Prediction of HLA-A*0201 restricted cytotoxic T lymphocyte epitopes in influenza A H1N1 virus and the similarity analysis of these epitopes with those

existing in other influenza viruses WANG Fei-peng, HE Xian-hui*

Institute of Tissue Transplantation and Immunology Jinan University, Guangzhou, China

*Corresponding author: thexh@jnu.edu.cn

WANG Fei-peng, HE Xian-hui* Key Laboratory for Regenerative Medicine of Ministry of

Education, Jinan University Guangzhou, China thexh@jnu.edu.cn

Abstract—In order to predict and compare the identities and differences of HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes among the influenza A virus ( H1N1), avian influenza virus (H5N1) and human influenza virus (H3N2). The SYFPEITHI, a T-cell epitope prediction tool, was adopted to predict the HLA-A*0201-restricted CTL epitopes in the three influenza viruses; DNAMAN was used to analyze the sequence homology of the proteins contained in the three influenza viruses, which were retrieved from GenBank; The results revealed that a large number of CTL epitopes were shared by the H1N1, H5N1 and H3N2 viruses although significant divergence existed in the protein sequence. This in silico analysis demonstrated that the antigen cross-reaction existed in the three kinds of viruses, which may partly explain why many people who ever experienced H3N2 only displayed mild symptoms when infected with influenza A H1N1 virus. It also suggests that human influenza vaccine inoculation may help attenuate the course of H1N1 induced disease before influenza A H1N1 vaccine is available for the population.

Keywords-H1N1; influenza A; epitope; epitope prediction; cross reaction

I. INTRODUCTION During April 2009, a novel swine-origin influenza virus

(formally named as influenza A H1N1 virus) was detected in Mexico and the United States and quickly transmitted in the population [1]. After a few weeks, the virus spread worldwide to 30 countries by animal-to-human and human-to-human transmission [2]. However, as time went on, influenza A H1N1 has not caused panic persistently and subsequently proved to be totally different from the virus circulating during the influenza pandemic in 1918 [3]. In China, though the transmission was rapid, the infected persons only showed mild symptoms [4]. It suggests that the people who infected with influenza A H1N1 virus seem to show some resistance to this virus and have the capacity to remove it or inhibit its replication in vivo. The reason underlying deserves carefully investigation.

H1N1 virus who mainly hosts in birds and some mammals belongs to the orthomyxoviridae family [5]. All H1N1 viruses have been isolated from wild birds, some serious illness caused by H1N1 mostly occurred in poultry but hardly infected human beings. However, with the mutation and the spread by birds and mammals, the pre-existing H1N1 virus has evolved into a new one that can lead to the human influenza pandemic. The current H1N1 virus is a completely new one produced by the gene rearrangement of the three viruses —swine influenza virus, avian influenza virus and human influenza virus. This novel virus has incubated in human beings for a long time without being detected until the outbreak of influenza pandemic in 2009 [6].

The current pandemic influenza A H1N1 virus contains the composition from human influenza virus, as well as those from swine and avian influenza viruses, which show high homology with the previous human influenza viruses. Although people can not prevent the H1N1 virus infection due to the lack of neutralizing antibodies against H1 and N1 antigens which are completely new to humans. It is possible that some similarity or homology may exist in the cytotoxic T lymphocyte （CTL） epitopes between human influenza H3N2 virus and influenza A H1N1 virus. Hence, the pre-existing memorial CTLs specific to epitopes from the human influenza virus in vivo is most likely to recognize the epitopes derived from influenza A H1N1 virus, and further eliminates the cells infected by the viruses in human leukocyte antigen (HLA)-restricted manner. It is therefore not difficult to understand why the persons infected with influenza A H1N1 virus merely display relatively mild symptoms. In the present study, we have compared the identity and similarity of CTL epitopes, which are restricted by HLA-A*0201 allele, among H1N1, avian virus H5N1 and human virus H3N2. Our results demonstrate that the novel H1N1 virus and human influenza H3N2 virus share many common CTL epitopes, suggesting that it may be helpful to attenuate the course of H1N1 induced disease by the human influenza H3N2 vaccine inoculation before the H1N1 vaccine is available for the whole population.

National Natural Science Foundation of China

978-1-4244-4713-8/10/$25.00 ©2010 IEEE

II. MATERIALS AND METHODS

A. Materials The protein sequences of H1N1, H5N1 and H3N2 viruses

were obtained from GenBank. The CTL epitope prediction software SYFPEITHI is available at world-wide web (http://www.syfpeithi.de/). DNAman V5.2.2 was a product of American Lynnon Biosoft. The known CTL epitopes were from Immune Epitope Database (IEDB) (http://www. immuneepitope.org/)

B. Methods 1) Acquisition of virus protein sequences: The newly

included protein sequences of influenza A H1N1 virus and the previous protein sequences of the H5N1 and H3N2 virus were downloaded from GenBank. DNAman was used to analyze the homology of these protein sequences.

2) Prediction of CTL epitopes: The SYFPEITHI was used to predict the HLA-A*0201-restricted CTL epitopes (nonamers) belonging to the three viruses (H1N1, H3N2 and H5N1).

III. RESULTS

A. Prediction of HLA-A*0201restricted CTL epitopes in influenza A H1N1 virus

Using the sequence alignment tool analyzed the sequences of the H1N1 virus from GenBank, we found that although the newly discovered sequences increased rapidly, most of the newly added sequences were highly homologous with the previous sequence (close to 100%), which indicates that remarkable mutation does not happen to this H1N1 virus. So, we choose the typical H1N1 protein sequences and use SYFPEITHI software to predict the HLA-A*0201 restricted CTL epitopes. The predicted CTL epitopes derived from hemagglutinin (HA), neuraminidase (NA) and matrix protein (MP) are summarized in Table I. Table II lists the epitopes of nucleoprotein (NP), polymerase B1 (PB1) and polymerase B2 (PB2) and Table III presents the epitopes from polymerase A (PA), non-structural protein (NS) and nuclear export protein (NEP). B. Analysis of protein sequence homology among H1N1,

H5N1 and H3N2 The DNAman is served to align the nine protein sequences

from the three viruses for analyzing their homology among them. The result is shown in Table IV. Except HA, NA and NS have low similarity, the homology of the other six proteins is more than ninety percent, which implies that higher antigenic cross-reactivity may exist among the three viruses.

C. Comparison of CTL epitope homology among H1N1, H5N1 and H3N2

We compared the homology of predicted CTL epitopes among H1N1, H5N1 and H3N2, and the results showed that there were a great number of identical epitopes among the three viruses (Table V and VI ), especially between H1N1 and

H3N2. Moreover, we also found that at least fourteen predicted epitopes has been identified experimentally and listed in the IEDB database (data not shown).

TABLE I. PREDICTION OF HLA-A*0201RESTRICTED CTL EPITOPES FROM HA, NA AND MP OF H1N1 VIRUS

HA（ACJ06667） NA（ACT35526） MP (ACI48769) 444ILLENERTL28 35TILEKNVTV 27 391GISNKVNSV27 542VLLVSLGAI26 388AIDGISNKV25 117ELKEQLSTV24 330RMATGLRNI24 7 VLFCAFTAL 23

58 LMSCPIGEV27 18 GMANLILQI25 98 AIYSKDNSV25 21 NLILQIGNI24 75 AAGQSVVSV24 388SIKQDIVGI24 187GINWLTIGI22 14 CMTIGMANL21

58 GILGFVFT30 51ILSPLTKGI25 3 LLTEVETYV23 130LIYNRMGTV23 116SLSYSAGAL22 123ALASCMGLI22 134RMGTVTTEV22 23 EIAQRLEDV21

Notes: the first number represents the start of the epitope sequence and the last number represents the prediction value; all the sequences in this table are the top 8 CTL epitopes from all the prediction epitopes.

TABLE II. PREDICTION OF HLA-A*0201RESTRICTED CTL EPITOPES FROM NP, PB1 AND PB2 OF H1N1 VIRUS

NP（ABR87894） PB1（ACI48762） PB2(ACR46984) 275CLPACVYGL26 373AMDSNTLE25 258FLARSALIL24 48KLSDYEGRL23 158GMDPRMCSL23 417NLPFEKATV23 55RLIQNSITI22 181AAVKGVGTI22

395LLIDGTASL33 162RLIDFLKDV27 247QIRGFVYFV26 410GMFNMLSTV26 413NMLSTVLGV26 418VLGVSILNL26 745KICSTIEEL25 65QLNPIDGPL23

344VLTGNLQTL28 724VLIGQGDVV25 373ILRKATRRL24 647ILVRGNSPV24 725LIGQGDVVL24 259LIIAARNIV23 272VSADPLASL23 279SLLEMCHST23

Notes: the first number represents the start of the epitope sequence and the last number represents the prediction value; all the sequences in this table are the top 8 CTL epitopes from all the prediction epitopes.

TABLE III. PREDICTION OF HLA-A*0201RESTRICTED CTL EPITOPES FROM PA, NS AND NEP OF H1N1 VIRUS

PA（ACR78592） NS（ACT21577） NEP (ACT21959) 664KLLLIVQAL28 218SLPPNFSSL26 282FLLMDALKL26 549LLRTAIGQV26 594SMIEAESSV26 675NLEPGTFDL25 694LINDPWVLL25 70ALLKHRFEI24

76TLRMTIASV28 51GLDIETATL27 42SLKGRGNTL26 136VIFNRLETL26 155AIVGEISPL26 111IIGPLCVRL25 184GLEWNGNTV23 103FMLMPRQKI22

105LLLEVEQEI26 37SLKIYRDSL24 101QALQLLLEV24 44SLGETVMRM22 79LIEEMRHRL22 30GMVTRFESL21 68QLGQKFEEI21 99FMQALQLLL21

Notes: the first number represents the start of the epitope sequence and the last number represents the prediction value; all the sequences in this table are the top 8 CTL epitopes from all the prediction epitopes.

TABLE IV. ANALYSIS OF PROTEIN SEQUENCE HOMOLOGY AMONG HA, NA, MP AND THE OTHER PROTEINS

H1N1 H5N1 H3N2

HA ACJ06667 ACR49182 ACV72364

60.05%

NA ACT35526 ACR49039 ABB59656

69.47%

MP ABR87893 ABF50834 ACT34416

96.57%

NP ABR87894 ACR49175 ACV49505

95.85%

NEP ACT21959 ACR48991 AAZ38755

93.11%

NS ABR87891 ACT75694 ACV49517

81.40%

PA ACJ06675 ACR49064 ACV49530

96.14%

PB1 ACI48762 ACR49086 ACV41545

98.55%

PB2 ACJ06674 ACR49134 ACV41492

98.33%

TABLE V. THE SIMILARITY OF HLA-A*0201 RESTRICTED CTL EPITOPES AMONG H1N1、H5N1AND H3N2.

H1N1 H5N1 H3N2

HA No same or similar antigen epitope

NA No same or similar antigen epitope

MP ABR87893 ABF50834 ACT34416 58GILGFVFTL30 51ILSPLTKGI25 3LLTEVETYV23 130LIYNRMGTV23 116SLSYSAGAL22 123ALASCMGLI22 134RMGTVTTEV22 23EIAQRLEDV21

58GMLGFVFTL30 51ILSPLTKGM21 3LLTEVETYV23

130LIYNRMGTV23 116ALSYSTGAL24 123ALASCMGLI22 134RMGTVTTEV22 23EIAQKLEDV22

58GILGFVFTL30 51ILSPLTKGI25 3LLTEVETYV23 130LIYNRMGAV22 116ALSYSAGAL22 123ALASCMGLI22 134RMGAVTTEV22 23EIAQRLEDV 21

NP ABR87894 ACR49175 ACV49505 373AMDSNTLEL25 258FLARSALIL24 48KLSDYEGRL23 158GMDPRMCSL23 417NLPFERATV23 55RLIQNSITI22 189IAMELIRMI22 256LIFLARSAL22

373AMDSNTLEL25 258FLARSALIL24 48KLSDYEGRL23 158GMDPRMCSL23 417NLPFERATI21 55RLIQNSITI22 189MVMELIRMI20 256LIFLARSAL22

373AMESSTLEL24 258FLARSALIL24 48KLSDYEGRL23 158GMDPRMCSL23 417NLPFDKPTI20 55RLIQNSLTI23 189MVMELIRMI20 256LIFLARSAL22

NEP

ACT21959 ACR48991 AAZ38755 105LLLEVEQEI26 37SLKIYRDSL24 101QALQLLLEV24 44SLGETVMRM22 79LIEEMRHRL22 30GMVTRFESL21 68QLGQKFEEI21 20QLGSSSEDL20

105LLLEVEQEI26 37SLKLYRDSL24 101QALQLLLEV24 44SLGEAVMRM22 79LIEEVRHRL22 30GMITQFESL23 68QLSQKFEEI21 20QLASSSEDL22

105LLFEVEQEI25 37SLKIYRDSL24 101QALQLLFEV23 44SLGEAVMRM22 79LIEEVRHRL22 30GMITQFESL23 68QLGQKFEEI21 20QLGSSSEDL20

Notes: the first number represents the start of the epitope sequence and the last number represents the prediction value; all the sequences in this table are the top 8 CTL epitopes from all the prediction epitopes; the sequence in italic and bold represents the different amino acid residues compared with

H1N1.

TABLE VI. THE SIMILARITY OF HLA-A*0201 RESTRICTED CTL EPITOPES AMONG H1N1、H5N1AND H3N2.

NS ABR87891 ACT75694 ACV49517 42SLKGRGNTL26 136VIFNRLETL26 155AIVGEISPL26 184GLEWNGNTV23 103FMLMPRQKI22 180ILIGGLEWN22 35RLRRDQKSL21 158GEISPLPSL21

42SLRGRGNTL27 131VIFDRLETL27 150AIVGEISPL26 179GLEWNDNTV23 98FMLMPKQKV24 175VLIGGLEWN21 35RLRRDQKSL21 153GEISPLPSL21

42SLRGRGSTL26 136VIFDRLETL27 155AIVGEISPL26 184GLEWNDNTV23 103FMLIPKQKV24 180VLIGGLEWN21 35RLRRDQKSL21 158GEISPLPSL21

PA ACJ06675 ACR49064 ACV49530 664KLLLIVQAL28 218SLPPNFSSL26 282FLLMDALKL26 594SMIEAESSV26 675NLEPGTFDL25 694LINDPWVLL25 70 ALLKHRFEI24 225SLENFRAYV24

664KLLLIAQAL24 218SLPPNFSSL26 282FLLMDALKL26 594SMIEAESSV26 675NLEPGTFDL25 694LINDPWVLL25 70ALLKHRFEI24 225SLENFRAYV24

664KLLLVVQAL28 218SLPPNFSCL24 282FLLMDALKL26 594SMIEAESSV26 675NLEPGTFDL25 694LINDPWVLL25 70ALLKHRFEI24 225CLENFRAYV22

PB1 ACI48762 ACR49086 ACV41545 395LLIDGTASL33 162RLIDFLKDV27 247QIRGFVYFV26 410GMFNMLSTV26 413NMLSTVLGV26 418VLGVSILNL26 745KICSTIEEL25 407MMMGMFNML22

395LLIDGAASL31 162RLIDFLKDV27 247QIRGFVYFV26 410GMFNMLSTV26 413NMLSTVLGV26 418VLGVSILNL26 745KICSTIEEL25 407MMMGMFNML22

395LLIDGTASL33 162RLIDFLKDV27 247QIRGFVYFV26 410GMFNMLSTV26 413NMLSTVLGV26 418VLGVSILNL26 745KICSTIEEL25 407MMMGMFNML22

PB2 ACJ06674 ACR49134 ACV41492 344VLTGNLQTL28 724VLIGQGDVV25 373ILRKATRRL24 647ILVRGNSPV24 725LIGQGDVVL24 259LIIAARNIV23 272VSADPLASL23 279SLLEMCHST23

344VLTGNLQTL28 724VLIGQGDVV25 373ILRKATRRL24 647LLVRGNSPV24 725LIGQGDVVL24 259LIIAARNIV23 272VSADPLASL23 279SLLEMCHST23

344VLTGNLQTL28 724VLIGQGDVV25 373ILRKATRRL24 647ILVRGNSPV24 725LIGQGDVVL24 259LIIAARNIV23 272VSADPLASL23 279SLLEMCHST23

Notes: the first number represents the start of the epitope sequence and the last number represents the prediction value; all the sequences in this table are the top 8 CTL epitopes from all the prediction epitopes; the sequence in italic and bold represents the different amino acid residues compared with H1N1

IV. DISCUSSION The antibodies which can neutralize the activity of H1 and

N1 are required for protecting people from influenza A H1N1 virus infection [7]. However, since the influenza A H1N1 is a virus which emerged recently and accumulated a large number of mutations, the human beings lack the specific antibodies against this virus. Thus, humans are extremely susceptible to the virus, and the virus can replicate rapidly in vivo, leading to apparent symptoms. Because of its quick spreading around the world, the H1N1 virus incurs the people’s panic as well as the high alert of the governments. However, as the time went on, it is found that most cases infected with H1N1 displayed mild syndrome; therefore, it is proposed that the patients infected with H1N1 virus can be isolated and treated at home. Base on this phenomenon, we put forward our own hypotheses: there were pre-existing specific immune memorial cells, especially the memorial cytotoxic lymphocytes (CTLs) specific to human influenza virus, which could cross-react with and recognize epitopes from H1N1 virus. When exposed to the specific antigens, these memorial CTLs could promptly kill the infected cells and prevent the spread of the virus in vivo, thereby attenuating the symptoms of H1N1 virus infection.

To confirm this hypothesis, we analyzed the protein sequences of influenza A virus (H1N1), highly pathogenic

avian influenza virus (H5N1) and human influenza virus (H3N2) and then compared the potential CTL epitopes in the three viruses. The results show that there are many completely identical CTL epitopes in spite of a great number of different CTL epitopes among the three viruses. Some predominant epitopes shared by H1N1 and H3N2 have been confirmed by experimental assay. For instance, the MP58-66 epitope (GILGFVFTL), which is a HLA-A*0201restricted epitope, is a dominant epitope contained in human influenza virus. In our previous study, the CTLs specific for MP58-66 epitope, which accounted for 0.1% of total CD8+ T cells, has been detected in Chinese population using the tetramer technology [8]. Similar level of MP58-66 epitope-specific CTLs is reported in Europe and the United States. This epitope was also identified in influenza A H1N1 virus, thus ,the cells infected with the H1N1 virus should present the antigen peptide through HLA-A*0201, which results in the recognition of it by the memorial CTLs specific for MP58-66 epitope and elimination of the infected cells. In summary, the pre-existing CTLs shared by H1N1 and H3N2 may play an important role in controlling the infection of influenza A H1N1 virus and attenuating the syndrome of the disease in those people who experienced human influenza H3N2 virus. This may partly explain why many adults who were infected by H1N1 virus had mild syndromes and why children are more susceptible to H1N1 infection and have serious symptoms. These results also suggest that the human influenza vaccine may not only prevents human influenza, but, to some extent, benefits the people by attenuating the influenza A H1N1 syndrome when influenza A H1N1 vaccine is not general available at this moment.

Moreover, the prediction result reveals that H1N1 virus shares some identical epitopes with the avian influenza H5N1 virus. This suggests that the people who ever suffered from avian influenza and then recovered are more likely to have a resistance to H1N1 virus. Although there is no specific statistical data related to this now, from the epidemic map published by Centers for Disease Control and Prevention (CDC), we can see that influenza A H1N1 was originally spreading from North America [9], while avian influenza almost did not happen there, it seems that there is certain correlation between them [10].

In conclusion, the pre-existing memory CTLs specific to previous human influenza virus may recognize the novel

influenza A H1N1 epitopes and should help inhibit the spreading of the H1N1 virus and accelerate the rate of clearance of the virus in vivo in the healthy population. Accordingly, under the circumstances of shortage of large number of vaccines against influenza A H1N1, the susceptible population, especially the children, should be firstly vaccinated with H1N1 vaccine to effectively control the spreading of influenza A H1N1 virus and further relieve the burden in the prevention and control of this influenza virus.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (No 30230350, No 30572199), as well as by “211 Engineering” Phase III Biotechnology and Biomedical Engineering Project of Jinan University.

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[10] http://www.pandemicflu.ac.cn/n3520543/n3521107/n3528367/n3528370/images/4449838.jpg