Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice.

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.

Methylglycol chitosan and a synthetic TLR4 agonist enhance immune responses to influenza vaccine administered sublingually.

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.

Effects of different adjuvants in the context of intramuscular and intranasal routes on humoral and cellular immune responses induced by detergent-split A/H3N2 influenza vaccines in mice.

Influenza A/H3N2 viruses have caused the most severe epidemics since 1968 despite current immunization programs with inactivated vaccines. We undertook a side-by-side preclinical evaluation of different adjuvants (Alum, AS03, and Protollin) and routes of administration (intramuscular [i.m.] and intranasal [i.n.]) for assessing their effect on the immunogenicity and cross-reactivity of inactivated split vaccines (A/H3N2/New York/55/2004). Humoral and T cell-mediated immune responses against the homologous virus and a heterologous drifted strain (A/H3N2/Wisconsin/67/2005) were measured in BALB/c mice at 2, 6, and 19 weeks postboost. The AS03- and Alum-adjuvanted i.m. vaccines induced at least an 8-fold increase over the nonadjuvanted vaccine in functional antibody titers against both the homotypic and heterotypic strains and low IgG2a and high IgG1 levels, suggesting a mixed Th1/Th2 response with a Th2 trend. The Protollin-adjuvanted i.n. vaccine induced the lowest IgG1/IgG2a ratio, which is indicative of a mixed Th1/Th2-type profile with a Th1 trend. This adjuvanted vaccine was the only vaccine to stimulate a mucosal IgA response. Whatever the timing after the boost, both hemagglutination inhibition (HAI) and microneutralization (MN) titers were higher with the AS03-adjuvanted i.m. vaccine than with the protollin-adjuvanted i.n. vaccine. Finally, the Alum-adjuvanted i.m. vaccine and the lower-dose Protollin-adjuvanted i.n. vaccine elicited significantly higher CD4(+) Th1 and Th2 responses and more gamma interferon (IFN-γ)-producing CD8(+) T cells than the nonadjuvanted vaccine. Our data indicate that the adjuvanted vaccines tested in this study can elicit stronger, more persistent, and broader immune responses against A/H3N2 strains than nonadjuvanted inactivated influenza vaccines.

Proteosome-adjuvanted intranasal influenza vaccines: advantages, progress and future considerations.

The development of a safe and effective non-live intranasal influenza vaccine has been an elusive target in vaccinology for many decades. It is perceived that intranasal immunization, by offering a more convenient and less invasive vaccination modality, will boost vaccination rates against influenza, a disease that continues to inflict a significant annual health and economic burden worldwide. Intranasal immunization may also confer additional immunoprotective benefits by eliciting mucosal secretory antibodies at the site of entry of the virus, which are typically more broadly cross-reactive and cross-protective compared with those induced by systemic routes of vaccination. This property is highly desirable for confering improved protection against variant strains of influenza virus. Here we review the current status of intranasal proteosome-based influenza vaccines that comprise commercial detergent-split influenza antigens and proteosome adjuvants derived from purified bacterial outer membrane proteins. We demonstrate that these vaccines exhibit the desired advantages expected from immunization via the intranasal route. Furthermore, in clinical trials proteosome-based influenza vaccines were shown to be safe and protective in humans. The future possibilities for commercializing intranasal proteosome-influenza vaccines are also discussed.

Oseltamivir-resistant pandemic A/H1N1 virus is as virulent as its wild-type counterpart in mice and ferrets.

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK alpha2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1-7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate.

A nasal proteosome adjuvant activates microglia and prevents amyloid deposition.

OBJECTIVE: We assessed whether peripheral activation of microglia by a nasal proteosome-based adjuvant (Protollin) that has been given safely to humans can prevent amyloid deposition in young mice and affect amyloid deposition and memory function in old mice with a large amyloid load. METHODS: Amyloid precursor protein (APP) transgenic (Tg) J20 mice received nasal treatment with Protollin weekly for 8 months beginning at age 5 months. Twenty-four-month-old J20 mice were treated weekly for 6 weeks. RESULTS: We found reduction in the level of fibrillar amyloid (93%), insoluble beta-amyloid (Abeta; 68%), and soluble Abeta (45%) fragments in 14-month-old mice treated with Protollin beginning at age 5 months. Twenty-four-month-old mice treated with nasal Protollin for 6 weeks had decreased soluble and insoluble Abeta (1-40) and (1-42) and improved memory function. Activated microglia (CD11b+ cells) colocalized with Abeta fibrils in the 24-month-old animals, and microglial activation correlated with the decrease in Abeta. No microglial activation was observed in 14-month-old mice, suggesting that once Abeta is cleared, there is downregulation of microglial activation. Both groups had reduction in astrocytosis. Protollin was observed in the nasal cavity and cervical lymph node but not in the brain. Activated CD11b+SRA+ (scavenger receptor A) cells were found in blood and cervical lymph node and increased interleukin-10 in cervical lymph node. No toxicity was associated with treatment. INTERPRETATION: Our results demonstrate a novel antibody-independent immunotherapy for both prevention and treatment of Alzheimer's disease that is mediated by peripheral activation of microglia with no apparent toxicity.

A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice.

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin (Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG1:IgG2a ratios and MV-specific IFNgamma production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced TH1/TH2-type response.

A nasal Proteosome influenza vaccine containing baculovirus-derived hemagglutinin induces protective mucosal and systemic immunity.

The potential for enhancing the immunogenicity of recombinant (baculovirus-derived) influenza hemagglutinin (rHA) was investigated by comparing the immune responses elicited in mice by an intranasal (i.n.) rHA formulated with Proteosomes, with those induced by intramuscular (i.m.) or i.n. rHA alone. The Proteosome-rHA vaccine induced mucosal responses in the respiratory tract, as well as high serum IgG and hemagglutination inhibition (HAI) titers. In contrast, rHA alone given i.m. induced serum IgG without mucosal responses and was ineffective at inducing either mucosal or systemic responses when given i.n. Only mice immunized with the Proteosome-rHA vaccine were completely protected from both death and acute morbidity following live virus challenge, indicating that the i.n. Proteosome-rHA vaccine induced more complete protective immunity than the same doses of unformulated rHA given i.n. or i.m.