Effect of viral membrane fusion activity on antibody induction by influenza H5N1 whole inactivated virus vaccine

Whole inactivated virus (WIV) influenza vaccines are more immunogenic in unprimed individuals than split-virus or subunit vaccines. In mice, this superior immunogenicity has been linked to the recognition of the viral ssRNA by endosomal TLR7 receptors in immune cells, leading to IFNα production and Th1-type antibody responses. Recent data suggest that viral membrane fusion in target cell endosomes is necessary for TLR7-mediated IFNα induction. If so, virus inactivation procedures that compromise the fusion activity of WIV vaccines, like formaldehyde (FA) treatment, could potentially harm vaccine efficacy. Therefore, we measured the effect of fusion inactivation of H5N1 WIV on TLR7 activation in vitro, and on antibody isotype responses in vivo. Fusion inactivation of WIV reduced, but did not block, TLR7-dependent IFNα induction in murine dendritic cells in vitro. In vivo, fusion-inactive WIV was as potent as fusion-active WIV in inducing total H5N1-specific serum IgG and IgG2c subtype antibodies in unprimed mice. Both vaccines induced only small amounts of IgG1. However, FA treatment of WIV did reduce the capacity of the vaccine to induce hemagglutination-inhibiting (HI) antibodies. This possibly relates to modification of epitopes that are targets for HI antibodies rather than to loss of fusion activity. Antibody affinity maturation was not negatively affected by fusion inactivation. In conclusion, fusion activity of H5N1 WIV does not play a major role in Th1-type antibody induction. Yet, to preserve the full immunogenicity of WIV, or possibly also other inactivated influenza vaccines, harsh treatment with formaldehyde should be avoided.     

The type of adjuvant in whole inactivated influenza a virus vaccines impacts vaccine-associated enhanced respiratory disease

Influenza A virus (IAV) causes a disease burden in the swine industry in the US and is a challenge to prevent due to substantial genetic and antigenic diversity of IAV that circulate in pig populations. Whole inactivated virus (WIV) vaccines formulated with oil-in-water (OW) adjuvant are commonly used in swine. However, WIV-OW are associated with vaccine-associated enhanced respiratory disease (VAERD) when the hemagglutinin and neuraminidase of the vaccine strain are mismatched with the challenge virus. Here, we assessed if different types of adjuvant in WIV vaccine formulations impacted VAERD outcome. WIV vaccines with a swine δ1-H1N2 were formulated with different commercial adjuvants: OW1, OW2, nano-emulsion squalene-based (NE) and gel polymer (GP). Pigs were vaccinated twice by the intramuscular route, 3 weeks apart, then challenged with an H1N1pdm09 three weeks post-boost and necropsied at 5 days post infection. All WIV vaccines elicited antibodies detected using the hemagglutination inhibition (HI) assay against the homologous vaccine virus, but not against the heterologous challenge virus; in contrast, all vaccinated groups had cross-reactive IgG antibody and IFN-γ responses against H1N1pdm09, with a higher magnitude observed in OW groups. Both OW groups demonstrated robust homologous HI titers and cross-reactivity against heterologous H1 viruses in the same genetic lineage. However, both OW groups had severe immunopathology consistent with VAERD after challenge when compared to NE, GP, and non-vaccinated challenge controls. None of the WIV formulations protected pigs from heterologous virus replication in the lungs or nasal cavity. Thus, although the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar H1, none tested here increased the breadth of protection against the antigenically-distinct challenge virus, and some impacted immunopathology after challenge.     

Alum boosts TH2-type antibody responses to whole-inactivated virus influenza vaccine in mice but does not confer superior protection

Clinical trials with pandemic influenza vaccine candidates have focused on aluminium hydroxide as an adjuvant to boost humoral immune responses. In this study we investigated the effect of aluminium hydroxide on the magnitude and type of immune response induced by whole-inactivated virus (WIV) vaccine. Balb/c mice were immunized once with a range of antigen doses (0.04-5 microg) of WIV produced from A/PR/8 virus, either alone or in combination with aluminium hydroxide. The hemagglutination inhibition (HI) titers of mice receiving WIV+aluminium hydroxide were 4-16-fold higher than HI titers in mice receiving the same dose of WIV alone, indicating the boosting effect of aluminium hydroxide. WIV induced a TH1 skewed humoral and cellular immune response, characterized by strong influenza-specific IgG2a responses and a high number of IFNgamma-secreting T cells. In contrast, immunization with WIV adsorbed to aluminium hydroxide resulted in skewing of this response to a TH2 phenotype (high IgG1 levels and a low number of IFNgamma-producing T cells). To assess the effect of the observed immune response skewing on viral clearance from the lungs mice immunized once with 1 microg WIV without or with aluminium hydroxide were challenged with A/PR/8 virus 4 weeks later. The immunized mice showed a significant decrease in viral lung titers compared to control mice receiving buffer. However, despite higher antibody titers, mice immunized with WIV adsorbed to aluminium hydroxide suffered from more severe weight loss and had significantly higher virus loads in their lung tissue than mice receiving WIV alone. Major difference between these groups of mice was the type of immune response induced, TH2 instead of TH1, indicating that a TH1 response plays a major role in viral clearance.     

Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine

We prepared a series of cationic lipid vesicles comprising a cationic cholesterol derivative, DC-Chol with or without a neutral phospholipid, DOPC or DOPE. The vesicles were tested for their ability to bind and adjuvant split inactivated influenza vaccines. We found that DC-Chol-containing liposomes are capable to strongly bind influenza vaccine antigens upon simple mixing with the vaccine. The resulting formulations induced robust anti-influenza immune responses both after s.c. and i.n. administration in BALB/c mice while neutral Cholesterol/DOPC liposomes displayed virtually no stable antigen binding and no adjuvant effect. The parenteral adjuvant effect of DC-Chol on trivalent split influenza vaccines was then confirmed in outbred mice and monkeys. Among the most potent formulations tested, a simple mixture of the vaccine with a microfluidized dispersion of DC-Chol in an aqueous buffer is being considered for further development to produce an improved influenza vaccine.     

Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin

Influenza virus is a highly infectious respiratory pathogen that results in severe morbidity and mortality. The current licensed trivalent vaccine formulations in the U.S. are made from virus grown in allantoic fluid from infected hen eggs that is then chemically inactivated and split into subunit components. These vaccines elicit antibodies, primarily to the viral hemagglutinin (HA), which are efficacious in healthy adults, but are limited in protecting high risk individuals, such as the elderly and immunocompromised. To address the need for improved influenza vaccines and the limitations of egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine against influenza infection. VLPs, based on the A/Fujian/411/2002 (H3N2) isolate, were purified from the supernatants of Spodoptera frugiperda Sf9 insect cells following infection of baculovirus vectors encoding an expression cassette comprised of only three influenza virus structural proteins, hemagglutinin (HA), neuraminidase (NA), and matrix (M1). Mice or ferrets were vaccinated intramuscularly with VLPs in a dose sparing experiment, based on HA concentration (3 microg-24 ng), and the immune responses were compared to responses elicited in animals vaccinated with recombinant HA (rHA) or inactivated whole influenza virions (WIV). All vaccinated animals had high titer anti-HA antibodies regardless of the vaccine immunogen and animals vaccinated with the highest doses of VLPs (3 microg and 600 ng) also had antibodies against NA. Purified rHA elicited primarily IgG1 antibodies, which is indicative of a T helper (Th) type 2 response, whereas mice vaccinated with the VLPs or WIV were associated with a dominant Th1 immune response (IgG2a and IgG2b). Interestingly, VLPs elicited antibodies that recognized a broader panel of antigenically distinct H3N2 viral isolates compared to rHA or WIV in a hemagglutination-inhibition (HAI) assay.     

Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization

Stabilization and ease of administration are two ways to substantially improve the use of current vaccines. In the present study an influenza whole inactivated virus (WIV) vaccine was freeze-dried or spray-freeze dried in the presence of inulin as a cryoprotectant. Only spray-freeze drying rendered powders compatible with administration to the lungs by insufflation. Pulmonary administration of the powder vaccine obtained by this method to BALB/c mice led to a transient influx of neutrophils and a concomitant decrease in the number of macrophages as did administration of liquid vaccine. Inflammatory reactions to both vaccines were mild and short-lived. Immunization studies showed that the immunogenic properties of WIV vaccine were not affected by drying. Pulmonary administration of the powder WIV vaccine induced a systemic immune response of the same magnitude as liquid vaccine while mucosal IgA responses were higher for powder WIV. In a challenge study where immunized mice were exposed to a lethal dose of live virus, two pulmonary doses of either liquid or powder WIV vaccine were equally effective as a single intramuscular injection of subunit vaccine in terms of reduction of the viral load in the lungs. To conclude, in the models employed for these studies the use of a dry powder WIV vaccine for pulmonary immunization was shown to be safe and efficient.     

Alum-adjuvanted H5N1 whole virion inactivated vaccine (WIV) enhanced inflammatory cytokine productions

Alum-adjuvanted H5 whole virion inactivated vaccine (WIV) was licensed for adults in Japan but induced marked febrile reactions with significantly stronger antibody responses in children. In this study, the mechanisms behind the different responses were investigated. Lymphocytes were obtained from 25 healthy subjects who were not immunized with H5 vaccine, to examine the innate immune impact of the various vaccine formulations, analyzing the cytokine production profile stimulated with alum adjuvant alone, alum-adjuvanted H5 WIIV, plain H5 WIV, and H5 split vaccine. Alum adjuvant did not induce cytokine production, but H5 split induced IFN-γ and TNF-α. H5 WIV induced IL-6, IL-17, TNF-α, MCP-1, IFN-γ, and IFN-α. An extremely low level of IL-1β was produced in response to H5 WIV, and alum-adjuvanted H5 WIV enhanced IL-1β production, with similar levels of other cytokines stimulated with H5 WIV. Enhanced production of cytokines induced by alum-adjuvanted H5 WIV may be related to the higher incidence of febrile reactions with stronger immune responses in children but it should be further investigated why efficient immune responses with febrile illness were observed only in young children.     

Intranasally administered Endocine™ formulated 2009 pandemic influenza H1N1 vaccine induces broad specific antibody responses and confers protection in ferrets

Influenza is a contagious respiratory disease caused by an influenza virus. Due to continuous antigenic drift of seasonal influenza viruses, influenza vaccines need to be adjusted before every influenza season. This allows annual vaccination with multivalent seasonal influenza vaccines, recommended especially for high-risk groups. There is a need for a seasonal influenza vaccine that induces broader and longer lasting protection upon easy administration. Endocine™ is a lipid-based mucosal adjuvant composed of endogenous lipids found ubiquitously in the human body. Intranasal administration of influenza antigens mixed with this adjuvant has been shown to induce local and systemic immunity as well as protective efficacy against homologous influenza virus challenge in mice. Here we used ferrets, an established animal model for human influenza virus infections, to further investigate the potential of Endocine™ as an adjuvant. Intranasal administration of inactivated pandemic H1N1/California/2009 split antigen or whole virus antigen mixed with Endocine™ induced high levels of serum hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers that were also cross reactive against distant swine viruses of the same subtype. HI and VN antibody titers were already demonstrated after a single nasal immunization. Upon intratracheal challenge with a homologous challenge virus (influenza virus H1N1/The Netherlands/602/2009) immunized ferrets were fully protected from virus replication in the lungs and largely protected against body weight loss, virus replication in the upper respiratory tract and pathological changes in the respiratory tract. Endocine™ formulated vaccines containing split antigen induced higher HI and VN antibody responses and better protection from body weight loss and virus shedding in the upper respiratory tract than the Endocine™ formulated vaccine containing whole virus antigen.     

Adjuvanted inactivated influenza A(H3N2) vaccines induce stronger immunogenicity in mice and confer higher protection in ferrets than unadjuvanted inactivated vaccines

Influenza viruses are major respiratory pathogens and the development of improved vaccines to prevent these infections is of high priority. Here, we evaluated split inactivated A(H3N2) vaccines (A/Uruguay/716/2007) combined or not with adjuvants (AS03, AS25 and Protollin) and administered by three different routes, intramuscular (i.m.), intranasal (i.n.) or intradermal (i.d.), both in BALB/c mice and in ferrets. Ferrets were challenged with the homologous strain A/Uruguay/716/2007 (H3N2) or the heterologous strain A/Perth/16/2009 (H3N2) 4 weeks after the second immunization with A/Uruguay/716/2007 vaccines. Temperature, weight loss and clinical signs were monitored on a daily basis and nasal washes were performed to evaluate viral titers in the upper respiratory tract. All adjuvanted vaccines induced stronger humoral immune responses than unadjuvanted ones in both mice and ferrets. In mice, the AS03- and AS25-adjuvanted i.m. vaccines generated a mixed Th1–Th2 response at 6 and 19 weeks after the last immunization as shown by the production of IgG1 and IgG2a antibodies as well as the production of IL-2, IL-4 and IFN-γ by CD4+ and CD8+ T cells. HAI and MN titers were also higher in those groups when compared to the i.n. Protollin-adjuvanted and unadjuvanted groups. The Protollin-adjuvanted i.n. vaccine induced a more Th1 oriented response with a significant production of IgA in bronchoalveolar lavages. In ferrets, the AS03- and AS25-adjuvanted i.m. vaccines also induced higher HAI and MN titers compared to the other groups. These vaccines also significantly decreased viral titers after challenge with both the homologous A/Uruguay/716/2007 (H3N2) and the heterologous A/Perth/16/2009 (H3N2) strains. In conclusion, adjuvanted influenza vaccines elicited stronger humoral response in mice and conferred greater protection in naive ferrets than unadjuvanted ones. Interestingly, the AS25 adjuvant system containing monophosphoryl-lipid-A appears particularly promising for developing more potent inactivated influenza vaccines.     

Bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses through intranasal administration

Administration of influenza vaccines through the intranasal (IN) route forms an attractive alternative to conventional intramuscular (IM) injection. It is not only a better accepted form of vaccine administration but it also has the potential to induce, in addition to systemic antibodies, local protective antibodies, i.e. S-IgA. Most commercially available vaccines however are inactivated non-replicating vaccines and have a low immunogenicity when administered intranasally. Local administration of these vaccines would therefore need an adjuvant to boost systemic and local antibody responses. Here we explored the use of a safe adjuvant system, i.e. bacterium-like particles (BLPs) derived from the food-grade bacterium in Lactococcus lactis, in the induction of protective antibody responses after intranasal immunization of mice. Supplementation of H1N1 split vaccine with BLPs significantly increased levels of serum influenza-specific IgG and hemagglutination-inhibiting antibodies: this was dependent on the dose of admixed BLPs and number of immunizations. Admixing BLPs further boosted local influenza-specific S-IgA antibody levels at lung and nasal mucosal sites, but also at distant mucosal sites such as the vaginal mucosal tissue. Mice immunized IN with BLP-adjuvanted vaccine and IM with non-adjuvanted vaccine were protected against weight loss upon homologous infection with H1N1 A/PR/8/34. Full protection against weight loss upon heterologous challenge with H1N1 A/PR/8/34 was seen in mice immunized IN with BLP-adjuvanted H1N1 A/New Caledonia-derived split virus vaccine, but not in those receiving the split virus vaccine IM. Mice immunized IN with BLP-adjuvanted vaccine had significantly lower lung viral titers upon homologous and heterologous challenge when compared to titers detected in mice immunized by IM injection of non-adjuvanted vaccine. Thus, adjuvantation of IN-administered influenza vaccines with BLPs effectively enhances systemic and local antibody responses leading to a superior protection against homologous and heterologous influenza infection compared to conventional IM immunization.     

Protective antibody responses against A(H1N1)pdm09 primed by infection and recalled by intranasal vaccination

This study investigated the effects of preceding infection and administration of whole inactivated virus (WIV) vaccine on immune responses against influenza virus challenge. Preceding infection alone provided minimal reduction in virus titer following viral challenge. Single administration of intranasal or subcutaneous WIV vaccine alone failed to reduce virus titers and induce antibody responses. Subcutaneous administration of A/Narita/1/09 (A/NRT)-WIV after A/NRT infection provided complete protection against infection and yielded low nasal IgA and high serum IgG antibody responses. Subcutaneous administration of A/NRT-WIV after A/Puerto Rico/8/34 (A/PR8) infection provided no protection. Conversely, intranasal administration of A/NRT-WIV after A/NRT infection provided complete protection and high nasal IgA and serum IgG antibody responses. While, intranasal administration of A/NRT-WIV after A/PR8 infection provided moderate reduction in viral titer with moderate increases in nasal IgA antibodies. These results indicate that intranasal vaccination is superior to subcutaneous vaccination in inducing protective immune responses after preceding heterologous infection.     

Immunogenic properties of plant-derived recombinant smallpox vaccine candidate pB5

The extracellular virion membrane protein B5 is a potent inducer of immune responses capable of protecting mice and primates against poxvirus infections. Here, we examined the antibody response induced in mice immunized intramuscularly (i.m.) or intranasally (i.n.) with plant-derived B5 (pB5) accompanied or not with plant total soluble protein (TSP) at various concentrations. Increasing amounts of TSP inhibited the pB5-specific response in both i.m.- and i.n.-immunized mice, with more dramatic effects in the latter. pB5 administered to mucosal surfaces induced specific IgG and IgA responses, whereas i.m. immunization produced high serum IgG titers and no IgA. A 6-fold increase in pB5 dosage administered i.n. led to an antibody response comparable to that obtained by i.m. injection. Our study addresses the quality/quantity issues of the pB5 subunit preparation and demonstrates the feasibility of mucosal administration of plant-derived smallpox subunit vaccine in obtaining a potent immune response. Overall, this work points to the practicability of needle-free mucosal administration of such vaccines in light of purity, dosage and adjuvant formulation.     

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.     

Use of a zwitterionic detergent for the preparation of an influenza virus vaccine: 2. Immunogenicity of Empigen-treated virus in animals

The immunogenicity of zwitterionic detergent-disrupted influenza virus vaccine preparations, intact virus vaccine and vaccine preparations obtained by treatment of the intact virus with Triton-X 100 or cetyl trimethyl ammonium bromide (CTAB) was studied in hamsters and mice. In all experiments the intact, inactivated virus vaccine induced greater serum haemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibody titres than the detergent-disrupted preparations. The serum HI antibody responses induced in hamsters and mice by Empigen-, Triton-, or CTAB-disrupted vaccines were similar, although more highly purified Empigen-disrupted preparations elicited marginally lower immune responses. In both animal species, all vaccine preparations afforded a similar degree of protection against homologous virus challenge.     

AIDS vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but not following intravenous challenge with cell-free virus.

The feline immunodeficiency virus (FIV) provides an excellent model system for AIDS vaccination studies. In the present experiments we investigated the immunogenicity and the protective activity of two inactivated vaccines prepared from a primary virus isolate. One vaccine was composed of whole virus inactivated with paraformaldehyde and then purified (WIV) and the other of viral proteins extracted with Tween-ether (TEV). Both vaccines elicited robust antiviral responses, but neither conferred appreciable levels of resistance against systemic challenge with the homologous virus. In addition, we tested whether the WIV vaccine, that had appeared more immunogenic, could protect against nontraumatic intravaginal exposure to FIV-infected cells. Although the proportions of control and vaccinated animals that became infected following mucosal challenge were similar, the vaccinees had significantly lower viral burdens than the controls, thus suggesting that immunisation with the WIV vaccine had limited FIV replication following intravaginal challenge.     

Protection against lethal enterovirus 71 infection in newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus.

Enterovirus 71 (EV71), the newest member of Enteroviridae, is notable for its etiological role in epidemics of severe neurological diseases in children. Developing effective vaccines is considered a top choice among all control measures. We compared the inactivated virus vaccine (10 microg protein/mouse) with subunit vaccines--VP1 DNA vaccine (100 microg/mouse) or recombinant VP1 protein (10 microg/mouse)--in its ability to elicit maternal antibody and to provide protection against lethal infection of EV71 in suckling mice. Prior to gestation, all three groups of vaccinated dams possessed similar levels of neutralizing antibody. With a challenge dose of 2300 LD(50) virus/mouse, suckling mice born to dams immunized with inactivated virus showed 80% survival. The subunit vaccines provided protection only at a lower challenge dosage of 230 LD(50) per mouse, with 40% survival for DNA vaccine and 80% survival for VP1 protein. The cytokine profile produced by splenocytes showed a high level of IL-4 in the inactivated virus group, high levels of IFN-gamma and IL-12 in the DNA vaccine group, and high levels of IL-10 and IFN-gamma in the VP1 protein group. Overall, the inactivated virus elicited a much greater magnitude of immune response than the subunit vaccines, including total IgG, all four IgG subtypes, and T-helper-cell responses; these antibodies were shown to be protective against lethal infection when passively transferred to susceptible newborn mice. Our data indicated that inactivated virus is the choice of vaccine preparation capable of fulfilling the demand for effective control, and that VP1 subunit vaccines remain promising vaccine strategies that require further refinement.     

An Advax-CpG55.2 adjuvanted recombinant hemagglutinin vaccine provides immunity against H7N9 influenza in adult and neonatal mice

There is a major unmet need for strategies to improve the immunogenicity and effectiveness of pandemic influenza vaccines, particularly in poor responder populations such as neonates. Recombinant protein approaches to pandemic influenza offer advantages over more traditional inactivated virus approaches, as they are free of problems such as egg adaptation or need for high level biosecurity containment for manufacture. However, a weakness of recombinant proteins is their low immunogenicity. We asked whether the use of an inulin polysaccharide adjuvant (Advax) alone or combined with a TLR9 agonist (CpG55.2) would enhance the immunogenicity and protection of a recombinant hemagglutinin vaccine against H7N9 influenza (rH7HA), including in neonatal mice. Advax adjuvant induced predominantly IgG1 responses against H7HA, whereas Advax-CpG55.2 adjuvant also induced IgG2a, IgG2b and IgG3 responses, consistent with the TLR9 agonist component inducing a Th1 bias. Advax-CpG55.2 adjuvanted rH7HA induced high serum neutralizing antibody titers in adult mice. In newborns it similarly overcame immune hypo-responsiveness and enhanced serum anti-rH7HA IgG levels in 7-day-old BALB/C and C57BL/6 mice. Immunized adult mice were protected against a lethal H7N9 virus challenge. When formulated with Advax-CpG55.2 adjuvant, greater protection was seen with rH7HA than with inactivated H7 whole virus antigen. Advax-CpG55.2 adjuvanted rH7HA represents a promising influenza vaccine platform for further development.     

Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice

In this study pulmonary vaccination with a new influenza subunit vaccine powder was evaluated. Vaccine powder was produced by spray-freeze drying (SFD) using the oligosaccharide inulin as stabilizer. Immune responses after pulmonary vaccination of BALB/c mice with vaccine powder were determined and compared to those induced by intramuscular and pulmonary vaccination with a conventional liquid subunit vaccine. All vaccinations were performed without adjuvant. Pulmonary vaccination with liquid subunit vaccine resulted in systemic humoral (IgG) immune responses similar to intramuscular immunization. In contrast, the vaccine powder delivered by the pulmonary route, induced not only systemic humoral (IgG) responses, but also cell-mediated (Il-4, IFN-γ) and mucosal immune responses (IgA, IgG). This study demonstrates that the combination of pulmonary antigen delivery and antigen powder production by SFD improves the immunogenic potential of (influenza subunit) antigen. In conclusion, vaccination with a non-adjuvanted SFD subunit vaccine powder by inhalation might be feasible and could be an alternative to conventional parenteral vaccine administration.     

Immunogenicity and safety of inactivated influenza vaccines in primed populations: a systematic literature review and meta-analysis

Several inactivated influenza vaccine formulations for systemic administration in man are currently available for annual (seasonal) immunization: split virus and subunit (either plain-aqueous, or virosomal, or adjuvanted by MF59). From a literature search covering the period 1978-2009, 33 articles could be identified, which described randomized clinical trials comparing at least two of the four vaccine formulations with respect to serum hemagglutination inhibition (HI) antibody response, local and systemic vaccine reactions and serious adverse events after vaccination, and employing seasonal vaccine components and doses. In total, 9121 vaccinees of all ages, either healthy or with underlying diseases, were involved. Most vaccinees were primed or had been vaccinated in previous years.For immunogenicity, homologous post-vaccination geometric mean HI titers (GMTs) were analyzed by a random effects model for continuous data. Unreported standard deviations (SD) were addressed by imputing assumed SD-values. Age and health state of the vaccinees appeared to have little influence on the outcome. The immunogenicity of split, aqueous and virosomal subunit formulations were similar, with geometric mean ratio values (GMR, quotient of paired GMT-values) varying around one (0.93-1.24). The MF59-adjuvanted subunit vaccine induced, on average, larger antibody titers than the non-adjuvanted vaccine formulations, but the absolute increase was small (GMR-values varying between 1.25 and 1.40).Vaccine reactions were analyzed using a random effects model for binary data. Local and systemic reactogenicity was similar among non-adjuvanted formulations. The adjuvanted subunit formulation was more frequently associated with local reactions than the non-adjuvanted formulations (rate ratio: 2.12, significant). Systemic reactions were similar among all vaccine formulations. The original articles emphasized the mild and transient character of the vaccine reactions and the absence of serious vaccine-related adverse events.This adequate amount of evidence led to the conclusion that all the currently available inactivated influenza vaccine formulations are safe, well tolerated and similarly effective to control seasonal influenza outbreaks across primed populations and age ranges.     

N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2 with a tripolyphosphate (TPP) solution, at ambient temperature and pH 7.4 while stirring. The nanoparticles had an average size of about 800 nm with a narrow size distribution and a positive surface charge. The nanoparticles showed a loading efficiency of 78% and a loading capacity of 13% (w/w). It was shown that more than 75% of the protein remained associated with the TMC nanoparticles upon incubation of the particles in PBS for 3h. The molecular weight and antigenicity of the entrapped hemagglutinin was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. Single i.n. or i.m. immunization with antigen-loaded TMC nanoparticles resulted in strong hemagglutination inhibition and total IgG responses. These responses were significantly higher than those achieved after i.m. administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The i.n. administered antigen-TMC nanoparticles induced higher immune responses compared to the other i.n. antigen formulations, and these responses were enhanced by i.n. booster vaccinations. Moreover, among the tested formulations only i.n. administered antigen-containing TMC nanoparticles induced significant IgA levels in nasal washes of all mice. In conclusion, these findings demonstrate that TMC nanoparticles are a potent new delivery system for i.n. administered influenza antigens.