Journal of Immunology Research
Volume 2018 (2018), Article ID 3174868, 12 pages
https://doi.org/10.1155/2018/3174868

Research Article

An Increased Frequency in HLA Class I Alleles and Haplotypes Suggests Genetic Susceptibility to Influenza A (H1N1) 2009 Pandemic: A Case-Control Study

Ramcés Falfán-Valencia,¹ Arun Narayanankutty,¹ Juan M. Reséndiz-Hernández,¹ Gloria Pérez-Rubio,¹ Alejandra Ramírez-Venegas,² Karol J. Nava-Quiroz,¹ Nora E. Bautista-Félix,² Gilberto Vargas-Alarcón,³ Manuel D. J. Castillejos-López,⁴ and Andrés Hernández⁴

¹HLA Laboratory, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, 14080 Ciudad de México, Mexico
²Tobacco Smoking and COPD Research Department, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, 14080 Ciudad de México, Mexico
³Molecular Biology Department, Instituto Nacional de Cardiología Ignacio Chávez, 14080 Ciudad de México, Mexico
⁴Hospital Epidemiological Surveillance Unit, Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, 14080 Ciudad de México, Mexico

Correspondence should be addressed to Ramcés Falfán-Valencia; dcb_rfalfanv@hotmail.com

Received 23 August 2017; Revised 26 December 2017; Accepted 11 January 2018; Published 25 February 2018

Academic Editor: Peirong Jiao

Copyright © 2018 Ramcés Falfán-Valencia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Background. The influenza A H1N1/09 pandemic infected a small number of exposed individuals, which suggests the involvement of genetic factors. There are scarce data available on classical HLA class I association with the influenza A H1N1/09 pandemic. Methods. We analyzed the frequency of classical HLA class I alleles and haplotypes in A H1N1/09 influenza in a case-control study including 138 influenza patients (INF-P) and 225 asymptomatic healthy contacts (INF-C) simultaneously recruited. HLA class I typing was performed by high-resolution sequence-based typing method. Results. Our analysis revealed higher frequency of C07:02:01, B39:06:02, C03:02:01, B44:03:01, B51:01:05, and B73:01 (; OR = 1.84–9.98) and of two haplotypes—A68:01:02-C07:02:01 (; OR = 23.99) and B35:01:01-C07:02.01 (, OR = 2.15)—in A H1N1/09 influenza subjects. A68:01:01 was exclusively present only in the INF-P group (5/138). A decrease in the frequency of C03:03:01, A11:01:01, B39:01:01, A24:02:01, C03:04:01, B51:01:01, and C07:01:01 (; OR = 0.12–0.52) and of haplotypes A02:01:01-B35:01:01-C04:01:01, A24:02:01-B35:01:01, B39:01:01-C07:02:01, and B40:02:01-C03:04:01 (; OR = 0.08–0.22) were observed in INF-P group. Conclusion. Selective classical HLA class I allele and haplotype combinations predispose individuals towards susceptibility or protection against the influenza A H1N1/09 pandemic. This work has significant implications for accessing population transmission risk for A H1N1/09 or a similar strain breakout in the future.

1. Introduction

Influenza A H1N1/09 pandemic, with its origin in Mexico, is the first human flu in 40 years to have caused such transmissibility and infectivity on a global scale [1, 2]. However, the mortality rates were merely marginal compared to those of previous global outbreaks. For example, the first well-known Spanish flu outbreak at 1918-1919 killed at least 40 million people globally, more than the total mortality rate attributed to World War I [3, 4]. The infection rate in various ethnic populations seems to be disproportionate along with the inability to identify any country-based transmission risk factors [2]. It has been previously shown that community and/or individual host factors can account for differential mortality rate [5]. Approximately 2/3 of global hospitalized subjects and 40% of fatal cases did not have any comorbidities or risk factors associated with influenza [6]. This may point to an underlying complex genetic component interplay associated with disease susceptibility. Several studies pointed towards the existence of a genetic component [7–9]; in this paper we analyze classical HLA class I region.

The differential specificity, selectivity, and diversity of human leukocyte antigen (HLA) directly reflect a delicate balance between interplay of molecular defense mechanisms against foreign antigens and autoimmunity acquired in the course of human evolution and migration. It is evident from various studies that selective combinations of HLA class I alleles exert quantitatively different effects on disease progression of infective or autoimmune origin [10–12]. HLA class I is involved in both innate (NK cells) and cell-mediated (CD8+ cells) immune response, significantly contributing towards viral clearance and decrease in the severity of influenza infection [4, 13].

Recent studies indicate that the NK cell frequency negatively correlates with the severity of influenza A H1N1/09 infection [13, 14]. NK cells attack virus-infected cells using a broad range of stimulatory and inhibitory receptor-ligand interactions. Quantitative interactions between selective HLA class I ligands and KIR, a major receptor of NK cell, variably influence the NK cell response towards viral-infected cells. Certain KIR3DL1/S1 allotypes, 3DL1/S1 and 2DL1 ligand-negative pairs, and 2DL2/L3 ligand-positive pairs are shown to be differentially enriched in severe cases of A H1N1/09 infection [15]. However, the less-studied KIRs are more polymorphic and diverse than are their classical HLA class I ligand counterparts. CD8+ effector T cell frequencies are shown to be normal regardless of patient disease status and severity [13]. However, it can be argued that there exist entirely different clones of CD8+ cells that correlate with H1N1 infection severity, and selective HLA class I alleles and combinations influence their generation/maintenance. This argument can be substantiated by the study showing that cytotoxic T lymphocytes established by seasonal influenza that cross-react against H1N1/09 are disproportionately distributed in a population [16]. Also, there are numerous reports of cross-reactive CTLs to heterosubtypic influenza A strains [17–20].

Currently, there are no studies in the Mexican population using direct sequencing on the relationship between HLA class I allele frequency and susceptibility to influenza virus A H1N1/09 infection. This is the first report that shows HLA class I allele association with susceptibility to influenza A H1N1/09 in Mexican mestizo population. Identification of alleles and haplotype associations with specific strains of influenza may help us to predetermine transmission rate of pandemic A H1N1/09 in various populations based on their HLA class I allele frequency.

2. Materials and Methods

2.1. Ethics Statement

This study was approved by the Institutional Committee for Science and Bioethics of the Instituto Nacional de Enfermedades Respiratorias (INER) Ismael Cosío Villegas (code B05-10). The study protocol was explained to all participants, and signed informed consent was duly obtained.

All the information was collected complying with Official Mexican Standards (Mexicana NOM-168-SSA1-1998); topics covered included age, gender, tobacco smoking, body mass index (BMI; patients with BMI > 30 kg/m²), and disease morbidity (pulmonary, hepatic, renal, cardiac, and neurological diseases, diabetes mellitus, hypertension, and cancer). The symptoms evaluated were fever, cough, rhinorrhea, dyspnea, nasal congestion, thoracic pain, headache, diarrhea, and vomiting. The start of antiviral therapy was evaluated in relation to the days with previous symptomatology. The laboratory parameters included were leukocyte titer, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), blood urea nitrogen (BUN), and arterial gases (with PaO₂ < 60 mmHg defined as severe disease). Pneumonia was verified by radiological findings. All patients admitted to the intensive care unit (ICU) and those put on assisted mechanical ventilation (AMV) were identified.

2.2. Study Subjects

A total of 196 subjects showing influenza-like illness were enrolled in the study during the first and second waves of influenza A H1N1/09 infection in Mexico (between April and December 2009). All the subjects included in the study were Mexican mestizos by ethnicity with at least 2 generations born and brought up in Mexico. The 196 study subjects were classified into two categories: 138 H1N1/09 positive (influenza A H1N1/09 positive subjects [INF-P]) and 58 H1N1/09 negative subjects; only the INF-P group was enrolled in the study. Another group of 225 subjects (asymptomatic healthy contacts [INF-C]) was also enrolled in parallel during the same period (Figure 1). Anti-influenza A H1N1 antibody titers were determined to ensure that those in the INF-C group had been exposed to the virus.

Figure 1: Selection criteria for both influenza A H1N1/09 patient (INF-P) and contact (INF-C) groups. INF-P: influenza patients; INF-C: influenza contacts; HI: hemagglutination inhibition; RT-PCR: RT-PCR: real-time polymerase chain reaction, also called quantitative PCR (qPCR).

2.3. Immunological and Viral Measurements

2.3.1. INF-P Group ()

Nasopharyngeal swabs were taken from the subjects and eluted in 800 μL saline by vigorously rotating the swab in the diluent. Each sample was analyzed using rapid QuickVue Influenza A + B test (Quidel, CA, USA) following the recommendations for collection and testing from US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). Positive cases for influenza A H1N1 confirmed by QuickVue were further validated using an RT-PCR-based RespiFinder 22 test (Maastricht, Netherlands) [21].

2.3.2. INF-C Group ()

Although not biologically related to any patients in the INF-P group, they were in personal contact with one or more of A H1N1-infected patients. However, they never showed any symptoms of flu or took any medication for it. Hemagglutination inhibition (HI) assay was performed using peripheral blood samples for each subject as previously described [22]. All subjects included in this group had an HI titer value greater than 1 : 16 dilutions, indicating direct contact with the virus. All INF-C subjects were under 60 years of age to avoid any cases of cross-reacting antibodies prevalent in older age groups from H1N1/1918-1919 infection [23]. As this cohort was not progressively monitored for the dynamics of anti-A H1N1/09 antibody titer over time, we will not show the results.

2.4. Anti-A H1N1/09 Antibody Titers

Serially diluted aliquots of serum samples (25 μL) in PBS were mixed with 25 μL aliquots of the A H1N1/09 virus strain (corresponding to four hemagglutination units). The serum/virus dilutions were incubated for 30 min at room temperature. 50 μL of 0.5% chicken erythrocytes was added, and after 30 min, the HI activity was evaluated. The serum HI antibody titer was established as the reciprocal of the last serum dilution with no hemagglutination activity. Those individuals with titers greater than 1 : 16 were considered positive for A/H1N1 infection/exposure.

2.5. Population HLA Frequency Data

HLA frequency data for Mexican mestizos were obtained from the http://www.allelefrequencies.net website [24]. Average HLA frequencies were computed using a weighted average of all reported studies, based on study sample size.

2.6. DNA Extraction and HLA Class I Typing

High-molecular-weight genomic DNA was extracted from peripheral blood using BDtract genomic DNA isolation kit (Maxim Biotech, San Francisco, CA, USA). All DNA samples were stored indefinitely at −20°C in Tris-EDTA buffer (10 mM Tris-HCl, 2 mM EDTA, pH 8.0) until further use.

2.7. HLA Class I Typing

Genomic DNA (50 ng/μL) was amplified using long-range PCR (LR-PCR) for each HLA-A, HLA-B, and HLA-C loci using AlleleSEQR core reagent pack according to the manufacturer’s protocol. HLA-A, HLA-B, and HLA-C alleles were determined by a sequence-based typing (SBT) method, using the AlleleSEQR HLA-A, AlleleSEQR HLA-B, and AlleleSEQR HLA-C plus sequencing kits, respectively (Atria Genetics Inc., San Francisco, CA, USA). The cycling conditions used were 95°C 10 min, 1 cycle; 96°C 20 sec, 60°C 30 sec, and 72°C 3 min for 36 cycles; and 4°C until the next step. The 2 kb amplicons were verified by 1% agarose gel electrophoresis. The product obtained was treated with ExoSAP-IT, a mixture of an exonuclease I/alkaline phosphatase, in order to remove unincorporated primers and dNTPs, using one cycle of 37°C 30 min, 80°C 15 min, and 4°C until the next step. Exons 2, 3, and 4 were amplified with specific degenerate primers for HLA-A, HLA-B, and HLA-C loci using the following cycling conditions: twenty-five cycles at 96°C 20 sec, 50°C 30 sec, and 60°C 2 min and 4°C until the next step. Samples were prepared for loading by adding 15 μL of formamide and denaturing at 95°C for 2 min.

Sequencing was performed on a ABI3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using mobile file v2.mob DT3130POP6{BD}, with an analysis module RapidSeq36_POP6_1, 1–1.5 kV injecting 5–10 seconds, collecting data for 1800 seconds and reviewing it with the Sequencing Analysis software v5.4. The interpretation and assignment of HLA alleles were performed using Assign v3.5 software (Conexio Genomics), with reference sequences updated to April 2012 from the IMGT database.

2.8. Statistical Analysis

Allele and haplotype frequencies of HLA class I were enumerated by direct counting in both INF-P and INF-C study groups. The observed and expected HLA class I alleles (each locus) were tested for Hardy-Weinberg equilibrium using a conventional Fisher’s exact test. Differential allele and haplotype frequency associations between different HLA class I allele variants were assessed using Fisher’s exact two-tailed test with a significance cutoff level of . Bonferroni correction was carried out by only taking into account alleles with allele frequency greater than 5% to compensate for overcorrection. Odd ratios and 95% confidence intervals were calculated using Epi Info 7.1.3.0 software. Allele frequency graphs were plotted using SigmaPlot 11. Finally, the haplotypes (2-locus and 3-locus) were constructed employing Arlequin v3.1 software [25] using the maximum likelihood method, with an iterative EM algorithm.

3. Results

We analyzed clinical data from 138 influenza patients and 225 healthy contacts, as described in Table 1. No statistically significant difference was observed for age between the study groups (43.26 versus 42.21, ). The male to female ratios of INF-P and INF-C were 1.3 : 1 and 1 : 1.14, respectively (). As described previously [6], a statistically significant difference was observed in the variables of weight (83.41 versus 71.0, ), height (1.64 versus 1.59, ), BMI (30.88 versus 27.85, ), and active smoking status (45.65% versus 30.61%, ). Comorbidities and symptomatology characteristics are described in Table 1. Eighty-nine patients (64.49%) had pneumonia, and 71 (51.44%) were admitted to the ICU, whereas thirty-one patients positive for influenza A H1N1/09 virus died. None of the alleles/haplotypes were directly associated with mortality or greater severity.

Table 1: Characteristics of influenza A H1N1/09 patients (INF-P) and contacts (INF-C) recruited during April to December 2009.

INF-P ()
INF-C ()

Age
43.26 ± 13.86
42.21 ± 12.03

Gender [male (%)/female (%)]
78 (56.52)/60 (43.48)
105 (46.67)/120 (53.33)

Weight (kg)
83.41 ± 20.45
71.00 ± 14.10
<0.01

Height (m)
1.64 ± 0.10
1.59 ± 0.09
<0.01

BMI
30.88 ± 6.92
27.85 ± 4.86
0.01

Smoking (Y/N, %)
45.65/54.35
30.61/69.39
0.02

Comorbidities (%)

    Neurological disease
25 (18.11)
33 (14.66)

    Asthma
2 (1.44)
6 (2.66)

    Cancer
1 (0.72)
0 (0.00)

    Hypertension
8 (5.79)
17 (7.55)

Symptomatology (%)

    Fever (>38°C)
104 (75.36)
0 (0.00)

    Cough
72 (52.17)
0 (0.00)

    Nasal congestion
17 (12.31)
0 (0.00)

    Rhinorrhea
39 (28.26)
0 (0.00)

    Dyspnea
101 (73.18)
0 (0.00)

Region of origin; (%)

    Central Mexico
106 (76.81)
155 (68.89)

    North Mexico
18 (13.04)
45 (20.00)

    Southeast Mexico
14 (10.14)
25 (11.11)

INF-P: influenza patients; INF-C: influenza contacts; : number of subjects; BMI: body mass index. value < 0.05 is considered significant.

In order to avoid population stratification effects, cases and contacts were compared with respect to their region of origin, grouping them as coming from central (states of Mexico, Hidalgo, Puebla, Morelos, Tlaxcala, and Mexico City), north (states of Chihuahua, Sonora, Durango, and Zacatecas), and southeast (states of Veracruz, Tabasco, Campeche, and Yucatan) regions (Figure 2). 76.81% of the INF-P and 68.9% of INF-C were from the central area of the country contributing to majority of subjects in both the study groups (). The northern states contributed 13.04% of INF-P and 20% of INF-C (). Finally, 10.14% of INF-P subjects were from the southeastern states of Mexico, compared to 11.11% INF-C () (Figure 2). No statistically significant differences were found in the population stratification based on region of origin between cases and contacts.

Figure 2: Depiction of influenza A H1N1/09 patient (INF-P) and contact (INF-C) organized to three strata based on their region of origin. INF-P: influenza patients; INF-C: influenza contacts.

3.1. Differential Frequency Distribution of HLA Class I Alleles

The HLA allele pairs of each locus from both groups satisfied the Hardy-Weinberg equilibrium (), accounting for common alleles producing common genotypes [i.e., both heterozygous (e.g., B35:01:01 and B39:01:01) and homozygous (e.g., A02:01:01) pairs].

Our analysis identified 31 HLA-A alleles in influenza patients (INF-P) and 38 in asymptomatic healthy contacts (INF-C) out of which 7 of the alleles in the INF-P group showed an allele frequency (AF) higher than 6%; the most common were A02:01:01 (24.6%), A68:01:02 (11.6%), A24:02:01 (7.9%), A03:01:01 (7.6%), A31:01:02 (7.2%), and A01:01:01 (6.5%). Interestingly, A68:01:01 allele was present exclusively in the INF-P (AF = 1.8%). Furthermore, we observed a lower frequency of the allele A24:02:01 (7.9% versus 18.6% ) in the INF-P compared to INF-C, respectively, suggesting a possible protective role against the A H1N1/09 virus infection (OR = 0.38). A lower frequency of A11:01:01 (0.4% versus 2.8%, ) was also observed in INF-C but did not reach statistical significance after Bonferroni correction (Table 2).

Table 2: HLA-A allele frequencies in patients with influenza A/H1N1 infection (INF-P) and asymptomatic healthy contacts (INF-C).

HLA-A
INF-P
INF-C

pC
OR
95% CI

AF
(%)

AF
(%)

A02:01:01
68
0.2464
24.638
120
0.2667
26.667

A68:01:02
32
0.1159
11.594
41
0.0911
9.111

A24:02:01
22
0.0797
7.971
84
0.1867
18.667

0.3774
0.2298–0.6197

A03:01:01
21
0.0761
7.609
27
0.0600
6.000

A31:01:02
20
0.0725
7.246
22
0.0489
4.889

A01:01:01
18
0.0652
6.522
23
0.0511
5.111

A02:06:01
17
0.0616
6.159
19
0.0422
4.222

A29:02:01
10
0.0362
3.623
10
0.0222
2.222

A26:01:01
8
0.0290
2.899
8
0.0178
1.778

A32:01:01
8
0.0290
2.899
7
0.0156
1.556

A30:02:01
7
0.0254
2.536
10
0.0222
2.222

A23:01:01
6
0.0217
2.174
14
0.0311
3.111

A26:01:02
5
0.0181
1.812
4
0.0089
0.889

A68:01:01
5
0.0181
1.812
0

Und
Und-Und

A01:03
3
0.0109
1.087
1
0.0022
0.222

A11:24:02
3
0.0109
1.087
0

A33:01:02
3
0.0109
1.087
11
0.0244
2.444

A66:02
3
0.0109
1.087
0

A68:02:01
3
0.0109
1.087
1
0.0022
0.222

A25:01:01
2
0.0072
0.725
3
0.0067
0.667

A33:01:01
2
0.0072
0.725
0

A02:04
1
0.0036
0.362
0

A02:05:01
1
0.0036
0.362
0

A11:01:01
1
0.0036
0.362
13
0.0289
2.889

0.1222
0.0159–0.9397

A23:01:02
1
0.0036
0.362
0

A23:02
1
0.0036
0.362
0

A24:02:06
1
0.0036
0.362
0

A30:01:01
1
0.0036
0.362
1
0.0022
0.222

A32:02:01
1
0.0036
0.362
1
0.0022
0.222

A68:03:01
1
0.0036
0.362
1
0.0022
0.222

A74:02:01
1
0.0036
0.362
0

A01:01:02
0

2
0.0044
0.444

A02:01:02
0

1
0.0022
0.222

A02:02:01
0

3
0.0067
0.667

A02:03:01
0

1
0.0022
0.222

A11:01:02
0

1
0.0022
0.222

A11:04
0

1
0.0022
0.222

A26:02:01
0

1
0.0022
0.222

A31:02
0

4
0.0089
0.889

A31:14N
0

1
0.0022
0.222

A36:01
0

4
0.0089
0.889

A66:01:01
0

3
0.0067
0.667

A68:01:03
0

1
0.002
0.222

A68:03:02
0

1
0.002
0.222

A68:05
0

1
0.002
0.222

A69:01
0

1
0.002
0.222

A74:01
0

2
0.004
0.444

A80:01
0

1
0.002
0.222

INF-P: influenza patients; INF-C: influenza contacts; HLA: human leukocyte antigen; AF: allele frequency; : number of subjects; OR: odds ratio; CI: confidence interval; pC: value after Bonferroni correction; Und: undefined; only showing value < 0.05, which is considered significant.

HLA-B locus typing identified 42 alleles in INF-P and 57 alleles in INF-C. The most frequent were B35:01:01, B15:01:01, and B07:02:01 in both study groups; in addition, B39:01:01 and B51:01:01 were common in the INF-C group. Increased frequency of B51:01:05 (, OR = 6.18) was detected in the INF-P group compared with INF-C. B39:06:02 (), B44:03:01 (), and B73:01 () were also increased in the INF-P group but failed to reach significance after Bonferroni correction (OR = 2.53–9.98). In contrast, a decreased frequency of the alleles B51:01:01 (, OR = 0.43) and B39:01:01 (, OR = 0.26) was observed in INF-P compared to INF-C (Table 3).

Table 3: HLA-B allele frequencies in patients with influenza A/H1N1 infection (INF-P) and asymptomatic healthy contacts (INF-C).

HLA-B
INF-P
INF-C

pC
OR
95% CI

AF
(%)

AF
(%)

B35:01:01
37
0.1341
13.406
64
0.1422
14.222

B15:01:01
26
0.0942
9.420
33
0.0733
7.333

B07:02:01
17
0.0616
6.159
24
0.0533
5.333

B48:01:01
17
0.0616
6.159
20
0.0444
4.444

B39:06:02
15
0.0543
5.435
10
0.0222
2.222

2.5287
(1.1197–5.7111)

B40:02:01
14
0.0507
5.073
26
0.0578
5.778

B51:01:05
11
0.0399
3.986
3
0.0067
0.667

6.1849
(1.7100–22.3706)

B44:03:01
10
0.0362
3.623
3
0.0067
0.667

5.6015
(1.5279–20.5356)

B52:01:02
10
0.0362
3.623
15
0.0333
3.333

B39:05:01
9
0.0326
3.261
8
0.0178
1.778

B51:01:01
9
0.0326
3.261
33
0.0733
7.333

0.4259
(0.2006–0.9043)

B08:01:01
8
0.0290
2.899
10
0.0222
2.222

B14:02:01
7
0.0254
2.536
11
0.0244
2.444

B39:01:01
6
0.0217
2.174
36
0.0800
8.000

0.2556
(0.1062–0.6147)

B58:01:01
6
0.0217
2.174
3
0.0067
0.667

B73:01
6
0.0217
2.174
1
0.0022
0.222

9.9778
(1.1948–83.3273)

B13:02:01
5
0.0181
1.812
4
0.0089
0.889

B18:01:01
5
0.0181
1.812
9
0.0200
2.000

B35:17
5
0.0181
1.812
4
0.0089
0.889

B38:01:01
4
0.0145
1.449
6
0.0133
1.333

B40:05
4
0.0145
1.449
1
0.0022
0.222

B44:02:01
4
0.0145
1.449
16
0.0356
3.556

B49:01:01
4
0.0145
1.449
7
0.0156
1.556

B14:06:02
3
0.0109
1.087

B15:03:01
3
0.0109
1.087
5
0.0111
1.111

B35:02:01
3
0.0109
1.087
6
0.0133
1.333

B39:06:01
3
0.0109
1.087
6
0.0133
1.333

B40:01:01
3
0.0109
1.087
7
0.0156
1.556

B14:01:01
2
0.0072
0.725
4
0.0089
0.889

B27:03
2
0.0072
0.725
6
0.0133
1.333

B27:05:02
2
0.0072
0.725
2
0.0044
0.444

B35:05:01
2
0.0072
0.725
0

B35:12:01
2
0.0072
0.725
9
0.0200
2.000

B39:02:02
2
0.0072
0.725
3
0.0067
0.667

B41:01:01
2
0.0072
0.725
4
0.0089
0.889

B53:01:01
2
0.0072
0.725
2
0.0044
0.444

B15:02:01
1
0.0036
0.362
4
0.0089
0.889

B15:16:01
1
0.0036
0.362
0

B35:01:02
1
0.0036
0.362
0

B45:01:01
1
0.0036
0.362
5
0.0111
1.111

B57:01:01
1
0.0036
0.362
6
0.0133
1.333

B78:02:02
1
0.0036
0.362
0

B13:01:01
0

1
0.0022
0.222

B15:05:01
0

1
0.0022
0.222

B15:15
0

2
0.0044
0.444

B35:01:04
0

1
0.0022
0.222

B35:40N
0

1
0.0022
0.222

B37:01:01
0

1
0.0022
0.222

B38:01:02
0

6
0.0133
1.333

B40:01:02
0

1
0.0022
0.222

B40:01:04
0

3
0.0067
0.667

B40:02:03
0

1
0.0022
0.222

B40:11:01
0

1
0.0022
0.222

B42:01:01
0

2
0.0044
0.444

B44:18:01
0

1
0.0022
0.222

B45:01
0

1
0.0022
0.222

B46:02
0

1
0.0022
0.222

B50:01:01
0

3
0.0067
0.667

B51:02:01
0

1
0.0022
0.222

B55:01:01
0

3
0.0067
0.667

B55:02:01
0

2
0.0044
0.444

B56:01:01
0

1
0.0022
0.222

INF-P: influenza patients; INF-C: influenza contacts; HLA: human leukocyte antigen; AF: allele frequency; : number of subjects; OR: odds ratio; CI: confidence interval; pC: value after Bonferroni correction; only showing value < 0.05, which is considered significant.

HLA-C locus typing identified 26 alleles in INF-P and 21 in INF-C. The molecular subtypes C07:02:01, C04:01:01, C01:02:01, and C08:01:01 represent more than 55% of the specificities detected in INF-P patients. We observed an increased frequency of C07:02:01 () and C02:02:02 () alleles in the INF-P group compared with INF-C. A decrease in the frequency of the subtype C03:04:01 () was detected in the INF-P group. C07:01:01 () and C03:03:01 () were also decreased but failed to reach Bonferroni significance; something similar happened with C03:02:01 and C08:01:02, which are statistically increased in the patients group ( and , resp.) (Table 4).

Table 4: HLA-C allele frequencies in patients with influenza A/H1N1 infection (INF-P) and asymptomatic healthy contacts (INF-C).

Allele
INF-P
INF-C

pC
OR
95% CI

AF
(%)

AF
(%)

C07:02:01
60
0.2174
21.739
59
0.1311
13.111

1.8409
1.2390–2.7351

C04:01:01
50
0.1812
18.116
75
0.1667
16.667

C01:02:01
22
0.0797
7.971
31
0.0689
6.889

C08:01:01
22
0.0797
7.971
26
0.0578
5.778

C07:01:01
14
0.0507
5.073
42
0.0933
9.333

0.5191
0.2780–0.9692

C03:04:01
12
0.0435
4.348
48
0.1067
10.667

0.3807
0.1985–0.7302

C03:02:01
11
0.0399
3.986
5
0.0111
1.111

3.6943
1.2697–10.7492

C08:02:01
11
0.0399
3.986
15
0.0333
3.333

C16:01:01
11
0.0399
3.986
17
0.0378
3.778

C06:02:01
10
0.0362
3.623
14
0.0311
3.111

C05:01:01
8
0.0290
2.899
18
0.0400
4.000

C12:03:01
8
0.0290
2.899
22
0.0489
4.889

C02:02:02
6
0.0217
2.174
0

Und
Und-Und

C08:01:02
5
0.0181
1.812
0

Und
Und-Und

C15:02:01
5
0.0181
1.812
21
0.0467
4.667

C03:03:01
4
0.0145
1.449
24
0.0533
5.333

0.261
0.0896–0.7605

C03:03:02
3
0.0109
1.087
0

C03:06
3
0.0109
1.087
5
0.0111
1.111

C14:02:01
3
0.0109
1.087
4
0.0089
0.889

C03:02:02
2
0.0072
0.725
0

C02:02:01
1
0.0036
0.362
10
0.0222
2.222

C03:07
1
0.0036
0.362
0

C05:01:02
1
0.0036
0.362
0

C07:04:01
1
0.0036
0.362
6
0.0133
1.333

C17:01:01
1
0.0036
0.362
6
0.0133
1.333

C18:01
1
0.0036
0.362
0

C03:05:01
0

1
0.0022
0.222

C07:01:03
0

1
0.0022
0.222

INF-P: influenza patients; INF-C: influenza contacts; HLA: human leukocyte antigen; AF: allele frequency; : number of subjects; OR: odds ratio; CI: confidence interval; pC: value after Bonferroni correction; only showing value < 0.05, which is considered significant.

3.2. Differential Frequency Distribution of Haplotypes (Loci A-B-C, A-B, A-C, and B-C)

We identified 211 HLA-A-B-C haplotypes in the INF-P and 276 combinations in the INF-C, respectively. In the INF-P group, only 10 combinations showed a haplotype frequency (HF) > 1%; the most common were A02:01:01-B35:01:01-C07:02:01 (2.54%), A68:01:02-B48:01:01-C08:01:01 (1.81%), A01:01:01-B08:01:01-C07:02:01 (1.45%), A03:01:01-B35:01:01-C04:01:01 (1.45%), A24:02:01-B15:01:01-C01:02:01 (1.45%), A02:01:01-B15:01:01-C07:02:01 (1.45%), and A32:01:01-B35:01:01-C04:01:01 (1.45%). Similarly, 16 haplotypes had an HF > 1% in the INF-C group; the most frequent were A02:01:01-B35:01:01-C04:01:01 (2.57%), A24:02:01-B35:01:01-C04:01:01 (2.64%), A02:01:01-B39:01:01-C07:02:01 (2.38%), A24:02:01-B35:01:01-C03:04:01 (1.99%), and A03:01:01-B07:02:01-C07:02:01 (1.33%).

We compared the HF of HLA-A-B-C loci observed in INF-P with that observed in INF-C (Table 5). Our analysis revealed that the combination A02:01:01-B35:01:01-C04:01:01 was diminished significantly in INF-P (, OR = 0.13, CI = 0.01–0.91); however, after Bonferroni correction (HF > 1%), value was not statistically significant (). Interestingly, 2 haplotypes were present at high frequency (~2.0%) in the INF-C group and completely absent among the INF-P; these include A24:02:01-B35:01:01-C03:04:01 and A24:02:01-B35:01:01-C04:01:01 (see Tables SIX–SXII, Supplementary Materials); it is not possible to make statistical comparisons between groups as the haplotype frequencies readily decline due to extensive sample size dilution. However, the last two haplotypes carry A24:02:01 and C03:04:01 alleles, both previously associated with protection in the individual locus analysis.

Table 5: HLA-A-B-C, A-B, B-C, and A-C haplotype frequencies in patients with influenza A/H1N1 infection (INF-P) and asymptomatic healthy contacts (INF-C).

Haplotype
INF-P
INF-C

pC
OR
95% CI

HF
%
HF
%

A02:01:01-B35:01:01-C04:01:01
0.004
0.36
0.026
2.57

0.13
0.01–0.91

A24:02:01-B35:01:01
0.012
1.23
0.046
4.59

0.22
0.06–0.74

B35:01:01-C07:02:01
0.035
3.54
0.002
0.23

16.88
2.14–132.60

B39:01:01-C07:02:01
0.006
0.63
0.042
4.22

0.17
0.04–0.71

B40:02:01-C03:04:01
0.004
0.36
0.042
4.21

0.08
0.01–0.62

A68:01:02-C07:02:01
0.05
4.96
0.002
0.22

23.99
3.14–183.51

Haplotypes with uncorrected value < 0.05, which is considered significant, are only presented. All values were obtained by Fisher exact two-tailed test. INF-P: influenza patients; INF-C: influenza contacts; HF: haplotype frequency; : number of subjects; OR: odds ratio; CI: confidence interval; pC: value after Bonferroni correction. For a complete list of haplotypes, check Tables SIX–SXII.

In a separate analysis of A-B, A-C, and B-C haplotypes, few combinations had frequencies greater than 2%. We observed that the haplotype frequencies of A24:02:01-B35:01:01, B39:01:01-C07:02:01, and B40:02:01-C03:04:01 were diminished in the INF-P compared to INF-C ( uncorrected). Two highly significant haplotypes—A68:01:02-C07:02:01 (, OR = 23.99, CI = 3.14–183.51) and B35:01:01-C07:02:01 (, OR = 16.88, CI = 2.14–132.60)—were observed in the INF-P, suggesting that these haplotypes are important genetic susceptibility factors predisposing them to symptomatic influenza A H1N1/09 viral infection. Three haplotypes (HF > 2.0%)—A68:01:02-B35:01:01, A68:01:02-B51:01:05, and B08:01:01-C07:02:01—are increased in the INF-P group and two among contacts, A24:02:01-C ∗ 03:04:01 and B51:01:01-C15:02:01. However, it is not possible to make statistical comparisons between groups. For a complete list of haplotypes, check the Supplementary Materials.

3.3. General Mexican Mestizo Population HLA Class I Allele Frequency Compared with INF-P and INF-C

Statistically significant alleles from the INF-P versus INF-C group were compared with population allele frequency for Mexican mestizos. However, B51:01:05, B41:03:01, and none of the HLA-C alleles were compared as there were no six-digit resolution data available. B39:01:01 showed statistically significant variation from the INF-C group (), indicating allele enrichment different from that of the general population (Figure 3). The sample size of HLA class I reported studies was significantly lower compared to sample size of our study. A stringent Fisher’s exact two-tailed test was used to calculate the values. This may have under-/overrepresented the actual allele frequencies.

Figure 3: Allele frequency distribution in general Mexican mestizo population compared to influenza A H1N1/09 patient (INF-P) and contact (INF-C). Statistically significant alleles in the comparison of INF-P to INF-C with available 6-digit allele frequency data for Mexican mestizo population are included. INF-P: influenza patients; INF-C: influenza contacts; HLA: human leukocyte antigen. value < 0.05; value < 0.01. All values were obtained by Fisher exact two-tailed test.

4. Discussion

In this study, we identified several classical HLA class I alleles and haplotypes that may confer susceptibility or protection against A H1N1/09 infection. We analyzed distribution of classical HLA class I (A, B, and C) loci in 138 patients with influenza A H1N1/09 infection and 225 asymptomatic contacts. As expected, we found enrichment of specific alleles and its combinations in the INF-P group.

According to the primary reports of the A H1N1/09 outbreak in Mexico, subjects aged between 10 and 19 years were at increased risk of infection [26]. However, our study subjects do not show this trend. INER has one of the most important respiratory critical care hospitals in Mexico. As a result, severe/complicated cases are referred more to this hospital, hence contributing towards offsets from the previous reports. Increased BMI and smoking were more frequent in influenza patients, which agrees with previous studies [6].

Our analysis revealed lower frequency of A24:02:01 in patients compared to asymptomatic contacts, suggesting a possible protective effect. This result is in disaccord with the study showing A24 (A24 family) positively correlating with severe A H1N1 infection and mortality [27]. However, that result was obtained employing database analysis of conserved proteomic regions-based experimental predictions of HLA binding affinity, showing that HLA alleles preferentially target conserved regions of viral proteins (HLA targeting efficiency) [28]. The study did not consider HLA-restricted NK cell-mediated viral clearance, which may have introduced a certain degree of bias. The HLA-A24 subtype is a potential ligand for KIR3DL1, rendering an inhibitory NK signal [29]. This may reduce the overreactivity of immune system to influenza A H1N1/09 virus, which is known to contribute to the significant pathobiology of the disease [15, 30]. Also, A24:02 is in linkage disequilibrium with B39:01 [31], which possesses high targeting efficiency scores. A24 comprises a large portion of the world population; this allele family is more common in certain indigenous group and constitutes more than half of the global population, especially in Asian countries and several Native American populations [32]. In Mexican mestizos, there exist heterogeneous A24 frequencies depending on the Amerindian contribution and the region studied [31]. In this study, only 8.33% of the patients had any allele of A24 serotype whereas the contact subjects reach 18% (A24:02:01). The general allele frequency of A24:02:01 in Mexican mestizos is 15% [33] (Figure 3).

Our HLA dataset contains six A68 alleles, out of which two belong to different supertypes: A68:01 to the A3 supertype and A68:02 to the A2 supertype. No association was found with A68:02:01 to A H1N1/09 infection, while A68:01:01, despite its low frequency (1.8%), was only present in patients. This is in concordance with the results of the HLA targeting efficiency study showing the A68:01 subtypes positively correlating with A H1N1/09 mortality rates but not A68:02 [27, 34]. Also, we did not find any relationship between A02:01:01 and for that matter any A02 subtypes against A H1N1 infection, which is considered to be a general protection factor against influenza [35].

Higher frequencies of B39:06:02 (statistically insignificant after Bonferroni correction) and B51:01:05 were observed in subjects suffering from influenza A H1N1. In spite of high targeting efficiency scores, B39:06:02 (Bw6) allele frequency was higher in A H1N1 patients compared to asymptomatic contacts. We suspect that this may be due to selective pressure exerted by certain KIR-HLA combinations favoring underreactivity of NK cells. Interestingly, the allele B39:06 has been observed in several Amerindians populations with a frequency of around 5% [31] and possesses high targeting efficiency score [27]. In contrast, B39:01:01, another Bw6 supertype, showed a protective effect; this observation may be due to strong linkage disequilibrium with A24:02 existing in Amerindian populations. A higher frequency of B51:01:05 was also found in INF-P patients. Another allele, HLA-B51:01:01 (not significant after Bonferroni correction), an ancestral allele of Amerindian origin [31], had low frequency in A H1N1/09 patients, pointing towards a possible protective role. In contrast with the HLA-A alleles, the HLA-B alleles bind more efficiently with the conserved regions of the A H1N1 viral proteins. Molecular subtypes of HLA-B39 (B39:01 and B39:06), common in Amerindian populations, are apparently associated with severe forms of the disease [27], which may be a result of these alleles in linkage disequilibrium with another region within or close to HLA locus.

High frequency of C03:02:01 was observed in A H1N1/09 patients, despite its very low frequency in the Mexican population [31]; however, this is not statistically significant after Bonferroni correction. On the other hand, C03:03:01, C03:04:01, and C07:01:01 showed low frequency in the INF-P group. HLA-C, compared to HLA-A or HLA-B, is less polymorphic and presents more restricted repertoire of peptides and low cell surface expression [31, 36]. Proportionally higher frequency of KIR2DL1 C2− C1+ and KIR2DL3 C1+ was reported in ICU A H1N1/09 patients (indigenous and nonindigenous), relative to healthy controls [15].

Relatively low frequency of the A02:01:01-B35:01:01-C04:01:01 haplotype was observed in the A H1N1/09 patients. This specific HLA haplotype represents more than 2.5% in the control group and has been found only in Hispanic and Mexican populations with the resolution employed in this study. We observed an increase in the frequency of A68:01:02-C07:02:01 haplotype in the patients. This finding is relevant because linkage disequilibrium is very high in the region rendering potential haplotypes and can potentially amplify the risk to disease. Considering the resolution employed in the study, it is difficult to compare functional and population-based allele/haplotype frequencies.

Our study was subjected to the following limitations; first, we did not consider risk factors associated with influenza other than obesity, smoking status, and pregnancy. Various diseases such as asthma, diabetics, immunodeficiency, and pneumococcal pneumonia are all risk factors for A H1N1/09 influenza [6, 37]. Second, the sample size of each group and the general allele frequency was low, which may have affected the power of statistical analysis to a certain degree. Third, the antibody titer observed in the contacts group may have contributions from seasonal influenza strains. The exact degree of contribution from cross-reacting antibodies from seasonal strains is largely unknown [38]. However, we expect that the contribution is minor and could not reach a threshold of 1 : 16 dilutions. Surveillance of circulating A H1N1/pdm09 viruses has revealed some genetic variations in the viral surface glycoproteins, hemagglutinin, and neuraminidase. However, until recently, the antigenicity of the circulating A H1N1/pdm09 viruses was similar to the vaccine strain (A/California/7/2009) in assays with panels of antiserum obtained from infected ferrets [39]. Despite these limitations, this is the first report showing HLA class I alleles and haplotypes associated with susceptibility to influenza A H1N1/09 in the Mexican population.

In summary, B51:01:05, C07:02:01, and C02:02:02 were all increased among A H1N1/09-infected patients (INF-P), while A24:02:01, B51:01:01, B39:01:01, and C03:04:01 were elevated in asymptomatic healthy contacts. B39:01:01 showed a statistically significant variation from the INF-C group compared to general Mexican mestizos. Haplotype analysis revealed enrichment of certain allele combinations out of which A68:01:02-C07:02:01 and B35:01:01-C07:02:01 were found to be important haplotypes susceptible to A H1N1/09 infectivity.

Our study validates that selective classical HLA class I alleles and haplotypes are favored for both susceptibility and protection against A H1N1/09 pandemic. From our results, we speculate that the selective pressure on HLA class I enrichment may be a result of specific KIR-HLA combinations and HLA targeting efficiency along with other factors. Further studies may be needed to validate the result and elucidate the role of HLA class I alleles in A H1N1/09 influenza.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by Consejo Nacional de Ciencia y Tecnología from México (Grant nos. SALUD-2009-C02-126699 and SALUD-2009-126731 to Ramcés Falfán-Valencia and Alejandra Ramírez-Venegas, resp.).

Supplementary Materials

Table SVI: Complete list of HLA-A alleles in Mexican mestizo population with and without infection by influenza A/H1N1 virus. Table SVII: Complete list of HLA-B alleles in Mexican mestizo population with and without infection by influenza A/H1N1 virus. Table SVIII: Complete list of HLA-C alleles in Mexican mestizo population with and without infection by influenza A/H1N1 virus. Table SIX: HLA-A-B-C haplotype frequencies in patients with influenza A/H1N1 infection and asymptomatic healthy contacts. Table SX: HLA-A-B haplotype frequencies in patients with influenza A/H1N1 infection and asymptomatic healthy contacts. Table SXI: HLA-B-C haplotype frequencies in patients with influenza A/H1N1 infection and asymptomatic healthy contacts. Table SXII: HLA-A-C haplotype frequencies in patients with influenza A/H1N1 infection and asymptomatic healthy contacts. (Supplementary Materials)

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