Bragstad
Karoline Bragstad y otros, 2010. (Ir a Personas. Cosas. Cronología. Geografía. Fuentes.)
Influenza Other Respir Viruses. 2011 Jan; 5(1): 13–23.
Published online 2010 Nov 3. doi: 10.1111/j.1750-2659.2010.00177.x
PMCID: PMC4941650
Pandemic influenza 1918 H1N1 and 1968 H3N2 DNA vaccines induce cross‐reactive immunity in ferrets against infection with viruses drifted for decades
Karoline Bragstad, 1 Cyril J. Martel, 2 Joakim S. Thomsen, 1 Kim L. Jensen, 2 Lars P. Nielsen, 1 Bent Aasted, 2 and Anders Fomsgaard 1
1Department of Virology, Statens Serum Institut, Copenhagen, Denmark.
2Faculty of Life Sciences, Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark.
Anders Fomsgaard, Chief of laboratory, Department of Virology, Statens Serum Institut, Room 327/85, Artillerivej 5, DK‐2300 Copenhagen S, Denmark. E‐mail: AFO@ssi.dkkd
Copyright © 2010 Blackwell Publishing Ltd
The most severe influenza to date was the 1918 H1N1 ‘Spanish flu’, which killed at least 50 million people worldwide during 1918 and 1919. 9 Based on preserved specimens, all genes have been genetically characterised and the entire virus has been reconstructed. 10 This provides a unique opportunity to elucidate the mechanisms of pathogenesis, but also any unique immunogenic properties of this first case of the pandemic strain. Recently, a lifelong specific immunity to the 1918 H1N1 virus was shown in some individuals born in or before 1915. 11 We hypothesise that employing the original pandemic 1918 H1N1 and 1968 H3N2 strains as DNA vaccines may induce similar long‐time protection, but also cross‐immune protection against long‐time drifted viruses within the same subtype.
The surprising cross‐reactive immunity observed after DNA vaccination with genes from pandemic 1918 H1N1 and 1968 H3N2 may partly be explained by the non‐adapted genes themselves 28 and/or by the intrinsic ability of the optimised DNA vaccines to induce relevant B‐cell and T‐cell immune responses. 29 In this study, we have only investigated the humoral immune response. The contribution of the cellular immunity will require further studies. The DNA vaccine encoding only the internal proteins NP and M from 1918 enabled ferrets to clear a challenge infection by the extensively drifted 1999 H1N1 virus more efficiently than the conventional trivalent protein vaccine homologous to the H1N1 challenge virus. Also, the 1918 H1N1 DNA vaccine was able to clear the contemporary virus infection despite no measurable cross‐reactive HI titres at the day of challenge. These results demonstrate that cross‐reactive immunity is mediated by mechanisms beyond neutralising antibodies. T‐cell immunity by the NP/M DNA vaccine may play an important role in this cross‐reactive immunity as the M and NP proteins are highly conserved. Therefore, the addition of NP and M genes in the DNA vaccines may improve cross‐protection by different immunological mechanisms similar to a natural infection. 30 , 31
The HA gene was included in the DNA vaccines to prevent and neutralise the infection whereas antibodies against the NA should prevent release of newly synthesised virus particles, as demonstrated by others. 32 , 33 The cross‐reactivity of NA antibodies towards drifted viruses may be explained by the high identity within the NA subtypes. 34 , 35 An NA DNA vaccine based on the Aichi 1968 (H3N2) virus has previously induced complete protection against homologous and heterologous virus challenge in mice. 36 Also, humans with immunity against human N1 virus are able to respond against the highly pathogenic avian H5N1 virus. 37 Cross‐reactivity within subtypes of influenza viruses is well known. Studies have shown that a vaccine prepared from the A/New Caledonia/20/99 (H1N1) virus was able to provide some degree of protection against a lethal 1918 recombinant virus challenge in mice, which could not be explained by either HI or neutralising antibodies. 28 People exposed to H1N1 viruses in the late 1940s had detectable antibodies against H1N1 in the 1978 outbreak. 38 Also, some people naturally infected with the 1918 H1N1 virus between 1928 and 1933 still have antibody titres against the 1918 viruses. 39 Therefore, a vaccine inducing cross‐reactivity would be of great value in preventing influenza.
The A/South Carolina/1/18 and A/New Caledonia/20/99 H1N1 viruses in this study are 18·4% different in the HA1 protein and possess eight substitutions at residues involved in the antigenic sites (defined by Caton, et al., 40 ) while the NAs differ by 13%. The A/Aichi/2/68 and A/Wisconsin/67/05 H3N2 viruses differ by 18·2% in the HA1 protein and by 13% in the NA protein. It is striking that the pandemic H3N2 DNA vaccines are able to induce cross‐reactivity against a strain that has discrepancy in 49 of a total of 129 residues involved in the HA antigenic sites. Cross‐protection and cross‐reactivity by DNA vaccines against viruses differing by 11–13% in the HA1 region have been demonstrated by others. 41 , 42 , 43 Altogether, the overall difference between the glycoproteins may play a minor role compared to the location of the discrepancies. 22 We speculate that the pandemic antigens may possess the ability to induce broad cross‐reacting recall antibody responses as these have not yet accumulated glycosylations camouflaging epitopes, and therefore, more conserved epitopes may be available for immune induction. 44 , 45 A recent paper by Rechert et al., 23 explains the protection against the novel 2009 H1N1 in elderly as pre‐exposure to a H1N1 virus with similar glycosylation patterns as the novel H1N1 virus. Glycosylation sequons exceeding the conserved ones might mask immunogenicity. We have shown that the pandemic HA genes in our DNA vaccines possess the least amount of glycosylations. These sequons have become conserved since then, while preceeding strains have gained additional sequons, mainly in the globular head. 22 , 23 , 24 Therefore, the cross‐reactivity of the pandemic DNA vaccine genes with more recent strains could be related to the limited number of glycosylation sites.
Influenza broad spectrum neutralising antibodies 46 as well as common antigenic sites 47 , 48 have recently been identified. Our results indicate the presence of common epitopes in the 1918, 1947 and 1999 H1N1 viruses. Thus, the 1918 H1N1 DNA vaccination, followed by 1947 H1N1 virus challenge, induced ELISA IgG antibodies cross‐reacting strongly with the 1999 H1N1 antigens. Unexpectedly, influenza 1999 H1N1‐specific IgG antibodies were also induced after 1918 H1N1 DNA vaccination, and in fact, the recall response towards the 1999 virus challenge was comparable to the response observed after vaccination with the conventional trivalent homologous protein vaccine. Likewise, vaccination with 1968 H3N2 DNA induced cross‐reactive recall IgG antibody responses towards contemporary 2005 H3N2 influenza antigen after infection with the 2005 H3N2 virus. Haemagglutinin inhibitory antibodies neutralising the influenza virus only bind a few specific epitopes on the HA protein 32 while total ELISA IgG antibodies have a broader range of binding sites, both on HA and NA. In this study, the DNA vaccines were more effective at inducing HI titres after vaccination than the conventional trivalent protein vaccine.
To our surprise, post‐challenge HI antibodies from ferrets vaccinated with 2005 H3N2 DNA were able to gradually cross‐react with the 1968 pandemic H3N2 virus. Post‐challenge sera from ferrets vaccinated with the conventional protein vaccine did not show this ability. The same trend was observed in the H1N1 experiment. Post‐1999 virus‐challenged ferrets vaccinated with both 1999 DNA and conventional protein vaccines developed cross‐reaction against the 1918‐like H1N1 swine influenza virus from 1931. The reason why the antibodies induced by the conventional trivalent protein vaccine are able to recognise the swine 1931 H1N1 virus and not the H3N2 from 1968 may be that the H1 protein has changed less over time than the H3 protein. 22 This could also explain the better virus clearance observed in the H1N1 experiments.
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