proposed. Here, we unravelled the interplay between antigen- and vector-specific immunity, using recombinant Semliki Forest virus (rSFV). Priming of mice with rSFV, encoding LacZ (SFVLacZ), or with empty rSFV strongly suppressed subsequent induction of ovalbumin or Human Papilloma virus (HPV) E6/E7-specific CTL activity by a booster with SFVeOVA or SFVeE6,7, respectively. Yet, priming with irradiated, i.e. replication-defective, SFVLacZ did not affect subsequent CTL induction, indicating that the interfering vector-specific immunity was directed against the viral replicase. However, immune responses against the strongly immunogenic nucleoprotein of influenza virus encoded by SFV were less severely affected by priming with SFVLacZ. Thus the outcome of heterologous prime-boost immunizations appears to depend on the immunogenicity of the respective antigens.     

IL-6 induces long-term protective immunity against a lethal challenge of influenza virus

The coadministration of cytokines can modulate immunity in DNA based viral vaccines. In order to determine the effects of various cytokines on long-term protection against the influenza virus, mice were intramuscularly coinoculated with plasmids that encoded either the granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin-4 (IL-4), interleukin-12 (IL-12), or the interleukin-6 (IL-6) gene, in the presence of two plasmids that encoded the nucleoprotein (NP) and the hemagglutinin (HA) gene of the influenza A virus. The coadministration of IL-4, IL-6 and IL-12 transiently enhanced antibody responses against influenza virus in early time points (4 to 7 week post immunization) after post inoculation. The expression of GMCSF gene resulted in the sustained elevation of antibody responses for at least 20 weeks post inoculation. However, NP-specific CTL responses decreased in these animals. Mice that received either the IL-12 or the IL-6 gene had enhanced NP-specific CTL responses. Remarkably, the coadministration of the IL-6 gene completely protected mice from a lethal challenge with influenza virus. Conversely, mice that received the IL-4 gene appeared to be more susceptible to lethal challenge than mice that were inoculated with the NP and the HA genes alone. These results demonstrate that the use of cytokines as molecular adjuvants when coadministered in influenza DNA vaccination must be specific. Our data also demonstrates that the coadministration of IL-6 should be considered to enhance the efficacy of influenza DNA vaccines.     

Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.     

The effect of pre-existing immunity on the capacity of influenza virosomes to induce cytotoxic T lymphocyte activity

Protein antigens encapsulated in virosomes generated from influenza virus can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. In the present study we determined, in a murine model system, whether pre-existing immunity against influenza virus hampers the induction of a CTL response. CTL induction was only slightly reduced by pre-injection of influenza virus-specific antibodies or pre-exposure to influenza virus. Both pretreatments resulted in the same level of reduction, suggesting that virus-specific antibodies rather than T cell responses account for the reduction. Furthermore, a booster immunization enhanced CTL activation, indicating that virosome-specific immunity induced by a prime immunization does not hamper the booster effect. In conclusion, CTL induction against virosome-encapsulated protein antigens is not significantly inhibited by pre-existing humoral or cellular immunity against influenza virus.     

Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A

An eight amino acid sequence (TELRTFSI) present in the carboxy terminal end (aa 577-584) of membrane-anchored GP, the major structural protein of Ebola virus, was identified as an H-2k-specific murine cytotoxic T cell epitope. Cytotoxic T lymphocytes (CTLs) to this epitope were induced by immunizing B10.BR mice intravenously with either irradiated Ebola virus or with irradiated Ebola virus encapsulated in liposomes containing lipid A. The CTL response induced by irradiated Ebola virus could not be sustained after the second round of in vitro stimulation of immune splenocytes with the peptide, unless the irradiated virus was encapsulated in liposomes containing lipid A. The identification of an Ebola GP-specific CTL epitope and the requirement of liposomal lipid A for CTL memory recall responses could prove to be a promising approach for developing a vaccine against Ebola virus infection.     

Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice

Mucosal immunity is critical for protection from viral infections. We attempted to activate mucosal cytotoxic T lymphocytes (CTLs) specific for influenza A virus nucleoprotein (NP) which play an important role in protective immunity. It has been shown that dendritic cells (DCs) activated by signaling via CD40-CD40 ligand (CD40L) interaction are required for the differentiation of naive CD8(+) T cells into antigen-specific CTLs in a non-mucosal environment. We herein inoculated mice intranasally with an anti-CD40 monoclonal antibody (anti-CD40 mAb) and NP366-374 peptide, corresponding to a CTL epitope on NP, encapsulated in liposome (liposomal NP366-374) to induce protective CTL responses against influenza A virus. Intranasal but not subcutaneous immunization with liposomal NP366-374 effectively induced mucosal immunity to reduce virus replication in the lung, suggesting that anti-CD40 mAb also functioned as a mucosal adjuvant. Interestingly, neither MHC class I- nor class II-deficient mice immunized intranasally with these materials were resistant to the infection. Since anti-CD40 mAb was considered to help replace CD4(+) T cells, another help of CD4(+) T cells are presumably required for the induction of CTL activity in the lung. This approach may prove promising for developing vaccines to induce mucosal CTL responses, and seems to highlight differences between mucosal and non-mucosal immunity.     

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes

DNA vaccination is highly efficient at inducing CD8⁺ T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8⁺ T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D^b-restricted CD8⁺ CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.     

Long- and short-time immunological memory in different strains of mice given nasally an adjuvant-combined nasal influenza vaccine

Immunological memory induced by nasal immunization with adjuvant-combined influenza vaccine was analyzed in different ages and strains of mice. The memory activities were assessed by secondary nasal-wash IgA and serum IgG antibody (Ab) responses and protection against challenge infection with a lethal dose of influenza virus. Mice were primed with 0.1 microg of vaccine and boosted with 0.1 or 1.0 microg vaccine 1 (short-term memory)- or 17 (long-term memory)-months later. Influenza-specific short-term memory responses in young adult BALB/c mice (2-month-old) were significantly higher than those of long-term memory activities in mice boosted at 19 months of age. However, those influenza-specific long-term memory responses provided protective immunity against influenza virus challenge and were higher than short-term memory in aged mice primed at 18-month-old and boosted 1 month later. These results show that the age at which initial nasal immunization is given is critically important in order to induce protective immunity in aged mice. Similar findings were noted in the C3H mouse strain; however, C57BL/6 mice failed to induce influenza-specific immune responses in both young adult and aged mice. These results indicate that low doses of cholera toxin B subunit (supplemented with 0.2% of hole toxin) combined nasal vaccine may required further improvement in order to provide protective immunity in human use.     

Role of an immunodominant T cell epitope of the P6 protein of nontypeable Haemophilus influenzae in murine protective immunity

Nontypeable Haemophilus influenzae (NTHI) is a common cause of otitis media in children and lower respiratory tract infection in adults with chronic lung disease. The highly conserved P6 protein of NTHI infection is under evaluation as a vaccine antigen in several animal models. To elucidate the role of cellular immune response to P6 in protective immunity, the goal of this study was to identify and characterize T cell epitope(s) on P6 and to investigate the role of these epitope(s) in eliciting antigen specific antibody responses and in mediating pulmonary clearance of NTHI. We report that T cells from BALB/c immunized with P6 recognize a single, immunodominant region, represented by 15 amino acids (residues 41-55) of the P6 protein. To verify the ability of this epitope to elicit T cell responses to the P6 protein, mice were immunized with a synthetic peptide corresponding to the sequence of dominant peptide. T cells isolated from mice primed in vivo with the peptide responded following in vitro stimulation with either the peptide or with the whole P6 molecule. Substitution of single amino acids and N or C terminal truncations of the dominant peptide resulted in complete abrogation of the response, implicating their importance to the T cell response. Furthermore, mucosal immunization of mice with a chimeric peptide that encompassed the dominant T cell epitope and a putative B cell epitope resulted in enhanced bacterial clearance following pulmonary challenge with NTHI. Collectively, these results establish that, in a mouse model, P6 contains a single immunodominant T cell epitope and this epitope plays an important role in protective immune responses induced by immunization with P6.     

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-na?ve human volunteers using the needle-free PowderJect system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 microg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg(335-343) tetramers, secreted IFN-gamma, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.     

Immunization with a soluble recombinant HIV protein entrapped in biodegradable microparticles induces HIV-specific CD8 cytotoxic T lymphocytes and CD4 Th1 cells

One of the major obstacles to the development of successful recombinant vaccines against human immunodeficiency virus (HIV) and other intracellular pathogens is the identification of a safe and effective vaccine delivery system for the induction of cell mediated immunity with soluble protein antigens. In this study it was demonstrated that immunization with a recombinant HIV envelope (env) protein entrapped in biodegradable poly(lactide-co-glycolide) (PLG) microparticles induced consistent HIV-specific CD4⁺ and CD8⁺ T-cell responses in mice. Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) responses were detected following a single systemic immunization with gp120 entrapped microparticles and when given by the intranasal (i.n.) route induced HIV-specific CD8⁺ CTL and secretory IgA. Furthermore immunization with gp120 entrapped in microparticles generated CD4⁺ T cells that secreted moderate to high levels of IFN-γ. Therefore, PLG microparticles are a safe and effective means of delivering antigen to the appropriate processing site for the generation of class I-restricted CTL, and are also capable of inducing Th1 cells.     

Antibody responses and protection against influenza virus infection in different congenic strains of mice immunized intranasally with adjuvant-combined A/Beijing/262/95 (H1N1) virus hemagglutinin or neuraminidase

Antibody (Ab) responses and protection against influenza virus infection in mice immunized intranasally with hemagglutinin (HA) or neuraminidase (NA) purified from the A/Beijing/262/95 (A/Beijing) (H1N1) virus were compared among B10 congenic mouse strains. Mice were immunized intranasally with 0.1, 0.3 or 1microg of HA or NA together with the cholera toxin adjuvant, and then boosted intranasally with 0.3 microg of the adjuvant-combined HA or NA 4 weeks later. Two weeks after the second immunization, the mice were challenged by an infection of the upper respiratory tract with the homologous virus. After 3 days, nasal wash and serum specimens were collected for virus and Ab titration. The HA immunization induced HA-specific IgG Ab responses against A/Beijing HA, which depended on the H-2 haplotype of the strain: The B10.A (H-2(a)), B10.D2 (H-2(d)), B10.BR (H-2(k)) and B10 (H-2(b)) strains were the highest, high, intermediate and low responders, respectively. The nasal IgA responses were induced in the B10.A, B10.D2 and B10.BR strains, but not in the B10 strain. In parallel with Ab responses, the B10.A, B10.BR and B10.D2 strains were conferred significant protection at any dose of primary immunization, but the B10 strain was provided protection only at 1microg of HA. On the other hand, the NA immunization induced NA-specific Ab responses, which depended on the the H-2 haplotype of the strain: the B10.A, B10.D2, B10 and B10.BR strains were the highest, high, intermediate and low responders, respectively. In parallel with Ab responses, all the strains were conferred significant protection at any dose of primary immunization. These results indicate that the MHC-restricted responsiveness of mice to HA is different from that to NA, suggesting that the use of high-HA dose or NA as a component of the nasal influenza A (H1N1 subtype) virus vaccine improves the protective efficacy against influenza among low responder populations.     

Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.     

A single intranasal immunization with inactivated influenza virus and alpha-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system

alpha-Galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of alpha-GalCer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and alpha-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given alpha-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, alpha-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and alpha-GalCer is a safe and effective means of preventing influenza infection.