Effects of route and coadministration of recombinant raccoon poxviruses on immune responses and protection against highly pathogenic avian influenza in mice

We previously demonstrated that recombinant raccoonpox (RCN) virus could serve as a vector for an influenza vaccine. RCN constructs expressing the hemagglutinin (HA) from H5N1 viruses were immunogenic in chickens. In the current study, we generated several recombinant RCN constructs expressing influenza (H5N1) antigens and a molecular adjuvant (Heat-Labile enterotoxin B from E. coli: RCN-LTB), demonstrated their expression in vitro, and evaluated their ability to protect mice against H5N1 virus challenge. RCN-HA provided strong protection when administered intradermally (ID), but not intranasally (IN). Conversely, the RCN-neuraminidase (NA) construct was highly efficacious by the IN route and elicited high titers of neutralizing antibodies in mice. Vaccination by combined ID (RCN-HA) and IN (RCN-NA) routes offered mice the best protection against an IN challenge with heterologous H5N1 virus. However, protection was reduced when the different RCN constructs were pre-mixed, perhaps due to reduced expression of antigen.     

Intramuscular immunization with a vesicular stomatitis virus recombinant expressing the influenza hemagglutinin provides post-exposure protection against lethal influenza challenge

Vaccines currently licensed for the prevention of seasonal influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least 2 weeks after immunization for the development of protective immunity. These vaccines do not induce protective responses quickly enough to blunt the effects of infection when administered after exposure. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly at least 24 h after delivery of the influenza challenge virus. To our knowledge this is the first vaccine that effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The induction of HA-specific immune responses by the vaccine is necessary for full protection from challenge, because animals immunized with an empty rVSV vector were not protected equally. Our results are consistent with a model in which vaccination induces an immediate antiviral cytokine response, followed by development of humoral and cellular immune responses which act to reduce pulmonary viral loads and accelerate recovery. Consistent with this model, mice vaccinated with the specific vaccine rVSV HA had high levels of IFN-α in the serum by 24 h after challenge/vaccination, developed serum neutralizing Ab to influenza 2 days prior to control animals, and had detectable anti-HA CD8 T cells present in the peripheral blood 3 days prior to control mice.     

Pandemic H1N1 influenza virus-like particles are immunogenic and provide protective immunity to pigs

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine.     

Nanoemulsion W805EC improves immune responses upon intranasal delivery of an inactivated pandemic H1N1 influenza vaccine

Currently available influenza vaccines provide suboptimal protection. In order to improve the quality of protective immune responses elicited following vaccination, we developed an oil-in-water nanoemulsion (NE)-based adjuvant for an intranasally-delivered inactivated influenza vaccine. Using a prime-boost vaccination regimen, we show that intranasal vaccines containing the W₈₀5EC NE elicited higher titers of serum hemagglutination inhibiting (HAI) antibody and influenza-specific IgG and IgA titers compared to vaccines that did not contain the NE. Similarly, vaccines containing the W₈₀5EC NE resulted in higher influenza-specific IgA levels in the bronchoalveolar lavage (BAL) fluid and nasal wash when compared to vaccines formulated without NE. The higher antibody titers in mice immunized with the NE-containing vaccines correlated with reduced viral loads in the lungs and nasal turbinates following a high dose viral challenge. Mice immunized with vaccines containing the W₈₀5EC NE also showed a reduction in body weight loss following challenge compared to mice immunized with equivalent vaccines produced without NE. Taken together, our results show that the W₈₀5EC NE substantially improves the magnitude of protective influenza-specific antibody responses and is a promising mucosal adjuvant for influenza vaccines and vaccines against other mucosal pathogens.     

Mucosal IgA responses in influenza virus infections; thoughts for vaccine design

The current challenge in influenza vaccine design is to induce long-lasting protection not only against the vaccine strain, but also against drifted (point mutations in the surface antigens HA or NA) and even shifted (exchange of genome segments) strains. Several immune mediators that can induce cross-protection have been described, such as CD4 T-cells, CD8 T-cells and antibodies, including IgA. However, most vaccines are now administered intramuscularly or subcutaneously and subsequently relatively little is known on the role of local, mucosal responses. Since local IgA responses have been shown to play an important role in responses to natural infection, and IgA responses in mice were shown to also be involved in cross-protection, the research on mucosal influenza vaccines is currently expanding. However, the functioning of the mucosal immune system, especially in the respiratory tract, is just beginning to be revealed. Here, the current knowledge on the induction of IgA, the role of influenza specific IgA producing B-cells in anti-influenza immunity as well as the role of humoral memory responses induced upon vaccination will be reviewed.     

Establishment of functional influenza virus-specific CD8+ T cell memory pools after intramuscular immunization

The emergence of the avian-origin influenza H7N9 virus and its pandemic potential has highlighted the ever-present need to develop vaccination approaches to induce cross-protective immunity. In this study, we examined the establishment of cross-reactive CD8⁺ T cell immunity in mice following immunization with live A/Puerto Rico/8/1934 (PR8; H1N1) influenza virus via two non-productive inoculation routes. We found that immunization via the intramuscular (IM) route established functional influenza-virus specific memory CD8⁺ T cell pools capable of cross-reactive recall responses. Epitope-specific primary, memory and recall CD8⁺ T-cell responses induced by the IM route, highly relevant to human influenza immunisations, were of comparable magnitude and quality to those elicited by the intraperitoneal (IP) priming, commonly used in mice. Furthermore, IM immunisation resulted in lower lung viral titres following heterologous challenge with A/Aichi/68 (X31; H3N2) compared to the IP route. Examining the ability of DCs from lymphoid organs to present viral antigen revealed that immune induction following IM immunization occurred in draining lymph nodes, while immunization via the IP route resulted in the priming of responses in distal lymphoid organs, indicative of a systemic distribution of antigen. No major differences in the pulmonary cytokine environment of immunized animals following X31 challenge were observed that could account for the improved heterologous protection induced by the IM route. However, while both routes induced similar levels of PR8-specific antibodies, higher levels of cross-reactive antibodies against X31 were induced following IM inoculation. Our data demonstrate how non-replicative routes of infection can induce efficient cross-reactive CD8⁺ T cell responses and strong strain-specific antibody responses, with the additional benefit from IM priming of enhanced heterosubtypic antibody production.     

Flagellin-HA vaccines protect ferrets and mice against H5N1 highly pathogenic avian influenza virus (HPAIV) infections

In order to meet the global demand for rapid production of pandemic influenza vaccines, we have developed a recombinant fusion vaccine platform in which the globular head of hemagglutinin (HA) antigen is genetically fused to bacterial flagellin (a TLR5 ligand). These flagellin-HA fusion vaccine candidates elicit highly protective immunity against a lethal challenge with 2009 pandemic H1N1 (Liu, et al. PLoS ONE 2011; 6:e20928) or H5N1 influenza A/Vietnam/1203/04 (A/VN) infections in mice (Song, et al. Vaccine 2009;27:5875–88). Here we provide the first evidence showing that two A/VN vaccine candidates elicited HA-specific IgG, reduced nasal virus shedding, and conferred full protection against a lethal A/VN infection in ferrets. Furthermore, we show that similar flagellin-HA vaccine candidates of two other H5N1 HPAIV are immunogenic and/or efficacious in mice. Vaccines of A/Indonesia/5/05 (A/IN) induced significant HAI titers to homologous and heterologous A/Anhui/1/05 (A/AN) H5N1 viruses. Two subcutaneous immunizations with doses of either 0.3 μg or 3 μg of A/IN candidates resulted in ≥2.5 log₁₀ unit reduction in day 5 lung virus titer and 90–100% protection against a lethal A/IN challenge in mice. Both R3.HA5 IN and R3.2xHA5 IN vaccines elicited robust neutralizing antibody responses that last for at least 9 months and demonstrated a significant anamnestic antibody response upon further booster immunization. Finally, we found that two vaccine candidates of A/AN induced significant HAI titers in mice. Taken together, our recombinant flagellin-HA platform has been successfully used to generate potent H5N1 HPAIV vaccine candidates. These promising preclinical results justify the advancement of these candidates into the clinic.     

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.     

Inactivated influenza H5N1 whole-virus vaccine with aluminum adjuvant induces homologous and heterologous protective immunities against lethal challenge with highly pathogenic H5N1 avian influenza viruses in a mouse model

In response to recent outbreaks of H5N1 highly pathogenic avian influenza virus (HPAIV), the development of an effective H5N1 influenza vaccine is urgently important. We assessed the efficacy of two inactivated H5N1 whole-virus vaccines, rgHK213/03 and rgVNJP1203/04, generated by reverse genetics in a mouse model in the presence or absence of aluminum hydroxide (alum) adjuvant. Mice immunized with rgHK213/03 vaccine produced sufficient levels of serum antibodies that were cross-reactive to recent heterologous HPAIV-H5N1 virus, A/Turkey/12/06. The vaccinated mice also elicited protective immunity against challenge with both homologous and heterologous HPAIV-H5N1 viruses. These immune responses were enhanced by addition of alum adjuvant, resulting in antigen sparing of vaccine. On the other hand, mice immunized with rgVNJP1203/04 vaccine had low levels of serum antibodies and less protective immunity than that elicited with rgHK213/03 vaccine regardless of addition of alum adjuvant. Our study suggests that rgHK213/03 vaccine is still useful as a backup vaccine for recent H5N1 viruses and that if rgVNJP1203/04 vaccine is employed, more vaccine antigen would be necessary to induce sufficient immunity.     

Protective efficacy of several vaccines against highly pathogenic H5N1 avian influenza virus under experimental conditions

Although several vaccines have been developed to protect against highly pathogenic avian influenza of subtype H5N1 'Asia' their efficiency has primarily been assessed individually. Thus, a direct comparison of their performance is still lacking. The following study was conducted to compare the protective efficacy of three commercially available inactivated vaccines based on influenza virus strains of subtypes H5N2 (vaccine A), H5N9 (vaccine B), and H5N3 (vaccine C), as well as two hemagglutinin expressing experimental vector vaccines (modified vaccinia virus Ankara-H5 and Newcastle disease virus-H5) against a lethal dose of highly pathogenic H5N1 avian influenza virus in chickens. To assess their potential as emergency vaccines, a single immunisation was performed for all vaccines, despite the recommendation of a double-vaccination schedule for commercial vaccines B and C. Overall, all vaccines induced clinical protection against challenge infection 3 weeks after immunisation. No mortality was observed in chickens immunised with vaccine A and viral shedding could not be detected. Immunisation with NDV-H5, vaccine C and MVA-H5 conferred also protection against lethal challenge. However, viral RNA was detected by real-time RT-PCR in swabs of 10%, 20% and 50% of animals, and 0%, 10% and 30% of animals, respectively, shed infectious virus. Immunisation with vaccine B was less protective since 50% of the vaccinated animals shed infectious virus after challenge and 20% of the chickens succumbed to disease. These results indicate that the NDV-H5 vectored vaccine is similarly effective as the best inactivated vaccine. Considering the advantage of live NDV which can be administered via spray or drinking water as well as the potential use of this H5 expressing vector vaccine for an easy DIVA (differentiating infected from vaccinated animals) strategy, NDV-H5 could represent an alternative for extensive vaccination against avian influenza in chickens.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.     

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent,cross-clade protection against H5N1 influenza virus in mice

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8⁺ T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.     

Ginseng and Salviae herbs play a role as immune activators and modulate immune responses during influenza virus infection

We have investigated the adjuvant roles of common herbal medicines (ginseng, Salviae) and their effects on early immune responses during influenza virus infection in a mouse model. Intranasal co-administration with inactivated influenza virus A (PR8) and ginseng or Salviae extract increased the levels of influenza virus specific antibodies and neutralizing activities compared to immunization with PR8 alone, and provided protective immunity. Salviae co-administration significantly enhanced IFN-gamma and IL-2 cytokine producing splenocytes while ginseng induced high levels of IL-4 and IL-5 cytokine producing cells after challenge infection. Cells expressing an early activation marker CD69 and levels of a pro-inflammatory cytokine IL-6 were highly elevated in lungs from na?ve mice during challenge virus infection, which might be a mechanism in lung inflammation leading to death. In contrast, immunized mice that were co-administered ginseng or Salviae modulated CD69 expressing immune cells, did not produce IL-6, and showed significant enhancement of influenza virus specific IgA antibody in lungs after challenge virus infection. Therefore, these results indicate that both ginseng and Salviae play a role as mucosal adjuvants against influenza virus as well as immuno-modulators during influenza virus infection.     

The effects of preexisting immunity to influenza on responses to influenza vectors in mice

The use of viral vectors as vaccine candidates has shown promise against a number of pathogens. However, preexisting immunity to these vectors is a concern that must be addressed when deciding which viruses are suitable for use. A number of properties, including the existence of antigenically distinct subtypes, make influenza viruses attractive candidates for use as viral vectors. Here, we evaluate the ability of influenza viral vectors containing inserts of foreign pathogens to elicit antibody and CD8⁺ T cell responses against these foreign antigens in the presence of preexisting immunity to influenza virus in mice. Specifically, responses to an H3N1-based vector expressing a 90 amino acid polypeptide derived from the protective antigen (PA) of Bacillus anthracis or an H1N1-based vector containing a CD8⁺ T cell epitope from the glycoprotein (GP) of lymphocytic choriomeningitis virus were evaluated following infections with either homosubtypic or heterosubtypic influenza viruses. We found that mice previously infected with influenza viruses, even those expressing HA and NA proteins of completely different subtypes, were severely compromised in their ability to mount an immune response against the inserted epitopes. This inhibition was demonstrated to be mediated by CD8⁺ T cells, which recognize multiple strains of influenza viruses. These CD8⁺ T cells were further shown to protect mice from a lethal challenge by a heterologous influenza subtype. The implication of these data for the use of influenza virus vectors and influenza vaccination in general are discussed.     

Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus

The pandemic virus of 2009 (2009 H1N1) continues to cause illness worldwide, especially in younger age groups. The widespread H1N1 virus infection further emphasizes the need for vaccine strategies that are effective against emerging pandemic viruses and are not dependent on the limitations of traditional egg-based technology. This report describes a recombinant influenza virus-like particle (VLP) vaccine consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza A/California/04/2009 (H1N1) virus. Influenza H1N1 VLPs with a diameter of approximately 120 nm were released into the culture medium from Sf9 insect cells infected with recombinant baculovirus coexpressing HA, NA, and M1 proteins. Purified recombinant H1N1 VLPs morphologically resembled influenza virions and exhibited biological characteristics of influenza virus, including HA and NA activities. In the ferret challenge model, 2009 influenza H1N1 VLPs elicited high-titer serum hemagglutination inhibition (HI) antibodies specific for the 2009 H1N1 virus and inhibited replication of the influenza virus in the upper and lower respiratory tract tissues following A/Mexico/4482/09 (H1N1) virus challenge. Moreover, a single 15 μg dose of H1N1 VLPs resulted in complete virus clearance in the ferret lung. These results provide support for the use of recombinant influenza VLP vaccine as an effective strategy against pandemic H1N1 virus.     

Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin

For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (LT) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, C3d, which is fused to the secreted form of hemagglutinin (sHA-mC3d3) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d3, the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d3-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination.     

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.     

Antibodies and CD4(+) T-cells mediate cross-protection against H5N1 influenza virus infection in mice after vaccination with a low pathogenic H5N2 strain

A H5N2 low pathogenic avian influenza virus (LPAIV) was isolated from a natural reservoir in Bavaria during a routine screen and was used as a vaccine strain to scrutinize the immune response involved in cross-protection after challenge infection with a H5N1 highly pathogenic avian influenza virus (HPAIV). The challenge virus was also isolated from a natural reservoir in Bavaria. Wild type, antibody deficient (muMT), CD4(-/-) and CD8(-/-) mice were infected with the apathogenic H5N2 vaccine strain and challenge infection with a 100-fold MLD(50) of the H5N1 strain was performed 80 days later. While 100% of the wild type and 100% of the CD8(-/-) mice stayed healthy, only 50% of the CD4(-/-) and none of the antibody deficient mice were protected. These results support the view that the humoral immune response and to certain extends the CD4(+) T helper cells are a prerequisite for cross-protective immunity against H5 influenza virus.     

Protection of chickens against H5N1 highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase

Attenuated vaccine strains of the alphaherpesvirus causing infectious laryngotracheitis of chickens (ILTV, gallid herpesvirus 1) can be used for mass application. Previously, we showed that live virus vaccination with recombinant ILTV expressing hemagglutinin of highly pathogenic avian influenza viruses (HPAIV) protected chickens against ILT and fowl plague caused by HPAIV carrying the corresponding hemagglutinin subtypes [Lüschow D, Werner O, Mettenleiter TC, Fuchs W. Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene. Vaccine 2001;19(30):4249–59; Veits J, Lüschow D, Kindermann K, Werner O, Teifke JP, Mettenleiter TC, et al. Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague. J Gen Virol 2003;84(12):3343–52]. However, protection against H5N1 HPAIV was not satisfactory. Therefore, a newly designed dUTPase-negative ILTV vector was used for rapid insertion of the H5-hemagglutinin, or N1-neuraminidase genes of a recent H5N1 HPAIV isolate. Compared to our previous constructs, protein expression was considerably enhanced by insertion of synthetic introns downstream of the human cytomegalovirus immediate-early promoter within the 5′-nontranslated region of the transgenes. Deletion of the viral dUTPase gene did not affect in vitro replication of the ILTV recombinants, but led to sufficient attenuation in vivo. After a single ocular immunization, all chickens developed H5- or N1-specific serum antibodies. Nevertheless, animals immunized with N1-ILTV died after subsequent H5N1 HPAIV challenge, although survival times were prolonged compared to non-vaccinated controls. In contrast, all chickens vaccinated with either H5-ILTV alone, or H5- and N1-ILTV simultaneously, survived without showing any clinical signs. Real-time RT-PCR indicated limited challenge virus replication after vaccination with H5-ILTV only, which was completely blocked after coimmunization with N1-ILTV. Thus, chickens can be protected from H5N1 HPAIV-induced disease by live vaccination with an attenuated hemagglutinin-expressing ILTV recombinant, and efficacy can be further increased by coadministration of an ILTV mutant expressing neuraminidase. Furthermore, chickens vaccinated with ILTV vectors can be easily differentiated from influenza virus-infected animals by the absence of serum antibodies against the AIV nucleoprotein.     

Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses

Immunization against conserved virus components induces broad, heterosubtypic protection against diverse influenza A viruses, providing a strategy for controlling unexpected outbreaks or pandemics until strain-matched vaccines become available. This study characterized immunization to nucleoprotein (NP) and matrix 2 (M2) by DNA priming followed by parenteral or mucosal boosting in mice and ferrets. DNA vaccination followed by boosting with antigen-matched recombinant adenovirus (rAd) or cold-adapted (ca) influenza virus provided robust protection against virulent H1N1 and H5N1 challenges. Compared to other boosts, mucosal rAd induced stronger IgA responses, more virus-specific activated T-cells in the lung, and better protection against morbidity following challenge even eight months post-boost. In ferrets, both mucosal and parenteral rAd boosting protected from lethal H5N1 challenge. These findings demonstrate potent protection by vaccination highly focused on conserved antigens and identify immune response measures in mice that differed among vaccinations and correlated with outcome.