HLA-B*35 as a new marker for susceptibility to Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) in patients with Human T-cell Lymphotropic Virus type 1 (HTLV-1) in Argentina

Paula Benencio CONICET-Universidad de Buenos Aires, Instituto en Investigaciones Biomédicaas en Retrovirus y SIDA (INBIRS) https://orcid.org/0000-0001-6471-748X Sindy A. Fraile Gonzalez CONICET-Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS) Nicolás Ducasa CONICET-Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS) Kimberly Page University of California San Francisco Department of Epidemiology and Biostatistics Carolina A. Berini (  caroberini@gmail.com ) CONICET-Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retovirus y SIDA (INBIRS) Mirna M. Biglione CONICET-Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retovirus y SIDA (INBIRS)


Background
Human T-cell Lymphotropic Virus type 1 (HTLV-1) was the rst human retrovirus to be discovered and is the etiological agent of Adult T Cell Leukemia/Lymphoma (ATLL), a progressive neurological disease called HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) (1,2) and Uveitis and a severity factor for Bronchiectasis and other diseases (3). The vast majority of HTLV-1-infected individuals are asymptomatic and around 1-2% of them will develop ATLL and less than 5%, HAM/TSP. Although the risk factors causing different HTLV-1 associated diseases are not fully understood, their pathogenesis is thought to be in part due to proviral load (PVL) and/or the host genetic factors (4,5,6).
The target cells of HTLV-1 are CD4 + T cells, and to a lesser extent CD8 + T cells, B cells, monocytes, macrophages and dendritic cells (6). The maintenance of HTLV-1 infection occurs mostly by clonal expansion of infected cells. In the case of ATLL, the expression of Tax protein in some of these clones with accumulated genomic abnormalities could help develop a pre-leukemic state in some individuals. The malignantly transformed HTLV-1 infected cells very often suppress Tax expression in favor of HTLV-1 basic leucine zipper factor (HBZ) expression, a negative regulator of Tax that is believed to aid immune evasion of the infected cells and further assist cancerous transformation (7). On the other hand, the increased number of HTLV-1 infected T-cells may also cause imbalance of the immune system, resulting in immune dysfunction or in ammatory diseases like myelopathy and uveitis (8). In this context, HAM/TSP pathogenesis is a hyperactive immune response induced by HTLV-1 infection that produces chronic in ammation in the central nervous system (CNS) with slowly progressive evolution. It is characterized by the production of elevated levels of proin ammatory cytokines, including IFN-and TNF, and by HTLV-1speci c CD8 + T cells in peripheral blood and spinal cord lesions (9)(10)(11).
Human Leukocyte Antigen (HLA) class I genes have been associated with susceptibility to disease in many human infections and it is among the host genetic factors that could be related to manifestation of ATLL and HAM/TSP (12,13). A unifying theory is that HLA alleles associated with ATLL show a limited recognition of HTLV-1 Tax peptide anchor motifs and epitopes capable of generating anti-HTLV-1 Tax CD8 + T cells while for HAM/TSP they induce strong cytotoxic T-lymphocyte (CTL) responses against the viral oncoprotein Tax (14,15).
Speci c HLA alleles have been linked to protection from developing these pathologies, whereas other HLA alleles have been correlated with an increased risk of developing them (Table 1). Some studies reported the HLA-A*02 allele to have a protective role both in ATLL and HAM/TSP disease in Jamaica, Brazil, Japan and Peru (15)(16)(17)(18)(19)(20). The same was the case for allele Cw-08 in Japan and Iran (15,19,21). HLA-A*26 and A*54 were associated with susceptibility to ATLL and HAM/TSP, respectively, in Japan, whereas A*36 was associated with susceptibility to ATLL in Jamaica (16,22). In contrast, HLA-B*5401 has been associated with an increased susceptibility to HAM/TSP in Japan and Iran (15,23). Allele C*07, on the other hand, has been associated to susceptibility to disease in Brazil, only in patients who did not possess A*02 (24).
Another perspective suggests that greater HLA diversity conveys selective advantage against disease because the immune response is elicited by a greater variety of antigens as described for human immunode ciency virus (HIV) and acquired immunode ciency syndrome (AIDS) (29). Since each HLA allele exposes a different set of amino acids in their peptide cleft, they will each be able to present peptides with different speci cities. This results in a heterogeneous capacity to activate T CD8 + lymphocytes to target the infected cells. Carrying mismatched alleles for each HLA gene thus confers the ability to present a wider range of peptides, and to then be more likely to activate cytotoxic T cells to eliminate infected cells. It has been reported that, in HIV infection, HLA class I heterozygotes progress more slowly to AIDS than do homozygotes and that the viral load is signi cantly lower due to rare HLA class I alleles (30,31). In relation to HTLV-1 infection, Goedert et al., showed that HLA class I diversityreduces the risk of ATLL presumably by limiting the proliferation of HTLV-1 infected cells in vivo and therefore decreasing the possibilities of developing the disease (27).
The aim of this study was to identify HLA class I alleles associated to protection or susceptibility to disease and to analyze its possible association with proviral load in individuals infected with HTLV-1 from Argentina.
The protocol was reviewed and approved by the Institutional Review Board as well as by the External Ethical Committee (NEXO AC IRB#0005349, protocol #1563). An informed consent was obtained from all individuals. The diagnosis of ATLL and HAM/TSP was performed in accordance with Tsukasaki and Osame criteria, respectively (17,19). Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated blood samples by a Ficoll-Hypaque density gradient (Ficoll Paque Plus, Sigma Aldrich, Saint Louis, USA) and DNA was extracted using a commercial kit (ADN PuriPrep-S kit, Inbio Highway, Tandil, Argentina). After serological screening, HTLV-1 infection was con rmed by an in-house nested polimerase chain reaction (n-PCR) as described elsewhere (32). Absolute quantitation of PVL was performed by real-time SYBR Green PCR, using an ABI Prism 7500 Prism System (Applied Biosystems, USA) as previously described (33,34).
HLA class I characterization was performed by sequence based typing (SBT). HLA-A exons 2 and 3 were ampli ed together while HLA-B/C exons 2 and 3 were ampli ed separately, as described elsewhere (35).
Amplicons were sequenced using the Big Dye Terminator sequencing kit (Applied Biosystems, USA) on a 3500xL Genetic Analyzer AB/HITACHI according to the manufacturer's instructions.
For the calculation of allele frequencies, we counted each individual allele for each locus across all samples. In the cases where an individual resulted homozygous for a locus, this situation was equivalent to a count of 2 for the corresponding allele.
Data analysis was performed using the Kruskal-Wallis non-parametric method; when two groups were compared the Chi2 test or Exact Fisher test, and one-way ANOVA were used. Epidat (version 4.2) and GraphPad Prism (version 6.03) software was applied and signi cant differences were de ned as p < 0.05.
Maximum-likelihood haplotype frequencies were estimated by an expectation maximization (EM) algorithm performed with Arlequin (Version 3.1).

Results
Among the 73 samples analyzed, there was no signi cant difference in gender (p = 0.135) nor age (p = 0.6497, mean age = 38.56, mean age NII = 28.69, mean age Ac = 36.91, mean age HAM/TSP = 43.74, mean age ATLL = 44.33) between asymptomatics and the group with HTLV-1 associated pathologies. Figure 1 presents the demographic characteristics of all studied individuals.
There were a total of 11 samples for which only one of the alleles for a speci c HLA I loci could be identi ed.
Overall, A*02 (28.77%), B*35 (20.55%) and C*07 (21.23%) were the most frequent in the population studied. Table 2 shows the frequencies of individual alleles in all the studied individuals.
In relation to HLA-A, the alleles A*23, A*24, A*32 and A*68 were observed among ATLL patients, but not in HAM/TSP, while the opposite case was found for A*11 and A*29 in HAM/TSP patients but not ATLL. Alleles HLA-A*02, A*31, and A*33, present both in individuals with ATLL and HAM/TSP, showed no signi cant differences between them (p = 0.071, p = 0.458, p = 0.414, respectively). However, there was a signi cant difference in the frequency of HLA-A*02 between asymptomatic carriers and those with ATLL (p = 0.042).
Alleles A*01 and A*36 were only found among NII; A*25 and A*26, among Ac; A*11, among HAM/TSP patients; and A*23, among ATLL patients. For the 43 HLA-C samples, the alleles C*01 and C*31 were only found in NII, while the alleles C*05, C*06, C*16 and C*18 were exclusive for asymptomatic carriers. There were not any ATLL-or HAM-exclusive alleles. Overall, patients that had developed either one of the associated pathologies exhibited a rather limited arrange of alleles: only C*03, C*07 and C*15 were identi ed in our analysis. Out of the 4 pathology cases (2 HAM/TSP and 2 ATLL) that presented C*07, all of them homozygotes for that loci, only one displayed also the allele A*02; the exact same was for the Ac group.
The mean PVL in asymptomatic carriers was 2.85 per 100 PBMCs, and 10.32 in individuals with HTLV-1 associated pathologies (16.36 for ATLL and 8.6 for HAM/TSP patients). PVL of patients with HTLV-1 associated pathologies was signi cantly higher than that of asymptomatic carriers (p = 0.0177) while there were no differences between the mean PVL of ATLL and HAM/TSP patients (p = 0.3135). Table 3 presents the values of PVL classi ed per HLA-A, HLA-B and HLA-C alleles. No signi cant differences were observed for HLA-A, B and C when comparing the PVLamong all the alleles of each loci by one-way ANOVA.
Most of the ATLL and HAM/TSP patients were homozygous for HLA-A (23/26) locus and HLA-C (7/8), in contrast to the HLA-B locus (6/32). Regarding heterozygosity, for the HLA-A locus there was a signi cant difference when comparing asymptomatics versus individuals with pathologies, heterozygosity being more frequent among asymptomatics (p = 0.023). We found all of the ATLL and HAM patients to be homozygous for HLA-C and all of the asymptomatic carriers to be homozygous for HLA-B.
Lastly, we performed an expectation-maximization algorithm analysis of all the haplotypes found in our research to estimate the maximum-likelihood haplotype frequencies. Table 4 shows the frequency of haplotypes that had no missing data at any loci.

Discussion
It has been proposed that the HTLVs have arisen as a consequence of inter-species transmissions that took place millions of years ago in Africa and that HTLV-1 was introduced in America during the multiple pre-Columbian Mongoloid migrations over the Bering Strait, and in the post-Columbian era from Japan and with the slave trade from Africa. Therefore, the different migration waves of infected populations resulted in an ethnic/geographical restriction in the American continent for HTLV-1 and 2. Moreover,Lou et al., described certain polymorphisms as possibly associated to susceptibility for HTLV-1 infection for ethnically related populations of Russia (HLA-A*02, A*24) and Japan (HLA-A*24 y A*26) (14,36). In the general population of Argentina the reported frequency of A*02, A*24 and A*26 was 24.95%, 11.25% and 4.02%, respectively (37).
In the studied population (Table 3) there was a high frequency of allele A*02 (28.77%), and contrary to the data reported in the general population, the frequencies of A*24 (6.16%) A*26 (0.68%) were lower. In the case of HLA-B and -C, the most common alleles in Argentina are B*35 (14.6%), B*44 (11.4%) and B*51 (7.9%), and C*07 (24.6%), C*04 (16.6%) and C*03 (10.4%) (26). Our own population largely matched these data, except for the cases of alleles B*44 and C*04, which were relatively uncommon (3.42% and 5.48%, respectively). Instead, alleles B*39 (13.01%) and C*15 (6.85%) were among the top found. It should be noted that all the alleles are present in a lower proportion in the general population than in our own due to the fact that there was a smaller variety of alleles found in the latter, which translates in a bigger proportion of the total distribution for each of them.
Regarding, the thirteen Peruvian and the four Paraguayan individuals sampled, the allele frequencies matched the reported prevalence in these populations (38,39). The most frequent alleles for HLA-A in the general population in Peru were A*02, A*24 and A*68, in decreasing order, which correlated with our own ndings. The same happened for HLA-B (most frequent HLA-B*35), and HLA-C, the most common allele being Cw*04. In our own population, we found 8 copies of said allele, 7 of which corresponded to Peruvian individuals. When it came to the four Paraguayan individuals tested, the most common alleles were HLA-A*02 and HLA-B*35, the same as for the general population in that country, although for the case of the indigenous Guaraní, the most common HLA-B alleles were HLA-B*15 and B*40.
Regarding the pathologies associated to HTLV-1 infection, it is known that most individuals remain asymptomatic throughout their lives. ATLL and HAM/TSP patients are more frequent in areas of high endemicity and they represent a small percentage of the infected population (up to 5%).
The reasons behind the development of pathologies during adulthood and their association to host genetic factors are still unclear even though many hypotheses have been proposed. HLA class I genes may have an effect on the progression towards ATLL and HAM/TSP due to its critical role in antigen presentation (14).
Various alleles have been described as either protective or susceptible for the development of ATLL or HAM/TSP. In Jamaica, Japan and Brazil the allele HLA-A*02 has been described as protective both for ATLL and HAM/TSP, in accordance with other studies which reported nding it signi cantly more frequent in asymptomatic carriers (14,18,36,40). In our population, A*02 was signi cantly more rare in ATLL patients when compared to HTLV-1 + asymptomatic carriers, suggesting as well a protective role for this allele in this group. This protective role could not be con rmed for HAM/TSP patients in this study. The allele HLA-A*03, previously described as protective in Jamaica, was only found in asymptomatic patients and healthy donors in the studied population and with a low frequency.
We did not found any associations linking HLA-A*26 to either protection or susceptibility to ATLL, although it should be pointed out that this allele is not frequently found in the Argentine population (4.02%) (37).
In the case of HLA-B allele distribution, B*35 was found to be signi cantly more frequent in patients with HAM/TSP than in asymptomatics, which could point to a possible association of this allele to the development of diseases. Although B*35 has been previously linked to progression to disease, viral load, heterosexual transmission and mother to child transmission in HIV-1 infected individuals and to disease progression in HBV, this is the rst report about this allele in relation to HTLV-1 infection (41)(42)(43).
Regarding HLA-C, our analysis yielded no results compatible with previous studies in Brazil (24), which reported a correlation between C*07 and progression to disease, neither in individuals who also exhibited HLA-A*02 or among those who did not.
Another aspect to be considered is that all of the alleles found solely in one group had a very low frequency; many of them were actually identi ed that one time ( Table 2). They were also very rare alleles for the general Argentine population (37). Thus, it is not possible to draw any conclusions regarding these ndings.
To this day, the therapies for HTLV-1 associated pathologies seek to reduce the proviral load. In the last decade, a real time quantitative PCR (qPCR) has been implemented for the quanti cation of proviral load (PVL) of HTLV-1/2 from cells of infected patients. Its determination is used as an indicator of the course of infection in asymptomatic carries in order to evaluate their predisposition to the development of pathology and to monitor treatment progression in ATLL and HAM patients (44). It has been reported that, although the PVL has been suggested to be directly related to the severity of the disease, the values among infected individuals often vary signi cantly (45). This corresponds with the dispersion of the values observed in this study. Previously reported values indicate that in asymptomatic carriers, the mean proviral load is 0.1-1 copy/100 PBMCs, while in patients with HAM/TSP is 5-10/100 PBMCs, exceeding sometimes 30 copies (45). Despite these differences observed in the PVL values and the technique used, all the reports conclude that there is a signi cant difference among asymptomatics and patients with pathologies as observed in our studied population. These results also indicate that there is a correlation towards disease progression (35).
Some studies have proposed that HLA allelic variants could determine the PVL levels of HTLV-1 infected individuals (13,26). Nonetheless, we couldn't nd any signi cant differences in the PVL of any allele to support these previous claims (Table 3).
It has been proposed that heterozygosis on HLA confers advantages on disease progression in AIDS, revealing a greater variety of the immune response (46,47). In accordance to this, heterozygosis for HLA-A was signi cantly more frequent among asymptomatics when compared to individuals with pathologies.
However, the opposite was true for HLA-B, for which homozygosis was more frequent in asymptomatic carriers than in patients with pathologies.
In conclusion, several HLA alleles identi ed in our study were associated with disease progression. Our results adds more evidence to the protective effect of HLA-A*02 allele on progression to ATLL, and draws attention to HLA-B*35 as a new allele to be considered in relation to susceptibility to HAM/TSP.
To this day, however, no allele or allele pattern has been identi ed to be exclusive to either asymptomatic individuals or those who develop pathologies, and to thus be of use when it comes to providing a predictive diagnosis. Were an allele like this to be found, in line with the rapidly evolving eld of precision medicine, it would mean the possibility to conduct a closer follow up of each asymptomatic HTLV-1 + carrier, for those patients that choose to learn the impact of their genetic background on the infection by HTLV-1.

Conclusions
We have found HLA-A*02 and HLA-B*35 to be associated to protection from ATLL and susceptibility to HAM/TSP, respectively. Whereas HLA-A*02 protection from ATLL has already been extensively described in other regions of the world, this is the rst report that links HLA-B*35 and an increased susceptibility to HAM/TSP.  Ac: asymptomatics, HAM/TSP: HTLV-1 associated myelopathy/Tropical Spastic Paraparesis, ATLL: Adult T cell leukemia/lymphoma, NII: Non-infected Individuals, HLA: Human leukocyte antigen.