http://www.jci.org/articles/view/32460
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Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals
<nobr> Laurel Yong-Hwa Lee </nobr><sup>1</sup>, <nobr> Do Lien Anh Ha </nobr><sup>2</sup>, <nobr> Cameron Simmons </nobr><sup>2</sup>, <nobr> Menno D. de Jong</nobr><sup> 2</sup>, <nobr> Nguyen Van Vinh Chau </nobr><sup>2</sup>, <nobr> Reto Schumacher </nobr><sup>1</sup>, <nobr> Yan Chun Peng</nobr><sup> 1</sup>, <nobr> Andrew J. McMichael </nobr><sup>1 </sup>, <nobr> Jeremy J. Farrar </nobr><sup>2</sup>, <nobr> Geoffrey L. Smith </nobr><sup>3</sup>, <nobr> Alain R.M. Townsend </nobr><sup>4</sup>, <nobr> Brigitte A. Askonas </nobr><sup>1</sup>, <nobr> Sarah Rowland-Jones </nobr><sup>1</sup> and <nobr> Tao Dong </nobr><sup>1</sup>
<sup>1</sup>MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
<sup>2</sup>Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
<sup>3</sup>Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.
<sup>4</sup>Molecular Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
Address correspondence to: Tao Dong or Andrew J. McMichael, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222336; Fax: 44-1865-222600. E-mail: tao.dong@imm.ox.ac.uk (T. Dong); andrew.mcmichael@ndm.ox.ac.uk (A.J. McMichael).
First published September 18, 2008
Received for publication April 20, 2007, and accepted in revised form July 30, 2008.
The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4<sup>+</sup> and CD8<sup>+</sup> memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells isolated from the majority of participants exhibited human influenza–specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4<sup>+</sup> and CD8<sup>+</sup> T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4<sup>+</sup> and CD8<sup>+</sup> T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell–mediated immunity may confer broad protection against avian and human influenza A
viruses.
Introduction
As transmission of highly virulent avian influenza A (H5N1) viruses to humans continues to occur in large parts of the world (385 reported cases including 243 deaths as of June 2008; ref. 1), there remains real concern that such a virus might mutate or reassort with a human influenza virus and hence acquire efficient human to human transmission and cause an influenza pandemic (2).
Currently available influenza vaccines induce antibodies against the viral surface glycoproteins HA and neuraminidase (NA), with the HA being by far the more important. While neutralizing antibodies can provide sterilizing immunity by blocking infection, this protection is only specific for the immunizing and closely related strains and is ineffective against heterologous strains with serologically distinct HAs. H5N1 influenza viruses continue to evolve and change antigenically (3, 4), and the WHO has reported the emergence of H5 HAs that are antigenically distinct from the H5 HAs used for vaccine production (5). H5N1 vaccine candidates developed so far are based on the clade 1 viruses (6–9). Induction of cross-reactive neutralizing antibodies against several clade 2 H5N1 strains by an adjuvant clade 1 vaccine has been documented recently (10). However, drastic antigenic changes are inevitable if and when the H5N1 virus adapts to transmit efficiently between humans. A pandemic strain may even be of another subtype. Thus, antibody-based vaccines developed against current H5N1 strains alone are unlikely to confer adequate protection against a pandemic strain, and there is an urgent need for another vaccine strategy.
An alternative or additional approach would be to stimulate T cell–mediated immunity, particularly virus-specific CD8<sup>+</sup> CTLs that target the highly conserved internal proteins, as well as CD4<sup>+</sup> T cells that provide help for the generation and maintenance of CD8 memory T cells (reviewed in refs. 11, 12). While CTLs do not prevent the establishment of infection, there is good evidence in mice and humans that T cells provide partial protection against influenza by promoting viral clearance and reducing the severity of symptoms (13–15). Influenza-specific CTLs mostly target internal proteins (16–21) and can provide partial protection across heterologous strains by targeting such conserved regions (22).
In view of the annual exposure to seasonal human influenza viruses (predominantly H3N2 and H1N1), most healthy adults may possess immunological memory against influenza virus. Indeed, influenza A virus–specific and mostly cross-reactive CD4 <sup>+</sup> (23, 24) and CD8 <sup>+</sup> (24–27) memory T cell responses have been demonstrated in humans. Recognition of target cells infected with swine or avian strains by T cell cultures derived from healthy individuals has also been reported (28, 29).
Despite the evidence supporting potential heterosubtypic protection that influenza A–specific T cells may confer, our understanding of T cell–mediated immunity to influenza infection remains incomplete, particularly in the context of human infection with avian influenza A (H5N1) viruses (30). Such limitation stems largely from the technology available for earlier studies. As the frequency of circulating influenza-specific T cells decline after recovery, in vitro stimulation of T cell cultures was necessary in most studies to expand the antigen-specific populations to a detectable level. A shortcoming of this method is that T cells expanded by culture in the presence of antigen may consist of preferentially expanded populations that do not truly represent the overall memory T cell repertoire in vivo. This limitation can now be overcome by using IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, in which fresh PBMCs are stimulated with overlapping peptides representing the whole virus proteome set out in a 3-dimensional matrix system. This can provide a more comprehensive picture of the virus-specific memory T cell populations circulating in peripheral blood.
In the present study, the overall influenza A virus–specific memory T cell response and the extent of cross-reactivity to H5N1 in healthy individuals from the United Kingdom (UK) and Viet Nam (n = 48 and 42, respectively) were evaluated ex vivo using the overlapping peptides spanning the full proteome of the influenza A/New York 232/2004 (H3N2) and A/Viet Nam/CL26/2004 (H5N1) strains. The H5N1 cross-reactive T cell responses were further examined against target cells infected with the recombinant vaccinia viruses (rVACVs) expressing the matrix protein 1 (M1) or nucleoprotein (NP) of the H5N1 strain. We report here that the majority of healthy individuals possessed influenza A–specific CD4<sup>+</sup> and CD8<sup>+</sup> memory T cell populations that were broadly cross-reactive to H5N1 internal proteins and discuss the relevance of these observations to a pre-pandemic vaccine strategy.
free full text
Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals
<nobr> Laurel Yong-Hwa Lee </nobr><sup>1</sup>, <nobr> Do Lien Anh Ha </nobr><sup>2</sup>, <nobr> Cameron Simmons </nobr><sup>2</sup>, <nobr> Menno D. de Jong</nobr><sup> 2</sup>, <nobr> Nguyen Van Vinh Chau </nobr><sup>2</sup>, <nobr> Reto Schumacher </nobr><sup>1</sup>, <nobr> Yan Chun Peng</nobr><sup> 1</sup>, <nobr> Andrew J. McMichael </nobr><sup>1 </sup>, <nobr> Jeremy J. Farrar </nobr><sup>2</sup>, <nobr> Geoffrey L. Smith </nobr><sup>3</sup>, <nobr> Alain R.M. Townsend </nobr><sup>4</sup>, <nobr> Brigitte A. Askonas </nobr><sup>1</sup>, <nobr> Sarah Rowland-Jones </nobr><sup>1</sup> and <nobr> Tao Dong </nobr><sup>1</sup>
<sup>1</sup>MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
<sup>2</sup>Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam.
<sup>3</sup>Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.
<sup>4</sup>Molecular Immunology Group, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
Address correspondence to: Tao Dong or Andrew J. McMichael, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Phone: 44-1865-222336; Fax: 44-1865-222600. E-mail: tao.dong@imm.ox.ac.uk (T. Dong); andrew.mcmichael@ndm.ox.ac.uk (A.J. McMichael).
First published September 18, 2008
Received for publication April 20, 2007, and accepted in revised form July 30, 2008.
The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4<sup>+</sup> and CD8<sup>+</sup> memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells isolated from the majority of participants exhibited human influenza–specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4<sup>+</sup> and CD8<sup>+</sup> T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4<sup>+</sup> and CD8<sup>+</sup> T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell–mediated immunity may confer broad protection against avian and human influenza A
viruses.
Introduction
As transmission of highly virulent avian influenza A (H5N1) viruses to humans continues to occur in large parts of the world (385 reported cases including 243 deaths as of June 2008; ref. 1), there remains real concern that such a virus might mutate or reassort with a human influenza virus and hence acquire efficient human to human transmission and cause an influenza pandemic (2).
Currently available influenza vaccines induce antibodies against the viral surface glycoproteins HA and neuraminidase (NA), with the HA being by far the more important. While neutralizing antibodies can provide sterilizing immunity by blocking infection, this protection is only specific for the immunizing and closely related strains and is ineffective against heterologous strains with serologically distinct HAs. H5N1 influenza viruses continue to evolve and change antigenically (3, 4), and the WHO has reported the emergence of H5 HAs that are antigenically distinct from the H5 HAs used for vaccine production (5). H5N1 vaccine candidates developed so far are based on the clade 1 viruses (6–9). Induction of cross-reactive neutralizing antibodies against several clade 2 H5N1 strains by an adjuvant clade 1 vaccine has been documented recently (10). However, drastic antigenic changes are inevitable if and when the H5N1 virus adapts to transmit efficiently between humans. A pandemic strain may even be of another subtype. Thus, antibody-based vaccines developed against current H5N1 strains alone are unlikely to confer adequate protection against a pandemic strain, and there is an urgent need for another vaccine strategy.
An alternative or additional approach would be to stimulate T cell–mediated immunity, particularly virus-specific CD8<sup>+</sup> CTLs that target the highly conserved internal proteins, as well as CD4<sup>+</sup> T cells that provide help for the generation and maintenance of CD8 memory T cells (reviewed in refs. 11, 12). While CTLs do not prevent the establishment of infection, there is good evidence in mice and humans that T cells provide partial protection against influenza by promoting viral clearance and reducing the severity of symptoms (13–15). Influenza-specific CTLs mostly target internal proteins (16–21) and can provide partial protection across heterologous strains by targeting such conserved regions (22).
In view of the annual exposure to seasonal human influenza viruses (predominantly H3N2 and H1N1), most healthy adults may possess immunological memory against influenza virus. Indeed, influenza A virus–specific and mostly cross-reactive CD4 <sup>+</sup> (23, 24) and CD8 <sup>+</sup> (24–27) memory T cell responses have been demonstrated in humans. Recognition of target cells infected with swine or avian strains by T cell cultures derived from healthy individuals has also been reported (28, 29).
Despite the evidence supporting potential heterosubtypic protection that influenza A–specific T cells may confer, our understanding of T cell–mediated immunity to influenza infection remains incomplete, particularly in the context of human infection with avian influenza A (H5N1) viruses (30). Such limitation stems largely from the technology available for earlier studies. As the frequency of circulating influenza-specific T cells decline after recovery, in vitro stimulation of T cell cultures was necessary in most studies to expand the antigen-specific populations to a detectable level. A shortcoming of this method is that T cells expanded by culture in the presence of antigen may consist of preferentially expanded populations that do not truly represent the overall memory T cell repertoire in vivo. This limitation can now be overcome by using IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, in which fresh PBMCs are stimulated with overlapping peptides representing the whole virus proteome set out in a 3-dimensional matrix system. This can provide a more comprehensive picture of the virus-specific memory T cell populations circulating in peripheral blood.
In the present study, the overall influenza A virus–specific memory T cell response and the extent of cross-reactivity to H5N1 in healthy individuals from the United Kingdom (UK) and Viet Nam (n = 48 and 42, respectively) were evaluated ex vivo using the overlapping peptides spanning the full proteome of the influenza A/New York 232/2004 (H3N2) and A/Viet Nam/CL26/2004 (H5N1) strains. The H5N1 cross-reactive T cell responses were further examined against target cells infected with the recombinant vaccinia viruses (rVACVs) expressing the matrix protein 1 (M1) or nucleoprotein (NP) of the H5N1 strain. We report here that the majority of healthy individuals possessed influenza A–specific CD4<sup>+</sup> and CD8<sup>+</sup> memory T cell populations that were broadly cross-reactive to H5N1 internal proteins and discuss the relevance of these observations to a pre-pandemic vaccine strategy.
