Joost H. C. M. Kreijtz

Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus.

ABSTRACT Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.

Immune responses to influenza virus infection

Influenza viruses cause annual outbreaks of respiratory tract infection with attack rates of 5-10%. This means that humans are infected repeatedly with intervals of, on average, 10-20 years. Upon each infection subjects develop innate and adaptive immune responses which aim at clearing the infection. Strain-specific antibody responses are induced, which exert selective pressure on circulating influenza viruses and which drive antigenic drift of seasonal influenza viruses, especially in the hemagglutinin molecule. This antigenic drift necessitates updating of seasonal influenza vaccines regularly in order to match the circulating strains. Upon infection also virus-specific T cell responses are induced, including CD4+ T helper cells and CD8+ cytotoxic T cells. These cells are mainly directed to conserved proteins and therefore display cross-reactivity with a variety of influenza A viruses of different subtypes. T cell mediated immunity therefore may contribute to so-called heterosubtypic immunity and may afford protection against antigenically distinct, potentially pandemic influenza viruses. At present, novel viral targets are identified that may help to develop broad-protective vaccines. Here we review the various arms of the immune response to influenza virus infections and their viral targets and discuss the possibility of developing universal vaccines. The development of such novel vaccines would imply that also new immune correlates of protection need to be established in order to facilitate assessment of vaccine efficacy.

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.

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.

Recombinant Modified Vaccinia Virus Ankara–Based Vaccine Induces Protective Immunity in Mice against Infection with Influenza Virus H5N1

Since 2003, the number of human cases of infections with highly pathogenic avian influenza viruses of the H5N1 subtype is still increasing, and, therefore, the development of safe and effective vaccines is considered a priority. However, the global production capacity of conventional vaccines is limited and insufficient for a worldwide vaccination campaign. In the present study, an alternative H5N1 vaccine candidate based on the replication-deficient modified vaccinia virus Ankara (MVA) was evaluated. C57BL/6J mice were immunized twice with MVA expressing the hemagglutinin (HA) gene from influenza virus A/Hongkong/156/97 (MVA-HA-HK/97) or A/Vietnam/1194/04 (MVA-HA-VN/04). Subsequently, recombinant MVA-induced protective immunity was assessed after challenge infection with 3 antigenically distinct strains of H5N1 influenza viruses: A/Hongkong/156/97, A/Vietnam/1194/04, and A/Indonesia/5/05. Our data suggest that recombinant MVA expressing the HA of influenza virus A/Vietnam/1194/04 is a promising alternative vaccine candidate that could be used for the induction of protective immunity against various H5N1 influenza strains.

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus.

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1). Prior infection with influenza virus A/Hong Kong/2/68 reduced clinical signs, body weight loss, mortality and virus replication in the lungs as compared to naive mice infected with HPAI virus A/Indonesia/5/05. Priming by infection with respiratory syncytial virus, a non-related virus did not have a beneficial effect on the outcome of A/H5N1 infections, indicating that adaptive immune responses were responsible for the protective effect. In mice primed by infection with influenza A/H3N2 virus cytotoxic T lymphocytes (CTL) specific for NP(366-374) epitope ASNENMDAM and PA(224-232) SCLENFRAYV were observed. A small proportion of these CTL was cross-reactive with the peptide variant derived from the influenza A/H5N1 virus (ASNENMEVM and SSLENFRAYV respectively) and upon challenge infection with the influenza A/H5N1 virus cross-reactive CTL were selectively expanded. These CTL, in addition to those directed to conserved epitopes, shared by the influenza A/H3N2 and A/H5N1 viruses, most likely contributed to accelerated clearance of the influenza A/H5N1 virus infection. Although also other arms of the adaptive immune response may contribute to heterosubtypic immunity, the induction of virus-specific CTL may be an attractive target for development of broad protective vaccines. Furthermore the existence of pre-existing heterosubtypic immunity may dampen the impact a future influenza pandemic may have.

Vaccination against human influenza A/H3N2 virus prevents the induction of heterosubtypic immunity against lethal infection with avian influenza A/H5N1 virus.

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains. Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza. The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.

Recombinant Modified Vaccinia Virus Ankara Expressing the Hemagglutinin Gene Confers Protection against Homologous and Heterologous H5N1 Influenza Virus Infections in Macaques

BACKGROUND Highly pathogenic avian influenza viruses of the H5N1 subtype have been responsible for an increasing number of infections in humans since 2003. More than 60% of infected individuals die, and new infections are reported frequently. In light of the pandemic threat caused by these events, the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the hemagglutinin (HA) gene of H5N1 viruses is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous H5N1 influenza virus in mice. METHODS In the present study, we evaluated a recombinant MVA vector expressing the HA gene of H5N1 influenza virus A/Vietnam/1194/04 (MVA-HA-VN/04) in nonhuman primates. Cynomolgus macaques were immunized twice and then were challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity. RESULTS Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia. CONCLUSION Therefore, MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic H5N1 avian influenza viruses in humans.

Yearly influenza vaccinations: a double- edged sword?

Yearly vaccination against seasonal influenza viruses is recommended for certain individuals at high risk of complications associated with influenza. It has been recommended in some countries, including the USA, that all children aged 6-59 months are vaccinated against seasonal influenza. However, it has been shown-mainly in animals-that infection with influenza A viruses can induce protective immunity to influenza A viruses of other unrelated subtypes. This so-called heterosubtypic immunity does not provide full protection, but can limit virus replication and reduce morbidity and mortality of the host. This type of immunity might be relevant to human beings when a new subtype of influenza A virus is introduced into the population, such as the new influenza A H1N1 virus responsible for the present influenza pandemic and the highly pathogenic avian influenza H5N1 viruses that are causing an ever increasing number of human infections with high mortality rates. Preventing infection with seasonal influenza viruses by vaccination might prevent the induction of heterosubtypic immunity to pandemic strains, which might be a disadvantage to immunologically naive people-eg, infants.

The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets

ABSTRACT 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.