Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)-specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.     

Induction of cross‐protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co‐administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti‐A/PR8 HA‐specific IgA and IgG response in nasal washings and serum, respectively. Virus‐specific cytotoxic T‐lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross‐protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow‐derived dendritic cells. These results suggest that mycelial extract‐adjuvanted vaccines can confer cross‐protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection. J. Med. Virol. 82:128–137, 2010. © 2009 Wiley‐Liss, Inc.     

Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3N2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein

Vaccination is a cost-effective way to control the influenza epidemic. Vaccines based on highly conserved antigens can provide protection against different influenza A strains and subtypes. In this study, the recombinant nucleoprotein (rNP) of the A/PR/8/34 (H1N1) influenza virus strain was effectively expressed using a prokaryotic expression system and then purified with a nickel-charged Sepharose affinity column as a candidate component for an influenza vaccine. The rNP was administered intranasally three times at 3-week intervals to female BALB/c mice in combination with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin). Twenty-one days after the last immunization, the mice were challenged with homologous or heterologous influenza viruses at a lethal dose. The results showed that intranasal immunization of 10 μg rNP with adjuvant completely protected the immunized mice against the homologous influenza virus, and immunization with 100 μg rNP in combination with adjuvant provided good cross-protection against heterologous H5N1 and H9N2 avian influenza viruses. The results indicate that such a vaccine administered intranasally can induce mucosal and cell-mediated immunity, thus having the potential to control epidemics caused by new emerging influenza viruses.     

Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant

NB-1008 is a surfactant-stabilized soybean oil-in-water nanoemulsion (NE) adjuvant with influenza virus antigen incorporated into the NE by simple mixing. Intranasal administration of the antigen with NE adjuvant efficiently produces both mucosal and serum antibody responses as well as a robust cellular Th1 immune response. To demonstrate the adjuvant effect of the W₈₀5EC NE, a killed commercial influenza vaccine for intramuscular administration (Fluzone or Fluvirin) was mixed with the W₈₀5EC NE adjuvant and administered intranasally to naïve ferrets. After a single intranasal immunization, the adjuvanted influenza vaccine elicited elevated serum hemagglutination inhibition (HAI) geometric mean titers (GMTs) ranging from 196 to 905 for the three hemagglutinin (HA) antigens present in the vaccine, which are approximately 19- to 90-fold higher titers at 1/50 the standard intramuscular commercial nonadjuvanted influenza vaccine dose. Seroconversion rates of 67% to 100% were achieved against each of the three viral strains present. The adjuvanted nasal influenza vaccine also produced significant cross immunity to five other H3N2 influenza virus strains not present in the vaccine and produced sterile immunity after challenge with homologous live virus. No safety issues were observed in 249 ferrets receiving the adjuvanted influenza vaccine. These findings demonstrate the ability of W₈₀5EC NE to adjuvant nasally administered influenza vaccine and provide a basis for studying the intranasal W₈₀5EC-adjuvanted influenza vaccine in humans.     

Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant.

Mucosal immunization by inactivated viruses often fails to evoke a sufficient immune response. Intensive efforts have been made to enhance the response by suitable adjuvants. We used the G+ nonpathogenic delipidated bacterium Bacillus firmus with pronounced immunostimulatory properties as an adjuvant for immunizing mice with inactivated influenza virus type A. BALB/c mice were immunized intratracheally with inactivated influenza A H1N1 and H3N2 viruses. The production of antibodies in sera and secretions was determined by the ELISA. The local situation in the lungs was assessed histologically and by testing the cytokine expression. The protective and cross-protective effect against infection was tested in in vivo experiments after infection with influenza virus A H1N1. B. firmus as adjuvant increased both systemic and mucosal antibody responses, improved protection against homologous virus and induced cross-protection against virus H1N1 after immunization with virus H3N2.     

Virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge

We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), and matrix 2 (M2), or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) after immunization of mice. VLP vaccine ( approximately 1 microg HA) were formulated with or without IL-12 as adjuvant and administered twice, at 2-week intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.     

Intranasal administration of adjuvant‐combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre‐pandemic vaccine combined with synthetic double‐stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant‐combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine‐specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA‐combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential. J. Med. Virol. 82:1754–1761, 2010. © 2010 Wiley‐Liss, Inc.     

Prophylactic effects of chitin microparticles on highly pathogenic H5N1 influenza virus

Highly pathogenic avian influenza virus (H5N1) is an emerging pathogen with the potential to cause great harm to humans, and there is concern about the potential for a new influenza pandemic. This virus is resistant to the antiviral effects of interferons and tumor necrosis factor-alpha. However, the mechanism of interferon-independent protective innate immunity is not well understood. The prophylactic effects of chitin microparticles as a stimulator of innate mucosal immunity against a recently obtained strain of H5N1 influenza virus infection were examined in mice. Clinical parameters and the survival rate of mice treated by intranasal application of chitin microparticles were significantly improved compared to non-treated mice after a lethal influenza virus challenge. Flow cytometric analysis revealed that the number of natural killer cells that expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and that had migrated into the cervical lymph node was markedly increased (26-fold) after intranasal treatment with chitin microparticles. In addition, the level of IL-6 and interferon-gamma-inducible protein-10 (IP-10) in the nasal mucosa after H5N1 influenza virus challenge was decreased by prophylactic treatment with chitin microparticles. These results suggest that prophylactic intranasal administration of chitin microparticles enhanced the local accumulation of natural killer cells and suppressed hyper-induction of cytokines, resulting in an innate immune response to prevent pathogenesis of H5N1 influenza virus.     

Vaccination against H9N2 avian influenza virus reduces bronchus‐associated lymphoid tissue formation in cynomolgus macaques after intranasal virus challenge infection

H9N2 avian influenza virus causes sporadic human infection. Since humans do not possess acquired immunity specific to this virus, we examined the pathogenicity of an H9N2 virus isolated from a human and then analyzed protective effects of a vaccine in cynomolgus macaques. After intranasal challenge with A/Hong Kong/1073/1999 (H9N2) (HK1073) isolated from a human patient, viruses were isolated from nasal and tracheal swabs in unvaccinated macaques with mild fever and body weight loss. A formalin‐inactivated H9N2 whole particle vaccine derived from our virus library was subcutaneously inoculated to macaques. Vaccination induced viral antigen‐specific IgG and neutralization activity in sera. After intranasal challenge with H9N2, the virus was detected only the day after inoculation in the vaccinated macaques. Without vaccination, many bronchus‐associated lymphoid tissues (BALTs) were formed in the lungs after infection, whereas the numbers of BALTs were smaller and the cytokine responses were weaker in the vaccinated macaques than those in the unvaccinated macaques. These findings indicate that the H9N2 avian influenza virus HK1073 is pathogenic in primates but seems to cause milder symptoms than does H7N9 influenza virus as found in our previous studies and that a formalin‐inactivated H9N2 whole particle vaccine induces protective immunity against H9N2 virus.     

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.     

Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure

The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection

Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract.     

Immunity to Influenza in Ferrets X. Intranasal Immunization of Ferrets with Inactivated Influenza A Virus Vaccines

The response of ferrets after intranasal inoculation of inactivated A/Hong Kong/68 (H3N2) influenza virus vaccines is reported. Normal ferrets given either saline vaccine in drops or freeze-dried vaccine in an aerosol intranasally did not produce detectable serum or nasal hemagglutination inhibiting antibody and were found to be completely susceptible to challenge infection with A/Hong Kong/68 virus. Intranasal saline vaccine did not produce an additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant. Ferrets primed by previous infection with A/PR/8/34 (H0N1) influenza virus, however, responded to intranasal immunization with saline A/Hong Kong/68 virus vaccine and produced serum and nasal antibody. These animals were found to be partially resistant to challenge infection, in contrast to similar animals given saline vaccine intramuscularly which were completely resistant to challenge infection. Primed ferrets did not respond after immunization with the freeze-dried aerosol vaccine, but this may have been due to a failure of the aerosol to be inhaled satisfactorily.     

蛋白体佐剂及用于鼠疫F1-Ⅴ抗原的免疫效果

目的以蛋白体(proteosomes)佐剂,非共价结合鼠疫F1-V重组蛋白为免疫原,探讨滴鼻免疫BALB/c小鼠后诱导的免疫应答和免疫保护效果。方法佐剂与鼠疫F1-V重组蛋白为免疫原非共价结合,滴鼻免疫BALB/c小鼠4次后,采用间接ELISA检测血清特异性抗F1-V的IgG和IgA抗体及抗体亚型分类,并检测鼻咽、肺、小肠及阴道灌洗液中特异性抗F1-V的黏液分泌型IgA;并用流式细胞术检测鼻相关淋巴组织淋巴细胞、脾淋巴细胞、肠系膜淋巴结及小肠PP结T淋巴细胞表型的变化。第4次免疫后7d,用100 LD_(50)的鼠疫141强毒株进行腹腔攻毒。结果(1)以蛋白体为佐剂的鼠疫F1-V抗原与单纯的F1-V组相比,蛋白体疫苗组诱导血清IgG、IgA抗体显著升高(P<0.01),同时蛋白体疫苗组能诱导鼻咽、肺、小肠和阴道内多个黏膜部位特异性IgA抗体的产生,尤其是肺和生殖道冲冼液内抗体升高极为显著(P<0.01);(2)蛋白体疫苗组主要引起IgG1型抗体,主要诱导T_H2型免疫反应;(3)蛋白体疫苗组NALT和SP中CD4~ /CD8~ 比值比PBS对照有显著增高(P<0.01),MLN和PP中CD4~ /CD8~ 比值与PBS对照差异无统计学意义(P>0.05)。(4)小鼠在100 LD_(50)的鼠疫141强毒株腹腔攻毒后鼠疫F1-V重组蛋白组小鼠免疫保护率为0,而蛋白体佐剂疫苗组小鼠免疫保护率为67%。结论以自制的蛋白体为鼠疫F1-V抗原的佐剂滴鼻免疫小鼠,蛋白体不仅提高鼠疫F1-V抗原的系统免疫应答,而且能诱导小鼠呼吸道、消化道和生殖道局部黏膜免疫应答。蛋白体佐剂鼠疫疫苗对100 LD_(50)的鼠疫141强毒株腹腔攻毒具有一定的免疫保护作用,这为鼠疫黏膜疫苗的研制提供候选材料,也为鼠疫黏膜疫苗深入研究奠定了基础。     

Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge

We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1) and M2, or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) following immunization of mice. VLP vaccine (approximately 1 microg HA) were formulated with or without IL-12 as adjuvant and administered twice, at two weeks intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.     

Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge

Mucosal surfaces are important for the induction of immunity against influenza virus. In a murine intranasal immunization model, we demonstrated that the attenuated Shigella flexneri Deltaasd strain 15D, carrying a DNA construct encoding the influenza virus hemagglutinin (HA), induces protective immunity against a lethal respiratory challenge with influenza A/WSN/33. Influenza virus-specific IFN-gamma T cells were detected among splenocytes, and anti-HA IgG was detected in serum post-immunization, albeit at low levels. Following influenza virus challenge, an accelerated anti-HA IgA antibody response was detected in bronchoalveolar lavage (BAL) washings from mice vaccinated with attenuated shigella containing the HA construct. These results suggest that S. flexneri Deltaasd strain 15D is a promising vector for mucosal DNA vaccine immunization against influenza virus and other mucosal pathogens.     

Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant

The increasing number of recent outbreaks of HPAI H5N1 in birds and humans brings out an urgent need to develop potent H5N1 vaccine regimens. Here we present a study on the intranasal vaccination of recombinant baculovirus surface-displayed hemagglutinin (BacHA) or inactivated whole H5N1 viral (IWV) vaccine with a recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant in a BALB/c mouse model. Two groups of mice were vaccinated with different doses (HA titer of log 2⁴ or log 2⁸) of either HA surface-displayed baculovirus or inactivated whole viral vaccine virus adjuvanted with different doses (2 μg or 10 μg) of rCTB. The vaccinations were repeated after 28 days. HA specific serum IgG and mucosal IgA antibodies were quantified by indirect ELISA, and serum neutralizing antibody titer were estimated by hemagglutination inhibition (HI) assay and virus neutralization titer assay. Functional protective efficacy of the vaccine was assessed by host challenge against HPAI H5N1 strains. The results revealed that mice co-administered with log 2⁸ HA titer of BacHA vaccine and adjuvanted with 10 μg of rCTB had a significantly enhanced serum IgG and mucosal IgA immune response and serum microneutralization titer compared with mice administered with unadjuvanted log 2⁴ or log 2⁸ HA titer of BacHA alone. Also vaccination with 10 μg of rCTB and log 2⁸ HA titer of BacHA elicited higher HA specific serum and mucosal antibody levels and serum HI titer than vaccination with log 2⁸ HA titer of inactivated H5N1 virus adjuvanted with the same dose of rCTB. The host challenge study also showed that 10 μg rCTB combined with log 2⁸ HA titer of BacHA provided 100% protection against 10MLD₅₀ of homologous and heterologous H5N1 strains. The study shows that the combination of rH5 HA expressed on baculovirus surface and rCTB mucosal adjuvant form an effective mucosal vaccine against H5N1 infection. This baculovirus surface-displayed vaccine is more efficacious than inactivated H5N1 influenza vaccine when administered by intranasal route and has no biosafety concerns associated with isolation, purification and production of the latter vaccine.