International Journal of Molecular Sciences Article Lipoprotein Profile in Immunological Non-Responders PLHIV after Antiretroviral Therapy Initiation Jenifer Masip 1,2,3,† , Rosa Jorba 1,2,3,† , Miguel López-Dupla 1,2,3,4 , Pere Domingo 5 , Yolanda María Pacheco 6 , Graciano García-Pardo 1,2,3 , Esteban Martínez 4,7 , Consuelo Viladés 1,2,3,4 , Sergi Veloso 1,2,3 , Verónica Alba 1,2,3,4 , Montserrat Olona 1,2,3,4 , Francesc Vidal 1,2,3,4 , Frederic Gómez-Bertomeu 1,2,3,4 , Joaquim Peraire 1,2,3,4, * and Anna Rull 1,2,3,4, * 1 Infection and Immunity Research Group (INIM), Universitat Rovira i Virgili, 43003 Tarragona, Spain; jenifer.masip@urv.cat (J.M.); rjorba.hj23.ics@gencat.cat (R.J.); jmlopezdupla.hj23.ics@gencat.cat (M.L.-D.); ggarciap.hj23.ics@gencat.cat (G.G.-P.); cvilades.hj23.ics@gencat.cat (C.V.); sveloso.hj23.ics@gencat.cat (S.V.); veronica.alba@urv.cat (V.A.); molona.hj23.ics@gencat.cat (M.O.); fvidalmarsal.hj23.ics@gencat.cat (F.V.); ffgomez.hj23.ics@gencat.cat (F.G.-B.) 2 Infection and Immunity Research Group (INIM), Institut Investigació Sanitària Pere Virgili (IISPV), 43003 Tarragona, Spain 3 Infection and Immunity Research Group (INIM), Hospital Universitari de Tarragona Joan XXIII, 43005 Tarragona, Spain 4 Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain; estebanm@clinic.cat 5 Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; pdomingo@santpau.cat 6 Immunology Lab, Institute of Biomedicine of Seville, University Hospital Virgen del Rocío/CSIC/University Citation: Masip, J.; Jorba, R.; of Seville, 41013 Seville, Spain; ypacheco-ibis@us.es López-Dupla, M.; Domingo, P.; 7 Infectious Diseases Service, Hospital Clinic-IDIBAPS, University of Barcelona, 08036 Barcelona, Spain Pacheco, Y.M.; García-Pardo, G.; * Correspondence: jjperaire.hj23.ics@gencat.cat (J.P.); anna.rull@iispv.cat (A.R.) Martínez, E.; Viladés, C.; Veloso, S.; † These authors contributed equally to this work. Alba, V.; et al. Lipoprotein Profile in Immunological Non-Responders Abstract: Nuclear magnetic resonance (NMR)-based advanced lipoprotein tests have demonstrated PLHIV after Antiretroviral Therapy that LDL and HDL particle numbers (LDL-P and HDL-P) are more powerful cardiovascular (CV) risk Initiation. Int. J. Mol. Sci. 2022, 23, biomarkers than conventional cholesterol markers. Of interest, in people living with HIV (PLHIV), 8071. https://doi.org/10.3390/ predictors of preclinical atherosclerosis and vascular dysfunction may be associated with impaired ijms23158071 immune function. We previously stated that immunological non-responders (INR) were at higher Academic Editors: Vladimir CV risk than immunological responders (IR) before starting antiretroviral therapy (ART). Using Titorenko, Jose Luis Liposcale® tests, we characterized the lipoprotein profile from the same cohort of PLHIV at month Sanchez-Quesada and Sonia 12 and month 36 after starting ART, intending to explore what happened with these indicators of CV Benítez González risk during viral suppression. ART initiation dissipates the differences in lipoprotein-based CV risk Received: 22 June 2022 markers between INR and IR, and only an increase in the number of HDL-P was found in INR + IR Accepted: 19 July 2022 when compared to controls (p = 0.047). Interestingly, CD4+ T-cell counts negatively correlated with Published: 22 July 2022 medium HDL-P concentrations at month 12 in all individuals (ρ = −0.335, p = 0.003). Longitudinal analyses showed an important increase in LDL-P and HDL-P at month 36 when compared to baseline Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in values in both IR and INR. A proper balance between a proatherogenic and atherogenic environment published maps and institutional affil- may be related to the reconstitution of CD4+ T-cell count in PLHIV. iations. Keywords: antiretroviral therapy; cardiovascular risk; immunological non-responder; lipoproteins; longitudinal analysis; nuclear magnetic resonance; people living with HIV Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and The incidence of non-acquired immunodeficiency syndrome (AIDS)-defining diseases conditions of the Creative Commons Attribution (CC BY) license (https:// (nonADDs), such as cardiovascular events (CVEs), is two-fold higher in persons living with creativecommons.org/licenses/by/ human immunodeficiency virus (PLHIV) than in the general population [1–3], and this 4.0/). incidence of disease is explained mainly by HIV-induced activation of inflammatory and Int. J. Mol. Sci. 2022, 23, 8071. https://doi.org/10.3390/ijms23158071 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 8071 2 of 12 coagulation pathways [4]. Moreover, combination antiretroviral therapies, from previous exposure to first-generation nucleoside analogue reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), were associated with an increased risk of cardiovascular disease (CVD) [5]. Treated HIV-infected adults have approximately double the levels of inflammatory markers, such as inflammatory interleukin-6, soluble CD14 and soluble CD163, which have been associated with an increased risk of coronary artery inflammation and atherosclerosis when compared to age- and sex-matched healthy populations [4,6,7]. Furthermore, in PLHIV, markers of preclinical atherosclerosis and vascular dysfunction may be associated with impaired immune function [8]. Indeed, in our previous group study, we stated that ART-naïve PLHIV with less than 200 CD4+ T-cell counts/µL, which revealed a poor immune response to ART (immunological non-responders), were at a higher cardiovascular (CV) risk than PLHIV who started ART with less than 200 CD4+ T-cell counts/µL, and reached more than 250 CD4+ T-cell counts/µL at month 36 on ART (immunological responders) [9]. Abnormal levels of blood lipids, such as high concentrations of low-density lipid cholesterol (LDL-C) and low levels of high-density lipid cholesterol (HDL-C), are tradi- tionally known as important predictors of cardiovascular risk. Of interest, the recent use of nuclear magnetic resonance (NMR)-derived lipoprotein subclasses (VLDL, LDL and HDL) for the characterization of lipoprotein particles, including size and particle num- ber, has improved the risk stratification for subclinical atherosclerosis in comparison to conventional lipid measurements [10–13]. Concretely, NMR-based advanced lipoprotein tests have demonstrated that LDL and HDL particle numbers (LDL-P and HDL-P) are more powerful cardiovascular risk biomarkers than conventional cholesterol markers [14]. Indeed, LDL-P, and more specifically the increased presence of small LDL-P, was found to be associated with a higher atherosclerotic risk [15], whereas an important representa- tion of HDL-P in circulation seemed to be inversely associated with carotid intima–media thickness [16]. Thus, the recent methodology based on a two-dimensional spectra (2D) from diffusion-ordered NMR spectroscopy (DOSY) experiments (Liposcale® test) provides not only lipid concentration but also lipoprotein size, and lipoprotein particle numbers of the main fractions and subclasses compared to liquid chromatography/tandem mass spectrometry (LC-MS/MS) which also offers a valuable tool for determining a lipid profile with potential diagnostic applications due to its sensitivity and rapid separation limited to lipid concentrations [17,18]. Thus, using a two-dimensional NMR technique (Liposcale® ) [9], we previously re- ported higher baseline values of both LDL particles/HDL particles and total particles/HDL particle ratios in INR than in IR [9]. These results support the idea that chronic activation of the innate immune system could be related to increased CVD in PLHIV with poor immune responses. However, information regarding the evolution of lipoprotein lipid content and particle distribution once ART was introduced to INR or, more importantly, how ART could improve or impair the suspected baseline CV risk is still scarce. Thus, in the current study, we aimed to characterize the lipoprotein evolution from INR and IR during ART and to corroborate whether LDL-P/HDL-P or total particles/HDL-P ratios, both indicators of CV risk, remain significantly higher in INR compared to IR during ART follow-up. For that reason, using the same 2D 1 H-NMR (Liposcale® ) and the same study cohort, we evaluated the lipoprotein profile at months 12 and 36 [9], and we also included a group of PLHIV starting ART with more than 200 CD4+ T-cell counts/µL to explore whether the ART-naïve CD4+ T-cell decrease could affect cardiovascular health in PLHIV once ART was introduced. 2. Results A total of 21 PLHIV with good recovery status at the initiation of ART (472 (383–563) cells/µL) were included in the study as a control group. Of the 53 PLHIV with low CD4+ T-cell counts at the initiation of ART (less than 200 cells/µL), 38 had a successful good recovery status at month 36 (IR). In IR, the total CD4+ T-cell counts were four times increased at Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 13 Int. J. Mol. Sci. 2022, 23, 8071 3 of 12 T-cell counts at the initiation of ART (less than 200 cells/µ L), 38 had a successful good recovery status at month 36 (IR). In IR, the total CD4+ T-cell counts were four times increased at 36 months (357 (303–461.8) cells/µ L) compared to baseline values (90 (45–171) 36 months cells/µ L) (p <(357 (303–461.8) 0.001), cells/µL) but the values compared remained to baseline significantly values lower than(90 the(45–171) cells/µL) control group at (p < 0.001), but the values remained significantly lower than the control the same time point (month 36) (752 (639–864) cells/µ L (p < 0.001)). However, group at the same 15 time point (month participants of the 5336)initiating (752 (639–864) cells/µL ART with (p < 0.001)). less than However, 200 cells/µ 15 participants L did not reach a goodof the 53 initiating ART with less than 200 cells/µL did not reach a good recovery status even after being on successful virological suppression (INR). In INR, recovery status the even after being on successful virological suppression (INR). In INR, the mean total CD4+ mean total CD4 T-cell counts at month 36 were also significantly increased (186 (117–220) + T-cell counts at month 36 were also significantly increased (186 (117–220) cells/µL) when cells/µ L) when compared to their baseline values (57 (27–99) cells/µ L, p < 0.001), although compared to their baseline values (57 (27–99) cells/µL, p < 0.001), although the values the values prevailed significantly lower when compared to the+ total CD4+ T-cell counts of prevailed significantly lower when compared to the total CD4 T-cell counts of both IR and both IR and controls (p < 0.001) (Figure 1A). controls (p < 0.001) (Figure 1A). CD4+ +T-cell Figure1.1.CD4 Figure T-cellcounts countsduring duringART. CD4+ T-cell ART. CD4+ T-cell counts counts(cells/µL) (cells/μL) at at month month1212andandmonth month3636in (A) control group starting first ART with more than 350 cells/µL, (B) immunological in (A) control group starting first ART with more than 350 cells/µ L, (B) immunological responders responders (IR)who whopresented presentedwithwitha aCD4 CD4 + T-cell count equal to or greater than 250 cell/µL at month 36 and (IR) + T-cell count equal to or greater than 250 cell/µ L at month 36 and (C) (C) immunological non-responders + T-cell immunological non-responders (INR) (INR) who didwho notdid notmore reach reach more than 250than cell/µ250 cell/µL L CD4 CD4 + T-cell at month 36. at Horizontal green dotted month 36. Horizontal linesdotted green represent baseline lines values represent of thevalues baseline CD4 T-cell + of thecounts+ in eachcounts CD4 T-cell group.in The eachWilcoxon group. Thepaired test was Wilcoxon performed paired test wastoperformed evaluate differences to evaluatebetween differencesmonth 12 and between month month 12 36. and **month p < 0.001. 36. ** p < 0.001. 2.1.Stable 2.1. StableARTARTDiminishes DiminishesDifferences DifferencesininLipoprotein-Based Lipoprotein-BasedCardiovascular CardiovascularRisk RiskMarkers Markers Previously,both Previously, bothtotal total particles/HDL-P particles/HDL-Pand andLDL-P/HDL-P LDL-P/HDL-Pratios ratiosconfirmed confirmedthat thatINR INR participants were at higher risk of suffering cardiovascular diseases than participants were at higher risk of suffering cardiovascular diseases than IR participants IR participants before starting before starting ART ART [9]. [9].Thus, Thus,both bothpredictive predictivecardiovascular cardiovascular markers markerswerewere evaluated again evaluated at month again 12 and at month 12month and month36, once 36, ART once was ARTinitiated, and were was initiated, andcompared to the control were compared to the group. group. control No significant differences No significant were observed differences among groups were observed amongatgroups monthat12month or at month 36. 12 or at monthThen, 36. we investigated whether the type of ART prescribed differentially affects the CV Then, risk bywe comparing investigated the whether ratios in the the type recategorized study cohort of ART prescribed based on the differentially ART affects re- the CV risk by comparing the ratios in the recategorized study cohort based on the ARTof ceived. At month 36, the percentage of antiretroviral therapies based on the combination nucleoside received. At analogue month 36,reverse transcriptase the percentage inhibitors (NRTIs) of antiretroviral therapiesplus nonnucleoside based analogue on the combination reverse transcriptase inhibitors (NNRTIs) was the primary modality of nucleoside analogue reverse transcriptase inhibitors (NRTIs) plus nonnucleoside for the treatment and management analogue of HIV infection reverse transcriptase in all groups, inhibitors (NNRTIs) which was was followedmodality the primary by ART schemes for the containing NRTIs plus protease inhibitors (PIs) (Figure 2A). Although treatment and management of HIV infection in all groups, which was followed by ART both ratios were higher in HIV-positive patients receiving NNRTI + PI, no statistically schemes containing NRTIs plus protease inhibitors (PIs) (Figure 2A). Although both ratios significant results werehigher were obtained in when compared HIV-positive to other patients ART schemes receiving NNRTI (Figure + PI, 2B). Of note, thesignificant no statistically NNRTI+PI was the less-frequent ART combination in the controls, IR and INR results were obtained when compared to other ART schemes (Figure 2B). Of note, the (Figure 2A). NNRTI+PI was the less-frequent ART combination in the controls, IR and INR (Figure 2A). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 13 Int.Int. J. Mol. Sci. 2022, 23, 8071 J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 13 4 of 12 A) B) A) NRTIs+NNRTIs B) NRTIs+NNRTIs NRTIs+PI NRTIs+PI NRTIs+NNRTIs NRTIs+NNRTIs NNRTIs+PI NNRTIs+PI 26.7% NRTIs+PI 26.7% NRTIs+PI Other Other 33.3% NNRTIs+PI 33.3% NNRTIs+PI 26.7% Other 26.7% Other 33.3% Control IR INR 33.3% 50 6.7% Total particles/ HDL-particles 6.7% Control IR INR 50 6.7% Total particles/ HDL-particles 33.3% 6.7% 33.3% 40 33.3% 33.3% 40 23.8% 21.1% 30 26.7% 23.8% 21.1% 33.3% 30 2.6% 26.7% 52.4% 47.4% 33.3% 9.5% 2.6% 20 52.4% 47.4% 6.7% 20 9.5% 28.9% 6.7% 14.3% 28.9% 33.3% 10 14.3% 33.3% 10 0 0 36 months 36 months Figure 2. Evaluation of cardiovascular risk by ART scheme. (A) Percentage distribution of the Figure 2.2.Evaluation Figure Evaluationofofcardiovascular cardiovascularriskrisk by by ART ART scheme. scheme. (A) (A) Percentage Percentage distribution distribution of theof the differentART different ARTschemes schemesincluding including the combinations of nucleoside analogue reverse transcriptase different ART schemes including thethe combinations combinations of nucleoside of nucleoside analogue analogue reverse reverse transcriptase transcriptase inhibitors(NRTIs) inhibitors inhibitors (NRTIs)plus (NRTIs) plus plusaaanonnucleoside nonnucleoside nonnucleoside reverse reverse reverse transcriptase transcriptase transcriptase inhibitor (NNRTI) inhibitor inhibitor (NNRTI) (NNRTI) ororprotease protease inhibitors or protease inhibitors (PIs) or the inhibitors (PIs) (PIs) ororthe thecombination combination combination of of of NNRTI NNRTI in +PI +PI +PI NNRTI the in the study in the study study group. group. group. (B) (B) Total (B) Total Total particle/HDL particle/HDL particle/HDL particle particle particle ratio at ratio ratio monthatat month month 3636in 36 in the inthe therecategorized recategorized recategorized study studystudy group group group (n =(n 74)=(nbased 74)= 74) basedbased on on combination on the the the combination combination of of ART ART of ART received. To received. analyzeTo received. Toanalyze the analyze the lipoproteinthelipoprotein profile lipoprotein evolution profile profile evolution evolution during during ART duringARTARTfollow-up, follow-up, data data follow-up, data at at month month at 12 and12 and 12month and data at month data dataatatmonth month3636werewerenormalized normalized byby dividing dividing each itemitem each by its byrespective baseline its respective value.value. baseline 36 were normalized by dividing each item by its respective baseline value. 2.2. 2.2.HDL HDLParticle ParticleSize SizeCorrelates Correlateswith CD4 with + T-Cell CD4 + Count + T-Cell at Month Count 12 (ART) at Month 12 (ART) 2.2. HDL Particle Size Correlates with CD4 T-Cell Count at Month 12 (ART) Regarding Regarding the lipid lipidconcentration andand lipoprotein sizing and particle number Regarding the the lipid concentration concentration lipoprotein and lipoprotein sizing sizing and and particle particle number number determina- determination, determination, only onlydifferences differences in the in HDL the HDLparticles were particles statistically were significant statistically at significantmonth at month 12tion, only differences between controls, IRIR inand theINR. HDLConcretely, particles were statistically thethe size of HDL significant particles at month was 12 between con- significantly 12 between trols, IR andcontrols, INR. and INR. Concretely, Concretely, theARTsizewith of HDL size particles ofwas HDL particles significantly was significantly higher in those indi- higher in those individuals starting less than 200 cells/µ L than in controls (21.38 higher viduals instarting those individuals ART starting thanART 200 with less than than 200 cells/µ L(21.38 than in controls (21.38 (17.87–24.34) µ mol/L andwith 19.56less (14.80–21.86) cells/µL in controls µ mol/L, respectively; (17.87–24.34) p = 0.047), probably due µmol/L (17.87–24.34) µ mol/L and 19.56 (14.80–21.86) µ mol/L, respectively; p = 0.047), probably due toand the 19.56 (14.80–21.86) differences µmol/L, in the number respectively; of medium p = 0.047), HDL particles probably (6.15 dueµ to (4.74–7.80) the and mol/L differences in to the differences in the number of medium HDL particles (6.15 (4.74–7.80) µ mol/L and the(4.26–6.44) 5.07 number ofµmedium HDL particles mol/L, respectively, (6.15 Indeed, p = 0.06). (4.74–7.80) µmol/L although the and 5.07of number (4.26–6.44) medium µmol/L, 5.07 (4.26–6.44) HDL particles was µ mol/L, respectively, p = 0.06).INR Indeed, although the numberµ mol/L) of medium respectively, p = significantly 0.06). Indeed, higher in IR and although the number at month 12 (6.64 (5.1–7.71) of medium HDL particles was signifi- HDL comparedparticles was to controls, significantly only higher theatdifferences in IR and between INR at month IR andµmol/L) 12 (6.64 (5.1–7.71) controls compared µ mol/L) were statistically cantly higher in IR and INR month 12 (6.64 (5.1–7.71) to controls, only compared significant to controls, (p = 0.015) only (FigureIR3A).the differences between IR and controls were statistically the differences between andCorrelation controls were analyses confirmed statistically the negative significant (p association = 0.015) (Figure 3A). significant between CD4 (p+ =T-cell 0.015) (Figure counts and 3A). HDL Correlation size at monthanalyses 12 ( confirmed = −0.335, p the = negative 0.003) association (Figure 3B). Correlation analyses confirmed the negative association between CD4+ T-cell counts and between CD4 + T-cell counts and HDL size at month 12 ( = −0.335, p = 0.003) (Figure 3B). At month 36, no statistically differences were found in lipid concentration. For both HDL size at month 12 (ρ = −0.335, p = 0.003) (Figure 3B). At month 36, no statistically At month sizing lipoprotein 36, noand statistically differences particle number were found in lipid concentration. For both determination. differences were found in lipid concentration. For both lipoprotein sizing and particle lipoprotein sizing and particle number determination. number determination. A) B) A) B) 8 ✱ Control (μmol/L) IR 8.5 8 ✱ INRControl (μmol/L) IR 8.5 6 INR 8.4 HDL size (μmol/L) Particles 6 8.4 HDL size (μmol/L) 8.3 (ρ=-0.335, P=0.003) Particles 4 8.3 (ρ=-0.335, P=0.003) HDL HDL 8.2 4 2 Medium 8.1 8.2 2 Medium 0 8.0 8.1 12 months 0 500 1000 0 8.0 + CD4 T-cell count (cells/μL) 12 months 0 500 1000 + Figure 3. The negative relationship between HDL particle size CD4andT-cell CD4count (cells/μL) + T-cell count. (A) HDL medium-size particle distribution (μmol/L) at month 12 in each studied group +(control group Figure 3. The starting negative relationship between HDL particle size and who CD4presented T-cell count. (A) HDL Figure first ART 3. The with more negative than 350 cells/µ relationship L, immunological between HDL particleresponders size and (IR) CD4+ T-cell count.CD4 (A)+ HDL medium-size T-cell count particle equal to or distribution greater than (µmol/L) 250 cells/µ Lat atmonth month 12 36 in andeach studied immunologicalgroup (control group non-responders medium-size particle distribution (μmol/L) at month 12 in each studied group (control group starting first ART with + starting first ARTmore with than more350 thancells/µL, immunological 350 cells/µ L, immunologicalresponders (IR) responders who (IR) presented who presentedCD4 CD4+ T-cell T-cell countcount equalequal to orto or greater greater thanthan 250 250 cells/µatL month cells/µL at month 36 36 and and immunologicalnon-responders immunological non-responders (INR) who did not reach more than 250 cell/µL CD4+ T-cell at month 36). Mann–Whitney nonparametric test * p < 0.05. (B) Spearman correlation analysis between HDL size (µmol/L) and CD4+ T-cell counts (cells/µL) in all individuals (n = 74) (ρ = −0.335, p = 0.003). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 13 Int. J. Mol. Sci. 2022, 23, 8071 5 of 12 (INR) who did not reach more than 250 cell/µ L CD4+ T-cell at month 36). Mann–Whitney nonparametric test * p < 0.05. (B) Spearman correlation analysis between HDL size (μmol/L) and CD4+ T-cell counts (cells/µ L) in all individuals (n = 74) ( = −0.335, p = 0.003). 1 2.3. Lipoprotein Profile Evolution in PLHIV Based on the 2D H-NMR Liposcale Test As mentioned 2.3. Lipoprotein before, the Profile Evolution baseline in PLHIV lipoprotein Based signature on the 2D 1H-NMR basedTest Liposcale on the two-dimensional spectra from the diffusion-ordered As mentioned before, the baseline NMR spectroscopy lipoprotein (DOSY) signature basedexperiment on the two-was previously described for dimensional INRfrom spectra andtheIRdiffusion-ordered participants [9].NMRAt baseline, spectroscopytotal particles/HDL-P (DOSY) experiment and LDL- was previously P/HDL-P described ratios for INR andand were 1.42-times IR participants 1.39-times[9]. At baseline, higher in INRtotal particles/HDL- than in IR, respectively. Thus, Ptoand LDL-P/HDL-P analyze ratios were the lipoprotein 1.42-times profile and during evolution 1.39-times ARThigher in INR than follow-up, in IR, the lipoprotein profile respectively. Thus, to analyze the lipoprotein profile evolution during ART follow-up, the obtained during ART was normalized by dividing each item (month 12 and month 36) by lipoprotein profile obtained during ART was normalized by dividing each item (month its respective baseline values. 12 and month 36) by its respective baseline values. 2.3.1.Lipoprotein Lipoprotein Profile Evolution + 2.3.1. Profile Evolution in in PLHIV PLHIV Starting Starting ARTART withwith MoreMore thanthan 350 CD4 350 CD4 + T- T-Cells/µL Cells/µIncreases L in the mean total cholesterol (1.27 times, p < 0.001) and total triglycerides (1.26 times, in Increases p= the0.013) mean were found when total cholesterol (1.27lipids times, pwere assessed < 0.001) and total with NMR in the control triglycerides group. (1.26 Concretely, times, a higher p = 0.013) were foundcontent of cholesterol when lipids were assessedin LDL with and NMRHDL in theparticles control was found group. to be Concretely, statistically a higher content significant of cholesterol between month in LDL 12 andand month HDL particles 36 (p was found = 0.013 and 0.030, re- tospectively) be statistically significant (Figure 4A). Thebetween month of distribution 12 LDL and month 36 (p =compared composition 0.013 and to 0.030, baseline values respectively) (Figure 4A). The distribution of LDL composition compared to baseline (LDL-P (p = 0.002)) was as follows: the large LDL-P was 1.29 times increased (p = 0.003), values (LDL-P (p = 0.002)) was as follows: the large LDL-P was 1.29 times increased (p = the medium LDL-P was 1.36 times increased (p = 0.002) and the small LDL-P was 1.31 times 0.003), the medium LDL-P was 1.36 times increased (p = 0.002) and the small LDL-P was increased 1.31 (p = 0.001). times increased In fact,Inall (p = 0.001). LDL-sized fact, particles all LDL-sized showed particles showeda asignificant significant increase be- increase between month 12 and month 36 (Figure 4C). However, the distributionofofHDL tween month 12 and month 36 (Figure 4C). However, the distribution HDL composition (HDL-P (p (HDL-P composition = 0.003))(p compared to baseline = 0.003)) compared values to baseline was was values as follows: as follows:thethelarge large HDL-P was 1.35 times HDL-P increased was 1.35 (p = 0.004), times increased the medium (p = 0.004), the mediumHDL-P HDL-P waswas1.22 1.22times increased (p = 0.003) times increased (pand = 0.003) and the the small small HDL-P HDL-P wastimes was 1.21 1.21 times increased increased (p =(p0.011). = 0.011).InInthis thiscase, case, only onlythethe large-sized large-sized HDL particles were found to be significantly increased between HDL particles were found to be significantly increased between month 12 and month 36 month 12 and month 36 (p = 0.021) (Figure 4D). (p = 0.021) (Figure 4D). Figure 4. Cholesterol and triglyceride content and lipoprotein particle size distribution. (A) Distribu- Figure 4. Cholesterol and triglyceride content and lipoprotein particle size distribution. (A) tion of cholesterol Distribution content of cholesterol in in content VLDL, VLDL,LDL LDL and HDLatatmonth and HDL month 12 and 12 and month month 36 in 36 in the the control group control starting first ART with more than 350 cells/µL. (B) Distribution of triglyceride content in VLDL, LDL and HDL at month 12 and month 36. (C) Lipoprotein particle size distribution of LDL particles (large, medium and small (nmol/L)) at month 12 and month 36 (D) Lipoprotein particle size distribution of HDL particles (large, medium and small (µmol/L)) at month 12 and month 36. Cholesterol content (mg/dL), triglyceride content (mg/dl), LDL particle size (mmol/L) and HDL particle size (µmol/L) at month 12 and month 36 were normalized by dividing each item by its respective baseline value to obtain the ration for each value represented by bars (mean ± SEM). The Wilcoxon paired test was performed to evaluate differences between month 12 and month 36 * p < 0.05. Cholesterol content (mg/dL), triglyceride content (mg/dl), LDL particle size (mmol/L) and HDL particle size (µ mol/L) at month 12 and month 36 were normalized by dividing each item by its respective baseline value to obtain the ration for each value represented by bars (mean ± SEM). The Wilcoxon paired test was performed to evaluate differences between month 12 and month 36 * p < 0.05. Int. J. Mol. Sci. 2022, 23, 8071 6 of 12 2.3.2. Lipoprotein Profile Evolution in PLHIV Starting ART with Less than 200 CD4+ T- Cells/µ L 2.3.2. Lipoprotein Profile Evolution in PLHIV Starting ART with Less than 200 CD4+ T-Cells/µL Immunological Responders (IR) Immunological Responders (IR) IR participants showed a higher mean total cholesterol at month 36 compared to baselineIRvalues participants due to showed a higher a significant mean increase total in the cholesterol content at month of cholesterol 36 lipoproteins in all compared to baseline (1.25 timesvalues due(p in VLDL to=a0.014), significant which increase in the was 1.40 content times in LDLof (p cholesterol in all < 0.001) and lipoproteins 1.36 times in HDL (p < 0.001)). However, no significant differences were found in the cholesterol times (1.25 times in VLDL (p = 0.014), which was 1.40 times in LDL (p < 0.001) and 1.36 and in HDL (p content triglyceride < 0.001)). However, between month no12significant and month differences 36 (Figurewere 5A,B). found in the Similar cholesterol to the control and triglyceride group, content the distribution of between large HDL month 12 and particles month 36 increased (Figure from month5A,B). 12 to Similar monthto36,the control group, the distribution of large HDL particles increased although the differences were not statistically significant (Figure 5D). from month 12 to month 36, although the differences were not statistically significant (Figure 5D). Cholesterol and Figure5.5. Cholesterol Figure and triglyceride triglyceridecontent contentandandlipoprotein particle lipoprotein size size particle distribution. (A) Distri- distribution. (A) bution of cholesterol content in VLDL, LDL and HDL at month 12 and Distribution of cholesterol content in VLDL, LDL and HDL at month 12 and month 36 inmonth 36 in immunological responders (IRs) immunological who presented responders (IRs) whowith a CD4+ T-cell presented with acount CD4+equal T-cellto or greater count equal than to or 250 cell/µL greater thanat 250 cell/µ36. month L at (B)month 36. (B) of Distribution Distribution triglycerideofcontent triglyceride content in VLDL, LDL in VLDL, and HDL LDL and 12 at month HDLandatmonth month36. 12(C) and month 36.particle Lipoprotein (C) Lipoprotein particle of size distribution size distribution LDL of LDLmedium particles (large, particlesand (large, smallmedium (nmol/L))andat small month(nmol/L)) at month 12 and month 12 Lipoprotein 36. (D) and month particle 36. (D) size Lipoprotein particle distribution sizeparticles of HDL distribution (large,ofmedium HDL particles and small (large, medium (µmol/L)) and small at month (μmol/L)) 12 and month 36. at Cholesterol month 12 and month content 36. Cholesterol (mg/dL), triglyceridecontent content (mg/dL), triglyceride content (mg/dL), LDL particle size (mmol/L) and HDL particle size (µ mol/L) (mg/dL), LDL particle size (mmol/L) and HDL particle size (µmol/L) at month 12 and month 36 were normalized by dividing each item by its respective baseline value and ratios are represented by bars (mean ± SEM). The Wilcoxon paired test was performed to evaluate differences between month 12 and month 36. Immunological Non-Responders (INR) Similar to controls and IR participants, in INR, the mean LDL cholesterol (p = 0.022) and HDL cholesterol (p = 0.001) were higher at month 36 when compared to baseline values (both were 1.53 times increased). Interestingly, in that case, a significant increase in the distribution of some LDL particles and some HDL particles was observed. As previously observed in controls, medium LDL particles (p = 0.047) and large HDL particles (p = 0.041) Immunological Non-Responders (INR) Similar to controls and IR participants, in INR, the mean LDL cholesterol (p = 0.022) and HDL cholesterol (p = 0.001) were higher at month 36 when compared to baseline values (both were 1.53 times increased). Interestingly, in that case, a significant increase Int. J. Mol. Sci. 2022, 23, 8071 in the distribution of some LDL particles and some HDL particles was observed.7 As of 12 previously observed in controls, medium LDL particles (p = 0.047) and large HDL particles (p = 0.041) were significantly higher at month 36 than at month 12 (Figure 6C,D). Of interest, small HDL particles (p = 0.047) were significantly higher at month 36 than at were significantly month higher at month 36 than at month 12 (Figure 6C,D). Of interest, small 12 (Figure 6D). HDL particles (p = 0.047) were significantly higher at month 36 than at month 12 (Figure 6D). Figure 6. Cholesterol and triglyceride content and lipoprotein particle size distribution. (A) Distri- Figure bution 6. of Cholesterol and triglyceride cholesterol content in VLDL, LDL content and at and HDL lipoprotein month 12 andparticle monthsize36distribution. in immunological (A) Distribution of cholesterol content in VLDL, LDL and HDL at month + 12 and non-responders (INR) who did not reach more than 250 cell/µL CD4 T-cell at month 36. (B) Distri- month 36 in immunological non-responders (INR) who did not reach more than 250 cell/µ L CD4 bution of triglyceride content in VLDL, LDL and HDL at month 12 and month 36. (C) Lipoprotein + T-cell at month 36. (B) Distribution of triglyceride content in VLDL, LDL and HDL at month 12 and month 36. (C) particle size distribution of LDL particles (large, medium and small (nmol/L)) at month 12 and Lipoprotein particle size distribution of LDL particles (large, medium and small (nmol/L)) at month month 36. (D) Lipoprotein particle size distribution of HDL particles (large, medium and small 12 and month 36. (D) Lipoprotein particle size distribution of HDL particles (large, medium and (µmol/L)) small at month (μmol/L)) 12 and12month at month 36. Cholesterol and month content (mg/dL), 36. Cholesterol triglyceride content (mg/dL), content (mg/dL), triglyceride content LDL-particle (mg/dL), size (mmol/L) LDL-particle and HDL-particle size (mmol/L) size (µmol/L) and HDL-particle size (µat month mol/L) at12 and month month 12 and36month were nor- 36 malized were by dividing normalized each item by dividing eachbyitem its respective baselinebaseline by its respective value and valueratios andare represented ratios by bars are represented by bars ± (mean (mean SEM). ± The SEM). The Wilcoxon Wilcoxon paired paired test wastest was performed performed to evaluate to evaluate differencesdifferences between between month 12 month 12 and36month and month 36 * p < 0.05. * p < 0.05. 3.3.Discussion Discussion Impaired immune function was associated with markers of preclinical atherosclerosis and vascular dysfunction in PLHIV [8]. Notably, advanced HIV infection (low CD4+ counts) linked to chronic inflammation and increased immune activation was associated with the alteration of metabolic parameters related to lipid metabolism and increased atherogenic risk [8,19]. Indeed, in naïve PLHIV, we previously reported an increased CV risk in INR compared to IR, using the lipoprotein signature measured by a 2D 1 H- NMR (Liposcale® ) [9]. Because ART was associated with an increased risk of coronary artery inflammation and atherosclerosis compared to the healthy population [4,6,7], we considered it critical to reevaluate the CVD risk from our previously studied cohort of INR and IR after starting ART. Thus, the new analyses were performed with a total of 53 PLHIV from the previous cohort of 64 individuals [9] who were successfully followed up with ART until month 36 and had an available sample for the 2D 1 H-NMR technique. Both INR and IR were compared to a group of PLHIV who initiated ART with optimal values Int. J. Mol. Sci. 2022, 23, 8071 8 of 12 of CD4+ T-cell count and maintained the same period of stable ART at follow-up. Our findings corroborated that the initiation of ART progressively increased LDL-P and LDL-C concentrations in HIV-positive patients. However, both large-sized HDL-P and small-sized HDL-P were noticeably increased from month 12 to month 36 in INR. Remarkably, at month 12, medium HDL particle concentrations were significantly higher in PLHIV starting ART with low CD4+ T-cell counts. The negative correlation between CD4+ T-cell count and the medium HDL-P could indicate the importance of HDL-P in poor immune recovery status, in agreement with previously published data [20]. Both total particles/HDL particles and LDL particles/HDL particles ratios provide additional predictive information over other used cardiovascular biomarkers [19,21]. No- tably, at month 12 and month 36, no significant differences were observed among the groups. The greater predictive value observed in naïve INRs was dissipated with the initiation of ART, suggesting that the introduction of ART could hit PLHIV alike with no dependence on immune status. Moreover, from the recommended ART combinations, the use of NNRTI + PI seemed to have a more negative impact on CVD, although the results were not statistically significant, probably due to the low number of individuals affected by this ART combination. Both LDL-P concentrations and LDL-C were increased between month 12 and month 36 in controls and INRs, although the differences were only significant in the control group. Of note, medium-sized LDL-P was significantly higher in controls and INRs, whereas large-sized LDL-P and small-sized LDPL-P were significantly higher only in the control group. Although IRs showed no significant differences between month 12 and month 36, high concentrations of both LDL-P and LDL-C were observed during all ART follow-ups. Higher concentrations of LDL-P and LDL-C are considered to be atherogenic. Previous prospective studies showed that the LDL-P number had a better discriminatory role in cardiometabolic risk than LDL-C concentrations since LDL-C could be a consequence of the initiation of the antiretroviral regimen [22]. However, a decrease in large-sized LDL-P followed by an increase in medium- or small-sized LDL-P was previously related to a two-fold and 40% increase in risk for coronary heart disease, respectively [23]. Indeed, small, dense LDL particles have been proven to confer greater atherogenic risk than larger, less dense LDL particles [15]. Large HDL-P concentrations increased from month 12 to month 36 in all HIV-positive patients, whereas in the INR, there was not only a progressive increase in large HDL-P concentrations but also in small HDL-P concentrations. Plasma concentrations of specific HDL subpopulations could be strongly associated with cardiovascular risk when compared to total HDL particle number [24]. Several recent studies have shown the atheroprotective properties of large HDL particles when compared to small and medium HDL particles [25], which typically reveal positive correlations with risk. Missing information regarding inflammatory-related parameters, immunological re- covery factors (CD4/CD8 ratio) and important cardiovascular clinical parameters were the major limitations of the present study. However, connecting information on lipidomics from previous omics studies was also challenging because of the interassay bias that was minimized by the normalization of the data. Moreover, it could also be interesting to continue the follow-up for a longer period for a better conclusion. Validation studies are needed. 4. Materials and Methods 4.1. Study Design and Participants This was a multicenter, prospective cohort study comprising all adult HIV-1-infected individuals who started first ART between 2009 and 2011 and were followed up at the HIV outpatient clinics of the participating hospitals: Hospital Joan XXIII (Tarragona), Hospital de la Santa Creu i Sant Pau (Barcelona) and Hospital Virgen del Rocío (Sevilla). Of the initial cohort of 64 participants [9], only 53 HIV patients had available follow-up datasets at both month 12 and month 36 on ART (Figure 7). According to the previous immune datasets at both month 12 and month 36 on ART (Figure 7). According to the previous immune recovery classification, the two groups were categorized based on their CD4+ T- cell count after 36 months of being on stable ART: the immunological responder group when their CD4+ T-cell count was equal to or greater than 250 cells/µ L (IR) and immunological non-responders (INR), if they did not reach this threshold. A group of 21 Int. J. Mol. Sci. 2022, 23, 8071 HIV individuals starting with first ART with more than 350 cells/µ L and then maintaining 9 of 12 the same cutoff of CD4+ T-cell count at month 36 was added as a control group. All the selected patients were required to fulfil the following inclusion criteria: aged ≥ 18 years, presence of HIV-1 infection, on an ART regimen during the 36 months of the recovery study andclassification, the two undetectable plasma groups HIV-1 viralwere load categorized at 36 months.based CD4+ that on theircriteria The exclusion T-cell count could36 after potentially months of bias the results being of the on stable study ART: thewere the presence of immunological active opportunistic responder group when their CD4 + T-cellcurrent infections, inflammatory count was equal to ordiseases greaterorthan conditions that could 250 cells/µL develop (IR) and a CVD, immunological non- responders (INR), if they did not reach this threshold. A group of 21 HIVT-cell changes in the ART regimen and the use of drugs known to affect/modify CD4⁺ individuals counts (antineoplastic starting with first ARTdrugs, steroids, with more than immune response 350 cells/µL modulators, and then and the maintaining colony- same cutoff stimulating + factors, among others). of CD4 T-cell count at month 36 was added as a control group. Figure 7. Figure 7. Flowchart Flowchart illustrating illustratingthe thestudy studydesign designandandanalysis. analysis.(A) (A)From the From initial the cohort initial cohortof of 6464 PLHIV PLHIV starting ART with less than 200 cells/µ L, 53 had available follow-up datasets at both starting ART with less than 200 cells/µL, 53 had available follow-up datasets at both month 12 andmonth 12 and month 36 on ART. The group of 53 PLHIV were categorized based on their CD4+ T-cell count month 36 on ART. The group of 53 PLHIV were categorized based on their CD4+ T-cell count after after 36 months of being on stable ART: immunological non-responders (INR) when their CD4+ T- 36 months of being on stable ART: immunological non-responders (INR) when their CD4+ T-cell count was less than 250 cells/µL and immunological responders (IR) when their CD4+ T-cell count was equal to or greater than 250 cells/µL. A group of 21 PLHIV starting ART with more than 350 cells/µL was added as a control group. Blood samples were obtained at baseline, and at month 12 and month 36 on ART. (B) Using Liposcale® tests (2D 1H-NMR), the lipoprotein profile (including cholesterol (chol.) and triglycerides (TG) concentrations, sizes, particle numbers for each lipoprotein and particle numbers of nine subclasses, namely large, medium, and small VLDL, LDL, and HDL, respectively) was obtained at month 12 and month 36 after starting ART. Before statistical analyses, data at month 12 and data at month 36 were normalized by dividing each item by its respective baseline value. Workflow created with BioRender.com. All the selected patients were required to fulfil the following inclusion criteria: aged ≥ 18 years, presence of HIV-1 infection, on an ART regimen during the 36 months of the study and undetectable plasma HIV-1 viral load at 36 months. The exclusion criteria that could potentially bias the results of the study were the presence of active opportunistic infections, current inflammatory diseases or conditions that could develop a CVD, changes in the ART regimen and the use of drugs known to affect/modify CD4+ T-cell counts (antineoplastic drugs, steroids, immune response modulators, and colony-stimulating factors, among others). 4.2. Laboratory Measurements Blood was drawn from a peripheral vein after an overnight fast, collected in ethylene- diaminetetraacetic acid (EDTA) tubes and immediately centrifuged for 15 min at 4 ◦ C and 1500× g. Plasma samples were then stored at −80 ◦ C at the BioBanc IISPV until further analysis. Int. J. Mol. Sci. 2022, 23, 8071 10 of 12 4.3. D 1H-NMR Liposcale Test The 2D 1 H-NMR Liposcale test (Biosfer Teslab, Reus, Spain) was previously used to characterize INR and IR participants before starting ART [9]. The Liposcale test provides lipid concentrations (triglycerides and cholesterol), sizes, and particle numbers for VLDL, LDL, and HDL classes, as well as the particle numbers of nine subclasses, namely large, medium, and small VLDL, LDL, and HDL, respectively. The test was based on two- dimensional spectra (2D) from diffusion-ordered NMR spectroscopy (DOSY) experiments to decompose the (CH3 ) proton resonances of the lipoprotein particles (BrukerAvance III 600 spectrometer (Bruker GmbH, Karlsruhe, Germany), operating at a proton frequency of 600.20 MHz (14.1 T), at 310 K) into nine Lorentzian functions [12]. The methyl signal was surface-fitted and the number of functions was increased to account for the nine lipoprotein subclasses. Cholesterol and triglyceride concentrations of the main lipoprotein fractions (VLDL, LDL and HDL) were predicted using partial least squares (PLS) regression models. The NMR functions were associated with a given lipoprotein class according to their associated NMR size. The main lipoproteins fractions were defined as VLDL (38.6–81.9 nm), LDL (18.9–26.5 nm) and HDL (7.8–11.5 nm). The mean particle size of every main fraction (VLDL, LDL and HDL) was derived by averaging the NMR area of each fraction by its associated size. Particle-weighted lipoprotein size was obtained by dividing each NMR area by its associated volume. For each lipoprotein class, the mean particle size was obtained by multiplying the NMR lipoprotein particle size by its fractional particle concentration relative to the total particle concentration of a given class [12]. The particle numbers of each lipoprotein main fraction were calculated by dividing the lipid volume by the particle volume of a given class. The lipid volumes were determined by using common conversion factors to convert concentration units obtained from the PLS model into volume units. The relative areas of the lipoprotein components used to decompose the 2D spectra were used to derive the particle numbers of the nine lipoprotein subclasses. 4.4. Statistical Analysis All data were tested for normality using the Kolmogorov–Smirnov test before the statistical analyses were applied. A descriptive data analysis of patient characteristics was carried out using percentages for categorical variables and medians and interquartile ranges (25th percentiles–75th percentiles) for continuous variables. The “times increased” data were calculated as a subtraction between the results at month 36 and the respective baseline values. Differences between the groups of the study were assessed through the nonparametric Kruskal–Wallis (KW) and/or Mann–Whitney test for continuous unpaired variables. CD4+ T-cell intragroup results among the different time points studied (baseline, month 12 and month 36) were compared using the Friedman and Wilcoxon test for paired samples. To analyze the lipoprotein profile evolution during ART follow-up, the Liposcale test results at month 12 and month 36 were normalized by dividing each item by its respective baseline value. Correlation analysis between CD4+ T-cell counts (cells/µL) and lipoprotein items was performed using the Spearman correlation test. The statistical software used was SPSS Software v22 (IBM, Madrid, Spain). Graphs were generated using GraphPad Prism (GraphPad Prism version 5.0 for Windows, GraphPad Software, San Diego, CA, USA). p values less than 0.05 were considered statistically significant. 5. Conclusions The present results corroborated the applicability of nuclear magnetic resonance (NMR)-based advanced lipoprotein tests in a population with chronic infection. The initiation of ART progressively increased HDL-P and LDL-P in HIV-positive patients whereas VLDL particles were only increased in IR compared to its baseline values. In INR, ART initiation dissipates the greater CVD risk previously observed at baseline (ART-naïve) when compared with IR. Concretely, in INR, ART follow-up increases large HDL but also large and small LDL-Ps, which denoted a conflict between atherogenic and proatherogenic factors. Additionally, there is a negative correlation between CD4+ T-cell counts and Int. J. Mol. Sci. 2022, 23, 8071 11 of 12 medium HDL-P concentrations, corroborating its immunomodulatory role but also its controversial function in CVD. A proper balance between a proatherogenic and atherogenic environment is related to the reconstitution of CD4+ T-cell count. Further investigations are needed to elucidate the mechanisms by which the different lipoproteins could be associated to immune response in PLHIV. Author Contributions: The author’s contributions are as follows: Conceptualization, J.M., R.J., F.V. and A.R.; methodology, J.M., R.J., V.A. and A.R.; software, J.M. and A.R.; validation, M.L.-D., C.V., S.V., G.G.-P., M.O., F.G.-B., and J.P.; formal analysis, J.M., C.V., V.A., M.O., F.V., J.P. and A.R.; investigation, J.M., R.J., F.V. and A.R.; resources, M.L.-D., P.D., Y.M.P., E.M., F.V. and A.R.; data curation, J.M., R.J., C.V., S.V. and M.O.; writing—original draft preparation, J.M. and A.R.; writing—review and editing, J.M., P.D., Y.M.P., E.M., F.V. and A.R.; visualization, J.M., R.J., J.P. and A.R.; supervision, R.J., F.V., J.P. and A.R.; project administration, F.V., J.P. and A.R.; funding acquisition, F.V., J.P. and A.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Fondo de Investigacion Sanitaria [PI16/00503, PI19/01337 and PI20/00326]-ISCIII-FEDER (co-funded by the European Regional Development Fund/European Social Fund; “A way to make Europe”/”Investing in your future”); Programa de Suport als Grups de Recerca AGAUR (2017SGR948), the SPANISH AIDS Research Network [RD16/0025/0006]-ISCIII- FEDER (Spain) and the Centro de Investigación Biomédica en Red de Enfermedades Infecciosas-ISCIII (CIBERINFEC) [CB21/13/00020], Madrid, Spain. JM is supported by the Universitat Rovira I Virgili under grant agreement “2019PMF-PIPF-18”, through the call “Martí Franquès Research Fellowship Programme”. FV is supported by grants from the Programa de Intensificación de Investigadores (INT20/00031)-ISCIII and by “Premi a la Trajectòria Investigadora dels Hospitals de l’ICS 2018”. AR is supported by a grant from IISPV through the project “2019/IISPV/05” (Boosting Young Talent), by GeSIDA through the “III Premio para Jóvenes Investigadores 2019” and by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CP19/00146” through the Miguel Servet Program. Institutional Review Board Statement: The studies involving human participants were reviewed, approved and performed according to the recommendations of the Ethical Committee for Clinical Research following the rules of Good Clinical Practice from the Institut d’Investigació Sanitària Pere Virgili (CEIm IISPV, ref. 041/2018). The CEIM IISPV is an independent committee made up of health and non-health professionals that supervise the correct compliance of the ethical principles governing clinical trials and research projects that are carried out in our region, specifically in terms of methodology, ethics and laws. Informed Consent Statement: Written informed consent has been obtained from each participant in agreement with the Declaration of Helsinki. Data Availability Statement: The data that support the finding of this study are available from the corresponding author upon reasonable request. Acknowledgments: This study would not have been possible without the collaboration of all the patients and medical and nursing staff who have taken part in the project. We also acknowledge the BioBanc IISPV (B.0000853 + B.0000854) integrated into the Spanish National Biobanks Platform (PT20/00197), the Biosfer Teslab (a spin-off company of Universitat Rovira i Virgili (URV)), the CERCA Programme (Generalitat de Catalunya) and the Institut d’Investigacions Sanitàries Pere Virgili (IISPV). Conflicts of Interest: The authors declare no conflict of interest. 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