Induction of Cross-Clade Antibody and T-Cell Responses by a Modified Vaccinia Virus Ankara-Based Influenza A(H5N1) Vaccine in a Randomized Phase 1/2a Clinical Trial.

Background: High-pathogenicity avian influenza viruses continue to circulate in poultry and wild birds and occasionally infect humans, sometimes with fatal outcomes. Development of vaccines is a priority to prepare for potential pandemics but is complicated by antigenic variation of the surface glycoprotein hemagglutinin. We report the immunological profile induced by human immunization with modified vaccinia virus Ankara (MVA) expressing the hemagglutinin gene of influenza A(H5N1) virus A/Vietnam/1194/04 (rMVA-H5). Methods: In a double-blinded phase 1/2a clinical trial, 79 individuals received 1 or 2 injections of rMVA-H5 or vector control. Twenty-seven study subjects received a booster immunization after 1 year. The breadth, magnitude, and properties of vaccine-induced antibody and T-cell responses were characterized. Results: rMVA-H5 induced broadly reactive antibody responses, demonstrated by protein microarray, hemagglutination inhibition, virus neutralization, and antibody-dependent cellular cytotoxicity assays. Antibodies cross-reacted with antigenically distinct H5 viruses, including the recently emerged subtypes H5N6 and H5N8 and the currently circulating subtype H5N1. In addition, the induction of T cells specific for H5 viruses of 2 different clades was demonstrated. Conclusions: rMVA-H5 induced immune responses that cross-reacted with H5 viruses of various clades. These findings validate rMVA-H5 as vaccine candidate against antigenically distinct H5 viruses. Clinical Trials Registration: NTR3401.

Differential Recognition of Influenza A Viruses by M158-66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues.

UNLABELLED: Natural influenza A virus infections elicit both virus-specific antibody and CD4(+) and CD8(+) T cell responses. Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158-66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158-66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8(+) T cell recognition of the M158-66 epitope. These data indicate that human influenza A viruses can impair recognition by M158-66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE: Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8(+) cytotoxic T lymphocytes (CTLs) are directed predominantly against conserved internal proteins and confer cross-protection, even against influenza A viruses of various subtypes. In some CTL epitopes, mutations occur that allow influenza A viruses to evade recognition by CTLs. However, the immunodominant HLA-A*0201-restricted M158-66 epitope does not tolerate mutations without loss of viral fitness. Here, we describe naturally occurring variations in amino acid residues outside the M158-66 epitope that influence the recognition of the epitope. These results provide novel insights into the epidemiology of influenza A viruses and their pathogenicity and may aid rational design of vaccines that aim at the induction of CTL responses.

Virus replication kinetics and pathogenesis of infection with H7N9 influenza virus in isogenic guinea pigs upon intratracheal inoculation.

Since 2013, avian influenza viruses of subtype H7N9 have been transmitted from poultry to humans in China and caused severe disease. Concerns persist over the pandemic potential of this virus and further understanding of immunity and transmission is required. The isogenic guinea pig model uniquely would allow for investigation into both. Eighteen female isogenic guinea pigs 12-16 weeks were inoculated intratracheally with either A/H7N9 virus (n=12) or PBS (n=6) and sacrificed on days 2 and 7 post-inoculation. Nasal and pharyngeal swabs were taken daily to assess viral replication kinetics and necropsies were performed to study pathogenesis. All animals showed peak virus titers in nasal secretions at day 2 post-inoculation and by day 7 post-inoculation infectious virus titers had decreased to just above detectable levels. At day 2, high virus titers were found in nasal turbinates and lungs and moderate titers in trachea and cerebrum. At day 7, infectious virus was detected in the nasal turbinates only. Histology showed moderate to severe inflammation in the entire respiratory tract and immunohistochemistry (IHC) demonstrated large numbers of viral antigen positive cells in the nasal epithelium at day 2 and fewer at day 7 post-inoculation. A moderate number of IHC positive cells was observed in the bronchi(oli) and alveoli at day 2 only. This study indicates that isogenic guinea pigs are a promising model to further study immunity to and transmission of H7N9 influenza virus.

Heterosubtypic immunity to H7N9 influenza virus in isogenic guinea pigs after infection with pandemic H1N1 virus.

Heterosubtypic immunity is defined as immune-mediated (partial) protection against an influenza virus induced by an influenza virus of another subtype to which the host has not previously been exposed. This cross-protective effect has not yet been demonstrated to the newly emerging avian influenza A viruses of the H7N9 subtype. Here, we assessed the induction of protective immunity to these viruses by infection with A(H1N1)pdm09 virus in a newly developed guinea pig model. To this end, ten female 12-16 week old strain 2 guinea pigs were inoculated intratracheally with either A(H1N1)pdm09 influenza virus or PBS (unprimed controls) followed 4 weeks later with an A/H7N9 influenza virus challenge. Nasal swabs were taken daily and animals from both groups were sacrificed on days 2 and 7 post inoculation (p.i.) with A/H7N9 virus and full necropsies were performed. Nasal virus excretion persisted until day 7 in unprimed control animals, whereas only two out of seven H1N1pdm09-primed animals excreted virus via the nose. Infectious virus was recovered from nasal turbinates, trachea and lung of all animals at day 2 p.i., but titers were lower for H1N1pdm09-primed animals, especially in the nasal turbinates. By day 7 p.i., relatively high virus titers were found in the nasal turbinates of all unprimed control animals but infectious virus was isolated from the nose of only one of four H1N1pdm09-primed animals. Animals of both groups developed inflammation of variable severity in the entire respiratory tract. Viral antigen positive cells were demonstrated in the nasal epithelium of both groups at day 2. The bronchi(oli) and alveoli of unprimed animals showed a moderate to strong positive signal at day 2, whereas H1N1pdm09-primed animals showed only minimal positivity. By day 7, only viral antigen positive cells were found after H7N9 virus infection in the nasal turbinates and the lungs of unprimed controls. Thus infection with H1N1pdm09 virus induced partially protective heterosubtypic immunity to H7N9 virus in (isogenic) guinea pigs that could not be attributed to cross-reactive virus neutralizing antibodies.

Market implementation of the MVA platform for pre-pandemic and pandemic influenza vaccines: A quantitative key opinion leader analysis.

A quantitative method is presented to rank strengths, weaknesses, opportunities, and threats (SWOT) of modified vaccinia virus Ankara (MVA) as a platform for pre-pandemic and pandemic influenza vaccines. Analytic hierarchy process (AHP) was applied to achieve pairwise comparisons among SWOT factors in order to prioritize them. Key opinion leaders (KOLs) in the influenza vaccine field were interviewed to collect a unique dataset to evaluate the market potential of this platform. The purpose of this study, to evaluate commercial potential of the MVA platform for the development of novel generation pandemic influenza vaccines, is accomplished by using a SWOT and AHP combined analytic method. Application of the SWOT-AHP model indicates that its strengths are considered more important by KOLs than its weaknesses, opportunities, and threats. Particularly, the inherent immunogenicity capability of MVA without the requirement of an adjuvant is the most important factor to increase commercial attractiveness of this platform. Concerns regarding vector vaccines and anti-vector immunity are considered its most important weakness, which might lower public health value of this platform. Furthermore, evaluation of the results of this study emphasizes equally important role that threats and opportunities of this platform play. This study further highlights unmet needs in the influenza vaccine market, which could be addressed by the implementation of the MVA platform. Broad use of MVA in clinical trials shows great promise for this vector as vaccine platform for pre-pandemic and pandemic influenza and threats by other respiratory viruses. Moreover, from the results of the clinical trials seem that MVA is particularly attractive for development of vaccines against pathogens for which no, or only insufficiently effective vaccines, are available.

A single immunization with modified vaccinia virus Ankara-based influenza virus H7 vaccine affords protection in the influenza A(H7N9) pneumonia ferret model.

Since the first reports in early 2013, >440 human cases of infection with avian influenza A(H7N9) have been reported including 122 fatalities. After the isolation of the first A(H7N9) viruses, the nucleotide sequences became publically available. Based on the coding sequence of the influenza virus A/Shanghai/2/2013 hemagglutinin gene, a codon-optimized gene was synthesized and cloned into a recombinant modified vaccinia virus Ankara (MVA). This MVA-H7-Sh2 viral vector was used to immunize ferrets and proved to be immunogenic, even after a single immunization. Subsequently, ferrets were challenged with influenza virus A/Anhui/1/2013 via the intratracheal route. Unprotected animals that were mock vaccinated or received empty vector developed interstitial pneumonia characterized by a marked alveolitis, accompanied by loss of appetite, weight loss, and heavy breathing. In contrast, animals vaccinated with MVA-H7-Sh2 were protected from severe disease.

Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine: a randomised, double-blind phase 1/2a clinical trial.

BACKGROUND: Modified vaccinia virus Ankara (MVA) is a promising viral vector platform for the development of an H5N1 influenza vaccine. Preclinical assessment of MVA-based H5N1 vaccines showed their immunogenicity and safety in different animal models. We aimed to assess the safety and immunogenicity of the MVA-haemagglutinin-based H5N1 vaccine MVA-H5-sfMR in healthy individuals. METHODS: In a single-centre, double-blind phase 1/2a study, young volunteers (aged 18-28 years) were randomly assigned with a computer-generated list in equal numbers to one of eight groups and were given one injection or two injections intramuscularly at an interval of 4 weeks of a standard dose (10(8) plaque forming units [pfu]) or a ten times lower dose (10(7) pfu) of the MVA-H5-sfMR (vector encoding the haemagglutinin gene of influenza A/Vietnam/1194/2004 virus [H5N1 subtype]) or MVA-F6-sfMR (empty vector) vaccine. Volunteers and physicians who examined and administered the vaccine were masked to vaccine assignment. Individuals who received the MVA-H5-sfMR vaccine were eligible for a booster immunisation 1 year after the first immunisation. Primary endpoint was safety. Secondary outcome was immunogenicity. The trial is registered with the Dutch Trial Register, number NTR3401. FINDINGS: 79 of 80 individuals who were enrolled completed the study. No serious adverse events were identified. 11 individuals reported severe headache and lightheadedness, erythema nodosum, respiratory illness (accompanied by influenza-like symptoms), sore throat, or injection-site reaction. Most of the volunteers had one or more local (itch, pain, redness, and swelling) and systemic reactions (rise in body temperature, headache, myalgia, arthralgia, chills, malaise, and fatigue) after the first, second, and booster immunisations. Individuals who received the 10(7) dose had fewer systemic reactions. The MVA-H5-sfMR vaccine at 10(8) pfu induced significantly higher antibody responses after one and two immunisations than did 10(7) pfu when assessed with haemagglutination inhibition geometric mean titre at 8 weeks against H5N1 A/Vietnam/1194/2004 (30·2 [SD 3·8] vs 9·2 [2·3] and 108·1 [2·4] vs 15·8 [3·2]). 27 of 39 eligible individuals were enrolled in the booster immunisation study. A single shot of MVA-H5-sfMR 10(8) pfu prime immunisation resulted in higher antibody responses after the booster immunisation than did two shots of MVA-H5-sfMR at the ten times lower dose. INTERPRETATION: The MVA-based H5N1 vaccine was well tolerated and immunogenic and therefore the vaccine candidates arising from the MVA platform hold great promise for rapid development in response to a future influenza pandemic threat. However, the immunogenicity of this vaccine needs to be compared with conventional H5N1 inactivated non-adjuvanted vaccine candidates in head-to-head clinical trials. FUNDING: European Research Council.

Novel G3/DT adjuvant promotes the induction of protective T cells responses after vaccination with a seasonal trivalent inactivated split-virion influenza vaccine.

Vaccines used against seasonal influenza are poorly effective against influenza A viruses of novel subtypes that may have pandemic potential. Furthermore, pre(pandemic) influenza vaccines are poorly immunogenic, which can be overcome by the use of adjuvants. A limited number of adjuvants has been approved for use in humans, however there is a need for alternative safe and effective adjuvants that can enhance the immunogenicity of influenza vaccines and that promote the induction of broad-protective T cell responses. Here we evaluated a novel nanoparticle, G3, as an adjuvant for a seasonal trivalent inactivated influenza vaccine in a mouse model. The G3 adjuvant was formulated with or without steviol glycosides (DT, for diterpenoid). The use of both formulations enhanced the virus-specific antibody response to all three vaccine strains considerably. The adjuvants were well tolerated without any signs of discomfort. To assess the protective potential of the vaccine-induced immune responses, an antigenically distinct influenza virus strain, A/Puerto Rico/8/34 (A/PR/8/34), was used for challenge infection. The vaccine-induced antibodies did not cross-react with strain A/PR/8/34 in HI and VN assays. However, mice immunized with the G3/DT-adjuvanted vaccine were partially protected against A/PR/8/34 infection, which correlated with the induction of anamnestic virus-specific CD8(+) T cell responses that were not observed with the use of G3 without DT. Both formulations induced maturation of human dendritic cells and promoted antigen presentation to a similar extent. In conclusion, G3/DT is a promising adjuvant formulation that not only potentiates the antibody response induced by influenza vaccines, but also induces T cell immunity which could afford broader protection against antigenically distinct influenza viruses.

Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases.

Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses.

Human cytotoxic T lymphocytes directed to seasonal influenza A viruses cross-react with the newly emerging H7N9 virus.

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus.

Infection of the upper respiratory tract with seasonal influenza A(H3N2) virus induces protective immunity in ferrets against infection with A(H1N1)pdm09 virus after intranasal, but not intratracheal, inoculation.

The clinical symptoms caused by infection with influenza A virus vary widely and depend on the strain causing the infection, the dose and route of inoculation, and the presence of preexisting immunity. In most cases, seasonal influenza A viruses cause relatively mild upper respiratory tract disease, while sometimes patients develop an acute severe pneumonia. Heterosubtypic immunity induced by previous infections with influenza A viruses may dampen the development of clinical symptoms caused by infection with influenza A viruses of another subtype, as is the case during influenza pandemics. Here we show that ferrets acquire protective immunity after infection of the upper respiratory tract with a seasonal influenza A(H3N2) virus against subsequent infection with influenza A(H1N1)pdm09 virus inoculated by the intranasal route. However, protective heterosubtypic immunity was afforded locally, since the prior infection with the A(H3N2) virus did not provide protection against the development of pneumonia induced after intratracheal inoculation with the A(H1N1)pdm09 virus. Interestingly, some of these animals developed more severe disease than that observed in naïve control animals. These findings are of interest in light of the development of so-called universal influenza vaccines that aim at the induction of cross-reactive T cell responses.

Human T-cells directed to seasonal influenza A virus cross-react with 2009 pandemic influenza A (H1N1) and swine-origin triple-reassortant H3N2 influenza viruses.

Virus-specific CD8(+) T-cells contribute to protective immunity against influenza A virus (IAV) infections. As the majority of these cells are directed to conserved viral proteins, they may afford protection against IAVs of various subtypes. The present study assessed the cross-reactivity of human CD8(+) T-lymphocytes, induced by infection with seasonal A (H1N1) or A (H3N2) influenza virus, with 2009 pandemic influenza A (H1N1) virus [A(H1N1)pdm09] and swine-origin triple-reassortant A (H3N2) [A(H3N2)v] viruses that are currently causing an increasing number of human cases in the USA. It was demonstrated that CD8(+) T-cells induced after seasonal IAV infections exerted lytic activity and produced gamma interferon upon in vitro restimulation with A(H1N1)pdm09 and A(H3N2)v influenza A viruses. Furthermore, CD8(+) T-cells directed to A(H1N1)pdm09 virus displayed a high degree of cross-reactivity with A(H3N2)v viruses. It was concluded that cross-reacting T-cells had the potential to afford protective immunity against A(H1N1)pdm09 viruses during the pandemic and offer some degree of protection against infection with A(H3N2)v viruses.

Evasion of influenza A viruses from innate and adaptive immune responses.

The influenza A virus is one of the leading causes of respiratory tract infections in humans. Upon infection with an influenza A virus, both innate and adaptive immune responses are induced. Here we discuss various strategies used by influenza A viruses to evade innate immune responses and recognition by components of the humoral and cellular immune response, which consequently may result in reduced clearing of the virus and virus-infected cells. Finally, we discuss how the current knowledge about immune evasion can be used to improve influenza A vaccination strategies.

Easy and efficient protocols for working with recombinant vaccinia virus MVA.

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient strain of vaccinia virus that is increasingly used as vector for expression of recombinant genes in the research laboratory and in biomedicine for vaccine development. Major benefits of MVA include the clear safety advantage compared to conventional vaccinia viruses, the longstanding experience in the genetic engineering of the virus, and the availability of established procedures for virus production at an industrial scale. MVA vectors can be handled under biosafety level 1 conditions, and a multitude of recombinant MVA vaccines has proven to be immunogenic and protective when delivering various heterologous antigens in animals and humans. In this chapter we provide convenient state-of-the-art protocols for generation, amplification, and purification of recombinant MVA viruses. Importantly, we include methodology for rigid quality control to obtain best possible vector viruses for further investigations including clinical evaluation.

Annual influenza vaccination affects the development of heterosubtypic immunity.

Annual vaccination of healthy children >6 months of age against seasonal influenza has been recommended by public health authorities of some countries. However, currently used seasonal vaccines provide only limited protection against (potentially) pandemic influenza viruses. Furthermore, we recently hypothesized that annual vaccination may hamper the development of cross-reactive immunity against influenza A viruses of novel subtypes, that would otherwise be induced by natural infection. Here we summarize our findings in animal models in which we demonstrated that vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity against highly pathogenic avian influenza A/H5N1 virus, otherwise induced by a prior infection with influenza A/H3N2 virus. The reduction of heterosubtypic immunity correlated with reduced virus-specific CD8+ T cell responses. An additional study was performed in humans, in which we collected peripheral blood mononuclear cells from annually vaccinated children with cystic fibrosis (CF) and age-matched unvaccinated healthy control children to study the virus-specific T cell response. An age-related increase of the virus-specific CD8+ T cell response was observed in unvaccinated children that was absent in vaccinated children with CF. These findings highlight the importance of the development of vaccines that provide protection against influenza A viruses of all subtypes.

The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets.

The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space-including along the olfactory route-and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.

Cross-protective immunity against influenza pH1N1 2009 viruses induced by seasonal influenza A (H3N2) virus is mediated by virus-specific T-cells.

Influenza A (H1N1) viruses of swine origin were introduced into the human population in 2009 and caused a pandemic. The disease burden in the elderly was relatively low, which was attributed to the presence of cross-reacting serum antibodies in this age group, which were raised against seasonal influenza A (H1N1) viruses that circulated before 1957. It has also been described how infection with heterosubtypic influenza viruses can induce some degree of protection against infection by a novel strain of influenza virus. Here, we assess the extent of protective immunity against infection with the 2009 influenza A (H1N1) pandemic influenza virus that is afforded by infection with a seasonal influenza A (H3N2) virus in mice. Mice that experienced a primary A (H3N2) influenza virus infection displayed reduced weight loss after challenge infection and cleared the 2009 influenza A (H1N1) virus infection more rapidly. To elucidate the correlates of protection of this heterosubtypic immunity to pandemic H1N1 virus infection, adoptive transfer experiments were carried out by using selected post-infection lymphocyte populations. Virus-specific CD8(+) T-cells in concert with CD4(+) T-cells were responsible for the observed protection. These findings may not only provide an explanation for epidemiological differences in the incidence of severe pandemic H1N1 infections, they also indicate that the induction of cross-reactive virus-specific CD8(+) and CD4(+) T-cell responses may be a suitable approach for the development of universal influenza vaccines.

Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation.

Most patients infected with highly pathogenic avian influenza A/H5N1 virus develop severe pneumonia resulting in acute respiratory distress syndrome, with extrarespiratory disease as an uncommon complication. Intranasal inoculation of ferrets with influenza A/H5N1 virus causes lesions in both the respiratory tract and extrarespiratory organs (primarily brain). However, the route of spread to extrarespiratory organs and the relative contribution of extrarespiratory disease to pathogenicity are largely unknown. In the present study, we characterized lesions in the respiratory tract and central nervous system (CNS) of ferrets (n = 8) inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). By 7 days after inoculation, only 3 of 8 ferrets had a mild or moderate bronchointerstitial pneumonia. In contrast, all 8 ferrets had moderate or severe CNS lesions, characterized by meningoencephalitis, choroiditis, and ependymitis, and centered on tissues adjoining the cerebrospinal fluid. These findings indicate that influenza A/H5N1 virus spread directly from nasal cavity to brain, and that CNS lesions contributed more than pulmonary lesions to the pathogenicity of influenza A/H5N1 virus infection in ferrets. In comparison, intratracheal inoculation of ferrets with the same virus reproducibly caused severe bronchointerstitial pneumonia. The method of virus inoculation requires careful consideration in the design of ferret experiments as a model for influenza A/H5N1 in humans.